Cysteine protease

Papain is a peptidase C1 family cysteine protease that finds application in the culinary, pharmaceutical, textile, and cosmetic sectors.

Murine papain-induced emphysema is a model that reproduces many of the features found in patients. Bone marrow-derived mononuclear cells (BMMC) have already been used to repair the alveolar epithelium in respiratory diseases, but not in the papain model. Thus, we hypothesized that BMMC could prevent the pathophysiological processes in papain-induced experimental emphysema. Female BALB/c mice received intratracheal instillation of 50 μL of saline (S groups) or papain (P groups, 10 IU/50 μl of saline) on days 1 and 7 of the experimental protocol. On the 14th day, 2 × 106 BMMC of male BALB/c mice (SC21 and PC21) or saline (SS21 and PS21) were injected by the jugular vein. Analyses were done on days 14 (S14 and P14) and 21 (SS21, PS21, SC21, and PC21) of the protocol. qPCR evaluated the presence of the Y chromosome in the lungs of BMMC recipient animals. Functional residual capacity (FRC), alveolar diameter, cellularity, elastic fiber content, concentrations of TNF-α, IL-1β, IL-6, MIP-2, KC, IFN-γ, apoptosis, mRNA expression of the dual oxidase (DUOX1 and DUOX2), production of H2O2 and DUOX activity were evaluated in lung tissue. We did not detect the Y chromosome in recipients' lungs. FRC, alveolar diameter, polymorphonuclear cells (PMN) and levels of KC, MIP-2, and IFN-γ increased in P14 and PS21 groups; the changes in the latter were reverted by BMMC. TNF-α, IL-1β e IL-6 were similar in all groups. The amount of elastic fibers was smaller in P14 and PS21 than in other groups, and BMMC did not increase it in PC21 mice. PS21 animals showed increased DUOX activity and mRNA expression for DUOX1 and 2. Cell therapy reverted the activity of DUOX and mRNA expression of DUOX1. BMMC reduced mRNA expression of DUOX2. Apoptosis index was elevated in PS21 mice, which was reduced by cell therapy in PC21. Static compliance, viscoelastic component of elastance and pressure to overcome viscoelasticity were increased in P14 and PS21 groups. These changes and the high resistive pressure found on day 21 were reverted by BMMC. In conclusion, BMMC showed potent anti-inflammatory, antiapoptotic, antioxidant, and restorative roles in papain-triggered pulmonary emphysema.[1]

The animals were sedated by inhalation of sevoflurane, weighed (model BR), and saline or papain was injected into the trachea using an insulin syringe. This procedure last about 3 min.[1]
Fifteen male BALB/c mice (20–25 g) were quickly euthanized by cervical dislocation. BMMC were aspirated from their femur and tibia by flushing the bone marrow cavity with Dulbecco's modified Eagle's medium (DMEM). After a homogeneous cell suspension was obtained, the cells were centrifuged (4,000 g for 10 min), re-suspended in DMEM and added to Ficoll-Hypaque (Histopaque 1083), centrifuged again (5,000 g, 30 min) and supplemented with sterile PBS. Cells were counted in a Neubauer chamber with Trypan Blue for evaluation of viability. Cell characterization was performed by flow cytometry using specific antibodies (Conget and Minguell, 1999; Maron-Gutierrez et al., 2011).[1]
Figure ​Figure11 shows that 60 female BALB/c mice (20–25 g) were randomly divided into six groups. In S14 (n = 10), SS21 (n = 10), and SC21 (n = 10) groups, mice were intratracheally (i.t.) injected with 50 μL of sterile saline solution (0.9% NaCl) on days 1 and 7 of the experimental protocol. In P14 (n = 10), PS21 (n = 10), and PC21 (n = 10) groups, mice were i.t. injected with 50 μL of sterile saline solution (0.9% NaCl) containing 10 IU of papain (0.2 IU/μL) on days 0 and 7 of the experimental protocol. Papain (USP 225310) had been previously activated in 0.1 M sodium phosphate buffer containing 10 mM EDTA, 0.4 NaCl and 5 mM dithiothreitol for 10 min at 40°C (Machado et al., 2014). On the 14th day, 2 × 106 BMMC from male BALB/c mice suspended in 50 μL of sterile saline solution (SC21 and PC21 groups) or 50 μL of sterile saline solution (0.9% NaCl) (SS21 and PS21 groups) were injected into the jugular vein. Histopathological parameters and pulmonary mechanics were analyzed on the 14th (S14 and P14 groups) and 21st (SS21, PS21, SC21, and PC21 groups) days of the protocol.[1]

[1]. Bone Marrow-Derived Mononuclear Cell Therapy in Papain-Induced Experimental Pulmonary Emphysema. Front Physiol. 2018 Feb 20;9:121.

Type II alveolar cells express the enzymes DUOX 1 and DUOX 2, which, just as in the airways, are found in the apical pole of cells. Although DUOX 1 and 2 are found in the alveoli, there is scanty available information about their participation in H2O2 production at this histological level (Fischer, 2009). H2O2 production by these enzymes at the alveolar level is considered low in relation to the generation of H2O2 seen in the airway epithelium (Fischer et al., 2007). The precise role of the DUOXs enzymes in the pathogenesis of pulmonary emphysema is controversial and require more enlightening studies. We evaluated the mRNA expression for DUOX1 and DUOX2 and observed an increased expression of mRNA for DUOX1 and DUOX2 in the lung tissue (Figure ​(Figure5)5) of the animals exposed to papain, in agreement with other groups (Ameziane-El-Hassani et al., 2005; Harper et al., 2005; Rigutto et al., 2009). Administration of 2 × 106 BMMC attenuated mRNA expression for DUOX1 and DUOX2 in lung tissue. In addition, we observed an increase in the H2O2 generation and in the calcium-stimulated activity of DUOX in the lung tissue of animals treated with papain compared to the other groups, reinforcing the hypothesis that 2 × 106 BMMC may behave as an antioxidant agent (Figure ​(Figure6).6). There are distinct roles for the NADPH oxidase in the airways (Fischer, 2009). Despite the high structural similarity of the two DUOX isoforms, the production of H2O2 by DUOX2 is higher than by DUOX1 in the pulmonary airway epithelium (Ameziane-El-Hassani et al., 2005; Rigutto et al., 2009). However, normal airway epithelium expresses DUOX1 at higher levels than DUOX 2 (Schwarzer et al., 2004; Harper et al., 2005). Therefore, the release of H2O2 by DUOX1 and DUOX2 may be similar in the airways of normal individuals. Some cytokines selectively regulate DUOX1 and DUOX2 expression levels; IFN-γ positively regulates DUOX2 expression (Harper et al., 2005). We found that IFN-γ was augmented in papain-exposed mice and was reduced by BMMC (Table ​(Table2),2), in line with DUOX activity (Figure ​(Figure6).6). Briefly, we emphasize the novelty of our results, since there is no description to date of the effects of cell therapy on the pathways of NADPH oxidases in a murine model of papain-induced pulmonary emphysema, specifically related to the regulation of DUOXs enzymes. In conclusion, 2 × 106 BMMCs showed potent anti-inflammatory, antiapoptotic, antioxidant and restorative effects in papain-triggered pulmonary emphysema, possibly by blocking DUOX1 and reducing DUOX2.

C9H14N4O3

226.2325

451.217

9001-73-4

5249653

White to off-white solid powder

1.5±0.1 g/cm3

29 °C

1.652

-1.47

3

4

5

16

254

0

CQOVPNPJLQNMDC-UHFFFAOYSA-N

InChI=1S/C9H14N4O3/c10-2-1-8(14)13-7(9(15)16)3-6-4-11-5-12-6/h4-5,7H,1-3,10H2,(H,11,12)(H,13,14)(H,15,16)

2-(3-azaniumylpropanoylamino)-3-(1H-imidazol-5-yl)propanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL
DMSO : ~25 mg/mL

Solubility in Formulation 1: 50 mg/mL (Infinity 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.)