Substance P antagonist CP-96345 blocks lung vascular leakage and inflammation more effectively than its stereoisomer CP-96344 in a mouse model of smoke inhalation and burn injury

Abstract

The recently developed murine model of smoke inhalation and burn (SB) injury was used to study the effect of the substance-P antagonist CP96345. C57BL/6 mice were pre-treated with an i.v. dose of a specific NK-1 receptor antagonist, CP9635, or its inactive enantiomer, CP96344, (10 mg/Kg) 1 h prior to SB injury per protocol (n = 5). Mice were anesthetized and exposed to cooled cotton smoke, 2X 30 s, followed by a 40% total body surface area flame burn per protocol. At 48 h after SB injury Evans Blue (EB) dye and myeloperoxidase (MPO) were measured in lung after vascular perfusion. Lungs were also analyzed for hemoglobin (Hb) and wet/dry weight ratio. In the current study, CP96345 pre-treatment caused a significant decrease in wet/dry weight ratio (23%, p = 0.048), EB (31%, p = 0.047), Hb (46%, p = 0.002), and MPO (54%, p = 0.037) levels following SB injury compared to animals with SB injury alone. CP-96344 pre-treatment caused an insignificant decrease in wet/dry weight ratio (14%, p = 0.18), EB (16%, p = 0.134), Hb (9%, p = 0.39), and an insignificant increase in MPO (4%, p = 0.79) as compared to mice that received SB injury alone. As expected, levels of EB, Hb, MPO, and wet/dry weight ratios were all significantly (p < 0.05) increased 48 h following SB injury alone compared to respective sham animals. In conclusion, the current study indicates that pre-treatment with a specific NK-1R antagonist CP-96345 attenuates the lung injury and inflammation induced by SB injury in mice.

Fig. 1. Effect of NK-1 antagonist pretreatment on plasma extravasation

A significant decrease (31%, *p = 0.047) in EB levels was observed in NK-1 antagonist pretreated mice (checked Bar) 48 hr following SB injury compared to animals with SB injury alone (black filled bar). EB levels were significantly (*p =0.0356) increased 48 hr following SB injury alone (black filled bar) compared to respective sham animals (open bar). The group treated with CP-96345 (speckled bar) had a statistically insignificant increase (16%, *p = 0.134) in EB level as compared to respective sham animals. EB levels were quantitated from an authentic standard curve with known concentrations of EB. Values are means ±SE of at least 5 mice/group.

Fig. 2. Effect of NK-1 antagonist pretreatment on edema

A significant decrease (23%, *p = 0.048) in wet/dry weight ratios was indicated in NK-1 antagonist CP-96345 pretreated mice (checked Bar) 48 hr following SB injury compared to animals with SB injury alone. (black filled bar). Wet/dry weight ratios were significantly increased (47%, *p = 0.018) 48 hr following SB injury alone (black filled bar) compared to respective sham animals (open bar). Pretreatment with NK-1 inactive enantiomer CP-96344 followed by SB injury (speckled bar) caused a statistically insignificant decrease (14%, *p=0.354) in wet/dry weight ratios compared to mice that received SB injury alone (black filled bar). Values are means ±SE of at least 6 mice/group.

Fig. 3. Effect of NK-1 antagonist pretreatment on Hemoglobin (Hb)

Significantly decreased Hb levels (46%, *p = 0.002) were seen in NK-1R antagonist pretreated mice after SB injury (checked bar). Increased Hb levels (68%, *p=0.037) were observed in SB injury alone (black filled bar) compared to sham (open bar). There was a low level insignificant decrease (9%, *p=0.39) in CP-96344 pretreated mice (specked bar) compared to the SB injury alone group (black filled bar). Values are means ±SE of at least 6 mice/group.

Fig. 4. Effect of NK-1 antagonist pretreatment on neutrophil infiltration and inflammation

Significantly decreased MPO levels (54%, * p = 0.037) were found in NK-1 antagonist pretreated mice (checked bar) following SB injury compared to animals with SB injury alone (black filled). Significantly increased MPO levels were indicated (227%, *p =0.005) 48 hr following SB injury alone (black filled bar) compared to respective sham animals (open bars). Pretreatment with inactive isomer CP-96344 followed by SB injury (speckled bar) led to a slight insignificant increase (4%, p=0.79) compared to animals pretreated with CP-96345 followed by SB injury (black filled bar). MPO levels were quantitated from an authentic standard curve with known amounts of MPO. Values are means ±SE of at least 5 mice/group.

Fig. 5. Proposed pathway depicting the role of NK-1 antagonist in lung injury following SB injury in mice

Neurogenic inflammation induced by SB induced noxious stimuli is caused in part by release of neuropeptides from nociceptor nerves, and so treatment with an NK-1 receptor antagonist will reduce plasma leakage, edema, and neutrophil infiltration.