IDO1 (IC50 = 71.8 nM)

Epacadostat (INCB 024360) has no effect on other related enzymes like IDO2 or tryptophan 2,3-dioxygenase (TDO), but it specifically inhibits human IDO1 in cellular experiments with an IC50 value of about 10 nM. In a comparable test utilizing mouse IDO1-transfected HEK293/MSR cells, epacadostat (INCB 024360) also shown considerable action against mouse IDO1, with an IC50 value of 52.4 nM±15.7 nM [1].
In cellular assays, INCB024360 selectively inhibits human IDO1 with IC(50) values of approximately 10nM, demonstrating little activity against other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO). In coculture systems of human allogeneic lymphocytes with dendritic cells (DCs) or tumor cells, INCB024360 inhibition of IDO1 promotes T and natural killer (NK)-cell growth, increases IFN-gamma production, and reduces conversion to regulatory T (T(reg))-like cells. IDO1 induction triggers DC apoptosis, whereas INCB024360 reverses this and increases the number of CD86(high) DCs, potentially representing a novel mechanism by which IDO1 inhibition activates T cells. Furthermore, IDO1 regulation differs in DCs versus tumor cells.

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INCB023843 and INCB024360 restored tryptophan levels to those seen in DMSO-treated controls and significantly impaired kynurenine generation in both cell lines with IC50 values of 172 and 76 nmol/L, respectively, for CT26 cells and 46 and 27 nmol/L, respectively, for PAN02 cells (Fig. 2B). Hydroxyamidines seem to be slightly less potent on cells expressing murine Ido than those expressing human Ido. For example, there was a >4-fold shift in potency between HEK293 cells transfected with human (15 nmol/L) and mouse Ido1 (66 nmol/L) for INCB024360 [2].

For a period of 12 days, female Balb/c mice with CT26 tumors were given an oral dose of 100 mg/kg of epacadostat twice a day. Kynurenine is potently inhibited in plasma, tumors, and lymph nodes by epacadostat (INCB 024360). 50 mg/kg Epacadostat (INCB 024360) decreased plasma kynurenine levels in naive C57BL/6 mice in less than an hour, and these levels stayed at least 50% suppressed for the duration of an 8-hour period[2].

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To investigate whether IDO1 inhibition would similarly reverse immune escape in vivo, we treated mice bearing IDO1-expressing PAN02 pancreatic carcinomas orally with INCB024360. The growth of tumors in syngeneic immunocompetent C57BL/6 mice was inhibited in a dose-dependent fashion, with 37% and 57% TGC, respectively, for 25 and 100 mg/kg INCB024360 (Figure 5A; P < .01). However, tumors growing in immunodeficient Balb/c nu/nu mice were not affected by similar doses of INCB024360 (Figure 5B). The inability of INCB024360 to elicit an antitumor response in the immunodeficient mice was not due to lesser impact on kyn generation, as the compound levels were similar between the 2 strains and kyn-to-trp ratios were, in fact, more affected in the immunodeficient mice (Figure 5C). Therefore, consistent with the proposed mechanism of action, INCB024360 suppresses kyn generation in vivo, and its antitumor activity is mediated by lymphocytes.[1]

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In naïve C57BL/6 mice, 50 mg/kg INCB024360 decreased plasma kynurenine levels within 1 hour and those levels stayed at least 50% suppressed through the 8-hour time course (Fig. 1A; P < 0.01). To confirm that the decreased kynurenine levels observed in wild-type mice resulted specifically from IDO1 inhibition, Ido1−/− mice were dosed as above. Consistent with specific inhibition of Ido1 no reduction in kynurenine levels was observed in the Ido1−/− mice where kynurenine (approximately 20-25% of kynurenine in wild-type mice) was generated by other tryptophan-catabolizing enzymes (e.g., Tdo or Ido2; Fig. 1A). This was seen despite similar compound exposures between mouse strains (Fig. 1A). Further, during maximal suppression of Ido by INCB024360 in wild-type mice, the plasma kynurenine levels were quite similar to those present at baseline in the Ido1−/− mice, suggesting >90% inhibition of Ido1 activity by INCB024360.[2]

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Balb/c mice bearing well-established CT26 colon carcinomas were implanted with s.c. pumps delivering 50 mg/kg/d (based on a mouse starting weight of 20 g) INCB023843 or INCB024360 or vehicle. Both agents inhibited CT26 tumor growth, with 57% and 54% TGC for INCB023843 and INCB024360, respectively (P < 0.05; day 25, Fig. 3A). Because INCB023843 and INCB024360 performed equivalently in multiple in vitro and in vivo assessments, the two compounds were used interchangeably in subsequent studies. [2]

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To investigate the potential for INCB024360 as an oral agent, we administered increasing doses to CT26 tumor–bearing mice. There was a dose-dependent inhibition of tumor growth with 34% and 57% TGC seen with 30 (P < 0.05) and 100 mg/kg (P < 0.01) bid, respectively (day 21, Fig. 3B). [2]

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In a manner similar to CT26 tumors in Balb/c mice, PAN02 tumors respond to INCB023843 (Fig. 5A) and INCB024360 (data not shown) in a dose-dependent fashion when grown in wild-type C57BL/6 mice.[2]

IDO Enzyme Assay [4].
Human IDO with an N-terminal His tag was expressed in E.coli and purified to homogeneity. IDO catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N’-formylkynurenine. The assays were performed at room temperature as described in the literature using 20 nM IDO and 2 mM D-Trp in the presence of 20 mM ascorbate, 3.5 µM methylene blue and 0.2 mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5). The initial reaction rates were recorded by continuously following the absorbance increase at 321 nm due to the formation of N’-formlylkynurenine. In order to determine mode of inhibition, Km and Vmax values were determined for D-Trp at several inhibitor concentrations. Ki values were determined using the following equation which describes the behavior of a competitive inhibitor. No effect on Vmax was observed, but Km was linearly related to the inhibitor concentration. This profile is indicative of competitive inhibition. Ki values were determined by linear regression of the following equation: Km,eff = Km (1 + [I]/Ki). See the following reference for more details on the determination of binding kinetics of ligands to IDO via absorption spectroscopy and Soret peak analyses, Sono, M., Taniguchi, T., Watanabe, Y., and Hayaishi, O. Indoleamine 2,3-Dioxygenase; Equilibrium Studies of the Tryptophan Binding to the Ferric, Ferrous and Co-bound Enzymes. J. Biol. Chem. (1980), 255, 1339-1345.
Mode of Inhibition - Binding Kinetics.[4]
For mode of inhibition analysis, the final D-Trp concentrations varied between 0.6 mM and 30 mM. The initial reaction rates of these reactions were fit to the Michaelis-Mention equation by nonlinear regression analysis (Graphpad Prism). A competitive Ki was determined by linear regression of a plot of Km vs. [inhibitor], such that Ki = -(x-intercept). For example compound 1 was determined to be a competitive inhibitor of IDO with respect to the substrate D-trp, as shown in Figure 1.

Cell Based Determinations of Tryptophan and Kynurenine[2]
Both CT26 and PAN02 cells were plated at 3 × 105 cells/well in 6-well plates, and allowed to adhere overnight. The next day, the media were replaced, and appropriate wells received recombinant murine IFNγ to a final concentration of 100 ng/mL, whereas an unstimulated control received diluent. At that time, cells received IDO inhibitors across a 10-point dilution scheme. After 48 h, media were collected and centrifuged to remove dead cells and stored at −20°C until liquid chromotography/mass spectrometry (LC/MS) analysis as described below for tissue and plasma samples in the Pharmacodynamic Analyses section.

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Cell-based IDO and TDO assays[5]
The HeLa cell–based kyn assay to determine inhibitory activity of INCB024360 was performed as described previously.27 For the DC-based kyn assay, DCs were differentiated from human monocytes (see “Lymphocyte and DC or HeLa cocultures” for details), and stimulated with 50 ng/mL human recombinant IFN-γ and 5 μg/mL lipopolysaccharide (LPS) from Salmonella typhimurium in complete RPMI 1640 for 2 days. Established and primary AML cells were also stimulated with 50 ng/mL human recombinant IFN-γ and 5 μg/mL LPS before kyn measurement. The determination of INCB024360 activity was performed similarly to the HeLa cell assay.[5]
To determine INCB024360 activity against IDO in recombinant cells, HEK293/MSR cells were transiently transfected with full-length human or mouse IDO1, or mouse IDO2 cDNA, with Transit-293 transfection reagent or Lipofectamine 2000 reagents. INCB024360 at different concentrations was added to the recovered transfected cells seeded at 2 × 104 cells per well in a 96-well plate (200 μL/well). The cells were incubated for 2 days, and kyn in the supernatants was measured as described in the HeLa cell assay. The tryptophan 2,3-dioxygenase (TDO) assay was performed similarly with HEK293/MSR cells transfected with a human TDO expression vector.[5]

Syngeneic Tumor Models[2]
For the CT26 model, 8-week-old female Balb/c or Balb/c nu/nu mice (Charles River) were inoculated s.c. with 1 × 106 tumor cells. For the PAN02 model, 8-week-old female C57BL/6 mice, Balb/c nu/nu (Charles River), or Ido1-deficient (Ido1-/-) mice were inoculated s.c. with 3 to 5 × 106 tumor cells. Tumor sizes were measured after becoming visible two or three times weekly in two dimensions using a caliper, and the volume presented in mm3 using the formula: V = 0.5(A × B2), where A and B are the long and short diameters of the tumor, respectively. Tumor-bearing animals were sorted into groups with similar mean tumor volumes prior to treatment, usually 100 to 200 mm3. Treatments are listed in each experiment. Each day of the oral dosing studies, free base INCB023843 and INCB024360 were reconstituted in 3% N,N–Dimethylacetamide, 10% (2-Hydroxypropyl) β-Cyclodextrin. For studies with s.c. pumps, INCB023843 and INCB024360 were reconstituted in 40% N,N–Dimethylacetamide, 60% propylene glycol. Body weights were monitored throughout the study as a gross measure of toxicity/morbidity. TGC, expressed in %, is calculated using the formula: 1-[(treated (day X) − treated (day Y)) / (vehicle (day X) − vehicle (day Y)], where X is the day of last or interim measurement and Y is the day dosing commenced. Data were analyzed using one-way ANOVA with Dunnett's posttest for statistical significance. Plasma concentration of INCB024360, tryptophan, and kynurenine were determined by LC/MS/MS analysis following retro-orbital or cardiac puncture blood collection. In certain experiments, tumors and tumor-draining lymph nodes (TDLN) were also harvested for the determination of INCB023843, INCB024360, tryptophan, and kynurenine.[2]

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Pharmacokinetic-Phamacodynamic Studies[2]
To determine the effect of IDO inhibition on plasma kynurenine, fed C57BL/6 wild-type or Ido1−/−-deficient mice (B6.129-Ido1tm1Alm/J) were administered a single oral dose of INCB023843 or INCB024360, at which point food was removed from the cages until after the 8-h time point. At various time points after dosing, mice were euthanized and blood was collected by cardiac puncture. To determine the effect of IDO inhibition on plasma kynurenine in a nonrodent species, fed male beagle dogs were administered a single dose of INCB023843, at which point food was removed from the cages until after the 12-h time point. Blood was collected at various time points after dosing. Plasma was analyzed for the presence of INCB023843, INCB024360, tryptophan, and kynurenine according to the methods below. Data were analyzed using one-way ANOVA with Dunnett's posttest for statistical significance.

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#1: Dissolved in 3% N,N–Dimethylacetamide, 10% (2-Hydroxypropyl) β-Cyclodextrin; 100 mg/kg; p.o. administration [2]; #2: Dissolved in 10% DMSO, 40% PEG 300, and 50% NaCl 0.9% [3]

Female C57BL/6 or Balb/c nu/nu mice bearing PAN02 pancreatic tumors

Following oral dose administration in the fasted state, the PK of epacadostat was characterized by a time of maximum observed concentration at approximately 2 hours and a biphasic disposition with an apparent terminal-phase disposition half-life of 2.9 hours, which appeared to be dose-independent. Systemic accumulation following BID dosing increased mean epacadostat maximum observed concentration (Cmax) and area under the concentration versus time curve over 1 steady-state dosing interval (AUC0–τ) by 16% and 33%, respectively, suggesting a relatively longer effective half-life of 4 to 6 hours. Increases in epacadostat Cmax and AUC0–τ were slightly less than proportional to dose within the range of 50 to 700 mg BID. Moderate intersubject variability was observed for epacadostat plasma exposures (Table 3). Administration of a high-fat meal with epacadostat 600 mg BID decreased the geometric mean Cmax by approximately 10% and increased the geometric mean area under the curve from 0 to 12 hours (AUC0–12h) by 22%. The 90% CIs of the geometric mean ratio point estimates for Cmax and AUC0–12h were 0.645 to 1.25 and 0.952 to 1.57, respectively, both including the value of 1 and indicating that the effect on epacadostat plasma exposures from a high-fat meal was not statistically significant.[4]

Dose-Limiting Toxicities [4]
Two patients experienced DLTs: grade 3 radiation pneumonitis and grade 3 fatigue occurred in 1 patient each at the 300- and 400-mg BID dose levels, respectively. In light of the projection from preclinical animal modeling data (30) that all BID doses administered during dose escalation (50–700 mg) would be efficacious, no other DLT occurred with dosing up to 700 mg BID; thus, the MTD was not established for epacadostat.

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Safety and Tolerability [4]
The median (range) duration of epacadostat exposure was 51.5 (7–284) days with a median (range) total daily dose of 800 (43.2–1400) mg. Irrespective of association with therapy, the most common AEs (all grades) occurring throughout the study period were fatigue (69.2%), nausea (65.4%), decreased appetite (53.8%), and vomiting (42.3%; Table 2). These AEs were managed by investigators using routine supportive care measures. Seven patients (13.5%) discontinued therapy because of AEs (50 mg once daily, n=1; 100 mg BID, n=1; 300 mg BID, n=2; 400 mg BID, n=3), including pain, hepatic infection, pneumonia, radiation recall pneumonitis (DLT), fatigue (DLT), dyspnea and hypoxia, and nausea and vomiting. Only radiation pneumonitis and fatigue were considered DLTs and possibly related, but these dose levels were expanded and determined to not exceed the MTD. Liver enzymes were monitored closely throughout treatment in all patients. No grade 4 elevations were observed in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels. Grade 3 AST/ALT elevation was observed in 1 patient but was attributed to biliary duct obstruction consistent with progressive disease. A second patient also experienced grade 3 AST/ALT elevations, but this was determined to be most likely related to acetaminophen ingestion over the maximum recommended daily dose for tumor fevers.

[1]. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood. 2010 Apr 29;115(17):3520-30.

[2]. Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase potently suppress systemic tryptophan catabolism and the growth of IDO-expressing tumors. Mol Cancer Ther. 2010 Feb;9(2):489-98.

[3]. LW106, a novel indoleamine 2,3-dioxygenase 1 inhibitor, suppresses tumour progression by limiting stroma-immune crosstalk and cancer stem cell enrichment in tumour micro-environment. Br J Pharmacol. 2018 Jul;175(14):3034-3049.

[4]. First-in-Human Phase I Study of the Oral Inhibitor of Indoleamine 2,3-Dioxygenase-1 Epacadostat (INCB024360) in Patients with Advanced Solid Malignancies. Clin Cancer Res. 2017 Jul 1;23(13):3269-3276.

Epacadostat has been used in trials studying the treatment of HL, Melanoma, Glioblastoma, Mucosal Melanoma, and Ovarian Carcinoma, among others.
Epacadostat is an orally available hydroxyamidine and inhibitor of indoleamine 2,3-dioxygenase (IDO1), with potential immunomodulating and antineoplastic activities. Epacadostat targets and binds to IDO1, an enzyme responsible for the oxidation of tryptophan into kynurenine. By inhibiting IDO1 and decreasing kynurenine in tumor cells, INCB024360 increases and restores the proliferation and activation of various immune cells, including dendritic cells (DCs), NK cells, and T-lymphocytes, as well as interferon (IFN) production, and a reduction in tumor-associated regulatory T cells (Tregs). Activation of the immune system, which is suppressed in many cancers, may inhibit the growth of IDO1-expressing tumor cells. IDO1 is overexpressed by a variety of tumor cell types and DCs.

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Indoleamine 2,3-dioxygenase-1 (IDO1; IDO) mediates oxidative cleavage of tryptophan, an amino acid essential for cell proliferation and survival. IDO1 inhibition is proposed to have therapeutic potential in immunodeficiency-associated abnormalities, including cancer. Here, we describe INCB024360, a novel IDO1 inhibitor, and investigate its roles in regulating various immune cells and therapeutic potential as an anticancer agent. In cellular assays, INCB024360 selectively inhibits human IDO1 with IC(50) values of approximately 10nM, demonstrating little activity against other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO). In coculture systems of human allogeneic lymphocytes with dendritic cells (DCs) or tumor cells, INCB024360 inhibition of IDO1 promotes T and natural killer (NK)-cell growth, increases IFN-gamma production, and reduces conversion to regulatory T (T(reg))-like cells. IDO1 induction triggers DC apoptosis, whereas INCB024360 reverses this and increases the number of CD86(high) DCs, potentially representing a novel mechanism by which IDO1 inhibition activates T cells. Furthermore, IDO1 regulation differs in DCs versus tumor cells. Consistent with its effects in vitro, administration of INCB024360 to tumor-bearing mice significantly inhibits tumor growth in a lymphocyte-dependent manner. Analysis of plasma kynurenine/tryptophan levels in patients with cancer affirms that the IDO pathway is activated in multiple tumor types. Collectively, the data suggest that selective inhibition of IDO1 may represent an attractive cancer therapeutic strategy via up-regulation of cellular immunity.[1]

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Malignant tumors arise, in part, because the immune system does not adequately recognize and destroy them. Expression of indoleamine-2,3-dioxygenase (IDO; IDO1), a rate-limiting enzyme in the catabolism of tryptophan into kynurenine, contributes to this immune evasion. Here we describe the effects of systemic IDO inhibition using orally active hydroxyamidine small molecule inhibitors. A single dose of INCB023843 or INCB024360 results in efficient and durable suppression of Ido1 activity in the plasma of treated mice and dogs, the former to levels seen in Ido1-deficient mice. Hydroxyamidines potently suppress tryptophan metabolism in vitro in CT26 colon carcinoma and PAN02 pancreatic carcinoma cells and in vivo in tumors and their draining lymph nodes. Repeated administration of these IDO1 inhibitors impedes tumor growth in a dose- and lymphocyte-dependent fashion and is well tolerated in efficacy and preclinical toxicology studies. Substantiating the fundamental role of tumor cell-derived IDO expression, hydroxyamidines control the growth of IDO-expressing tumors in Ido1-deficient mice. These activities can be attributed, at least partially, to the increased immunoreactivity of lymphocytes found in tumors and their draining lymph nodes and to the reduction in tumor-associated regulatory T cells. INCB024360, a potent IDO1 inhibitor with desirable pharmaceutical properties, is poised to start clinical trials in cancer patients.[2]

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Background and purpose: Indoleamine 2,3-dioxygenase 1 (IDO1) is emerging as an important new therapeutic target for treatment of malignant tumours characterized by dysregulated tryptophan metabolism. However, the antitumour efficacy of existing small-molecule inhibitors of IDO1 is still unsatisfactory and the underlying mechanism remains largely undefined. Hence, we discovered a novel potent small-molecule inhibitor of IDO1, LW106, and studied its antitumour effects and the underlying mechanisms in two tumour models. Experimental approach: C57BL6 mice, athymic nude mice or Ido1-/- mice were inoculated with IDO1-expressing and -nonexpressing tumour cells and treated with vehicle, epacadostat or increasing doses of LW106. Xenografted tumours, plasma, spleens and other vital organs were harvested and subjected to kynurenine/tryptophan measurement and flow cytometric, histological and immunohistochemical analyses. Key results: LW106 dose-dependently inhibited the outgrowth of xenografted tumours that were inoculated in C57BL6 mice but not nude mice or Ido1-/- mice, showing a stronger antitumour efficacy than epacadostat, an existing IDO1 inhibitor. LW106 substantially elevated intratumoural infiltration of proliferative Teff cells, while reducing recruitment of proliferative Treg cells and non-haematopoietic stromal cells such as endothelial cells and cancer-associated fibroblasts. LW106 treatment resulted in a reduced subpopulation of cancer stem cells (CSCs) in xenografted tumours in which fewer proliferative/invasive tumour cells and more apoptotic tumour cells were observed. Conclusions and implications: LW106 inhibits tumour outgrowth by limiting stroma-immune crosstalk and CSC enrichment in the tumour micro-environment. LW106 has potential as a immunotherapeutic agent for use in combination with immune checkpoint inhibitors and (or) chemotherapeutic drugs for cancer treatment.[3]

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Purpose: Indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the degradation of tryptophan to N-formyl-kynurenine. Overexpressed in many solid malignancies, IDO1 can promote tumor escape from host immunosurveillance. This first-in-human phase I study investigated the maximum tolerated dose, safety, pharmacokinetics, pharmacodynamics, and antitumor activity of epacadostat (INCB024360), a potent and selective inhibitor of IDO1.Experimental Design: Fifty-two patients with advanced solid malignancies were treated with epacadostat [50 mg once daily or 50, 100, 300, 400, 500, 600, or 700 mg twice daily (BID)] in a dose-escalation 3 + 3 design and evaluated in 28-day cycles. Treatment was continued until disease progression or unacceptable toxicity.Results: One dose-limiting toxicity (DLT) occurred at the dose of 300 mg BID (grade 3, radiation pneumonitis); another DLT occurred at 400 mg BID (grade 3, fatigue). The most common adverse events in >20% of patients overall were fatigue, nausea, decreased appetite, vomiting, constipation, abdominal pain, diarrhea, dyspnea, back pain, and cough. Treatment produced significant dose-dependent reductions in plasma kynurenine levels and in the plasma kynurenine/tryptophan ratio at all doses and in all patients. Near maximal changes were observed at doses of ≥100 mg BID with >80% to 90% inhibition of IDO1 achieved throughout the dosing period. Although no objective responses were detected, stable disease lasting ≥16 weeks was observed in 7 of 52 patients.Conclusions: Epacadostat was generally well tolerated, effectively normalized kynurenine levels, and produced maximal inhibition of IDO1 activity at doses of ≥100 mg BID. Studies investigating epacadostat in combination with other immunomodulatory drugs are ongoing. Clin Cancer Res; 23(13); 3269-76. [4]

C11H13BRFN7O4S

438.23

436.991

C, 30.15; H, 2.99; Br, 18.23; F, 4.34; N, 22.37; O, 14.60; S, 7.32

1204669-58-8

1204669-58-8 (INCB024360); 914471-09-3 (INCB14943); 1204669-37-3 (INCB024360);

135564890

White to gray solid

2.0±0.1 g/cm3

672.3±65.0 °C at 760 mmHg

360.4±34.3 °C

0.0±2.2 mmHg at 25°C

1.742

3.92

5

11

8

25

563

0

BrC1=C(C([H])=C([H])C(=C1[H])/N=C(/C1C(=NON=1)N([H])C([H])([H])C([H])([H])N([H])S(N([H])[H])(=O)=O)\N([H])O[H])F

FBKMWOJEPMPVTQ-UHFFFAOYSA-N

InChI=1S/C11H13BrFN7O4S/c12-7-5-6(1-2-8(7)13)17-11(18-21)9-10(20-24-19-9)15-3-4-16-25(14,22)23/h1-2,5,16,21H,3-4H2,(H,15,20)(H,17,18)(H2,14,22,23)

(Z)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-((2-(sulfamoylamino)ethyl)amino)-1,2,5-oxadiazole-3-carboximidamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.62 mg/mL (5.98 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: ≥ 2.62 mg/mL (5.98 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (5.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.70 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 5: ≥ 2.5 mg/mL (5.70 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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Solubility in Formulation 6: 10%DMSO+90%PEG400: 30mg/mL

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