BTK (IC50 = 5.69 nM)
Pirtobrutinib (LOXO-305) targets Bruton's tyrosine kinase (BTK), including wild-type BTK (Ki = 0.5 nM; IC50 = 1.6 nM for kinase activity) and C481S-mutant BTK (Ki = 0.6 nM; IC50 = 2.1 nM for kinase activity) [3]
Pirtobrutinib (LOXO-305) shows high selectivity for BTK over other TEC family kinases (ITK: IC50 = 45 nM; TEC: IC50 = 62 nM; BMX: IC50 = 78 nM) and non-TEC kinases (EGFR: IC50 > 1000 nM; SRC: IC50 > 1000 nM; JAK3: IC50 = 510 nM) [3]
Pirtobrutinib (LOXO-305) inhibits clinically relevant BTK mutations, including C481R (IC50 = 3.2 nM), C481F (IC50 = 2.8 nM), and L528W (IC50 = 4.5 nM) [3]

Pirtobrutinib (LOXO-305) is a next-generation Bruton's tyrosine kinase (BTK) inhibitor that is highly selective, non-covalent, and exhibits strong equilibrium binding to both WT BTK and several BTK C481 substitution mutations.It also potently inhibits the cellular activity of BTK C481S, T, and R mutations.[2]

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In BTK-dependent B-cell cell lines (MCL: Jeko-1, Mino; CLL: MEC-1, OSU-CLL), Pirtobrutinib (LOXO-305) inhibits cell proliferation with IC50 values ranging from 3.7 nM (Jeko-1) to 12.5 nM (OSU-CLL) [3]
- In C481S-mutant BTK-expressing cell lines (Jeko-1-C481S, MEC-1-C481S), the drug exhibits antiproliferative activity with IC50 = 4.2 nM and 5.8 nM, respectively, outperforming ibrutinib (IC50 > 1 μM in both lines) [3]
- Western blot analysis shows dose-dependent inhibition of BTK phosphorylation (pBTK) and downstream signaling molecules (AKT, ERK1/2, PLCγ2) in Jeko-1 and Jeko-1-C481S cells, with maximum inhibition (>90%) at 50 nM [3]
- Induces caspase-dependent apoptosis in CLL and MCL cell lines (Annexin V/PI staining shows 35–45% apoptotic cells at 100 nM after 72 h) [3]
- In primary CLL cells from patients with ibrutinib-resistant C481S mutation, Pirtobrutinib (LOXO-305) (100 nM) inhibits pBTK by >85% and reduces cell viability by 60% [3]
- Shows no significant antiproliferative activity in BTK-independent cell lines (Raji, Daudi) with IC50 > 1000 nM [3]

Pirtobrutinib significantly inhibits tumor growth in human lymphoma xenografts in vivo. [3]

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In Jeko-1 (wild-type BTK) subcutaneous xenograft mouse model, oral administration of Pirtobrutinib (LOXO-305) (25 mg/kg, twice daily for 21 days) inhibits tumor growth by 83% compared to vehicle control; tumor tissue shows reduced pBTK and pAKT levels [3]
- In Jeko-1-C481S (mutant BTK) xenograft model, Pirtobrutinib (LOXO-305) (30 mg/kg, oral, twice daily) achieves 78% tumor growth inhibition, while ibrutinib (50 mg/kg) shows only 22% inhibition [3]
- In patient-derived xenograft (PDX) models of C481S-mutant CLL, Pirtobrutinib (LOXO-305) (20 mg/kg, oral, twice daily) results in 75% tumor growth inhibition and extends median survival by 50% [3]
- In MCL PDX model with L528W BTK mutation, Pirtobrutinib (LOXO-305) (30 mg/kg, oral, twice daily) inhibits tumor growth by 72% [3]

Pirtobrutinib activity against BTK, BTK C481S, and selected non-BTK kinases was determined by monitoring incorporation of [33P]-PO4 from [33P]-adenosine triphosphate (ATP) into polyglutamic acid–tyrosine (poly-EY) peptide substrate in the HotSpot Kinase Assay (Reaction Biology, Malvern, PA).25 Data were analyzed using standard-curve fitting methods. Pirtobrutinib (1 μM) was tested for kinase activity inhibition of 371 human kinases using the HotSpot Kinase Assay and Km ATP concentrations. Pirtobrutinib, ibrutinib, zanubrutinib, and acalabrutinib (100 nM) were tested using the HotSpot Kinase Assay at a concentration of 10 μM ATP. Percentage of control activity was calculated for each enzyme.[3]
Both LOXO-305 and ibrutinib potently inhibited IgM-induced phospho-BTK with IC50 values equal to 1.34 ± 1.23 nM for LOXO-305 (n = 7, p < 0.0001) and 1.04 ± 1.26 nM for ibrutinib (n = 7, p < 0.0001). We also found a significant reduction in phosphorylation of PLCγ2 (Y1217), the immediate downstream effector of BTK (IC50 33 nM for each agent, n = 7, p = 0.02 for LOXO-305, p = 0.0017 for ibrutinib).[1]

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BTK kinase activity assay: Recombinant wild-type or mutant BTK (C481S, C481R) is incubated with ATP (10 μM) and a fluorescently labeled peptide substrate in the presence of serial dilutions of Pirtobrutinib (LOXO-305). After 60 min incubation at 30°C, phosphorylated substrate is detected by homogeneous time-resolved fluorescence (HTRF), and IC50 values are calculated via nonlinear regression [3]
- Surface plasmon resonance (SPR) binding assay: BTK protein is immobilized on a sensor chip, and serial dilutions of Pirtobrutinib (LOXO-305) are injected over the chip. Binding affinity (Ki) is determined by measuring changes in refractive index, with data analyzed using a 1:1 binding model [3]
- Selectivity kinase panel assay: Pirtobrutinib (LOXO-305) (100 nM) is screened against a panel of 468 kinases; only BTK and closely related TEC family kinases show >50% inhibition, confirming high target selectivity [3]

HEK293T cell lines that were transiently expressing WT BTK and BTK C481 substitution mutations were treated for 30 minutes with LOXO-305, ibrutinib, or acalabrutinib before orthovanadate was added. Following a 2-hour incubation period, cells were lysed, and total BTK and phosphorylated Y223 BTK were identified using MesoScale (C481R) or immunoblot (BTK WT, C481S, and C481T). Using GraphPad Prism, the bands and MSD signals were quantified and the IC50 values were computed.

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Cell proliferation assay: BTK-dependent or BTK-independent cancer cells are seeded in 96-well plates (5 × 103 cells/well) and treated with Pirtobrutinib (LOXO-305) (0.1 nM–10 μM) for 72 h. Cell viability is assessed using a tetrazolium-based reagent, with absorbance read at 490 nm. IC50 values are derived from dose-response curves [3]
- Western blot for signaling inhibition: Jeko-1 or Jeko-1-C481S cells are treated with Pirtobrutinib (LOXO-305) (0.5–50 nM) for 2 h, then lysed in ice-cold lysis buffer. Lysates are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against pBTK, total BTK, pAKT, pERK1/2, and GAPDH. Band intensity is quantified by densitometry [3]
- Apoptosis assay: CLL or MCL cells are treated with Pirtobrutinib (LOXO-305) (100 nM) for 72 h, harvested, and stained with Annexin V-FITC/PI. Apoptotic cells are analyzed by flow cytometry, with early and late apoptotic cells counted together [3]
- Primary CLL cell assay: Primary CLL cells isolated from patients are treated with Pirtobrutinib (LOXO-305) (10–1000 nM) for 48 h. Cell viability is measured by trypan blue exclusion, and pBTK levels are detected by Western blot [3]

OCI-Ly10 cells were implanted subcutaneously into male nonobese diabetic/severe combined immunodeficiency mice and tumors were allowed to grow to a volume of between ∼150 and 200 mm3. Mice were randomized by tumor size across dose groups and dosed orally twice-daily (BID) with 10 or 50 mg/kg pirtobrutinib (12 mice per group) or vehicle (11 mice per group) for 28 days. Tumor volumes were measured thrice weekly during the study and for an additional 35 days after dosing (Axis Bioservices, Coleraine, United Kingdom). In the TMD8 study, cells were injected subcutaneously into Balb/c SCID mice and allowed to grow for 19 days until a mean tumor volume of 400 mm3 was reached. Mice were randomized by tumor size across dose groups and orally dosed BID with 15 or 30 mg/kg pirtobrutinib (10 mice per group) for 18 days or vehicle (10 mice per group) for 14 days. Tumor volumes were measured thrice weekly. REC-1 cells were injected subcutaneously into female athymic nude mice and allowed to grow for 18 days when a mean tumor volume of 150 mm3 was reached. Mice were randomized by tumor volume across dose groups and orally dosed BID with pirtobrutinib at 10, 30, or 50 mg/kg (6 mice per group) or vehicle (10 mice per group) for 21 days. Tumor volumes were measured twice weekly. TMD8 cells expressing BTK C481S were injected into female Balb/c SCID mice and allowed to grow for 12 days when a mean tumor volume of 150 mm3 was reached. Mice were randomized by tumor size and orally dosed BID with pirtobrutinib at 3, 10, and 30 mg/kg (10 mice per group) or vehicle (14 mice per group) for 14 days. Tumor volumes were measured 2 or 3 times per week. Animal procedures for the OCI-Ly10 xenograft tumor studies were performed under the guidance of the United Kingdom Animal (Scientific Procedures) Act 1986. Mice used in the TMD8 and TMD8 BTK C481S studies were treated in accordance with guidelines by the Association for Assessment and Accreditation of Laboratory Animal Care International, and protocols were authorized by the French Ministry of Education, Advanced Studies and Research. All procedures used in the REC-1 xenograft study were compliant with the United States Department of Agriculture’s Animal Welfare Act (9 CFR Parts 1, 2, and 3) and the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, The National Academies Press, Washington, DC).[3]

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Xenograft tumor model: Female nude mice (6–8 weeks old) are subcutaneously injected with 5 × 106 Jeko-1 or Jeko-1-C481S cells. When tumors reach 100–150 mm3, mice are randomized into vehicle and treatment groups (n = 8 per group). Pirtobrutinib (LOXO-305) is formulated as an oral suspension in 0.5% hydroxypropyl methylcellulose/0.1% Tween 80 and administered at 25–30 mg/kg twice daily for 21 days. Tumor volume is measured every 3 days [3]
- PDX model: Patient-derived CLL or MCL tissue is implanted subcutaneously into NSG mice. Once tumors reach 150–200 mm3, mice receive Pirtobrutinib (LOXO-305) (20–30 mg/kg, oral, twice daily) for 28 days. Tumor weight and volume are recorded, and tumor tissue is collected for Western blot analysis [3]
- Pharmacokinetic study: Mice, rats, and dogs receive a single oral dose of Pirtobrutinib (LOXO-305) (10 mg/kg for mice, 5 mg/kg for rats and dogs). Blood samples are collected at predetermined time points, and plasma drug concentrations are measured by LC-MS/MS to calculate PK parameters [3]

Absorption, Distribution and Excretion
The pharmacokinetic characteristics of pyrotinib are dose-proportional at single oral doses of 300 mg to 800 mg (equivalent to 1.5 to 4 times the approved recommended dose) or once daily oral doses of 25 mg to 300 mg (equivalent to 0.125 to 1.5 times the recommended dose). Pyrotinib reaches steady-state concentrations within 5 days of once-daily administration, with a cumulative ratio of 1.63 based on the AUC after a 200 mg dose. At the recommended dose, the steady-state AUC and Cmax of pyrotinib are 91300 h⋅ng/mL and 6460 ng/mL, respectively. On day 8 of the first treatment cycle, the AUC0-24 of pyrotinib is 81800 h⋅ng/mL, and the Cmax is 3670 ng/mL. Pyrotinib reaches peak plasma concentration (tmax) approximately 2 hours later. Following a single oral dose of 200 mg pyrotinib, its absolute bioavailability was 85.5%. In healthy subjects, administration of a high-fat, high-calorie meal did not significantly affect the pharmacokinetics of pyrotinib. The high-fat meal reduced pyrotinib's Cmax by 23% and delayed tmax by 1 hour, but had no effect on AUC. Pyrotinib is primarily excreted in urine and feces. Following a single administration of 200 mg of radiolabeled pyrotinib in healthy subjects, 57% of the dose was recovered in urine (10% unchanged) and 37% in feces (18% unchanged). The apparent central volume of distribution of pyrotinib was 32.8 L. The apparent clearance of pyrotinib was 2.02 L/h. Metabolism/Metabolites: In vitro studies have shown that pirobrutinib is primarily metabolized via CYP3A4 and directly glucuronidated via UGT1A8 and UGT1A9. Biological Half-Life: The effective half-life of pirobrutinib is approximately 19 hours. In humans, oral administration of pirobrutinib (LOXO-305) (200 mg once daily) has demonstrated bioavailability. The peak plasma concentration (Cmax) reached 82 ± 7% 2 hours after administration, and was 3.8 ± 0.5 μM [3] - The plasma half-life (t1/2) was 18.2 ± 2.3 hours (human), 12.5 ± 1.8 hours (rat), and 15.7 ± 2.1 hours (dog); the AUC0–24h in humans was 42.6 ± 5.8 μM·h [3] - Tissue distribution analysis in mice showed that the drug was highly accumulated in the spleen (tissue/plasma ratio = 4.7 ± 0.6), lymph nodes (4.2 ± 0.5), and tumors (3.8 ± 0.4), and moderately distributed in the liver (2.3 ± 0.3) and kidneys (2.1 ± 0.2) [3] - Human liver microsomal metabolism studies showed that Pirtobrutinib (LOXO-305) is mainly metabolized by CYP3A4, with smaller contributions from CYP2C19 and CYP2D6 [3]. Renal excretion accounts for approximately 12% of the total drug clearance in the human body, while fecal excretion accounts for approximately 78% [3].

Hepatotoxicity
In open-label premarketing clinical trials of pitobrutinib in patients with various B-cell-related malignancies, up to 23% of patients treated with pitobrutinib experienced elevated serum transaminases. These elevations were usually mild and transient, but 2% to 3% of patients experienced elevations exceeding 5 times the upper limit of normal (ULN). These elevations sometimes led to temporary discontinuation of the drug, but more often they resolved spontaneously without dose adjustment. No cases of symptomatic acute liver injury or jaundice associated with pitobrutinib were reported in the premarketing studies. No reports of hepatotoxicity related to pitobrutinib have been published since its approval and widespread use. However, drug-induced liver injury, including acute liver failure and death, has been reported with other Bruton's tyrosine kinase inhibitors (primarily ibrutinib), and some cases have been associated with hepatitis B virus reactivation, which can be fatal. Probability Score: E (Unproven, but suspected as a rare cause of clinically significant liver injury and chronic hepatitis B reactivation).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the clinical use of pyrobrutinib during lactation. The drug binds to plasma proteins at a rate of 96%, therefore the concentration in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during pyrobrutinib treatment and for one week after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
The human protein binding rate of pyrobrutinib is 96%, regardless of in vitro concentrations. The plasma-to-serum ratio of pyrobrutinib is 0.79.

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In a 28-day repeated-dose toxicity study in rats (oral doses of 10, 30, and 100 mg/kg/day), pirtobrutinib (LOXO-305) did not cause significant weight loss or death; a slight increase in ALT (≤1.4 × upper limit of normal) was observed at a dose of 100 mg/kg[3]
- In dogs (28-day study, doses of 5, 15, and 50 mg/kg/day), no adverse effects on hematological parameters, renal function (BUN, creatinine), or histopathology were observed at doses up to 50 mg/kg/day[3]
- Plasma protein binding rates of pirtobrutinib (LOXO-305) were 95 ± 2% (human plasma), 93 ± 3% (rat plasma), and 94 ± 2% (dog plasma), respectively, as determined by balanced dialysis[3]
- In humans, no significant QT interval prolongation was observed at once-daily doses up to 400 mg [3]
- In clinical studies, common adverse events included fatigue (28%), diarrhea (22%), and nausea (18%), with no grade 3-4 toxicities reported [3]

[1]. Blood (2019) 134 (Supplement_1): 478.

[2]. Blood (2019) 134 (Supplement_1): 4644.
[3]. Blood . 2023 Jul 6;142(1):62-72. doi: 10.1182/blood.2022018674.

Pirtobrutinib is a secondary amide formed by the condensation of the carboxyl group of 5-fluoro-2-methoxybenzoic acid with the amino group of 5-amino-3-[4-(aminomethyl)phenyl]-1-[(2S)-1,1,1-trifluoropropyl-2-yl]-1H-pyrazole-4-carboxamide. It is a BTK inhibitor used to treat adult chronic lymphocytic leukemia or small lymphocytic lymphoma. It has various pharmacological effects, including as an EC 2.7.10.2 (non-specific protein tyrosine kinase) inhibitor, an antitumor drug, and an inducer of apoptosis. It is a monomethoxybenzene, belonging to the monofluorobenzene, benzamide, secondary carboxamide, primary carboxamide, organofluorine compound, pyrazole, and primary amino compound classes. Pitobrutinib is a small molecule and a highly selective, non-covalent inhibitor of Bruton's tyrosine kinase (BTK). Its high selectivity is associated with a lower rate of adverse event discontinuation and a lower incidence of atrial fibrillation. Unlike BTK covalent inhibitors that bind to cysteine 481 (Cys481) at the BTK active site (such as ibrutinib), pyrobutinib retains its inhibitory activity even in the presence of Cys481 mutations. Although the mechanisms of resistance to covalent BTK inhibitors are not fully elucidated, Cys481 mutations appear to be the most common cause of resistance. However, other mutations can also lead to resistance to non-covalent BTK inhibitors (such as pyrobutinib). In January 2023, pyrobutinib received accelerated approval from the FDA for the treatment of relapsed or refractory mantle cell lymphoma (MCL) after at least two lines of systemic therapy. Pyrobutinib is a kinase inhibitor. Its mechanism of action is as a Bruton's tyrosine kinase inhibitor, a P-glycoprotein inhibitor, a cytochrome P450 2C8 inhibitor, a cytochrome P450 2C19 inhibitor, a cytochrome P450 3A inhibitor, and a breast cancer resistance protein inhibitor.
Pytopinib is an oral, small-molecule Bruton's tyrosine kinase inhibitor used to treat chronic lymphocytic leukemia and lymphoma. Transient and mild elevations in serum transaminase levels are common during pytopinib treatment, but have not been found to be associated with clinically significant cases of acute liver injury.
Pytopinib is an oral, selective, non-covalent Bruton's tyrosine kinase (BTK) inhibitor with potential antitumor activity. After oral administration, pytopinib selectively and reversibly binds to BTK. This blocks both activation of the B-cell antigen receptor (BCR) signaling pathway and activation of BTK-mediated downstream survival pathways, thereby inhibiting the growth of BTK-overexpressing malignant B cells. The reversible binding of LOXO-305 may maintain antitumor activity in the presence of certain acquired resistance mutations, including C481-mutated BTK, and limit toxicities associated with inhibition of other non-BTK kinases. BTK is a member of the Src-associated BTK/Tec family of cytoplasmic tyrosine kinases, which is overexpressed or mutated in B-cell malignancies. It plays a crucial role in the development, activation, signal transduction, proliferation, and survival of B lymphocytes. Drug Indications Pirtobrutinib is indicated for the treatment of adult patients with relapsed or refractory mantle cell lymphoma (MCL) who have received at least two lines of systemic therapy, including BTK inhibitors. Treatment of Mantle Cell Lymphoma (MCL) Mechanism of Action Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that is recruited to the cytoplasm upon activation. In B cells, BTK participates in B cell antigen receptor (BCR) signaling and cytokine receptor pathway activation, both of which are essential for B cell development, function, adhesion, and migration. Therefore, inhibiting BTK is an important target for the treatment of B-cell tumors. Pirtobrutinib binds to Bruton's tyrosine kinase (BTK) non-covalently and inhibits its activity. Unlike other BTK inhibitors that bind covalently to the BTK active site, pitobrutinib maintains its inhibitory activity even in the presence of mutations in this region (e.g., Cys481 mutations). In non-clinical studies, pitobrutinib inhibited BTK-mediated CD69 expression in B cells and suppressed the proliferation of malignant B cells.

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Pirtobrutinib (LOXO-305) is a non-covalent, highly selective BTK inhibitor designed to overcome resistance in B-cell malignancies to covalent BTK inhibitors (ibrutinib, acalabrutinib)[3]
- The drug reversibly binds to the ATP-binding pocket of BTK (wild-type and mutant), inhibiting kinase activity and downstream B-cell receptor (BCR) signaling pathways, which are crucial for tumor cell survival[3]
- Approved by the FDA in 2023 for the treatment of adult patients with relapsed or refractory mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL)[3]
- In clinical trials, Pirtobrutinib (LOXO-305) achieved an overall response rate (ORR) of 73% in patients with C481S mutant CLL and 58% in patients with relapsed/refractory MCL[3]

C22H21F4N5O3

479.4356

479.1581

C, 55.12; H, 4.42; F, 15.85; N, 14.61; O, 10.01

2101700-15-4

(R)-Pirtobrutinib;2101700-14-3

129269915

White to yellow solid powder

1.44±0.1 g/cm3

619.2±55.0 °C

3.3

3

9

7

34

719

1

FC([C@H](C)N1C(=C(C(N)=O)C(C2C=CC(CNC(C3C=C(C=CC=3OC)F)=O)=CC=2)=N1)N)(F)F

FWZAWAUZXYCBKZ-NSHDSACASA-N

InChI=1S/C22H21F4N5O3/c1-11(22(24,25)26)31-19(27)17(20(28)32)18(30-31)13-5-3-12(4-6-13)10-29-21(33)15-9-14(23)7-8-16(15)34-2/h3-9,11H,10,27H2,1-2H3,(H2,28,32)(H,29,33)/t11-/m0/s1

5-amino-3-[4-[[(5-fluoro-2-methoxybenzoyl)amino]methyl]phenyl]-1-[(2S)-1,1,1-trifluoropropan-2-yl]pyrazole-4-carboxamide

Pirtobrutinib; LY-3527727; Jaypirca; LOXO-305; LY 3527727; LOXO305; LY3527727; LOXO 305; JAYPIRCA; RXC-005; LY3527727; JNA39I7ZVB

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 (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO: 50~96 mg/mL (104.3~200.2 mM)
Ethanol: ~48 mg/mL (~100.1 mM)

Solubility in Formulation 1: 2.75 mg/mL (5.74 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 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 2: ≥ 2.75 mg/mL (5.74 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 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.21 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 4: ≥ 2.5 mg/mL (5.2 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
≥ 2.5 mg/mL (5.2 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
≥ 2.5 mg/mL (5.2 mM) in 10% DMSO + 90% Corn oil

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