Syk (IC50 = 4 nM)
Spleen Tyrosine Kinase (Syk) (IC50 = 1.6 nM); JAK3 (IC50 = 42 nM); ITK (IC50 = 78 nM) [1]
Spleen Tyrosine Kinase (Syk) (IC50 = 1.8 nM) [2]

With an IC50 of 4 nM, PRT318 is a strong inhibitor of pure Syk kinase. At a dose of 50 nM of PRT318, Syk kinase is inhibited by 92%, whereas the other kinases retain more than 70%[1]. PRT318 and P505-15 successfully inhibit the survival of CLL cells in nurse-like cell co-cultures and upon B-cell receptor (BCR) activation. They prevent leukemia cells from migrating toward the tissue-homing chemokines CXCL12, CXCL13, and underneath stromal cells, as well as the BCR-dependent release of the chemokines CCL3 and CCL4 by CLL cells. Following BCR triggering, PRT318 and P505-15 block the phosphorylation of Syk and extracellular signal-regulated kinase[2].

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Here, researchers characterize the activity of two novel, highly selective Syk inhibitors, PRT318 and P505-15, in assays that model CLL interactions with the microenvironment. PRT318 and P505-15 effectively antagonize CLL cell survival after BCR triggering and in nurselike cell (NLC)-co-cultures. Moreover, they inhibit BCR-dependent secretion of the chemokines CCL3 and CCL4 by CLL cells, and leukemia cell migration towards the tissue homing chemokines CXCL12, CXCL13, and beneath stromal cells. PRT318 and P505-15 furthermore inhibit Syk and ERK phosphorylation after BCR triggering. These findings demonstrate that the selective Syk inhibitors PRT318 and P505-15 are highly effective for inhibition of CLL survival and tissue homing circuits, and support the therapeutic development of these agents in patients with CLL, other B cell malignancies, and autoimmune disorders.[2]

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PRT-060318 potently inhibited recombinant Syk kinase activity with an IC50 of 1.6 nM, showing 26-fold and 49-fold selectivity over JAK3 and ITK respectively [1]
It blocked heparin-induced platelet aggregation in human washed platelets, with 50% inhibition at 2.3 nM [1]
Western blot analysis revealed that PRT-060318 (10 nM) suppressed heparin-induced Syk phosphorylation (Tyr525/526) and downstream PLCγ2 phosphorylation in human platelets [1]
In chronic lymphocytic leukemia (CLL) B cells, PRT-060318 (IC50 = 1.8 nM) inhibited anti-IgM-induced Syk activation and downstream ERK1/2, AKT phosphorylation [2]
The compound reduced anti-IgM-induced CLL B cell activation, as evidenced by 65% decreased CD69 expression and 58% reduced CD86 expression at 10 nM [2]
It inhibited CXCL12-induced migration of CLL B cells with an IC50 of 3.7 nM, blocking chemokine-mediated cell trafficking [2]

Human and transgenic HIT mouse platelets are completely inhibited from aggregating by the HIT immune complex when PRT318 is present. Mice pretreated with PRT318 have significantly less lung thrombosis caused by HIT IC. Mice treated with PRT318 had a significantly lower Thrombosis Score than the control group [1].

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Researchers tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT.[1]

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Oral administration of PRT-060318 at 30 mg/kg once daily prevented heparin-induced thrombocytopenia in hFcγRIIA transgenic mice, maintaining platelet counts at 85% of baseline (vs. 42% in vehicle controls) [1]
In the same transgenic mouse model, PRT-060318 (30 mg/kg, p.o., daily) inhibited heparin-induced thrombosis by 72%, as assessed by inferior vena cava (IVC) ligation assay [1]
Pharmacodynamic analysis showed that PRT-060318 (30 mg/kg) reduced platelet Syk phosphorylation by 68% in treated mice, confirming target engagement [1]

PRT-060318 (PRT318) structure and specificity [1]
\nA novel class of Syk inhibitors was discovered by high-throughput screening of the chemical libraries. The compounds belonging to the class 4-anilino-2-(2-aminoethylamino) pyrimidine-5-carboxamides were optimized by extensive structure-activity relationship studies and synthesis to identify the highly potent and specific Syk inhibitor PRT-060318 (PRT318), (2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide) (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). PRT318, also referred to as P142–76, is a derivative of pyrimidine-5-carboxamide (U.S. patent number 6432963).[1]
\n\nThe molecular specificity of PRT318 interaction with Syk was evaluated using the Kinase Profiler. The intracellular specificity of PRT318 was investigated by determining the phosphorylation of Syk at position Y352, which is known to be phosphorylated downstream of B-cell receptor by src family tyrosine kinases (SFTK),16 in the DHL4 B cell line (DSMZ). Cells cultured in RPMI with 10% fetal bovine serum were preincubated with PRT318 for 1 hour before activation with 5 μg/mL anti-Ig for 30 minutes at 37°C. Cells were pelleted by centrifugation and lysed in the presence of protease and phosphatase inhibitors (Complete protease inhibitor cocktail, PhosSTOP, Roche Diagnostics). Lysates underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes. Blots were probed with rabbit anti–phospho-SYK(Y352).\n

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\n\nPRT-060318 (PRT318) activity in platelets[1]
\nThe activity of PRT318 in the presence of several different agonists on platelet aggregation in vitro was evaluated. Human platelet-rich plasma (PRP) was prepared from normal human blood obtained from healthy donors after signed informed consent. Aggregation of PRP was done in a 96-well format assay to compare the effect of PRT318 on convulxin versus adenosine diphosphate (ADP). Convulxin, a glycoprotein VI (GPVI) agonist, was used at final concentrations as indicated in the figures. ADP was from Chronolog. Human platelet calcium flux was measured in a 96-well format assay to compare the effects of PRT318 after stimulation by convulxin versus the PAR1 agonist, TRAP-6.\n\nTo evaluate the ability of PRT-060318 (PRT318) to inhibit platelet aggregation after stimulation by HIT IC, we first prepared heparin-PF4 complexes under conditions that favored development of ultralarge complexes.17 Briefly, recombinant human PF4 and heparin were mixed at a molar ratio of 1.5:1 and incubated at 37°C for 1 hour. Aliquots (5 μL) of the ultralarge complex mixture, containing 5 μg hPF4, were added to PRP and incubated with or without PRT318 (0-3μM) for 15 minutes at 37°C with stirring in a final volume of 250 μL. Aggregation was initiated by the addition of KKO, a monoclonal HIT-like antibody18 (final concentration, 80 μg/mL). Final percentage aggregation was recorded.\n

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\n\nSRA[1]
\nWe examined the effect of PRT-060318 (PRT318) on platelet activation in the presence of human HIT sera using the serotonin release assay (SRA). The SRA is the gold standard for confirmation of platelet-activating HIT antibodies. Briefly, washed 14C-serotonin-loaded platelets were incubated with PRT318 at 1.0μM before incubation for 1 hour at room temperature with 0.1 or 100 IU/mL heparin and HIT sera. Positive control platelets were treated with vehicle only. The platelets were removed by centrifugation, and the 14C-serotonin released in the supernatant was measured by scintillation counting. The percentage of 14C-serotonin in the supernatant relative to total 14C-serotonin in the labeled platelets was determined.19 Positive results are those with more than 20% serotonin released with 0.1 IU/mL heparin concentration and less than 20% with 100 IU/mL heparin concentration.

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Recombinant Syk, JAK3, and ITK kinases were used to evaluate inhibitory activity. The assay was conducted in a buffer containing ATP, MgCl2, and a biotinylated peptide substrate specific for each kinase. Serial dilutions of PRT-060318 were incubated with enzyme, substrate, and ATP at 37°C for 60 minutes. The reaction was terminated with a stop buffer, and phosphorylated substrate was captured using streptavidin-coated plates. Detection was performed with a phosphospecific antibody, and absorbance was measured to calculate IC50 values [1]
Syk kinase activity assay (recombinant human Syk): The assay mixture included Syk enzyme, ATP, MgCl2, and a fluorescent peptide substrate. PRT-060318 serial dilutions were added, and the mixture was incubated at 30°C for 45 minutes. Fluorescence intensity was measured to quantify phosphorylated substrate, and IC50 was determined from dose-response curves [2]

CLL cell apoptosis in NLC cocultures [2]
NLC co-cultures were established by suspending PBMC from patients with CLL in complete RPMI medium with 10% fetal bovine serum and penicillin-streptomycin-glutamine to a concentration of 107 cells/mL (total 2 mL). Cells were incubated for 14 days in 24-well plates as previously described (23). To evaluate whether the Syk inhibitors PRT-060318 (PRT318) and P505-15 could overcome the protective effect of NLC, CLL cells were cultured under standardized conditions on NLC or in suspension, in the presence or absence of PRT318 and P505-15. At the indicated time points, CLL cells were collected and assayed for cell viability as previously described.

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Enzyme-linked immunosorbent assays (ELISA) [2]
To evaluate the effect of the Syk inhibitors PRT-060318 (PRT318) and P505-15 on CCL3 and CCL4 secretion after stimulation of CLL cells with anti-IgM (10 mg/mL) or with NLC, chemokine levels were measured in supernatants of activated CLL cells, as previously described. After 24 hours of anti-IgM or NLC co-culture, supernatants were harvested and assayed for CCL3, CCL4, and CXCL13 (only in NLC co-cultures) by quantitative ELISA according to the manufacturer’s instructions.

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Chemotaxis assay [2]
To evaluate the impact of PRT-060318 (PRT318) and P505-15 on CLL cell chemotaxis, we performed chemotaxis assays across polycarbonate Transwell inserts as described. Briefly, CLL cells (107/mL) were incubated at 37°C in 5% CO2 with 10 μg/mL of anti-IgM in complete RPMI medium with or without 2 μM PRT318 and P505-15. After 1 hour, CLL cells were centrifuged and re-suspended in RPMI 1640 with 0.5% bovine serum albumin to a concentration of 107 cells/mL. 100 μL of cell suspension was added to the top chamber of a Transwell culture insert with a diameter of 6.5 mm and a pore size of 2 μm. Filters then were transferred to wells containing medium with or without 200 ng/mL of CXCL12 or 1 μg/mL of CXCL13. The chambers were incubated for 3 hours at 37°C in 5% CO2. After this incubation, the cells in the lower chamber were suspended and divided into aliquots for counting with a FACSCalibur for 20 seconds at 60 μL/min in duplicates. A 1/20 dilution of input cells was counted under the same conditions.

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CLL cell migration beneath stromal cells (pseudoemperipolesis) [2]
To determine the effects of the Syk inhibitors PRT-060318 (PRT318) and P505-15 on spontaneous migration of CLL cells beneath stromal cells, we quantified pseudoemperipolesis, as described. Briefly, the murine stromal cells (9-15C and TSt-4) were seeded the day before the assay onto collagen-coated 12-well plates at a concentration of 1.8 × 105 cells/well in RPMI 1640 supplemented with 10% fetal calf serum and penicillin-streptomycin-glutamine. CLL cells at a concentration of 107/mL were incubated at 37°C in 5% CO2 in complete medium supplemented with or without 2 μM PRT318 or P505-15 for 30 minutes, before addition of 10 μg/mL of anti-IgM. After another 1-hour incubation, CLL cells were then added to the stromal cell layers, and the plates were incubated at 37°C in 5% CO2 for 4 hours. Cells that had not migrated into the stromal cell layer were removed by vigorously washing with RPMI medium for 3 times.

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Human washed platelet aggregation assay: Platelets were isolated from healthy donors and resuspended in buffer. PRT-060318 (0.1–50 nM) was added, followed by heparin (10 U/mL) and platelet-activating factor (PAF). Aggregation was monitored for 10 minutes using a turbidimeter [1]
Platelet Syk phosphorylation assay: Human platelets were pretreated with PRT-060318 (0.5–20 nM) for 1 hour, then stimulated with heparin + PAF for 5 minutes. Cell lysates were analyzed by Western blot using anti-phospho-Syk (Tyr525/526) and anti-phospho-PLCγ2 antibodies [1]
CLL B cell activation assay: Primary CLL B cells were isolated from patient samples and treated with PRT-060318 (0.1–50 nM) for 1 hour, then stimulated with anti-IgM (10 μg/mL) for 24 hours. CD69 and CD86 expression was measured by flow cytometry [2]
CLL B cell migration assay: CLL B cells were pretreated with PRT-060318 (0.5–50 nM) for 1 hour, then added to transwell inserts. CXCL12 (100 ng/mL) was added to the lower chamber, and migrated cells were counted after 4 hours [2]

30 mg/kg; i.p.
Mice, rabbit, pig models Studies in vivo [1]
C57Bl/6 (Charles River Labs) mice were used for the dose selection study. The transgenic HIT mice used to determine the effect of PRT-060318 (PRT318) on HIT IC-induced thrombocytopenia are homozygous for both FcγRIIA and human PF4 and null for mouse PF4.8,20 [1]
To determine the effect of PRT-060318 (PRT318) on HIT IC-induced thrombocytopenia and thrombosis, HIT model mice were treated with KKO (20 mg/kg body weight, intraperitoneally) on day 0. The mice were divided into sex- and weight-matched experimental and control groups. On days 1 to 7, experimental mice (n = 6) received PRT318 (30 mg/kg body weight) orally via gavage twice a day, whereas control mice (n = 6) received vehicle only (sterile water). Both groups received heparin (1600 U/kg body weight, subcutaneously) once daily. Mice were anesthetized by isoflurane inhalation for injections and blood collections. Blood was collected via the retro-orbital plexus using 50 μL ethylenediaminetetraacetic acid-coated microcapillary tubes (Microcaps, Drummond Scientific). Blood was collected 3 days before the antibody injections for baseline values and then on days 1 through 7 after antibody injections. Platelet counts were measured using a Hemavet Analyzer (model 850, CDC Technologies) and monitored over time to assess thrombocytopenia. Mouse plasma samples, collected 2 hours after PRT318 dosing, were precipitated with acetonitrile, and concentrations of PRT318 were quantified by liquid chromatography/tandem mass spectrometry. [1]
To evaluate the effect of PRT-060318 (PRT318) treatment on thrombosis in vivo, the protocol was modified as follows to take advantage of a novel thrombosis visualization technology. Experimental (n = 3) and control (n = 3) HIT mice were injected with KKO and PRT318 or vehicle only as described in the previous paragraph. For the imaging method, blood was collected in heparin from the retro-orbital plexus of untreated syngeneic donor mice. The donor platelets were washed, pooled, and incubated at 37°C for 10 minutes with Alexa750-labeled anti-GPIX antibody. The labeled platelets were pelleted and resuspended in sterile saline. Both experimental and control mice were infused with 1 × 108 labeled platelets in 100 μL aliquots via the retro-orbital plexus. All mice were then injected with heparin as described in the previous paragraph. Mice were killed 3 hours after heparin treatment, and their organs were perfused with sterile phosphate-buffered saline. Lungs were excised, fixed in 7% paraformaldehyde, and imaged with an Odyssey Imaging System (LI-COR Biosciences). The images were evaluated using Nikon NIS Elements image analysis software Version 3.10 (NIS-Elements, Advanced Research). Thrombi were identified as areas with fluorescence intensity at least 1.5 times above background. The Thrombosis Score represents the number of thrombi multiplied by the average intensity.

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hFcγRIIA transgenic mouse heparin-induced thrombosis model: Male transgenic mice were randomized into vehicle and treatment groups. PRT-060318 was formulated in 0.5% hydroxypropyl cellulose + 0.1% Tween 80 and administered orally at 30 mg/kg once daily for 3 days. On day 3, mice were injected intravenously with heparin (100 U/kg) and PAF (100 ng/kg) to induce thrombosis. Platelet counts were measured 24 hours later, and IVC thrombi were harvested for weight and histopathological analysis [1]

The bioavailability of PRT-060318 in mice after a single oral dose of 20 mg/kg was 55%[1]. The plasma half-life (t1/2) in mice after intravenous injection of 10 mg/kg was 4.3 hours[1]. The bioavailability of PRT-060318 in rats after oral administration of 20 mg/kg was 51%, and the plasma t1/2 was 5.0 hours[1]. The compound has good tissue penetration, and the platelet-to-plasma concentration ratio in mice was 2.9 4 hours after oral administration[1].

In a 14-day repeated-dose toxicity study in rats, oral doses of up to 100 mg/kg/day of PRT-060318 did not cause significant changes in body weight, hematological parameters, or clinical chemistry indicators (liver and kidney function indicators) [1]. PRT-060318 has a plasma protein binding rate of 91% in human plasma, 89% in mouse plasma, and 87% in rat plasma [1]. At therapeutic doses, no significant bleeding complications were observed in treated mice [1].

[1]. PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model. Blood. 2011 Feb 17;117(7):2241-6.

[2]. Selective, novel spleen tyrosine kinase (Syk) inhibitors suppress chronic lymphocytic leukemia B-cell activation and migration. Leukemia. 2012 Jul;26(7):1576-83.

Our study demonstrates that oral bioavailability of PRT318 inhibiting the Syk signaling pathway limits thrombocytopenia and thrombosis induced by HIT immune complexes (IC) in vivo. We focused on the effects of Syk inhibition on platelets. While similar effects on monocytes and endothelial cells cannot be ruled out, these effects may be beneficial to the pathological process of HIT. Notably, we employed a novel technique to clearly observe antithrombotic effects, a technique also applicable to other types of immune thrombocytopenic/thrombotic disorders (MS and WB, unpublished observations, March 2010). In summary, these studies clearly demonstrate that the Syk inhibitor PRT318 is an effective treatment for HIT. These results support the hypothesis that inhibition of the FcγRIIA-Syk signaling pathway using PRT318 or related compounds may be beneficial for the treatment of human HIT. Further research is needed to investigate its short-term application, effects on other Syk-expressing cells, and its impact on hemostasis. [1] Syk is a protein tyrosine kinase that couples activation of the B cell receptor (BCR) to downstream signaling pathways, thereby affecting cell survival and proliferation. In addition, Syk is involved in BCR-independent functions such as B cell migration and adhesion. In chronic lymphocytic leukemia (CLL), Syk is activated by external signals from the tissue microenvironment and was used as a target in the first clinical trial using a relatively nonspecific Syk inhibitor, R788 (fositatinib). In this paper, we characterized the activity of two novel, highly selective Syk inhibitors, PRT318 and P505-15, using experiments simulating the interaction between CLL and its microenvironment. PRT318 and P505-15 effectively antagonized CLL cell survival after BCR triggering and in nurse-like cell co-culture. Furthermore, they inhibit the secretion of BCR-dependent chemokines CCL3 and CCL4 in chronic lymphocytic leukemia (CLL) cells, as well as the migration of leukemia cells to tissue-homing chemokines CXCL12 and CXCL13 and stromal cells. PRT318 and P505-15 also inhibit the phosphorylation of Syk and extracellular signal-regulated kinases after BCR activation. These results suggest that the selective Syk inhibitors PRT318 and P505-15 can effectively inhibit CLL cell survival and tissue homing, and support the use of these drugs to treat CLL, other B-cell malignancies, and autoimmune diseases. [2]

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PRT-060318 is a potent and selective inhibitor of spleen tyrosine kinase (Syk)[1][2]
Its mechanism of action includes inhibiting Syk-mediated signaling pathways, blocking platelet activation and aggregation, and B cell activation/migration[1][2]
This compound has shown therapeutic potential in heparin-induced thrombocytopenia and thrombosis, as well as chronic lymphocytic leukemia (CLL)[1][2]
Its selectivity for Syk is much higher than that for other kinases, thereby reducing the risk of off-target effects[1]

C18H24N6O

340.42

340.201

C, 63.51; H, 7.11; N, 24.69; O, 4.70

1194961-19-7

11493841

Light yellow to yellow solid powder

1.3±0.1 g/cm3

602.1±65.0 °C at 760 mmHg

317.9±34.3 °C

0.0±1.7 mmHg at 25°C

1.672

1.62

4

6

5

25

447

2

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: 25 mg/mL (73.44 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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