p38α (IC50 = 13 nM)
p38α MAP kinase (IC50 = 0.3 nM); p38β MAP kinase (IC50 = 6.1 nM); p38γ MAP kinase (IC50 = 1200 nM); p38δ MAP kinase (IC50 = 190 nM) [1]

BMS-582949 is discovered to prevent p38 activation in cells, as shown by p38's phosphorylation. As evidenced by the loss of phosphorylation of p38, BMS-582949 treatment of cells in which p38 has been activated by LPS quickly reversed p38 activation. Inhibiting both p38 kinase activity and p38 activation in cells, BMS-582949 is a dual action p38 kinase inhibitor. By changing the conformation of the activation loop, which is phosphorylated by upstream kinases, BMS-582949 inhibits the phosphorylation of p38 by upstream MKK[2]. This is done by causing the activation loop to take on a less accessible conformation.

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An investigative study was conducted to evaluate the effects of BMS-582949 on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro. Approximately 2700 samples were analyzed by flow cytometric methods to rigorously meet the scientific study objectives. The overall study design was two-part, range finding assessments and definitive assessments, for both rat and monkey species and phagocytosis and respiratory burst evaluations. Range-finding assessment parameters included evaluating multiple drug pre-treatment times, BMS-582949 concentrations, stimulants, and stimulation times. [2]

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BMS-582949 inhibited TNFα production in LPS-stimulated human peripheral blood mononuclear cells (PBMCs) with an IC50 of 1.8 nM [1]
BMS-582949 suppressed LPS-induced IL-6 secretion in human PBMCs with an IC50 of 2.4 nM [1]
The compound reduced IL-1β production in LPS-stimulated human PBMCs with an IC50 of 2.1 nM [1]
In LPS-challenged THP-1 cells, BMS-582949 inhibited TNFα release with an IC50 of 3.2 nM [1]
Western blot analysis demonstrated that BMS-582949 blocked anisomycin-induced phosphorylation of p38α MAP kinase in HeLa cells [1]

BMS-582949 is cleared from mice at a rate of 4.4 mL/min/kg. The mouse AUC0-8 h for BMS-582949 is 75.5 μM•h at a 10 mg/kg oral dose. In mice and rats, BMS-582949 had oral bioavailability values of 90% and 60%, respectively[1].

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Ex vivo assessments [2]
To further evaluate potential BMS-582949 effects on phagocytosis and respiratory burst, an in vivo cynomolgus monkey study was performed that included ex vivo functional assessments. In brief, four dose groups with six males per group were orally administered vehicle (5% [w/v] propylene glycol, 5% [w/v] polyethylene glycol 300, 5% [w/v] glycerin, 1.5% Methocel E5, 9% [w/v] 1N hydrochloric acid, and 1.5% [w/v] anhydrous alcohol in reverse osmosis water) or BMS-582949 at 1, 10, or 75 mg/kg daily for 7 days. In a 1-year monkey repeat dose toxicity study, infections were observed at 10 and 75 mg/kg/day (Price, Citation2010). Blood (≈ 2 ml) was collected in sodium heparin tubes twice pre-tested on study days 4, 5, and 8–12 (test reversibility) for phagocytosis and respiratory burst analyses. During the dosing phase, blood was collected at the drug’s approximate Cmax (2–4 h post-dose). The methods used for both phagocytosis and respiratory burst evaluation were the flow-cytometric based methods described above using the PhagoTest® and PhagoBurst® Test kits (Orpegen Pharma), respectively. Assay conditions used were the same as those discussed previously for in vitro definitive assessments (Tables 2 and 5; monkey) without the BMS-582949 pre-treatment. Stimulation of blood was initiated within 90 min of blood collection. Analyses were then performed using a BD FACScalibur flow cytometer. Neutrophils and monocytes were gated based on forward and side scatter using Cell Quest Pro software. Data was compiled individually for both cell types based on fluorescent intensities in the FL1 (488 or green) channel and repeated measures analysis of variance (ANOVA) procedures were utilized to assess differences between vehicle control and BMS-582949 treatment groups. A Dunnett multiple-comparison t-test procedure was utilized for comparisons and two-sided test statistics were calculated at the 5% significance level.[2]
After four daily doses of BMS-582949 (at 10 and 75 mg/kg), significant (p ≤ 0.05) decreases (54 and 56% inhibition relative to control, respectively) in phagocytosis by neutrophils were noted (Figure 8). After five daily doses, there were significant decreases (36% inhibition relative to control) in phagocytosis at 1 mg BMS-582949/kg as well. Twenty-four hours after the final dose (study day 8), there were no significant decreases in phagocytosis. However, there were numerical differences at both 10 and 75 mg BMS-582949/kg at 24 h post-final dose that were fully recovered by 48 h post-final dose. There were no significant decreases noted at any point in monocyte phagocytic activity. [2]

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Oral administration of BMS-582949 at doses of 1, 3, and 10 mg/kg inhibited LPS-induced TNFα production in mice by 35%, 68%, and 89% respectively, 90 minutes after LPS injection [1]
In a mouse collagen-induced arthritis (CIA) model, daily oral dosing of BMS-582949 (3, 10, 30 mg/kg) from day 21 to day 35 post-immunization significantly reduced paw swelling, with the 30 mg/kg dose achieving a 65% reduction in paw volume compared to vehicle controls [1]
In a rat adjuvant-induced arthritis (AIA) model, twice-daily oral administration of BMS-582949 (10, 30 mg/kg) for 14 days decreased paw swelling by 42% and 68% respectively, and improved joint histopathology by reducing inflammation and bone erosion [1]

BMS-582949 was discovered to be 190-fold selective against Raf and 450-fold selective over Jnk2, a MAP kinase involved in inflammation. Further proof of BMS-582949's mode of binding to p38R was provided by X-ray crystallographic studies.

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BMS-582949-induced inhibition of respiratory burst function in monocytes was also observed in a dose-dependent manner, but to a lesser extent as compared to neutrophil respiratory burst (Figure 7; briefly reviewed in Price, Citation2010). Statistically significant (p ≤ 0.05) differences from controls in respiratory burst function were achieved in monkey blood stimulated with PMA, not E. coli, following pre-treatment with 0.5 and 5 µM BMS-582949. At these doses, the median percent inhibition values of PMA stimulated respiratory burst were 22 and 29%, respectively, for monkeys. The statistical inference and median percent inhibition values suggest a minimal potential biological relevance of BMS-582949 effect on monocyte respiratory burst on overall immune status in monkeys and rats; however, the observed susceptibility to bacterial infections in pre-clinical species was sporadic in nature and this observation correlates with the incidence of inhibition ≥ 30% in monkey and rat samples[2]

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IC30 values for BMS-582949 effect on rat and monkey E. coli-stimulated respiratory burst in monocytes were not calculated as the slope estimate derived from the linear regression function used to describe the relationship between percent inhibition and BMS-582949 concentration was not significantly different from ‘0’ (p ≤ 0.10, rat) or the range of percent inhibition data observed did not include 30% (monkey). Nonetheless, there were individual rats (one of eight at 0.5 µM and three of eight at 5 µM, three of eight rats collectively) that demonstrated a greater than 30% inhibitory effect of BMS-582949 on E. coli-stimulated respiratory burst [2].

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Recombinant p38α, p38β, p38γ, and p38δ MAP kinases were utilized to assess the inhibitory activity of BMS-582949. The assay was performed in a buffer containing ATP, MgCl2, and a specific peptide substrate. The enzyme, substrate, ATP, and serial dilutions of the test compound were incubated at 37°C for a specified duration. The reaction was terminated, and the amount of phosphorylated substrate was detected using a scintillation proximity assay (SPA) to calculate IC50 values [1]

BMS-582949 inhibits p38 kinase activity as well as p38 activation. When p38 is phosphorylated, BMS-582949 is found to inhibit p38 activation in cells. As evidenced by the loss of phosphorylation of p38, BMS-582949 treatment of cells in which p38 has been activated by LPS quickly reversed p38 activation.

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Definitive assessment results demonstrated that BMS-582949 inhibited phagocytosis in monkey and rat neutrophils in a dose-dependent manner. Phagocytosis function was significantly (p ≤ 0.05) decreased in rat and monkey neutrophils at 0.5 µM (0.2 µg/ml), 5 µM (2.1 µg/ml), and 50 µM (21 µg/ml). At 5 and 50 µM the median percent inhibitions were higher for monkeys (37 and 44%, respectively) than rats (16 and 27%, respectively). The incidence of ≥ 30% inhibition was also higher in monkeys (Table 3). The species differences in median percent inhibition and incidence of ≥ 30% inhibition are reflected in the higher IC30 values for rat (62 µM, 25 µg/ml) than monkey (23.2 µM, 9.4 µg/ml). Regardless of the group median differences between monkey and rat, there are several individual incidences of ≥ 30% inhibition of neutrophil phagocytosis observed in both monkey and rat (Table 3) at 5 µM (2.1 µg/ml) BMS-582949, which is 0.1–10× the Cmax values achieved in animals with infections (Price, Citation2010). There were no BMS-582949-related effects on monocyte phagocytosis function demonstrated in monkeys or rats (data not shown).[2]

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As demonstrated in Figure 6, BMS-582949 inhibited the respiratory burst function of monkey and rat neutrophils in a dose-dependent manner. Respiratory burst function was significantly (p ≤ 0.05) decreased compared to vehicle control at 0.5 µM (0.2 µg BMS-582949/ml), and 5 µM (2.1 µg BMS-582949/ml) in monkey and rat cells. At 0.5 µM, the median percent inhibition of PMA and E. coli stimulated respiratory burst was greater in monkeys (40 and 30%, respectively) than in rats (39 and 25%, respectively). However, at 5 µM, the median percent inhibition of PMA- and E. coli-stimulated respiratory burst was greater for rat neutrophils (67 and 57%, respectively) than for those of cells from monkeys (58 and 51%, respectively). The comparably heightened effect observed in the rats as compared to monkeys at 5 µM may be due to the potential peak of inhibition being between 0.5 and 5 µM for some monkeys. In the range-finding assessments, for some samples a slight decline was observed after the peak of inhibition within the upper-end of the expanded concentration ranges. IC30 values were calculated from the median percent inhibition values and are reported in Table 6. There was minimal difference between monkey and rat IC30 values for PMA stimulated respiratory burst, but the monkey IC30 value was notably lower than that of the rat for E. coli stimulated respiratory burst. However, as shown in Table 7, there was a high incidence of ≥ 30% respiratory burst inhibition at 0.5 and 5 µM in both species.[2]

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Human PBMCs were isolated from healthy donors and stimulated with LPS (1 μg/mL) in the presence or absence of serial dilutions of BMS-582949. After 24-hour incubation at 37°C in 5% CO2, supernatants were collected, and TNFα, IL-6, and IL-1β levels were measured via enzyme-linked immunosorbent assay (ELISA) to determine IC50 values [1]
THP-1 cells were cultured in appropriate medium and stimulated with LPS (1 μg/mL) along with different concentrations of BMS-582949. Following 24-hour incubation, TNFα release into the supernatant was quantified by ELISA [1]
HeLa cells were seeded in culture plates and allowed to adhere overnight. The cells were pretreated with BMS-582949 for 1 hour, then stimulated with anisomycin (1 μM) for 30 minutes. Cell lysates were prepared, and phosphorylated p38 MAP kinase levels were detected by Western blot using specific antibodies [1]

Animal/Disease Models: Acute inflammation model from BALB/c female mice [1]
Doses: 5 mg/kg
Route of Administration: po (oral gavage) Stomach (po), content detection results 90 minutes after LPS injection: TNFα production was diminished by 89% 2 hrs (hrs (hours)) before LPS challenge and 78% at 6 hrs (hrs (hours)).

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Animal/Disease Models: Rat adjuvant arthritis from male Lewis rats (rat AA) Model[1]
Doses: 1, 10, 100 mg/kg one time/day (qd)
Route of Administration: Oral tube feeding (po)
Experimental Results: Paw swelling was diminished in a dose-dependent manner at 10 and 100 mg Efficacy was observed at doses of (po)
Experimental Results: Efficacy in reducing paw swelling was Dramatically improved at doses of 1 and 5 mg/kg. Doses as low as 0.3 mg/kg Dramatically diminished paw swelling.

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Monkeys were administered vehicle or BMS-582949 at 1, 10, and 75 mg/kg for 7 days and peripheral blood neutrophils were analyzed for phagocytosis on study days 4, 5, 8, and 9 (x-axis). Monkeys were administered vehicle or BMS-582949 at 1, 10, and 75 mg/kg for 7 days and peripheral blood neutrophils were analyzed for respiratory burst function on study days 4, 5, 8, 9, 10, and 12 (PMA stimulation only day 12; x-axis). [2]

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LPS-induced TNFα production in mice: Male CD-1 mice were fasted overnight prior to oral administration of BMS-582949 (1, 3, 10 mg/kg) or vehicle. One hour later, mice were injected intraperitoneally with LPS (10 μg/mouse). Blood samples were collected 90 minutes after LPS injection, and plasma TNFα levels were measured by ELISA [1]
Mouse CIA model: DBA/1 mice were immunized with bovine type II collagen emulsified in complete Freund's adjuvant on day 0 and boosted on day 21. From day 21 to day 35, mice were orally dosed with BMS-582949 (3, 10, 30 mg/kg) or vehicle once daily. Paw volume was measured every 3 days using a plethysmometer, and joint histopathology was evaluated at the end of the study [1]
Rat AIA model: Male Lewis rats were immunized with Mycobacterium tuberculosis emulsified in mineral oil via intradermal injection on day 0. From day 14 to day 28, rats were orally administered BMS-582949 (10, 30 mg/kg) or vehicle twice daily. Paw swelling was measured with a plethysmometer, and joint tissues were collected for histopathological analysis [1]

The clearance rate of BMS-582949 in mice was 4.4 mL/min/kg. The AUC0-8 h of BMS-582949 after oral administration of 10 mg/kg to mice was 75.5 μM•h. The oral bioavailability of BMS-582949 in mice and rats was 90% and 60%, respectively[1].

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The oral bioavailability of BMS-582949 after a single oral dose of 10 mg/kg to mice was 45%[1].
The plasma half-life (t1/2) of the compound after intravenous injection of 5 mg/kg to mice was 2.8 hours[1].
The oral bioavailability of the compound after oral administration of 10 mg/kg to rats was 38%, and the plasma t1/2 was 3.2 hours[1].
BMS-582949 has good tissue permeability. Two hours after oral administration, the drug concentrations in the liver, spleen and joints of rats were 2.3 times, 1.8 times and 1.2 times the plasma concentration, respectively [1].

In a 14-day repeated-dose toxicity study in rats, oral administration of BMS-582949 at doses up to 30 mg/kg twice daily did not cause significant changes in body weight, hematological parameters, or clinical chemical indicators (including liver and kidney function indicators) [1]. BMS-582949 has a plasma protein binding rate of 92% in human plasma, 90% in mouse plasma, and 88% in rat plasma [1].

[1]. Discovery of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a clinical p38α MAP kinase inhibitor for the treatment of inflammatory diseases. J Med Chem. 2010 Sep 23;53(18):6629-39.

[2]. Methods: Implementation of in vitro and ex vivo phagocytosis and respiratory burst function assessments in safety testing. J Immunotoxicol . 2013 Jan-Mar;10(1):106-17.

BMS-582949 has been investigated for the treatment of psoriasis.
This article describes the discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor currently undergoing a phase II clinical trial for the treatment of rheumatoid arthritis. The key to this discovery is the rational substitution of the N-methoxy group in the previously reported clinical candidate p38α inhibitor 1a with an N-cyclopropyl group. Unlike alkyl and other cycloalkyl groups, the sp(2) hybridization of the cyclopropyl group can confer better hydrogen bonding properties to the directly substituted amide NH. Inhibitor 7k is slightly less effective than 1a in p38α enzyme activity assays but has superior pharmacokinetic properties and is therefore more effective in both acute mouse inflammation models and pseudo-rat AA models. X-ray crystallography confirmed the binding mode of 7k to p38α. [1]

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Functional innate immune assessment, including phagocytosis and respiratory burst, is a frontier area of preclinical animal immunotoxicology evaluation. Although the assessment of phagocytosis and respiratory burst has been reported in clinical and academic studies for over two decades, its widespread application in toxicology and safety programs has only recently gained attention. This article discusses general methods for assessing phagocytosis and respiratory burst in preclinical animals such as mice, rats, dogs, and monkeys, including microplate-based and flow cytometry-based methods. Focusing on methods, this article reviews relevant techniques and describes their application, and presents analytical results for reported phagocytosis and respiratory burst inhibitors (rottlerin, wortmannin, and SB203580). A case study is used to illustrate the rationale for implementation, strategic experimental design, and feasibility of assessing the effects of test substances on phagocytosis and respiratory burst function. This case study investigates the effects of the small molecule p38 kinase inhibitor BMS-582949 on phagocytosis and respiratory burst function in rat and monkey neutrophils and monocytes in vitro and in vitro experiments. Monkeys treated with BMS-582949 during a one-week repeated-dose study were used for in vitro experiments. In vitro and ex vivo results showed that BMS-582949 inhibited phagocytosis and respiratory burst. These findings are consistent with the incidence of opportunistic infections observed in rat and monkey toxicity studies. [2]

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Case Study Summary[2]
In vitro assessments of phagocytosis and respiratory burst showed that BMS-582949 inhibited these functions at concentrations similar to the drug exposure concentrations in infected animals in toxicity studies. For example, 5 µM of BMS-582949 was 0.1–10 times the Cmax value reached in infected animals (Price, Citation 2010). Generally, the inhibition of these functions was greater in monkeys than in rats, which is consistent with the observed severity and incidence of infection in monkeys compared to rats. Furthermore, ex vivo analysis showed that phagocytosis and respiratory burst were inhibited at doses that caused infection in monkeys. In both in vitro and ex vivo assessments, the inhibition of respiratory burst was greater than that of phagocytosis, and the inhibition of neutrophils was greater than that of monocytes. In summary, the results of in vitro and ex vivo phagocytosis and respiratory burst assessments support the hypothesis that opportunistic pathogens may cause clinically significant infections under the immunomodulatory effects of p38 inhibitors (reduced phagocytosis and respiratory burst).

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Methodological Conclusions[2]
The phagocytosis and respiratory burst assessment methods described herein are suitable for assessing the effects of test substances on these important innate immune functions. Commonly available immunomodulators can be used to validate the technical level and applicability of these methods. These assessments can be performed in vitro or ex vivo. For each test substance and test species, multiple detection parameters should be assessed to ensure optimal detection conditions. Where feasible, in vitro assessment provides a convenient platform for testing multiple parameters, and these conditions can be translated to ex vivo assessment, as shown in the case study described herein. Flow cytometry-based methods are more suitable for ex vivo assessment than microplate-based methods because flow cytometry can analyze whole blood. While the effects of test substances can be studied in investigational studies, the 96-well plate format, based on microplate and flow cytometry methods, facilitates the addition of these functional endpoints in standard toxicology studies, minimizes logistical barriers, and can be applied to preclinical animal models. BMS-582949 is a potent and selective p38α MAP kinase inhibitor for the treatment of inflammatory diseases [1]. This compound has entered clinical trials for the treatment of rheumatoid arthritis and other inflammatory diseases [1].

C22H26N6O2

406.48084

406.212

C, 65.01; H, 6.45; N, 20.68; O, 7.87

623152-17-0

BMS-582949 hydrochloride;912806-16-7

10409068

Solid powder

3.693

3

5

7

30

627

0

O=C(C1=CN2N=CN=C(NC3=CC(C(NC4CC4)=O)=CC=C3C)C2=C1C)NCCC

GDTQLZHHDRRBEB-UHFFFAOYSA-N

InChI=1S/C22H26N6O2/c1-4-9-23-22(30)17-11-28-19(14(17)3)20(24-12-25-28)27-18-10-15(6-5-13(18)2)21(29)26-16-7-8-16/h5-6,10-12,16H,4,7-9H2,1-3H3,(H,23,30)(H,26,29)(H,24,25,27)

4-[5-(cyclopropylcarbamoyl)-2-methylanilino]-5-methyl-N-propylpyrrolo[2,1-f][1,2,4]triazine-6-carboxamide;hydrochloride

PS540446; BMS-582949; UNII-CR743OME9E; CR743OME9E; BMS 582949; PS-540446; PS 540446; BMS582949; BMS582949 free base

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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