## 2 Results and Discussion
### Anti-Inflammatory Effect in TNBS-Induced Colitis  
Due to the lack of a anti-inflammatory effect in opiorphin and its analog Pal-KKQRFSR in the mouse model of trinitrobenzesulfonic acid (TNBS)-induced colitis [15; 16], only sialorphin analogs \begin{table}
\begin{tabular}{c c c c} \hline
**Peptide** & **Inhibitor** & \(\mathbf{1000\times k}\) **[1/min]** & \(\mathbf{t_{1/2}}\) **[min]** \\ \hline \multicolumn{4}{c}{Without inhibitor *} & \(25.35\pm 1.05\) & \(27\pm 1\) \\ \hline \multirow{2}{*}{**I**} & Sialorphin * & \multirow{2}{*}{\(8.84\pm 0.27\)**} & \multirow{2}{*}{\(78\pm 2\)**} \\  & (Gln-His-Asn-Pro-Arg) & & \\ \hline \multirow{2}{*}{**II**} & Opiorphin * & \multirow{2}{*}{\(7.38\pm 0.20\)**} & \multirow{2}{*}{\(93\pm 2\)**} \\  & (Gln-Arg-Phe-Ser-Arg) & & \\ \hline
**V** & Cys-Gln-Arg-Phe-Ser-Arg & & \(3.47\pm 0.12\)** & \(199\pm 7\)** \\ \hline
**VI** & Cys-Leu-Val-Val-Tyr-Pro-Trp-Thr & & \(6.43\pm 0.17\)** & \(107\pm 3\)** \\ \hline
**VII** & (Cys-Gln-His-Asn-Pro-Arg)\({}_{2}\) & \(4.75\pm 0.08\)** & \(145\pm 2\)** \\ \hline
**VIII** & (Cys-Gln-Arg-Phe-Ser-Arg)\({}_{2}\) & \(1.53\pm 0.01\)** & \(451\pm 2\)** \\ \hline \multirow{2}{*}{**X**} & Cys-Gln-His-Asn-Pro-Arg & & \\  & Cys-Leu-Val-Val-Tyr-Pro-Trp-Thr & & \\ \hline \multirow{2}{*}{**XI**} & Cys-Gln-Arg-Phe-Ser-Arg & & \\  & \multirow{2}{*}{\(1.13\pm 0.04\)**} & \multirow{2}{*}{\(612\pm 22\)**} \\  & Cys-Leu-Val-Val-Tyr-Pro-Trp-Thr & & \\ \hline \end{tabular} Data are the mean \(\pm\) SEM. **\(p<0.001\)**, compared to \"without inhibitor\". * Data published in [PERSON] et al. [24]. The vertical lines in the sequences of peptide X and peptide XI mean the disulfide bridges between Cys residues.  
\end{table} 
Table 2: Degradation rates (k) and half-lives (\(\mathbf{t_{1/2}}\)) of Met-enkephalin incubated with neutral endopeptidase (NEP) alone and with peptides. Figure 1: The stabilities of sialorphin, spinorphin and their analog (peptide **X**) in human plasma. The means of the % of remaining peptide with a standard deviation are presented. * data published in [PERSON] et al. [21]. were considered for in vivo testing in the current study. To investigate the anti-inflammatory potential of the inhibitor in the GI tract, we tested the effect of the heterodimer (peptide **X**) that demonstrated favorable properties in vitro in the degradation of Met-enkephalin by NEP, in the mouse model of TNBS-induced colitis. The intracolonic (_i.c._) instillation of TNBS caused reproducible colitis in mice, characterized by the loss of bodyweight (data not shown), increased macroscopic colon damage scores and augmented myeloperoxidase (MPO) activity. The administration of peptide **X** (1 mg/kg, i.p., B.I.D) significantly attenuated TNBS-induced colitis (Figure 2), as demonstrated by a decreased total macroscopic score (\(3.65\pm 0.34\) vs. \(4.90\pm 0.64\) for TNBS-treated mice, Figure 2B), colonic wall thickness (\(1.16\pm 0.07\) vs. \(1.45\pm 0.09\) for TNBS-treated mice, Figure 2C), and MPO activity (\(6.32\pm 1.03\) vs. \(33.57\pm 9.20\) for TNBS-treated mice, Figure 2D). Peptide **X** did not statistically significantly reduce the degree of ulcer scores compared to the TNBS-treated mice (\(0.93\pm 0.15\) vs. \(1.23\pm 0.25\) for TNBS-treated mice, Figure 2E). The shortening of the colon was more pronounced in TNBS-treated mice than in peptide **X**-treated animals (\(8.09\pm 0.16\) vs. \(6.66\pm 0.31\) for TNBS-treated mice, Figure 2F). Peptide **X** showed anti-inflammatory activity comparable to sialorphin [16] and its analog Pal-KKQHNPR [15] in terms of macroscopic score. The shortening of the colon was at the same level in Pal-KKQHNPR-treated animals as in peptide **X**-treated animals. Greater reduction in the activity of MPO was observed in peptide **X**-treated animals than in sialorphin [15] or its analog Pal-KKQHNPR [16] vs. TNBS-treated mice. Figure 2: Peptide **X** injected i.p., twice daily over 3 days at the dose of 1 mg/kg attenuated TNBS-induced colitis in mice.