p38α (IC50 = 13 nM)

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]

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]

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].

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]

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]

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].

[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.
The discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38α inhibitor. Unlike alkyl and other cycloalkyls, the sp(2) character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38α enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38α was confirmed by X-ray crystallographic analysis.[1]

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Functional innate immune assessments, including phagocytosis and respiratory burst, are at the forefront of immunotoxicology evaluation in pre-clinical animal species. Although in the clinic and in academic science, phagocytosis, and respiratory burst assessments have been reported for over two decades, the implementation of phagocytosis and respiratory burst analyses in toxicology safety programs is just recently gaining publicity. Discussed herein are general methods, both microtiter plate-based and flow cytometric-based, for assessing phagocytosis and respiratory burst in pre-clinical species including mouse, rat, dog, and monkey. This methods-centric discussion includes a review of technologies and descriptions of method applications, with examples of results from analyses testing reported inhibitors (rottlerin, wortmannin, and SB203580) of phagocytosis and respiratory burst. Justification of implementation, strategic experimental design planning, and feasibility aspects of evaluating test article effects on phagocytosis and respiratory burst function are described within the context of a case study. The case study involves investigation of the effects of a small molecule p38 kinase inhibitor, BMS-582949, on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro, as well as ex vivo in these innate immune cells from monkeys administered BMS-582949 during a 1-week repeat dose investigative study. The results of the in vitro and ex vivo assessments demonstrated that BMS-582949 inhibited phagocytosis and respiratory burst. These findings correlated with incidences of opportunistic infections observed in rat and monkey toxicity studies.[2]

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Case study summary[2]
In vitro phagocytosis and respiratory burst assessments demonstrated that BMS-582949 inhibits these functions at concentrations which were similar to drug exposures in animals that had infections in toxicity studies. For example, 5 µM BMS-582949 is 0.1–10× the Cmax values achieved in animals with infections (Price, Citation2010). In general, inhibition of these functions was greater in monkeys than rats, which correlates with the observation that severity and incidence of observed infections were higher in monkeys compared to rats. Moreover, ex vivo analyses demonstrated that phagocytosis and respiratory burst were inhibited at doses that resulted in infections in a 1-year monkey study. In both the in vitro and ex vivo assessments, respiratory burst was inhibited more than phagocytosis and inhibition was greater in neutrophils than monocytes. In summary, the in vitro and ex vivo phagocytosis and respiratory burst assessment results supported the hypothesis that in the context of immunomodulation (decreased phagocytosis and respiratory burst) by a p38 inhibitor, opportunistic pathogens may manifest clinically-relevant infections.

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Methodology conclusions[2]
The methods described herein for assessing phagocytosis and respiratory burst are suitable for evaluating test article effects on these important innate immune functions. Common, commercially available immunomodulators can be used to verify the technical expertise and suitability of these methods. These assessments can be performed in vitro or ex vivo. For each test article and species tested, multiple assay parameters should be evaluated to ensure optimal assay conditions. If feasible, in vitro assessments provide a facile platform for testing numerous parameters and these conditions can translate into ex vivo evaluations as demonstrated by the case study described herein. Flow cytometric-based methods are more amenable to ex vivo assessments compared to plate-based methods as whole blood can be analyzed by flow cytometric-based methods. Although investigation of test article effects can be performed in investigative studies, the 96-well format of the plate- and flow cytometric-based methods facilitate addition of these functional end-points in standard toxicology studies with minimal logistical obstacles and can be utilized across pre-clinical species.

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.)