2019 年 67 巻 9 号 p. 897-903
The word “theranostics,” a portmanteau word made by combining “therapeutics” and “diagnostics,” refers to a personalized medicine concept. Recently, the word, “radiotheranostics,” has also been used in nuclear medicine as a term that refer to the use of radioisotopes for combined imaging and therapy. For radiotheranostics, a diagnostic probe and a corresponding therapeutic probe can be prepared by introducing diagnostic and therapeutic radioisotopes into the same precursor. These diagnostic and therapeutic probes can be designed to show equivalent pharmacokinetics, which is important for radiotheranostics. As imaging can predict the absorbed radiation dose and thus the therapeutic and side effects, radiotheranostics can help achieve the goal of personalized medicine. In this review, I discuss the use of radiolabeled probes targeting bone metastases, sigma-1 receptor, and αVβ3 integrin for radiotheranostics.
“Molecular imaging” refers to techniques of noninvasively recognition and determination of molecular processes caused by changes and interactions in the living body. This type of imaging greatly contributes to clinical diagnosis in nuclear medicine, drug discovery, and life sciences. Radiolabeled probes are useful in the molecular imaging. Diagnostic radiolabeled probes are injected into patients and accumulate in target lesions where the radioisotopes (RIs) in the radiolabeled probes decay with emission of radiation. The imaging data for diagnosis can be obtained by gamma scintigraphy, single photon emission computed tomography (SPECT), or positron emission tomography (PET). Quantitative imaging data shows the distribution of the radiolabeled probes within the patients’ bodies and provides information for diagnosis of lesions, such as the expression levels of target molecules.
In recent years, the word “theranostics,” a portmanteau of the words “therapeutics” and “diagnostics,” has been used in the field of oncology. “Theranostics” describes agents or techniques that couple diagnostic imaging with targeted therapy. The system of theranostics consists of an imaging component to investigate the lesions before treatment and a corresponding therapeutic component to treat the same lesions. Recently, the word “radiotheranostics” has also been used in nuclear medicine as a term that refers to the use of RIs in combined imaging and therapy.1,2) Radioisotopes can effectively be used to establish a theranostics system. A diagnostic probe and a corresponding therapeutic probe with equivalent pharmacokinetics for radiotheranostics can be prepared by introducing a diagnostic RI and a therapeutic RI into the same precursor. The equivalent pharmacokinetics of the two probes makes radiotheranostics possible. Because quantitative analyses based on diagnostic imaging data can strictly predict the absorbed radiation dose in the lesion sites and normal tissues, the therapeutic effects in the lesion namely and side effects in normal tissues can also be predicted. Radiotheranostics can greatly contribute to the realization of personalized medicine because it enables appropriate selection of patients and optimization of therapeutic doses.
In this review, I discuss my previous studies aimed at developing radiolabeled probes for use in radiotheranostics.
Bone is a common tissue in which cancer metastases often appear because bone contains many growth factors and is a good environment for tumors, which is consistent with Paget’s “seed and soil” theory.3–5) Nuclear medicine imaging using radiopharmaceuticals has been used as the most sensitive diagnosis method of bone metastases because it can diagnose those bone metastases before detection by anatomical X-ray imaging.6–9) As radiopharmaceuticals for diagnoses of bone metastases, [99 mTc]Tc-bisphosphonate complexes, such as [99 mTc]Tc-methylenediphosphonate (MDP, Fig. 1A) and [99 mTc]Tc-hydroxymethylenediphosphonate (HMDP, Fig. 1B), have been used worldwide because of simple imaging methods using conventional gamma cameras or SPECT and the convenient physical characteristics of 99 mTc, such as suitable gamma energy for imaging, moderate half-life (6.01 h) for clinical usage, and in-house generator produced radionuclide.
Most patients with bone metastases suffer severe pain, which decreases their patients’ QOL.10) Thus, palliation of pain is important for these patients. To decrease cancer-induced bone pain, analgesic drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs) and opioids, have been used to treat patients according to the WHO three-step ladder. Unfortunately, however, those drugs are often insufficient to effectively alleviate incidental pain. Localized radiation therapy and surgical removal of lesion sites are effective methods for treatment of metastatic bone pain, but, the majority of patients with severe bone pain have multiple metastatic sites. It therefore often makes application of these therapeutics difficult. On the other hand, targeted radionuclide therapy using bone-seeking radiopharmaceuticals has gathered attention because of their effectiveness for treatment of multiple metastatic sites and few side effects.
A classic bone-seeking radiopharmaceutical as an element for palliation therapy is [89Sr]SrCl2 (Metastron®),11) and a newer one is [223Ra]RaCl2 (Xofigo®), which was recently approved as the first alpha-particle emitting bone-seeking radiopharmaceutical for castration-resistant prostate cancer patients with bone metastases in many countries because it showed great treatment outcomes in a worldwide phase III study.12) Another bone-seeking radiopharmaceutical, [153Sm]Sm-ethylenediaminetetramethylene phosphonic acid (EDTMP, Fig. 1C) complex, has also been approved by the Food and Drug Administration (FDA) as an agent for palliation of bone pain. These bone-seeking radiopharmaceuticals for palliation therapy and the above-mentioned [99 mTc]Tc-bisphosphonate complexes for bone imaging highly accumulate in metastatic bone lesions. Therefore, the accumulation of therapeutic bone-seeking radiopharmaceuticals in lesion sites can be predicted by using bone scintigraphy.13) The combination of the above-mentioned agents therapeutic bone seeking radiopharmaceuticals and [99 mTc]Tc-bisphosphonate complexes may be useful as “radiotheranostics,” however they do not always show the same complete biodistribution. [99 mTc]Tc-MDP and [99 mTc]Tc-HMDP have not yet been optimized from a chemical and pharmaceutical perspective because these complexes are not well-defined single-chemical species but rather mixtures of short-chain and long-chain oligomers.9,14) Moreover, the phosphonate groups in [99 mTc]Tc-MDP and [99 mTc]Tc-HMDP are used both as ligands for coordination and as carriers to hydroxyapatite (HA) in bone,15) which may decrease the inherent affinity of MDP and HMDP for bone. To overcome these drawbacks, a more logical design strategy has been proposed on the basis of the conjugation of a stable radiometal complex with a carrier molecule to bone. To develop better tracers for radiotheranostics, I designed, synthesized, and evaluated radiometal complex-conjugated bisphosphonate compounds.16–24) This drug design allows the ligand and carrier function to work independently and effectively.
In these radiometal complex-conjugated bisphosphonate compounds, [186Re]Re-mercaptoacetylglycylglycylglycine (MAG3)-conjugated bisphosphonate, [186Re]Re-MAG3-HBP (Fig. 1D), showed superior characteristics as a bone-seeking radiopharmaceutical.17) Rhenium, which has similar chemical properties to technetium because technetium and rhenium are both members of group 7 elements of the periodic table, has two useful radionuclides, 186Re (T1/2 = 3.72 d, β−max = 1.07 MeV, γ = 137 keV) and 188Re (T1/2 = 17.0 h, β−max = 2.12 MeV, γ = 155 keV), for radionuclide therapy.25) A corresponding 99 mTc-labeled compound, [99 mTc]Tc-MAG3-HBP (Fig. 1D), showed equivalent biodistribution to that of [186Re]Re-MAG3-HBP.19) In the therapeutic experiments, [186Re]Re-MAG3-HBP showed significant palliation effects and inhibition of tumor growth in a bone metastasis rat model20) (Fig. 2). Therefore, the combination of [99 mTc]Tc-MAG3-conjugated bisphosphonate for diagnosis and [186Re]Re-MAG3-HBP for therapy could be one of the best candidates for radiotheranostics of bone metastases.
Data are expressed as tumor volume relative to that on day of treatment (mean ± S.E.M. for 5–7 rats). Significance was determined using 1-way ANOVA followed by the Dunnett post hoc test (* p < 0.05 vs. no treatment). This research was originally published in JNM. Kazuma Ogawa et al. Therapeutic effects of a 186Re-complex-conjugated bisphosphonate for the palliation of metastatic bone pain in an animal model. J. Nucl. Med.; 48, 122–127, 2007 © SNMMI.
It has been reported that major non-collagenous bone proteins, such as osteopontin and bone sialoprotein, contain many acidic amino acids (aspartic acid (Asp) or glutamic acid (Glu)) in their amino acid sequences, whose offers HA binding ability,26–28) and polyglutamic acid peptides and polyaspartic acid peptides could be carriers of drugs to bone because of their high affinity for HA.29–31) To evaluate acidic amino acid peptides as a carriers of radiometals to bone lesions, I designed [67Ga]Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated L-Asp or D-Asp peptides ([67Ga]Ga-DOTA-Dn, Fig. 3A or [67Ga]Ga-DOTA-dn, Fig. 3B), which have varying peptide lengths (n = 2, 5, 8, 11, or 14).32,33) An attractive radionuclide for PET clinically is 68Ga (T1/2 = 67.7 min) because of its radiophysical properties.34) Since it is a 68Ge/68Ga generator-produced radionuclide, an on-site cyclotron is not required. Although I am interested in 68Ga, 67Ga (T1/2 = 3.26 d) was used in these acidic amino acid peptide carrier studies as an alternative radionuclide because of its long half-life. An excellent chelator for radiotheranostics is the DOTA ligand because it forms stable complexes with trivalent metals, such as 67/68Ga and 111In for imaging and 90Y, 177Lu, and 225Ac for therapy. These studies showed that the binding affinities to HA of [67Ga]Ga-DOTA-Dn and [67Ga]Ga-DOTA-dn increased with increasing length of the aspartate peptide. In biodistribution experiments of normal mice, [67Ga]Ga-DOTA-Dn and [67Ga]Ga-DOTA-dn (n = 11 or 14) selectively and highly accumulated in bone. These results indicate that not only bisphosphonate molecules but also acidic amino acid peptides are useful as carriers of radiometals to bone lesions.
Not only clinically used [99 mTc]Tc-bisphosphonate complexes but also my synthesized probes can accumulate in metastatic osteoblastic lesions well, however it is difficult to accumulate in metastatic osteolytic lesions, because the bone accumulation mechanism of these probes is based on a high affinity for HA.35) To resolve the problem, I designed, synthesized, and evaluated Ga-DOTA-D11-c(RGDfK)36) (Fig. 3C), which contains an aspartic acid peptide linker and c(RGDfK), could accumulate in primary tumors, osteoblastic bone metastases, and osteolytic bone metastases simultaneously because the aspartic acid peptide linker enables radioactivity localization in osteoblastic bone metastatic lesions and the arginine-glycine-aspartic acid (RGD) peptide site enables accumulation of radioactivity in primary tumor and osteolytic bone metastatic lesions.37–39) As expected, [67Ga]Ga-DOTA-D11-c(RGDfK) showed high binding affinity to both HA and αVβ3 integrin in vitro and was highly accumulated in both bone and tumor in U87MG tumor-bearing mice (Fig. 4). [68Ga]Ga-DOTA-D11-c(RGDfK) could be the first PET probe to enable simultaneous diagnosis of primary tumors, osteoblastic bone metastatic, and osteoclastic bone metastatic lesions. The combination of [68Ga]Ga-DOTA-D11-c(RGDfK) and a therapeutic radiometal, such as 90Y and 177Lu, labeled DOTA-D11-c(RGDfK), is expected to be useful for radiotheranostics.
Arrows indicate the site where tumor cells were injected. Reprinted with permission from Ref. 36. Copyright © 2015 American Chemical Society. (Color figure can be accessed in the online version.)
Sigma receptors were reported as a new subtype of the opioid receptor in 197640); subsequently, they were reclassified as original receptors with at least two subtypes: sigma-1; and sigma-2 41). The sigma-1 receptor comprises 223 amino acids and its molecular size is 25.3 kDa. This receptor is mainly located on the endoplasmic reticulum membrane in the cell and works to maintain cellular homeostasis as a molecular chaperone.42,43) Since the sigma-1 receptor is related to functions of the central nervous system, such as signal transduction, memory, recognition, and emotion, determining the expression level of the sigma-1 receptor should be useful for diagnosis of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s diseases, and amyotrophic lateral sclerosis.44–46) The sigma-1 receptor is also highly expressed in various cancer cells.47) Sigma-1 receptor agonists and antagonists have been candidates as drugs for the sigma-1 related diseases mentioned above.48,49) Therefore, imaging using radiolabeled probes to determine the sigma-1 receptor expression could become a companion diagnostic test of therapeutic agents targeting this receptor. In this review, I introduced some radiolabeled sigma-1 receptor targeting probes.
It has been found previously that vesamicol derivatives with high affinity for vesicular acetylcholine transporter also have high affinity for the sigma receptors. Vesamicol analogs containing iodine on the benzene ring were synthesized and evaluated.50,51) As a result, (+)-2-[4-(4-iodophenyl)piperidino] cyclohexanol [(+)-pIV, Fig. 3A] showed higher affinity for the sigma-1 receptor than that of (+)-pentazocine or haloperidol as sigma-1 receptor ligands.51)
123I has been widely used clinically as a radionuclide for SPECT. Although I am interested in 123I (t1/2 = 12.3 h)-labeled probes for SPECT imaging, 125I (t1/2 = 59.4 d) was used as an alternative radionuclide because of its long half-life. (+)-[125I]pIV was prepared via the iododestannylation reaction and evaluated by using DU-145 tumor-bearing mice. DU-145 is a human prostate cancer cell line overexpressing the sigma-1 receptor. Testing showed that (+)-[125I]pIV was highly accumulated in the tumor, and showed high the tumor/blood and tumor/muscle ratios of radioactivity. In blocking experiments, co-administration of an excess amount of a sigma-1 receptor ligand significantly decreased the tumor accumulation of (+)-[125I]pIV. This finding suggested that cancer accumulation of (+)-[125I]pIV was sigma-1 receptor specific.52) However, radioactivity accumulation in nonspecific tissues, especially the liver, was also high, and the radioactivity was retained.
Thus, the purpose of the next study was to create a radioiodine labeled sigma-1 receptor ligand with better kinetic characteristics. I hypothesized that the high accumulation of (+)-[125I]pIV in the liver was derived from its high lipophilicity. (+)-4-[1-(2-Hydroxycyclohexyl)piperidine-4-yl]-2-iodophenol [(+)-IV-OH, Fig. 3B] was designed, synthesized, and evaluated.53) Lower lipophilicity of (+)-[125I]IV-OH was confirmed by measuring the 1-octanol/water partition coefficient. The log P value of (+)-[125I]IV-OH (1.13 ± 0.01) was lower than that of (+)-[125I]pIV (2.08 ± 0.02). Although (+)-IV-OH had lower affinity than (+)-pIV in an in vitro binding assay, biodistribution experiments using DU-145 tumor-bearing mice showed that (+)-[125I]IV-OH had comparable high uptake at 1 h post-injection in tumor. In most tissues, (+)-[125I]pIV tended to be retained but (+)-[125I]IV-OH was cleared. As expected, the radioactivity in the liver after injection of (+)-[125I]IV-OH was significantly lower at all-time points relative to radioactivity of (+)-[125I]pIV.
Bromine-76 (76Br) is also a promising radioisotope because it is a positron emitter for PET, has a relatively long half-life (t1/2 = 16.1 h), and has chemical properties similar to those of iodine. Thus, I prepared and evaluated the radiobromine-labeled vesamicol analogs, (+)-[77Br]pBrV (Fig. 3A) and (+)-[77Br]BrV-OH (Fig. 5B), by using 77Br which has a longer half-life (57.0 h) instead of 76Br.54,55) These probes showed properties similar to those of the corresponding radioiodine-labeled probes.
Recently, receptor radionuclide therapy for somatostatin receptor-positive tumors has been performed clinically.56) I tried to apply the receptor radionuclide therapy to the sigma-1 receptor. 131I is a therapeutic radionuclides for clinical use, and [131I]NaI has been used for therapy of thyroid cancer patients. Thus, (+)-[131I]pIV was prepared and its therapeutic efficacy was evaluated in DU-145-bearing cancer mice. The results showed that (+)-[131I]pIV (7.4 MBq single administration) significantly inhibited tumor growth relative to tumor growth in the untreated control group.52)
α-Particle emitting radionuclides have gained much attention in radionuclide therapy because they have high linear energy transfer. Among alpha-emitting radionuclides, astatine-211 (211At) is a candidate for clinical use in the future. 211At has an appropriate half-life for alpha therapy (t1/2 = 7.21 h) and emits high energy of α-particles (5.9, 7.4 MeV). Astatine has no stable isotopes. Even 210At, which has the longest life among astatine isotopes, has a half-life of 8.1 h. For this reason, the properties of astatine have not yet been fully characterized. However, because astatine is a halogen, it exhibits chemical properties similar to those of iodine. Thus, (+)-[211At]pAtV (Fig. 5A) was prepared by a standard halogenation reaction using the corresponding tributylstannyl precursor and was evaluated.57) HPLC analysis of [211At]pAtV was performed and compared with (+)-pIV because of its lack of stable isotopes of astatine. The (+)-[211At]pAtV and (+)-pIV retention times in the HPLC analyses were consistent with similar log P values from partition coefficient experiments for (+)-[211At]pAtV and (+)-[125I]pIV (2.14 ± 0.02 and 2.08 ± 0.02, respectively). As the result of biodistribution experiments in DU-145 tumor-bearing mice by the double tracer method using (+)-[211At]pAtV and (+)-[125I]pIV, (+)-[211At]pAtV in DU-145 tumor-bearing mice showed biodistributions very similar to that of (+)-[125I]pIV. Tumor accumulations of both (+)-[211At]pAtV and (+)-[125I]pIV were significantly inhibited by co-injection of an excess amount of sigma-1 ligand, SA4503 (10 µmol/kg).
These results indicate that radiotheranostics via coupling (+)-[123I]pIV for SPECT, (+)-[76Br]pIV for PET, and (+)-[131I]pIV and (+)-[211At]pAtV for radionuclide therapy could be useful. Moreover, further studies for development of radiohalogen labeled sigma-1 receptor ligands are now on going.58)
RGD peptides possess affinity for αvβ3 integrin,59) a cell-adhesion molecule representative subtype present in heterodimeric transmembrane receptors that regulates angiogenesis.60) Since αvβ3 integrin is overexpressed on angiogenetic endothelial cells61) and some types of cancer cells,62) RGD peptides are used as carriers of RI to αvβ3 integrin in tumors.63) Radiolabeled RGD peptides have been investigated for not only imaging and determination of αvβ3 integrin expression but also for radionuclide therapy.64–67) However, 211At-labeled RGD peptides have never to date been reported. Thus, I aimed in this study to determine their radiotheranostics potential by coupling 211At-labeled and 123I-labeled RGD peptides.
For radioiodine labeling of RGD peptides, the simplest and most effective method is to introduce a radioiodine at the 3-position on the tyrosine residue in c(RGDyK) via the chloramine-T method, which typically results in high radiochemical yields.68) Although direct labeling of antibodies with radioiodine is also effective via the chloramine-T method,69) direct labeling of antibodies with 211At has been shown to be impractical.70) Thus, 211At might not be introduced into the tyrosine residue of c(RGDyK) via the normal chloramine-T method. As methods for 211At-labeling of antibodies, both N-succinimidyl [211At]astatobenzoate ([211At]SAB)-conjugated antibodies and N-succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE)-conjugated antibodies followed by 211At labeling via the astatodestannylation reaction have been reported.71,72) These methods, namely 211At labeling via conjugation of [211At]SAB or ATE with the ε-amino group of lysine residues or the N-terminus of amino acid sequences, are applicable to both proteins and peptides. Since the same strategy is applicable to RGD peptides, I tried to synthesize 125I- and 77Br-labeled RGD peptides via conjugation of N-succinimidyl [125I]iodobenzoate ([125I]SIB) or N-succinimidyl [77Br]bromobenzoate ([77Br]SBrB) with the ε-amino group of the lysine residue in the c(RGDfK) peptide as a preliminary study of 211At-labeled RGD peptide.68) Radiolabeling with both 125I and 77Br was successful, but the biodistribution of the labeled RGD peptides was not favorable owing to low tumor uptake and high uptake in the intestine, which can be due to the increased lipophilicity of these peptides. The molecular sizes of peptides are much smaller than those of antibodies, so the biodistribution of labeled RGD peptides must be drastically affected by introduction of molecules for radiolabeling. Next, labeled RGD peptides containing a hydrophilic linker between the c(RGDfK) peptide and a radiolabeled site were prepared to decrease the lipophilicity of the labeled RGD peptides. Although these labeled RGD peptides containing a hydrophilic linker improved their biodistribution, the improvement was not sufficient. Therefore, I assumed that another strategy for preparing 211At-labeled RGD peptides would be necessary. As a new method for 211At-labeling of RGD peptides, I tried to introduce a tributylstannyl group (for subsequent halogen labeling) into the phenylalanine residue of the RGD peptide. Actually, an 211At-labeled RGD peptide, [211At]c[RGDf(4-At)K], was prepared according to Chart 1.73) The 211At- and 125I-labeled RGD peptides, [211At]c[RGDf(4-At)K] and [125I]c[RGDf(4-I)K], showed very similar biodistributions based on their high stability in vivo and their high affinity for αvβ3 integrin. This result shows that their use in a radiotheranostics system is possible. Specifically, [123I]c[RGDf(4-I)K] SPECT imaging can predict the therapeutic effects and side effects of [211At]c[RGDf(4-At)K] α-targeted therapy as a radiotheranostics system.
(a) 30% HFIP (b) diphenylphosphoryl azide (DPPA), NaHCO3, Pd[P(C6H5)3]4, reflux, 48 h (c) trifluoroacetic acid (TFA), triisopropylsilane (TIS), H2O. (d) [(nBu)3Sn]2, tris(dibenzylideneacetone)dipalladium(0) (e) N-chlorosuccinimide (NCS), AcOH, [211At]At−
The potential of radiotheranostics has been receiving increased attention and has been successfully used in nuclear medicine and clinical oncology. Further development is anticipated, and it is hoped that my research findings will contribute to future progress in radiotheranostics.
This review of the author’s work was written by the author upon receiving the 2019 Pharmaceutical Society of Japan Award for Divisional Scientific Promotion.
The author declares no conflict of interest.
