# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 6 In Vivo Testing of d-AA-Containing HDPs
### Targeting Bacterial Cells  
Compared to cancer treatment, diastereomer HDPs have had a longer history of usage in fighting bacterial infections, which included actual clinical applications such as the natural diastereomer gramicidin as discussed earlier. This section focuses on non-topical administration routes because the topical application of AMPs is well documented.  
[PERSON] et al. [46] compared a diastereomer AMP to its l-AA parent, which were both consisted of only Leu and Lys, for the treatment of mice infected with gentamicin-sensitive _P. aeruginosa_ and gentamicin-resistant _A. baumannii_ bacteria. To reduce the influence of the immune system, the mice were rendered transiently neutropenic by cyclophosphamide treatment. The pathogen-inoculated mice became weak and less active after just one day and death occurred between 2 to 4 days. The diastereomer but not its l-AA parent peptide cured the infection in nearly 80% of instances and was equally effective against both gentamicin-sensitive _P. aeruginosa_ and gentamicin-resistant _A. baumannii_ bacteria. Although gentamicin was slightly more effective than the diastereomer against the gentamicin-sensitive _P. aeruginosa_, it was completely useless against the gentamicin-resistant _A. baumannii_. Physical improvement after diastereomer administration was observed between 3 to 5 days after the bacterial challenge. Mortality was monitored for at least 10 days after treatment and all survivors recovered their physical activity. Both the diastereomer and all-L enantiomer peptides were tested for acute toxicity, and mortality occurred when the peptides were injected intravenously at 6 mg/kg dissolved in 0.25 mL buffer. Interestingly, when dissolved in 0.9 mL of buffer, no mortality occurred despite the use of the same 6 mg/kg dose of peptide. Blood chemistry tests revealed no abnormality when the peptide was used in safe doses.  
[PERSON] et al. [1] combined peptide library screening with structure-based approach to design a family of diastereomers targeted to a number of commonly faced pathogenic bacteria named ESKAPE pathogens. One of the designer diastereomers termed 17 BIPHE2 exhibited activity against all ESKAPE pathogens in vitro and could prevent staphylococcal biofilm formation in a catheter-associated infection mouse model. A hemolytic assay showed that the majority of the diastereomers had 50% hemolysis (HL50) at a concentration greater than 500 \(\upmu\)M, whereas the most potent 17 BIPHE2 showed an HL50 of 225 \(\upmu\)M; in contrast, the mean MIC value was just 3.1 \(\upmu\)M against the ESKAPE pathogens.  
[PERSON] et al. [117] tested 6 analogous all-d enantiomers against the extensively drug-resistant _Mycobacterium tuberculosis_ (XDR-TB). The study used human monocytic cells THP-1 as macrophage models in both in vitro and ex vivo settings and the results demonstrated the effectiveness of all six peptides at neutralizing the XDR-TB bacterial colony in vitro. Importantly, the peptides were effective at breaking down the heavily aggregated XDR-TB colonies. The author postulated but did not prove that the breakdown of the bacterial aggregates was due to the amphipatible peptides acting as detergents and reducing the hydrophobic interactions between the highly lipidic cell walls of the mycobacteria. The peptides were unable to eradicate intracellular mycobacteria, although the intracellular growth of the bacteria was substantially inhibited.