# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 6 In Vivo Testing of d-AA-Containing HDPs
### Targeting Cancer Cells  
[PERSON] et al. [13] performed an experiment demonstrating the high in vivo effectiveness of diastereomer HDPs. Metastatic cancer-bearing mice treated with a diastereomer consisting entirely of Leu and Lys survived for no less than 165 days, after which the experiment was terminated. For the entire duration of the test, no change in the HDP dosage was required to maintain negligible tumor growth and strikingly there was zero mortality in the treated animals for the entire duration of the experiment, which is in strike contrast to the 100% mortality after 75 days in the control group. Furthermore, there were no noteworthy side effects of the peptide, and blood tests showed that the peptide did not adversely affect the healthy control mice either. The diastereomer peptide was shown to act via membrane disruption although partial apoptotic activity could not be ruled out. The above results are significant for several reasons as follows:The benefit of long-term survival with nearly no side effects was demonstrated. The tumor was unable to develop resistance against the diastereomeric peptide tested by [PERSON] et al. [13] even after prolonged exposure. This finding is in line with a previous observation by [PERSON] et al. [2], who reported that no acquisition of resistance to HDPs by cancer has ever been documented. Furthermore, l-AA AMPs have been shown to induce resistance in bacteria when applied using graded doses for a prolonged period of time [8]. This finding may be due to the greater mutation potential of prokaryotes than that of eukaryotic cancer cells. However, a gradient exposure study performed with an all-d AMP in _E. coli_ and _S. aureus_ showed that resistance did not develop against the all-d AMP [9]. This result indicated that the inclusion of d-AAs not only stabilizes the peptide against serum proteases but also likely protects them against bacterial proteases. Thus, d-AA inclusion as a method of boosting HDP resistance against pathogen evasion is an avenue that cannot be overlooked. The diastereomeric peptide tested by [PERSON] et al. [13] was suitable for systemic application and was not conjugated to any homing motifs, nor did it depend on any delivery vesicles. The development of anticancer HDPs suited for systemic administration is important because this application remains the only route for combating metastatic cancer. Previous systemic administration of membrane lytic peptides almost invariably required a homing domain. Mostly, for in vivo experiments, anticancer HDPs were not administered systemically but instead the experiments used local or intratumoral injection and most significantly nearly all previous tests had little to no influence on metastatic cancer [13]. Therefore, this study provided a proof of concept that diastereomeric anticancer HDPs can be very effective as long-term metastatic cancer management agents. The results demonstrated in a practical setting that these diastereomeric HDPs are able to overcome many of the issues faced by HDPs in clinical applications such as short serum life [10], sensitivity to proteases [47], often inadequate selectivity and potentially damaging antigenicity [11; 12]. A similar achievement was presented by [PERSON] et al. [113] where two diastereomers composed solely of Leu and Lys (i.e., similar to the diastereomer also tested by [PERSON] et al. [13]) were tested for their capacity to inhibit metastatic melanoma in mice. One of the two tested peptides exhibited highly selective toxicity towards cancer cells with minimal side effects when systemically administered. No weakness or loss of weight was observed throughout the entire experiment and postmortem histopathological evaluation revealed that despite the intravenous injection, the diastereomer did not cause damage to any organs. [PERSON] et al. [114] tested three diastereomers in human prostate carcinoma xenograft-bearing mice. The tested diastereomers were rich in histidine, thus making them sensitive to environmental pH.