PRT062607 Hydrochloride (P505-15 Hydrochloride) is a highly specific and potent inhibitor of purified Syk (IC₅₀ 1-2 nM).

IC50: 1 nM (Syk), 81 nM (Fgr), 88 nM (MLK1), 123 nM (Yes)^[1]

PRT062607 (P505-15) is a novel, highly specific, and potent orally available small-molecule inhibitor of Syk. The potency of PRT062607 against its target kinase Syk is initially tested in two different purified kinase assays. Using a FRET assay, half-maximal Syk inhibition required 6±0.2 nM (mean±S.E.M.). Similar potency is observed when tested in a radioactive enzyme assay, with a resulting Syk IC₅₀ of 2.1±0.4 nM (mean±S.E.M.). In human whole blood, PRT062607 potently inhibits B cell antigen receptor-mediated B cell signaling and activation (IC₅₀ 0.27 and 0.28 μM, respectively) and Fcε receptor 1-mediated basophil degranulation (IC₅₀ 0.15 μM)^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

In the mouse CAIA model, oral administration of PRT062607 (P505-15) results in an average inhibition of paw inflammation, as measured by daily scoring of inflammation compared with vehicle controls, of 12, 44, and 87% with average plasma concentration (C average over 24 h) assessed at the end of the study of 0.38, 0.95, and 1.47 μM, respectively. In mice treated with 30 mg/kg PRT062607, the damage to the joints is significantly reduced and seemed indistinguishable from normal mice. In the rat CIA model, the high dose of PRT062607 (15 mg/kg b.i.d.) completely suppresses inflammation in a majority of the animals (seven of eight), by the end of the study (mean inflammation score±S.E.M.=0.63±1.1; p<0.0001 versus vehicle)^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

429.91

C₁₉H₂₄ClN₉O

1370261-97-4

Room temperature in continental US; may vary elsewhere.

4°C, sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

H₂O : ≥ 50 mg/mL (116.30 mM)

DMSO : ≥ 33 mg/mL (76.76 mM)

* “≥” means soluble, but saturation unknown.

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： PBS

  Solubility: 50 mg/mL (116.30 mM); Clear solution; Need ultrasonic
• 2.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (5.82 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (5.82 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 3.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (5.82 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (5.82 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 4.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (5.82 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (5.82 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Coffey G, et al. Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis. J Pharmacol Exp Ther. 2012 Feb;340(2):350-9.

Potency of Syk inhibition is determined by using a fluorescence resonance energy transfer (FRET) assay. The extent of substrate phosphorylation by Syk is measured in the presence of various PRT062607 (P505-15) concentrations. Syk activity is determined by a fluorescent antibody specific for phosphorylated tyrosine by using the increase of FRET. Twelve concentrations are tested for dose response. Specificity and potency of kinase inhibition is determined by evaluation of PRT062607 in the Kinase Profiler panel of 270 independent purified kinase assays. For profiling, PRT062607 is tested in duplicate at two concentrations at a fixed concentration of ATP. Subsequently, IC₅₀ determinations using the radioactive assays are carried out at an ATP concentration optimized for each individual kinase. All radioactive ATP incorporation enzyme assays are performed^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

SUDHL4 cells (10⁶ cells) are stimulated for 30 min with F(ab)′2 anti-human IgM antibody. Signaling is terminated by resuspending cells in ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1% nonidet P-40, and 0.5% sodium deoxycholate) containing freshly added protease and phosphatase inhibitor tablets. Protein lysates are resolved by SDS-polyacrylamide gel electrophoresis under reducing conditions and probed with the indicated antibodies. Secondary antibody conjugated to horseradish peroxidase and enhanced chemiluminescence detection reagent are used for the detection of specific protein. Where indicated, cells are pretreated for 1 h at 37°C with PRT062607 or vehicle control before stimulation. Densitometry of exposed films is performed by using the AlphaImager 2200. Ba/F3 cells are seeded at 5000 cells per well in a 384-well tissue culture plate containing media with various concentrations of PRT062607 or staurosporin. After 3 days, viability of cells is quantitated by CellTiter Glo following the manufacturer-supplied protocols^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Female BALB/c mice receive a single oral dose of 15 or 30 mg/kg PRT062607 and are anesthetized with a subcutaneous ketamine cocktail, and blood is harvested via cardiac puncture at 0.5, 1, 2, 3, 4, 5, 6, 8, and 24 h postdose, (n=3/time point; n=8 vehicle controls). Blood is dispensed into three heparin-containing tubes, one for determination of drug concentration and the remaining two for ex vivo stimulation with isotype control or anti-mouse IgD antibody for 10 min. Blood is processed for intracellular phospho-flow cytometry to evaluate BCR signaling as described earlier; mouse B cells are detected by using CD45R-B220 PerCP-conjugated antibody. Plasma samples are analyzed for PRT062607 concentration by using a liquid chromatography tandem mass spectrometer. The analytical range is 2 to 5000 ng/mL.
Rats^[1]
Collagen-induced arthritis (CIA) is induced in female, 7-week old Lewis rats by subcutaneous injection of bovine collagen II emulsified with incomplete Freund’s adjuvant at the base of the tail. On day 10, the rats are boosted with a second subcutaneous injection. PRT062607 administration is initiated when at least one hind paw is inflamed (inflammation score 1), and enrollment into a treatment group is designated as day 1 of the study for each individual animal (n=8/dose group). Progression of disease is evidenced by increased edema and erythema of one or both ankle joints, followed by the involvement of the metatarsal and interphalangeal joints. Fully developed arthritis is observed approximately 4 to 7 days after the onset of inflammation. Hind paws (sum of two paws) are scored daily for progression of inflammation. Inflammation scores for each paw are determined based on the following scale: 0, normal; 1, mild, but definite redness and swelling of the ankle possibly limited to individual digits; 2, moderate redness and swelling of ankle; 3, severe redness and swelling of the entire paw including digits; and 4, maximally inflamed limb with involvement of multiple joints. Ankle thickness measurements are obtained by a caliper device. At study termination, rats are anesthetized, and blood is harvested by cardiac puncture for complete blood counts and drug plasma concentration.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Coffey G, et al. Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis. J Pharmacol Exp Ther. 2012 Feb;340(2):350-9.