# The Metabolic Stability of Antimicrobial Peptides IK8 in Plasma and Liver S9
## 4 Discussion  
One of the major factors confining the clinical utility of AMPs resides in their instability, especially their susceptibility to the degradation by proteases [22]. In this paper, we aimed to elucidate the relationship between configurations of IK8 and its stability through analyzing metabolites. The results suggested that the stability of three configurations of IK8 in five species of plasma was IK8-L \(<\) IK8-2D \(<\) IK8-D, which was the same as that in liver S9. On the one hand, as incubating in the plasma of various species, four metabolites were produced for IK8-L, among which the maximum metabolite was M4 (RIKIRIK-NH\({}_{2}\)); one metabolite was produced for IK8-2D, which was also M4 (RIKIRIK-NH\({}_{2}\)). On the other hand, as for incubation in liver S9 of various species, nine metabolites were produced for IK8-L. Apart from dog liver S9, M4 (RIKIRIK-NH\({}_{2}\)) was the most abundant metabolite in liver S9 of the other four species; two metabolites were produced for IK8-2D. Further, a shared phenomenon was revealed that regardless of whether in plasma or liver S9, no metabolites of IK8-D were detected. Incidentally, our major work in this paper focused on identifying the metabolites with retention time after 16 min; therefore, the possible metabolites before this time remain to be determined. Interestingly, only peptide bond hydrolytic metabolites of IK8-L were found in plasma incubation solution (M1, M2, M3, and M4). However, in liver S9 incubation solution, except for peptide bond hydrolysis metabolites, metabolites with terminal amide bonds hydrolyzed to carboxyl groups were also found (M5, M6, and M7), which was employed to remove the carboxyteminal negative charge and particularly render a high net positive charge for enhanced antimicrobial activities. In view of these results, we believed that the introduction of D-amino acid plays a crucial role in promoting the stability of the polypeptide, which is consistent with [PERSON]'s findings [20]. Importantly, according to our published report [21], the substitution of peptide IK8-L with D-amino acids considerably strengthened antimicrobial activities. Moreover, among the three configuration peptides referred in this study, IK8-D demonstrated the most potent antimicrobial activities against five types of microorganisms (_S. epidermidis_, _S. aureus_, _E. coli_, _P. aeruginosa_, and _C. albicans_), with the lowest geometric mean of minimum inhibitory concentrations (MIC) value of 4.3 mg/L, while the values of IK8-L and IK8-2D under identical testing conditions were 23.4 mg/L and 12.5 mg/L, respectively. Figure 9: Relevant structure information of M7: (**a**) the structure of M7 is inferred as IRIKIRIK; (**b**) primary mass spectrum of M7 ([M + 2H]\({}^{2+}\)); (**c**) secondary mass spectrum of M7 ([M + 3H]\({}^{3+}\)); (**d**) the assignment of ion fragments in secondary mass spectrum. In terms of toxicity study, several years ago, we initially used 4% (by volume) rabbit blood to evaluate the hemolytic activities of these three IK8 forms [21], and the results suggested that they all exhibited minimal hemolysis against the highly sensitive erythrocytes at their corresponding MIC values, indicating prominent hemocompatibility. Meanwhile, only slight differences existed in hemolytic activities among three enantiomeric forms of IK8, with the HC\({}_{10}\) values (defined as the lowest peptide concentration that induced 10% or more hemolysis) of IK8-L, IK8-2D and IK8-D observed to be 2000, 1600, and 1750 mg/L, respectively. To further verify the safety, the cytotoxic test of three IK8 configurations on human keratinocytes (HaCat cells) was subsequently carried out via the MTT assay [23], and the obtained statistic revealed that viability percentages of human HaCaT cells after incubation with these three IK8 forms were all more than 90%, up to 500 mg/L, which significantly exceeded the maximal MIC of three IK8 forms toward numerous bacteria such as _S. epidermidis_ and _E. coli_.