Differences in the Renal Accumulation of Radiogallium-Labeled (Glu)14 Peptides Containing Different Optical Isomers of Glutamic Acid

Acidic amino acid peptides have a high affinity for bone. Previously, we demonstrated that radiogallium complex-conjugated oligo-acidic amino acids possess promising properties as bone-seeking radiopharmaceuticals. Here, to elucidate the effect of stereoisomers of Glu in Glu-containing peptides [(Glu)14] on their accumulation in the kidney, the biodistributions of [67Ga]Ga-N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid-conjugated (l-Glu)14 ([67Ga]Ga-HBED-CC-(l-Glu)14), [67Ga]Ga-HBED-CC-(d-Glu)14, [67Ga]Ga-HBED-CC-(dl-Glu)14, and [67Ga]Ga-HBED-CC-(d-Glu-l-Glu)7 were compared. Although the accumulation of these compounds in the bone was comparable, their kidney accumulation and retention were strikingly different, with [67Ga]Ga-HBED-CC-(d-Glu-l-Glu)7 exhibiting the lowest level of kidney accumulation among these compounds. Repeated d- and l-peptides may be a useful method for reducing renal accumulation in some cases.


Introduction
Bone scintigraphy using bone-seeking radiopharmaceuticals, such as [ 99m Tc]Tc-MDP, has been a longstanding and effective method for detecting bone metastases because of its high sensitivity [1][2][3].[ 99m Tc]Tc-MDP is a multinuclear complex that consists of a bisphosphonate compound with a remarkable affinity for bone, which is coupled with 99m Tc, a gamma ray-emitting radionuclide used for imaging.In the case of [ 99m Tc]Tc-MDP, 99m Tc seamlessly coordinates with the bisphosphonate, serving as a carrier that specifically targets bone metastases [4].
In our investigations, the bone accumulation patterns were comparable among [ 67   [21].In addition, a recent study reported by our group revealed that [ 67 Ga]Ga-HBED-CC-(L-Glu) 14 exhibited a higher kidney accumulation than [ 67 Ga]Ga-HBED-CC-(DL-Gla) 14 [22].That is to say, Glu peptides are more highly accumulated in the kidney than other acidic acid peptides.There could be differences in renal accumulation depending on the optical activity of Glu.However, the mechanism underlying these differences remains unclear.
In this study, to elucidate the effect of stereoisomers of Glu in radiogallium complexconjugated (Glu) n on their accumulation in the kidney and to find a strategy to reduce the renal accumulation in some peptide-based radiolabeled compounds, the biodistribution of [ 67 Ga]Ga-HBED-CC-(L-Glu) 14 , [ 67 Ga]Ga-HBED-CC-(D-Glu) 14 , [ 67 Ga]Ga-HBED-CC-(DL-Glu) 14 (which is composed of racemic Glu), and [ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu) 7 (which is composed of repeated D-Glu and L-Glu) was compared.The structure of them is shown in Figure 1.Although we were interested in 68 Ga (t 1/2 = 68 min) for PET imaging, 67 Ga (t 1/2 = 3.3 days) was used in this essential study as an alternative radionuclide because of its long half-life.
HBED-CC-(DL-Gla)14 [22].That is to say, Glu pept kidney than other acidic acid peptides.There cou depending on the optical activity of Glu.Howev ferences remains unclear.
In this study, to elucidate the effect of stereoi conjugated (Glu)n on their accumulation in the ki renal accumulation in some peptide-based radio of [ 67 Ga]Ga-HBED-CC-(L-Glu)14, [ 67 Ga]Ga-HBED Glu)14 (which is composed of racemic Glu), (which is composed of repeated D-Glu and L-Glu is shown in Figure 1.Although we were intereste 67 Ga (t1/2 = 3.3 days) was used in this essential stud of its long half-life.

Biodistribution Experiments
The accumulations of [ 67 Ga]Ga-HBED-CC-(L-Glu)14, [ 67 Ga]Ga-HBED-CC-(D-Glu)14, [ 67 Ga]Ga-HBED-CC-(DL-Glu)14, and [ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu)7 in the bones and kidneys of normal mice are reported in Figure 3, and detailed data pertaining to their biodistribution are displayed in Tables S1-S3 and a previous paper [22].All 67 Ga-labeled peptides accumulated and were retained in the bone at high levels, and their accumulation rates were similar (Figure 3a).In turn, the renal accumulation of the tracers was largely dependent on the optical isomer of the constituent amino acid, Glu (Figure 3b).In all [ 67 Ga]Ga-HBED-CC-(Glu)14 compounds, radioactivity in all tissues except the bones and kidneys was low (Tables S1-S3).

Biodistribution Experiments
The accumulations of [ 67 Ga]Ga-HBED-CC-(L-Glu) 14 , [ 67 Ga]Ga-HBED-CC-(D-Glu) 14 , [ 67 Ga]Ga-HBED-CC-(DL-Glu) 14 , and [ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu) 7 in the bones and kidneys of normal mice are reported in Figure 3, and detailed data pertaining to their biodistribution are displayed in Tables S1-S3 and a previous paper [22].All 67 Ga-labeled peptides accumulated and were retained in the bone at high levels, and their accumulation rates were similar (Figure 3a).In turn, the renal accumulation of the tracers was largely dependent on the optical isomer of the constituent amino acid, Glu (Figure 3b).In all [ 67 Ga]Ga-HBED-CC-(Glu) 14 compounds, radioactivity in all tissues except the bones and kidneys was low (Tables S1-S3).

Biodistribution Experiments
The accumulations of [ 67 Ga]Ga-HBED-CC-(L-Glu)14, [ 67 Ga]Ga-HBED-CC-(D-Glu)14, [ 67 Ga]Ga-HBED-CC-(DL-Glu)14, and [ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu)7 in the bones and kidneys of normal mice are reported in Figure 3, and detailed data pertaining to their biodistribution are displayed in Tables S1-S3 and a previous paper [22].All 67 Ga-labeled peptides accumulated and were retained in the bone at high levels, and their accumulation rates were similar (Figure 3a).In turn, the renal accumulation of the tracers was largely dependent on the optical isomer of the constituent amino acid, Glu (Figure 3b).In all [ 67 Ga]Ga-HBED-CC-(Glu)14 compounds, radioactivity in all tissues except the bones and kidneys was low (Tables S1-S3).

Discussion
Generally, peptides composed of D-type amino acids are more stable in vivo than peptides composed of L-type amino acids because peptidases recognize and metabolize the latter [26].Therefore, studies have often been performed to improve the stability of peptides by replacing L-amino acids with D-amino acids [27].In our previous study, repeated D-Asp, i.e., (D-Asp) n , was initially synthesized in an attempt to achieve a higher bone accumulation compared to (L-Asp) n as bone-targeting carriers.However, the accumulation of [ 67 Ga]Ga-DOTA-(D-Asp) n and [ 67 Ga]Ga-DOTA-(L-Asp) n in bone was hardly different because the two types of tracers showed extremely rapid clearance from the blood [21].Similarly, in the present study, [ 67 Ga]Ga-HBED-CC-(L-Glu) 14 , [ 67 Ga]Ga-HBED-CC-(D-Glu) 14 , [ 67 Ga]Ga-HBED-CC-(DL-Glu) 14 , and [ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu) 7 exhibited a similar bone accumulation (Figure 3) caused by rapid clearance and a similar affinity for hydroxyapatite as expected.
Several studies have investigated the differences in the renal accumulation of peptides composed of L-amino acids and D-amino acids.One study reported that the antimicrobial peptide composed of D-amino acid named danalexin resulted in prolonged retention in the rat kidney compared with the counterpart peptide, ranalexin, which is composed of Lamino acid [28].That article indicates that the observed prolonged retention is attributable to the increased stability of the peptide and its increased resistance to peptidolysis.In turn, the initial renal uptake of D-amino acid peptides compared with L-amino acid peptides tends to be lower.This difference is primarily caused by the altered stereochemistry of D-amino acids, which affects their recognition and transport by renal uptake mechanisms, such as specific transporters and receptors [29].In this study, [ 67 Ga]Ga-HBED-CC-(L-Glu) 14 exhibited a very high initial uptake into the kidney, with the accumulation gradually decreasing thereafter in a time-dependent manner.In contrast, [ 67 Ga]Ga-HBED-CC-(D-Glu) 14 showed a moderate uptake in the kidney and the accumulation was retained.These results are consistent with the above-mentioned descriptions provided in previous articles.
[ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu) 7 was synthesized as a peptide in which the D-Glu and L-Glu were alternately repeated.[ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu) 7 exhibited the lowest radioactivity in the kidney among all [ 67 Ga]Ga-HBED-CC-(Glu) 14 compounds, although the explanation for this observation remains unknown.It is known that poly-Lglutamic acid could be α-helical or have random coil peptide conformation depending on conditions [30].The presence of D-type amino acids induces conformational preferences not followed by peptides consisting of naturally abundant L-type amino acids [31].Therefore, we assume that repeating the D-Glu and L-Glu may have a very different conformation from oligo-L-glutamic acid, making it less susceptible to transporter recognition for kidney uptake.This is only speculation; further studies are needed to elucidate the mechanism.
Because [ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu) 7 showed an equivalent bone accumulation compared with the remaining [ 67 Ga]Ga-HBED-CC conjugated (Glu) 14 compounds, [ 67 Ga]Ga-HBED-CC-(D-Glu-L-Glu) 7 possessed the most ideal pharmacokinetics as a boneseeking radiopharmaceutical.Although the compounds assessed in this study targeted hydroxyapatite in the bone and, therefore, no difference in target directivity should exist between D-Glu and L-Glu, the D-and L-peptides may not be easily interchangeable when targeting receptors and enzymes, etc.This is the reason for the limited number of targets that can be used.If target directivity is maintained, repeated D-and L-peptides may be a useful method for reducing the renal accumulation of peptides.However, the reduction of renal uptake using repeated D-and L-peptides has only been confirmed for Glu-peptides in this study.Therefore, future studies are needed to determine whether this strategy can be applied to other peptides.
A typical method of reducing the renal accumulation of radiopharmaceuticals includes coinfusion of lysine and arginine [32].This method is used in clinical [ 177 Lu]Lu-DOTATATE for radiation protection of kidneys [33].Administration of the mixture of lysine and arginine is a relatively safe and effective method.However, the long time required to administer the mixture of lysine and arginine and its high dosage volume are problems.Another method to reduce renal radioactivity is introducing a cleavable linkage by enzymes on the renal brush border membrane [34,35].This method is an excellent scientific approach; however, the drug design is complex.The method suggested in this study may overcome these problems.However, as mentioned, it is expected to be used in limited cases.It may be an option for reducing renal accumulation of radiopharmaceuticals.
[ 67 Ga]Ga-DOTA-(L-Glu) 14 also showed a very high initial uptake in the kidney, and its accumulation was retained in a previous report [20].The difference in retention observed between [ 67 Ga]Ga-HBED-CC-(L-Glu) 14 and [ 67 Ga]Ga-DOTA-(L-Glu) 14 may have been depended on the difference in the lipophilicity of their complexes.In turn, the renal accumulation and retention of [ 67 Ga]Ga-DOTA-(L-Asp) 14 and [ 67 Ga]Ga-DOTA-(D-Asp) 14 were not high and were similar.The mechanisms of uptake of oligo-aspartic acid and oligo-glutamic acid may be different.

Animals
Experiments with animals were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University.The animal experimental protocols used were approved by the Committee on Animal Experimentation of Kanazawa University (AP-204165, 5 April 2023).The animals were housed with free access to food and water at 23 • C with a 12 h alternating light/dark schedule.