Apical sodium-dependent bile acid transporter (ASBT) (IC50 = 42±3 nM for human ASBT)
Apical Sodium-Dependent Bile Acid Transporter (ASBT, also known as IBAT) (no definite IC₅₀, Ki, or EC₅₀ data provided; described as a "highly potent" inhibitor) [1]
Apical Sodium-Dependent Bile Acid Transporter (ASBT) (used to study its regulatory role in hepatic cysteine sulfinic acid decarboxylase (CSAD) expression) [2]
Apical Sodium-Dependent Bile Acid Transporter (ASBT) (target for treating pruritus in Primary Biliary Cholangitis (PBC)) [3]
Linerixibat (GSK2330672) is a highly potent and nonabsorbable inhibitor of the apical sodium-dependent bile acid transporter (ASBT), also referred to as the ileal bile acid transporter (IBAT). This transporter is responsible for reabsorbing bile salts from the lumen of the gastrointestinal tract. By inhibiting ASBT in the ileum, linerixibat reduces the enterohepatic circulation of bile acids, leading to decreased serum bile acid levels and increased fecal bile acid excretion. No IC50 or Ki values are provided in the available search results.

Compound 56, also known as zwitterionic, non-hygroscopic crystalline salt form, has good solubility (>7 mg/mL) at pH 7.4, great thermal stability, and does not generate artifacts or reactivity [1].
GSK2330672 is a highly potent, nonabsorbable ASBT inhibitor with excellent aqueous solubility, selectivity, and developability properties for evaluation in safety studies and ultimately humans. GSK2330672 will be a valuable clinical tool for exploring the therapeutic utility of a nonabsorbable ASBT inhibitor for treatment of patients with type 2 diabetes.[1]

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Linerixibat (GSK2330672) was identified through extensive structure-activity relationship (SAR) studies starting from the first-generation inhibitor 264W94. Lead optimization efforts focused on identifying a potent, nonabsorbable ASBT inhibitor with excellent developability properties. The compound demonstrated the ability to lower glucose in animal models of type 2 diabetes. No detailed in vitro assay data (e.g., IC50 values, cell-based activity) are provided in the available search results.

In animal models of type 2 diabetes, treatment with Linerixibat (GSK2330672; 0.05–10 mg/kg; side wall gavage; twice daily for 14 days; gravimetric ZDF content) lowers diabetes [1].
GSK2330672 results in potent inhibition of ASBT and very low oral absorption in the rat. GSK2330672 shows potent mouse and rat ASBT activity and was stable in GI stability assays. GSK2330672 is stable in the rodent GI tract and potently induced fecal bile acid excretion in mice, leading us to select these three compounds for mechanistic and efficacy studies in vivo in lean rats and Zucker Diabetic Fatty (ZDF) rats, respectively[1].

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1. Antihyperglycemic effect in type 2 diabetes animal models: Linerixibat (GSK2330672), as a highly potent and nonabsorbable ASBT inhibitor, significantly lowered glucose levels in an animal model of type 2 diabetes, demonstrating its potential therapeutic utility for type 2 diabetes [1]
2. Regulation of hepatic CSAD expression in mice: Male C57BL/6J mice were treated with Linerixibat (GSK2330672) (2 mg/kg twice a day) via oral gavage for 1 week. After overnight fasting and tissue collection, analysis showed that Linerixibat (GSK2330672) significantly induced the expression of hepatic CSAD mRNA and protein, indicating that ASBT inhibition can upregulate CSAD expression in mouse liver [2]
3. Efficacy and safety in PBC patients (Phase 2b study): Linerixibat (GSK2330672) is being evaluated in a Phase 2b study for the treatment of pruritus in PBC patients. The study aims to determine the optimal dose and dosing frequency to improve pruritus and assess effects on underlying PBC. Preliminary results showed that the drug was generally well-tolerated in a small prior study, with no serious adverse events reported. It is developed as a tablet to block intestinal bile acid reabsorption, thereby promoting the excretion of pruritus-causing chemicals in stool [3]

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In patients with type 2 diabetes taking metformin, linerixibat (GSK2330672) at 90 mg twice daily for 14 days reduced fasting plasma glucose by -1.21 mmol/L (95% CI: -2.14, -0.28) and weighted-mean glucose area under the curve (AUC0-24h) by -1.33 mmol/L (95% CI: -2.30, -0.36) compared with placebo. Fasting and weighted-mean insulin AUC0-24h were also reduced. Linerixibat reduced LDL cholesterol by approximately 40%, as well as non-HDL cholesterol, total cholesterol, and apolipoprotein B concentrations. There was no change in HDL cholesterol, but a trend toward increased fasting triglyceride levels was observed. In the Phase 2b GLIMMER trial for primary biliary cholangitis patients with pruritus (N=147), linerixibat groups exhibited ≥2-point mean reductions in mean worst daily itch score from baseline at week 16; however, differences from placebo were not significant in the primary intent-to-treat analysis. Post hoc analysis of monthly itch score over the treatment period showed significant differences between placebo and linerixibat 180 mg once daily (P = .0424), 40 mg twice daily (P = .0105), and 90 mg twice daily (P = .0370). A significant relationship between total daily dose and response was observed in the per protocol population (P = .0542).

Method for Determination of Human, Mouse, and Rat ASBT Inhibition[1]
In preparation for measurement of bile acid uptake into cells expressing ASBT, HEK293 cells were cultured in DMEM/F12 supplemented with 10% FBS. Twenty-four h prior to running an experiment, cells were harvested when at a confluence of 80–90%. Cells were seeded in poly d-lysine coated plates at 50000 cells per well, and ASBT Bacmam virus was added such that each well contains 3.67 × 106 pfu (73.4 pfu/cell). Each assay plate was covered with Breathe Easy Seal and placed in an incubator for 24 h to allow expression of the transporter. On the day of the uptake experiment, 10 mM HEPES was added to Hank’s Balanced Salt Solution, and the pH was adjusted to 7.4 with TRIS (HBSSH). The assay buffer was prepared by adding 100 pM [3H]-taurocholate and 10 μM cold taurocholate to room temperature HBSSH. A separate washing buffer was prepared by adding 10 μM cold taurocholate to HBSSH (∼30 mL per assay plate) and placed on ice. Using 100% DMSO, 8-point, 3-fold dilution curves for each test compound was prepared starting at 200 μM. Similarly, an 8-point dose response curve was prepared of the control compound 1 starting at 1.8 mM. Drug plates were created by adding 3 μL of each concentration to a v-bottom 96-well plate then diluted 60-fold with 177 μL of assay buffer. Plates were removed from the incubator and allowed to cool to 25 °C. Media was aspirated, and wells were washed once with 300 μL of HBSSH. Then 50 μL of each dose response curve concentration was added in triplicate by column to the assay plates, reserving column 10 for control (assay buffer + 1.67% DMSO) and columns 11 and 12 for the control compound. Plates were incubated at ambient temperature for 90 min then the plates were aspirated then washed 1× with 300 μL of wash buffer. Then 220 μL of Microscint 20 was added to each well, and the plates were sealed. The amount of [3H]-taurocholate in cell lysate was quantitated using a microplate scintillation counter on the following day. Percent inhibition of uptake was determined using the following formula at each drug concentration: 100 × (1 – ((T1 – C2)/(C1 – C2))); where T1 is average cpm for the test compound, C1 is average cpm observed in the absence of any added inhibitor, and C2 is average cpm observed in the presence of a substance known to elicit 100% inhibition of uptake (30 μM control compound). IC50s can be generated using the formula, y = (Vmax × xn)/(Kn + xn).

Method for Determination of MDCK Permeability[1]
Passive permeability was measured in vitro using stably transfected human Multi-Drug Resistance 1–Madin–Darby Canine Kidney (hMDR1-MDCK) cells incubated under conditions relevant to intestinal absorption. Briefly, hMDR1-MDCK cells were seeded at 6.6 × 105 cells/well onto 12-well polycarbonate Transwells filter membranes with 0.4 μm pore size and maintained in Dulbecco’s Modified Eagle’s Media containing 10% fetal bovine serum (DMEM-FBS) at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity. After three days, media was removed from both the apical and basolateral chambers and replaced with transport buffer (HBSS containing 25 mM glucose and 25 mM HEPES) containing the P-gp inhibitor GF120918A at a final concentration of 2 μM. After a 30 min equilibration, the transport buffer was removed from the apical chambers and replaced with fasted-state simulated intestinal fluid (FaSSIF) containing 3 μM test compound, 2 μM GF120918A, 25 mM glucose, and 250 μM Lucifer Yellow CH. Next, the transport buffer was removed from the basolateral chambers and replaced with transport buffer containing 1% (w/v) human serum albumin and 2 μM GF120918A. After 60 min incubation at 37 °C, samples were collected from the apical (donor) and basolateral (receiver) compartments and added to acetonitrile (1:1 and 1:2 (v/v), respectively). Receiver samples were then centrifuged and the supernatants were removed and analyzed by LC-MS/MS. The final DMSO concentration in all dose solutions was 0.3% (v/v). Each treatment was performed in duplicate. Propranolol, a high permeability marker compound, and amprenavir, a marker compound for P-gp activity, were included in separate wells as controls for the assay. Cell monolayer integrity was assessed by measuring Lucifer yellow transport via a fluorescence-based assay.

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Rat Luminal Contents Stability Assay[1]
A 10% (w/v) homogenate of the luminal contents from rat cecum and colon in phosphate buffered saline (PBS, pH 7.4) was prepared as follows. Two male SD rats were fasted overnight and euthanized by CO2 asphyxiation, followed by exsanguination. The large intestine and cecum were removed from both animals and cut lengthwise. The luminal contents were removed, pooled into a preweighed 50 mL conical tube, diluted with PBS (10 mL/g sample weight), and gently mixed by inversion. The homogenate was placed on wet ice until use. Test compounds (10 μM final concentration) were added to a 4 mL glass screwcap vial containing 3 mL of the homogenate of the luminal contents from rat cecum and colon. Immediately after the addition of test compound, the vial was gently mixed and 3 × 100 μL aliquots were removed (t = 0) and placed into a 96-deepwell plate containing 400 μL of stopping solution (80% acetonitrile/20% methanol). Next, the glass vial was purged under a gentle stream of nitrogen gas for approximately 30 s, capped, and placed in a 37 °C shaking water bath. At t = 2, 4, and 24 h, 3 × 100 μL aliquots were removed from the vial and placed into a 96-deepwell plate containing 400 μL of stopping solution. The vial was purged under a gentle stream of nitrogen gas for approximately 30 s, capped, and placed in a 37 °C shaking water bath after each time point. Samples were covered and stored at −10 °C until LC-MS/MS analysis.

Animal/Disease Models: Male Zucker diabetic fat (ZDF) rat [1]
Doses: 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg twice (two times) daily; Results lasting 14 days: Glycated hemoglobin (HbA1c) diminished by 1.30-1.64%, non-fasting blood glucose diminished by more than 50% to less than 200 mg/dL, and plasma insulin increased Dramatically.

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Rat Oral Absorption Assay[1]
Male Sprague–Dawley (SD) rats (271–303 g; Charles River Laboratories, Raleigh, NC) were housed with free access to standard chow (PMI 5002 block chow) and water, unless otherwise noted. Animals for intravenous treatment groups were surgically implanted with a jugular and femoral vein cannula. Animals for oral treatment groups were surgically implanted with a jugular and portal vein cannula. Food was withheld from rats overnight prior to dosing and was returned at approximately 4 h postdose.Oral treatment groups received test compounds formulated as a homogeneous suspension in 0.5% HPMC/0.1% Tween via oral gavage at a dose of 10 mg/kg. Blood samples were collected from both jugular and portal vein cannulae at 0.25, 0.5, 1, 2, 4, and 8 h postdose. Plasma samples were prepared and stored at −70 °C until analysis.

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Rat Fecal Drug Recovery[1]
Fecal Recovery[1]
Male SD rats (Charles River Laboratories, Raleigh, NC) were administered test compounds formulated as a homogeneous suspension in 0.1% HPMC:0.5% Tween via oral gavage at a dose of 10 mg/kg. Fecal samples were collected across the following intervals: 0–6, 6–12, 12–24, 24–36, 36–48, 48–60, and 60–72 h postdose. After each collection interval, the samples were capped and stored at −70 °C until analysis. Prior to analysis, the samples were diluted with 5 volumes of 20% EtOH:80% H2O, soaked overnight at 10 °C, and then homogenized using a Polytron hand-held homogenizer. The homogenates were extracted with 3 volumes of acetonitrile and then centrifuged for 15 min at 2304g and 4 °C. Aliquots of each acetonitrile supernatant was transferred to clean 96-well plates and diluted with an equal volume of water. Drug concentrations were quantified via LC-MS/MS.

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Animals for Efficacy Studies[1]
Male C57BL/6J mice were purchased from Jackson Laboratories at seven weeks old and fed with normal rodent diet. Male Zucker Diabetic Fatty (ZDF/GmiCrl-fa/fa) mice were purchased from Charles River and had free access to rodent food. All animals were housed under controlled conditions (12/12 light–dark cycle, 24 °C and 50% relative humidity). All procedures performed were in compliance with the Animal Welfare Act and U.S. Department of Agriculture regulations and were approved by the GlaxoSmithKline Animal Care and Use Committee.

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Fecal Collection in Mice[1]
Male C57BL/6J mice were dosed with vehicle (0.5% hydroxypropyl methylcellulose (HPMC), 0.1% Tween80) or six doses (0.0001, 0.001, 0.01, 0.1, 1, and 10 mg/kg) of compounds at 0700 and 1500 for one day, and fecal samples were collected for 24 h (0700–0700). Animals were used for up to five studies with one week washout between studies.

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Fecal Collection in Rat[1]
Male ZDF rats arrived at seven weeks of age (±3 days). After a one-week acclimation period, rats were assigned to different treatment groups (n = 6–8/group) based upon baseline glucose/vehicle (0.5% hydroxypropyl methylcellulose (HPMC), 0.1%Tween80); one vehicle group for each compound) and six doses (0.05, 0.1, 0.5, 1, 5, and 10 mg/kg) of compounds 20, 45, and 56. All treatments were given via oral gavage twice a day. Fecal samples were collected for 24 h on day 7 of treatment.

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Metabolic Effects in ZDF Rats[1]
Male ZDF rats arrived at seven weeks of age (±3 days). After a one-week acclimation period, rats were anesthetized with isoflurane (Abbott Laboratories, IL) and tail-vein blood samples were collected at 0900 without fasting. To ensure balanced treatment groups, ZDF rats were assigned to six treatment groups based upon baseline glucose/vehicle (0.5% hydroxypropyl methylcellulose (HPMC), 0.1%Tween80) and selected doses of compounds (0.05, 0.1, 0.5, 1, 5, and 10 mg/kg or 0.001, 0.01, 0.1, 1, and 10 mg/kg for compounds 20 and 45 or 56, respectively). All treatments were given via oral gavage twice a day, and animals were followed for two weeks with blood samples collected from tail vein on day 14 at 0900 without fasting. Plasma samples were stored at −80 °C for further analyses.

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Measurement of Clinical Chemistry Parameters[1]
Plasma glucose and bile acids in fecal extraction were measured using the Olympus AU640 clinical chemistry analyzer. Glucose test reagents were manufactured by Beckman Coulter. Bile acids reagents were manufactured by Trinity Biotech. HbA1c was measured by the Primus Affinity Ultra2 HPLC system using Primus Affinity Assay reagents. Insulin, total GLP-1 (tGLP-1), PYY, and GIP were assayed using the Meso Scale Discovery (MSD) assay kits. Total Glp-1 was assayed by the MSD Total Glp-1 assay kit and analyzed on an MSD Sector Imager 6000.

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Fecal Bile Acid Extraction[1]
Fecal samples were air-dried for five days and extracted in methanol–KOH (300 mM) at 60 °C for 24 h. Fecal extract was then mixed with 150 mM Mg2SO4 (1:1). After centrifugation, the supernatant was saved and submitted for bile acids measurement as described above.

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1. Type 2 diabetes animal model experiment: Specific animal species (e.g., mice, rats) were used to establish a type 2 diabetes model. Linerixibat (GSK2330672) was administered via an appropriate route (likely oral, consistent with its nonabsorbable property and later tablet formulation). The dosing frequency and duration were set to evaluate glucose-lowering effects. Glucose levels were measured at specified time points to assess therapeutic efficacy [1]
2. Mouse hepatic CSAD regulation experiment: Male C57BL/6J mice were used (n=5 per group). Linerixibat (GSK2330672) was dissolved in a suitable vehicle (not specified) and administered via oral gavage at a dose of 2 mg/kg twice a day for 1 week. After the treatment period, mice were fasted overnight, then euthanized, and liver tissues were collected. Liver samples were used for mRNA detection (e.g., RT-PCR) and protein detection (e.g., Western blot) to analyze CSAD expression levels [2]

1. Non-absorbable properties: Linerixibat (GSK2330672) is explicitly described as a "non-absorbable" inhibitor. After oral administration, it is not absorbed into the systemic circulation and mainly remains in the gastrointestinal tract, exerting a local inhibitory effect on ASBT without systemic distribution.[1] 2. Local gastrointestinal effects: Due to its non-absorbable properties, the drug specifically acts on ASBT located on the apical membrane of intestinal epithelial cells, blocking the reabsorption of bile acids from the intestinal lumen. It is mainly excreted in feces (inferred from its non-absorbable properties).[1]

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Linerixibat (GSK2330672) is designed as a nonabsorbable compound with minimal systemic exposure. In a Phase 1 study of healthy Japanese volunteers receiving single oral doses of 10-180 mg, the drug was undetectable in the plasma of the majority of participants. This pharmacokinetic profile is consistent with its intended mechanism of action, which relies on local activity in the gastrointestinal tract rather than systemic circulation. No specific values for Cmax, Tmax, AUC, half-life, clearance, or volume of distribution are reported in the available search results, though these parameters were measured in clinical trials. The compound is administered orally as a solution (1.5 mg/g preserved solution in amber glass bottles) or as a powder diluted in vehicle.

1. Adverse events in clinical studies: In a previous small study of patients with primary biliary cholangitis (PBC), the most common adverse event with linecitabine (GSK2330672) was diarrhea (7 cases in the drug group and 1 case in the placebo group), while headache was more common in the placebo group (7 cases in the placebo group and 6 cases in the drug group). No serious adverse events were reported. [3]

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In a Phase 1 study of healthy Japanese volunteers (single oral doses of 10-180 mg), linerixibat (GSK2330672) showed no serious adverse events and no adverse events leading to study discontinuation or withdrawal. Drug-related adverse events were all mild and resolved without intervention, including gastrointestinal symptoms (mostly diarrhea) and positive fecal occult blood tests. In the Phase 2b GLIMMER trial in PBC patients (n=147), consistent with the mechanism of action, diarrhea was the most frequent adverse event, and its incidence increased with dose. In Phase 1/2 studies of type 2 diabetes patients, the most common adverse events associated with GSK2330672 were gastrointestinal, mostly diarrhea (occurring in 22-100% of subjects), which appeared to be independent of dose. In a Phase 1 first-in-human study, exclusion criteria included fecal occult blood positivity at screening, history of inflammatory bowel disease, chronic diarrhea, Crohn‘s disease, or diarrhea related to malabsorption syndromes. Subjects with QTc ≥450 msec were also excluded. No deaths or severe toxicity were reported in any of the available studies.

[1]. Discovery of a highly potent, nonabsorbable apical sodium-dependent bile acid transporter inhibitor (GSK2330672) for treatment of type 2 diabetes. J Med Chem. 2013 Jun 27;56(12):5094-114.

[2]. HNF4α Regulates CSAD to Couple Hepatic Taurine Production to Bile Acid Synthesis in Mice. Gene Expr. 2018 Aug 22;18(3):187-196.

[3]. Linerixibat (GSK2330672) granted Orphan Status. September 24, 2019. https://pbc-society.ca/gsk-gsk2330672-linerixibat-gets-orphan-status/

GSK2330672 has been studied for the treatment of type 2 diabetes.

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Drug indications
Treatment of primary biliary cholangitis

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1. Treatment indications: Linerixibat (GSK2330672) was originally developed for the treatment of type 2 diabetes, targeting ASBT, which regulates glucose metabolism[1]; later, it was repurposed for the treatment of pruritus in primary biliary cholangitis (PBC) to meet the unmet need of PBC patients for effective antipruritic treatment (because the first-line PBC treatment drug ursodeoxycholic acid is ineffective for pruritus)[3]
2. Mechanism of action: Linerixibat (GSK2330672) inhibits ASBT-mediated bile acid reabsorption in the intestinal lumen. For type 2 diabetes, this inhibitory effect can regulate hepatic cholesterol metabolism and glucose homeostasis; for pruritus associated with primary biliary cholangitis (PBC), it reduces systemic bile acid accumulation (one of the main causes of pruritus) by promoting bile acid excretion in feces [1, 3]. 3. Current Status of Drug Development: Linerixibat (GSK2330672) received Orphan Drug Designation from the U.S. Food and Drug Administration (FDA) in September 2019. Orphan Drug Designation is granted to drugs for rare diseases, which can shorten the development cycle and accelerate patient access. The drug is being developed as an oral tablet and is being evaluated in a Phase IIb study to determine the optimal dose, dosing frequency, safety, and tolerability in patients with moderate to severe pruritus of primary biliary cholangitis (PBC) [3]
4. Clinical study design (Phase IIb): The Phase IIb study of Linerixibat (GSK2330672) consisted of 7 field visits and 1 final telephone follow-up, conducted by the study physician/nurse. In Canada, the study was conducted at 5 research centers (Montreal, Winnipeg, Calgary, Edmonton, and London). Participants received reimbursement for travel expenses and compensation for meals/tea for two visits (each visit lasted 2–5 hours) [3]
5. Role in bile acid metabolism studies: Linerixibat (GSK2330672) was used as a tool to study the link between bile acid reabsorption and hepatic taurine production. It induced CSAD expression in mouse liver, confirming that ASBT inhibition couples bile acid metabolism with taurine synthesis [2]

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Linerixibat (GSK2330672) was discovered through a lead optimization effort starting from the first-generation ASBT inhibitor 264W94. Extensive SAR studies culminated in the identification of this compound, which showed excellent developability properties for evaluating the potential therapeutic utility of a nonabsorbable ASBT inhibitor for treatment of patients with type 2 diabetes. The compound has been investigated in multiple clinical trials, including first-in-human studies (NCT01416324), studies in type 2 diabetes patients on metformin (NCT02202161), studies in primary biliary cholangitis patients with pruritus (NCT01899703), and the Phase 2b GLIMMER trial (NCT02966834). The pharmacodynamic effects include reduction in total serum bile acids and increase in serum 7α-hydroxy-4-cholesten-3-one (C4), a key intermediate in bile acid synthesis, with increasing doses. A well-tolerated dose was identified for Phase 3 investigation for cholestatic pruritus in PBC.

C28H38N2O7S

546.675527095795

546.24

C, 61.52; H, 7.01; N, 5.12; O, 20.49; S, 5.87

1345982-69-5

53492727

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

5.708

4

9

13

38

870

2

O=C(O)CC(NCC1=C(OC)C=C(C2=C1)[C@@H](C3=CC=CC=C3)N[C@](CC)(CCCC)CS2(=O)=O)CC(O)=O

CZGVOBIGEBDYTP-VSGBNLITSA-N

InChI=1S/C28H38N2O7S/c1-4-6-12-28(5-2)18-38(35,36)24-13-20(17-29-21(14-25(31)32)15-26(33)34)23(37-3)16-22(24)27(30-28)19-10-8-7-9-11-19/h7-11,13,16,21,27,29-30H,4-6,12,14-15,17-18H2,1-3H3,(H,31,32)(H,33,34)/t27-,28-/m1/s1

3-[[(3R,5R)-3-butyl-3-ethyl-7-methoxy-1,1-dioxo-5-phenyl-4,5-dihydro-2H-1lambda6,4-benzothiazepin-8-yl]methylamino]pentanedioic acid

1345982-69-5; Linerixibat; GSK2330672; GSK-2330672; Iinerixibat; Linerixibat [USAN]; CHEMBL2387408; Linerixibat (USAN);

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 : ~50 mg/mL (~91.46 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.57 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (4.57 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 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: ≥ 2.5 mg/mL (4.57 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.

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