The physiological differences between normal and tumor tissues provide an opportunity for the development of novel diagnostic and therapeutic agents specifically targeting cancer cells. However, the acidic extracellular environment in tumors has not been properly exploited, probably due to a lack of compounds whose properties change dramatically in the range of pH 6.0–7.5. The significance of the current work is that it proposes an innovative and novel method to target tumors based on an intrinsic physiological property - the acidic extracellular environment, and thereby addresses an important problem in the diagnosis and monitoring or response to therapy of cancer. The method is based on the pH-selective interaction of the pHLIP peptide with cell membranes. In this study, we have demonstrated and validated a novel, pH-selective PET tracer - 64Cu-DOTA-pHLIP. This is the first time a peptide-based PET agent has been employed for the delineation of the pHe of tumors.
Our data show that 64
Cu-DOTA-pHLIP is stable in tumors. None of the animals in any of the studies showed any adverse effects due to the administration of any of the pHLIP constructs. The biodistribution and PET imaging data demonstrated retention of radioactivity in the tumors over 24 h. The tumor uptake of 64
Cu-DOTA-pHLIP correlates well with in vivo
fluorescence imaging studies, which demonstrated that Cy5.5-pHLIP and Alexa750-pHLIP stayed in tumors for several (>4) days (25
). This long retention might be explained by the fact that when the peptide is inserted into the cell membrane it is protected from attack by proteases, allowing it to accumulate in tumor tissue in significant amounts.
There are two main factors that might contribute to the accumulation of pHLIP peptides in non-tumor tissue. While at normal, physiological pH the probability of pHLIP insertion into the cell membrane is low, the peptide still interacts with the surface of the membrane through its hydrophobic motif. Although this produces a small background signal, it also allows for a longer blood circulation of pHLIP, enhancing the probability of delivering functional imaging moieties or therapeutic cargo to the site of disease. The background signal decreases with time, while the signal in tumors remains static or is enhanced. A second reason for unwanted background is the relative instability of the 64
Cu-DOTA chelation. 64
Cu has been shown to dissociate in vivo
from DOTA and DOTA-conjugates, undergoing subsequent metabolism and trans-chelation to superoxide dismutase and other proteins, resulting in increased accumulation in the blood and liver (27
). This could account, in part, for the main differences in biodistribution between Cy5-pHLIP and 64
Cu-DOTA-pHLIP, particularly the difference in liver uptake. In contrast to the PET results, the fluorescence data showed a very low uptake of Cy5.5-pHLIP by the liver (25
), since the NIR dyes were conjugated covalently to the N-terminus of the peptide. We therefore believe that the apparent liver uptake of 64
Cu-DOTA-pHLIP could be significantly decreased by optimizing the copper-chelating moiety. The cross-bridged cyclam chelator, CBTE2A, has demonstrated improved in vivo
stability and consequently a reduction in transchelation (35
), but requires elevated temperatures for copper complexation that may not compatible with the pHLIP peptide construct.
The control peptide, 64
Cu-DOTA-K-pHLIP, which had just two amino acid residues replaced, demonstrated ~40% less PC-3 tumor uptake as early as 1 hour post-injection; we did anticipated that the mutant peptide would have lower uptake in the PC-3 model (the less acidic tumor) than the parent pHLIP. Also, small-animal PET imaging experiments showed that control peptide did not accumulate in tumors, consistent with previous fluorescence studies (25
). Also we observed lowering of K-pHLIP level in blood and tissues, even though the equivalent mass of labeled K-pHLIP was administered and the data is normalized to the injected dose (%ID/g). We assume that rapid excretion of the mutant-pHLIP could be associated with its reduced affinity to the membrane at normal pH, in contrast to the parent pHLIP. Our thermodynamic studies indicated that pHLIP has high affinity to the membrane at normal pH (ΔGbinding
is about −7 kcal/mol at 37°C) (26
), which is probably associated with the prolongated peptide circulation in the blood. The pHLIP affinity to the cell membrane increases in an environment of low pH, at which point pHLIP inserts into the membrane and adopts a stable transmembrane configuration. In contrast to pHLIP, the mutant peptide K-pHLIP cannot insert into the cell membrane to form a transmembrane alpha-helix (25
), so it can be assumed that the tumor uptake we observed for 64
Cu-DOTA-K-pHLIP in the acute biodistribution studies must be due to the passive diffusion of the construct into the tumor interstitium. Also, as stated, we cannot exclude the possibility that the creation of 64
Cu bound to serum proteins by exchange with 64
Cu-DOTA-pHLIP contributes to the background, especially at early time points. Therefore, even though it is known that K-pHLIP does not target acidity the use of K-pHLIP may not be an appropriate control system.
The selectivity of the pHLIP peptide was demonstrated by the modulation of pHe
in LNCaP-bearing mice in which more acidic LNCaP tumors had greater uptake of 64
Cu-DOTA-pHLIP than the less-acidic (bicarb-modulated) LNCaP tumors. As can be seen from , it is clear that administering sodium bicarbonate significantly altered the uptake of the peptide only in the tumor and kidney. Unlike with k-pHLIP () there were no significant differences in the uptake by the skin, which cannot be explained at this time. The use of bicarbonate is known to have a profound effect on pHe
) For example, Raghunand et al, showed with 31
P-magnetic resonance spectroscopy (MRS) that the pHe
of MCF-7 human breast cancer xenografts can be effectively and significantly raised with sodium bicarbonate in drinking water.(12
) This was achieved with the mice drinking ad libitum
water containing 200 mM NaHCO3
for periods up to 90 days continuously, without any changes in subjective parameters and weight gain compared to control mice. In this current study the average pHe
in the non-modulated LnCaP tumors was more acidic than the average pHe
of the tumors in mice having received 7 days of bicarbonated water (6.62 ± 0.35 vs. 6.94 ± 0.56, respectively p = 0.28). This correlated well with the data from the acute biodistribution where 64
Cu-DOTA-pHLIP uptake in the non-modulated LnCaP tumors was significantly greater that the uptake in the tumors in mice having received 7 days of bicarbonated water (4.50 ± 1.71 vs. 1.31 ± 0.60, respectively, p = 0.005).
Apart from the liver and blood uptake, the quantitative PET data and images are in general agreement with the biodistribution studies and the previous reported fluorescence studies (25
), including uptake of the peptide by the kidney. The kidney has acidic regions and is a major site of catabolism of low-molecular-weight proteins. The kidney uptake can be reduced by providing mice with bicarbonate-buffered drinking water at pH 8.0 (12
), or drugs like acetozalomide, a carbonic anhydrase inhibitor that causes urinary alkalization. This reduction in kidney uptake with bicarbonate-buffered drinking water was confirmed in our own study presented in .
In summary, we have synthesized and evaluated the first generation of novel pHe
sensitive peptide PET agents for the delineation of low pHe in tumors. This is the first report of this novel class of PET imaging agents. Although the biokinetics of the agent are not optimal, additional strategies are currently under development to enhance tumor accumulation while reducing unwanted background uptake. This first generation agent offers the possibility of designing a new class of non-invasive pH-selective PET imaging agents that will be useful for the imaging of a broad range of disease states. Multimodal (diagnostic + therapeutic) pHLIP peptides with an N-terminus imaging label and C-terminus chemotherapeutic cargo (23
) would afford the opportunity to monitor drug delivery, providing a key tool in efforts to predict therapeutic outcome.