99mTc Labeling of Highly Potent Small Peptides - Bioconjugate

Bioconjugate Chem. 1997, 8, 621−636

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REVIEWS 99mTc

Labeling of Highly Potent Small Peptides

Shuang Liu,* D. Scott Edwards, and John A. Barrett Radiopharmaceuticals Division, The DuPont Merck Pharmaceutical Company, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received April 21, 1997 INTRODUCTION

Peptides are compounds that contain amino acids (Raminocarboxylic acids) linked by amide (peptide) bonds. Designed by nature for stimulating, inhibiting, or regulating numerous life functions, peptides for a long time have been considered ideal agents for therapeutic applications (1). Although the 111In-labeled peptide was first explored for use in nuclear medicine in 1981 (2), it was not until recently that 99mTc-labeled peptides have emerged as an important class of radiopharmaceuticals in diagnostic nuclear medicine. Of particular interest are 99mTc-labeled chemotactic peptides (3-11), small peptides based on platelet factor 4 (12), and tuftsin receptor antagonists (13-15) for imaging focal sites of infection, somatostatin analogs for imaging tumors (16-20), and platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonists for imaging thrombi (21-34). In the past two decades, monoclonal antibodies (MAbs) or their fragments, F(ab′) or F(ab′)2, have been widely used as vehicles to carry radionuclides of diagnostic or therapeutic characteristics to a specific target such as a tumor (35-40). Although considerable progress has been made in this area (40-43), clinical studies with radiolabeled MAbs have often demonstrated limited accumulation in the tumor, and relatively slow blood clearance due to their high molecular weight, resulting in only modest target-to-background ratios. The poor match between the pharmacokinetics and the physical half-life of 99mTc has limited the development of 99mTc-labeled MAbs and their fragments as commercial target-specific radiopharmaceuticals. The difference between proteins and peptides is their sizes. The term “peptides” is usually used to refer to those compounds containing fewer than 100 amino acids with a molecular mass of about 10 000 Da (44). Small peptides refers to peptides with fewer than 30 amino acids or molecular mass of 70% of pulmonary emboli arise from DVT in the lower extremities (132). However, existing diagnostic modalities are inadequate to diagnose and determine the morphology of the evolving thrombus (133-137). Thus, the development of agents that will not only detect the location but, in addition, determine the age of the thrombi is a critical unmet need in diagnostic nuclear medicine. A venous thrombus is an intravascular deposit predominantly comprising fibrin, and aggregates of platelets and red blood cells. Platelet aggregation is mediated by fibrinogen, which binds via the Arg-Gly-Asp (RGD) tripeptide sequence to the GPIIb/IIIa receptor expressed on activated platelets. Much of the early work in thrombus imaging was focused on the radiolabeling of fibrinogen (138, 139). In 1976, it was reported that platelets can be labeled very efficiently with [111In]oxine (140). Since then, 111In-labeled platelets have been used to detect left ventricular thrombi (141, 142), pulmonary emboli (143), and DVT (144, 145). However, imaging using 111In-labeled platelets suffers from several limitations. The technique is too complex and time-consuming for routine clinical use (146). It usually takes 24-72 h to get useful images. The 111In-labeled platelets cannot detect small thrombi such as coronary artery thrombi due to the relatively high background (blood pool) activity (147). The slow blood clearance and poor T/B severely limit the use of 111In-labeled platelets. Platelet activation and deposition are the initial events in acute thrombus formation and may continue for a variable period as thrombus organization proceeds (31). Activated platelets express the GPIIb/IIIa receptor, which recognizes proteins and peptides bearing the RGD tripeptide sequence, while nonactivated platelets express virtually none of the receptor in its active conformation. A number of RGD-containing small peptides (33, 148151), which are antagonists of the platelet GPIIb/IIIa receptor, have been synthesized and studied for their antithrombotic activities. These small peptides represent a rapidly growing class of antithrombotics. Recently, DeGrado and co-workers (152-155) reported a series of very potent cyclic GPIIb/IIIa receptor antagonists, two (I and II) of which are shown in Figure 3. Peptides I and II have very high selectivity and binding affinity for the GPIIb/IIIa receptor, with IC50 values in the nanomolar range for inhibition of both platelet aggregation and fibrinogen binding (153). This makes them candidates of choice as targeting molecules to carry

Figure 3. Cyclic GPIIb/IIIa receptor antagonists.

the radionuclide (99mTc) to thrombi. Since the RGD tripeptide sequence is vital for maintaining biological activity, the functionalized cyclic peptides III-V were prepared by incorporation of a lysine residue between the arginine residue and the Mamb [mamb ) m-(aminomethyl)benzoic acid] or by attachment of a 6-aminocaproic acid linker on the benzene ring of the Mamb moiety (156, 157). The use of the 6-aminocaproic acid linker keeps the Tc center far from the RGD sequence to minimize the effect of 99mTc labeling on the receptor binding affinity. Since the GPIIb/IIIa receptor is expressed only on activated platelets, the 99mTc-labeled cyclic GPIIb/IIIa receptor antagonists should only be bound to platelets intimately involved in the thrombolic event. The rapid blood clearance of small peptides allows the use of 99mTc and permits earlier imaging of rapidly growing thrombi. The preformed chelate approach has been used to label several cyclic GPIIb/IIIa receptor antagonists with 99mTc (25). It was found that both the cyclic peptide and the BFCA contribute to the differences in physical and biological properties of the [99mTc]BFCA-peptide complex (25, 26). In a canine arteriovenous shunt (AV shunt) model four complexes, [99mTcO(L1-III)]-, [99mTcO(L2III)]-, [99mTcO(L1-V)]-, [99mTcO(L2-V)]-, were found to be incorporated into the growing thrombus under both arterial and venous conditions. The differences in the uptake of these complexes under both arterial and venous conditions are related to the receptor binding affinity of the peptide and the rate of clearance of technetium complexes from the blood circulation. In a canine DVT model, the complex [99mTcO(L1-V)]- was incorporated in the growing thrombus with images clearly detectable within 15 min postinjection. At 2 h postinjection, thrombus-to-blood and thrombus-to-muscle ratios were approximately 7:1 and 10:1, respectively. This clearly demonstrated that the complex [99mTcO(L1-V)]- has the potential for rapid diagnosis of thromboembolic events occurring under both arterial and venous conditions. The indirect labeling approach (Chart 3) has been used to synthesize the complex [99mTcO(L1-V)]- (117). The synthesis of the complex [99mTcO(L1-V)]- was carried out using the ligand exchange method, in which the (EOE)2H2L1-V conjugate was allowed to react with [99mTc]glucoheptonate. It usually requires at least 200 µg of (EOE)2H2L1-V conjugate in each reaction vial to achieve a successful 99mTc labeling. This is consistent with the low labeling efficiency of N2S2 diamidedithiols.

626 Bioconjugate Chem., Vol. 8, No. 5, 1997 Chart 4.

Liu et al.

99m

Tc Labeling of HYNIC-V

Figure 4. Proposed structures for ternary ligand technetium complexes.

HYNIC was also used to label the peptide V with 99mTc (24) (Chart 4). The advantage of using HYNIC as the BFCA is its high labeling efficiency and the choice of various coligands, which allows control of the hydrophilicity and pharmacokinetics of the 99mTc-labeled small peptides. Among various coligands, tricine gives the best radiolabeling efficiency. For example, very high specific activity (e20 000 mCi/µmol) can be achieved for the complex [99mTc(HYNIC-V)(tricine)2]. However, it was found that the complex [99mTc(HYNIC-V)(tricine)2] is not stable, particularly in dilute solutions, and exists as multiple species, which we have attributed to different bonding modalities of the hydrazine moiety of the HYNIC-V and the tricine coligands. Although the animal studies in a canine arteriovenous (AV) shunt and a DVT model show that [99mTc(HYNIC-V)(tricine)2] is able to image both arterial and venous thrombi (23, 29), it would still be difficult to develop for clinical use because of the solution instability and the presence of the many isomeric forms. To prepare [99mTc]HYNIC-V complexes with higher solution stability and less isomerism, EDDA (ethylenediamine-N,N′-diacetic acid), which is more symmetrical and potentially tetradentate, was used as the coligand for the radiolabeling (Chart 4). It was found that EDDA forms a complex, [99mTc(HYNIC-V)(EDDA)], with much higher solution stability and less isomerism (24). However, the HPLC data showed that the complex [99mTc(HYNIC-V)(EDDA)] exists in at least three isomeric forms. In an alternative approach (Figure 4), a water soluble phosphine (TPPTS, TPPDS, and TPPMS) is used as an additional coligand to form a ternary ligand system (27). These monodentate phosphines react readily with [99mTc(HYNIC-V)(tricine)2], replace one of its two tricine coligands, and form complexes, [99mTc(HYNIC-V)(tricine)(L)] (RP444, L ) TPPTS; RP445, L ) TPPDS; RP446, L )

Figure 5. Representative DVT images of complexes RP444, RP445, and RP446 at 15, 60, and 120 min postinfusion. The bar to the right of the images indicates the scale from 0 (white) to 506 (greatest/black). The images have not been filtered.

TPPMS) in high yield and high specific activity (g20 000 Ci/mmol). RP444, RP445, and RP446 are formed as equal mixtures of two isomeric forms, which is due to the resolution of diastereomers resulting from the chiral centers on the peptide backbone and the chirality of the technetium chelate (158). The composition of these complexes was determined to be 1:1:1:1 for Tc:HYNICV:L:tricine through a series of mixed ligand experiments on the tracer (99mTc) level. This composition is maintained over a wide range of relative ligand ratios. In the canine AV shunt model (28), RP444, RP445, and RP446 were adequately incorporated into the arterial and venous portions of the growing thrombus (7.8-9.9 and 0.2-3.7% ID/g, respectively). Figure 5 shows representative unfiltered images of RP444, RP445, and RP446 in the DVT imaging model at 15, 60, and 120 min postinfusion. All three complexes had thrombus uptake (%ID/g ) 2.86 ( 0.4, 3.4 ( 0.9, 3.38 ( 1.1 for RP444, RP445, and RP446, respectively) that far exceeded that of the negative control, [99mTc]albumin. They also show similar blood clearance with a t1/2 values of approximately 90 min. Visualization of DVT can be as early as 15 min postinjection and improves over time with the thrombus/ muscle ratios of 9.7 ( 1.9, 13.8 ( 3.6, and 9.4 ( 2 for RP444, RP445, and RP446, respectively, at 120 min postinjection. The administration of RP444, RP445, and RP446 did not alter platelet function, hemodynamics, or the coagulation cascade. Therefore, all three complexes have the capability to detect rapidly growing venous and arterial thrombi. RP444 was selected for clinical studies

Reviews

as a new thrombus imaging agent for both arterial thrombi and DVT. Obviously, the use of this new ternary ligand system offers several major advantages. First, bonding of the phosphine coligand to the Tc dramatically reduces the number of isomeric forms of the [99mTc]HYNIC-V complexes. Second, the solution stability of [99mTc]HYNICV complexes is dramatically improved. RP444 was found to be stable for at least 6 h under dilute conditions, while the HPLC-purified corresponding 99Tc analog, [99Tc(HYNIC-V)(tricine)(TPPTS)], remains stable in aqueous solution for more than 6 months without any decomposition (158). It is amazing that three different ligands (HYNIC-V, tricine, and phosphine) combine with Tc and form technetium complexes with only two detectable isomers and with extremely high solution stability. Finally, the hydrophilicity of [99mTc]HYNIC-V complexes with this ternary ligand system can be tuned either by altering the number of sulfonato groups or by using water soluble phosphines with other polar functionality. The tricine coligand can also be substituted by other potentially tetradentate aminocarboxylates such as dicine [N-bis(hydroxymethyl)methylglycine] and bicine [N,Nbis(hydroxyethyl)glycine]. However, the specific activity of [99mTc]HYNIC-V complexes using dicine and bicine as coligands is not as high as that of the corresponding tricine complexes. This is consistent with the literature results reported by Abrams and co-workers (93-96), who found that the [99mTc]tricine precursor complex has the highest reactivity with hydrazines. Using the combination of a polydentate aminocarboxylate and a phosphine ligand, HYNIC-conjugated peptides and other HYNICderivatized small molecules can be readily labeled with 99mTc in high specific activity and high stability for potential use as radiopharmaceuticals. The technetium oxidation state and the exact nature of bonding between these three ligands (HYNIC, tricine, and phosphine) and the Tc are not yet clear. It has been proposed that the technetium oxidation state in the [99mTc]tricine complex is 5+ (92, 93). However, the oxidation state might change when the HYNIC group and phosphine coligand are bonded to the Tc center. Furthermore, the technetium oxidation state largely depends on how the HYNIC group binds to the Tc and how one counts the charge on the HYNIC ligand. Complexes containing Tc-hydrazido and Tc-diazenido bonds have been previously reported and characterized by X-ray crystallography (159-165). Recently, Davison and co-workers (166, 167) reported a series of technetium(III) and rhenium(III) complexes of 2-hydrazinopyridine. It was found that unlike phenylhydrazine, 2-hydrazinopyridine has several bonding modalities: neutral bidentate pyridyldiazene (166, 167), neutral monodentate pyridiniumdiazenido (166), and anionic monodentate pyridyldiazenido (167). Various Tc/Re complexes can be prepared depending upon the starting material and reaction conditons. For example, the reaction of NH4[TcO4] with 2-hydrazinopyridine dihydrochloride gives [TcCl3(HNdNC5H4N)(NdNC5H4NH)]. The reaction of [ReCl3(HNdNC5H4N)(NdNC5H4NH)] with triphenylphosphine (PPh3) in the presence of a base such as diisopropylethylamine produces the complex [ReCl2(PPh3)(NdNC5H4N)(NdNC5H4NH)], in which the NdNC5H4N moiety was found to be an anionic monodentate pyridyldiazenido (166). We also found that NH4[TcO4] or [n-Bu4N][TcOCl4] reacts with HYPY‚2HCl in the presence of excess PPh3 to give the complex [Tc(NdNC5H4N)(PPh3)2Cl2]. The NMR data suggest that the NdNC5H4N moiety is likely bidentate pyridyldiazenido (158). When the HYNIC binds to the Tc, it likely forms either

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a terminal Tc-diazenido or Tc-hydrazido bond. If one assumes that the coordination geometry around the Tc in the ternary ligand complexes is distorted octahedral, the tricine coligand is expected to be tetradentate with the carboxylate O, amine N, and hydroxy O occupying equatorial positions and one of the two remaining hydroxy O’s occupying the apical site of the Tc (Figure 4). Efforts are continuing to better understand the coordination chemistry of this unique, versatile, and complex ternary ligand system. Lister-James and co-workers (31-34) also used 99mTclabeled IIb/IIIa receptor antagonists for the development of new thrombus imaging agents. P280 is a small oligopeptide made of two identical, linked, cyclic 13 amino acid monomers. Each monomer contains a (S-aminopropylcysteine)-Gly-Asp tripeptide sequence, which is mimetic of the RGD sequence found in molecules such as fibrinogen and fibronectin. P280 has high affinity for the GPIIb/IIIa receptor with an IC50 of 87 nM for inhibition of human platelet aggregation (31). Each monomer contains a Cys(Acm)-Gly-Cys(Acm) tripeptide sequence, which forms an N2S2 diamidedithiol bonding unit for 99mTc labeling. Because of the low labeling efficiency of N2S2 diamidedithiols and the presence of protecting group on the two thiol groups, the 99mTc labeling of P280 requires the use of a large amount of peptide (g250 µg) and heating at 100 °C for 15 min when prepared by ligand exchange with [99mTc]glucoheptonate. The specific activity for the [99mTc]P280 complex is low (60 mCi/µmol) compared to that of RP444 (g20 000 mCi/µmol). [99mTc]P280 was reported to give excellent images of DVT in a canine DVT model. However, the thrombus uptake at 4 h postinjection is only 0.0059 ( 0.0025 %ID/g (31), which is much lower than that of RP444 (2.8 ( 0.4 %ID/g) (28). The low thrombus uptake is most likely related to the lower binding affinity of P280 (IC50 ) 87 nM) for the GPIIb/IIIa receptor and faster blood clearance 99mTcP280 (t1/2R ) 1.6 min and t1/2β )20 min) as compared to the binding affinity of peptide V (IC50 ) 6 nM) and the blood clearance of RP444 (t1/2 ) 90 min). P748 is another platelet GPIIb/IIIa receptor antagonist under investigation for imaging pulmonary embolism (33, 44). P748 has a higher receptor binding affinity (IC50 ) 28 nM against aggregation of human platelets) than P280 (IC50 ) 87 nM), and [99mTc]P748 has slower blood clearance with t1/2R ) 9.6 min and t1/2β ) 145 min. It is not surprising that [99mTc]P748 shows a higher uptake (%ID/g ) 0.018 ( 0.0068 at 4 h postinjection) and a higher T/B than [99mTc]P280. Unlike P280, P748 uses an N3S aminediamidethiol chelating unit. Because of the asymmetric character of the chelator, two epimers are seen, one of which is predominant. It was reported that P748 can be easily labeled by ligand exchange with [99mTc]glucoheptonate at room temperature. The reported highest specific activity for [99mTc]P748 is about 2000 mCi/µmol. Apparently, the presence of the amine N donor and a unprotected thiol in the chelator dramatically improves the radiolabeling efficiency. Infection/Inflammation Imaging. Imaging focal sites of infection or inflammation using radiolabeled peptides is another important area in diagnostic nuclear medicine. Accurate and rapid localization of infectious and inflammatory foci facilitate elucidation of the cause of disease and the implementation of a tailored therapeutic regimen (169). Current imaging procedures such as X-ray computed tomography (CT), ultrasound (US), conventional radiography, and magnetic resonance imaging (MRI) rely primarily on focal changes in tissue density to define lesions. In the early stage of an inflammatory lesion, lesion localization using these pro-

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cedures may be difficult because the tissue changes associated with necrosis have not occurred (6). For a number of years, imaging of inflammation and infection has been performed using either [67Ga]citrate or 111In-labeled white blood cells (WBCs) (170, 171). 99mTc-labeled nonspecific polyclonal IgG’s were also used for detection of infection foci (92, 94, 172-177). Although these agents are well-accepted and efficacious, it usually takes 24 h to obtain diagnostically useful images. Recently, two new radiopharmaceuticals were introduced for imaging sites of infection: [99mTc]hexamethylpropylene amine oxime (HMPAO)-WBCs (178-180) and [99mTc]albumin colloid-WBCs (181). Imaging using both agents can be completed on the same day, but these procedures may impose significant risks to laboratory personnel and patients, particularly with the increasing prevalence of human immunodeficiency virus in the population (6, 182, 183). WBCs, particularly polymorphonuclear leukocytes (PMNLs) and monocytes, accumulate in high concentrations at sites of infection. Therefore, most attention has been directed toward radiolabeling small molecules that bind to both circulating granulocytes and leukocytes. These include chemotactic peptides, analogs of N-formylmethionylleucylphenylalanine (fMLF) (3-11), small peptides based on platelet factor 4 (12), and tuftsin receptor antagonists (13-15). The chemotactic peptide fMLF is a bacterial product that initiates leukocyte chemotaxis by binding to highaffinity receptors on inflammatory cells. Fischman and co-workers (4-11) have investigated a series of radiolabeled chemotactic peptides as infection/inflammation imaging agents. These peptides were modified with HYNIC (for 99mTc labeling) or DTPA (for 111In labeling) at the C terminus. The 99mTc labeling of HYNIC-modified chemotactic peptides can be achieved using various coligands such as glucoheptonate, mannitol, and glucamine (5). It was reported that very high specific activity (g20 000 mCi/µmol) could be achieved using glucoheptonate as the coligand. Different biodistributions were observed for various coligands (5). Animal studies have shown evidence of binding to leukocytes in vivo and localization at sites of infection (6-11). In a rabbit infection model, a 99mTc-labeled fMLF analog gave a target to background ratio greater than or equal to that achieved with [111In]WBCs (11). We also labeled a chemotactic peptide-HYNIC conjugate (fMLFK-HYNIC) using tricine or glucoheptonate as coligand (183). It was found that tricine and glucoheptonate form technetium complexes, [99mTc(fMLFKHYNIC)(L)2] (L ) tricine and glucoheptonate), with many isomeric forms. These technetium complexes are not stable in the absence of excess coligand. The combination of tricine and TPPTS forms a stable ternary ligand complex [99mTc(fMLFK-HYNIC)(tricine)(TPPTS)], which was evaluated in two animal (guinea pig and rabbit) models of focal infection. It was found that the ternary ligand complex was superior with a T/B of 5-7:1 at 4 h postinjection, reflecting an increased clearance from the normal muscle. In the rabbit infection model a transient decrease in WBC count of 35% was observed in all three groups during the first 30 min postinjection (183). A similar neutropenic response was also reported for the 99m Tc-labeled chemotactic peptides, [99mTc]RP050 and 99mTc]RP056 (184). [ Apparently, the 99mTc labeling of these highly potent agonists suffers a drawback, severe reduction of peripheral leukocyte count because of the unlabeled peptide even at very low doses. There are two approaches to avoid this problem. The first approach is to separate the

Liu et al.

radiolabeled peptide from the excess unlabeled peptide using HPLC. This results in a product almost at its theoretical specific activity but is inconvenient for routine clinical use. The alternative approach is to use antagonist peptides, which bind to the receptor but do not trigger the neutropenic effect, as targeting molecules. However, the receptor binding affinities of the antagonists tested to date appear to be much lower than that of the agonists (185). Platelet factor 4 (PF-4) is a 29 kDa homotetrameric protein for which receptors have been identified on PMNLs, monocytes, endothelium, fibroblasts, and hepatocytes. Peptide P483 (acetyl-Lys-Lys-Lys-Lys-Lys-CysGly-Cys-Gly-Gly-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-LysLeu-Leu-Glu-Ser) is an analog of the C-terminal tridecapeptide of human PF-4. It contains a Cys-GlyCys tripeptide chelating unit for 99mTc labeling and a pentalysine sequence on the N terminus to promote renal clearance (12). It was reported that complexation of [99mTc]P483 with heparin substantially enhanced the binding to WBCs and resulted in improved uptake in sites of infection in a rabbit infection model (12). The in vitro distribution in human blood suggests that [99mTc]P483H associates with specific WBCs, particularly monocytes. In the rabbit infection model, [99mTc]P483H showed slightly higher infection uptake (0.062 ( 0.022 %ID/g) than [111In]oxine-WBCs (0.051 ( 0.008 %ID/g), and a 6-fold higher T/B, probably due to the rapid blood clearance (12). Tuftsin is a tetrapeptide (TKPR) derived from the Fc portion of IgG. It promotes phagocytosis and chemotaxis of neutrophils and monocyte/macophages. Recently, a 99mTc-labeled tuftsin receptor antagonist [Pic-SC(Acm)GTKPPR; Pic ) picolinic acid] was used for imaging inflammation (13). The Pic-SC(Acm)G sequence forms an N3S chelating unit for 99mTc bonding. Radiolabeling was achieved by ligand exchange with [99mTc]glucoheptonate. In rats with infectious (Escherichia coli) inflammation (13), the 99mTc-labeled tuftsin antagonist was able to give excellent images with the T/B of 3.6, 5.0, and 16.2 at 0.5, 3, and 17 h postinjection, respectively. Another 99mTc-labeled tuftsin receptor antagonist is [99mTc]RP128 [RP128 ) dimethylGSC(Acm)G-TKPPR]. In an inflammatory bowel disease (IBD) model, [99mTc]RP128 showed much better imaging quality than [111In]oxine-WBCs (14). The target (inflamed terminal colon) to background (proximal noninflamed colon) ratios of 2.14, 2.51, 2.90, and 1.90 were obtained at 0.5, 1, 3, and 18 h postinjection, respectively. Both agents were rapidly excreted via the renal system. Tumor Imaging. There is an unmet need for the development of new target-specific tumor-imaging agents. Radiolabeled receptor-based biomolecules such as small peptides are of particular interest because they have the potential to detect primary sites, identify occult metastatic lesions, guide surgical intervention, stage tumors, and predict efficacy of therapeutic agents. When labeled with a suitable radionuclide, they can also be used as radiotherapeutic agents. The peptide that has attracted greatest interest is somatostatin, a tetradecapeptide which exhibits an inhibitory effect on the secretion of numerous hormones, including growth hormone, thyrotrophin, insulin, glucogon, vasoactive intestinal peptide (VIP), and secretin. Somatostatin receptors are overexpressed on a number of human tumors and their metastases (186-189), thereby serving well as the target for tumor imaging. Although the first report of in vivo imaging with a somatostatin analog appeared in 1976 (190), further development was hampered due to rapid degradation of the native peptide

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Reviews

by plasma and tissue proteases (191). For this reason, analogs of somatostatin have been synthesized using D-amino acids to prolong the in vivo half-life by inhibiting the action of amino and carboxypeptidases. Octreotide (Sandostatin, SMS 201-995) is a metabolically stable analog of somatostatin and has been successfully used for the treatment of acromegaly and GEP tumors (192194). Derivatives of octreotide have been radiolabeled with γ-emitting radionuclides such as 123I (195, 196) and 111In (197-204), and these radiotracers have been successfully used to detect somatostatin receptor-positive tumors by γ-scintigraphy. [111In]DTPA-octreotide (OctreoScan) has been approved in many countries in Europe and North America and has become one of the most commonly used radiopharmaceuticals in clinical tumor imaging. However, this agent suffers several drawbacks such as the long half-life (t1/2 ) 67 h, γ ) 173 keV, 89%, and 247 keV, 94%) and the high cost of 111In. For diagnostic purposes, 99m Tc is more desirable because of its low cost, easy availability, and ideal nuclear characteristics, which better match the rapid blood clerance and fast tumor accumulation of octreotide. In the past several years, various somatostatin analogs have been labeled with 99mTc using different BFCAs, including N2S2 diamidedithiols (19, 20), N3S triamidethiols (19, 20), N3S aminediamidethiols (19, 20), PnAO (propyleneamine oxime) (16), and tetraamines (17). Of particular interest are 99mTc-labeled peptides P587 and P829. Both peptides contain the Tyr-(D-Trp)-Lys-Val sequence, which is responsible for somatostatin receptor binding. The cyclic peptide backbone does not contain the S-S disulfide linkage and is not susceptible to reductive cleavage. In P587, the Gly-Gly-Cys tripeptide constitutes an N3S triamidethiol chelator for 99mTc bonding, while P829 uses the (β-Dap)-Lys-Cys tripeptide sequence to form an N3S aminediamidethiol chelating unit. Studies in CA20948 tumor-bearing rats showed that the tumor uptake of [99mTc]P587 and [99mTc]P829 is comparable to or better than that of [111In]DTPA-octreotide (19). [99mTc]P829 is excreted predominantly through the renal system and has been selected for clinical trials. HYNIC was also used to label somatostatin analogs with 99mTc (205, 206). It was conjugated to the N terminus of octreotide either directly or via a β-alanine or diaminobutanesuccinyl linker. The use of tricine as coligand produced a very high specific activity (∼6000 mCi/µmol). In a tumor bearing mouse model (AR4-2J), all three 99mTc-labeled HYNIC-octreotide conjugates showed comparable tumor uptake to [111In]DTPA-octreotide (205, 206). The observed high blood activity is probably related to the instability of [99mTc]HYNICoctreotide-tricine complexes and plasma protein binding. Somatostatin analogs (e.g. BIM-23014, RC-160, and Sandostatin), which contain a S-S disulfide bond in the cyclic peptide backbone, have also been radiolabeled by the direct labeling approach (1, 18, 207, 208). It is believed that when the S-S bond is reduced, the thiolate S atoms bond to the Tc center and form stable 99mTc complexes (18). It was also reported that these 99mTclabeled somatostatin analogs have not shown any apparent loss of biological activity, nor have they shown any abnormal blood clearance in experimental animals compared to that with [111In]DTPA-octreotide (1, 207, 208). However, several critical questions remain to be answered for this approach. First, how many peptides are bonded to the Tc center? If only one peptide binds to the Tc, which tripeptide sequence (Cys-AA-AA or AAAA-Cys) is involved in Tc bonding? How many 99mTc

species are in the radiolabeled kit? What is the impact of 99mTc labeling on the receptor binding affinity of the cyclic peptide? CONCLUSIONS

From the discussion above, it is apparent that significant progress has been made in the development of peptide-based target specific radiopharmaceuticals. 99mTc labeling of biologically active peptides stemmed from the studies of 99mTc-labeled antibodies for diagnosis or therapy of various tumors. In a very short period of time, 99mTclabeled peptides have become an important class of imaging agents for the detection of not only tumors but also thrombosis and infection/inflammation. 99mTclabeled peptides such as RP444, [99mTc]P280, [99mTc]P483H, [99mTc]P748, [99mTc]P829, and [99mTc]RP128 are now being evaluated in clinical trials. Results from preeclinical studies have lived up to their expectations: high specificity, high uptake in the target organ, and high T/B due to rapid blood clearance of the 99mTc-labeled small peptides. Hopefully, some of these 99mTc-labeled small peptides will soon become commercial products and serve the nuclear medicine community for decades. Radiolabeled small peptides continue to attract interest because of their favorable physical and chemical characteristics compared to antibodies and their fragments. Small peptides, once the receptor binding sequence is identified, can be easily synthesized and modified according to their pharmacokinetic requirements. Ideally, a peptide-based radiopharmaceutical should have the following characteristics: rapid uptake by target tissues, metabolic stability, and fast renal clearance. This is particularly important for infection and tumor imaging because rapid renal clearance minimizes the accumulation of radioactivity in the abdominal area and results in improved T/B. The isotope of choice for radiolabeling will continue to be 99mTc rather than 111In for diagnostic radiopharmaceuticals because of cost and convenience. The experience learned from 99mTc labeling of small peptides can be applied to other highly potent receptor binding small molecules. In addition, these biologically active small peptides can also be used for the development of therapeutic radiopharmaceuticals when labeled with appropriate radionuclides such as 186Re. In the area of tumor imaging, 99mTc-labeled substance P (SP) and vasoactive intestinal peptide (VIP) as well as their analogs may become new classes of tumor-imaging agents. [111In-DTPA-Arg1]SP has been successfully used to visualize SP-positive processes such as adjuvant arthritis and atransplantable pancreatic tumor (209). Studies using [123I]VIP have clearly demonstrated its ultility in localizing intestinal adenocarcinoma and endocrine tumors as well as metastatic tumor sites in humans (210). Of course, the next logical step is 99mTc labeling of substance P (SP) and vasoactive intestinal peptide (VIP) analogs with favorable pharmacokinetics. 99mTc labeling of highly potent small peptides is different from that of simple organic chelators. Because of the possible side-effects, the BFCA-peptide conjugate has to be labeled at very low concentrations (usually 5-20 µg/mL, corresponding to 5 × 10-6 to 2 × 10-5 M for a BFCA-peptide conjugate with molecular mass of 1000 Da and IC50 values in the nanomolar range) to achieve high specific activity. One of the challenges for inorganic chemists is to design new ligand systems that have very high labeling efficiency and form technetium complexes with minimal isomerism. It should be noted that the development of new 99mTclabeling technologies and synthesis of new biologically active peptides with high receptor binding affinities are

630 Bioconjugate Chem., Vol. 8, No. 5, 1997

equally important. Improvement of the pharmacokinetics of 99mTc-labeled peptides can be achieved by modification of both the peptide and the Tc chelate. The development of 99mTc-labeled peptide radiopharmaceuticals is a multidisciplinary effort and requires the collaboration from scientists in several areas, including organic chemistry, inorganic chemistry, biochemistry, biology, formulation chemistry, and nuclear medicine. As Jurrisson (211) stated: “Without their joint efforts nuclear medicine would not be where it is today, nor will it progress”. ACKNOWLEDGMENT

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