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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD

Created: ; Last Update: July 27, 2009.

Chemical name:64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7-Tris-acetic acid-10-maleimidoethylacetamide-ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG
Abbreviated name:64Cu-DOTA-pHLIP
Agent Category:Peptide
Target:Low extracellular pH (pHe)
Target Category:Other
Method of detection:Position emission tomography (PET)
Source of signal / contrast:64Cu
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No structure available.



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 (pHe) 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 pHe in tumors (3). The half-life of 64Cu is 12.74 hours.

Tumor microenvironment is characterized by low pHe (4, 5). Almost all solid tumors have a neutral to alkaline intracellular pH (pHi), but they develop an acidic pHe (known as the Warburg effect, Nobel Prize in 1931). The average pHe could be as low as 6.0 (6-8). A pH gradient (pHi > pHe) exists across the cell membrane in tumors. This gradient is contrary to that found in normal tissues, in which pHi is lower than pHe (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 pHe 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 pHe 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 pHe (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 pHe of tumors will therefore hinder weakly basic drugs from reaching their intracellular targets, thereby reducing cytotoxicity (15). Conversely, the acid pHe 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 pHe (13-15). On the contrary, the actions of the weak acids are enhanced by the low pHe. 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 pHe 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 pHe leads to the development of targeted therapeutic approaches (5, 6). Low pHe 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 pHe in prostate tumors (3). The pHLIP interacts with the surface of membranes as an unstructured peptide at neutral pHe, but at acidic pHe (<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 pHe in prostate tumor xenografts (3).



The synthesis of 64Cu-DOTA-pHLIP and 64Cu-DOTA-K-pHLIP was described in detail by Vāvere et al. (3). The sequence of pHLIP is ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG. K-pHLIP is a mutant version of pHLIP in which the key Asp residues of the transmembrane part of pHLIP are replaced by positively charged Lys residues (ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG). Because of the replacement, K-pHLIP has lost its pH-sensitive nature, and it is used as a control in the study. Both peptides were prepared with solid-phase peptide synthesis and purified with reverse-phase chromatography. The NH2 terminus of the peptides was first covalently conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7-Tris-acetic acid-10-maleimidoethylacetamide, a maleimide-containing derivative of DOTA. The conjugates were then radiolabeled by incubation with 64Cu at a ratio of 1:1 (µg:mCi) in 0.5 mol/L ammonium acetate (pH 5.5) for 30 min at 25°C. After purification, the labeled solutions were reported to be stable overnight without heating. The specific activity of 64Cu-DOTA-pHLIP and 64Cu-DOTA-K-pHLIP was 58.8 and 63.4 GBq/µmol (1,591 and 1,713 mCi/µmol), respectively. The radiochemical purity was >95% for both conjugates.

In Vitro Studies: Testing in Cells and Tissues


No references are currently available.

Animal Studies



Vāvere et al. analyzed the acute biodistribution of 64Cu-DOTA-pHLIP in PC-3 and LNCaP xenograft-bearing mice (3). The tumor xenografts were implanted in the right flank of male athymic mice. 64Cu-DOTA-pHLIP (~1.04 GBq (28 µCi)) was injected intravenously to mice with tumors that were 0.5–0.75 cm3 in volume. An additional group of PC-3–bearing mice (n = 5) received an equivalent mass of 64Cu-DOTA-K-pHLIP as a control. Animals were euthanized at different time points after injection (1, 4, 24, and 48 h; n = 4–5 mice). To investigate the tumor accumulation of 64Cu-DOTA-pHLIP after modulation of the tumor pH, a cohort of LNCaP-bearing mice (n = 8) was split into two groups, with the first group receiving 150 mmol/L bicarbonated water (pH 8.0) ad libitum for 7 days (modulated group), and the second group receiving regular drinking water (nonmodulated group). Analysis showed that there was no significant difference between PC-3 and LNCaP tumor models for the 64Cu-DOTA-pHLIP distribution in the blood and blood-rich organs such as liver, lung, heart, and spleen. In the PC-3 tumors, 64Cu-DOTA-pHLIP accumulation reached the maximum (2.78 ± 0.19% injected dose per gram (ID/g)) after 4 h. In the LNCaP tumors, 64Cu-DOTA-pHLIP accumulation reached the maximum (4.50 ± 1.71% ID/g) after 1 h and was still high with 3.23 ± 0.55% ID/g at 24 h and 1.74 ± 1.41% ID/g at 48 h after injection, respectively. The distribution of the control, 64Cu-DOTA-K-pHLIP, exhibited a pattern similar to that of the 64Cu-DOTA-pHLIP, although accumulation was lower in all tissues. However, the amount of 64Cu-DOTA-K-pHLIP was ~40% less than that of 64Cu-DOTA-pHLIP in the PC-3 tumors at 1 h after injection. As the 64Cu-DOTA-pHLIP was given to the LNCaP-bearing mice with or without modulation, no difference was observed between the two groups for its accumulation in the 17 collected organs except for the tumor and kidney. The uptake of 64Cu-DOTA-pHLIP was significantly higher (P = 0.005) in the nonmodulated tumors than in the modulated tumors (4.50 ± 1.71 versus 1.31 ± 0.60% ID/g, respectively). In the kidney, 64Cu-DOTA-pHLIP uptake was 5.20 ± 1.06 and 3.78 ± 0.29% ID/g (P < 0.005) in the nonmodulated and modulated mice, respectively.

Vāvere et al. performed small-animal PET imaging with 64Cu-DOTA-pHLIP on the two tumor models (PC-3, n = 4 mice; LNCaP, n = 10 mice) (3). 64Cu-DOTA-pHLIP (7.4 MBq (200 µCi)) was injected via tail vein. Images were evaluated by analyzing the standardized uptake value (SUV) of the tumors and muscles. In the PC-3 model, the tumor/muscle ratios derived from the SUVs at 1, 4, and 24 h were 1.45 ± 0.09, 2.67 ± 0.40, and 4.64 ± 1.08, respectively, showing a gradual increase in tumor uptake with concurrent washout from nontarget organs. The tumor/muscle ratios in the LNCaP tumors were significantly higher (P = 0.0001) than in the PC-3 tumors, with values of 3.44 ± 0.50, 5.56 ± 0.21, and 6.55 ± 1.98 for the 1, 4, and 24 h time points, respectively. 64Cu-DOTA-K-pHLIP did not exhibit any targeting ability (data not shown by the authors).

The differences in the 64Cu-DOTA-pHLIP PET imaging between LNCaP and PC-3 tumors, and between modulated and nonmodulated LNCaP tumors, were mirrored in the pH values determined with magnetic resonance spectroscopy (MRS). Based on the 31P magnetic resonance-observable chemical shift of 3-aminopropylphosphonate (3-APP), MRS showed that the LNCaP tumors had a more acidic average pHe (6.78 ± 0.29) than the PC-3 tumors (7.23 ± 0.10) (P = 0.039). The average pHe was also more acidic in the nonmodulated LNCaP tumors than in the modulated LNCaP tumors (6.62 ± 0.35 versus 6.94 ± 0.56, respectively), although the pHe difference did not reach statistical significance (P = 0.28) (3). However, MRS measures the volume-average pHe of tumors and is not a direct measure of the pH that causes pHLIP insertion. Vāvere et al. concluded that 64Cu-DOTA-pHLIP offers the possibility of designing a new class of noninvasive pH-selective PET imaging agents that will be useful for the imaging of a broad range of disease states (3).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


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