JAK3 (IC50 = 33.1 nM)

Ritlecitinib is a powerful JAK3-selective inhibitor that has an IC50 of 33.1 nM for inhibiting JAK3 kinase activity, but no activity (IC50>10,000 nM) against JAK1, JAK2, and TYK2. With IC50 values of 244, 340, 407, and 266 nM, respectively, ritlecitinib suppresses the phosphorylation of STAT5 induced by IL-2, IL-4, IL-7, and IL-15. With an IC50 of 355 nM, ritlecitinib also prevents IL-21-induced STAT3 phosphorylation. Ritlecitinib inhibits Th1 and Th17 differentiation (measured by IFNγ after 5 days under Th1 circumstances and IL-17 production after 6 days under Th17 settings) in T-cell differentiation assays, according to functional assessment (IC50 values: 30 nM and 167 nM, respectively). Additionally, ritlecitinib inhibits Th1 and Th17 function as demonstrated by the suppression of IFNγ production (IC50=48 nM) and IL-17 production (IC50=269 nM) in cells that have undergone prior differentiation and resting before PF-06651600 treatment[1].

Ritlecitinib reduces paw swelling in the rat adjuvant-induced arthritis (AIA) model, with an unbound EC50 of 169 nM. In the experimental autoimmune encephalomyelitis (EAE) mouse model, ritlecitinib, administered either therapeutically at 30 or 100 mg/kg or prophylactically at 20 or 60 mg/kg, significantly reduces the severity of the disease. Ritlecitinib's effectiveness in treating inflammatory and autoimmune diseases in these two rodent models shows that JAK3-selective inhibition alone may be enough to modify disease in humans[1].

JAK enzyme assays.[1]
Human Janus kinase (JAK) activity was determined using a microfluidic assay to monitor phosphorylation of a synthetic peptide by the recombinant human kinase domain of each of the four members of the JAK family, JAK1, JAK2, JAK3, and TYK2. GSTtagged recombinant human kinase domains of JAK1, JAK2, and JAK3 were purchased from Life Technologies. His-tagged recombinant human TYK2 kinase domain was expressed in SF21/baculovirus and purified using a two-step affinity (Ni-NTA) and size-exclusion (SEC S200) purification method. Test compounds were solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. Compounds were diluted in DMSO to create an 11-point half log dilution series with a top concentration of 600 µM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 in the assay, resulting in a final assay compound concentration range of 10 µM to 100 pM and a final assay concentration of 1.7% DMSO. Test compounds and controls solubilized in 100% DMSO were added (250 nL) to a 384 well polypropylene plate (Matrical) using a non contact acoustic dispenser. Kinase assays were carried out at room temperature in a 15 µL reaction buffer containing 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20 and 1mM Dithiothreitol (DTT). Reaction mixtures contained 1 µM of a fluorescently labeled synthetic peptide, at a concentration less than the apparent Michaelis-Menten constant (Km) (5FAM-KKSRGDYMTMQID for JAK1 and TYK2 and FITC-KGGEEEEYFELVKK for JAK2 and JAK3). Reaction mixtures contained adenosine triphosphate (ATP) at either a level equal to the apparent Km for ATP (40 µM for JAK1, 4 µM for JAK2, 4 µM for JAK3 and 12 µM for TYK2) or at 1 mM ATP. Compound was added to the buffer containing ATP and substrate and immediately after this step the enzyme was added to begin the reaction. The assays were stopped with 15 µL of a buffer containing 180 mM HEPES, pH=7.4, 20 mM EDTA, 0.2% Coating Reagent, resulting in a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument or Caliper Life Science EZ Reader instrument and each well was sampled using appropriate separation conditions to determine the level of phosphorylation[1].

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Binding and inactivation kinetics of JAK3.[1]
The kinetics of PF-06651600 binding to and inactivation of JAK3 and a panel of kinases that contained a Cys in the equivalent position as Cys909 in JAK3 were measured using a time-resolved Förster resonance energy transfer (TRFRET) assay based on the LanthaScreen Eu Kinase Binding Assay (Invitrogen/Life Technologies). Kinase-specific reagents and assay validation can be found at: https://www.thermofisher.com/us/en/home/industrial/pharma-biopharma/drug-discoverydevelopment/target-and-lead-identification-and-validation/kinasebiology/kinase-activityassays/lanthascreentm-eu-kinase-binding-assay/lanthascreen-eu-kinase-binding-assay-validationtable.html. Assay buffer was 20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.01% BSA, 1 mM DTT, 0.0005% Tween 20, and 2% DMSO. Inactivation kinetic reactions were performed by preparing 15 µL of a 1.33Χ solution of (final concentrations) 2 nM Eu-Ab, 1-8 nM kinase (optimal concentrations of each kinase were empirically determined) and a variable concentration of PF06651600, and pre-incubating this for a variable amount of time (detailed below). This was then combined this with 5 µL of 4X solution of the validated probe (150 nM, final concentration). For all kinases, the following experiments were performed: (A) [PF-06651500] = 0, 4.9, 14.8, 44.4, 133.3, and 400 nM; pre-incubation time = 2 h. (B) [PF-06651500] = 0, 0.5, 1.0, 2.0, 4.0, and 8.0 µM; pre-incubation time = 120 s. For JAK3, the following additional experiments were performed: [PF-06651500] = 0, 0.66, 1.98, 5.93, 17.8, 53.3, 160, 480 nM; pre-incubation time of (C) 30 s, (D) 60 s, and (E) 1.5 h. The assays were read using an EnVision plate reader. The excitation wavelength was 340 nm, and the output monitored was the emission ratio, calculated by dividing the signal from the emission peak of the probe (665 nm) by that of the europium (615 nm). Measurements were taken every 120 s for 1.5 h. Recombinant TXK kinase (N-terminal GST-fusion protein) failed to produce a suitable TR-FRET signal in combination with anti-GST Eu-antibody (Life Technologies) and probe. Thus, an alternative approach was taken to measure kinact/Ki , utilizing the classic pyruvate kinase/lactate dehydrogenase (PK/LDH) coupled enzyme assay. In the PK/LDH assay, ADP, which is a product of the kinase reaction, is measured by coupling its production first to the dephosphorylation of phosphoenolpyruvate (PEP) to form pyruvate, which is coupled to NADH-dependent reduction of pyruvate to form lactate. The concomitant oxidation of NADH to form NAD+ is monitored spectrophotometrically by loss of absorbance at 340 nm. The TXK kinase buffer was: 50 mM HEPES, pH 7.5, 10 mM Mg2Cl, 0.01% Triton X-100, 1 mM DTT, and 1% DMSO. Also included (PK/LDH assay reagents): 0.25 mM NADH (Sigma, N8129), 2.5 mM phosphoenolpyruvate (PEP), 12 U/mL pyruvate kinase (PK) and 18 U/mL lactate dehydrogenase (LDH). Final substrate concentrations were 100 µM each ATP and Srctide peptide (sequence: GEPLYWSFPAKKK). In each experiment, 10 µL of PF-06651600 solution (concentration during preincubation: 0, 10.4167, 20.83, 41.67, 83.3, 333.3, 666.7, 1333.3, 2666.7 nM) was combined with 20 µL of solution containing TXK kinase (34 nM concentration during pre-incubation) + PK/LDH solution reagents. After a variably timed pre-incubation period (15 min, 30 min, 1 h, 1.5 h, and 2 h), 10 µL of 4Χ ATP/peptide substrate was added to initiate the reaction.

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Human serum albumin binding of PF-06651600. [1]
Human recombinant serum albumin (HSA) expressed in Saccharomyces cerevisiae was obtained as a 1.5 mM solution and used by dilution into Dulbecco's phosphate-buffered saline (PBS) without further processing. HSA was incubated with compounds diluted into PBS from 8 mM stock solutions made in DMSO as indicated in the legend to fig. S3 for 22 h, after which the protein was subjected to direct mass analysis by liquid chromatography-mass spectrometry using an Agilent 1100 HPLC system and a Waters LCT Premier XE mass spectrometer as described previously1 . Mass distribution of the protein was extracted from the spectra averaged over the protein peak using MaxEnt 1 software (incorporated into the Waters MassLynx program) run for 20 iterations with a target mass range of 66,000-67,500 Da.

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Covalent Binding in Human Hepatocytes.[1]
Pooled cryopreserved human hepatocytes were suspended in Williams’ E Media (GIBCO, ThermoFisherScientific) at a final concentration of 750,000 cells/mL (n=2). Cell viability based on trypan blue exclusion was >80%. The cell suspensions (4 mL) were incubated at 37oC for up to 4 h with radiolabeled substrate (1 µM). Aliquots were removed at 240 min, quenched with acetonitrile, centrifuged at 3500 rpm for 15 min and exhaustively extracted (N=7) with a combination of organic solvents (acetonitrile: methanol, acetonitrile and acetonitrile:0.1% formic acid in water). Supernatant fractions were monitored until radioactivity levels were below twice the level of background (80 dpm). NaOH (1M) was added to the remaining protein pellet and placed in a water bath overnight to dissolve and the total radioactivity was determined by liquid scintillation counting. Protein concentrations were determined using the Bradford Protein assay.

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LC-MS/MS of JAK3 occupancy and p-STAT5 inhibition in human whole blood.[1]
Occupancy of JAK3 by an irreversible covalent inhibitor was measured utilizing LC/MS-MS surrogate tryptic peptide analysis. Human JAK3 protein sequence (Uniprot accession number P52333) was used to verify the sequence of a tryptic peptide containing 909Cys, the covalent molecule binding target. A favorably sized peptide with the sequence LVMEYLPSGCLR is generated by tryptic cleavage of the JAK3 protein using MS grade Trypsin/Lys-C enzyme mix. Bound-to-inhibitor and free peptide was measured by MRM mode IA-LC-MS/MS analysis which included an online anti-peptide antibody immuno-affinity enrichment step. Double immuno-precipitation at the protein and peptide level provided ample sample enrichment for free and bound LVME peptide in PBMC samples generated from human whole blood. Biotinylated antihuman JAK3 polyclonal capture antibody was obtained from Santa Cruz. Extended sequence stable isotope labeled (SIL) peptide containing a tryptic cleavage site was custom synthesized by New England Peptide...

Th1 cell differentiation.[1]
Cryopreserved human normal peripheral blood CD4+/CD45RA+/CD25- naïve T cells were purchased from Allcells. Frozen CD4+/CD45RA+/CD25- naïve T cells were thawed in a water bath (37o C), and washed once with RPMI1640 medium. Cells were resuspended at 200,000 cells/mL in RPMI medium containing 10% fetal bovine serum (FBS), IL-2 (10 ng/mL), IL-12 (5 ng/mL), anti-IL-4 (5 ng/mL), anti-CD3 (10 µg/mL) and anti-CD28 (0.1 µg/mL) antibodies. To evaluate the effect of PF-06651600 during the differentiation phase of Th1 cells, resuspended naïve CD4+ T cells were cultured for 5 days in the presence of 11 different concentrations of JAK inhibitors (0.2% DMSO final). Supernatants were harvested and the concentrations of IFNγ were measured with MSD according to manufacturer’s instructions. To study the effect of PF06651600 on Th1 cells post differentiation skewed Th1 cells were resuspended in RPMI medium containing 10% FBS and cultured under cytokine free conditions for 7 h. Cells were then harvested and cultured in the presence of 10% FBS, IL-2 (10 ng/mL), IL-12 (5 ng/mL), anti-IL-4 antibody (5 ng/mL), plus 11 different concentrations of PF-06651600 in 96-well plates for 2 additional days. The concentration of IFNγ was determined as described above.

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Th17 cell differentiation.[1]
Human CD4+ T cells were purified from buffy coat with RosetteSep CD4+ T Cell Enrichment Cocktail and skewed for 6 days with cytokine cocktails (25 ng/mL of IL-6, 25 ng/mL of IL-23, 12.5 ng/mL of IL-1β, 25 ng/mL of IL21, 5 ng/mL of TGFβ1, 10 µg/ml of anti-CD3 antibody (pre-coated on plate surface) and 1 µg/mL of anti-CD28 antibody) in the presence of JAK inhibitors at 10 different concentrations. Supernatants were harvested and the concentrations of IL-17A were determined with MSD assay following the protocol provided by the manufacturer. To study the effect of PF-06651600 on Th17 cells post differentiation, skewed Th17 cells were washed, rested with X-VIVO 15 medium (Lonza) for overnight and resuspended in medium containing the same concentrations of cytokines as during skewing but without anti-CD3 or anti-CD28 antibodies, in the presence of PF-06651600 at 10 different concentrations for 2 additional days. On Day 9, supernatant was harvested from each well and IL-17A was determined as described above.

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JAK3 half-life in human T cells.[1]
CD4+ cells were purified by negative selection from Buffy coat (SeraCare) and cryopreserved. Ten million cells were thawed in RPMI supplemented with 10% FBS (Sigma) 100 U/mL penicillin/100 µg/mL streptomycin (Gibco), 2 mM L-glutamin, 100 µM Non-essential amino acid, 1 mM sodium pyruvate, 20 mM HEPES and 2 ng/ml IL-2. After 2 h at 37 °C in 5% CO2 CD3/CD28 magnetic beads were added (ratio of 3 beads/cell). Seventy two hours later the beads were removed and 800 million cells were used for the pulse chase experiments. Each data point utilized 40 million cells. Cells were supplemented with media supplemented with 500 µCi S35 labeled Cys/Met at 37 °C in 5% CO2 for 30 minutes followed by two washes in PBS. The cells were then incubated for various length of time in media supplemented with 200 µM Cys/Met and lysed in cold lysis buffer before being stored at - 80 °C. The lysates were thawed on ice and subjected to immunoprecipitation with anti-JAK3 antibodies overnight at 4 °C. Protein G magnetic beads were used to capture the anti-JAK3 antibodies. JAK3 was then eluted in 60 µl Laemmli with 5% BME and subjected to electrophoresis and quantification on phosphorimager.

Rat adjuvant induced arthritis.[1]
The effect of JAK3 inhibition by PF-06651600 was evaluated in vivo using a therapeutic dosing paradigm in a rat adjuvant-induced arthritis. The efficacy of this molecule was evaluated in three separate studies using successively lower doses. Arthritis was induced by immunization of female Lewis rats (8 to 10 weeks old; Charles River Laboratories) via intradermal injection at the base of the tail with complete Freund’s adjuvant with three 50 µL injections (15 mg/mL Mycobacterium tuberculosis in incomplete Freund’s adjuvant (Sigma Aldrich). Seven days after the initial immunization, the baseline hind paw volume of the immunized rats was measured via plethysmograph (Buxco Inc). The rats were monitored daily for signs of arthritis including change in body weight and hind paw volume measurement. When individual hind paw volume measurements indicated an increase of 0.2 mL (or greater) in a single hind paw, animals were randomly assigned to a treatment group. Daily treatment with PF-06651600 was administered via oral gavage. Treatment groups for Experiment 1 were: 80, 15, or 6 mg/kg or vehicle (2% Tween 80 /0.5% methylcellulose/deionized water). Treatment groups for Experiment 2 were: 30, 10, and 3 mg/kg or vehicle (0.5% methylcellulose / de-ionized water/ 1 mEQ hydrochloric acid). Treatment groups for Experiment 3 were: 10, 1, 0.3 and 0.1 mg/kg or vehicle (0.5% methylcellulose/de-ionized water/ 1 mEQ hydrochloric acid). Dosing began once individuals were enrolled into respective groups. Treatment continued for 7 days. At the conclusion of the study, whole blood was taken at 15 minutes post dose (peak concentration in plasma) for analysis of STAT phosphorylation, and plasma was taken for exposure concentration in PF-06651600 dosed groups.

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Mouse experimental autoimmune encephalomyelitis. [1]
Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice (Taconic Farms, 10 weeks old) at Hooke Laboratories. Mice were injected subcutaneously at 0.1 ml per site with the emulsion component (containing MOG35-55) of Hooke Kit™ MOG35-55/CFA Emulsion PTX (Hooke Laboratories). The pertussis toxin (PTX) component of the kit was diluted with PBS and administered intraperitoneally in a volume of 0.1 ml at 122 ng /dose (therapeutic dosing study) or 167 ng /dose (prophylactic dosing study) within 2 h of the injection of emulsion, and again at 111 ng/dose (therapeutic dosing study) or 156 ng /dose (prophylactic dosing study) 24 h after the injection of emulsion. For the therapeutic study, as each mouse developed clinical signs of EAE (minimum score of 0.5) they were assigned to one of the experimental groups (n=15 per group) in a balanced manner, to achieve groups with similar time of EAE onset and similar scores. Treatment started on the first day of disease for each mouse and lasted for 14 days. For the prophylactic dosing study, mice were assigned to groups (n=10 per group) on Day -1 in a balanced manner to achieve similar average body weight between the groups at the start of the study. Prophylactic dosing started on Day 0 and continued until Day 28. Dosing was blinded and consisted of oral (per os; PO) twice daily (BID) administrations of PF-06651600 or vehicle (0.5% MethylCellulose/ 1 Molar equivalent hydrogen chloride). The positive control group was dosed PO once daily (QD) in the morning with fingolimod (FTY720, Gilenya), the most commonly used positive control in these models, and PO QD in the afternoon with vehicle in order to control for dosing stress in comparison with the BID treated groups. There were no more than 14 hours between evening and morning doses and no less than 10 h between morning and evening doses. Body weight was measured 3 times per week and EAE scores were assessed daily beginning on day 7 (the seventh day after immunization). EAE was scored on a scale of 0 to 5 until the termination of the study. Scoring was performed in a blinded fashion by a person unaware of both treatment and of previous scores for each mouse. At the conclusion of the study, plasma was taken for exposure concentration of PF-06651600 at peak (15 minutes after dosing) and trough (10 h and 14 h after dosing) time points.

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2% Tween 80 /0.5% methylcellulose/deionized water

Female Lewis rat model (8 to 10 weeks old)

Absorption, Distribution and Excretion
Up to 200 mg, the AUC0-tau and Cmax of ritlecitinib increase in an approximately dose-proportional manner, and steady state is reached approximately by day 4. Ritlecitinib has an absolute oral bioavailability of approximately 64%, and 1 hour after an oral dose is administered, peak plasma concentrations are achieved. Food does not have a clinically significant impact on the systemic exposures of ritlecitinib. The co-administration of a high-fat meal and a 100 mg ritlecitinib capsule reduced Cmax by 32% and increased AUCinf by 11%. Ritlecitinib was administered without regard to meals during clinical trials.
Ritlecitinib is mainly excreted through urine and feces. Approximately 66% and 20% of radiolabeled ritlecitinib are excreted in the urine and feces, respectively. Approximately 4% of the ritlecitinib dose is excreted unchanged drug in urine.
Ritlecitinib is predicted to have a volume of distribution of 1.3 L/kg.
Ritlecitinib is predicted to have a blood clearance of 5.6 mL/min/kg.

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Metabolism / Metabolites
Ritlecitinib is metabolized by cytochrome P450 (CYP) and glutathione-S-transferase (GST) enzymes. The GST enzymes participating in the metabolism of ritlecitinib include cytosolic GST A1/3, M1/3/5, P1, S1, T2, Z1 and microsomal GST 1/2/3, and the CYP enzymes participating in this process include CYP3A, CYP2C8, CYP1A2, and CYP2C9. No single route contributes to more than 25% of the total metabolism of ritlecitinib.

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Biological Half-Life
Ritlecitinib has a terminal half-life that ranges from 1.3 to 2.3 hours.

Hepatotoxicity
In the prelicensure clinical trials in alopecia areata, serum aminotransferase elevations occurred in 1% to 3% of ritlecitinib treated subjects, but similar rates were found in placebo recipients. The elevations were typically mild and transient, and values above 5 times the upper limit of normal (ULN) occurred in less than 1% of patients. The elevations rarely led to early discontinuations, and often resolved even without dose adjustment. In prelicensure studies in alopecia areata and other autoimmune conditions, there were no instances of liver related severe adverse events or clinically apparent liver injury attributed to ritlecitinib. Since approval and more widescale availability of ritlecitinib, there have been no published reports of hepatotoxicity associated with its use.
Finally, ritlecitinib is an immune modulatory agent and has the potential of causing reactivation of viral infections including hepatitis B. Other JAK inhibitors have been implicated in rare instances of reactivation of hepatitis B, although the episodes were usually asymptomatic and self-limited in course. The risk of reactivation of hepatitis B in patients with HBsAg or with anti-HBc without HBsAg who are treated with ritlecitinib has not been defined.
Likelihood score: E* (unlikely cause of idiosyncratic clinically apparent liver injury, but is a potential cause of reactivation of hepatitis B).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of ritlecitinib during breastfeeding. Because of the risk of serious adverse effects, including malignancy, the manufacturer recommends that breastfeeding be discontinued during ritlecitinib therapy and for 14 hours after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Ritlecitinib is 14% bound to plasma proteins.

[1]. Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition. ACS Chem Biol. 2016 Dec 16;11(12):3442-3451.

Pharmacodynamics
Ritlecitinib is a kinase inhibitor that promotes the decrease of absolute lymphocyte levels, T lymphocytes (CD3) and T lymphocyte subsets (CD4 and CD8) in a dose-dependent manner. Ritlecitinib also promotes a decrease in NK cells (CD16/56), which remain stable up to week 48 after initiating treatment. In patients treated with 50 mg of ritlecitinib once a day, the decrease in median lymphocyte levels remains consistent up to week 48. At 12 times the mean maximum exposure of the 50 mg dose given to patients with alopecia areata once a day, ritlecitinib did not cause a clinically relevant effect on the QTc interval.[] The use of ritlecitinib is associated with the development of serious infections, malignancies (including non-melanoma skin cancer), major adverse cardiovascular events, thromboembolic events, and hypersensitivity. In the postmarketing safety study of another JAK inhibitor in patients with rheumatoid arthritis over 50 years of age with at least one cardiovascular risk factor, JAK inhibitors were associated with a higher rate of all-cause mortality, including sudden cardiovascular death, compared to TNF blockers.

C15H19N5O

285.34

285.158

C, 63.14; H, 6.71; N, 24.54; O, 5.61

1792180-81-4

(2R,5S)-Ritlecitinib;1792180-79-0; 2192215-81-7 ; 1792180-81-4; 2489392-29-0; 2140301-97-7 (malonate)

118115473

White to yellow solid

1.3±0.1 g/cm3

1.658

1.29

2

4

3

21

402

2

O=C(C=C)N1C[C@@H](CC[C@@H]1C)NC1C2C=CNC=2N=CN=1

CBRJPFGIXUFMTM-WDEREUQCSA-N

InChI=1S/C15H19N5O/c1-3-13(21)20-8-11(5-4-10(20)2)19-15-12-6-7-16-14(12)17-9-18-15/h3,6-7,9-11H,1,4-5,8H2,2H3,(H2,16,17,18,19)/t10-,11+/m0/s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.29 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 6.67 mg/mL (23.38 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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