64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7-Tris-acetic acid-10-maleimidoethylacetamide-ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG

Excerpt

The pH low insertion peptide (pHLIP) is a peptide of 37 amino acids that inserts across the cell membrane as an α-helix when the extracellular pH (pH_e) is acidic (1-3). pHLIP labeled with ⁶⁴Cu (⁶⁴Cu-DOTA-pHLIP) was developed by Vāvere et al. as a positron emission tomography (PET) tracer for delineation of the low pH_e in tumors (3). The half-life of ⁶⁴Cu is 12.74 hours.

Tumor microenvironment is characterized by low pH_e (4, 5). Almost all solid tumors have a neutral to alkaline intracellular pH (pH_i), but they develop an acidic pH_e (known as the Warburg effect, Nobel Prize in 1931). The average pH_e could be as low as 6.0 (6-8). A pH gradient (pH_i > pH_e) exists across the cell membrane in tumors. This gradient is contrary to that found in normal tissues, in which pH_i is lower than pH_e (7.2–7.4) (6-8). Diffusion of the H⁺ ions along concentration gradients from tumors into adjacent normal tissues creates a peritumoral acid gradient (9). The mechanisms responsible for the low pH_e include anaerobic glycolysis because of hypoxia, aerobic glycolysis (the Warburg effect), increased metabolic CO₂ production associated with uncontrolled cell growth, and increased activity of ion pumps on the cell membrane (4, 6).

Low pH_e affects many aspects of tumor physiology. It is one of the driving forces in the clonal selection leading to invasive and metastatic diseases (10, 11). Rofstad et al. have shown that lowering culture pH to 6.8 results in a promotion of in vivo metastasis of treated tumor cells compared with controls (cultured at pH 7.4) after tail vein injection in mice (12). Exposure of tumor cells to an acidic environment leads to increased expression of various factors that contribute to tumor progression (11). Tumor cells are able to maintain a high proliferation rate in the acidic environment, whereas the peritumoral acid gradient limits immune response to tumor antigens and induces normal cell apoptosis, extracellular matrix degradation, and angiogenesis (6, 10). The passage of noncarrier-mediated weak drugs through the cell membranes is also influenced by the acidic pH_e (13-15). Typically, the drugs in an uncharged state (lipophilic form) pass more efficiently through the cell membranes. This leads to the hypothesis of ‘ion-trapping’ that weakly basic drugs will concentrate in more acidic compartments (13, 14). The acid pH_e of tumors will therefore hinder weakly basic drugs from reaching their intracellular targets, thereby reducing cytotoxicity (15). Conversely, the acid pH_e of tumors will improve uptake of weak acids into the relatively neutral intracellular space (16). The currently used chemotherapeutic drugs such as mitoxantrone, doxorubicin, daunorubicin, anthracyclines, anthraquinones and vinca alkaloids are all weak bases (pKa 5.5–6.8), while cyclophosphamide, 5-fluorouracil and chlorambucil are weak acids (pKa 7.8–8.8) (14). Both in vitro and in vivo studies have shown that the activities of those weak bases are inhibited by the low pH_e (13-15). On the contrary, the actions of the weak acids are enhanced by the low pH_e. The pH gradient in tumors exerts a protective effect upon the cells from weak-base drugs as well as acts to potentiate the action of weak acid drugs (16). Studies have consistently shown that selective tumor alkalinization in vivo is likely to result in an enhancement in the anti-tumor activity of weakly basic chemotherapeutic drugs (17, 18). Low pH_e has also been shown to impair the effectiveness of some drugs such as paclitaxel in that their chemical structures do not predict pH-dependent ionization (6). In addition, radiation therapies are known to be significantly less effective at the hypoxic and acidic regions of tumor (19).

An understanding of the mechanisms involved in tumor-specific low pH_e leads to the development of targeted therapeutic approaches (5, 6). Low pH_e is also considered a promising marker for tumor targeting detection (3, 7). Vāvere et al. have synthesized and described the use of ⁶⁴Cu-DOTA-pHLIP for delineation of low pH_e in prostate tumors (3). The pHLIP interacts with the surface of membranes as an unstructured peptide at neutral pH_e, but at acidic pH_e (<7.0) it inserts across the membrane and forms a stable transmembrane α-helix (1, 20-22). The pHLIP affinity for membranes at low pH (5.0) is 20 times higher than that at high pH (8.0). Studies by Zoonens et al. showed that the pHLIP could translocate cell-impermeable cargo molecules across a cell membrane and release them in the cytoplasm (23). The process is mediated by the formation of a transmembrane α-helix because of increased peptide hydrophobicity associated with the protonation of Asp residues at low pH (21, 22). Replacement of the two key Asp residues located in the transmembrane part of pHLIP with Lys or Asn leads to the loss of pH-sensitive membrane insertion (2). With in vivo PET imaging, Vāvere et al. have shown that ⁶⁴Cu-DOTA-pHLIP delineates low pH_e in prostate tumor xenografts (3).