hLXRα (EC50 = 190 nM); hLXRβ(EC50 = 30 nM)[4]

In vitro, GW3965 hydrochloride induces GBM cell death with increased effectiveness in tumor cells that express EGFRvIII. GW3965 hydrochloride decreases LDLR levels while upregulating the expression of the E3 ubiquitin ligase IDOL and the cholesterol transporter gene ABCA1[2]. Platelet aggregation and calcium mobilization induced by collagen or CRP are inhibited by LXR ligands. When platelets are activated with 1 μg/mL CRP, GW3965 hydrochloride (1 or 5 μM) exhibits a slight inhibitory effect on fibrinogen binding and P-selectin exposure. GW3965 hydrochloride (10 μM) and T0901317 (40 μM) at greater concentrations, however, decrease the amounts of fibrinogen and P-selectin on the platelet surface[3].

---

The LXR Agonist GW3965 Promotes GBM Cell Death In Vitro with Enhanced Efficacy in EGFRvIII-Expressing Tumor Cells [2]
Intracellular cholesterol levels can be regulated through (1) uptake of LDL through LDLR, (2) efflux of cholesterol through ABCA1 or ABCG1 transporters, and (3) hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase)–dependent synthesis. Given the ability of pharmacologic LXR activation to limit intracellular cholesterol availability, we hypothesized that synthetic LXR agonists might inhibit the growth and survival of GBM cells. Indeed, treatment of U87 and U87-EGFRvIII GBM cells for 4 days with the LXR agonist GW3965 resulted in dose-dependent inhibition of growth and promotion of tumor cell death. Moreover, consistent with the enhanced dependence of EGFRvIII-bearing tumor cells on exogenous cholesterol, these cells exhibited markedly greater cell death than did those of the parental U87 cell line (Fig. 4A–D). Remarkably, tumor cell death was dose-dependently rescued by the addition of LDL (Fig. 4E–G), strongly suggesting that the tumoricidal effects of GW3965 were mediated through alteration of cellular cholesterol availability. [2]

---

To uncover the mechanism by which GW3965 induced tumor cell death, real-time PCR and immunoblot analyses for the LXR target genes ABCA1 and IDOL were performed. GW3965 treatment promoted dose-dependent increases in ABCA1 and IDOL, with a concomitant decrease in LDLR protein level (Fig. 5A–D; Supplementary Fig. S5). Unfortunately, no antibodies capable of detecting endogenous IDOL expression are available. The regulation of cholesterol efflux via ABCA1 is a 1-step process; ABCA1 is a direct transcriptional target of LXR. In contrast, LDLR regulation by LXR requires transcription and translation of IDOL, followed by ubiquitin-mediated degradation of LDLR. GW3965-mediated LDLR degradation in GBM cells took longer, and required a higher drug dose than did ABCA1 induction (Fig. 5C and D). The effects of GW3965 on ABCA1 and LDLR expression were confirmed across a panel of GBM and other cancer cell lines for which LDLR levels were linked with high levels of EGFR phosphorylation (Fig. 5E). Of interest, the dose of GW3965 required to promote cell death (Fig. 4B–D) correlated well with that required to accomplish LDLR degradation (Fig. 5C and D). Together, these results suggest that decrease of LDLR levels is required for the tumoricidal activity of GW3965. [2]

To directly test whether LDLR degradation was required for GBM cell death in response to GW3965, we measured the effect of lentiviral LDLR shRNA knockdown or scrambled control on sensitivity to the drug. Low-dose GW3965 (1 or 2 μM) induced ABCA1 but did not diminish LDLR expression or cause GBM cell death (Fig. 6A–C). Lentiviral delivery of LDLR shRNA resulted in LDLR knockdown, potently promoting tumor cell death upon low-dose GW3965 treatment (Fig. 6A–C). To examine the role of IDOL-mediated LDLR degradation in promoting this apoptotic response (Fig. 6D), we measured the effect of adenoviral delivery of IDOL on sensitizing U87-EGFRvIII GBM cells to low-dose GW3965. Phenocopying the effect of LDLR knockdown, IDOL overexpression potently sensitized GBM cells to low-dose GW3965 (Fig. 6E and F). Neither LDLR knockdown alone nor IDOL overexpression alone was sufficient to promote GBM cell death (Fig. 6). These findings indicate that IDOL-mediated degradation of LDLR is an important component of the mechanism of GW3965-induced GBM cell death. However, the observation that targeting LDLR alone is not sufficient to elicit GBM cell death indicates that additional mechanisms, such as the promotion of ABCA1-dependent cholesterol efflux, also contribute[2].

---

GW3965 caused LXR to associate with signaling components proximal to the collagen receptor, GPVI, suggesting a potential mechanism of LXR action in platelets that leads to diminished platelet responses. Activation of platelets at sites of atherosclerotic lesions results in thrombosis preceding myocardial infarction and stroke. Using an in vivo model of thrombosis in mice, we show that GW3965 has antithrombotic effects, reducing the size and the stability of thrombi. The athero-protective effects of GW3965, together with its novel antiplatelet/thrombotic effects, indicate LXR as a potential target for prevention of athero-thrombotic disease. [3]

---

In an effort to further increase the LXRα activity of the tertiary amines, an array of 1280 carboxamides were synthesized using Rink amide linker and screened for activity in the LXRα/SRC1 LiSA at 1 μM. Six carboxamides (6−11) were identified from the array with activity less than 1 μM in the LXRα/SRC1 LiSA (Table 1). Several of these analogues contained a m-trifluoromethyl functionality in the benzylamine substituent (9−11), with the 2-chloro-3-trifluoromethylbenzylamine 11 identified as the most potent analogue with an EC50 of 45 nM in the LXRα/SRC1 LiSA. As was seen in the earlier series of carboxamides, 11 showed a reduction in potency when tested in the cell-based LXRα-GAL4 reporter gene assay. Since cellular potency had been improved through conversion of amide 4 to carboxylic acid 5, the corresponding carboxylic acid 12 was synthesized using the Sasrin linker. Carboxylic acid 12/GW3965 showed an EC50 of 125 nM in the LXRα/SRC1 LiSA with comparable efficacy to EPC (1) for recruitment of the SRC1 peptide. To our delight, GW3965/12 maintained its potency in the LXRα cell-based reporter gene assay with an EC50 of 190 nM (Table 1, Figure 1B). When screened against a panel of nuclear receptors, 12 showed cross reactivity with only LXRβ (Figure 1A) and the pregnane X receptor (PXR) (data not shown). Full dose−response analysis on LXRβ- and PXR-GAL4 chimeras showed 12 was >10-fold selective for activation of LXR compared to PXR (Figure 1B). Thus, carboxylic acid 12 is a potent LXR agonist with good cellular activity and excellent selectivity over other nuclear receptors [4].

The CNS of non-pathological animals does not experience the elevation in neuroactive steroids that GW3965 hydrochloride causes in the spinal cord, cerebellum, and cerebral cortex of STZ-rats. When diabetic animals are treated with GW3965 hydrochloride, their spinal cords express more myelin basic protein and have higher levels of dihydroprogesterone[1]. In vivo, GW3965 hydrochloride (40 mg/kg, po) significantly increases GBM cell death by a factor of 25 and robustly promotes ABCA1 and decreases LDLR expression[2]. Additionally, it inhibits tumor growth by 59%. In vivo bleeding duration is prolonged and platelet thrombus development is regulated by GW3965 hydrochloride (2 mg/kg, IV)[3].

---

It was observed that the treatment with either Ro5-4864 (i.e., a ligand of TSPO) or with GW3965 (i.e., a ligand of LXRs) induced an increase of neuroactive steroids in the spinal cord, the cerebellum and the cerebral cortex of STZ-rats, but not in the CNS of non-pathological animals. Interestingly, the pattern of induction was different among the three CNS areas analyzed and between the two pharmacological tools. In particular, the activation of LXRs might represent a promising neuroprotective strategy, because the treatment with GW3965, at variance to Ro5-4864 treatment, did not induce significant changes in the plasma levels of neuroactive steroids. This suggests that activation of LXRs may selectively increase the CNS levels of neuroactive steroids avoiding possible endocrine side effects exerted by the systemic treatment with these molecules. Interestingly GW3965 treatment induced an increase of dihydroprogesterone in the spinal cord of diabetic animals in association with an increase of myelin basic protein expression. Thus we demonstrated that LXR activation was able to rescue CNS symptoms of diabetes. [1]

---

An LXR Agonist Inhibits GBM Tumor Growth In Vivo [2]
To test the therapeutic potential of LXR agonists in the treatment of GBM, we determined the efficacy of GW3965 at blocking growth and promoting tumor cell death in vivo. U87/EGFRvIII cells were implanted subcutaneously in mice that were then treated with GW3965 (40 mg/kg daily by oral gavage) for 12 days. GW3965 treatment strongly induced ABCA1 expression and reduced LDLR expression (Fig. 7A). Remarkably, this activity was accompanied by a 59% inhibition of tumor growth (Fig. 7B and C) and a 25-fold increase in GBM cell apoptosis (Fig. 7D and E). These data show that an LXR agonist potently inhibits GBM growth and promotes tumor cell death in vivo.

Tertiary amine 3 was identified from a high-throughput screen of the GlaxoSmithKline compound file using a cell-free ligand-sensing assay (LiSA) for human LXRα. The LXRα LiSA measures the ligand-dependent recruitment of a 24 amino acid fragment of the steroid receptor coactivator 1 (SRC1) to the ligand-binding domain of the receptor[4].

---

Human washed platelet preparation [3]
Human blood was obtained from consenting healthy volunteers. A total of 50 mL of blood was collected into a syringe containing 3 mL of anticoagulant 4% (weight/volume) sodium citrate and mixed with 7 mL of acid citrate dextrose (ACD), and washed platelets were prepared by differential centrifugation as described previously.35 Platelets were resuspended in modified Tyrode-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer (134mM NaCl, 0.34mM Na2HPO4, 2.9mM KCl, 12mM NaHCO3, 20mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 5mM glucose, and 1mM MgCl2, pH 7.3) and rested for 30 minutes at 30°C before experiments.

---

Platelet aggregation and dense granule secretion assays [3]
Platelets (4 × 108 cells/mL) were incubated with luciferin at 37°C for 2 minutes and then stimulated with an agonist in an optical lumi-aggregometer with continuous stirring (1200 rpm). Platelet aggregation was determined by measuring changes in optical density as described previously35 and dense granule secretion by measuring changes in the adenosine triphosphate (ATP) concentration using the luciferin-luciferase system kit.

---

Measurement of [Ca2+]i by spectrofluorimetry [3]
Mobilization of calcium from intracellular stores was measured in platelets preloaded with the fluorescent dye Fluo-4NW as described previously.39 Platelets were stimulated with CRP or thrombin and calcium release was measured using a Fluoroskan ascent plate reader with excitation at 485 nm and emission measured at 530 nm.

---

Flow cytometric measurement of LXR-β within platelets [3]
To measure LXR within platelets, washed platelets were fixed with 2% formyl saline, permeabilized using BD Phosflow perm buffer III, washed, resuspended in HEPES buffer saline, and incubated with antibodies to LXR-β. Platelets were then washed, resuspended in HBS, and incubated with Cy3-labeled secondary antibody, fixed using 0.2% formyl saline and analyzed by flow cytometry. Negative controls were set using an appropriate isotype control.

---

Flow cytometric analysis: α-granule secretion and fibrinogen binding to integrin αIIbβ3 [3]
Flow cytometry was used to examine affinity up-regulation of the integrin αIIbβ3 and platelet α-granule secretion by detecting levels of fibrinogen binding to αIIbβ3 and P-selectin exposure on platelet surface, respectively. After stimulation with CRP, platelets were incubated at room temperature for 20 minutes with fluorescein isothiocyanate-labeled fibrinogen and PE/Cy5 antihuman-CD62P (P-selectin). Reactions were stopped by 100-fold dilution in 0.2% (volume/volume) formyl saline. Flow cytometric acquisition was performed using a FACSCalibur device, and data were collected from 5000 events analyzed using the CellQuest Pro Software Version 3.3. Negative controls were set using an appropriate IgG1 κ-isotype matched control for the anti-CD62P antibody and ethyleneglycoltetraacetic acid (10μM) for the fibrinogen binding.

---

In vitro thrombus formation [3]
Whole citrated blood was incubated with the lipophilic dye 3,3-dihexyloxacarbocyanine iodide and perfused through collagen-coated (400 μg/mL) Vena8Biochip at a shear rate of 20 dyn/cm2. Thrombi Z-images were taken every 30 seconds using a Nikon eclipse (TE2000-U) microscope, and thrombus fluorescence intensity was calculated using the Slidebook, Version 5.

A total of 6 × 103 cells were seeded into 6-well plates in 5% FBS for 24 hours, then changed to 1% LPDS medium and treated with GW3965 in time course manner. Cells were washed once using PBS; then total RNA was extracted using TRIzol reagent according to its protocol (Invitrogen). Next, 800 ng RNA was complementarily synthesized to cDNA and amplified using real-time PCR (Bio-Rad), and its values were normalized against the internal control gene 36B4 (RPLP0) for each replicate. The primers used were as follows: ABCA1 forward: 5′-AACAGTTTGTGGCCCTTTTG-3′, reverse: 5′-AGTTCCAGGCTGGGGTACTT-3′; IDOL forward: 5′-CGAGGACTGCCTCAACCA-3′, reverse: 5′-TGCAGTCCAAAATAGTCAACTTCT-3′; 36B4 forward: 5′-AATGGCAGCATCTACAAC-CC-3′, reverse: 5′-TCGTTTGTACCCGTTGATGA-3′[2].

Age and weight matched male C57BL/6J mice were housed 5 mice /cage at 21°C and 50% relative humidity with a 12-hr light:dark cycle. Mice were fed a standard rodent chow (PMI Feeds, 5001) and were provided food and water ad libitum. C57BL/6J mice were dosed by oral gavage twice daily with GW3965A at 10mg/kg or vehicle (0.5% Methyl Cellulose) for 3, 7 or 14 days. Blood was collected under isofluorane anesthesia via cardiac puncture. Serum lipid measurements were obtained with an automated chemistry analyzer. Changes in ABCA1 mRNA expression were measured using the ABI7700 Sequence Detector. RNA was isolated from GW3965 tissues from treated animals using Trizol reagent. Fold changes are based upon the cycle threshold values obtained with vehicle treated control samples. All procedures performed were in compliance with the Animal Welfare ACT and U.S. Department of Agriculture regulation and were approved by the GlaxoSmithKline Institutional Animal Care and Use Committee[4].

---

Induction of diabetes and experimental treatments [1]
Diabetes was induced in two-month-old male rats by a single i.p. injection of freshly prepared STZ (65 mg/kg) in 0.09 M citrate buffer, pH 4.8. Control animals were injected with 0.09 M citrate buffer at pH 4.8. Hyperglycemia was confirmed 48 h after streptozotocin injection by measuring tail vein blood glucose levels using a glucometer OneTouch Ultra2. Only animals with mean plasma glucose levels over 300 mg/ml were classified as diabetic. Glycemia was also assessed before treatment with Ro5-4864 or GW3965 and before death. Two months after STZ injection, diabetic animals were treated once a week with Ro5-4864 (3 mg/kg) or GW3965 (50 mg/kg). Thus, they received four subcutaneous injections in a month. Control diabetic rats received 200 μl of vehicle (sesame oil). Four-month-old non-diabetic male rats were injected, following the same experimental schedule, with Ro5-4864, GW3965 or vehicle. Rats were killed 24 h after the last treatment.

---

Xenograft Model [2]
Isogenic human malignant glioma cells (U87, U87-EGFRvIII) and human primary GBM model GBM39 were implanted into immunodeficient SCID/Beige mice for subcutaneous xenograft studies. SCID/Beige mice were bred and kept under defined-flora pathogen-free conditions.

---

Tail-bleeding assay [3]
Fifteen 7- to 8-week-old C57BL/6 mice were anesthetized using ketamine (80 mg/kg) and xylazine (5 mg/kg) administered via the intraperitoneal route before a tail biopsy. The time to cessation of bleeding was measured up to 20 minutes.

---

Intravital microscopy and laser-induced injury [3]
Intravital microscopy and data analysis were performed as previously described. Briefly, 8 C57BL/6 mice were anesthetized by intraperitoneal injection of ketamine (125 mg/kg), xylazine (12.5 mg/kg), and atropine (0.25 mg/kg). Mouse circulation was accessed via a cannulus placed in the jugular vein, and platelets were marked with Alexa-488-conjugated anti-GPIb antibody. After exteriorization of the testicles and the surrounding cremaster muscle, injury on the cremaster arteriole wall was induced with a Micropoint Ablation Laser Paint . Thrombi were observed using an upright Olympus BX microscope. Images were captured prior to and after the injury by a Hamamatsu charge-coupled device camera in 640 × 480 format and analyzed using Slidebook software Version 5.0.

---

Dissolved in 0.5% Methyl Cellulose; ≤10 mg/kg; oral gavage [1]

C57BL/6 mice

In mice, 12/GW3965 showed 70% oral bioavailability with Cmax = 12.7 μg/mL and t1/2 = 2 h after dosing at 10 mg/kg. Analysis of the pharmacokinetic data indicated that the serum levels of 12/GW3965 were 5-fold above its EC50 in cells for up to 7 h after dosing. The pharmacological activity of 12/GW3965 was evaluated in C57BL/6 mice by dosing at 10 mg/kg bid for 14 days. By day 3, ABCA1 expression was increased 8-fold in the small intestine and 7-fold in peripheral macrophages (Figure 2A), while plasma levels of HDLc increased 30% at day 3 and was maintained until day 14 (Figure 2B). Thus, 12/GW3965 is an orally active LXR agonist that upregulates ABCA1 expression and raises circulating levels of HDL in C57BL/6 mice.[4]

[1]. LXR and TSPO as new therapeutic targets to increase the levels of neuroactive steroids in the central nervous system of diabetic animals. Neurochemistry International (2012), 60(6), 616-621.

[2]. An LXR Agonist Promotes Glioblastoma Cell Death through Inhibition of an EGFR/AKT/SREBP-1/LDLR-Dependent Pathway. Cancer Discovery (2011), 1(5), 442-456.

[3]. LXR as a novel antithrombotic target. Blood (2011), 117(21), 5751-5761.

[4]. Identification of a nonsteroidal liver X receptor agonist through parallel array synthesis of tertiary amines. J Med Chem. 2002 May 9;45(10):1963-6.

Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)–mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1–dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.[2]

---

A potent, selective, orally active LXR agonist was identified from focused libraries of tertiary amines. GW3965 (12) recruits the steroid receptor coactivator 1 to human LXRalpha in a cell-free ligand-sensing assay with an EC(50) of 125 nM and profiles as a full agonist on hLXRalpha and hLXRbeta in cell-based reporter gene assays with EC(50)'s of 190 and 30 nM, respectively. After oral dosing at 10 mg/kg to C57BL/6 mice, 12 increased expression of the reverse cholesterol transporter ABCA1 in the small intestine and peripheral macrophages and increased the plasma concentrations of HDL cholesterol by 30%. 12 will be a valuable chemical tool to investigate the role of LXR in the regulation of reverse cholesterol transport and lipid metabolism.[4]

---

Neuroactive steroid levels are decreased in the central nervous system (CNS) of streptozotocin (STZ) diabetic rats. In agreement, they exert protective effects in this experimental model, counteracting degenerative events occurring in the CNS. Therefore, an interesting therapeutic strategy could be to increase their levels directly in the CNS. In this study we have evaluated whether activation of translocator protein-18 kDa (TSPO) or liver X receptors (LXRs) may affect the levels of neuroactive steroids present in the CNS of diabetic and non-diabetic animals. We observed that the treatment with either Ro5-4864 (i.e., a ligand of TSPO) or with GW3965 (i.e., a ligand of LXRs) induced an increase of neuroactive steroids in the spinal cord, the cerebellum and the cerebral cortex of STZ-rats, but not in the CNS of non-pathological animals. Interestingly, the pattern of induction was different among the three CNS areas analyzed and between the two pharmacological tools. In particular, the activation of LXRs might represent a promising neuroprotective strategy, because the treatment with GW3965, at variance to Ro5-4864 treatment, did not induce significant changes in the plasma levels of neuroactive steroids. This suggests that activation of LXRs may selectively increase the CNS levels of neuroactive steroids avoiding possible endocrine side effects exerted by the systemic treatment with these molecules. Interestingly GW3965 treatment induced an increase of dihydroprogesterone in the spinal cord of diabetic animals in association with an increase of myelin basic protein expression. Thus we demonstrated that LXR activation was able to rescue CNS symptoms of diabetes. [1]

---

Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)–dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1–dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)–mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1–dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients. [2]

---

Liver X receptors (LXRs) are transcription factors involved in the regulation of cholesterol homeostasis. LXR ligands have athero-protective properties independent of their effects on cholesterol metabolism. Platelets are involved in the initiation of atherosclerosis and despite being anucleate express nuclear receptors. We hypothesized that the athero-protective effects of LXR ligands could be in part mediated through platelets and therefore explored the potential role of LXR in platelets. Our results show that LXR-β is present in human platelets and the LXR ligands, GW3965 and T0901317, modulated nongenomically platelet aggregation stimulated by a range of agonists. GW3965 caused LXR to associate with signaling components proximal to the collagen receptor, GPVI, suggesting a potential mechanism of LXR action in platelets that leads to diminished platelet responses. Activation of platelets at sites of atherosclerotic lesions results in thrombosis preceding myocardial infarction and stroke. Using an in vivo model of thrombosis in mice, we show that GW3965 has antithrombotic effects, reducing the size and the stability of thrombi. The athero-protective effects of GW3965, together with its novel antiplatelet/thrombotic [3]

---

A potent, selective, orally active LXR agonist was identified from focused libraries of tertiary amines. GW3965 (12) recruits the steroid receptor coactivator 1 to human LXRalpha in a cell-free ligand-sensing assay with an EC(50) of 125 nM and profiles as a full agonist on hLXRalpha and hLXRbeta in cell-based reporter gene assays with EC(50)'s of 190 and 30 nM, respectively. After oral dosing at 10 mg/kg to C57BL/6 mice, 12 increased expression of the reverse cholesterol transporter ABCA1 in the small intestine and peripheral macrophages and increased the plasma concentrations of HDL cholesterol by 30%. 12 will be a valuable chemical tool to investigate the role of LXR in the regulation of reverse cholesterol transport and lipid metabolism. [4]

C33H31CLF3NO3.HCL

618.51

617.171

C, 64.08; H, 5.22; Cl, 11.46; F, 9.21; N, 2.26; O, 7.76

405911-17-3

GW3965;405911-09-3

16078973

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

8.891

2

7

13

42

753

0

ClC1C(C(F)(F)F)=C([H])C([H])=C([H])C=1C([H])([H])N(C([H])([H])C([H])([H])C([H])([H])OC1=C([H])C([H])=C([H])C(C([H])([H])C(=O)O[H])=C1[H])C([H])([H])C([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C([H])=C([H])C=1[H].Cl[H]

NMPUWJFHNOUNQU-UHFFFAOYSA-N

InChI=1S/C33H31ClF3NO3.ClH/c34-32-27(15-8-17-30(32)33(35,36)37)22-38(18-9-19-41-28-16-7-10-24(20-28)21-31(39)40)23-29(25-11-3-1-4-12-25)26-13-5-2-6-14-26;/h1-8,10-17,20,29H,9,18-19,21-23H2,(H,39,40);1H

2-(3-(3-((2-chloro-3-(trifluoromethyl)benzyl)(2,2-diphenylethyl)amino)propoxy)phenyl)acetic acid hydrochloride

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.04 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.

---

Solubility in Formulation 2: 2.5 mg/mL (4.04 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.04 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 10 mg/mL (16.17 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

---

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