BTK (IC50 = 3 nM)
Acalabrutinib (ACP-196) targets Bruton's tyrosine kinase (Btk), covalently binding to Cys481 of Btk (no IC50/Ki/EC50 values for Btk inhibition provided) [1]
Acalabrutinib (ACP-196) targets Bruton's tyrosine kinase (Btk) (no IC50/Ki/EC50 values for Btk inhibition provided); it does not inhibit EGFR (no inhibition at 10 μM), Itk or Txk, while ibrutinib inhibits EGFR with EC50 of 47-66 nM [3]

Acalabrutinib inhibits tyrosine phosphorylation of downstream targets of ERK, IKB, and AKT in the in vitro signaling assay on primary human CLL cells. IC50 values on nine kinases with a cysteine residue in the same location as BTK show that acalabrutinib has a higher selectivity for BTK. Crucially, acalabrutinib does not inhibit EGFR, ITK, or TEC like ibrutinib does. EGFR phosphorylation at tyrosine residues Y1068 and Y1773 is unaffected by acalabrutinib. Acalabrutinib exhibits nearly no inhibition or a much higher IC50 (>1000 nM) on the kinase activities of ITK, EGFR, ERBB2, ERBB4, JAK3, BLK, FGR, FYN, HCK, LCK, LYN, SRC, and YES1 when compared to ibrutinib[1].

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1. Acalabrutinib (ACP-196) is a novel irreversible second-generation BTK inhibitor with higher potency and selectivity than ibrutinib [1]
2. In two immunophenotypically confirmed canine B-cell lymphoma cell lines (CLBL-1 and 17-71), treatment with Acalabrutinib (ACP-196) at concentrations as low as 10 nM for 1 hour potently inhibited the activation of Btk and its downstream effectors ERK 1/2 and PLCγ2 [2]
3. In a competitive binding assay against a panel of 395 non-mutant kinases (1 μM), Acalabrutinib (ACP-196) exhibited higher selectivity for Btk compared with ibrutinib and CC-292; IC50 determinations on 9 kinases with a Cys in the same position as Btk confirmed Acalabrutinib (ACP-196) as the most selective, attributed to reduced intrinsic reactivity of its electrophile [3]
4. In human whole blood, Acalabrutinib (ACP-196) and ibrutinib showed robust and equipotent inhibitory activity on B-cell receptor (BCR)-induced responses in the low nM range, while CC-292 was 10-20 fold less potent [3]
5. Phosphoflow assays on EGFR-expressing cell lines confirmed no EGFR inhibition by Acalabrutinib (ACP-196) at 10 μM, whereas ibrutinib inhibited EGFR with EC50 of 47-66 nM [3]

ACP-196, when given orally to mice, inhibits anti-IgM-induced CD86 expression in CD19+ splenocytes in a dose-dependent manner; the ED50 for ACP-196 is 0.34 mg/kg, while that of ibrutinib is 0.91 mg/kg. The length of Btk inhibition following a single oral dose of 25 mg/kg is compared using a comparable model. At three hours after dosage, ACP-196 inhibits CD86 expression by more than 90%[3].

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Acalabrutinib (ACP-196) in preclinical research[1]
Acalabrutinib was evaluated in several animal models of B cell non-Hodgkin lymphoma (NHL). These studies provided preclinical in vivo data necessary to move acalabrutinib into human trials. In a study of canine model of B cell NHL, 12 dogs with B cell NHL were orally administered acalabrutinib at escalating dosages of 2.5 mg/kg every 24 h (6 dogs), 5 mg/kg every 24 h (5 dogs), or 10 mg/kg every 12 h (1 dog). As a result, 3 dogs achieved a partial remission (PR), 3 dogs had stable disease (SD), whereas the remaining 6 dogs had progression of disease (PD). This study therefore showed that acalabrutinib has single agent biologic activity in a spontaneous large animal model of NHL.[1]
The in vivo effects of acalabrutinib against CLL cells were demonstrated in the NSG mouse model with xenografts of human CLL. Acalabrutinib significantly inhibited proliferation of human CLL cells in the spleens of NSG mice at all dose levels, as measured for the expression of Ki67 (P = 0.002). Tumor burden decreased with the treatment of acalabrutinib in a dose-dependent manner. Acalabrutinib inhibited BCR signaling by reduced phosphorylation of PLCγ2. Acalabrutinib transiently increased CLL cell counts in the peripheral blood. Therefore, the novel BTK inhibitor acalabrutinib shows in vivo efficacy against human CLL cells xenografted to the NSG mouse model.
Two murine models were used in another in vivo study. In the TCL1 adoptive transfer model, acalabrutinib inhibited BCR signaling by decreased autophosphorylation of BTK and reduction in surface expression of the BCR activation markers CD86 and CD69. Most interestingly, acalabrutinib treatment increased survival significantly over mice receiving vehicle (median 81 vs 59 days, P = 0.02). The second murine model was the NSG xenograft model. Acalabrutinib treatment significantly decreased the phosphorylation of PLCγ2 and ERK (P = 0.02), reduced tumor cell proliferation (P = 0.02), and tumor burden (P = 0.04). Acalabrutinib was shown to be a potent inhibitor of BTK in both murine models of human CLL.

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1. In mice, oral administration of Acalabrutinib (ACP-196) resulted in dose-dependent inhibition of anti-IgM-induced CD86 expression in CD19+ splenocytes with an ED50 of 0.34 mg/kg, compared to 0.91 mg/kg for ibrutinib [3]
2. In a mouse model evaluating the duration of Btk inhibition after a single oral dose of 25 mg/kg, Acalabrutinib (ACP-196) and ibrutinib inhibited CD86 expression >90% at 3h and ∼50% at 24h postdose; in contrast, CC-292 inhibited ∼50% at 3h and ∼20% at 24h postdose [3]
3. In an ongoing clinical trial with 12 companion dogs with spontaneously occurring B-cell non-Hodgkin lymphoma (NHL), Acalabrutinib (ACP-196) was orally administered at dosages of 2.5 mg/kg every 24 hours (6 dogs), 5 mg/kg every 24 hours (5 dogs), or 10 mg/kg every 12 hours (1 dog):
- Btk occupancy in peripheral blood and lymphoma cells was assessed using a biotin-tagged probe derived from Acalabrutinib (ACP-196); at 2.5 mg/kg, full Btk occupancy was achieved in peripheral B cells 3h after dosing for all dogs except one with high peripheral B-cell count, and 83-99% Btk target occupancy was observed at 24 hours postdose for all dogs [2]
- Partial response (per modified RECIST) was achieved in 2 dogs in the 2.5 mg/kg group and 1 dog in the 10 mg/kg group; one responder in the 2.5 mg/kg group had relapse and was dose-escalated to 10 mg/kg q12 on day 42, re-establishing partial response from relapse [2]
- 3 dogs achieved stable disease for >28 days, 6 dogs developed progressive disease within 28 days and discontinued the study [2]
4. In healthy volunteers, oral administration of Acalabrutinib (ACP-196) at 100 mg QD showed >90% Btk target coverage over a 24h period; Btk occupancy and regulation of PD markers (CD69 and CD86) correlated with PK parameters for exposure [3]
5. In chronic lymphocytic leukemia (CLL) patients, after 7 days of dosing with Acalabrutinib (ACP-196) at 200 mg QD, 94% Btk target occupancy was observed (compared with ∼80% for ibrutinib at 420 mg QD) [3]

ACP-196's decreased intrinsic reactivity of its electrophile was linked to its high selectivity for Btk when tested against a panel of 395 non-mutant kinases, according to a previous study. In contrast to ibrutinib, ACP-196 was unable to inhibit EGFR, Itk, or Txk. Further confirmation of ibrutinib's EGFR inhibition without ACP-196 inhibition was obtained through phosphoflow assays conducted on EGFR-expressing cell lines.

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Compared to ibrutinib and CC-292, ACP-196 demonstrated higher selectivity for Btk when profiled against a panel of 395 non-mutant kinases (1 μM) in a competitive binding assay. IC50 determinations on 9 kinases with a Cys in the same position as Btk showed ACP-196 to be the most selective. The improved selectivity is related to the reduced intrinsic reactivity of ACP-196's electrophile. Importantly, unlike ibrutinib, ACP-196 did not inhibit EGFR, Itk or Txk. Phosphoflow assays on EGFR expressing cell lines confirmed ibrutinib's EGFR inhibition (EC50: 47-66 nM) with no inhibition observed for ACP-196 at 10 μM. These data may explain the ibrutinib-related incidence of diarrhea and rash. Ibrutinib's potency on Itk and Txk may explain why it interferes with cell-mediated anti-tumor activities of therapeutic CD20 antibodies and immune-mediated killing in the tumor microenvironment. In human whole blood, ACP-196 and ibrutinib showed robust and equipotent inhibitory activity on B-cell receptor induced responses in the low nM range, whereas CC-292 was 10-20 fold less potent.[3]

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1. A competitive binding assay was conducted to profile the selectivity of Acalabrutinib (ACP-196) against a panel of 395 non-mutant kinases at a concentration of 1 μM; the assay compared the binding affinity of Acalabrutinib (ACP-196) with ibrutinib and CC-292 to evaluate relative selectivity for Btk [3]
2. IC50 determinations were performed on 9 kinases that possess a Cys residue in the same position as Btk; the assay measured the inhibitory concentration of Acalabrutinib (ACP-196), ibrutinib and CC-292 required to inhibit each kinase, to confirm the selectivity of Acalabrutinib (ACP-196) [3]
3. A phosphoflow assay was carried out on EGFR-expressing cell lines to assess the inhibitory effect of Acalabrutinib (ACP-196) and ibrutinib on EGFR activity; the assay quantified EGFR inhibition levels at different concentrations, with Acalabrutinib (ACP-196) tested up to 10 μM and ibrutinib tested to determine its EC50 (47-66 nM) [3]
4. An ELISA-based Btk target occupancy assay was developed to measure target coverage of Acalabrutinib (ACP-196) in preclinical and clinical studies; the assay utilized specific reagents to detect the binding of Acalabrutinib (ACP-196) to Btk in biological samples (peripheral blood, lymphoma cells, etc.) [3]

The EGFR-expressing cell lines used in the phosphoflow tests EGFR inhibition by ibrutinib was further validated without any ACP-196 inhibition being seen.

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Recent recognition of B-cell receptor (BCR) signaling as a critical factor in the progression of B-cell malignancies, including non-Hodgkin lymphoma (NHL), has resulted in the development of numerous targeted therapeutics that inhibit this signaling pathway. Ibrutinib, a small molecule inhibitor of Bruton tyrosine kinase (Btk) a key signaling molecule in the BCR pathway, has demonstrated significant clinical activity in a broad range of B-cell cancers. ACP-196 is a second generation Btk inhibitor with increased target selectivity and enhanced in vivo potency compared with ibrutinib and, thus, may represent an improvement over its predecessor. With the following studies, we sought to evaluate ACP-196 in canine models of B-cell NHL with the ultimate goal of providing the preclinical data necessary to move ACP-196 into human clinical trials. Using two immunophenotypically confirmed canine B-cell lymphoma cell lines, CLBL-1 and 17-71, we demonstrate potent in vitro inhibition of activation of Btk and the downstream effectors ERK 1/2 and PLCγ2 following 1 hour of treatment with ACP-196 at concentrations as low as 10nM.[2]

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1. Canine B-cell lymphoma cell lines (CLBL-1 and 17-71) with confirmed immunophenotype were treated with Acalabrutinib (ACP-196) at concentrations as low as 10 nM for 1 hour; the activation levels of Btk, ERK 1/2 and PLCγ2 (downstream effectors of Btk) were detected and quantified to evaluate the inhibitory effect of Acalabrutinib (ACP-196) on BCR signaling pathway [2]
2. Human whole blood samples were used to assess the inhibitory activity of Acalabrutinib (ACP-196), ibrutinib and CC-292 on B-cell receptor (BCR)-induced responses; the assay measured the functional responses of B cells after stimulation with BCR agonists in the presence of different concentrations of the inhibitors, with Acalabrutinib (ACP-196) and ibrutinib showing potent activity in the low nM range [3]

canine model of B cell NHL
2.5, 5, 10 mg/kg.
orally administered

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In vivo studies were performed in companion dogs as part of an ongoing clinical trial. Twelve dogs with immunophenotypically confirmed, spontaneously occurring B-cell NHL were orally administered ACP-196 at dosages of 2.5mg/kg every 24 hours (6 dogs), 5mg/kg every 24 hours (5 dogs), or 10mg/kg every12 hours (1 dog). Btk occupancy in peripheral blood and lymphoma cells was assessed using a biotin-tagged probe derived from ACP-196. Using this assay we found that at 2.5mg/kg full Btk occupancy was achieved in peripheral B cells 3h after dosing for all dogs, except for a single dog with high peripheral B-cell count. At 24 hours after dosing, 83-99% Btk target occupancy was observed for all dogs. Partial response, as assessed by a modified RECIST scheme, was achieved in 2 dogs in the 2.5mg/kg group and the dog in the 10mg/kg group. Upon relapse, one of the responders in the 2.5mg/kg group was dose escalated to 10mg/kg q12 on day 42 and partial response from relapse was reestablished. Of the remaining 9 dogs, 3 achieved stable disease for > 28 days and 6 discontinued the study after developing progressive disease within 28 days of starting treatment. In total, to date, 3 dogs achieved a partial response, 3 dogs stable disease, and 6 dogs progressive disease. ACP-196 was well tolerated with only mild anorexia noted in some dogs. These data demonstrate that ACP-196 has single agent biologic activity in a spontaneous large animal model of human NHL. Studies in dogs with NHL are ongoing to define regimens prior to initiation of human phase I clinical trials. Additional cohorts are planned combining ACP-196 with a phosphatidylinositide 3-kinase (PI3K) delta-specific inhibitor.[2]

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In vivo, oral administration of ACP-196 in mice resulted in dose-dependent inhibition of anti-IgM-induced CD86 expression in CD19+ splenocytes with an ED50 of 0.34 mg/kg compared to 0.91 mg/kg for ibrutinib. A similar model was used to compare the duration of Btk inhibition after a single oral dose of 25 mg/kg. ACP-196 and ibrutinib inhibited CD86 expression >90% at 3h and ∼50% at 24h postdose. In contrast, CC-292 inhibited ∼50% at 3h and ∼20% at 24h postdose. An ELISA based Btk target occupancy assay was developed to measure target coverage in preclinical and clinical studies. In healthy volunteers, ACP-196 at an oral dose of 100 mg QD showed >90% target coverage over a 24h period. Btk occupancy and regulation of the PD markers (CD69 and CD86) correlated with PK parameters for exposure. In CLL patients, after 7 days of dosing with ACP-196 at 200 mg QD, 94% Btk target occupancy was observed compared with ∼80% reported for ibrutinib at 420 mg QD [3].

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1. Mouse model for Btk inhibition evaluation: Acalabrutinib (ACP-196) was administered to mice via oral route at different doses (including 25 mg/kg as a single dose and graded doses to determine ED50); anti-IgM was used to induce CD86 expression in CD19+ splenocytes; CD86 expression levels were measured at different time points (3h and 24h postdose) to assess the potency and duration of Btk inhibition by Acalabrutinib (ACP-196), with ibrutinib and CC-292 as comparators [3]
2. Canine model of spontaneous B-cell NHL: 12 companion dogs with immunophenotypically confirmed B-cell NHL were enrolled in an ongoing clinical trial; Acalabrutinib (ACP-196) was administered orally at three dosage regimens: 2.5 mg/kg once daily (6 dogs), 5 mg/kg once daily (5 dogs), 10 mg/kg twice daily (1 dog); Btk occupancy in peripheral blood and lymphoma cells was assessed at 3h and 24h postdose using a biotin-tagged probe derived from Acalabrutinib (ACP-196); tumor response was evaluated per modified RECIST criteria over time, with follow-up for response (partial response, stable disease, progressive disease) and dose escalation (10 mg/kg q12) for relapsed patients [2]

Absorption, Distribution and Excretion
The geometric mean absolute bioavailability of acalabrutinib is 25%, and the median time to peak concentration (Tmax) is 0.75 hours. Following a single 100 mg dose of radiolabeled acalabrutinib in healthy subjects, 84% of the dose was recovered in feces, and 12% in urine. Of the radiolabeled acalabrutinib, 34.7% was recovered as metabolite ACP-5862; 8.6% was recovered as unmetabolized acalabrutinib; 10.8% was recovered as a mixture of metabolites M7, M8, M9, M10, and M11; 5.9% as metabolite M25; and 2.5% as metabolite M3. The mean steady-state volume of distribution is approximately 34 L.
Based on population pharmacokinetic analysis, the mean apparent oral clearance (CL/F) of acalabrutinib was 159 L/hr, with similar pharmacokinetic characteristics in patients and healthy subjects.
Metabolism/Metabolites
Acalabrutinib is primarily metabolized by the CYP3A enzyme. ACP-5862 was identified as the major active metabolite in plasma, with a geometric mean exposure (AUC) approximately 2–3 times that of acalabrutinib. In terms of BTK inhibition, ACP-5862 is approximately 50% less potent than acalabrutinib.
Biological Half-Life
Following a single oral dose of 100 mg acalabrutinib, the median terminal elimination half-life of the drug is 0.9 hours (range 0.6 to 2.8 hours). The half-life of the active metabolite ACP-5862 is approximately 6.9 hours.

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1. In healthy volunteers, the regulation of BTK occupancy and PD biomarkers (CD69 and CD86) by Acalabrutinib (ACP-196) (100 mg orally once daily) was associated with PK parameters of exposure[3]

Hepatotoxicity
In open-label clinical trials of acalatinib in patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma, 19% to 23% of patients experienced elevated serum transaminases during treatment, with 2% to 3% experiencing elevations exceeding 5 times the upper limit of normal (ULN). These elevations were transient and spontaneously resolved, but occasionally led to premature discontinuation of the drug. In 610 patients in the pre-registration trial treated with acalatinib, no clinically significant liver injury related to the drug was observed, but one case resulted in acute liver failure and death due to hepatitis B virus reactivation. Similar reactivation cases have been reported with another Bruton's tyrosine kinase inhibitor, ibrutinib. Clinical experience with acalatinib is limited, and the incidence of clinically significant liver injury and hepatitis B virus reactivation remains unclear. Most cases occurred in patients taking multiple immunosuppressants, not just those taking acalatinib alone. Probability score: D (Possibly a rare cause of hepatitis B virus reactivation).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of acalabrutinib during lactation. Because acalabrutinib binds to plasma proteins at a rate exceeding 97%, and the half-lives of the drug and its metabolites are both less than 7 hours, its concentration in breast milk is likely to be low. However, the protein binding rate of the active metabolite is unclear; therefore, the manufacturer recommends discontinuing breastfeeding during acalabrutinib treatment and for at least 2 weeks after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
The reversible binding rate of acalabrutinib to human plasma proteins is approximately 97.5%. The mean in vitro plasma-to-serum ratio is approximately 0.7. In vitro experiments showed that at physiological concentrations, acalabrutinib bound 93.7% to human serum albumin and 41.1% to α-1-acid glycoprotein.

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1. In canine clinical trials of acalabrutinib (ACP-196), the drug was well tolerated, with only mild anorexia observed in some dogs [2]
2. Known toxicities of Ibrutinib (a first-generation BTK inhibitor) include atrial fibrillation, diarrhea, rash, arthralgia, and bleeding events; these toxicities may be related to its off-target effects (e.g., inhibition of EGFR, Itk, and Txk), while acalabrutinib (ACP-196) has higher selectivity and does not inhibit EGFR, Itk, or Txk, suggesting that its potential off-target toxicity is lower [3]

[1]. J Hematol Oncol . 2016 Mar 9:9:21.

[2].Cancer Res (2014) 74 (19_Supplement): 1744.

[3]. Cancer Res (2015) 75 (15_Supplement): 2596.

Pharmacodynamics
Acalatinib is a Bruton's tyrosine kinase inhibitor that inhibits the proliferation, migration, chemotaxis, and adhesion of B cells. It is taken once every 12 hours and may cause other adverse reactions such as atrial fibrillation, other malignancies, cytopenia, bleeding, and infection.

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1. Acalabrutinib (ACP-196) is a novel, irreversible second-generation BTK inhibitor with superior potency and selectivity compared to ibrutinib (the first BTK inhibitor approved for the treatment of chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenström macroglobulinemia)[1]
2. B-cell receptor (BCR) signaling is a key factor in the progression of B-cell malignancies; Acalabrutinib (ACP-196) inhibits BCR signaling by targeting Btk, a key molecule in the BCR signaling pathway[2]
3. Acalabrutinib (ACP-196)'s increased selectivity is related to the reduced intrinsic reactivity of its electrophilic reagents; unlike ibrutinib, it does not inhibit EGFR, Itk, or Txk, which may avoid ibrutinib-related toxicities (diarrhea, rash) and interference with CD20 antibody-mediated antitumor activity[3]
4. Acalabrutinib Acalabrutinib (ACP-196) is currently undergoing clinical trial evaluation for B-cell malignancies; more cohort studies are planned to combine Acalabrutinib (ACP-196) with a phosphatidylinositol 3-kinase (PI3K) δ-specific inhibitor in a canine non-Hodgkin lymphoma model [2]. 5. In patients with chronic lymphocytic leukemia (CLL), Acalabrutinib (ACP-196) occupancy at 200 mg once daily for 7 days was higher than that at 420 mg once daily (approximately 80%) [3].

C26H23N7O2

465.51

465.191

C, 67.08; H, 4.98; N, 21.06; O, 6.87

1420477-60-6

Acalabrutinib-d4;2699608-18-7;Acalabrutinib-d3; 1420477-60-6; 2058091-99-7 (citrate); 2242394-65-4; 2058091-96-4 (phosphate); 2058091-93-1 (3 hydrate); 2058092-05-8 (sulfate); 2058091-94-2 (fumarate); 2058091-97-5 (tartrate)

71226662

Yellow solid powder

1.4±0.1 g/cm3

1.715

0.77

2

6

4

35

845

1

O=C(C#CC([H])([H])[H])N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C1=NC(C2C([H])=C([H])C(C(N([H])C3=C([H])C([H])=C([H])C([H])=N3)=O)=C([H])C=2[H])=C2C(N([H])[H])=NC([H])=C([H])N12

WDENQIQQYWYTPO-IBGZPJMESA-N

InChI=1S/C26H23N7O2/c1-2-6-21(34)32-15-5-7-19(32)25-31-22(23-24(27)29-14-16-33(23)25)17-9-11-18(12-10-17)26(35)30-20-8-3-4-13-28-20/h3-4,8-14,16,19H,5,7,15H2,1H3,(H2,27,29)(H,28,30,35)/t19-/m0/s1

4-[8-amino-3-[(2S)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-a]pyrazin-1-yl]-N-pyridin-2-ylbenzamide

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 (4.47 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 (4.47 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 (4.47 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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Solubility in Formulation 4: 2% DMSO+30% PEG 300+2% Tween 80+ddH2O: 6mg/mL

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