JAK1/Janus-associated kinase 1

Itacitinib (INCB039110) is a potent and selective inhibitor of JAK1, with >20-fold selectivity for JAK1 over JAK2 and >100-fold over JAK3 and TYK2. Myelofibrosis research employs itacitinib [1].

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Itacitinib reduces IL-6 production by macrophages [3]
Recent evidence shows that host macrophages are the major producers of IL-6 after CAR T-cell treatment. Therefore, we queried whether itacitinib would prevent IL-6 production by host macrophages. First, murine bone marrow–derived macrophages were expanded in vitro with granulocyte-colony stimulating factor, and itacitinib was added to the cultures at day 6. LPS was added to the culture at day 7 to activate the macrophages. Prophylactic treatment with itacitinib reduced IL-6 production in a dose-dependent manner, indicating that the activity of itacitinib in reducing production of inflammatory cytokines is not exclusive to T-cells (Fig. 2A). We also observed a non-significant trend towards reduction on several other cytokines (i.e. IL-10, IL-12p70, KC/GRO, Supplementary Fig. S2)

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Itacitinib reduces CAR T-cell cytokine production [3]
Whereas cytokine production by the host immune system is a primary cause of CRS, production of inflammatory cytokines by CAR T-cells is also a major contributor. Having shown that itacitinib is capable of reducing inflammatory cytokine production by the host immune system, we next wanted to examine whether itacitinib reduces inflammatory cytokine production by CAR T-cells. Human CD19-CAR T-cells were expanded under concentrations of tocilizumab or itacitinib equivalent to human doses achieved in clinical trials. After a 3-day expansion, CAR T-cells were co-cultured with CD19 expressing NAMALWA target cells, and 6 hours later supernatants were collected to quantify cytokines. Itacitinib, but not tocilizumab, was able to significantly reduce the levels of many inflammatory cytokines (i.e., IL-2, IFN-γ, IL-6, and IL-8) (Fig. 3). As an internal control (background), we also measured cytokines produced by non-transduced T-cells (Fig. 3).

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Itacitinib at clinically relevant concentrations does not affect PBMC proliferation [3]
As T-cells play an important antitumor role, the effect of itacitinib on T-cells was studied to determine if treatment may result in broad immunosuppression potentially interfering with normal T-cell proliferation and function. T-cells were obtained from freshly isolated human PBMCs of healthy adults. Following activation with anti-CD3/anti-CD28 coated beads, T-cells were expanded in the presence of increasing concentrations of itacitinib, and proliferation was measured by flow cytometry. When compared with the dimethyl sulfoxide (DMSO) control, concentrations of itacitinib relevant to the IC50 (50–100 nM) did not have a significant effect on anti-CD3/anti-CD28–induced expansion of human T-cells (Fig. 4A), demonstrating that itacitinib treatment has no negative impact on the ability of T-cells to proliferate. As an internal control, and consistent with the disruption of the JAK/STAT signaling activity, higher concentrations of itacitinib (1000 nM) blocked T-cell proliferation (Fig. 4A).

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Itacitinib does not affect CAR T-cell proliferation [3]
To study the effect of itacitinib specifically on CAR T-cell expansion and cytolytic activity, GD2-CAR T-cells were expanded with anti-CD3/anti-CD28 coated beads in the presence of increasing itacitinib concentrations, ranging from 100 to 500 nM. Once again, when compared with the DMSO control, itacitinib at 100–250 nM did not have a significant effect on CD3/CD28-induced expansion of GD2-CAR T-cells, whereas 500 nM was enough to block CAR T-cell proliferation (Fig. 4B). Importantly, GD2-CAR T-cells expanded in the presence of low (100 and 250 nM) itacitinib concentrations were able to efficiently lyse GD2-expressing tumor cells (Fig. 4C). As a negative (background) control, we also measured target cell lysis by non-transduced (nonspecific) T-cells. As expected, GD2-CAR T-cells expanded in the presence of a high dose of itacitinib (500 nM) and were unable to induce the lysis of target cells beyond background levels (Fig. 4C). Taken together, these data indicate that IC50 relevant doses of itacitinib do not interfere with CAR T-cell proliferation or antitumor activity in vitro. Next, we queried whether itacitinib has an effect on CAR T-cell antigen-specific proliferation. EGFR-CAR T-cells were expanded with anti-CD3/CD28 beads for 2 weeks and then restimulated with EGFR beads in the presence of itacitinib or DMSO controls. Once again, doses of itacitinib relevant to the IC50 (100 or 250 nM) did not affect CAR T-cell antigen-specific proliferation (Fig. 4D).

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Itacitinib does not affect CD19-CAR T-cell cytolytic activity in vitro [3]
To study the effect of itacitinib on human CAR T-cells targeting the CD19 antigen, we treated CD19-CAR T-cells with either itacitinib or anti–IL-6 receptor (tocilizumab) for 3 days and then measured their cytolytic activity against CD19-expressing target cells. Whereas concentrations of tocilizumab that mimic human pharmacologic activity significantly reduced the cytolytic activity of CAR T-cells, 100 nM of itacitinib showed no significant effect when compared with control CAR T-cells (Fig. 6A). CAR T-cells expanded with a higher dose of itacitinib (300 nM, approximately five-fold higher that the cellular IC50) showed a modest but statistically significant inhibition on the antitumor activity (Fig. 6A). As internal controls, the antitumor activity of non-transfected PBMCs was also tested. Non-transduced T-cells were unable to specifically lyse the target cells and were unaffected by neither itacitinib nor tocilizumab treatment beyond background levels (Fig. 6B).

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In vitro activity: Itacitinib (also known as INCB39110) is a potent, selective and orally bioavailable inhibitor of JAK1 (Janus-associated kinase 1) with >20-fold selectivity for JAK1 over JAK2 and >100-fold over JAK3 and TYK2 (IC50 for JAK1, 2, 3, and TYK2 are 2, 63, >2000, and 795 nM, respectively). It has potential antineoplastic activity and is currently in Phase II clinical trials for the treatment of myelofibrosis, rheumatoid arthritis and plaque psoriasis. Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. Kinase Assay: Itacitinib has >20-fold selectivity for JAK1 over JAK2 and >100-fold over JAK3 and TYK2 (IC50 for JAK1, 2, 3, and TYK2 are 2, 63, >2000, and 795 nM, respectively).

Itacitinib is able to inhibit tumor growth in human pancreatic xenograft models in mice at clinically relevant doses, both as monotherapy and in combination with cytotoxic agents such as gemcitabine.

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Itacitinib reduces cytokine levels in murine models of acute hyperinflammation [3]
As CRS is the most common side effect associated with CAR T-cell treatment, we investigated whether Itacitinib is able to reduce the levels of cytokines associated with acute hyperactivation leading to CRS. Therefore, we conducted experiments in which naïve animals were challenged with ConA, a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Similar to individuals experiencing CRS, animals receiving ConA have elevated serum levels of multiple inflammatory cytokines as well as behavioral changes such as fever, malaise, hypotension, hypoxia, capillary leak, multi-organ toxicity, and potentially death. To study the effect of itacitinib in this model, corresponding animals were prophylactically dosed with 60 or 120 mg/kg of itacitinib to achieve JAK1 inhibition coverage equivalent to that observed in clinical trials. When compared with vehicle-dosed animals, itacitinib was able to significantly reduce serum levels of many of the cytokines implicated in CRS (i.e., IL-6, IL-12, and IFN-γ) in a dose-dependent manner (Fig. 1A). As expected, itacitinib did not have a significant effect on cytokines independent of the JAK1 pathway (i.e., IL-5, Fig. 1A). However, not all JAK-mediated cytokines were significantly decreased (i.e. IL-4, Fig 1A). Additionally, itacitinib was also able to dose-dependently reduce CRS-implicated cytokines in a therapeutic mode, where animals were dosed with itacitinib 30 minutes after ConA challenge (Fig. 1B).

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To confirm the capacity of itacitinib to reduce hyperinflammation, naïve animals were challenged with anti-CD3 to induce nonspecific T-cell activation and cytokine response. Once again, corresponding animals were either prophylactically or therapeutically dosed with 120 mg/kg of Itacitinib. Compared with vehicle-treated mice, itacitinib was able to significantly reduce serum levels of many of the cytokines implicated in CRS, but have no effect on cytokines independent of the JAK1 pathway (Supplementary Fig. S1A) [3].

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Having determined that Itacitinib is able to reduce IL-6 production in vitro, we next expanded our studies to an in vivo context to assess the effect of itacitinib on activated macrophages in mice. Mice were prophylactically treated with itacitinib or vehicle for 3 days to achieve steady state before receiving an intraperitoneal injection of LPS. Two hours after injection, cytokines were measured from intraperitoneal lavages. As seen in Fig. 2B, doses of itacitinib that mimic the JAK1 inhibition coverage achieved in clinical trials significantly reduced IL-6 production by activated macrophages in vivo. Thus, data from both in vitro and in vivo systems indicate that itacitinib is capable of downregulating the major cellular source of inflammatory IL-6 during an experimentally induced model of CRS [3].

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Itacitinib does not impair T-cell antitumor activity in vivo [3]
To further test the effect of Itacitinib on antigen-specific T-cell proliferation and in vivo antitumor activity, we isolated splenocytes from OT-1 mice, transgenic for the TCR Vα2Vβ5 specific for the peptide OVA257–264 (SIINFEKL) (36). OT-1 T-cells were expanded with the SIINFEKL peptide in the presence of increasing concentrations of itacitinib, and their expansion was measured by flow cytometry. As expected, itacitinib concentrations relevant to the IC50 have a minimal effect on the expansion rate (Supplementary Fig. S3A). Itacitinib concentrations relevant to maximum free concentrations (189 and 244 nM, in the absence or presence of potent CYP3A4 inhibitors, respectively) induced a modest reduction on T-cell proliferation (Supplementary Fig. S3A). To evaluate the effect of itacitinib on the in vivo antitumor efficacy of OT-1 CD8, we conducted experiments involving adoptive transfer of OVA-specific CD8 cells into C57BL/6 mice previously transplanted with an OVA-expressing tumor cell line (EG7). Five days after tumor challenge, corresponding animals were orally dosed b.i.d. with vehicle or 60 or 120 mg/kg of itacitinib for 2 weeks. Eight days after tumor inoculation, animals received an adoptive transfer of OVA-specific OT-1 naïve CD8 cells. When compared with the control group, adoptive transfer of OT-1 CD8 was able to significantly reduce tumor growth (Fig. 4). Importantly, itacitinib doses equivalent to the JAK1 coverage target doses do not affect the antitumor efficacy of OT-1 cells (Fig. 5).

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Itacitinib does not affect CD19-CAR T-cell antitumor activity in vivo [3]
To test the effect of Itacitinib in vivo, we studied the effect of oral Itacitinib on the antitumor efficacy of CD19-CAR T-cells. Immunodeficient mice (NSG) were challenged with CD19+ nalm6-luciferase expressing human lymphoma cells. Once the tumor was engrafted (day 4), the mice received an adoptive transfer of 3 × 106 human CD19-CAR T-cells. In this model, CAR T-cells were able to control tumor growth, measured by luciferin expression (Fig. 7). Importantly, daily oral itacitinib dosing (120 mg/kg) of the animals did not affect the antitumor activity of adoptively transferred CAR T-cells, thus indicating that, at targeted doses, itacitinib does not have a significant effect on the antitumor activity of CAR T-cells.

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Finally, to confirm that itacitinib would not affect CAR T-cell activity on a more aggressive model, immunodeficient mice (NSG) were challenged with 5 × 106 CD19+ NAMALWA-luciferase expressing human lymphoma cells. Once the tumor was engrafted (day 7), the mice received an adoptive transfer of human CD19-CAR T-cells. When compared with animals receiving control cells, animals receiving an adoptive transfer of CAR T-cells had a significant tumor growth delay, measured both by luciferin expression (Supplementary Fig. S4B) and expansion of survival (P = 0.0014) (Supplementary Fig. S4C). Even in this aggressive tumor model, daily oral itacitinib dosing of the animals did not affect the antitumor activity of adoptively transferred CAR T-cells (P = 0.1860), thus confirming the safety of prophylactic itacitinib dosing to prevent CRS [3].

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Combined Janus kinase 1 (JAK1) and JAK2 inhibition therapy effectively reduces splenomegaly and symptom burden related to myelofibrosis but is associated with dose-dependent anemia and thrombocytopenia. In this open-label phase II study, we evaluated the efficacy and safety of three dose levels of Itacitinib/INCB039110, a potent and selective oral JAK1 inhibitor, in patients with intermediate- or high-risk myelofibrosis and a platelet count ≥50×109/L. Of 10, 45, and 32 patients enrolled in the 100 mg twice-daily, 200 mg twice-daily, and 600 mg once-daily cohorts, respectively, 50.0%, 64.4%, and 68.8% completed week 24. A ≥50% reduction in total symptom score was achieved by 35.7% and 28.6% of patients in the 200 mg twice-daily cohort and 32.3% and 35.5% in the 600 mg once-daily cohort at week 12 (primary end point) and 24, respectively. By contrast, two patients (20%) in the 100 mg twice-daily cohort had ≥50% total symptom score reduction at weeks 12 and 24. For the 200 mg twice-daily and 600 mg once-daily cohorts, the median spleen volume reductions at week 12 were 14.2% and 17.4%, respectively. Furthermore, 21/39 (53.8%) patients who required red blood cell transfusions during the 12 weeks preceding treatment initiation achieved a ≥50% reduction in the number of red blood cell units transfused during study weeks 1-24. Only one patient discontinued for grade 3 thrombocytopenia. Non-hematologic adverse events were largely grade 1 or 2; the most common was fatigue. Treatment with INCB039110 resulted in clinically meaningful symptom relief, modest spleen volume reduction, and limited myelosuppression [1].

Janus kinase (JAK) inhibitors (also termed Jakinibs) constitute a family of small drugs that target various isoforms of JAKs (JAK1, JAK2, JAK3 and/or tyrosine kinase 2 (Tyk2)). They exert anti-inflammatory properties linked, in part, to the modulation of the activation state of pro-inflammatory M1 macrophages. The exact impact of JAK inhibitors on a wider spectrum of activation states of macrophages is however still to be determined, especially in the context of disorders involving concomitant activation of pro-inflammatory M1 macrophages and profibrotic M2 macrophages. This is especially the case in autoimmune pulmonary fibrosis like scleroderma-associated interstitial lung disease (ILD), in which M1 and M2 macrophages play a key pathogenic role. In this study, we directly compared the anti-inflammatory and anti-fibrotic effects of three JAK inhibitors (ruxolitinib (JAK2/1 inhibitor); tofacitinib (JAK3/2 inhibitor) and itacitinib (JAK1 inhibitor)) on five different activation states of primary human monocyte-derived macrophages (MDM). These three JAK inhibitors exert anti-inflammatory properties towards macrophages, as demonstrated by the down-expression of key polarization markers (CD86, MHCII, TLR4) and the limited secretion of key pro-inflammatory cytokines (CXCL10, IL-6 and TNFα) in M1 macrophages activated by IFNγ and LPS or by IFNγ alone. We also highlighted that these JAK inhibitors can limit M2a activation of macrophages induced by IL-4 and IL-13, as notably demonstrated by the down-regulation of the M2a associated surface marker CD206 and of the secretion of CCL18. Moreover, these JAK inhibitors reduced the expression of markers such as CXCL13, MARCO and SOCS3 in alternatively activated macrophages induced by IL-10 and dexamethasone (M2c + dex) or IL-10 alone (M2c MDM). For all polarization states, Jakinibs with inhibitory properties over JAK2 had the highest effects, at both 1 μM or 0.1 μM. [2]

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Kinase biochemical profiling [4]
Enzyme assays were performed using a homogeneous time-resolved fluorescence assay with recombinant epitope tagged kinase domains (JAK1, 837–1142; JAK2, 828–1132; JAK3, 718–1124; TYK2, 873–1187) and peptide substrate (Biotin-EQEDEPEGDYFEWLE). Each enzyme reaction was carried out with or without test compound (11-point dilution), JAK enzyme, 500 nM peptide, adenosine triphosphate (ATP; 1 mM), and 2.0% dimethyl sulfoxide in assay buffer. The 50% inhibitory concentration (IC50) was calculated as the compound concentration required for inhibition of 50% of the fluorescent signal. Additional activity against a panel of 60 non-JAK family kinases was assessed with standard screening conditions testing 100 nM Itacitinib/INCB039110 using the respective Km concentrations for ATP for each individual kinase. Significant inhibition was defined as more than or equal to 30% (average of duplicate assays) compared with control values.

Validation of polarization markers and respective toxicity of the considered JAK inhibitors for two concentrations relevant of human plasma levels[2]
CXCL10, IL-6, IL1Ra and TNFα were all significantly over-expressed in the IFNγ-induced M1i MDM and in the (IFNγ + LPS)-induced M1Li MDM (Fig. 1A-B) in comparison with M0 unstimulated MDM. CCL18, PDGFbb, PPARγ and tenascin C (TenaC) were significantly over-expressed in the (IL-4/IL-13)-induced M2a MDM in comparison with M0 unstimulated MDM (Fig. 1C-D). CXCL13, IL-10, MARCO and SOCS3 were all upregulated in the IL-10-induced M2c MDM and in the (IL-10/Dexamethasone)-induced M2c + Dex (Fig. 1E).

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T-cell proliferation assay [3]
Peripheral blood mononuclear cells (PBMCs) were prepared from human whole blood samples using a Ficoll-Hypaque separation method, and T-cells were then obtained from the PBMCs by centrifugal elutriation. T-cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% HEPES, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol and 100 μg/mL streptomycin, and 100 units/mL penicillin (complete RPMI or CM). T-cells were activated with Dynabeads (immobilized agonist antibodies against CD3/CD28) at a 3:1 ratio, resuspended at a density of 0.5 × 106 cells/mL in 24-well plates and treated with Itacitinib at various concentrations (from 50 to 1000 nM). The plates were incubated at 37°C in 5% CO2 atmosphere for 10 days, and the proliferation was determined every other day by bead-based methods. Cultures were replenished every other day with fresh CM.

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Cytotoxicity assays [3]
Luciferase expressing SY5Y neuroblastoma cells (GD-2+) were plated in a 96-well plate at 50,000 cells/well. Twenty-four hours later, 150,000 CAR T-cells were added to corresponding wells in a final volume of 200 μL. Target cells alone were seeded in parallel to quantify the maximum luciferase expression (relative luminescent units; RLUmax). Seventeen hours later 100 μL of luciferin substrate was added to the co-culture. Luminescence was measured after a 10-minute incubation using an EnVision plate reader. The percent cell lysis was obtained using the following calculation: [1 − (RLUexperimental)/(RLUmax)] × 100. The experiment was performed in triplicates.

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Monocyte chemotatic protein (MCP)-1 assay [4]
Human PBMCs were preincubated with Itacitinib for 10 min at 37 °C, 5% CO2 and cultured at 1.5 × 106 cells/ml in RPMI media. Wells were stimulated by adding 30 ng/ml of human recombinant IL-6 and incubated for 48 h at 37 °C, 5% CO2. Supernatants were harvested and analyzed for levels of human MCP-1 by commercial ELISA. Itacitinib IC50 determination was performed by curve fitting using GraphPad Prism 5.0 software.

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cell proliferation assay [4]
Human T cells were then obtained from PBMCs and maintained in RPMI with 10% fetal bovine serum. For IL-2 stimulated cell proliferation analysis, cells were first treated with 10 μg/ml of phytohemagglutinin for 3 days to stimulate expression of IL-2 receptors. Cells were subsequently washed and resuspended in RPMI media at 6000 cells per well and treated with Itacitinib in the presence of 100 U/ml human IL-2. The plates were incubated at 37 °C in 5% CO2 for 3 days and proliferation determined by adding CellTiter-Glo® Reagent and detecting luminescence. Itacitinib IC50 determination was performed using the GraphPad Prism 5.0 software.

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IL-17/IL-22 cytokine analysis [4]
Human PBMCs were were maintained in RPMI supplemented with 10% fetal bovine serum, and T cell were activated with 1 μg/ml anti-CD3 and 5 μg/ml anti-CD28 antibodies. After 2 days, the cells were washed and recultured with IL-23 (100 ng/ml), IL-2 (10 ng/ml) and Itacitinib. Cells were incubated for an additional 4 days at 37 °C. IL-17 and IL-22 concentrations in the supernatants were measured by ELISA.

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Human phosphorylated STAT3 (pSTAT3) whole blood assay [4]
Blood from healthy human volunteers were collected into heparinized tubes. Blood was incubated with various Itacitinib concentrations for 10 min at 37 °C. Cells were subsequently stimulated with 100 ng/ml of IL-6 for 15 min at 37 °C. Red blood cells were lysed using hypotonic conditions, and the supernatant was removed by centrifugation. White blood cells were pelleted and lysed to make total cellular extracts. The extracts were analyzed for pSTAT3 using a commercial phospho-STAT3 specific ELISA.

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Emerging evidence has demonstrated the importance of tumor-associated inflammation in the development and progression of pancreatic ductal adenocarcinoma. Specifically, constitutive activation of the inflammation-related IL-6/JAK/STAT3 signaling pathway has been reported in pancreatic tumors and has been suggested to be a poor prognostic factor for overall survival in patients with advanced disease. The aim of this study was to assess the effects of inhibition of IL-6/JAK/STAT3 signaling on pancreatic cell growth in vitro and tumor growth in vivo. INCB039110, a JAK1 selective inhibitor currently in multiple Phase 2 clinical trials, was used to block the IL-6/JAK/STAT3 signaling pathway in cells. We show that while inhibition of IL-6/JAK/STAT3 signaling by INCB039110 showed no effects on the proliferation of pancreatic cell lines grown under conventional 2D monolayer cell culture conditions, this inhibitor demonstrated growth inhibitory activity against the pancreatic tumor cell lines, Hs700T and BxPC-3, in a 3D-spheroid culture system. Importantly, addition of cytokines that stimulated JAK/STAT3 signaling in these cells significantly promoted the growth of the spheroids, and this could be completely reversed by INCB039110. Furthermore, JAK1 inhibition enhanced the cytotoxicity induced by gemcitabine in both Hs700T and BxPC-3 spheroids in a combination study. The results were confirmed with another novel Jak1 selective inhibitor, INCB052793 which is currently in a Phase 1 clinical trial in patients with advanced malignancies. [2]

Based on these in vitro results, we also explored the effects of JAK2/1 inhibition by ruxolitinib in vivo, on mouse macrophages in a model of HOCl-induced ILD, that mimics scleroderma-associated ILD. In this model, we showed that ruxolitinib significantly prevented the upregulation of pro-inflammatory M1 markers (TNFα, CXCL10, NOS2) and pro-fibrotic M2 markers (Arg1 and Chi3L3). These results were associated with an improvement of skin and pulmonary involvement. Overall, our results suggest that the combined anti-inflammatory and anti-fibrotic properties of JAK2/1 inhibitors could be relevant to target lung macrophages in autoimmune and inflammatory pulmonary disorders that have no efficient disease modifying drugs to date.[2]

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Itacitinib modulation of mouse CRS models [3]
CRS was induced in BALB/c animals by intravenous injection (IV) of Concanavalin-A (ConA; 20 mg/kg) or 100 μg of an anti-CD3ε antibody (clone 145–2C11). Corresponding animals were orally dosed with 60 or 120 mg/kg of Itacitinib 60 minutes prior CRS induction (prophylactic) or 30 minutes after (therapeutic) CRS induction. Two hours after ConA or anti-CD3 injection, mice were sacrificed and blood was collected into K2EDTA tubes for cytokine measurement.

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Activated macrophage models [3]
Spleens were collected from 6- to 8-week-old C57BL-6 female mice and mechanically dissociated. Splenocytes were cultured in RPMI medium supplemented with 10% fetal bovine serum and macrophage colony-stimulating factor. On day 6, cells were treated with Itacitinib and then treated with 5 ng/mL lipopolysaccharide (LPS) on day 7. The following day, supernatant was collected and cytokines were measured as described below. Six- to 8-week-old female C57BL/6 mice were prophylactically orally dosed with vehicle, or 60 or 120 mg/kg of itacitinib twice a day (b.i.d.) for 3 days. Mice then received intraperitoneal injections of LPS (5 μg per animal). Two hours after injection, mice were euthanized and 3 mL of sterile saline was injected into the peritoneum to perform a peritoneal lavage. Subsequent cytokine measurements were performed as described below.

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OT-1 T-cell expansion and antitumor activity [3]
The OT-1 strain is transgenic for the TCR VαVβ5 specific for the OVA257–264 peptide (SIINFEKL) and restricted to H2-Kb. Naïve OT-1 CD8 cells were isolated from OT-1 spleens by negative selection using Naïve CD8a+ T Cell Isolation Kit.

To measure OT-1 CD8+ cell expansion, cells were CFSE-labeled (2 μM) and resuspended at 5 × 106 cells/mL in complete RPMI, 20 IU IL-2, 2 μg/mL of SIINFEKL peptide. and increasing Itacitinib concentrations, from 0 to 1000 nM. Three to five days later, OT-1 cells were collected, stained with an APC-anti-CD8 antibody, and the number of divisions was calculated by measuring the relative carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity of the different conditions by flow cytometry.

To study the effect of Itacitinib on the antitumor activity of CD8 OT-1 cells, C57BL/6 animals received subcutaneous injections of 0.5 × 106 OVA-expressing EG7 tumor cells into the shaved right flank. At day 7, corresponding animals were orally dosed with itacitinib b.i.d. at 60 or 120 mg/kg, and at day 10, corresponding animals received an intravenous (tail vein) injection of 5 × 106 naïve OT-1 CD8 cells. Tumor sizes were measured on two perpendicular axes using a digital caliper at least three times a week. Tumor volumes were calculated with the formula:
V=(W⁢2×L)/2, Where V is tumor volume, W is tumor width, and L is tumor length.

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Antitumor activity (Nalm6 model) [3]
To study whether JAK1 inhibition affects the antitumor effect of CAR T-cells, a xenograft model was used as previously reported. Briefly, 6- to 10-week-old immunodeficient NOD-SCID γc−/− (NSG) mice were injected intravenously via tail vein with 2.5 × 106 luciferase expressing Nalm6 acute lymphoblastic leukemia cells. Corresponding animals received 120 mg/kg of Itacitinib per 10 days (orally, b.i.d.), starting 1 day after the tumor challenge. Mice were then randomized into groups for adoptive transfer of 3 × 106 CAR T-cells, non-transduced cells, or vehicle intravenously at day 4. Anesthetized mice were imaged using a Xenogen IVIS Spectrum system once a week. Mice were given an intraperitoneal injection of D-luciferin (150 mg/kg). Total flux was quantified using Living Image 4.4 by drawing rectangles of identical area around mice, reaching from head to 50% of the tail length. Background bioluminescence was subtracted for each image individually. Animals were monitored daily, and survival rates were compared between groups.

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Rat adjuvant induced arthritis model [4]
Disease was elicited as previously described (Fridman, 2010). Briefly, Lewis rats were injected at the base of the tail with 100 μl of an emulsion of Complete Freund's Adjuvant. Each rat paw was scored following visual observation using a rating of 0–3, (0 = normal; 1 = redness and minimal swelling of digits; 2 = moderate swelling of the digits and/or paw; 3 = severe swelling of digits and/or paw). Individual animal paw scores are combined and recorded as a sum of all four paws and group means of these totals are reported. Animals were randomized across treatment groups following onset of inflammatory joint swelling —usually occurring 2 weeks after adjuvant injection. At the termination, hind paws were analyzed for histological analyses. Formalin fixed hind paws/ankles were decalcified using 5% formic acid, paraffin embedded, cut in the sagittal plane and then stained. All tissues were examined microscopically by a board certified veterinary pathologist. Adjuvant arthritic ankles were scored on a scale of 0–7 for each of the following four parameters (inflammation, bone resorption, pannus infiltration and cartilage damage). A summed score incorporating all four histological parameters was also determined for each joint (see Supplementary Table 1).

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Mouse oxazolone induced colitis model [4]
Male BALB/c mice (BALB/cAnNCrl strain# 028) were commercially purchased. On day 0, mice were sensitized by applying oxazolone (150 μL, 3% in acetone/olive oil, 4:1 v/v) to their preshaved rostral back. The animals were re-sensitized with oxazolone on Day 5. Mice were fasted before intra-rectal oxazolone challenge. Distal colitis was induced by intracolonic instillation of oxazolone solution (1 mg in 0.1 ml, 40% ethanol) after which, animals were kept in a vertical position for 30 s to ensure that the solution remained in the colon. Sham control mice received 0.1 ml of 40% ethanol alone. Diarrhea was quantified on a 0–3 rating scale, (0 = normal; 1 = soft but still formed; 2 = very soft; 3 = diarrhea). Fecal occult blood was detected on a 0 to 3 scale using S–Y occult blood paper, (0 = negative; 1 = positive; 2 = visible blood traces; 3 = rectal bleeding). On Day 8, the colon length and weight measured of euthanized mice. Furthermore, when the abdominal cavity was opened adhesions between the colon and other organs were noted as was the presence of colonic ulceration. Macroscopic scoring was performed on a 0 to 9 scale . Normalized colon weight represents the increase in tissue relative to sham control mice.

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Interleukin-10 knockout (IL-10) spontaneous colitis model [4]
Female IL-10 homozygote knockout mice on the BALB/c strain background were provided by Taconic USA. Itacitinib treatment was initiated from 6 weeks of age. Diarrhea was quantified on a 0 to 3 rating scale, (0 = normal; 1 = soft but still formed; 2 = very soft; 3 = diarrhea). At study termination, colon length and weight were quantified. Histopathology was performed by a board certified veterinary pathologist. Disease pathology was scored on a 0 to 12 scale with equal weighting (0 – normal, 3 – extensive) for the size and frequency of inflammatory infiltrates, erosions/ulceration and transmural inflammation.

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Mouse trinitrobenzenesulfonic acid (TNBS) induced colitis model [4]
Male BALB/c (BALB/cAnNCrl strain# 028) mice were commercially purchased (Charles River Laboratories). A cohort of animals underwent colon cannulation. Briefly, animals were fasted for 4 h prior to surgery. A ventral midline incision into the abdomen was made and the catheter inserted into the colon, passed through the abdominal wall, tunneled subcutaneously to the dorsal incision, exteriorized in the scapular region and secured. Surgery was performed a minimum of 10 days prior to the start of the experiment. Distal colitis was induced by intracolonic instillation of TNBS (1 mg in 0.1 ml, 50% ethanol). Itacitinib was administered by oral gavage or intracolonic cannula. Diarrhea was quantified on a 0 to 3 rating scale, (0 = normal; 1 = soft but still formed; 2 = very soft; 3 = diarrhea) on days 3–5 post TNBS sensitization.

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Mouse acute graft vs. host disease (GvHD) model [4]
Male C57BL/6 and BALB/c were commercially purchased. BALB/c mice received a single 8 Gy dose of total body irradiation on day −1, followed by an intravenous injection of a combination of splenocytes and T-cell depleted bone marrow cells on day 0. Animals received cell transfers from either BALB/c mice (syngeneic control) or from C57BL/6 mice to induce GvHD, and were monitored for engraftment by flow cytometry analysis of cells collected via retro-orbital bleeds twice weekly. Itacitinib was administered by oral gavage twice-daily beginning on day −3 (prophylactic) or day 14 (therapeutic). Animals were weighed daily and scored for GvHD progression on a 0 to 2 rating scale, (0 = normal, 1 = moderate, 2 = severe) in weight loss, posture, activity, fur texture, and skin integrity (see Supplemental Table 4). Colon samples were sectioned at 5 μm and stained with the following CD4+, CD8+, CD3+/pSTAT+ and total pSTAT3+. Slides were digitally scanned using an Aperio AT2 and image analyses performed using Visiopharm software. Tissue homogenate IL-1β, TNFα, IFNγ concentrations were quantified by ELISA.

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N/A

Human pancreatic xenograft models in mice

Pharmacokinetics of itacitinib in mice following multiple oral dosing [4]
The pharmacokinetics of itacitinib in female BALB/c mice was determined following twice daily oral doses at 20, 40, and 80 mg/kg for 12 days (Fig. 4). The mean peak plasma concentration (Cmax) and area under the curve values increased with the dose although not proportionally.

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Pharmacokinetics studies [4]
The oral absorption of itacitinib was determined in commercially purchased female BALB/c mice (BALB/cAnNCrl strain# 028, Charles River Laboratories). Itacitinib was formulated in 0.5% methylcellulose and administered by oral gavage at 20, 40, or 80 mg/kg twice daily for 12 days. Retro-orbital blood samples were collected at 1, 2, 8, and 16 h post-dose on the last day. All blood samples were collected using EDTA as the anticoagulant and centrifuged to obtain plasma samples. Plasma concentrations of itacitinib were determined by liquid chromatography coupled to tandem mass spectrometry using a positive interface on a Sciex API-4000 mass spectrometer and multiple reaction monitoring. The plasma concentration-time data was used to determine the pharmacokinetic parameters by standard non compartmental methods using WinNonlin® version 5.0.1.

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This article presents the population pharmacokinetic (PopPK) analysis and exposure-response analyses for the primary efficacy end point-acute graft-versus-host disease (aGVHD) day 28 response-and select safety measures (incidence of thrombocytopenia, hypertriglyceridemia, and cytomegalovirus infection) from a phase 3 randomized, double-blind study comparing itacitinib plus corticosteroids versus placebo plus corticosteroids for the treatment of aGVHD. The PopPK data set contained sparse data from patients with aGVHD and select enriched data from healthy volunteers. The structural model was a 2-compartment model with first-order elimination and dose-dependent nonlinear absorption with dual first-order absorption pathways with lag times. Strong cytochrome P450 (CYP) 3A inhibitor coadministration, moderate renal impairment, and participant population (healthy volunteers vs patients with aGVHD) were covariates on apparent clearance. Participant population was also a covariate on apparent intercompartmental clearance and lag time of the secondary absorption compartment. Apparent clearance decreased 42% with coadministration of strong CYP3A inhibitors. Simulations supported the following dose reductions with concomitant use of a strong CYP3A inhibitor: 300 mg once daily to 200 mg once daily, 400 mg once daily to 300 mg once daily, and 600 mg once daily to 400 mg once daily. No dose adjustment is recommended for any other covariate based on the magnitude of impact when they were retained in the model. The exposure-response relationship was characterized between itacitinib exposure and probability of aGVHD day 28 response using a linear logistic regression model. Both itacitinib exposure and aGVHD risk status were significant predictors of response. There was no relationship between itacitinib exposure and thrombocytopenia, hypertriglyceridemia, or cytomegalovirus infection. https://pubmed.ncbi.nlm.nih.gov/36601737/

Safety and tolerability [1]
The median (range) exposure to INCB039110 was 102 (23–519) days in the 100 mg twice-daily cohort, 268 (22–535) days in the 200 mg twice-daily cohort, and 197 (58–343) days in the 600 mg once-daily cohort. The most common non-hematologic adverse events regardless of causality are shown in Table 2; most were grade 1 or 2. Grade ≥3 non-hematologic adverse events that occurred in more than one patient were: pneumonia, dyspnea, and hypertension (3 patients each), and congestive heart failure, rectal hemorrhage, asthenia, pyrexia, urinary tract infection, hyperkalemia, increased alkaline phosphatase, and acute renal failure (2 patients each). These 25 events occurred in 18 unique patients. Although infections were common (44.8%), including upper respiratory tract infections in 19.5% of patients (Online Supplementary Table S1), most were mild or moderate, and only four cases (1 each of bronchitis, folliculitis, Herpes simplex, and urinary tract infection) were considered treatment-related by the investigator. All of these four cases were grade 2 and not considered serious, and all resolved without changes to study treatment. Two patients (both in the 600 mg once-daily cohort) died during the study: a 62-year old patient died of pneumonia after approximately 5 months on therapy, and a 61-year old patient died of unspecified causes potentially related to disease progression after slightly less than 4 months on therapy. Both deaths were considered by the investigator to be unrelated to treatment.

[1]. Primary analysis of a phase II open-label trial of INCB039110, a selective JAK1 inhibitor, in patients with myelofibrosis. Haematologica. 2017 Feb;102(2):327-335.

[2]. Combined Anti-Fibrotic and Anti-Inflammatory Properties of JAK-inhibitors on Macrophages in Vitro and in Vivo: Perspectives for Scleroderma-Associated Interstitial Lung Disease. Biochem Pharmacol. 2020 Jun 17;114103.

[3]. Itacitinib (INCB039110), a JAK1 inhibitor, Reduces Cytokines Associated with Cytokine Release Syndrome Induced by CAR T-Cell Therapy. Clin Cancer Res. 2020 Sep 30;26(23):6299–6309.

[4]. Preclinical characterization of itacitinib (INCB039110), a novel selective inhibitor of JAK1, for the treatment of inflammatory diseases. Eur J Pharmacol. 2020 Oct 15:885:173505.

Itacitinib Adipate is the adipate salt form of itacitinib, an orally bioavailable inhibitor of Janus-associated kinase 1 (JAK1) with potential antineoplastic and immunomodulating activities. Upon oral administration, itacitinib selectively inhibits JAK-1, thereby inhibiting the phosphorylation of signal transducer and activator of transcription (STAT) proteins and the production of proinflammatory factors induced by other cytokines, including interleukin-23 (IL-23) and interleukin-6 (IL-6). The JAK-STAT pathway plays a key role in the signaling of many cytokines and growth factors and is involved in cellular proliferation, growth, hematopoiesis, and the immune response; JAK kinases may be upregulated in inflammatory diseases, myeloproliferative disorders, and various malignancies.

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Itacitinib has been used in trials studying the treatment of Melanoma, Carcinoma, Metastatic Cancer, Endometrial Cancer, and B-cell Malignancies, among others.
Itacitinib is an orally bioavailable inhibitor of Janus-associated kinase 1 (JAK1) with potential antineoplastic and immunomodulating activities. Upon oral administration, itacitinib selectively inhibits JAK-1, thereby inhibiting the phosphorylation of signal transducer and activator of transcription (STAT) proteins and the production of proinflammatory factors induced by other cytokines, including interleukin-23 (IL-23) and interleukin-6 (IL-6). The JAK-STAT pathway plays a key role in the signaling of many cytokines and growth factors and is involved in cellular proliferation, growth, hematopoiesis, and the immune response; JAK kinases may be upregulated in inflammatory diseases, myeloproliferative disorders, and various malignancies.

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Drug Indication
Treatment of acute graft versus host disease.

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Janus kinase (JAK) inhibitors (also termed Jakinibs) constitute a family of small drugs that target various isoforms of JAKs (JAK1, JAK2, JAK3 and/or tyrosine kinase 2 (Tyk2)). They exert anti-inflammatory properties linked, in part, to the modulation of the activation state of pro-inflammatory M1 macrophages. The exact impact of JAK inhibitors on a wider spectrum of activation states of macrophages is however still to be determined, especially in the context of disorders involving concomitant activation of pro-inflammatory M1 macrophages and profibrotic M2 macrophages. This is especially the case in autoimmune pulmonary fibrosis like scleroderma-associated interstitial lung disease (ILD), in which M1 and M2 macrophages play a key pathogenic role. In this study, we directly compared the anti-inflammatory and anti-fibrotic effects of three JAK inhibitors (ruxolitinib (JAK2/1 inhibitor); tofacitinib (JAK3/2 inhibitor) and itacitinib (JAK1 inhibitor)) on five different activation states of primary human monocyte-derived macrophages (MDM). These three JAK inhibitors exert anti-inflammatory properties towards macrophages, as demonstrated by the down-expression of key polarization markers (CD86, MHCII, TLR4) and the limited secretion of key pro-inflammatory cytokines (CXCL10, IL-6 and TNFα) in M1 macrophages activated by IFNγ and LPS or by IFNγ alone. We also highlighted that these JAK inhibitors can limit M2a activation of macrophages induced by IL-4 and IL-13, as notably demonstrated by the down-regulation of the M2a associated surface marker CD206 and of the secretion of CCL18. Moreover, these JAK inhibitors reduced the expression of markers such as CXCL13, MARCO and SOCS3 in alternatively activated macrophages induced by IL-10 and dexamethasone (M2c+dex) or IL-10 alone (M2c MDM). For all polarization states, Jakinibs with inhibitory properties over JAK2 had the highest effects, at both 1 μM or 0.1 μM. Based on these in vitro results, we also explored the effects of JAK2/1 inhibition by ruxolitinib in vivo, on mouse macrophages in a model of HOCl-induced ILD, that mimics scleroderma-associated ILD. In this model, we showed that ruxolitinib significantly prevented the upregulation of pro-inflammatory M1 markers (TNFα, CXCL10, NOS2) and pro-fibrotic M2 markers (Arg1 and Chi3L3). These results were associated with an improvement of skin and pulmonary involvement. Overall, our results suggest that the combined anti-inflammatory and anti-fibrotic properties of JAK2/1 inhibitors could be relevant to target lung macrophages in autoimmune and inflammatory pulmonary disorders that have no efficient disease modifying drugs to date. [2]

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Purpose:
T-cells engineered to express a chimeric antigen receptor (CAR T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse-free survival in patients with B-cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening side effect often associated with CAR T-cell therapy. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including interferon-γ and interleukin-6.

Experimental Design:
Many cytokines implicated in CRS are known to signal through the Janus kinase–signal transducers and activators of transcription (JAK-STAT) pathway. Here we study the effect of blocking JAK pathway signaling on CAR T-cell proliferation, anti-tumor activity and cytokine levels in in vitro and in vivo models.

Results:
We report that itacitinib, a potent, selective JAK1 inhibitor, was able to significantly and dose-dependently reduce levels of multiple cytokines implicated in CRS in several in vitro and in vivo models. Importantly, we also report that at clinically relevant doses that mimic human JAK1 pharmacologic inhibition, itacitinib did not significantly inhibit proliferation or antitumor killing capacity of three different human CAR T-cell constructs (GD2, EGFR, and CD19). Finally, in an in vivo model, antitumor activity of CD19-CAR T-cells adoptively transferred into CD19+ tumor bearing immuno-deficient animals was unabated by oral itacitinib treatment.

Conclusion:
Together, these data suggest that itacitinib has potential as a prophylactic agent for the prevention of CAR T-cell–induced CRS, and a phase II clinical trial of itacitinib for prevention of CRS induced by CAR T-cell therapy has been initiated (NCT04071366).[3]

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Pharmacological modulation of the Janus kinase (JAK) family has achieved clinically meaningful therapeutic outcomes for the treatment of inflammatory and hematopoietic diseases. Several JAK1 selective compounds are being investigated clinically to determine their anti-inflammatory potential. We used recombinant enzymes and primary human lymphocytes to assess the JAK1 specificity of itacitinib (INCB039110) and study inhibition of signal transducers and activators of transcription (STAT) signaling. Rodent models of arthritis and inflammatory bowel disease were subsequently explored to elucidate the efficacy of orally administered itacitinib on inflammatory pathogenesis. Itacitinib is a potent and selective JAK1 inhibitor when profiled against the other JAK family members. Upon oral administration in rodents, itacitinib achieved dose-dependent pharmacokinetic exposures that highly correlated with STAT3 pharmacodynamic pathway inhibition. Itacitinib ameliorated symptoms and pathology of established experimentally-induced arthritis in a dose-dependent manner. Furthermore, itacitinib effectively delayed disease onset, reduced symptom severity, and accelerated recovery in three distinct mouse models of inflammatory bowel disease. Low dose itacitinib administered via cannula directly into the colon was highly efficacious in TNBS-induced colitis but with minimal systemic drug exposure, suggesting localized JAK1 inhibition is sufficient for disease amelioration. Itacitinib treatment in an acute graft-versus-host disease (GvHD) model rapidly reduced inflammatory markers within lymphocytes and target tissue, resulting in a marked improvement in disease symptoms. This is the first manuscript describing itacitinib as a potent and selective JAK1 inhibitor with anti-inflammatory activity across multiple preclinical disease models. These data support the scientific rationale for ongoing clinical trials studying itacitinib in select GvHD patient populations.[4]

C₃₂H₃₃F₄N₉O₅

699.66

699.254

C, 54.93; H, 4.75; F, 10.86; N, 18.02; O, 11.43

1334302-63-4

Itacitinib;1334298-90-6

67390313

White to light yellow solid powder

3

15

10

50

1090

0

CEGWJIQFBNFMHQ-UHFFFAOYSA-N

InChI=1S/C26H23F4N9O.C6H10O4/c27-20-18(1-7-32-22(20)26(28,29)30)24(40)37-9-3-17(4-10-37)38-13-25(14-38,5-6-31)39-12-16(11-36-39)21-19-2-8-33-23(19)35-15-34-21;7-5(8)3-1-2-4-6(9)10/h1-2,7-8,11-12,15,17H,3-5,9-10,13-14H2,(H,33,34,35);1-4H2,(H,7,8)(H,9,10)

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.57 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 (3.57 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 (3.57 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.)