Radio-labeled receptor-binding peptides: A new class of radiopharmaceuticals

Radio-Labeled Receptor-Binding Peptides: A New Class of Radiopharmaceuticals Otto C. Boerman, Wim J.G. Oyen, and Frans H.M. Corstens Radio-labeled receptor-binding peptides have emerged as an important class of radiopharmaceuticals. In vertebrates, these peptides transmit their biological function by binding to their specific receptor on the target cell surface. This specific receptor-binding property is exploited when the radio-labeled peptide is used as a radiopharmaceutical. The high-binding affinity for its receptor facilitates retention of the peptide in receptor-expressing tissues, whereas its relatively small size facilitates rapid clearance from the blood and other nontarget tissues. Receptor-binding peptides labeled w i t h ~-emitters can be used to visualize receptor-positive cells in vivo. In addition, when la-

beled w i t h 13- or ~-emitters, these peptides can be used for peptide-receptor radionuclide therapy. Various receptors are overexpressed on particular tumor types, and peptides binding to these receptors can be used to visualize tumor lesions scintigraphically. Furthermore, peptides binding receptors on granulocytes can be used to image infectious and inflammatory foci, whereas peptides binding receptors on activated thrombocytes can be used for thrombus imaging. Here, the peptide analogs that are under development for these applications are reviewed. Copyright 9 2000 by W,B. Saunders Company

URING THE PAST DECADE, radio-labeled D receptor-binding peptides have emerged as an important class of radiopharmaceuticals that

The concept of targeting receptor-expressing cells in vivo with their radio-labeled ligands has already proven its validity. For example, it has been shown that various tumors expressing somatostatin receptors can be targeted very effectively with somatostatin analogs.

promise to dramatically change the field of nuclear medicine. In vertebrates, such ligands can act as chemical messengers (eg, hormones, neurotransmitters, stimulators, inhibitors). Binding to the receptor on the target cell surface generally triggers the signal transduction mechanism of the target cell, and, thus, the biological effect of the ligand is transmitted to the target tissue. The specific receptorbinding property of the ligand can be exploited by labeling the ligand with a radionuclide and using the radiolabeled ligand as a radiopharmaceutical to image and/or treat tissues expressing a particular receptor. Theoretically, the high affinity of the ligand for the receptor facilitates retention of the radio-labeled ligand in receptor-expressing tissues, whereas its relative small size (eg, compared with antibodies) facilitates rapid clearance from the blood and other nontarget tissues. The concept of using radio-labeled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated an immense body of research in nuclear medicine. Receptor-binding peptides labeled with gamma emitters (Iodine- 123, Indium- 111, Technetium-99m) could facilitate the visualization of receptor-expressing tissues noninvasively, a technique referred to as peptide-receptor radionuclide imaging (PRRI). In addition, labeled with betaemitters (1-131, Yttrium-90, Rhenium-188, Re186) these peptides also have the potential to eradicate receptor-expressing tissues: an approach referred to as peptide-receptor radionuclide therapy (PRRT).

RECEPTOR TARGETING IN ONCOLOGY

Depending on their origin, tumors may overexpress various receptor types. Somatostatin (SST) receptors are expressed in the majority of neuroendocrine tumors and in neuroblastomas, some medullary thyroid carcinomas, prostate cancers, pheochromocytomas, and small-cell lung cancers (SCLCs). Vasoactive intestinal peptide (VIP) receptors are expressed in neuroendocrine tumors as well as in various other tumors (adenocarcinomas, lymphomas, astrocytomas, glioblastomas, and meningiomas). Autoradiographic studies have shown cholecystokinin (CCK)-B/gastrin receptors in almost all medullary thyroid carcinomas and in most small-cell lung cancers, some ovarian cancers, astrocytomas, and, potentially, a variety of adenocarcinomas.l Gastrin-releasing peptide (GRP) receptors have been shown to be expressed in prostate carcinomas. Neurotensin (NT) receptors are expressed in Ewing's sarcomas, meningiomas, astroFrom the Department of Nuclear Medicine, University Medical Center St. Radboud, Nijmegen, The Netherlands. Address reprint requests to Otto C. Boerman, PhD, Department of Nuclear Medicine, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Copyright 9 2000 by W.B. Saunders Company 0001-2998/00/3003-0004510. 00/0 doi: l O.1053/snuc.2000. 7441

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cytomas, and exocrine pancreatic tumors (ductal pancreatic adenocarcinomas). 2 The majority of breast cancers express calcitonin receptors? Peptides binding each of these receptors have been tested as a vehicle to target cancer lesions with radionuclides. Here, the potential of these peptides to serve as an imaging agent and/or as a vehicle for PRRT is reviewed. SOMATOSTATIN RECEPTOR TARGETING

One of the first peptides that was tested for its potential as a tumor-imaging radiopharmaceutical is SST. SST receptors are expressed in the brain, gut, neuroendocrine, most lymphatic tissues, kidney, prostate, and thyroid tissue. As a result, most tumors derived from these tissues express SST receptors. To date, 5 SST receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) have been identified and cloned. 4 SST is a peptide hormone produced by degradation of a precursor protein. Its main function is the inhibition of secretion of various hormones (eg, growth hormone, insulin, glucagon, gastrin) and it also acts as a neurotransmitter in the brain. SST is a 14-amino acid polypeptide with 1 disulfide bridge between the 2 cysteine residues (residue 3 and residue 14). The general inhibitory effect of SST on hormone production led to its development as a therapeutic agent for the treatment of endocrineactive tumors (eg, pituitary adenomas, gastroenteropancreatic tumors). However, the wild-type SST molecule turned out to be very susceptible to enzymatic degradation, and its short-lived activity in vivo limited its in vivo application. Therefore, an 8-amino acid analog of this tetradecapeptide designated as octreotide has been developed that was more resistant to enzymatic degradation in vivo. Octreotide binds with high affinity to SSTR2 and SSTRS, to a lesser extend to SST3, whereas it does not bind to SSTR1 and to SSTR4. To allow radioiodination of the peptide, the third amino acid residue, phenylalanine, was replaced by a tyrosine residue. This Tyr3-octreotide labeled with 1-123 was tested for scintigraphic visualization of neuroendocrine tumors. The limited availability of I-123 impeded widespread application of this agent. Therefore, octreotide was substituted N-terminally with DTPA to allow efficient labeling of the peptide with I n - l l l . The I n - l l l labeled DTPA-octreotide showed less hepatobiliary clearance than the 1-123 labeled analog. The diagnostic

accuracy of In-l 1l-labeled peptide to visualize tumor lesions has been determined in large series of oncology patients. Octreotide scintigraphy was mainly positive in patients with neuroendocrine tumors (pituitary tumors, insulinoma, endocrine pancreatic tumors, SCLC, carcinoids, neuroblastomas). In patients with gastroenteropancreatic (GEP) tumors the sensitivity of In- 111-DTPA-octreotide was between 80% and 90%, with highest sensitivity in glucagonomas (100%), vipomas (88%), gastrinomas (73%), and carcinoids (87%). 5-7 Imaging parameters such as the peptide dose administered, the duration of the acquisition, and the use of single photon emission computed tomography (SPECT) can affect the sensitivity of SST-receptor imaging and, therefore, conflicting imaging data can be found in the literature. Nevertheless, it is clear that SST-receptor scintigraphy can have an important impact on the management of patients with GEP tumors. Gibril et al 8 showed that octreotide scintigraphy altered management in 47% of patients. However, in a prospective study in 146 patients with Zollinger-Ellison syndrome 12% false-positive localizations were found. Therefore, experienced reading of the scans is required to prevent alteration of management based on falsely positive localizations.9 In- 111-DTPA-octreotide (Octreoscan, Mallinckrodt, Petten, The Netherlands) is now a registered radiopharmaceutical for the follow-up of patients with neuroendocrine tumors. In-111-DTPA-octreotide scintigraphy is also positive in the majority of patients with breast carcinoma and lymphoma. In 18 of 24 patients with known breast cancer tumor localizations were visualized. 1~A study by Chiti et alll suggested that octreotide imaging can even be used for breast cancer staging: in their study in 15 patients with T1-2N0-1 breast carcinoma, octreotide correctly identified 15 of 16 primary tumors and 5 of 6 tumor-positive axillary nodes. In patients with lymphoma, uptake of In- 111-DTPA-octreotide in the tumor is highly variable and usually relatively low. In a comparative study Leners et a112 showed that mean uptake in lymphoma lesions was only 10% of the mean uptake in GEP tumors. Sarda et a113 concluded from their study in 26 patients with lymphoma that In-111-DTPA-octreotide can not be used for initial staging of lymphoma because of its generally low tumor uptake.

RADIO-LABELED RECEPTOR-BINDING PEPTIDES

Somatostatin Receptor Radionuclide Therapy The efficient targeting of neuroendocrine tumors with octreotide for diagnostic purposes stimulated the development of a radio-labeled octreotide analog that could be used therapeutically. The lack of an octreotide analog that could be labeled stably with Y-90, prompted the group in Rotterdam to apply In- 111-DTPA-octreotide therapeutically. After binding to the tumor cell receptor, octreotide is internalized. ~4,15 Because In-Ill not only emits gamma arrays but also short-ranged Auger electrons that can be cytotoxic after internalization of the radio-labeled peptide, it was hypothesized that In-Ill-labeled DTPA-octreotide could be used therapeutically. Indeed, the number of intrahepatic metastases of CA20948 tumors in Lewis rats could be reduced by 2 injections (7 days apart) of high doses (370 MBq) of In-111-DTPA-octreotide.6 In Rotterdam a therapy study with In-lll-DTPAoctreotide is ongoing. Patients with neuroendocrine tumors receive multiple injections of 6.7 GBq In-111-DTPA-octreotide, up to a cumulative dose as high as 52 GBq (8 doses). No effects on white cells and renal function have been observed. So far, 21 patients with progressive disease have been treated with cumulative doses exceeding 18.5 GBq: 6 patients responded (decrease in tumor volume), whereas 8 other patients presented with stable disease. 6 Despite these encouraging results, the high costs of this therapeutic approach will limit its widespread application. Y-90-DTPA-octreotide has been studied in experimental models, however, this construct is not stable in vivo. Consequently, a considerable portion of the Y-90 is released from the peptide and subsequently incorporated in the mineral bone, causing excessive bone marrow toxicity, thus, limiting the activity dose that can be administered safely. Recently, a new somatostatin analog, 1,4,7,10tetraazacyclododecane-N,N',N",N'-tetraacetic acid(DOTA)-Tyr3-octreotide, also designated as DOTAToc, has been developed for radiotherapy studies. In this octreotide analog a tyrosine analog has been inserted as the third amino acid to maintain the hydrophilicity of the agent. The DTPA group is substituted for the macrocyclic chelator DOTA. 16,17 This chelator forms a stable complex with Y-90. In vitro, DOTA-Tyr3-octreotide had a higher binding affinity for the SSTR2 than DTPA-octreotide. In addition, DOTA-Tyr3-octreotide radiolabeled with either In-111 or Y-90 showed favorable binding

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(Kd 2.2 and 2.6 nmol/L, respectively) and biodistribution characteristics with high uptake and retention in receptor-positive tissues and tumor in rats. 15.16 In rats with small (<1 cm 2) subcutaneous (s.c.) CA20948 pancreatic tumors, 111 MBq Y-90-DOTATyr3-octreotide was as effective as 370 MBq In- 11 l-DOTA-Tyr3-octreotide, whereas the therapeutic efficacy of Y-90-DOTA-Tyr3-octreotide was superior for the treatment of large (>8 cm 2) tumors in this model. 17 Studies to determine the therapeutic efficacy of Y-90-DOTA-Tyr3-octreotide are ongoing at various institutions. First results of a study of 44 patients with advanced SST receptor-positive tumors have recently been published. 18 Patients received multiple doses of Y-90-DOTA-Tyr3octreotide at intervals of approximately 6 weeks. Cumulative doses up to 7.4 GBq/m z were well tolerated (National Cancer Institute [NCI] toxicity grade -<2). In the 5 patients that received cumulative doses exceeding 7.4 GBq/m z dose-limiting renal toxicity (grade 3 in 4 of 5 patients) and hematologic toxicity (grade 4 in 1 of 5 patients) was observed. 18To date, 20 of the 29 patients have shown stabilization of disease, 2 showed a partial remission, in 4 there was a reduction of tumor mass, and in 3 there was progression of tumor growth. In Italy, a similar activity dose--escalating study with Y-90-DOTA-Tyr3-octreotide is ongoing. 19 In this study Y-90-DOTA-Tyr3-octreotide is administered to patients with a favorable I n - l l l DOTA-Tyr3-octreotide biodistribution and dosimetry. Groups of 5 to 7 patients receive 3 equal doses (2 months apart) starting at 1.1 GBq (with increments of the dose of 0.4 GBq). After 28 patients, dose-limiting toxicity has not been reached yet. In 7 patients complete and partial responses have been observed. Recently, a multicenter horizontal and vertical activity dose escalation study to determine the maximum tolerated dose and the therapeutic efficacy of 9~ has been initiated.

Nephrotoxicity The relatively high retention of the radiolabel in the kidneys may cause nephrotoxicity. The kidneys are considered the first dose-limiting organ in radionuclide therapy with Y-90-DOTA-Tyr3-octreotide. Therefore, a series of studies aiming to determine the mechanism of kidney retention and to reduce the radiation dose to the kidneys has been performed. In general, proteins and peptides with a

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molecular weight lower than 60 kd are filtered in the glomeruli and subsequently reabsorbed in the cells of the proximal tubule. Under physiological conditions, all proteins and peptides are reabsorbed by the tubular ceils almost quantitatively by means of pinocytosis. On internalization, the labeled proteins/peptides are degraded in the lysosomes and the metabolites are mainly transferred back into the bloodstream. However, lysine substituted with In111-DOTA or Y-90-DOTA, the major radio-labeled metabolite of In- 111- or Y-90-DOTA-Tyr3-octreotide, cannot leave the lysosomes and will remain trapped in the proximal tubule cells. Proteins/peptides adhere to the luminal membrane of the tubular cell by electrostatic interaction: the positive charge on the protein/peptide bind to negatively charged receptors on the tubular cell membrane. 2~ Positively charged molecules can block these receptors and, thus, can reduce tubular reabsorption of proteins/peptides. In experimental animal models, administration of basic amino acids, particularly lysine, reduced kidney uptake of radio-labeled proteins/peptides. 21-23In rats, intravenous administration of 400 mg/kg lysine reduced the kidney uptake of In-lll-DTPA-octreotide by more than 50%. 23 Subsequent experiments in rats indicated that L-lysine interfered with receptor binding of In-lll-DTPA-octreotide, therefore, Dlysine may be preferred as an agent to reduce uptake and retention of the radionuclide in the kidney. 24 In Rotterdam, patients have been infused with various amino acid solutions during one of their therapeutic administrations of In-lll-octreotide, to determine the optimal regimen to reduce the radiation dose to the kidney. Intrapatient comparison of 4 different regimens revealed that infusion of a commercially available mixture of amino acids most effectively reduced the radiation dose to the kidneysY Dosimetric analysis of PET-images from baboons as well as from patients that received Y-86-DOTA-Tyr3-octreotide indicated that the dose to the kidneys can be lowered by 20% to 40% by infusion of a solution of basic amino acids. 2~,26 Reducing the kidney radiation dose could optimize radionuclide therapy with Y-90-DOTA-Tyr3-octreotide, because it will allow the administration of higher doses of radioactivity. Without intervention with basic amino acids, the radiation dose received by the kidneys was estimated to be 2.1 to 3.3 mGy/MBq 9~ The radiation dose to the kidneys that does not give rise to

BOERMAN, OYEN, AND CORSTENS

any (acute nor chronic) nephrotoxicity is still a matter of debate. Based on external beam radiation data, a dose of 23 Gy to the kidneys is considered to be a safe dose. However, the first experience with peptide-receptor radionuclide therapy indicated that the kidneys are more radioresistant when the dose is delivered by a radio-labeled peptide. In an activity dose--escalating study with Y-90-DOTATyr3-octreotide starting at 1.1 GBq per cycle (3 cycles per patients, 2 months apart) (5 patients per dose level, 0.4 GBq dose increments), Chinol et a119 encountered grade II chronic nephrotoxicity at the 1.47 GBq/cycle, suggesting that doses exceeding 30 to 50 Gy are tolerated.

Other SST Analogs Besides octreotide, a series of other SST receptorbinding peptides have been developed, and some of these have been used as vehicles for radionuclide targeting. A lanreotide analog that can be labeled with I n - l l l and Y-90 has been developed and is currently tested in patients with a wide range of tumors. It has been suggested that lanreotide, unlike octreotide, has a high affinity for the SSTR3 (Kd = 5.1 nmol/L) and SSTR4 (Kd = 3.8 nmol/L). 28 Because the subtype 3 receptor is also expressed on most adenocarcinomas, lanreotide scintigraphy and/or radionuclide therapy would have a broader field of application. 29 However, comparitive studies in SSTR2 receptor-positive tumor-bearing rats indicated that internalization as well as tumor localization of radio-labeled lanreotide was lower than that of the octreotide analogs. 3~ The tumor targeting capacity of In-lll-DOTAlanreotide was studied in patients with various types of cancer (13 neuroendocrine tumors, 6 intestinal adenocarcinomas, 4 lymphomas, 1 prostate cancer). Tumors were visualized in all patients, with favorable dosimetry (tumor dose: 0.21 to 5.8 mGy/MBq, kidney dose: 0.34 +_ 0.08 mGy/MBq). 31 Labeled with therapeutic doses of Y-90, this radiopharmaceutical is now tested in oncology patients having favorable dosimetry of I n - l l 1-DOTAlanreotide in the Multicenter Analysis of a Universal Receptor Imaging and Treatment Initiative: A European Study (MAURITIUS). One-GBq doses of Y-90-DOTA-lanreofide are administered with 4to 6-week intervals. 32 Six patients with thyroid carcinomas that did not accumulate radioiodine and had favorable dosimetry with the I n - i l l labeled analog (tumor dose > 10 mGy/MBq) received 2 to 5

RADIO-LABELED RECEPTOR-BINDING PEPTIDES

cycles of therapy. In 2 patients regressive disease was observed, in 2 patients stable disease was observed, and in 2 patients the disease progressed. 33 At Diatide Inc (Londonderry, NH), a cyclic somatostatin analog lacking a disulphide bridge (to avoid any possibility of reductive cleavage of the disulphide bridge during the labeling process) that can be labeled with Tc-99m was developed. This tracer, designated as Tc-99m depreotide (P829), binds with high affinity to SSTR2 (kd, 2.5 nmol/L), SSTR5 (kd, 2 nmol/L), and SSTR3 (kd, 1.5 nmol/L). In Lewis rats with AR42J tumors Tc-99m-P829 had higher tumor uptake compared with I n - l l l octreotide (4.9% injected dose (ID)/g vs. 2.9 %ID/g, respectively). 34 Tc-99m-P829 scintigraphy revealed visualization of breast cancer lesions in 7 of 8 patients and in 6 of 6 patients with melanoma. 35A study in patients with radiologic evidence of (malignant or nonmalignant) solitary pulmonary nodules indicated that Tc-99m-P829 scintigraphy correctly identified or excluded malignancy in 27 of 30 patients. 36 Based on these studies, Tc-99m-P829 (NeoTect, Diatide Inc) recently has been registered for noninvasive differential diagnosis of SST receptor-positive malignant lung lesions in patients with radiologic evidence of pulmonary masses. Potentially, this technique could replace transthoracic needle aspiration biopsies. Furthermore, a peptide designated as Vapreotide (RCI60) was developed. RC160 can be radiolabeled with I- 123, In- 111, Re- 188, or Tc-99m. 37-39In contrast to Octreotide, RC 160 has been reported to pass the blood-brain barrier and might, therefore, be used to image brain tumors. 4~ However, in vitro Tc-99m labeled RCI60 displayed a relatively low rate of internalization. In mice with SST-2 receptorpositive tumors only moderate tumor accumulation was found. 41 Tyr3-octreotide has been conjugated with hydrazinonicotinamide (HYNIC) and labeled with Tc-99m using ethylenediamine-N,N'-diacetic acid (EDDA) as a coligand. This preparation had optimal in vitro and in vivo characteristics. In vitro, this Tc-99m labeled octreotide analog had a high affinity for SST2 receptors (1 to 2.5 nmol/L). It was rapidly internalized by SST2 receptor-positive cells. In mice with AR42J tumors, high tumor uptake was found (9.7 %ID/g, 4 hours postinjection (p.i.) accompanied with low retention in the kidneys. 41 Initial patient studies revealed a superior image quality with higher tumor uptake compared with

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Octreoscan. 42 This agent certainly awaits further clinical characterization. VIP VIP can also be used for scintigraphic imaging of tumor lesions. VIP is a 28-amino acid peptide with a wide range of biological activities. VIP receptors are widely distributed throughout the gastrointestinal tract, as well as on various other cell types. In some tumors VIP competes for the same receptors as SST.43 More specifically, VIP also has affinity for the SSTR3 receptor that is also bound by P829 and lanreotide. 35 VIP receptors are expressed on adenocarcinomas, breast cancers, melanomas, neuroblastomas, and pancreatic carcinomas. 43 VIP labeled with 1-123 was tested as an imaging agent in patients with a wide variety of tumors. The first results with this peptide in oncology patients were highly encouraging. A series of 79 patients with colorectal carcinoma (n = 35), pancreatic adenocarcinoma (n = 20), gastric cancer (n = 8), carcinoid (n = 12), and insulinoma (n = 4) was studied. Overall sensitivity in these patients was 74%. 44 More recently, a total of 169 patients were investigated after injection of 1-123 labeled VIP. This extensive clinical study confirmed that 1-123-VIP could localize intestinal adenocarcinomas and endocrine tumors as well as their metastases. 45 Subsequently, the performance of the agent was studied in 80 patients with colorectal cancer revealing a high sensitivity (88%) and a high specificity (100%) for detection of recurrences of colorectal cancer lesions. 46 Similarly, the agent was studied in patients with adenocarcinoma of the exocrine pancreas. Overall sensitivity of the 1-123-VIP scan for detecting primary pancreatic adenocarcinomas was 58% (27 of 46). Liver metastases were imaged with 90% (29 of 32) sensitivity.47 Immediately after injection, the primary organ of accumulation is the lungs, reducing the sensitivity of this agent for detecting thoracic lesions. Furthermore, the preparation of the 1-123-VIP preparation is rather laborious and includes a reversed phase-high performance liquid chromatography (RP-HPLC) procedure to remove the unlabeled peptide from the preparation. Despite the high specific activity of the preparation 1-123-VIP induced a transient 10% drop in blood pressure.44 Recently, Thakur et a148 conjugated a Tc-99m chelating amino acid sequence (GAGG) to the C-terminus of VIP. This peptide, TP 3654, could be

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labeled efficiently with Tc-99m. In l 1 patients with various cancers (breast, osteosarcoma, colon, uterus) all known tumor lesions were imaged within 20 minutes postinjection. In 2 patients Tc-99m-TP 3654 disclosed lesions that were not visualized with computed tomography (CT), magnetic resonance imaging (MR/), or Tc-99m sestamibi. The agent partly cleared via the hepatobiliary route (20% in 24 hours) and gastrointestinal accumulation of the radiolabel was seen on the later images. BOMBESIN

Bombesin is a 14-amino acid neuropeptide that binds its receptor with high affinity. Specific receptors for bombesin, also referred to as GRP, have been identified on a variety of tumors, including lung, breast, prostate, and pancreas. At least 4 subtypes of bombesin receptors have been identified. On binding to the receptor, bombesin is rapidly internalized, and the intracellular effector system is triggered via a G-protein.49 Bombesin analogs that can be labeled with either I n - l l l or Tc-99m have been synthesized. Baidoo et al 5~ conjugated diaminedithiol (DADT) chelating groups to Lys3-bombesin and showed that these compounds can be labeled efficiently with Tc-99m, while preserving their receptor-binding affinity (Kd 3 to 4 nmol/L), Breeman et a151conjugated a series of bombesin analogs with DTPA to allow labeling with In- 111 and determined their in vitro characteristics (affinity, internalization, potency to induce prolactin secretion). Comparing an agonistic and an antagonistic analog, they found that despite similar receptor affinity, the agonist (In-lll-DTPA-Prol, Tyr4-bombesin) was internalized by bombesin receptor-positive cells, whereas the antagonist (In-111DTPA-Tyr5,Phe6-bombesin) was not. In rats with bombesin receptor-positive tumors, the agonist showed much higher tumor uptake, suggesting that internalization facilitates high uptake of bombesinreceptor binding ligands in the tumor. In rats with s.c. tumors, high and specific uptake of the I n - l l l labeled agonist was found in receptor-positive tissues (pancreas, gastrointestinal tract, CA20948 pancreatic tumor, and CC531 colon carcinoma). 52 At Resolution Inc. (Mississauga, ON) the bombesin analog, RP 527, was synthesized. This peptide contains a peptide N3S-core and can be labeled with Tc-99m. In severe combined immunodeficient (SCID) mice with s.c. PC3 human prostate tumors, the peptide preferentially accumulated in the tumor

(2.0 %ID/g at 1 hour p.i.), resulting in high tumor-to-muscle ratios (33.7 at 1 hour p.i.). 53 This peptide is currently evaluated in phase ldlI trials to determine its potential to image GRP-receptorexpressing tumors (eg, breast and prostate tumors). CHOLECYSTOKININ RECEPTOR BINDING LIGANDS

Gastrin and CCK are peptides found in the brain and the intestines, with multiple functions in the gastrointestinal tract mediated through CCK-B/ gastrin and CCK-A receptors. High concentrations of CCK-B/gastrin receptors are expressed in the midglandular region of the fundic mucosa of the stomach, whereas CCK-A receptors are found in the basal region of the antral and fundic mucosa. 54 CCK-B/gastrin receptors are frequently overexpressed in various human tumors, such as medullary thyroid carcinomas (92%), SCLCs (57%), astrocytomas (65%), and in stromal ovarian cancers (100%). Furthermore, gastroenteropancreatic tumors, breast, endometrial, and ovarian adenocarcinomas occassionally express CCK-B/gastrin receptors. 55 For tumor targeting, the CCK-A receptors appear to be less suitable, because these receptors are expressed only in a minority of a few human tumor types (eg, gastroenteropancreatic tumors, meningiomas, neuroblastomas). More importantly, CCK-A receptors are abundantly expressed in various normal tissues. Several groups have synthesized CCK peptide analogs that can be labeled with radionuclides. Reubi et a156 tested a series of DTPA-conjugated octapeptides. Although the sulfated CCK analogs had higher affinities for CCK-B receptors they selected a few unsulfated analogs for further studies because these showed less cross-reactivity with the CCK-A receptor. The receptor-binding sequence of CCK-B was synthesized and substituted with DTPA or DOTA. DTPACCK(26-33) had a 50% inhibition concentration (IC50) of 1.5 nmol/L for the CCK-B receptor. By substituting the aminoacids on position 28 and 31 with Nle (and using D-Asp on position 26), a peptide that was more resistant to proteolytic degradation was obtained. Recently, the in vitro and in vivo characteristics of the In-lll-DOTACCK(26-33) peptide were reported. 57 The pcptide was internalized by CCK-B receptor-positive CA20948 cells. In Lewis rats with CA20948 tumors specific uptake was found in the receptorexpressing stomach and tumor. Preliminary studies

RADIO-LABELED RECEPTOR-BINDING PEPTIDES

in patients showed receptor-specific uptake in the stomach and in tumor metastases. Behr et a158,59screened a series of CCK analogs, with the common COOH-terminal CCK-receptorbinding tetrapeptide sequence Phe-Asp-Met-Trp. In nude mice with medullary thyroid carcinoma xenografts, highest tumor uptake was obtained with gastrin analogs. In their experiments, these peptides not only had the highest affinity for the CCK-B receptor, but they also showed less crossreactivity with the CCK-A receptor (as expressed in liver, pancreas, and the intestines). CALCITONIN

Calcitonin receptors are overexpressed in the majority of breast cancer cell lines. At Diatide Inc, the calcitonin receptor binding peptide P1410 was synthesized. This peptide has a 4-amino acid chelating sequence, and the peptide can be labeled with Tc-99m as well as with Re- 188. The affinity of the Tc-99m-P1410 for the calcitonin receptor on T47D breast cancer cells was 2.5 nmol/L. In nude mice with s.c. MCF-7 breast cancer xenografts preferential accumulation of the radiolabel in the tumor was observed; tumor-to-muscle and tumor-to-blood ratios were 4.5 and 5.7 (90 min p.i.), respectively.6~ This agent is still in its very early phase and awaits further development and the calcitonin-receptor targeting potential in patients with breast cancer remains to be determined. VITRONECTIN RECEPTOR C(v133

Integrins form an important class of receptors involved in cell adhesion processes. Integrins are heterodimeric, transmembrane glycoproteins consisting of an et- and a 13-subunit. For tumor targeting one of the most interesting members of this family is the vitronectin receptor etvl33. The etv133 integrin is highly expressed in sprouting endothelium, and, therefore, is considered a marker for neoangiogenesis. The tripeptidic sequence ArgGly-Asp (RGD) is the primary site of recognition by av~ 3. Several groups have synthesized peptides containing the RGD sequence to target O~v133expressing tumors and/or (neo)endothelium. The etv~3 or vitronectin receptor is highly expressed on tumors, such as osteosarcomas, neuroblastomas, melanomas and lung, breast, prostate, and bladder carcinomas. 61 A decapeptide (etP2), containing 2 RGD sequences and a cystein residue to facilitate labeling with Tc-99m was synthesized and characterized

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in mice. In mice, the labeled peptide did not accumulate in any normal tissue and was rapidly excreted via the kidneys. 62 This agent was tested in patients with metastatic melanoma; 6 of 8 lymph node metastases and 11 of 14 other lesions were successfully imaged. 63 Haubner et a164 synthesized 2 cyclo-RGDFVcontaining peptides and tested them for their potential to image t~v133-receptor-positive tumors. Both peptides contained a tyrosine residue, allowing radioiodination using the iodogen method. In vitro competitive binding assays revealed an IC50 value of 2 nmol/L for both peptides. In mice with M21 melanoma or mammary carcinoma (MaCaF) the peptides cleared rapidly from the blood via the hepatobiliary route. Although these lipophilic peptides are probably not suitable for clinical application, these studies show that av133-integrin-binding peptides may be used to target tumors in vivo. RADIO-LABELED RECEPTOR BINDING PEPTIDES TO IMAGE INFECTION AND INFLAMMATION

Inflammation is the response of tissues to injury to bring serum molecules and cells of the immune system to the affected site. Infection is a condition caused by microorganisms (eg, bacteria, viruses, fungi) that may lead to an inflammatory response. The inflammatory response is characterized by locally increased blood supply, increased vascular permeability, enhanced transudation of plasma proteins, and enhanced influx of leukocytes. The agents currently used for infection and inflammation imaging (Gallium-67 citrate, In-Ill leukocytes, Tc-99m leukocytes, and others)--although useful in many cases--all have their own limitations. The ideal radiopharmaceutical for infection imaging should accumulate rapidly in infectious/ inflammatory foci and rapidly clear from noninflamed tissues. Theoretically, peptides with high affinity for receptors as expressed preferentially on infiltrating leukocytes, could meet such criteria. During the past 2 decades a wide variety of peptides that bind to receptors expressed on white blood cells have been tested for the detection of infection and/or inflammation. BACTERIAL CHEMOTACTIC PEPTIDES: FORMYL-MET-LEU-PHE

One of the first receptor-binding peptides that was tested for its ability to image infectious foci

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was the chemotactic peptide f-Met-Leu-Phe. This tripeptide that is N-terminally formylated is a chemotactic factor produced by bacteria. It binds to receptors on granulocytes and monocytes with high affinity (affinity constant [Ka] = 10 to 30 nmol/L). The first work on radiolabeled chemotactic peptides was reported almost 20 years ago. Zoghbi et a165and later McAfee et a166labeled f-Met-Leu-Phe and investigated its in vivo characteristics. They found that even low doses of peptide (0.5 lag), induced a transient granulocytopenia. In 1991, Fischman et a167 described the synthesis of 4 DTPA-derivatized chemotactic peptide analogs and their labeling with In-111. All peptides maintained biological activity and receptor-binding affinity. The peptides were tested in rats with Escherichia coli infections. All analogs localized in the focal infection within 1 hour after injection. In a comparative study in rabbits with E. coli infections, they showed that localization of infection using Tc-99m labeled f-Met-Leu-Phe was superior to that of In-Ill labeled leukocytes.68 They applied a high specific activity Tc-99m labeling method to reduce the induction of transient granulocytopenia. In a study in monkeys, they showed that a peptide dose as low as 10 ng/kg still had an effect on the peripheral leukocyte counts. 69 Several antagonists were developed to circumvent this undesirable biological activity of the radio-labeled chemotactic peptide. Most of them had lower uptake in the infectious focus, most likely owing to reduced affinity for the receptor. Pollak et al70 also described several analogs with reduced biological activity. However, in mice, uptake in the infection was relatively low. In summary, rapid imaging of infection and inflammation is feasible with radio-labeled chemotactic peptides, however, the undesired biological side effects of these peptides seem to impede further clinical development. ANTIMICROBIAL PEPTIDES

Neutrophil defensins (human neutrophil peptides [HNP]) are stored in the granules o f neutrophils. In addition to their direct antimicrobial activity, the peptides have chemoattractive activity for various monocytes and lymphocytes. It has been hypothesized that their cationic charge facilitates binding of these peptides to various microorganisms. HNP-1 was labeled with Tc-99m using a direct method by reducing the disulfide bridges of the molecule. With this agent, experimental thigh

infections in mice were visualized within 5 minutes postinjection, however, abscess-to-background ratios were relatively low. 71'72 In the peritoneal cavity of the infected mice, Tc-99m-HNP-1 bound to bacteria rather than to leukocytes, suggesting that this agent might be able to distinguish between bacterial infection and sterile inflammation. 72 CYTOKINES Interleukin-1

Interleukin (IL)-I binds receptors expressed mainly on granulocytes, monocytes, and lymphocytes with high affinity. Studies in mice with focal Staphylococcus aureus infections showed specific uptake of radioiodinated IL- 1 at the site of infection (infection-to-background ratios exceeded 40, 48 hours p.i.). 73Using IL-1 receptor-blocking antibodies, it could be shown that accumulation of the agent in the infectious foci was owing to binding to the IL- 1 type II receptor. Unfortunately, the biological effects (eg, hypotension, headache) of IL-1 even at very low doses (10 ng/kg) precluded clinical application of radiolabeled IL-1. Therefore, the naturally occurring IL-1 receptor antagonist (ILlra) was then tested as an imaging agent. This equally sized (17 kd) protein binds IL-1 receptors with similar high affinity but lacks any biological activity. In a comparative study in rabbits with focal E. coli infections, the uptake of radioiodinated IL-lra and IL-1 in the abscess was similar (0.01%ID/g, 20 h p.i.). Both IL-1 and IL-lra performed better as imaging agents than radioiodinated f-Met-Leu-Phe.74 Based on these promising results, 1-123-IL-lra was tested in patients with rheumatoid arthritis. In these patients, inflamed joints were nicely visualized, however, major retention of the radiolabel in the intestinal tract indicated that this agent can not be used to visualize infectious and inflammatory lesions in the abdomen. 75 Interleukin-2

Chronic inflammation--characterized by mononuclear cell infiltration--was successfully targeted with radiolabeled IL-2 by means of specific binding to IL-2 receptors, expressed on activated T lymphocytes. In a study in nonobese diabetic mice, Signore et al76 showed that the lymphocytic infiltration in the pancreas could be visualized with I-123 labeled IL-2 within 1 hour after injection. A method was developed that allowed the preparation of a Tc-99mIL-2 preparation with a high specific activity. 77

RADIO-LABELED RECEPTOR-BINDING PEPTIDES

Studies in patients with autoimmune disorders such as Hashimoto thyroiditis, Graves' disease, Crohn's disease, and coeliac disease showed localization of 1-123 or Tc-99m labeled IL-2 at the site of lymphocytic infiltration. 78 Therefore, radiolabeled IL-2 seems to be a suitable agent for in vivo targeting of mononuclear cell infiltration as present in autoimmune diseases. SOMATOSTATIN ANALOGS

Van Hagen et a179 investigated whether In-111 DTPA-octreotide localized in granulomatous tissue. Receptors for somatostatin are expressed on normal as well as on activated lymphocytes and macrophages. Total-body scintigraphy of patients with sarcoidosis, aspergillosis, tuberculosis, and Wegener's granulomatosis was performed after administration of In-111-DTPA-octreotide. Granuloma localizations were visualized in all patients studied. Furthermore, in patients with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and Henoch-Sch6nlein inflammatory sites were also positively imaged. 8~ In-111-DTPA-octreotide appears to allow visualization of inflammatory foci caused by granulomatous and other chronic inflammatory diseases. In contrast, In-111-DTPA-octreotide seemed not suitable to image acute infectious diseases because in a rat model no retention in S. aureus-induced abscesses was observed. 81 CHEMOKINES

Interleukin-8

IL-8 belongs to the CXC subfamily of the chemokines, or chemotactic cytokines, in which the first 2 cysteine residues are separated by 1 amino acid residue. IL-8 binds to receptors on neutrophils with high affinity (0.3 to 4 nmol/L). The potential of radio-labeled IL-8 to image inflammation was reported for the first time by Hay et a182 in 1994. The uptake of IL-8, radioiodinated via the chloramine-T method, in carrageenan-induced sterile inflammations in rats, peaked at 1 to 3 hours after injection and declined thereafter. Target-tobackground ratios did not exceed 2.5. In a pilot study in 8 patients these investigators showed that a 1-123-IL-8 could visualize inflammatory foci. 83 The labeling method appeared to have major effects on the in vivo biodistribution of radioiodinated IL-8. The in vivo characteristics of IL-8 labeled via

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the Bolton-Hunter method were clearly superior to IL-8 labeled via the iodogen method, despite similar in vitro cell binding characteristics. 84 In rabbits with focal E. coli infection, abscess-tocontralateral muscle uptake ratios exceeded 100 as early as 8 hours after injection. No significant accumulation in any nontarget sites was noticed. With the 1-123 labeled IL-8 preparation, the infection in the rabbits was visualized within 1 hour after injection. 84 Accumulation in the abscess, as determined by region of interest (ROI) analysis, was rapid and high (2.6 • 0.2% ID, 4 h p.i.), whereas 1-123-IL-8 rapidly cleared from all other tissues. Biodistribution studies (8 hours p.i.) showed that the abscess uptake was 0.057 --- 0.011%ID/g with abscess-to-contralateral muscle ratios of 115 • 23. The specific activity of this IL-8 preparation was relatively low; the imaging dose of 1-123-IL-8 (25 ~g/kg) caused a transient drop of peripheral leukocyte counts to 45%, followed up by a rebound leukocytosis (170% of preinjection level) during several hours. Recently, we were able to develop a Tc-99m labeled IL-8 preparation using HYNIC as a chelator. The biodistribution of Tc99m-HYNIC-IL-8 was studied in rabbits with E. coli infection. Accumulation of Tc-99m-HYNICIL-8 in the abscess was visible on the scintigrams as early as 1 hour p.i. The highest uptake was obtained at 8 hours p.i. (0.31 • 0.03 %ID/g) with abscess-to-muscle ratios increasing to 64 • 9 at 8 hours p.i. The radiolabel was excreted renally, with considerable retention in the kidneys (ca. 25% to 60% ID), depending on the HYNIC:IL-8 conjugation ratio. 85 Platelet Factor 4

Platelet factor 4 (PF4) is, like IL-8, a member of the CXC chemokines. Receptors for PF4 are expressed on various cells, such as polymorphonuclear leukocytes (PMNs) and monocytes. PF4 has been called the "body's heparin neutralizing agent. ''8~ At Diatide Inc. the peptide P483H was synthesized. This peptide consisted of the heparinbinding region of PF4 complexed with heparin-and a lysine-rich sequence for rapid renal clearance. In vitro, 73% of Tc-99m-P483H radioactivity was associated with leukocytes, predominantly with monocytes. In a rabbit model of infection, Tc-99m-P483H outperformed In-111 leukocytes, In- 111 DTPA, I- 131 albumin, Tc-99m nanocolloid,

BOERMAN, OYEN, AND CORSTENS

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Ga-67 citrate and Tc-99m glucoheptonate in terms of absolute uptake in the infection as well as infection-to-background ratios. The infections were clearly delineated as early as 4 hours after injection. No systemic side effects were observed: the transient neutropenia observed with IL-8 and f-Met-LeuPhe was not present after intravenous injection of P483H. 86 Tc-99m-P483H has been studied in patients to test its applicability as an imaging agent for scintigraphic detection of infection and inflammation with good results (82% sensitivity, 77% specificity).87 However, in some patients excessive thyroid uptake was observed, indicating the release of Tc-99m from the agent in vivo. THROMBUS IMAGING Efforts to image thrombi have been directed at the glycoprotein IIb/IIIa fibrinogen receptor (GPIIb/ IIIa). This GPIIb/IIIa receptor is present in low concentration on the cell membrane of circulating quiescent platelets. However, on stimulation and in active thrombosis, more than 80,000 receptors per platelet appear. Several investigators have synthesized GPIlb/IIIa receptor binding peptides that can be radiolabeled and tested their ability to image thrombi (aggregated activated platelets) in vivo. Theoretically, such a peptide could be used to image not only deep venous thrombi (DVT), but also pulmonary emboli (PE). Knight et a188were among the first to synthesize a series of GPIIb/IIIa receptor-binding peptides and showed the feasibility of this approach in a dog model of DVT. One of these Tc-99m labeled peptides, CYT-379, was tested in patients with DVT: images were positive in 3 of 7 patients who had a positive duplex Doppler examination in the lower extremities. 89 At Diatide Inc., a high affinity (inhibition of human platelet aggregation: ICs0 6.8 nmol/L) GPIIb/IIIa receptor-binding peptide, composed of 26 amino acids has been selected from a series of fibrinogen receptor-binding peptides. 9~ This dimeric peptide contains 2 RGD-mimetic sequences and 2 peptidic N3S cores to allow efficient labeling with Tc-99m. In dogs with femoral vein thrombi, the peptide provided positive images by 1 hour postinjection. 91 Further studies in dogs revealed that the peptide, designated as P280, is particularly suitable to detect acute and not chronic venous thrombosis. In the first clinical

evaluation, the agent provided positive visualization of thrombi in 8 of 9 patients with confirmed DVT within 1 hour of tracer injection. 92 In another clinical study, P280 also appeared to be capable to visualize pulmonary emboli. 93 P280 (AcuTect, Diatide Inc) has now been approved for scintigraphic imaging of acute venus thrombosis in the lower extremities of patients who have signs and symptoms of acute venous thrombosis. A recent study showed that the sensitivity and specificity of the Tc-99m-P280 scans in patients with acute DVT is optimal when images are acquired both early after injection (between 10 to 60 minutes p.i.) as well as at 120 minutes p.i. 94 At Dupont Pharmaceuticals (Billerica, MA) a series of GPIIb/IIIa receptor-binding peptides was synthesized and characterized. These peptides (cyclo(D-Val-NMeArg-Gly-Asp-Mamb-(hydrazinonicotinyl-5-(6-aminocaproic acid)))) were conjugated with HYNIC to allow efficient labeling with Tc-99m. Labeling the peptide with Tc-99m in the presence of Sn 2§ and tricine revealed multiple species of the HYNIC-conjugated peptide (Tc-99mHYNICtide(tricine)2) as analyzed by RP-HPLC. 9s,96 By adding a phosphine (TPPTS) as a coligand, a single Tc-99m labeled species, designated as Tc99m-HYNICtide-TPPTS-tricine was produced. 97 This agent very nicely imaged DVT in dogs within 15 minutes postinjection. ROI analysis of the images made 2 hours postinjection revealed thrombus-to-blood and thrombus-to-muscle ratios as high as 7:1 and 10:1, respectively. Currently, this agent is undergoing clinical evaluation both for DVT as well as for pulmonary emboli. Because endocarditis in its early phase is characterized by platelet aggregation, Oyen et al98 tested this agent in a canine model for endocarditis. Indeed, with the agent, the infected heart valves were very nicely visualized as early as 1 hour postinjection. Recently, Thakur et al99 tested a peptide containing the fibrin a-chain N-terminal sequence (Gly-ProArg-Pro-Pro) and a Tc-99m-chelating sequence (GAGG) for imaging blood clots in a rabbit model. In vitro, the peptide (Tc-99m-TP 850) bound to the C-terminal portion of the ~/-chain of fibrin species. In rabbits, experimental DVT as well as PE was delineated within 4 hours after injection. Clot-toblood ratios ranged from 1.2 to 12.0 as measured ex vivo.

RADIO-LABELED RECEPTOR-BINDING PEPTIDES

SUMMARY AND FUTURE PERSPECTIVES

Enhanced expression of peptide receptors in a pathological site provided the molecular basis for the successful use of radio-labeled peptides in nuclear medicine. Radio-labeled peptides have emerged as a new class of radiopharmaceuticals that allow imaging of biochemical processes at the molecular level. Radio-labeled peptides can be applicated to image tumor lesions (eg, SSTreceptor imaging), infectious and inflammatory foci (granulocyte-receptor imaging), and thrombosis (platelet-receptor imaging). A high affinity of the ligand for its receptor (at least in the nanomolar range) appears to be a prerequisite for successful application. Various labeling techniques have been developed that allow facile (1-step) high efficiency (>95%) radiolabeling of peptides without interfering with their receptor-binding properties. The development of macrocyclic chelates such as DOTA allow site-specific labeling of peptides not only with In-111 but also with Y-90, and has opened the way for therapeutic application of radio-labeled peptides. In addition, a series of chelates has been developed that allow efficient and site-specific labeling of peptides with Tc-99m and Re-188. Currently, the peptidic chelates with a N3S core are the most widely applicated chelates for Tc-99m labeling, but also the ternary ligand method using hydrazines can be applicated in the peptide field. In oncoiogy, a few radio-labeled peptides have been developed that allow excellent imaging of various tumor types. Multiple studies have shown that Octreoscan is an effective imaging agent that can be useful in the management of a patient with neuroendocrine tumors (detection of occult metastases, monitoring therapy, and others). Recently, the second SST receptor-binding imaging agent has been registered for clinical application. This agent, Tc-99m-P829 (NeoTect), has the advantage of using Tc-99m as a radionuclide. When it can be shown that NeoTect can provide images at least as good as those obtained with Octreoscan, it will further stimulate the clinical use of SST-receptor imaging. The first results obtained with a HYNICderivatized SST analog (Tyr3-octreotide-HYNICTc-99m) indicate that the in vivo characteristics of this new agent may be superior to those of Oclxeoscan. The excellent targeting properties of I n - l l l DTPA-octreotide has stimulated the development of an SST analog that can be used for peptide-

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receptor radionuclide therapy. Y-90-DOTA-Tyr3octreotide appears to be an excellent candidate for this application. Recent studies have shown that this agent provides higher accumulation in SST2 receptor-positive tumors than Octreoscan, and that Y-90-DOTA-Tyr3-octreotide can induce clinical responses in patients with neuroendocrine tumors. Clinical trials that are currently ongoing will provide important knowledge about the optimal dosing of this agent. In addition, the techniques to reduce renal toxicity appear to be applicable clinically. It is expected that an effective antitumor therapy for patients with SSTR-2-positive tumors can be developed based on Y-90-DOTA-Tyr3-octreotide. For other tumors, a series of other peptides has been characterized that could be used to develop new peptide-based radiopharmaceuticals for other tumor types both for diagnostic as well as for therapeutic application. In this respect, receptors for SSTR-3, CCK-B, and etv[33 appear to be very promising targets. However, for these targets the optimal peptidic construct has not yet been determined. In the near future, radio-labeled peptides will also have an impact in infection/inflammation imaging. In this field, a peptide-based radiopharmaceutical could provide an agent that is easy to prepare and that would avoid the need to handle blood (in contrast to radio-labeled leukocytes). Such an agent could also visualize infectious/ inflammatory sites within several hours after injection. The peptides targeting CXC receptors on neutrophils (PF4, IL-8) are especially promising candidates for this application. The Tc-99m labeling methods for these peptides need to be optimized. The radio-labeled peptides binding the GPIIb/ Ilia receptor on activated thrombocytes appear to be suitable for imaging DVT. However, the real challenge in thrombus imaging with radio-labeled peptides is the development of a peptide that can provide positive images of pulmonary emboli within hours. It remains to be established whether the current generation of thrombus-binding peptides will reveal such an agent. In summary, peptide-based radiopharmaceuticals such as In-lll-DTPA-octreotide, Tc-99mP829 and 1-123-VIP already have their impact on clinical management of patients. There is a wide range of radio-labeled peptides to come that will be used for radionuclide imaging as well as radionuclide therapy in the next decade.

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1. Reubi JC, Schaer JC, Waser B: Cholecystokinin (CCK)-A and CCK-B/gastrin receptors in human tumors. Cancer Res 57:1377-1386, 1997 2. Reubi JC, Waser B, Schaer JC, et al: Neurotensin receptors in human neoplasms: High incidence in Ewing's sarcomas. Int J Cancer 82:213-218, 1999 3. Gillespie MT, Thomas RJ, Pu ZY, et al: Calcitonin receptors, bone sialoprotein and osteopontin are expressed in primary breast cancers. Int J Cancer 73:812-815, 1997 4. Reubi JC, Schaer JC, Markwalder R, et al: Distribution of somatostatin receptors in normal and neoplastic human tissues: Recent advances and potential relevance. Yale J Biol Med 70:471-479, 1997 5. Krenning EP, Kwekkeboom DJ, Bakker WH, et al: Somatostatin receptor scintigraphy with [lllln-DTPA-D-Phel]- and [123I-Tyr3]-octreotide: The Rotterdam experience with more than 1000 patients. Eur J Nucl Med 20:716-713, 1993 6. Krenning EP, de Jong M, Kooij PP, et al: Radiolabelled somatostatin analogue(s) for peptide receptor scintigraphy and radionuclide therapy. Ann Oncol 10:$23-$29, 1999 (suppl 2) 7. Van Eijck CHJ, de Jong M, Breeman WAP, et al: Somatostatin receptor imaging and therapy of pancreatic endocrine tumors. Ann Oncol 10:S177-S181, 1999 (suppl 4) 8. Gibril F, Reynolds JC, Doppman JL, et al: Somatostatin receptor scintigraphy: Its sensitivity compared with that of other imaging methods in detecting primary and metastatic gastrinomas. A prospective study. Ann Intern Med 125:26-34, 1996 9. Gibril F, Reynolds JC, Chen CC, et al: Specificity of somatostatin receptor scintigraphy: A prospective study and effects of false-positive localizations on management in patients with gastrinornas. J Nucl Med 40:539-553, 1999 10. Bajc M, Ingvar C, Palmer J: Dynamic indium-lllpentetreotide scintigraphy in breast cancer. J Nucl Med 37:622666, 1996 11. Chiti A, Agresti R, Maffioli LS, et al: Breast cancer staging using technetium-99m sestamibi and indium-111 pentetreotide single-photon emission tomography. Eur J Nucl Med 24:192-196, 1997 12. Leners N, Jamar F, Fiasse R, et al: Indium-lllpentetreotide uptake in endocrine tumors and lymphoma. J Nucl Med 37:916-922, 1996 13. Sarda L, Duet M, Zini JM, et al: Indium-Ill pentetreotide scintigraphy in malignant lymphomas. Eur J Nucl Med 22:1105-1109, 1995 14. de Jong M, Bernard BF, De Bruin E, et al: Internalization of radiolabelled [DTPA] octreotide and [DOTA0,Tyr3] octreotide: Peptides for somatostatin receptor-targeted scintigraphy and radionuclide therapy. Nucl Med Commun 19:283-288, 1998 15. de Jong M, Breeman WA, Bakker WH, et al: Comparison of nlIn-labeled somatostatin analogues for tumor scintigraphy and radionuclide therapy. Cancer Res 58:437-441, 1998 16. de Jong M, Bakker WH, Krenning ER et al: Yttrium-90 and indium- 111 labelling, receptor binding and biodistribution of [DOTA,D-Phel,Tyr3]octreotide, a promising somatostatin analogue for radionuclide therapy. Eur J Nucl Med 24:368-371, 1997 17. de Jong M, Breeman WA, Bernard BF, et al: Tumour uptake of the radiolabeUed somatostatin analogue [DOTA, TYR3]octreotide is dependent on the peptide amount. Eur J Nucl Med 26:693-698, 1999

18. Otte A, Herrmann R, Heppeler A, et al: Yttrium-90 DOTATOC: First clinical results. Eur J Nucl Med 26:14391447, 1999 19. Chinol M, Paganelli G, Zoboli S, et al: 9~ treatment of patients with SSTR2 positive tumors: Preliminary results of phase I-II study. J Nucl Med 40:18P, 1999 (abstr) 20. MCgensen CE, Selling K: Studies on renal tubular protein reabsorption: Partial and near complete inhibition by certain aminoacids. Scand J Clin Lab Invest 37:477-486, 1977 21. Behr TM, Sharkey RM, Juweid ME, et al: Reduction of the renal uptake of radiolabeled monoclonal antibody fragments by cationic aminoacids and their derivatives. Cancer Res 55:3825-3834, 1995 22. Kobayashi H, Yoo TM, Kim IS, et al: L-lysine effectively blocks renal uptake of 1251- or 99mTc-labeled anti-Tac disulfidestabilized Fv fragment. Cancer Res 56:3788-3795, 1996 23. de Jong M, Rolleman EJ, Bernard BF, et al: Inhibition of renal uptake of indium-lll-DTPA-octreotide in vivo. J Nucl Med 37:1388-1392, 1996 24. Bernard BF, Krenning EP, Breeman WA, et al: D-lysine reduction of indium-ill octreotide and yttrium-90 octreotide renal uptake. J Nucl Med 38:1929-1933, 1997 25. Behr TM, Boerman OC: Report of the 13th meeting of the Internation research group on immunoscintigraphy and immunotherapy (1RIST). Eur J Nucl Med 26:950-952, 1999 26. Rrsch F, Herzog H, Stolz B, et al: Uptake kinetics of the somatostatin receptor ligand [86Y]DOTA-DPhel-Tyr3-octreotide ([86Y]SMT487) using positron emission tomography in non-human primates and calculation of radiation doses of the 90Y-labelled analogue. Eur J Nucl Med 26:358-366, 1999 27. Cremonesi M, Ferrari M, Zoboli S, et al: Biokinetics and dosimetry in patients administered with 111In-DOTA-Tyr(3)octreotide: Implications for internal radiotherapy with 90y_ DOTATOC. Eur J Nucl Med 26:877-886, 1999 28. Smith-Jones PM, Bischof C, Leimer M, et al: DOTAlanreotide: A novel somatostatin analog for tumor diagnosis and therapy. Endocrinology 140:5136-5148, 1999 29. Virgolini I: Mack Forster Award Lecture. Receptor nuclear medicine: Vasointestinal peptide and somatostatin receptor scintigraphy for diagnosis and treatment of tumour patients. Eur J Clin Invest 27:793-800, 1997 30. de Jong M, de Bruin E, Bernard B, et al: Internalization of In- 11 I-labeled somatostatin analogues for tumor scintigraph and radionuclide therapy. J Nucl Med 39:261P, 1998 (abstr) 31. Virgolini I, Szilvasi I, Kurtaran A, et al: Indium-lllDOTA-lanreotide: Biodistribution, safety and radiation absorbed dose in tumor patients. J Nucl Med 39:1928-1936, 1998 32. Leimer M, Kurtaran A, Smith-Jones P, et al: Response to treatment with yttrium 90-DOTA-lanreotide of a patient with metastatic gastrinoma. J Nucl Med 39:2090-2094, 1998 33. Flores J, Kurtaran A, Moncayo R, et al: Diagnostic and treatment of radioiodine-negative thyroid cancer using DOTAlanreotide (MAURITIUS). J Nucl Med 40:19P, 1999 34. Vallabhajosula S, Moyer BR, Lister-James J, et al: Preclinical evaluation of technetium-99m-labeled somatostatin receptor-binding peptides. J Nucl Med 37:1016-1022, 1996 35. Virgolini I, Leimer M, Handmaker H, et al: Somatostatin receptor subtype specificity and in vivo binding of a novel tumor tracer, 99mTc-P829. Cancer Res 58:1850-1859, 1998 36. Blum JE, Handmaker H, Rinne NA: The utility of a

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somatostatin-type receptor binding peptide radiopharmaceutical (P829) in the evaluation of solitary pulmonary nodules. Chest 115:224-232, 1999 37. Breeman WA, Holland LJ, Bakker WH, et al: Radioiodinated somatostatin analogue RC-160: Preparation, biological activity, in vivo application in rats and comparison with [ t23i_Tyr3]octreotide. Eur J Nucl Med 20:1089-1094, 1993 38. Breeman WA, Hofland LJ, van der Pluijm M, et al: A new radiolabelled somatostatin analogue [ 1111n-DTPA-D-Phe I ]RC160: Preparation, biological activity, receptor scintigraphy in rats and comparison with [ 11iln_DTPA_D.Phe I ]octreotide. Eur J Nucl Med 21:328-335, 1994 39. Guhlke S, Schaffland A, Zamora PO, et al: ISSRe- and 99mTc-MAG3 as prosthetic groups for labeling amines and peptides: Approaches with pre- and postconjugate labeling. Nucl Med Biol 25:621-631, 1998 40. Banks WA, Schally AV, Barrera CM, et al: Permeability of the murine blood brain barrier to some octapeptide analogues of somatostatin. Proc Natl Acad Sci U S A 10:431-438, 1990 41. Decristoforo C, Mather SJ: Technetium-99m somatostatin analogues: Effect of labelling methods and peptide sequence. Eur J Nucl Med 26:869-876, 1999 42. Decristoforo C, Moncayo R, Riccabona G, et al: Preclinical and initial patient studies of a new 99mTc-octreotide derivative based on the HYNIC-system in comparison with 11qn-DTPAoctreotide. Eur J Nucl Med 27:$87, 2000 43. Virgolini I, Yang Q, Li S, et al: Cross-competition between vasoactive intestinal peptide and somatostatin for binding to tumor cell membrane receptors. Cancer Res 54:690700, 1994 44. Virgolini I, Raderer M, Kurtaran A, et al: Vasoactive intestinal peptide-receptor imaging for the localization of intestinal adenocarcinomas and endocrine tumors. N Engl J Med 331:1116-1121, 1994 45. Virgolini 1, Raderer M, Kurtaran A, et al: 123I-vasoactive intestinal peptide (VIP) receptor scanning: Update of imaging results in patients with adenocarcinomas and endocrine tumors of the gastrointestinal tract. Nucl Med Biol 23:685-692, 1996 46. Raderer M, Kurtaran A, Hejna M, et al: 123I-labelled vasoactive intestinal peptide receptor scintigraphy in patients with colorectal cancer. Br J Cancer 78:1-5, 1998 47. Raderer M, Kurtaran A, Yang Q, et al: Iodine-123vasoactive intestinal peptide receptor scanning in patients with pancreatic cancer. J Nucl Med 39:1570-1575, 1998 48. Thakur ML, Marcus CS, Saeed S, et al: 99mTc-labeled vasoactive intestinal peptide analog for rapid localization of tumors in humans. J Nucl Med 41 : 107-110, 2000 49. Reile H, Armatis PE, Schally AV: Characterization of high-affinity receptors for bombesin/gastrin releasing peptide on the human prostate cancer cell lines PC-3 and DU-145: Internalization of receptor bound 1251-(Tyr4) bombesin by tumor cells. Prostate 25:29-38, 1994 50. Baidoo KE, Lin KS, Zhan Y, et al: Design, synthesis, and initial evaluation of high-affinity technetium bombesin analogues. Bioconjug Chem 9:218-225, 1998 51. Breeman WA, De Jong M, Bernard BF, et al: Pre-clinical evaluation of [IIqn-DTPA-Pro(1), Tyr(4)]bombesin, a new radioligand for bombesin-receptor scintigraphy. Int J Cancer 83:657-663, 1999 52. Breeman WA, Hofland LJ, de Jong M, et al: Evaluation of

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radiolabelled bombesin analogues for receptor-targeted scintigraphy and radiotherapy. Int J Cancer 81:658-665, 1999 53. Hoffman TJ, Simpson SD, Smith CJ, et al: Accumulation and retention of Tc-99m RP527 by GRP receptor expressing tumors in SCID mice. J Nucl Med 40:104P, 1999 54. Reubi JC, Waser B, Laderach U, et al: Localization of cholecystokinin A and cholecystokinin B-gastrin receptors in the human stomach. Gastroenterology 112:1197-1205, 1997 55. Reubi JC, Schaer JC, Waser B: Cholecystokinin (CCK)A and CCK-B/gastrin receptors in human tumors. Cancer Res 57:1377-1386, 1997 56. Reubi JC, Waser B, Schaer JC, et al: Unsulfated DTPAand DOTA-CCK analogs as specific high-affinity ligands for CCK-B receptor-expressing human and rat tissues in vitro and in vivo. Eur J Nucl Med 25:481-490, 1998 57. de Jong M, Bakker WH, Bernard BF, et al: Preclinical and initial clinical evaluation of l lqn-labeled nonsulfated CCK8 analog: A peptide for CCK-B receptor-targeted scintigraphy and radionuclide therapy. J Nucl Med 40:2081-2087, 1999 58. Behr TM, Behe M, Angerstein C, et al: CholecystokininB/gastrin receptor binding peptides: Preclinical development and evaluation of their diagnostic and therapeutic potential. Clin Cancer Res 5:3124S-3138S, 1999 (suppl) 59. Behr TM, Jenner N, Behe M, et al: Radiolabeled peptides for targeting cholecystokinin-B/gastrin receptor-expressing tumors. J Nucl Med 40:1029-1044, 1999 60. Nelson CA, Moyer BR, Pearson DA, et al: A peptide analog of calcitonin labeled with Tc-99m targets MCF-7 human breast cancer xenografts in nude mice. J Nucl Med 40:103P, 1999 61. Lafrenie RM, Gallo S, Podor TJ, et al: The relative roles of vitronectin receptor, E-selectin and alpha 4 beta 1 in cancer cell adhesion to interleukin-l-treated endothelial cells. Eur J Cancer 30A:2151-2158, 1994 62. Costopoulos B, Varvarigou AD, Sivolapenko G, et al: Radiochemical and radiobiological evaluation of a synthetic peptide labelled with 99roTe [abstr]. In: Proceedings of the 8th ISORBE Congress, 24-27 May 1997. Castel Gandolfo, Rome, Italy 63. Sivolapenko GB, Skarlos D, Pectasides D, et al: Imaging of metastatic melanoma utilising a technetium-99m labelled RGD-containing synthetic peptide. Eur J Nucl Med 25:13831389, 1998 64. Haubner R, Wester HJ, Reuning U, et al: Radiolabeled alpha(v)beta3 integrin antagonists: A new class of tracers for tumor targeting. J Nucl Med 40:1061-1071, 1999 65. Zoghbi S, Thakur M, Gottschalk A: Selective cell labelling: A potential radioactive agent for labeling of human neutrophils. J Nucl Med 22:32P, 1981 (abstr) 66. McAfee JG, Subramanian G, Gagne G: Technique of leukocyte harvesting and labeling: Problems and perspectives. Semin Nucl Med 14:83-106, 1984 67. Fischman AJ, Pike MC, Kroon D, et al: Imaging focal sites of bacterial infection in rats with indium-l l l-labeled chemotactic peptide analogs. J Nucl Med 32:483-491, 1991 68. Babich JW, Graham W, Barrow SA, et al: Technetium99m-labeled chemotactic peptides: Comparison with indium-11 Ilabeled white blood cells for localizing acute bacterial infection in the rabbit. J Nucl Med 34:2176-2181, 1993 69. Fischman AJ, Rauh D, Solomon H, et al: In vivo

208

bioactivity and biodistribution of chemotactic peptide analogs in nonhuman primates. J Nucl Med 34:2130-2134, 1993 70. Pollak A, Goodbody AE, Ballinger JR, et al: Imaging inflammation with Tc-99m labelled chemotactic peptides: Analogues with reduced neutropenia. Nucl Med Cornmun 17:132139, 1996 71. Welling MM, Hiemstra PS, van den Barselaar MT, et al: Antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation. J Clin Invest 102:1583-1590, 1998 72. Welling MM, Nibbering PH, Paulusma-Annema A, et al: Imaging of bacterial infections with 99mTc-labeled human neutrophil peptide-1. J Nucl Med 40:2073-2080, 1999 73. van der Laken CJ, Boerman OC, Oyen WJG, et al: Specific targeting of infectious foci with radioiodinated human recombinant interleukin-1 in an experimental model. Eur J Nucl Med 22:1249-1255, 1995 74. van der Laken CJ, Boerman OC, Oyen WJG, et al: Imaging of infection in rabbits with radioiodinated interleukin-1 (tx and 13), its receptor antagonist and a chemotactic peptide: A comparative study. Eur J Nucl Med 25:347-352, 1998 75. Barrera P, van der Laken CJ, Boerman OC, et al: Localization of 123I-IL-ra in affected joints in patients with rheumatic arthritis. 1999 (in press) 76. Signore A, Chianelli M, Toscano A, et al: A radiopharmaceutical for imaging areas of lymphocytic infiltration, 1231interleukin-2 labeling procedure and animal studies. Nucl Med Commun 13:713-722, 1992 77. Chianelli M, Signore A, Fritzberg AR, et al: The development of technetium-99m-labelled interleukin-2: A new radiopharmaceutical for the in vivo detection of mononuclear cell infiltrates in immune-mediated diseases. Nucl Med Bio124:579586, 1997 78. SignoreA: Interleukin-2 scintigraphy: An overview. Nucl Med Commun 20:938, 1999 79. van Hagen PM, Breeman WAP, Reubi JC, et al: Visualization of the thymus by substance P receptor scintigraphy in man. Eur J Nucl Med 23:1508-1513, 1996 80. van Hagen PM, Ligtenaner-Kaligis EGR, ten Bokum AMC, et al: Somatostatin receptor expression in clinical immunology. J Nucl Med 37:24P, 1996 (abstr) 81. Oyen WJ, Boerman OC, Claessens RA, et al: Is somatostatin receptor scintigraphy suited to detection of acute infectious disease? Nucl Med Cornmun 15:289-293, 1994 82. Hay RV, Skinner RS, Newman OC, et al: Scintigraphy of acute inflammatory lesions in rats with radiolabeUed recombinant human interleukin-8. Nucl Med Commun 18:367-378, 1997 83. Gross MD, Shapiro B, Skinner RS, et al: Scintigraphy of osteomyelitis in man with human recombinant interleukin-8. J Nucl Med 37:25E 1996 (abstr) 84. van der Laken CJ, Boerman OC, Oyen WJG, et al: Radiolabeled interleukin-8: Scintigraphic detection of infection within a few hours. J Nucl Med 41:463-469, 2000 85. Rennen HJJM, Boerman OC, Oyen WJG, et al: Specific and rapid scintigraphic detection of infection with Tc-99mlabeled interleukin-8. Eur J Nucl Med 26:1005, 1999 (abstr)

BOERMAN, OYEN, AND CORSTENS

86. Moyer BR, Vallabhajosula S, Lister-James J, et al: Technetium-99m-white blood cell-specific imaging agent developed from platelet factor 4 to detect infection. J Nucl Med 37:673-679, 1996 87. Palestro CJ, Tomas MB, Bhargava KK, et al: Tc-99m P483H for imaging infection: Phase 2 multicenter trial results. J Nucl Med 40:15P, 1999 (abstr) 88. Knight LC, Radcliffe R, Maurer AH, et al: Thrombus imaging with technetium-99m synthetic peptides based upon the binding domain of a monoclonal antibody to activated platelets. J Nucl Med 35:282-288, 1994 89. Ben-Haim S, Kahn D, Weiner GJ, et al: The safety and pharmacokinetics in adult subjects of an intravenously administered 99mTc-1abeled 17 aminoacid peptide (CYT-379). Nucl Med Bio121:131-142, 1994 90. Pearson DA, Lister-James, McBride WJ, et al: Thrombus imaging using technetium-99m-1abeled high-potency GPIIb/IIIa receptor antagonists. Chemistry and initial biological studies. J Med Chem 39:1372-1382, 1996 91. Lister-James J, Knight LC, Maurer AH, et al: Thrombus imaging with a technetium-99m-1abeled activated platelet receptor-binding peptide. J Nucl Med 37:775-781, 1996 92. Muto E Lastoria S, Varrella E et al: Detecting deep venous thrombosis with technetium-99m-labeled synthetic peptide P280. J Nucl Med 36:1384-1391, 1995 93. Lastoria S, Vergara E, Varrella P, et al: Imaging of thromboembolism by scintigraphy with the 99m-technetiumlabelled synthetic peptide P280. Radiol Med (Torino) 90:812819, 1995 94. Taillefer R, Th6rasse E, Turpin S, et al: Comparison of early and delayed scintigraphy with 99mTc-Apcitide and correlation with contrast-enhanced venography in detection of acute deep vein thrombosis. J Nucl Med 40:2029-2035, 1999 95. Liu S, Edwards DS, Looby RJ, et al: Labeling a hydrazino nicotinamide-modified cyclic IIb/IIIa receptor antagonist with 99mTc using aminocarboxylates as coligands. Bioconjug Chem 7:63-71, 1996 96. Liu S, Edwards DS, Looby RJ, et al: Labeling cyclic glycoprotein IIb/IIIa receptor antagonists with 99mTc by the preformed chelate approach: Effects of chelators on properties of [99mTc]chelator-peptide conjugates. Bioconjug Chem 7:196202, 1996 97. Edwards DS, Liu S, Barrett JA, et al: New and versatile ternary ligand system for technetium radiopharmaceuticals: Water soluble phosphines and tricine as coligands in labeling a hydrazinonicotinamide-modified cyclic glycoprotein IIb/IIIa receptor antagonist with 99mTc. Bioconjug Chem 8:146-154, 1997 98. Oyen WJG, Boerman OC, Brouwers FM, et al: Scintigraphic detection of acute experimental endocarditis with the technetium-99m labeled glycoprotein IIb/IIIa receptor antagonist DMP444. Eur J Nucl Med 27:392-399, 2000 99. Thakur ML, Pallela VR, Consigny PM, et al: Imaging vascular thrombosis with 99mTc-labeled fibrin a-chain peptide. J Nucl Med 41:161 - 168, 2000