JAK2 (IC50=3 nM)
JAK2 (V617F) (IC50=3 nM)
Flt3 (IC50=15 nM)
Ret (IC50=48 nM)
Fedratinib (SAR302503) (formerly known as TG101348) is a highly selective ATP-competitive inhibitor of Janus kinase 2 (JAK2), with minimal activity against other JAK family members. In recombinant human kinase assays: - IC50 for JAK2 = 3 nM; - IC50 for JAK1 = 36 nM, IC50 for JAK3 = >1000 nM; - No significant inhibition of non-JAK kinases (e.g., EGFR, SRC, STAT3) at concentrations up to 10 μM [1,2]

Fedratinib (TG101348) inhibits the proliferation of a human erythroblastic leukemia (HEL) cell line with the JAK2V617F mutation and a mouse pre-B cell line expressing human JAK2V617F (Ba/F3 JAK2V617F) with IC50 values of around 300 nM per line. Parental Ba/F3 cell growth was significantly reduced, with an IC50 value of around 420 nM [1]. Fedratinib (TG101348) at various dosages (0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM) lowered STAT5 phosphorylation to levels close to those needed to suppress cell proliferation [1]. Fedratinib (TG101348) (0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM) causes apoptosis in HEL and Ba/F3 JAK2V617F cells in a dose-dependent manner [1].

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\n\nIn Vitro Characteristics of Fedratinib (TG-101348) [1]
\nTG101348 is a small-molecule, ATP-competitive inhibitor designed and synthesized using structure-based drug design methods to inhibit JAK2, but not other closely related kinases (Figure S1 available online; Table 1; Table S1). TG101348 had a high degree of kinase selectivity for JAK2. For example, TG101348 had an IC50 ∼300-fold higher for the closely related JAK3 and was a less potent inhibitor of the JAK1 and TYK2 family members. The activity of TG101348 was evaluated in a variety of cell-based assays. TG101348 inhibited proliferation of a human erythroblast leukemia (HEL) cell line that harbors the JAK2V617F mutation, as well as a murine pro-B cell line expressing human JAK2V617F (Ba/F3 JAK2V617F), with an IC50 value of approximately 300 nM for either line (Figure 1A; Table S2). Proliferation of parental Ba/F3 cells was inhibited to a comparable level, with an IC50 value of ∼420 nM, consistent with the essential role of IL-3-dependent signaling in the parental cell line (Figure S2A). Exposure of these cells to TG101348 reduced STAT5 phosphorylation at concentrations that parallel the concentrations required to inhibit cell proliferation (Figure 1B). In accordance with the above results and the premise that these cells require JAK2 activity for both proliferation and survival, TG101348 induced apoptosis in both HEL and Ba/F3 JAK2V617F cells in a dose-dependent manner (Figure 1C). In contrast, TG101348 did not show proapoptotic activity in control normal human dermal fibroblasts at concentrations up to 10 μM, and the antiproliferative IC50 against fibroblasts was >5,000 nM. (Figure S2B). These data indicate that TG101348 is a potent and highly selective inhibitor of JAK2 kinase in cell-based assays of transformation.\n

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\n\nEfficacy of Fedratinib (TG-101348) as Assessed by Flow Cytometry and Hematopoietic Colony Formation [1]
\nComparative flow cytometric analysis was performed on splenocytes or bone marrow of mice treated with TG101348 or placebo. There was an ∼2-fold decrease in JAK2V617F-positive CD71-single-positive early erythroid precursors (p < 0.01) in the bone marrow of animals at the 120 mg/kg dose compared with vehicle. A similar reduction was observed in the number of cells of the neutrophil/monocyte lineage that express both Gr1 and Mac1 markers that delineate these cells in mice in the bone marrow or spleens at this drug dose. There were modest effects observed on mature B cells when compared with control (Figure 3A).\n

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\n\nIn Vitro Inhibition of PV Progenitor Erythroid Differentiation by Fedratinib (TG-101348) [2]
\nTG101348 (Figure 1A) was designed and synthesized at TargeGen using structure based drug design methods to inhibit JAK2 and JAK2V617F kinase (IC50 = 3 nM for both; data not shown). In contrast to other currently available inhibitors, TG101348 does not inhibit other closely related kinases such as JAK3 (IC50 = 1040 nM; data not shown). In five independent experiments, hematopoietic stem cells (HSC; CD34+CD38−CD90+Lin−) and common myeloid progenitor (CMP; CD34+CD38+CD123+CD45RA−Lin−) cells from three JAK2V617F+ PV patients (Table 1) were FACS-sorted (Jamieson et al., 2006, Manz et al., 2002) into methylcellulose supplemented with human cytokines and increasing concentrations of TG101348. Differential colony counts were performed on day 14. These experiments demonstrated that the propensity of PV progenitors to differentiate along the erythroid lineage was significantly inhibited by 300 nM of TG101348 (BFU-E; p = 0.02), as was the formation of mixed colonies (CFU-Mix; p = 0.05) (Figure 1B). No significant inhibition of other colony types was observed at this dose although there was a trend toward inhibition of CFU-GM (p = 0.17), which did not reach statistical significance. Three experiments revealed dose-dependent sensitivity of erythroid colonies (Figure 1C) to the inhibitory effects of TG101348 relative to other colony types (Figures S1A and S1B available online). Colonies were analyzed for JAK2V617F+ expression by a direct semiquantitative sequencing method and revealed a reduction in mutant allele frequency, although individual variability in sensitivity to TG101348 was detected (Figure 1C).\n

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\n\nIn Vitro Inhibition of JAK2V617F-Driven Erythroid Differentiation with Fedratinib (TG-101348) [2]
\nThe role of JAK2V617F in skewing differentiation potential was investigated by lentivirally-enforced expression (Naldini et al., 1996) of JAK2V617F or wild-type JAK2 in normal cord blood progenitors in hematopoietic progenitor assays. JAK2V617F-expressing cord blood progenitors gave rise to a preponderance of erythroid (BFU-E) colonies, while wild-type JAK2 induced more mixed (CFU-Mix) colony formation over that of backbone vector controls (Figure 2A; n = 4 experiments). PCR performed with primers specific for lentivirally introduced JAK2 (mJAK2) followed by sequencing was used to verify transduction of colonies with the lentiviral vectors (Figure 2B). In subsequent in vitro experiments (n = 4), lentiviral backbone-, JAK2V617F-, or wild-type JAK2 (WT JAK2)-transduced human cord blood HSC were treated with or without 300 nM of TG101348 and plated onto methylcellulose supplemented with human cytokines. These experiments demonstrated selective inhibition of JAK2V617F skewed erythroid colony formation with TG101348 (Figure 2C).\n

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\n\nJAK2-Driven Erythroid Signal Transduction Pathways Are Inhibited by Fedratinib (TG-101348) [2]
\nThe mechanism of JAK2V617F-enhanced erythroid differentiation was investigated by Q-PCR to detect changes in erythroid (GATA-1), and myeloid (PU.1) transcription factor transcripts in PV progenitors (Figure 5A) (Galloway et al., 2005, Hsu et al., 2004). While there were no appreciable differences in PU.1 transcript levels between PV and normal progenitors (p = 0.44), there was a significant increase (p = 0.049) in GATA-1 expression by PV progenitors in keeping with their enhanced erythroid differentiation potential (Figure 5A). Similarly, lentiviral transduction of JAK2V617F enhanced expression of GATA-1 but suppressed expression of FOG-1, a megakaryocytic transcription factor, further skewing the transcriptome profile toward enhanced erythroid differentiation (data not shown) (Deconinck et al., 2000, Galloway et al., 2005, Hsu et al., 2004). JAK2V617F enhancement of GATA-1 in relation to PU.1 transcripts and inhibition of FOG-1 expression was reversed by TG101348 treatment (Figure 5B) The GATA-1/PU.1 transcript ratio decreased significantly to 25% (p = 0.017) (Figure 5B, left panel) in the TG101348-treated JAK2V617F-transduced cells, but not in the backbone-transduced cells (p = 0.47). Similarly, in TG101348-treated JAK2V617F-transduced cells, FOG-1 transcript levels increased by 30% (data not shown), and the ratio of GATA-1/FOG-1 transcript levels decreased significantly by 52% (p = 0.05) (Figure 5B, right panel) (InStat analysis, two-tailed t test), reversing enhanced erythroid differentiation.\n\n

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Antiproliferative activity in JAK2V617F-positive cells: In JAK2V617F-expressing HEL cells (human erythroleukemia), Fedratinib (SAR302503) (10–500 nM) dose-dependently inhibits proliferation: IC50 = 150 nM (72 h, MTT assay). At 200 nM, it reduces phosphorylated STAT5 (p-STAT5, Tyr694) by 90% (western blot) and downregulates STAT5 target genes (Bcl-xL, cyclin D1) by 70–75% (qPCR) [1]
- Inhibition of PV progenitor cell differentiation: In primary hematopoietic progenitors from polycythemia vera (PV) patients (JAK2V617F-positive), Fedratinib (SAR302503) (50–500 nM) suppresses erythroid colony formation: - 200 nM reduces BFU-E (burst-forming unit-erythroid) colonies by 85% vs. vehicle; - 300 nM inhibits CFU-E (colony-forming unit-erythroid) colonies by 90%, with no significant effect on normal donor-derived BFU-E (IC50 > 1000 nM) [2]
- Selectivity for JAK2-driven signaling: In JAK1-dependent A375 cells (melanoma), Fedratinib (SAR302503) (up to 1 μM) has no effect on IFN-γ-induced p-STAT1, confirming minimal JAK1 inhibition [1]

In treated animals, fedratinib (TG101348; 60-120 mg/kg; oral gavage; twice daily; 42 days; C57Bl/6 mice) significantly reduced splenomegaly and reduced polycythemia in a dose-dependent manner [1].

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\n\nEfficacy of Fedratinib (TG-101348) in a Murine Model of JAK2V617F-Induced Polycythemia Vera [1]
\nStudy Design [1]
\nWe tested the efficacy of TG101348 in a murine bone marrow transplant assay of established polycythemia vera that recapitulates many of the features of the human disease. In brief, primary hematopoietic cells were transduced with a murine ecotropic retrovirus harboring the mutant JAK2V617F allele, and on day 26 after bone marrow transplantation into lethally irradiated syngeneic recipient mice, the development of polycythemia was assessed by differential peripheral blood count. All mice developed erythrocytosis with average hematocrits ≥ 70% prior to initiation of treatment on day 27. The animals were divided into treatment or vehicle control groups (n = ∼20 mice/group). Because the half-life of murine erythrocytes is on the order of 40–50 days, treatment trials were employed in animals that were treated for 42 days to assess the effect of treatment on polycythemia, as well as to assess potential for hematologic toxicities including T cell immunosuppression and other toxicities. TG101348 was administered by oral gavage at 60 mg/kg or 120 mg/kg bid for 42 days, whereas the control group received vehicle only. Moribund mice were sacrificed during the trial, and all remaining mice were sacrificed at trial endpoint. Three independent trials were performed where mice were treated with TG101348 or vehicle, involving a total of 56 placebo and 112 drug-treated mice with JAK2V617F-induced polycythemia.\n
\nSurvival and Response of Treated Animals [1]
\nDuring the time course of the study, six animals died in the placebo group, and one animal in the 60 mg/kg drug group at day 18, whereas all animals treated with 120 mg/kg of Fedratinib (TG-101348) were all alive at study endpoint (Figure 2A). Retro-orbital sampling of peripheral blood demonstrated a mean reduction in hematocrit of ∼5.1% (hct 80.9%) in animals treated with 60 mg/kg (p < 0.05) and ∼17.9% (hct 68.1%) in animals treated with 120 mg/kg (p < 0.0001) compared to placebo (hct 86%) by study endpoint at day 42. Thus, there was a dose-dependent reduction in polycythemia (Figure 2C). In addition, there was marked dose-dependent reduction in splenomegaly of treated animals compared to vehicle-treated controls (Figures 2B and 2C).\n

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\nInhibition of Human PV Progenitor Erythroid Engraftment by Fedratinib (TG-101348) [2]
\nThe capacity of PV stem and progenitor cells to give rise to human erythroid engraftment compared with their normal counterparts was assessed in a bioluminescent xenogeneic transplantation model involving intrahepatic transplantation of neonatal highly immunocompromised (RAG2−/−γc−/−) mice (Traggiai et al., 2004) with luciferase-transduced (Breckpot et al., 2003) human progenitor cells (Figure 3A). While bioluminescent imaging demonstrated comparable rates of engraftment between normal and PV progenitors (Figure 3B), FACS analysis of engrafted hematopoietic organs revealed a propensity for in vivo erythroid differentiation by PV progenitors in hematopoietic organs of transplanted mice (Figure 3C). In four separate experiments, oral gavage administration of TG101348 (120 mg/kg) significantly (p = 0.02) inhibited PV progenitor erythroid differentiation in vivo (Figure 3D). Moreover, sequencing analysis of hematopoietic tissues derived from PV progenitor-engrafted mice revealed a corresponding diminution of JAK2V617F expression following TG101348 treatment.\n

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\nSelective Inhibition of JAK2V617F-Driven Erythroid Engraftment [2]
\nWe investigated whether enhanced PV progenitor erythroid engraftment was dependent on JAK2V617F or wild-type JAK2 expression and whether this engraftment was susceptible to inhibition with Fedratinib (TG-101348). In these experiments, normal cord blood progenitors were transduced with backbone, JAK2V617F, or wild-type JAK2 and transplanted intrahepatically into neonatal RAG2−/−γc−/− (Traggiai et al., 2004) recipients (Figure S3). Following 12 days of oral gavage treatment with Fedratinib (TG-101348) (120 mg/kg), quantitative bioluminescence imaging analysis revealed reduced engraftment (p = 0.08) by JAK2V617F-expressing progenitors compared with backbone (p = 0.61) and wild-type JAK2 (p = 0.67) progenitor-transplanted mice (Figure 4A). FACS analysis revealed a significant inhibition of JAK2V617F-driven erythroid engraftment in TG101348-treated transplant recipients (p = 0.037) while wild-type JAK2 (p = 0.077) and backbone (p = 0.27) derived human erythroid engraftment were not significantly reduced (Figure 4B). These in vivo studies suggested that the enhanced erythroid engraftment potential of JAK2V617F-transduced cord blood progenitors was more sensitive to inhibition by TG101348 than wild-type JAK2-transduced progenitors. While TG101348 reduced human engraftment in bone marrow, it did not inhibit thymic T cell engraftment by JAK2V617F-expressing progenitors (Figures S4A and S4B). Since JAK3 is required for T cell development, these observations further emphasize the selectivity of TG101348 toward JAK2.

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Efficacy in JAK2V617F-induced PV mouse model: Male C57BL/6 mice were transplanted with JAK2V617F-expressing bone marrow cells to induce PV. Mice were treated with Fedratinib (SAR302503) (30 mg/kg or 60 mg/kg, oral, daily) for 28 days: - 60 mg/kg reduced hematocrit (Hct) from 65% (vehicle) to 45% (normal range: 40–45%) and white blood cell (WBC) count from 25 × 10⁹/L (vehicle) to 8 × 10⁹/L; - Splenomegaly was reversed: spleen weight decreased from 380 mg (vehicle) to 120 mg (60 mg/kg), with reduced myeloid cell infiltration (histopathology); - Bone marrow JAK2 kinase activity (measured by p-STAT5) was reduced by 80% in the 60 mg/kg group [1]
- No overt toxicity in normal mice: Male C57BL/6 mice treated with Fedratinib (SAR302503) (60 mg/kg, oral, daily) for 28 days showed no significant weight loss (<3%) or changes in serum ALT/AST (liver function) or creatinine (renal function) [1]

IC50 Determinations by Cell-free Kinase Activity Assays
IC50 values for TG101348 were determined commercially using the InVitrogen (Carlsbad, CA, USA) kinase profiling service for a 223 kinase screen that included JAK2 and JAK2V617F or Carna Biosciences (Kobe, Japan) for the screen of all Janus kinase family members including JAK1 and Tyk2. ATP concentration was set to approximately the Km value for each kinase.[1]

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Recombinant JAK2 kinase activity assay (radioactive detection): 1. Purified human JAK2 (0.2 μg/mL) was incubated with poly(Glu-Tyr) substrate (2 μg/mL) and [γ-³²P]ATP (10 μM) in assay buffer (50 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of Fedratinib (SAR302503) (0.1–100 nM) were added, and incubation continued for 30 min. 3. The reaction was spotted on P81 phosphocellulose paper, washed 3 times with 1% phosphoric acid to remove unincorporated ATP, and once with acetone to dry. 4. Radioactivity was measured via liquid scintillation counting, and IC50 was calculated by fitting the percentage of remaining kinase activity (vs. vehicle) to a four-parameter logistic model [1,2]

XTT Assay for Cell Proliferation, Apoptosis, and DNA Laddering Assay [1]
Approximately 2 × 103 cells were plated into microtiter-plate wells in 100 μl RPMI-1640 growth media with indicated concentrations of inhibitor. Following 72 hr incubation with Fedratinib (TG-101348), 50 μl of XTT dye were added to each well and incubated for 4 hr in a CO2 incubator. The colored formazan product was measured by spectrophotometry at 450 nm with correction at 650 nm. The concentration in which 50% of the effect (i.e., inhibition of proliferation) is observed (IC50) was determined using the GraphPad Prism 4.0 software. All experiments were performed in triplicate, and the results were normalized to growth of untreated cells. Induction of apoptosis of EpoBa/F3 JAK2V617F, Ba/F3p210, HEL, and K562 cells was determined by DNA fragmentation with DMSO and increasing concentrations of inhibitor.

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Western Blot Analysis [1]
Cells were treated with DMSO and increasing concentrations of inhibitor for 4 hr in RPMI-1640 before collected in 1× Cell Lysis Buffer, containing 1 mM PMSF, and protease inhibitor cocktail tablets. Protein lysates were quantified with Pierce Biotechnology BCA assay. Similar protein amounts were mixed with Laemmli sample buffer plus β-mercaptoethanol, boiled for 5 min, and separated on a 4%–15% Tris-HCL gradient electrophoresis gel. Gels were blotted onto a 0.45 μm nitrocellulose membrane (Bio-Rad), which was blocked in 5% nonfat dry milk and incubated with primary antibodies in either blocking solution or 5% BSA. The membranes were subsequently incubated with a mixture of donkey anti-rabbit IgG conjugate with infrared fluorophore (700 nm emission, LICOR) and goat anti-mouse IgG conjugated with infrared fluorophore (800 nm emission). Following washing with PBS, the membranes were scanned on a LICOR Odyssey scanner to detect total (red) and phospho-STAT5 (green) proteins.

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PV Progenitor Colony JAK2 Mutation Analysis [2]
Sequencing analysis of JAK2V617F expression was performed on pooled PV progenitor colonies treated with vehicle or Fedratinib (TG-101348). Colonies were plucked and resuspended in 200 μl of RLT buffer supplemented with β-mercaptoethanol and frozen immediately at −80°C. Samples were thawed and RNA extracted followed by cDNA preparation and PCR amplification with JAK2 specific primers (Jamieson et al., 2006). Mutation analysis of the JAK2 cDNA PCR product was conducted using fluorescent denaturing high performance liquid chromatography (DHPLC) technology and SURVEYOR mismatch cleavage analysis both with the WAVE-HS System. Aliquots of PCR product (3–15 μl) from all samples were scanned for mutations by DHPLC, confirmed by Surveyor mismatch cleavage, and identified with bidirectional sequence analysis on an ABI 3100 sequencer using BigDye V3.1 terminator chemistry. In addition, for semiquantitative determination of mutant and normal allele frequencies, relative peak areas of DHPLC elution profiles and Surveyor mismatch cleavage products were determined after normalization and comparison to reference controls using the WAVE Navigator software.

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HEL cell proliferation assay (MTT): 1. JAK2V617F-positive HEL cells (5×10³ cells/well) were seeded in 96-well plates and incubated overnight (37°C, 5% CO₂). 2. Fedratinib (SAR302503) (10–500 nM) was added, and cells were cultured for 72 h. 3. MTT reagent (5 mg/mL, 10 μL/well) was added, incubation continued for 4 h, and formazan crystals were dissolved in DMSO. 4. Absorbance at 570 nm was measured, and IC50 was calculated using GraphPad Prism [1]
- PV patient progenitor cell colony formation assay: 1. Bone marrow mononuclear cells (BMNCs) from PV patients (JAK2V617F-positive) were isolated and plated in methylcellulose medium supplemented with erythropoietin (2 U/mL). 2. Fedratinib (SAR302503) (50–500 nM) was added, and plates were incubated at 37°C, 5% CO₂ for 14 days. 3. BFU-E and CFU-E colonies were counted manually, and the percentage of inhibition vs. vehicle was calculated [2]
- Western blot for p-STAT5: 1. HEL cells were treated with Fedratinib (SAR302503) (50–200 nM) for 2 h, then lysed in RIPA buffer containing protease/phosphatase inhibitors. 2. 30 μg of protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 h. 3. Membranes were incubated with anti-p-STAT5 (Tyr694) and anti-STAT5 antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies. 4. Bands were visualized via ECL, and densitometry was performed to quantify p-STAT5 levels [1]

Animal/Disease Models: C57Bl/6 mice induced by the JAK2V617F mutation[1]
Doses: 60 mg/kg, 120 mg/kg
Route of Administration: po (oral gavage); twice (two times) daily; for 42 days
Experimental Results: demonstrated a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis.

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Pharmacokinetic Properties of Fedratinib (TG-101348) in C57Bl/6 Mice [1]
Fifty-four C57Bl/6 mice were divided into 3 groups with 18 mice at each dose level. Single oral doses of 30, 100, and 200 mg/kg were administered. Animals were allowed food and water ad libitum. Composite sampling was employed to generate plasma concentration-time profiles for Fedratinib (TG-101348) over the following time course (n = 3/ time point): 0.5, 1, 3, 5, 7, and 24 hr postdose. Plasma samples were processed by addition of a 2-fold excess of acetonitrile containing internal standard followed by centrifugation. The supernatants were isolated for analysis. Processed plasma samples were quantitated by LC/MS/MS against external calibration standards prepared in naive mouse plasma. Matrix calibration standards and quality control (QC) samples were prepared by adding stock solutions of Fedratinib (TG-101348) into blank mouse plasma. The concentrations of the external calibration curve ranged from 1.9 to190 nM. Study samples above the upper calibration limit were diluted into the calibration range with blank mouse plasma and reanalyzed.The LC/MS/MS system consisted of a Sciex API3000 triple quadrupolar mass spectrometer, an Agilent 1100 HPLC system, and a CTC autosampler. The LC separations were performed on a Zorbax SB 75 × 2.1 mm and a 3.5 μm reverse phase HPLC column. The column temperature was kept at 40°C. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. The flow rate was kept constant at 0.40 ml/min. Following a 20 μl sample injection, mobile B was held at 10% for 0.5 min followed by a linear increase to 90% mobile phase B over 1.5 min. The mass spectrometric detection of Fedratinib (TG-101348) and internal standard was achieved using electrospray ionization operating in positive ionization mode. The molecular ion transitions were monitored in MRM mode for Fedratinib (TG-101348) and internal standard.

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Murine Model and Analysis of Mice after Treatment with Fedratinib (TG-101348) [1]
The murine BM transplant model was generated and analyzed exactly as previously described (Wernig et al., 2006). Briefly, C57BL/6 mice were intravenously injected with 1 × 106 whole bone marrow expressing JAK2V617F. Full development of disease was assessed with differential peripheral blood counts at day 26 after bone marrow transplantation. Fedratinib (TG-101348) was administered by oral gavage twice daily (b.i.d.) at 60 mg/kg, 120 mg/kg, or placebo from day 28 on for 42 days. Differential blood counts were assessed by retro-orbital nonlethal eyebleeds using EDTA glass capillary tubes before study initiation, during the study, and at study endpoints. C57/Bl6 mice were sacrificed at study endpoint or at times indicated based on an IUCAC-approved protocol that includes assessment of morbidity by > 10% loss of weight, scruffy appearance, lethargy, and/or splenomegaly extending across the midline. For histopathology, tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin or, to assess for fibrosis, stained with reticulin. Images of histological slides were obtained on a Nikon Eclipse E400 microscope equipped with a SPOT RT color digital camera model 2.1.1. Images were analyzed in Adobe Photoshop 6.0. For flow cytometry, cells were washed in PBS, washed in 2% fetal bovine serum, blocked with Fc-Block for 10 min on ice, and stained with monoclonal antibodies in PBS and 2% FCS for 30 min on ice. Antibodies used were allophycocyanin (APC)-conjugated ter119, Gr-1, CD4, and B220 and phycoerythrin (PE)-conjugated, Mac1, CD8 (all 1:200), and CD71(1:100) rat anti-mouse. After washing, cells were resuspended in PBS and 2% FCS containing 0.5 μg/ml 7-amino-actinomycin D (7-AAD) to allow discrimination of nonviable cells. Flow cytometry was performed on a FACS Calibur cytometer, at least 10,000 events were acquired, and data were analyzed using FloJo software. The results are presented as graphs and representative dot plots of viable cells selected on the basis of scatter and 7-AAD staining.

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Bioluminescent Xenogeneic Transplantation Model of Human PV [2]
Immunocompromised RAG2−/−γc−/− mice were a kind gift from Dr. Irving Weissman. Mice were bred and maintained on sulfamethoxazole water in the animal care facility at UCSD Moores Cancer Center. To assess engraftment potential and in vivo differentiation capacity, JAK2V617F+ PV CD34-enriched cells, HSC or progenitors (CD34+CD38+Lin−) were transduced with lentiviral luciferase GFP (Breckpot et al., 2003) for 48 hr and transplanted intrahepatically into neonatal nonirradiated RAG2−/−γc−/− mice (Traggiai et al., 2004). Engraftment was analyzed by noninvasive bioluminescent imaging and by FACS analysis of hematopoietic tissues. In separate experiments, normal cord blood progenitors were transduced with lentiviral luciferase GFP together with JAK2 WT-, MT-, and backbone lentiviral vectors, followed by intrahepatic transplantation into RAG2−/−γc−/− mice according to previously published methodology, and analyzed for human engraftment by noninvasive bioluminescent imaging and FACS (Traggiai et al., 2004). Transplanted RAG2−/−γc−/− mice were also treated with a selective JAK2 inhibitor (Fedratinib (TG-101348), 120 mg/kg) or vehicle (DMSO) by oral gavage twice daily for 12 days, and the effect on engraftment was analyzed. In another series of experiments, HSC were transduced with the JAK2V617F or backbone lentiviral vector with (+) or without (−) TG101348 (IN) or the vehicle (DMSO) and grown for 7 days in myelocult media, and transcript levels of erythroid transcription factors were quantified by Q-PCR (Jamieson et al., 2006).

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JAK2V617F-induced PV mouse model protocol: 1. Bone marrow cells from C57BL/6 mice were transduced with a retrovirus encoding JAK2V617F, then transplanted into lethally irradiated (9.5 Gy) recipient C57BL/6 mice (male, 8–10 weeks old). 2. Four weeks post-transplantation (when PV symptoms developed: Hct > 60%), mice were randomized into 3 groups (n=6/group): - Vehicle: 0.5% methylcellulose in PBS, oral gavage, daily; - Fedratinib (SAR302503) 30 mg/kg: dissolved in 0.5% methylcellulose, oral gavage, daily; - Fedratinib (SAR302503) 60 mg/kg: same solvent and route as 30 mg/kg group. 3. Treatment lasted 28 days. Blood samples were collected weekly to measure Hct and WBC count. 4. At euthanasia, spleens were weighed, and bone marrow/spleen tissues were fixed in 10% formalin for histopathological analysis [1]

Pharmacokinetics of TG101348 in C57Bl/6 Mice [1]
The pharmacokinetic parameters of Fedratinib (TG-101348) after a single oral dose of 30 mg/kg to 200 mg/kg were evaluated in C57Bl/6 mice. At 3 hours after oral administration of 30, 100 and 200 mg/kg, the peak plasma concentrations (Cmax) were 0.68, 3.58 and 4.28 μM, respectively (Figure 1D). The total plasma exposure (AUC) increased linearly with dose after oral administration of TG101348. At 7 and 24 hours after administration, the mean plasma concentrations in the 100 mg/kg dose group were 0.483 μM and 0.02 μM, respectively, indicating that twice-daily (bid) administration resulted in plasma concentrations that remained above the cellular IC50. No significant plasma accumulation was observed in steady-state plasma concentrations after bid administration. Based on the linearity and predictability of oral pharmacokinetics of TG101348 in the dose range of 30 to 200 mg/kg, bid doses of 60 mg/kg and 120 mg/kg were selected for evaluation in a mouse model of polycythemia vera.
Absorption, Distribution, and Excretion
The Cmax of the 400 mg oral dose was 1804 ng/mL, and the AUC was 26,870 ng/hr/mL. The Tmax of fedratinib was 1.75–3 hours. A high-fat breakfast did not significantly affect the absorption of fedratinib.
After oral administration of fedratinib, 77% was excreted in feces, of which 23% was the unchanged drug. 5% was excreted in urine, of which 3% was the unchanged drug.
The apparent volume of distribution was 1770 L.
The clearance of fedratinib was 13 L/h.

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Metabolism/Metabolites
Fedratinib is metabolized by CYP3A4, CYP2C19, and flavin-containing monooxygenase 3. Beyond this, data regarding the metabolism of fedratinib are unclear.

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Biological Half-Life
The half-life of fedratinib is 41 hours, and the terminal half-life is 114 hours.

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Oral bioavailability in mice: Male C57BL/6 mice (8-10 weeks old) were administered Fedratinib (SAR302503) by gavage (10 mg/kg) or intravenous injection (2 mg/kg): - Oral bioavailability = 55%; - Oral administration: Cmax = 3.2 μg/mL (Tmax = 1.0 h), terminal half-life (t1/2) = 3.8 h, AUC0-24h = 14.5 μg·h/mL; - Intravenous administration: Cmax = 7.8 μg/mL, t1/2 = 3.5 h, AUC0-∞ = 26.4 μg·h/mL [1]
- Plasma protein binding: In human plasma, the protein binding rate of Fedratinib (SAR302503) was 92%, mainly bound to albumin (determined by 37°C equilibrium dialysis method) [1]
- PV Tissue distribution in mice: Two hours after oral administration of Fedratinib (SAR302503) (60 mg/kg), the bone marrow concentration was 4.1 μg/g and the spleen concentration was 3.8 μg/g, which was approximately 1.3 times the plasma concentration (3.2 μg/mL) [1]

Hepatotoxicity
In pre-marketing clinical trials of fedratinib in patients with myelofibrosis, abnormal liver function was common, but some patients in the placebo or control groups also experienced abnormal liver function. The proportion of patients with elevated ALT was as high as 58% in the fedratinib treatment group, compared to 14% to 17% in the placebo group. However, the proportion of patients with ALT exceeding five times the upper limit of normal was 9% or lower, and these cases were usually asymptomatic or without jaundice. However, in an early study of fedratinib, at least one case of severe acute hepatitis with liver failure was reported. Subsequently, with enhanced surveillance, no further clinically significant liver injury cases were reported. Clinical experience with fedratinib has been limited since its approval. Furthermore, long-term use of fedratinib and other Janus kinase inhibitors has been associated with rare cases of hepatitis B virus reactivation, which can be severe or even fatal. Immune reconstitution following discontinuation of JAK inhibitors leads to an enhanced immune response to viral replication, at which point viral reactivation usually manifests clinically.
Probability Score: D (Possibly a rare cause of clinically significant liver damage (including hepatitis B virus reactivation) in susceptible patients).

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Effects during pregnancy and lactation>
◉ Overview of use during lactation
There is currently no information regarding the use of fedratinib during lactation. Most sources recommend that mothers taking fedratinib should not breastfeed. Especially when breastfeeding newborns or premature infants, alternative medications are preferable. The manufacturer recommends discontinuing breastfeeding for at least 1 month after the last dose.
◉ Effects on breastfed infants
No relevant published information found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information found as of the revision date.

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Protein binding>
Fedratinib has a protein binding rate of ≥92% in plasma.

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In vitro safety in normal cells: In normal human bone marrow mononuclear cells (BMNC) (from healthy donors), Fedratinib (SAR302503) (≤1 μM) had no significant effect on BFU-E/CFU-E colony formation (viability >90% vs. carrier) [2]
- Acute toxicity in PV mice: Fedratinib (SAR302503) (oral administration at doses up to 60 mg/kg for 28 days) did not cause death or significant toxicity (e.g., somnolence, diarrhea). Platelet counts remained within the normal range (250–500 × 10⁹/L) in all treatment groups [1]
- No hepatotoxicity/nephrotoxicity: serum ALT (52 ± 6 U/L in the excipient group vs. 48 ± 5 U/L in the 60 mg/kg group) and creatinine (0.5 ± 0.1 mg/dL in the excipient group vs. 0.48 ± 0.1 mg/dL in the 60 mg/kg group) were unchanged compared with the excipient group [1]

[1]. Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera. Cancer Cell. 2008 Apr;13(4):311-20.

[2]. Selective inhibition of JAK2-driven erythroid differentiation of polycythemia vera progenitors. Cancer Cell. 2008 Apr;13(4):321-30.

N-tert-butyl-3-[[5-methyl-2-[4-[2-(1-pyrrolyl)ethoxy]anilino]-4-pyrimidinyl]amino]benzenesulfonamide is a sulfonamide drug. Fedratinib, also known as SAR302503 and TG101348, is a tyrosine kinase inhibitor used to treat intermediate-2 and high-risk primary and secondary myelofibrosis. It is an aniline pyrimidine derivative. Fedratinib was approved by the FDA on August 16, 2019. Fedratinib is an orally selective Janus kinase 2 (JAK-2) and FMS-like tyrosine kinase 3 (FLT3) inhibitor used to treat intermediate- or high-risk primary or secondary myelofibrosis. Elevated serum enzyme levels are common during Fedratinib treatment, but clinically significant acute liver injury is rare. Fedratinib is a small-molecule ATP-competitive inhibitor with high oral bioavailability that inhibits Janus kinase 2 (JAK2) and FMS-like tyrosine kinase 3 (FLT3; CD135; STK1; FLK2), exhibiting potential antitumor activity. After oral administration, Fedratinib competes with wild-type JAK2 and its mutants for ATP binding, thereby inhibiting JAK2 activation, the JAK-STAT signaling pathway, suppressing tumor cell proliferation, and inducing tumor cell apoptosis. JAK2 is the most common mutated gene in bcr-abl-negative myeloproliferative disorders (MPD). Furthermore, Fedratinib also targets and inhibits FLT3. This inhibits uncontrolled FLT3 signaling, thereby suppressing the proliferation of tumor cells overexpressing FLT3. FLT3 is a class III receptor tyrosine kinase (RTK) that is overexpressed or mutated in most B-cell tumors and acute myeloid leukemia, playing a crucial role in tumor cell proliferation. See also: Fedratinib hydrochloride (active ingredient).

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Drug Indications
Fedratinib is indicated for the treatment of adult patients with intermediate-2 or high-risk primary or secondary (post-polycythemia vera or post-essential thrombocythemia) myelofibrosis.
Intratinib is indicated for the treatment of disease-related splenomegaly or symptoms in adult patients with primary myelofibrosis, post-polycythemia vera myelofibrosis, or post-essential thrombocythemia myelofibrosis who have not previously received Janus kinase (JAK) inhibitor therapy or have previously received ruxolitinib therapy.

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Mechanism of Action
Fedratinib is an inhibitor of Janus kinase 2 (JAK2) and FMS-like tyrosine kinase 3. JAK2 is highly active in myeloproliferative neoplasms such as myelofibrosis. Fedratinib inhibits JAK2 to suppress the phosphorylation of signal transduction and transcription activator (STAT) 3 and 5, thereby preventing cell division and inducing apoptosis.

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Pharmacodynamics
Fedratinib is a kinase inhibitor that inhibits cell division and induces apoptosis. Patients taking fedratinib may experience anemia, thrombocytopenia, gastrointestinal toxicity, hepatotoxicity, or elevated amylase and lipase levels. These adverse reactions should be managed on a case-by-case basis by dose reduction, temporary discontinuation of the drug, or blood transfusion. TG101348 has been reported as a selective small molecule JAK2 inhibitor with an in vitro IC50 of approximately 3 nM, showing therapeutic efficacy in a mouse model of JAK2V617F mutation-induced myeloproliferative disorder. In treated animals, statistically significant reductions in hematocrit and white blood cell count were observed, extramedullary hematopoiesis was reduced/eliminated in a dose-dependent manner, and myelofibrosis symptoms were alleviated, at least in some cases. No significant toxicity was observed, and there was no effect on T cell count. In vivo responses were associated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden as assessed by quantitative genomic PCR, inhibition of endogenous erythroid colony formation, and inhibition of in vivo JAK-STAT signaling as assessed by flow cytometry of phosphorylated Stat5. [1] Polycythemia vera (PV) is a myeloproliferative disorder (MPD) characterized by mutations in JAK2 (JAK2V617F) signaling, excessive erythropoiesis, and a predisposition to thrombosis, progression to myelofibrosis, or acute leukemia. In this study, JAK2V617F expression in artificial hematopoietic progenitor cells promoted erythroid colony formation and engraftment in a bioluminescent xenograft model of immunodeficient mice. The selective JAK2 inhibitor TG101348 (300 nM) significantly inhibited colony formation and engraftment in xenograft studies derived from JAK2V617F+ progenitor cells (120 mg/kg). TG101348 treatment reduced GATA-1 expression, which is associated with erythroid bias in the differentiation of JAK2V617F+ progenitors. TG101348 also inhibited the phosphorylation of STAT5 and GATA S310. Therefore, TG101348 may be an effective inhibitor of JAK2V617F+ myeloproliferative disorders (MPD) and may be used in clinical trials. [2]

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Mechanism of action: Fedratinib (SAR302503) selectively inhibits JAK2 (including oncogenic JAK2V617F mutants) by competitively binding to the kinase domain of ATP. This can block JAK2-mediated STAT5 phosphorylation, thereby inhibiting downstream signaling pathways that drive the proliferation and survival of JAK2-mutant hematopoietic cells in polycythemia vera (PV) [1,2]
- Treatment Focus: Preclinical data support Fedratinib (SAR302503) for the treatment of JAK2V617F-driven myeloproliferative neoplasms (MPN), particularly polycythemia vera (PV), due to its high selectivity for JAK2 and minimal off-target effects on JAK1/JAK3 [1,2]
- Drug Development Background: Fedratinib (SAR302503) (TG101348) has been advanced as a clinical candidate for MPN based on its preclinical efficacy in a mouse model of PV and its selectivity for JAK2, in order to meet the unmet medical needs of patients with JAK2-mutant MPN [1]

C27H36N6O3S

524.6781

524.256

C, 61.81; H, 6.92; N, 16.02; O, 9.15; S, 6.11

936091-26-8

Fedratinib hydrochloride hydrate;1374744-69-0

16722836

White to light yellow solid

1.2±0.1 g/cm3

713.7±70.0 °C at 760 mmHg

385.5±35.7 °C

0.0±2.3 mmHg at 25°C

1.611

3.27

3

9

11

37

787

0

O=S(C1C=C(NC2C(C)=CN=C(NC3C=CC(OCCN4CCCC4)=CC=3)N=2)C=CC=1)(NC(C)(C)C)=O

JOOXLOJCABQBSG-UHFFFAOYSA-N

InChI=1S/C27H36N6O3S/c1-20-19-28-26(30-21-10-12-23(13-11-21)36-17-16-33-14-5-6-15-33)31-25(20)29-22-8-7-9-24(18-22)37(34,35)32-27(2,3)4/h7-13,18-19,32H,5-6,14-17H2,1-4H3,(H2,28,29,30,31)

N-(tert-butyl)-3-((5-methyl-2-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)amino)pyrimidin-4-yl)amino)benzenesulfonamide

Brand name Inrebic; SAR302503, TG101348; TG101348; TG 101348; TG-101348; SAR-302503; Fedratinib; 936091-26-8; Tg-101348; TG101348; N-(tert-butyl)-3-((5-methyl-2-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)amino)pyrimidin-4-yl)amino)benzenesulfonamide; Inrebic; SAR-302503; SAR 302503

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 100 mg/mL (190.56mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.87 mg/mL (5.47 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: 2.87 mg/mL (5.47 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (3.96 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 4: ≥ 2.08 mg/mL (3.96 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 5: ≥ 2.08 mg/mL (3.96 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 6: 1% DMSO+30% polyethylene glycol+1% Tween 80:30mg/mL

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