# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 6 In Vivo Testing of d-AA-Containing HDPs
### Targeting Cancer Cells
Because solid tumors have high metabolic rates and poor vasculature, their local environment tends to be anaerobic and acidic because ATP hydrolysis results in the production of lactic acid and because of the poor washout of the acidic products [115]. A drug that can react to environmental pH would have the advantage of selectivity by becoming active in only abnormal pH conditions. Because the tested diastereomers of [PERSON] et al. [114] possessed a significant percentage of histidine residues, they tended to become positively-charged only in acidic environments; thus, the peptides were more prone to bind to membranes only in the vicinity of tumors and had a decreased probability of non-specific toxicity. The peptides were administered both intratumorally and systemically. For both routes, the peptides were effective at reducing both the tumor weight and the tumor blood vessel growth. One of the three tested diastereomers exhibited acute systemic toxicity at a concentration above 8 mg/kg. However, the other two had no observable toxicity at concentrations of up to 30 and 20 mg/kg. [PERSON] et al. [116] also demonstrated a strong systemic anti-metastatic cancer effect in which an all-d enantiomer AMP was conjugated to tumor blood vessel homing motifs consisting either Cys-Asn-Gly-Arg-Cys (CNGRC) or doubly cyclized RGD (RGD-4C). The peptide displayed no lytic activity against mammalian cells but induced apoptosis by disrupting the negatively-chargedmitochondrial membrane once internalized into the cytoplasm. The peptide conjugate significantly prolonged the survival of metastatic cancer-bearing mice. In addition to systemic administration, d-AA-containing HDPs have been tested for treating cancer in model animals by other administration routes. Although these processes did not provide coverage as thorough as for systemic administration, impressive results were nevertheless achieved and included multiple instances of complete tumor ablation, which essentially cured the animal of cancer. Additionally, peptides used in these studies were not administered systemically nor were there evidence that the studied peptides cannot be systemically administered or further modified to make them suitable for systemic use. [PERSON] et al. [105] tested a series of magainin analogues for their inhibitory activity against ovarian cancer when injected intraperitoneally. One of the analogues, an all-d enantiomer, proved to be as effective as doxorubicin at inhibiting tumor growth and was the most active of the analogues tested. Some side effects were observed in the form of local irritation, resulting in adhesions between the serosal surfaces of the abdominal organs; however the study did not discuss the severity of the side effects as compared to those of doxorubicin. It is unlikely that the HDP side effects were comparable to those of doxorubicin. [PERSON] et al. [44] tested a diastereomer on a prostate cancer xenograft-bearing mice. The diastereomer was able to completely arrest tumor growth when administered intratumorally, whereas its l-AA parent peptide was able to only slow tumor growth. In fact, the diastereomer was so effective that the tumor completely disappeared in 40% of the test animals. Figure S6 shows the atrophied tumor at the end of the test. [PERSON] et al. [95] conjugated the integrin homing domain (RGD) to the AMP tachymelin. The construct was able to induce tumor apoptosis via disruption of the negatively-charged mitochondrial membrane when injected intraperitoneally into a melanoma xenograft-bearing mice.