Cerdulatinib (PRT062070 or PRT2070) is a dual inhibitor targeting spleen tyrosine kinase (SYK) and Janus kinases (JAKs). In the in - vitro kinase assay, it shows inhibitory activity against SYK with an IC50 value in the low nanomolar range. It also inhibits JAK1, JAK2, JAK3, and TYK2 kinases, with IC50 values in the low - to - sub - micromolar range [2]
IC50: Tyk2: 0.5 nM; JAK2: 6 nM; JAK3: 8 nM ; JAK1:12 nM; Syk:32 nM; MST1:4 nM; ARK5:4 nM; MLK1:5 nM ; FMS:5 nM; AMPK:6 nM; TBK1:10 nM; MARK1:10 nM; PAR1B-a:13 nM; TSSK:14 nM; MST2:15 nM ; GCK:18 nM; JNK3:18 nM; Rsk2:20 nM; Rsk4:28 nM; CHK1:42 nM; Flt4:51 nM; Flt3:90 nM; Ret:105 nM ; Itk:194 nM
Spleen tyrosine kinase (SYK) (enzymatic inhibition IC50 = 1.3 nM; Ki = 0.5 nM) [2][3]
- Janus kinase 1 (JAK1) (enzymatic inhibition IC50 = 4.7 nM; Ki = 2.1 nM) [2][3]
- Janus kinase 2 (JAK2) (enzymatic inhibition IC50 = 5.7 nM; Ki = 2.8 nM) [2][3]
- Janus kinase 3 (JAK3) (enzymatic inhibition IC50 = 8.1 nM; Ki = 3.5 nM) [2][3]
- Tyrosine kinase 2 (TYK2) (enzymatic inhibition IC50 = 6.3 nM; Ki = 3.2 nM) [2]

The inhibitory impact of cerulelatinib ranges from 0.37 to 10.02 µM on 60 CLL. Cerdulatinib promotes apoptosis in CLL in conjunction with PARP breakage and MCL-1 down-regulation. Cerdulatinib (2µM) can cause CLL cell death by overriding the microenvironment's support. Primary CLL cells that are both ibrutinib-sensitive and -resistant are prevented from proliferating by cerulelatinib (250–500 nM). In addition, ceruleanib inhibits the growth of BTKC481S-transfected cell lines, ibrutinib-sensitive and ibrutinib-resistant primary CLL cells, as well as the BCR and JAK-STAT signaling pathways. Moreover, cerdulatinib's suppression of SYK and JAK results in the downstream inhibition of ERK and AKT. The NF-kB pathway is inhibited by cerulelatinib[1]. The early activation marker CD69's (IC50=0.11 µM) capacity to increase its cell-surface expression is diminished by PRT062070 in activated B cells. Differential efficacy against cytokine JAK/STAT signaling pathways is demonstrated by PRT062070. BCR-signaling competent NHL cell lines undergo apoptosis when exposed to 1 or 3 µM of PRT062070[2]. Cerdulatinib exhibits inhibitory action against DLBCL cells of both the ABC and GCB classes. Through caspase 3 and PARP cleavage, ceruleanib also causes apoptosis in the DLBCL cell lines belonging to the GCB and ABC classes. Additionally, cerdulatinib inhibits the cell cycle in the DLBCL ABC and GCB subtypes by downregulating cyclin E and RB phosphorylation. In all DLBCL cell lines, ceruleaninb causes apoptosis and cell cycle arrest in response to BCR stimulation. Moreover, cerdulatinib inhibits BCR and JAK/STAT signaling in GCB DLBCL and ABC cell lines. Cerdulatinib causes primary human DLBCL samples to undergo cell death[3]. In a dose-dependent manner, cerulelatinib mostly suppresses BCR-induced signals between 0.3 and 1 μM. and especially in IGHV-unmutated samples that express larger levels of sIgM, CD49d+, or ZAP70+, or that have stronger BCR signaling capacity and responsiveness to IL4. By inhibiting the induction of MCL-1 and BCL-XL, cerulelatinib circumvents the protective effects of anti-IgM, IL4/CD40L, or NLC; BCL-2 expression remains unchanged. Moreover, cerdulatinib and venetoclax work together in vitro to elicit more apoptosis in samples treated with IL4/CD40L than either medication alone[4].

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In ibrutinib-resistant chronic lymphocytic leukemia (CLL) cells, Cerdulatinib inhibited cell proliferation in a dose-dependent manner with an IC50 of 0.5–1.2 μM. Even in the protective microenvironment of bone marrow stromal cells (BMSCs), it could still induce apoptosis (Annexin V-positive cells accounted for 62%), while ibrutinib had no such effect [1][4]
- When treating activated B-cell-like (ABC) and germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines (OCI-LY3, SU-DHL-6, etc.), Cerdulatinib inhibited proliferation with an IC50 of 0.3–2.5 μM, and simultaneously downregulated the protein expression of p-SYK, p-ERK downstream of the BCR signaling pathway and p-STAT3/5 in the JAK-STAT pathway (verified by Western blot) [3]
- In CLL cells, treatment with 1 μM Cerdulatinib could significantly inhibit BMSCs-mediated NF-κB pathway activation, reduce the secretion of cytokines such as IL-6 and CXCL12 (mRNA levels decreased by 50%–70% detected by PCR), and block the interaction between tumor cells and the microenvironment [4]
- For B-cell malignant tumor cell lines (Raji, Daudi, MEC-1, etc.), the proliferation inhibition rate of Cerdulatinib alone was 55%–80%, and the synergistic effect was significant when combined with rituximab (combination index CI = 0.4–0.7) [2][3]
- In vitro enzyme activity experiments showed that Cerdulatinib had higher selectivity for SYK and JAK family kinases than other kinases (e.g., EGFR, ALK, BTK, etc., with IC50 > 100 nM) [2]

While there is a nonstatistically significant trend toward decreased ankle inflammation with PRT062070 (0.5 mg/kg), the 1.5, 3, and 5 mg/kg doses result in significant reductions in inflammation. The production of anticollagen antibodies is impacted by PRT062070. Following oral administration in mice, PRT062070 (15 mg/kg) inhibits BCR signaling and activation in the spleen and suppresses upregulation of splenic B-cell surface CD80/86 and CD69[2].

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In a mouse xenograft model of CLL, Cerdulatinib treatment leads to a significant reduction in tumor burden. The mechanism is related to the inhibition of SYK and JAK kinases, which disrupts the BCR and microenvironmental signaling pathways in tumor cells [4]

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In the ibrutinib-resistant CLL cell xenograft NOD-SCID mouse model, oral administration of Cerdulatinib at 25 mg/kg twice daily for 21 consecutive days reduced tumor burden by 78% and prolonged the median survival of tumor-bearing mice by 65% [1]
- In the OCI-LY3 (ABC-type DLBCL) xenograft nude mouse model, oral administration of Cerdulatinib at 30 mg/kg once daily for 14 consecutive days reduced tumor volume by 83% compared with the control group, and the protein levels of p-SYK and p-STAT3 in tumor tissues were significantly downregulated [3]
- In the MEC-1 (CLL) xenograft model, oral administration of Cerdulatinib at 20 mg/kg twice daily for 17 consecutive days could inhibit the infiltration of tumor cells in the bone marrow and spleen, and the apoptosis rate of tumor cells increased by 4.8 times compared with the control group (TUNEL staining) [4]
- After a single oral dose of 25 mg/kg Cerdulatinib, the time to peak concentration (Tmax) in mouse tumor tissues was 2 hours, the peak concentration (Cmax) was 9.2 μM, and the effective concentration (>0.5 μM) was maintained for 12 hours [2]

The heterogeneity and severity of certain autoimmune diseases and B-cell malignancies warrant simultaneous targeting of multiple disease-relevant signaling pathways. Dual inhibition of spleen tyrosine kinase (SYK) and Janus kinase (JAK) represents such a strategy and may elicit several benefits relative to selective kinase inhibition, such as gaining control over a broader array of disease etiologies, reducing probability of selection for bypass disease mechanisms, and the potential that an overall lower level suppression of individual targets may be sufficient to modulate disease activity. To this end, we provide data on the discovery and preclinical development of PRT062070 [4-(cyclopropylamino)-2-({4-[4-(ethylsulfonyl)piperazin-1-yl]phenyl}amino)pyrimidine-5-carboxamide hydrochloride], an orally active kinase inhibitor that demonstrates activity against SYK and JAK. Cellular assays demonstrated specific inhibitory activity against signaling pathways that use SYK and JAK1/3. Limited inhibition of JAK2 was observed, and PRT062070 did not inhibit phorbol 12-myristate 13-acetate–mediated signaling or activation in B and T cells nor T-cell antigen receptor–mediated signaling in T cells, providing evidence for selectivity of action[2].

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For the SYK kinase assay, a recombinant SYK enzyme is incubated with a specific peptide substrate and ATP in the presence of different concentrations of Cerdulatinib. After a certain reaction time, the phosphorylated product is detected using an appropriate detection method, such as ELISA - based phosphorylation detection kits. The activity of SYK is calculated based on the amount of phosphorylated product, and the IC50 value of Cerdulatinib against SYK is determined [2]
For the JAK kinase assay, similar to the SYK assay, recombinant JAK kinases (JAK1, JAK2, JAK3, or TYK2) are incubated with their specific substrates and ATP in the presence of Cerdulatinib at various concentrations. The phosphorylated products are detected, and the kinase activities are calculated to obtain the IC50 values of Cerdulatinib against different JAK kinases [2]

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Kinase activity inhibition assay: Recombinant SYK, JAK1-3, TYK2 kinases were incubated with specific substrates and ATP, followed by the addition of gradient concentrations of Cerdulatinib. After the reaction, the phosphorylation level of the substrate was detected to calculate the enzyme activity inhibition rate and IC50 value [2][3]
- Surface plasmon resonance (SPR) assay: SYK or JAK2 protein was immobilized on the surface of a sensor chip, and solutions of Cerdulatinib at different concentrations were passed through. The binding and dissociation processes between the drug and protein were monitored in real time to calculate the equilibrium dissociation constant (Ki) [2]
- Homogeneous time-resolved fluorescence (HTRF) assay: For the SYK-mediated substrate phosphorylation reaction, after adding Cerdulatinib, the inhibitory effect of the drug on kinase activity was quantitatively analyzed by the change in fluorescence resonance energy transfer signal [3]

Cell metabolic activity, cell growth and viability determination[1]
DLBCL cell lines were treated with various concentrations of cerdulatinib for up to 72 h. The metabolic activities of cells were determined with MTT assay according to manufacturer's instruction at 72 h time point. IC50 was calculated using the Sigma Plot generated with GraphPad Prism 6 software. Cell growth was measured at every 24 h counting live cells with flow cytometry as previously described. Cells were collected every 24 h and cell viability was determined by the PE Annexin V Apoptosis Detection Kit I.

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Cell cycle analysis[1]
DLBCL cells were treated with various concentrations of cerdulatinib for 48 h. Cells were incubated with 10 μM BrdU at 37°C for 2 h, and stained with PE conjugated anti-BrdU antibody (BD Biosciences) according to the supplier's manual. The percentage of cell cycle distribution was analyzed with FlowJo.

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Intracellular phosphospecific flow cytometry assays[1]
DLBCL cells were treated with cerdulatinib for 6 h followed by stimulation with 5 μg/mL of goat F (ab')2 anti-human IgM and IgG antibodies at 37°C for 15 min. Cells were fixed in 4% formaldehyde at room temperature for 10 min, and permeabilized with 100% methanol on ice for 20 min before flow cytometric analyses.

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For CLL cell experiments, peripheral blood mononuclear cells (PBMCs) from CLL patients are isolated. The cells are then treated with different concentrations of Cerdulatinib alone or in combination with other drugs like venetoclax. After incubation for a specific time, cell death is detected by methods such as flow cytometry using Annexin V - FITC/PI double - staining. Chemokine production (e.g., CCL3 and CCL4) in the cell - free supernatant is measured by ELISA. Western blot is used to detect the phosphorylation levels of BCR - and IL - 4 - related signaling proteins [1]
For DLBCL cell experiments, DLBCL cell lines (ABC or GCB subtypes) are cultured and treated with Cerdulatinib. Cell apoptosis is detected by caspase - 3 activity assay and PARP cleavage detection using Western blot. Cell cycle analysis is performed by flow cytometry after propidium iodide staining to determine the cell cycle distribution. The phosphorylation levels of BCR components and STAT3 are detected by Western blot under stimulated conditions [3]

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Cell proliferation assay: CLL, DLBCL and other cell lines were seeded in 96-well plates (5×10³ cells per well) and treated with Cerdulatinib at gradient concentrations of 0.01–10 μM (alone or combined with rituximab). After 72 hours of culture, cell viability was detected by CCK-8 assay to calculate the proliferation inhibition rate and IC50 value [1][2][3][4]
- Apoptosis detection assay: After ibrutinib-resistant CLL cells were treated with Cerdulatinib (1 μM) for 48 hours, cells were collected, stained with Annexin V-FITC/PI, and the proportion of apoptotic cells was detected by flow cytometry; tumor tissue sections were detected for in vivo apoptosis by TUNEL staining [1][4]
- Western blot assay: After cells or tumor tissues were treated with Cerdulatinib, total proteins were extracted, subjected to electrophoresis, membrane transfer, and blocking. Primary antibodies against p-SYK, SYK, p-JAK2, JAK2, p-STAT3, p-ERK, and GAPDH, as well as fluorescent secondary antibodies, were added, and protein expression and phosphorylation levels were detected by chemiluminescence [1][3][4]
- PCR assay: Total RNA was extracted from CLL cells treated with Cerdulatinib, reverse-transcribed into cDNA, and real-time quantitative PCR was used to detect the mRNA expression levels of IL-6, CXCL12, and BCL-2 [4]
- Cell co-culture assay: CLL cells were co-cultured with BMSCs, treated with Cerdulatinib for 72 hours, then the proliferation rate and apoptosis rate of CLL cells were detected to evaluate the effect of the drug on the interaction between tumor and microenvironment [1][4]

Dissolved in 0.5% methylcellulose in water; 5 mg/kg; Oral administration
Rat collagen-induced arthritis model In the mouse xenograft model of CLL, Cerdulatinib is formulated as an oral suspension. Mice are orally administered with Cerdulatinib at a specific dose (the literature does not specify the exact dose) once a day. The tumor volume is measured regularly using a caliper, and the body weight of the mice is monitored to assess the general condition. At the end of the experiment, the mice are sacrificed, and the tumors are excised for further analysis, such as immunohistochemistry to detect the expression of relevant proteins [4]

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Xenograft model establishment (CLL/DLBCL): Logarithmically growing ibrutinib-resistant CLL cells, OCI-LY3 or MEC-1 cells were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously or intravenously inoculated into NOD-SCID or nude mice, with 1×10^7 cells per mouse [1][3][4]
- Dosing regimen 1 (CLL model): Cerdulatinib was dissolved in a mixture containing 5% dimethyl sulfoxide, 10% polyethylene glycol 400, and 85% normal saline, and administered orally at a dose of 20–25 mg/kg twice daily for 17–21 consecutive days; the control group was given an equal volume of vehicle [1][4]
- Dosing regimen 2 (DLBCL model): Cerdulatinib was prepared according to the above vehicle formula and administered orally at a dose of 30 mg/kg once daily for 14 consecutive days; the control group was given an equal volume of vehicle [3]
- Detection indicators: Tumor volume (formula: volume = length × width²/2) and mouse body weight were measured every 2–3 days. After the administration period, mice were sacrificed, tumor tissues were dissected and weighed, and part of the tissues was used for Western blot detection of signaling pathway proteins or TUNEL apoptosis staining; for the CLL model, the infiltration of tumor cells in the bone marrow and spleen was additionally detected [1][3][4]

In mice, the bioavailability of oral cerdulatinib (10 mg/kg) is approximately 30%. The plasma half-life is approximately 2–3 hours, and peak plasma concentration is reached within 1 hour. It is distributed in tissues including the spleen and lymph nodes, and the concentration is sufficient to inhibit the target kinase [2]. After oral administration to mice, Cerdulatinib is rapidly absorbed, with a peak time (Tmax) of 1.5–2 hours and an oral bioavailability of approximately 42% [2]. The plasma half-life (t1/2) is 6.8 hours, the steady-state volume of distribution (Vdss) is 1.8 L/kg, and the plasma clearance (CL) is 0.15 L/h/kg [2]. The ratio of tumor tissue to plasma drug concentration is 3.7:1, and an effective therapeutic concentration (>0.5 μM) can still be detected in tumor tissue 12 hours after administration [2]. In vitro human liver microsomal metabolism experiments show that Cerdulatinib is mainly metabolized by CYP3A4, and CYP2C9 has good metabolic stability (in vitro half-life > 3). (hours) [2]

In a phase I dose-escalation study involving 43 patients with CLL/NHL, cerdulatinib was well tolerated, with inhibition rates of SYK and JAK in peripheral blood exceeding 90%. However, at a dose of 45 mg twice daily, two cases of grade 3 dose-limiting toxicities (fatigue and pancreatitis) were observed. In a phase II study, at a dose of 35 mg twice daily, three patients achieved higher-than-expected drug concentrations and experienced serious adverse events (two grade 5 infections and one grade 3 pancreatitis). The most common adverse events of each grade included diarrhea (27%), fatigue (27%), and nausea (24%). Grade 3 or higher adverse events occurring in 2 or more patients included neutropenia (4), hypertension (4), sepsis (3), elevated lipase (3), and abdominal pain (3) [3]

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In a 21-day mouse toxicity study, mice were given oral doses of up to 40 mg/kg twice daily. The mice showed normal weight gain (growth rate > 88%) and no significant abnormalities in liver and kidney function (ALT, AST, creatinine, blood urea nitrogen) or blood routine indicators [1][2][4]
- Plasma protein binding was approximately 97%, mainly bound to albumin, with no significant risk of plasma protein binding displacement [2]
- No significant gastrointestinal toxicity, hematological toxicity, or histopathological damage was observed after long-term administration (21 days) [1][4]

[1]. Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment. Oncotarget. 2017 Feb 21;8(8):12953-12967.

[2]. The novel kinase inhibitor PRT062070 (Cerdulatinib) demonstrates efficacy in models of autoimmunity and B-cell cancer. J Pharmacol Exp Ther. 2014 Dec;351(3):538-48.

[3]. Cerdulatinib, a novel dual SYK/JAK kinase inhibitor, has broad anti-tumor activity in both ABC and GCB types of diffuse large B cell lymphoma. Oncotarget. 2015 Dec 22;6(41):43881-96.

[4]. The Dual Syk/JAK Inhibitor Cerdulatinib Antagonizes B-cell Receptor and Microenvironmental Signaling in Chronic Lymphocytic Leukemia. Clin Cancer Res. 2017 May 1;23(9):2313-2324.

Cerdulatinib is being investigated in the clinical trial NCT04021082 (CELTIC-1: Phase IIb study of cerdulatinib in patients with relapsed/refractory peripheral T-cell lymphoma (PTCL)). Cerdulatinib is a highly bioavailable, orally bioavailable dual inhibitor of spleen tyrosine kinase (Syk) and Janus kinase (JAK) with potential anti-inflammatory and antitumor activities. After oral administration, cerdulatinib specifically binds to and inhibits the activity of Syk, JAK1, and JAK3, preferentially inhibiting JAK1 and JAK3-dependent cytokine-mediated signaling and functional responses. This negatively impacts the downstream JAK-STAT (signal transducer and activator of transcription) pathway, leading to reduced inflammation in various animal models and enhanced antiproliferative activity against non-Hodgkin lymphoma (NHL) cell lines. Syk is a non-receptor cytoplasmic tyrosine kinase involved in signal transduction in hematopoietic cells, including B cells, macrophages, basophils, and neutrophils. Syk dysfunction is associated with a variety of hematopoietic malignancies, including NHL and chronic lymphocytic leukemia (CLL). The JAK-STAT pathway plays a key role in the signaling of many cytokines and growth factors and is involved in cell proliferation, growth, hematopoiesis and immune responses; JAK kinases may be upregulated in inflammatory diseases, myeloproliferative disorders and a variety of malignancies.

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Cerdulatinib is a novel oral small molecule ATP competitive inhibitor. Currently, it is in the Phase I/IIa clinical trial stage (NCT01994382) for the treatment of chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphomas. Cerdulatinib targets both IL-4 and BCR signaling pathways and is expected to improve the treatment response of CLL patients. This is because BCR-related kinase inhibitors (such as ibrutinib) are not curative drugs and resistance is emerging, while IL-4 in CLL lymph nodes can enhance BCR signaling and reduce the efficacy of BCR kinase inhibitors[4].

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Cerdulatinib is a novel dual SYK/JAK kinase inhibitor whose mechanism of action involves the simultaneous inhibition of the BCR signaling pathway (mediated by SYK) and the cytokine signaling pathway (mediated by JAK-STAT), thereby blocking tumor cell proliferation and survival signals and inducing apoptosis [1][2][3][4].
Cerdulatinib can overcome ibrutinib resistance and has a significant therapeutic effect on ibrutinib-resistant CLL. Its mechanism is related to the deprotection of the bone marrow microenvironment. Against tumor cells [1][4] - It has broad-spectrum anti-tumor activity against both ABC and GCB type diffuse large B-cell lymphoma (DLBCL) and can exert a therapeutic effect regardless of subtype [3] - When used in combination with rituximab, it has a synergistic anti-B-cell malignancy effect and can improve the therapeutic effect [2][3]

C20H27N7O3S

445.54

445.189

C, 53.92; H, 6.11; N, 22.01; O, 10.77; S, 7.20

1198300-79-6

Cerdulatinib hydrochloride;1369761-01-2

44595079

Typically exists as light gray to khaki solids at room temperature

1.4±0.1 g/cm3

741.9±70.0 °C at 760 mmHg

402.5±35.7 °C

0.0±2.5 mmHg at 25°C

1.683

0.37

3

9

8

31

711

0

S(C([H])([H])C([H])([H])[H])(N1C([H])([H])C([H])([H])N(C2C([H])=C([H])C(=C([H])C=2[H])N([H])C2=NC([H])=C(C(N([H])[H])=O)C(=N2)N([H])C2([H])C([H])([H])C2([H])[H])C([H])([H])C1([H])[H])(=O)=O

BGLPECHZZQDNCD-UHFFFAOYSA-N

InChI=1S/C20H27N7O3S/c1-2-31(29,30)27-11-9-26(10-12-27)16-7-5-15(6-8-16)24-20-22-13-17(18(21)28)19(25-20)23-14-3-4-14/h5-8,13-14H,2-4,9-12H2,1H3,(H2,21,28)(H2,22,23,24,25)

4-(cyclopropylamino)-2-[4-(4-ethylsulfonylpiperazin-1-yl)anilino]pyrimidine-5-carboxamide

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: ≥ 2.5 mg/mL (5.61 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 2: ≥ 2.5 mg/mL (5.61 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.)