Radiolabeled peptides in diagnosis and therapy

Radiolabeled Peptides in Diagnosis and Therapy R o n a l d E. W e i n e r a n d M a t h e w L. T h a k u r

During the past few years, there has been exponential growth in the development of radiolabeled peptides for diagnosis and therapy. This is because the peptides can be synthesized easily and inexpensively, they have fast clearance and rapid tissue penetration, and they are less likely to be immunogenic. More importantly, most peptides have a high affinity for characteristic receptor molecules that are overexpressed on malignant mammalian cells. Peptides can be labeled with a variety of radionuclides intended for specific applications, diagnostic or therapeutic, by using both conventional and novel chelating moieties, many of which can be incorporated during the solid state synthesis of a chosen peptide. High specific-activity peptides can be prepared and used to minimize unwanted physiologic effects, and known sequences of amino acids can be modified to slow their in vivo catabolic rate. These character-

istics have paved the way for the preparation of a large number of radiolabeled peptides for a variety of clinical and experimental applications. This article briefly discusses the peptide chemistry; it also summarizes the preparation of radiolabeled peptides and outlines their applications in imaging vascular thrombosis, detecting infection and inflammation, and localizing tumors. Their therapeutic applications in oncology are also presented and the future directions outlined. Peptides t h a t have been approved for human use, such as AcuTect (Diatide, Londonderry, NH) or OctreoScan (Mallinckrodt, St. Louis, MO), or those that have made it to clinical trials, are emphasized. Also discussed are selected promising agents that are still in preclinical investigation. Copyright 9 2001 by W.B. Saunders Company

ORE THAN 40 years ago, Nobel laureate Bruce Merrifield first described resin-based peptide synthesis. Since then, new methods of synthesizing longer peptides in large quantities and purifying, characterizing, and optimizing them have resulted in an explosion in the number of potentially useful peptides and in the interest in their commercial development. The use of radiolabeled peptides is only approximately a decade old for diagnostic and therapeutic applications. This momentum supercedes the radioimmunoscintigraphic and radioimmunotherapeutic use of mono clonal antibodies. The Food and Drug Administration (FDA) first approved an HIIn-labeled murine monoclonal antibody for diagnosis of the recurrence of colorectal and ovarian cancer in 1993.1 However, radiolabeled monoclonal antibodies have many disadvantages, including very slow blood clearance. For example, with 111In-labeled ProstaScint (Cytogen, Princeton, NJ) a whole immunoglobulin G (IgG) for the diagnosis of recurrent prostate cancer, imaging is usually initiated 5 days after radiopharmaceutical injection. Additionally, these large molecules (150,000 daltons) have difficulty penetrating the poorly perfused tumor mass, leading to low tumor uptake. 2 Furthermore, antibodies, because of their murine origin, may produce human antimouse antibodies (HAMA). A HAMA response limits repeat injections because the antibody-HAMA complex is quickly removed from the blood, thereby eliminating the tracer from its intended use. In contrast peptides, a chain of amino acids coupled by peptide bonds, CONH, are small, usually classified as containing less than 50 amino acids. At 100 amino acids or greater, the string of amino acids is considered a protein. 3"4 Because the average amino acid residue weight is approximately 110 daltons, by definition, the maximum molecular weight (MW) of a peptide is -5,500 daltons.

This low MW renders peptides low in antigenicity, fast in clearance, and rapid in tissue penetration. In contrast with monoclonal antibodies, peptides can be produced easily and inexpensively.5,6 In recent years, a wide variety of peptides have been identified that have high affinity for characteristic receptors that are overexpressed on a large number of cell types. Table 1 shows a selective list of peptides with their function and target cells. There are over 850 well-characterized endogenous peptides. 7 Thus, radiolabeled peptides offer the possibility of a wide array of applications in diagnostic and therapeutic medicine. Peptides, however also have their dark side. Their advantages tend to diminish as their size gets closer to 5,000 d, the magic number. As the MW of the peptide increases, the penetration by diffusion decreases. Peptides are built by adding amino acids one at a time, increasing the cost of their preparation, although not prohibitively. Endogenous peptides have physiologic effects, which may cause unwanted side effects. Thus, it is preferable to use high specific-activity compounds or antagonists that would exert high receptor specificity but would have no physiologic effect. To add an imaging radionuclide, a chelate is frequently covalently coupled to the peptide. This process can adversely influence biologic activity or receptor specificity of a peptide. Distorting peptide conformation can influence target binding.8 Most peptides have a very short biologic half-life because of rapid proteolysis by circulating enzymes. Enzymes usually have very defined specificities, eg, degrading a peptide from either the C- or N-terminus (exopeptidases) or binding and cleaving only the naturally occurring L-amino acids. For example, an octreotide was developed to enhance the antitumor growth effects of somatostatin (Fig 1A). The hormone was modified by increasing its extremely short blood half-life (2 to 4 minutes), and octreotide was the result (Fig 1B). With the longer half-life (1.5 to 2 hours), localization and imaging of tumors with radiolabeled octreotide was possible. There is little metabolism of 111In-OctreoScan,9 possibly because D-amino acids were inserted in the sequence, and both the - and C-terminal amino acids were modified (Fig 1C). Amino acid or peptide mimetics (structures that mimic) can also slow the degradation. Judicious use of D-amino acids and the end-capping process can induce resistance to in vivo enzymatic degradation. In

M

From the Deportment of Diagnostic Imaging and Therapeutics, University of Connecticut Health Center, Farmington, CT; and the Department of Radiology, Thomas Jefferson University Hospital, Philadelphia, PA. Address reprint requests to Ron Weiner, PhD, University of Connecticut Health Center, Division of Nuclear Medicine MC-2804, 263 Farmington Ave, Farmington, CT 06030. Copyright 9 2001 by W.B. Saunders Company 0001-2998/01/3104-0004535.00/0 doi:10.1053/snuc.2001.27045

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Seminars in Nuclear Medicine, Vol XXXI, No 4 (October), 2001: pp 296-311

RADIOLABELED PEPTIDES

297

Table 1. Naturally Occurring Peptides, Their Function, and the Target Disease and/or Cell-Expressing Receptors Peptide

Function

Tar~let Disease/Receptor

ACTH Bombesin

Pituitary gland hormone CNS & GI tract activity; suppresses feeding in rats

Brain natriuretic peptides Calcitonin CCK-B/gastrin

Ca regulation Gallbladder contraction/acid secretion

Epidermal growth factor Gastrin-releasing peptide Glucagon a-MSH Neurotensin Oxytocin

Somatostatin & analogues

Growth promoter Gastrin secretion Liver glucose release Regulation of skin pigment GI and cardiac activity, neuromodulator Uterus contracting and lactation stimulating; crosses blood-brain barrier Includes secretin, glucagon, calcitonin, parathyroid hormone Growth hormone release inhibiting factor

Substance P

Vasoactive

Vasopressin VIP

Antidiuretic hormone Vasodilator, growth promoter, immunomodulator

PACAP

Glioblastomas, SCLC, prostate, breast, gastric, colon, and pancreatic cancer Cardiac tissue CRF receptors SCLC, GI tumors, ovarian cancer, medullary thyroid, homology to VIP and PACAP receptors Breast cancer GRP/neuromedin B, see bombesin Endocrine pancreatic B cells Melanoma cells SCLC, colon cancer, meningioma, prostate cancer

Variety of tumors; subtypes 1 & 2 Neuroendocrine, SCLC, breast cancer, lymphocytes, subtypes 1-5 Glial tumors, medullary thyroid, breast cancer, astrocytomas Subtypes V1, V2, and V3, SCLC Subtypes 1 & 2, epithelial tumors, breast, colon, nSCLC, pancreatic, prostate, bladder and ovarian cancer

Abbreviations: ACTH, (adrenocorticotrop (h) in); CCK = cholecystokinin; GRP, gastrin-releasing peptide; CRF, Calcitonin-relaasing factor; PACAP, pituitary adenylate cyclase activating peptide; VIP, vasoactive intestinal peptide; ~-MSH, ~-melanocyte stimulating hormone; SCLC, small cell lung cancer; nSCLC, non--small cell lung cancer. Data from Okarvi, a Reubi,sa,14s,l~ and Heasly) 4B

addition to size and susceptibility to enzymatic degradation, lipophilicity is also an important parameter that contributes to pharmacokinetics of a given peptide. Lipophilic peptides can be sequestered by the liver and may be eliminated by hepatobiliary excretion. Hydrophilic peptides may be removed rapidly by the kidneys. Cyclization of peptides can exert restricted conformational mobility and enhance biologic activity. Peptides should be radiolabeled at a certain position that will not interfere with their

Ala-Gly-Cys-Lys-Asn-Phe~.

Ph~

binding to their biologic target. Amino acid spacers can be introduced between the parent peptide and a chelating moiety to eliminate steric hindrance, which will maintain the biologic activity of a peptide subsequent to radiolabeling. I~ Thus, a variety of techniques are available to hone the original peptide to be a useful diagnostic or therapeutic agent. This article concentrates on 3 areas in which peptides have made or are likely to make a contribution: in clot detection; inflammation/infection detection; and oncology, both in diagnosis and therapy. We emphasize agents that have already been used in human trials. Selected promising agents that have undergone only animal studies are also included.

Trp THROMBUS

A

~ Thr/ Cys-Ser-Thr-Phe ~

D-Phe-C~ - - Phe \ /

B ,s D-I

Thr(ol)-Cys~ Thr Fig 1. Schematic drawing of (A) somstoststln, (B) octreotlde (Ssndostatin), and (C) pentstreotlde (OctreoScsn) DTPA. Amino acids in bold srs directly Involved In somstoststln receptor binding. Amino acids are all L-Isomers except where noted or an amino alcohol, Thr(ol). DTPA Is coupled to the psptlde via an smlde bond.

IMAGING

Background For more than 25 years, the detection of thrombi and emboli by using agents that allow hot spot imaging has been a goal in radiopharmaceutical development. 12-14 Hemostasis, the process that stops bleeding when a blood vessel is injured, is exceedingly complex. 15-17 This process maintains a precarious balance between the thrombogenic substances (which stimulate coagulation) and antithrombotics (which inhibit this process) until hemostasis is needed. Disruptions in this system can tip the balance and result either in excessive or inadequate clot formation. Once the biochemical process of clotting is activated, there is a cascade of events. Most clotting factors are enzymes that, once activated, become proteases and cleave a portion of the next molecule in the

298

cascade. When blood vessel ruptures and the subendothelium is exposed, a platelet plug is formed. Next, platelets release stored adenosine diphosphate and thromboxane A 2, which stimulates the platelets to aggregate. This activation also exposes the glycoprotein (GP) IIb/IIIa receptor on the platelet's surface. Fibrinogen present in the blood adheres to this receptor and forms a bridge between platelets, promoting further clumping. This extrinsic pathway is initiated by tissue factors released by injured tissue. The end cascade is responsible for changing fibrinogen (inactive form) to fibrin monomer (active form) by enzymatically cleaving a very small portion of the fibrinogen molecule. The fibrin monomers form strands coupling the fibrin ends (head to tail). Lastly, fibronectin binds to fibrin strands and has another site that covalently cross links the fibrin to form a strong interconnected mesh. In low-flow environments, eg, the venous system, the thrombi consist of a large fibrin mesh containing red blood cells with some platelets and other cells. In contrast, in high shear en'dronments, eg, stenotic arteries, platelets dominate in the clot. A thrombus is developed at a site of vessel injury, while emboli break off from the thrombus and flow distally until they occlude a narrow vessel. Both deep vein thrombosis (DVT) and pulmonary emboli (PE) are common clinical conditions associated with significant mortality and morbidity. It is estimated that in the United States, more than 2 to 5 million patients have one or more episodes of DVT each year. In addition, it is estimated that 500,000 to 600,000 cases of PE occur, and 50,000 to 200,000 of these result in death. 1s'19 A large fraction (70% to 90%) of DVT becomes a life-threatening PE, making an accurate and timely diagnosis a challenging clinical problem. 2~ At present, compression ultrasonography is a common technique to detect occlusive thrombi in the thigh. However, it is much less accurate for thrombi in the calf22 and also has poor accuracy in asymptomatic patients at high risk. 23 It is also very operator-dependent and is difficult to perform in obese patients or patients with swollen legs. 24 Contrast venography, considered the gold standard, is highly accurate but invasive, has a high incidence of side effects, and requires special expertise, which limits repeated use to assess the influence of therapy. 25 This technique is practically inadequate or difficult to interpret in 10% to 30% of cases and is not reliable in differentiating acute recurrent DVT from old, nonacute DVT. One of the most commonly used radiopharmaceuticals for thrombus detection was lzSI-labeled fibrinogen. 12'26 The labeled fibrinogen procedure was a nonimaging counting technique in which a handheld probe was placed on the calf and thigh for radioactivity measurement over several days. Because of the high background (long blood half-life --4 days), early diagnosis was not possible. Labeling fibrinogen with nuclides suitable for imaging, eg, 123I or 99mTc, did not yield any improvement. Revised FDA guidelines made this agent less cost-effective, and it is no longer commercially available. Platelets were labeled with n l I n to take advantage of platelet participation in cloI~s.26 1lain_labeled platelets have a tong blood survival (--8 days) that resulted in high background activity and required more than 48 hours before clots could be imaged. In the 1980s, a wide variety of monoclonal antibodies were developed, directed against a host of platelet and fibrin antigens. 12'1'*'26'27Most of the antiplatelet antibodies were directed against the GP IIb/IIIa receptor on the activated platelets. 12'26 However, the complexity of labeling techniques and the

WEINER AND THAKUR

loss of antibody specificity prevented the technique from achieving clinical usefulness. The GP IIb/IIIa receptor is a heterodimeric transmembrane molecule consisting of a and 15 subunits. 16 There are 40,000 to 80,000 GP IIb/IIIa molecules on the platelet's surface and additional molecules are stored in granules, which can be brought to the surface during activation. Fibronectin and vitronectin also interact with the fibrinogen binding site. The site is located on the extracellular portion of the tx and [3 subunits. Fibrinogen binding is Ca2+-dependent and binds via two Arg-Gly-Asp (RGD) sequences on the c~ and "y chains. Three companies, Diatide (Londonderry, NH), DuPont Pharmaceuticals (Wilmington, DE), and Draximage (Montreal, Canada), have been developing 99mTc-labeled peptides for clot detection. 28-3~Diatide and DuPont have directed their approaches to the GP IIb/IIIa receptor by using RGD or RGD analogue peptide Sequences (Table 2). In contrast, Draximage has developed a peptide that binds to the fibronectin site on fibrin. Each peptide has been tested in patients, but the Diatide product, originally designated P280 and now called AcuTect, has recently been approved for human use. 31 Other peptides are still in preclinical studies.

AcuTect This peptide is a dimer that contains an RGD mimetic sequence, S-aminopropyl-L-cysteine (apc)-GD (Fig 2A). The RGD sequence contains a cyclize methionine group that confers rigidity and a D-amino acid, which enhances resistance to proteolysis. To label with 99mTC, glucoheptonate is used as the coligand added to peptide solution, and it is heated at 100~ for 15 minutes. 3a The

Table 2. Peptides for Clot Detection

Peptide

Target Receptor

Acu Tect (P280)

Platelets (GP lib/Ilia)

DMP-444

Platelets (GP lib/Ilia)

Bitistatin

Platelets (GP lib/Ilia)

Fibrin-Binding Domain Peptide

Fibronectin site on fibrin

TP 1201

Platelets (CD36)

TP 1301

Platelets (CD 36)

TP 850

~-chain of fibrin

Function Fibrinogen-binding site to form platelet bridge Fibrinogen-binding site to form platelet bridge Fibrinogen-binding site to form platelet bridge Fibronectin-binding site to crosslink fibrin monomers Exists in molecular domain of thrombospondin Exists in molecular domain of thrombospondin Inhibits fibrin polymerization

Data from Okarvi, 3 Ezov et al, 4~ and Thakur et al. 41

RADIOLABELED PEPTIDES

299

A r

O~

/

9~rr bindi~ aite-)

GPIlb/IIlabindingsite

~

r GPHb/IIlabindingsite

I"

~-~

/

~T~

Tcbimlinlsite

Adlv-A~',-td''J~- Gly'Gly'CY~ACm)'GIy'Cy~Acm)'Gly'GIy'Nr "'" " -" "-~ i-i i'l 14 O O

B

"1

CO

/

NH-CO-Aba-Pro-Pro-Arg-Pro-Oly Fig 2. The sequence and proposed structure of the clot-binding peptlde (A) AcuTect (P280) and (B) TP 850. Unusual amino acids: Apc, smlnopropyl-L-cys, cyclized Met; Acm, scetamldomethyl protecting group; Aba, aminobutyrlc acid. a1'41

concentration of AcuTect to achieve a 50% inhibition of fibrinogen (IC5o) binding to platelets was 6.8 nmol/L. In contrast, the IC5o was 12,900 nmoi/L for vitronectin binding to platelets. The phase III clinical studies comparing AcuTect with contrast venography have been reported.3~ Patients within 10 days from the onset of DVT signs and symptoms were injected with 740 to 925 MBq (20 to 25 mCi) of 99mTc*P280 containing 75 to 100 lag. Whole-body images showed rapid renal and gastrointestinal (GI) excretion. At 120 minutes postinjection (PI), activity was predominantly in the liver, GI, and bladder. The kidneys excreted 60% to 75% of the activity, and the liver excreted the rest. At 1 hour PI, there was only 5% of the injected dose (ID) in the circulation. Soft tissue uptake was minimal. Serial anterior and posterior images of the pelvis, thigh, knee, and calf were obtained. Their data suggested that specificity could be improved if 2 or more sets of sequential images were combined. In a comparison with contrast venography, blinded readers determined the sensitivity, specificity, and agreement rates as 73%, 68%, and 69%, respectively. The sensitivity seemed to be a bit better in the calf (83%) versus the knee (69%) and thigh (63%). The other values were similar. When patients who presented within 3 days of onset of DVT symptoms were included in the analysis, the overall values were improved. If the clinical information was taken into consideration, the sensitivity, specificity, and agreement rates were 91%, 84%, and 87%, respectively. Combining all this information with the disease prevalence of 20% to 50%, AcuTect scintigraphy provided a high

negative predictive value of 90% to 97%. Anticoagulant therapy did not appear to influence the agreement rate. Two different sets of images, one obtained early and the other at 1 to 2 hours after injection, gave the best detection rate. These data were encouraging, indicating that this peptide may finally serve as a replacement for 125I-fibrinogen. Whereas DVT is a difficult clinical problem that has long awaited a solution, the detection of PE has been even more difficult. A very preliminary study in only 10 patients has shown that AcuTect may be useful for this disease.32 A much larger controlled trial is required. DMP-444

The DuPont peptide, [cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5(6-(6-hydrazino nicotinamido)hexanamide)))(HYNICtide)], which includes the coligands tricine and trisodium tfiphenylphos-phine3,3',3"-trisulfonate, is designated DMP-444. 3 The molecule consists of a rigid RGD analogue, a linker, 6-aminocaproic acid (Aca), attached to the rigid m-(aminomethyl)benzoic acid (Mamb), and for 99mTCbinding, a hydrazinonicotyl (HYNIC) group is coupled to the Aca. The unlabeled peptide has an IC5o of 6 nmol/L for human platelets, comparable with AcuTect. Preparation is also similar and requires heating at 80~ for 30 minutes. Until the phase III studies are reported, only the abstracted data are available. DMP-444 has low soft-tissue uptake and rapid renal washout. 33'34 The spleen is the target organ, with an absorbed radiation dose of 0.055 mSv/MBq (0.2 rad/mCi). The peptide had no effect on coagulation parameters in the blood and platelet function. In a comparison with venography, 86 patients were injected with 740 to 925 MBq (20 to 25 mCi) of 99mTc-DMP444. 28.35 Images were obtained at 1 to 3 hours and 4 to 6 hours PI. When they interpreted blindly, without clinical information, the sensitivity, specificity, and agreement rates were high (90%, 91%, and 91%, respectively). In another comparison with venography in 19 patients undergoing knee replacement, DMP-444 scintigraphy was highly sensitive (91%) but not specific (75%), even though most DVT were present in the calf.35'36 In the 26 patients, heparin delayed the positive scan by approximately 3 hours. 35

Bitistatin

Knight et al have investigated a new class of large peptides (5 to 9 kd) called disintegrins. 37 They are found in viper venom and bind to the GP IIb/IIIa platelet receptor with high affinity. These peptides were labeled with ]23I and were studied in dogs. The best disintegrin was bitistatin. It localized in the spleen but not the liver, making it is easier to image clots in the lungs. There was low soft-tissue uptake compared with similar peptides. Its IC5o for human platelet aggregation was intermediate (165 nmol/L). However, at 4 hours PI, this peptide had the highest incorporation in both venous clots (0.21% ID/g) and PE (0.64% ID/g) compared with all other agents, including labeled fibrinogen and platelets. More recently, this peptide has been labeled with 99mTCby using the HYNIC approach similar to DMP-444. 38 The dissociation constant (Kd) for binding to activated platelets was 32 nmol/L, comparable with 125I-bitistatin. At 4 hours PI, the 99mTcpeptide

300

had very high incorporation in both venous clots (0.79% ID/g) and PE (0.89% ID/g). DVT and PE were detectable as early as 30 and 60 minutes PI, respectively, for venous clots and PE. Bitistatin had very high lesion-to-background ratios: DVT-to-blood (18), PE-tomuscle (284), and PE-toqung (34). Blood clearance is rapid, with a half-life of 8.9 minutes (55% of activity), and the remainder half-life is 5.8 hours. The activity distribution was - 6 0 / 4 0 cells versus plasma at 1 hour. These results compared favorably with those obtained with 99mTc-P280 studied in this model. Thus, bitistatin appears to be a very promising agent. This peptide is too large for efficient synthesis, but the recombinant peptide has been prepared. 39

Fibrin-Binding Domain The initial fibrin-binding domain (FBD) peptide consists of 5 repeated sequences, which contain 20 cysteine residues with a MW of 31 kd. 4~ These cys form 10 disulfide bonds, which constrain the sequence into 5 flngerlike loops per sequence ("5-finger"). A variety of binding sequences labeled with H lln were tested in rats, but a 12-kd sequence ("2-finger") was found to be superior. This sequence had somewhat smaller IC5o of - 4 laM (versus - 2 pM for 5-finger) for binding to fibrin but had a faster clearance (8-hour blood values for 31-kd segments were 2 to 3 times higher). Heparin did not affect the uptake of FBD in the clots. Rabbit studies showed that clot-to-blood ratios of --40:1 could be achieved at 24 hours. It is produced by recombinant gene technology and has a sequence identical to a portion of fibronectin. The initial studies were performed with m l n - F B D but were quickly switched to 99mTc-FBD,but the nature of the 99mTc-FBD preparation has not been published. In the phase II trial, 41 patients were injected with 15 to 25 mCi of 99mTccontaining 250 to 500 lag of peptide. 3~ The DVT diagnosis was determined by ultrasound and the d-dimer test. Anterior and posterior images of the calves, knees, thighs, and pelvis were obtained at 0.5 and 2 hours, and 4 to 6 hours. Thirty-eight patients were diagnosed with "definite" DVT and 3 with "probable" DVT. The sensitivity of 99mTc-FBD was low at 0.5 hours but was comparable (73% to 83%) if 2-hour results were compared with 4-hour or 6-hour imaging sets. When patients with probable DVT were included in the comparison, the sensitivity improved to 88% to 93%.

Promising Compounds Thakur et al4a have explored another approach. These investigators studied a peptide from the N-terminal of the c~ chain (head) of fibrin that binds to the C-terminal -,/ chain (tail) (Fig 2B), designated TP 850. Rabbit biodistribution showed mainly kidney excretion with less liver uptake. Blood washout was very rapid with a half-life of - 4 min (20%) and - 1 3 min (80%). The IC5o for binding of TP 850 to activated rabbit platelets was 167 pM, which was the lowest of all compounds tested. Both 99mTCpeptide uptake in venous clots and clot-to-blood ratios were modest and ranged from 0.01% to 0.09% ID/g and 1.2 to 12, respectively. In contrast with the other agents actively investigated, this agent has the

WEINER AND THAKUR

potential to detect both acute and aged clots (chronic DVT and PE). TP 850 depends on fibrin binding and not on the accumulation of activated platelets. I N F L A M M A T I O N DETECTION A G E N T S

Background Currently, 111In-labeled white blood cells (111In-WBC) and, more recently, 99mTc-labeled WBC (99mTc-WBC),with the use of similar methods, have supplemented 67Ga-citrate for imaging acute inflammations and infections.42 Whereas m l n * W B C has proved very useful for imaging infection/inflammation, as a technique, there are certain limitations. The radiopharmaceutical dose is limited to 500 pCi because of the high absorbed dose to the spleen, approximately 30 rad/mCi. For the preparation of alaln or 99mTc-WBC, blood must be removed from the patient, and the cells must be separated, labeled, and then reinjected. The procedure is time-consuming (11/2 to 2 hours), and most community hospitals lack the required labeling facilities. Practically, this adds 1 to 2 hours transportation time if the blood is sent to a radiopharmacy for labeling. Probably the most serious problem for both types of tracers is with imaging at 24 hours PI.42 99mTc-WBC permits the use of higher activities of the tracer and provides a nuclide with better imaging characteristics than alaln. However, it has not eliminated the preparation problems. The 99mTClabel is less stable than rain, although infections can be detected as early as 4 hours PI. 43"44However, for unequivocal diagnosis, imaging at 24 hours must also be performed. 45 Early uptake by the lungs, and the renal and hepatobiliary excretion with subsequent elimination of 99mTcinto the GI tract, complicate the use of 99mTc-WBC. Because of the problems associated with both tracers, investigations have continued to develop a variety of new agents. The main thrust of these new methods has been to target antigens or receptors expressed on the WBC in vivo (Table 3). Two of the peptides, P483H and RP-t28, have entered clinical trials. Three other compounds; chemotactic peptide, human neutrophil peptide, and elastase inhibitor, have unique characteristics but have only been studied in animals.

P483H This peptide, developed at Diatide, contains a heparin-binding sequence from the C-terminus of platelet factor-4 (PF-4) that facilitates cell binding. A lysine-rich sequence was added to the N-terminus to enhance renal clearance. 46 The sequence is shown in Fig 3A. PF-4, a 29-kd protein secreted by activated platelets, binds heparin. This complex binds to circulating and inflammation-activated WBC. The preparation is simple and just requires a short incubation after the addition of 99mTc. Heparin binding to the peptide is neces-sary for the P483 localization in inflammatory lesions. Preliminary studies were performed 47 in 30 patients given 3 different peptide concentrations: 29, 145, and 290 pg with 585 to 740 MBq of 99mTC,imaged at 15 minutes and 60 to 120 minutes. Activity is cleared by the kidneys and liver and is seen in the bowel at 120 minutes. In the images, unexplainable high lung uptake was seen. The peptide concentration did not appear to influence either the distribution or the accuracy of the results, although the number

301

RADIOLABELED PEPTIDES

Table 3. Peptides for Inflammation Detection Peptide Chemotactic

peptide P483H

RP-128

Elastase inhibitor

Target Receptor Formyl poptide receptor on PMN Specific PMN binding site unknown Tuftsin-binding

Function Stimulates chemotaxis Unknown

Stimulates

site on PMN,

chemotaxis &

monos and

phagocytosis

macrophages Noutrophit elastase

Prevents widespread tissue destruction by

elastase Human

Cation-binding

neutrophil peptide-1

Bactericidal

sites on bacteria C5a receptors on

TP 750

PMN

RP-128

Stimulates chemotaxis

Data from Okarvi, a Moyer at al, 4a Caveliors et al, 4a Dorian et al, as Rao et al, s9 Hancock et al, 94 and Rusckowski at al. 97

of patients in each category was small. Only 17 patients had a positive diagnosis. Even though the numbers were small, the sensitivity, specificity, and accuracy were 82%, 77%, and 80%, respectively. This is somewhat lower than the reported values for 99mTcWBC.42 There were 3 false-negative diagnoses: diverticulitis, femoral osteomyelitis, and otitis. There were also 3 false-positives: tumor, Charcot's joint, and loose prosthesis. Seventeen patients were compared directly with ~]qn*WBC and had comparable accuracies (76% [P483H] v 82%) from the final diagnoses.

RP-128, a small peptide, is directed to the tuftsin receptor. 4s Tuftsin, a leukokinin-derived tetrapeptide, stimulates chemotaxis and phagocytosis of polymorphonuclear (PMN) leukocytes, monocytes, and macrophages. RP-128 consists of a 5-amino acid receptor antagonist linked by a Gly to a N3S amino acid 99mTC binder (Fig 3B). This antagonist has a 4-fold greater affinity to the receptors than the tuftsin itself. The preparation requires a 30minute incubation with 99mTcO4. Two very small studies of clinical efficacy have been reported. '.s'49 In 8 healthy individuals, the injection of an average of 282 MBq caused no adverse events and caused no change in vital signs, hematologic parameters, or biochemical parameters. 48 At 4 hours PI, 64% of the activity was excreted, primarily through the kidneys, and at 24 hours only 1% was left in the circulation. The bladder wall received the maximum absorbed dose, 0.076 mGy/ MBq. Almost no activity was bound to blood cells. RP-128 was tested in 10 patients with rheumatoid arthritis by using a dose of 475 MBq. The correlation of the radioactivity uptake with pain, swelling, or joint erosion was modest. Among those 3 criteria, however, the best correlation for peptide uptake was with erosion. In this small number of lesions, the sensitivity, specificity, and accuracy were 73%, 64%, and 68%, respectively. However, there was a very high percentage (36%) of false-positives. Also, these results did not compare favorably with the use of endothelial leakage indicator, 99mTc-IgG, which showed a sensitivity in the range of 87% to 92%. Because RP-128 has little GI excretion, it was tested for the detection of active Crohn's disease. 49 Unfortunately, meager data were reported. Patients identified only by histology and endoscopy with active disease were imaged within 4 hours PI. In these patients, little binding of RP-128 to WBC binding in the blood was noted. It would seem, therefore, that the localization mechanism needs to be further clarified. Also, studies of the N3S influence on receptor binding would be informative. Bracco Inc, owner of rights to RP-I28, at present does not plan to pursue further clinical trials.

Chemotactic Peptide Analogues

A acetyI-Lys-Lys-Lys-Lys-Lys-Cys-Gly-Cys-Gly-Gly-Pro-LeuTyr-Lys-Lys-lle-Ile-Lys-Lys-Leu-Leu-Glu-Ser.

B

OH

O

CO

I Gly-Thr-Lys-Pro-Pro-Arg-OH Fig 3. The sequence and proposed structure of Infection detection agents (A) Dlatlde's P483 and (B) Resolution Pharmaceuticals' Psptlde, RP-128 (rights recently sold to Brecco Inc). 46,4a

The most extensively studied chemotactic peptides have been derivatives of N~-formyl-Met Leu-Phe. This peptide is an analogue of peptides secreted by bacteria that promote the migration of both PMN and monocytes to the site of bacterial invasion.5~ These peptide derivatives, mainly labeled by the HYNIC technique, have been studied in a number of different animal models.55"~s In a rabbit model, it was concluded that the 99mTcwas transported to the inflammation site mostly by proteins. However, at the site, ~90% of the activity was cell-bound, and the low radioactivity in most tissues led to high target-to-background (T/B) radioactivity ratios of ~26 at 17 hours PI. 52"54 Nonetheless, in a more relevant model, a rhesus monkey, a thigh inflammation was barley visible at 3 hours PI and was not visible at 15 hours. 52 The peptides also caused neutropenia. More recent work has been directed to increasing the stability of the 99mTc complex and chemically modifying the peptide to improve antagonist formyl peptide receptor binding.55'56's9'6~

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Antimicrobial Peptides A recent novel approach has been to use radiolabeled antimicrobial peptides. 61-63 These peptides are synthesized in PMN and stored in granules; they contribute to bacterial killing by binding electrostatically to the membranes and inducing membrane permeability. 64 These peptides should hopefully be able to differentiate between an infectious and noninfectious inflammation and monitor the effectiveness of antibiotic intervention. Human neutrophil peptide-l, 3 kilodaltons, has been shown to have the typical rapid blood clearance washout and rapid uptake in bacterial-induced lesions in mice. 61'62 The uptake peaked at 15 minutes PI but then declined. The T/B ratio values were very modest, with a maximum of 4 at 15 minutes. The biodistribution showed the excretion pathway was equally distributed between the kidney and GI tract. The high kidney, liver, and GI activity present would make detection of an inflammation in the abdomen difficult. When activity was recovered from a peritoneal infection model, there was a 1,000-fold greater 99mTc binding to bacteria than to WBC. However, a large fraction of the activity, 40% of 99mTC, was present in the washing fluid. In addition, statistical analysis showed a higher correlation with macrophage concentrations (r = 0.97) than bacterial concentrations (r = 0.81). It must be noted that WBC concentrations were 100-fold higher than the number of bacteria, which could explain the better correlation with macrophages. Thus, this peptide may have difficulty distinguishing between infectious and noninfectious lesions. Shorter (1.2 to 1.7 kd) active segments from another antimicrobial peptide, ubiquicidin, which would be easier to synthesize, showed similar results. 65"66

Human Neutrophil Elastase Elastase is an enzyme secreted by activated PMN in response to inflammatory stimuli. To prevent widespread tissue destruction by this enzyme, a high-affinity (-pmol/L) inhibitor is present in tissues. With the use of phage display technology, peptide analogues of this inhibitor have been developed. 67'68 One of these large peptides (--6 kd, designated EPI-HNE-2) has been labeled with 99mTc and studied in a monkey model of inflammation/ infection. Blood clearance was typically rapid, with a half-life of 7 minutes. Infections were visible at --3 hours PI, and T/B were --2.5 at 4 hours. Uptake appeared to be specific. A recent investigation to reduce tissue background showed that labeling methods for this peptide had a large effect on the biodistribution and renal excretion. 68 ONCOLOGY

Diagnostic Agents Table 1 gives examples of 16 peptides whose receptor specificity and target disease have been well characterized.

111In-OctreoScan The diagnostic use of mIn-OctreoScan has been extensively reviewed, and the reader is directed to these reviews for further details. 69-71 Somatostatin is a hormone that inhibits the release of

growth hormone, insulin, glucagon, and gastrin (Fig 1A).72 Somatostatin receptors (SSTR) have been identified on many cells of neuroendocrine origin, eg, anterior pituitary and the islet cells of the pancreas. Lymphocytes and monocytes also express SSTR. At present, 5 subtypes of the SSTR have been cloned. More importantly, tumors of neuroendocrine origin have increased expression of these receptors. Somatostatin was initially studied as an antitumor agent because of its antiproliferative effects on cultured tumor cells, on animal tumor models, and on neuroendocrine tumors in humans. However, its extremely short blood half-life (2 to 4 minutes) allowed little tumor uptake. Somatostatin was modified to yield an 8-peptide analogue (1,400 d). This analogue, octreotide, with a longer half-life (1.5 to 2 hours), yielded higher tumor uptake (Fig 1B). With this longer half-life, the localization and imaging of tumors with radiolabeled octreotide was possible. Initially, Krenning et a173 investigated the use of radioiodinated Tyr-octreotide to detect neuroendocrine tumors. Subsequently, diethylenetriamine pentaacetic acid (DTPA) octreotide was developed, which permitted the use of rain as a tracer (Fig 1C). 74 In 1994, rain-labeled DTPA octreotide (OctreoScan) became the first peptide-based agent approved by the FDA.

Normal Localization This radiopharmaceutical is rapidly cleared from the blood, leaving only 33% of the ID in the circulation at 10 minutes PI, and it is excreted swiftly into urine.9 Twenty-five percent of the injected radioactivity is present in the urine after 3 hours, 50% after 6 hours, and 90% after 48 hours. Hepatobiliary excretion is minor; less than 2% appears in feces after 72 hours. After intravenous administration, accumulation of mIn-OctreoScan is observed in the thyroid gland, pituitary gland, liver, spleen, kidney, and bladder. Gallbladder and intestinal activity are seen only at later time points. The spleen is the critical organ, followed by the kidneys and bone marrow, receiving 0.67, 0.49, and 0.03 mGy/ MBq, respectively. Initially, there is little metabolism of lalInOctreoScan. At 4 hours, the rain is still bound to the intact peptide in the blood, and only 10% of the total activity in the urine is nonpeptide-bound.

Indications OctreoScan localizes in a wide variety of tumors of neuroendocrine origin, eg, carcinoids, islet cell tumors, certain central nervous system (CNS) tumors, paragangliomas, pheochromocytomas, and gastrinomas.7~ Localization of these tumors is important because these diseases can be successfully treated with surgery. Figure 4 shows the images of a patient with a gastrinoma before and after tumor resection. In addition, identification of OctreoScan-positive lesions indicates patients that could benefit by octreotide therapy, v6 There is a strong correlation between a positive image in neuroendocrine tumors and an in vitro assay of SSTR in excised tumor tissue. 69 This suggests that mInOctreoScan is an in vivo indicator of SSTR. However, octreotide has a high affinity for only the 2 and 5 receptor subtype. 77 It appears that SSTR2 is the most frequently expressed subtype. 76'78 Because of the presence of SSTR in renal cell cancer, breast tumors, Hodgkin's, and non-Hodgkin's lymphoma, the usefulness

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A

D

E

B

(3

Fig 4. 1111n-OcteoSean Images of patient before (A-C) and after (D and E) surgery for removal of a gastrlnoma lesion. (A) Anterior, (13) posterior abdominal planar, and (C) transverse single photon emission computed tomography (SPECT) Image (24 hours PI, 6 mCI) showing anterior mldllne lesion. (D) Anterior and (E) posterior whole-body planar Images (4 hours PI, 6 mCI) after surgery.

of Ilqn-OctreoScan has been examined to image these tumors.79s2 A number of studies have appeared on the efficacy of OctreoScan in chronic infections where lymphocytes predominate.83-8S ~HIn-OctreoScan is well established for the cost-effective diagnosis of tumors of neuroendocrine origin, eg, paragangliomas, gastroenteropancreatic (GEP) tumors, carcinoids, and gastrinomas. 7~ It has limited diagnostic usefulness with pituitary tumors, islet cell tumors, medullar thyroid cancers, neuroblastomas, and pheochromocytomas. 7~ The sensitivity of 1HInOctreoScan ranges from 60% to 90%. 69'74 It has been argued that the lower sensitivity may be caused by the absence of SSTR in some lesions. 86 The T/B in various neuroendocrine tumors correlated directly with the mRNA assay for SSTR2. s7 In a careful study with a large number of patients (146) with gastrinomas, the specificity was 86%. 75 The main concern was false-positives that might alter patient management (eg, require surgical resection). False-positives occurred in about 12% of all patients. Extraabdominal (2/3) false-positive lesions were more common than intra-abdominal (1/3), with thyroid, breast, and granulomatous lung lesions the most frequent causes. A comparable specificity (81%) and a high sensitivity (96%) were also recently reported in another large group (253) of GEP tumors, ss OctreoScan can detect primary lesions in both small cell lung cancer (SCLC) and non-small cell lung cancer (nSCLC). 89-92 However, SSTR has been detected only on SCLC and not on nSCLC cells. 72"9~ At present, there is no consensus that this scintigraphy would add additional diagnostic information to alter

patient management,s9"93 In the diagnosis of SCLC, it is important to differentiate between patients with limited versus extensive disease. One study showed that OctreoScan imaging led to upstaging for 5 of 14 patients. 91 In contrast, Kirsch et als9 found the technique highly sensitive, but it did not provide any additional information not already provided by conventional imaging techniques. In Hodgkin's and non-Hodgkin's lymphoma, studies suggest that OctreoScan will not be able to challenge the dominance of 67Ga-citrate. For example, in Hodgkin's disease, sensitivity was high (88%) in the neck and chest but very low in the abdomen (13%). 81 In non-Hodgkin's, sensitivity was low (35%). In a more recent study with low-grade non-Hodgkin's in which 67Ga has poor sensitivity, the sensitivity for OctreoScan was not sufficient to be useful in initial staging. 79 In addition, a high percentage (38%) of lesions identified by other imaging modalities were missed by Hlln-OctreoScan scintigraphy. Specificity was very high (98% to 100%), suggesting that SSTR may not be expressed on all lesions. In sarcoidosis, an initial study showed that OctreoScan could detect lesions and monitor disease progression.84 In a direct comparison with 67Ga, Lebtahi et a183 showed that the accuracy of Hqn-OctreoScan scintigraphy compared favorably in overall patient evaluation. The patient numbers in both studies were small (46 and 18), and additional investigations are needed.

99mTc-NeoTect Because =~qn-OctreoScan could detect both SCLC and nSCLC with high sensitivity, Diatide Inc developed a 99mTc-based somatostatin analogue for lung cancer detection (Fig 5A). The objective was to provide a noninvasive method to differentiate malignant versus benign single pulmonary nodules (SPN) or masses detected on computed tomography (CT) or chest x-ray. The detection of SPN usually requires a thoracotomy or needle biopsy because only 28% to 39% of these are determined t o be malignant.94 Both thoracotomy or needle biopsy are associated with significant morbidity and cost. Positron emission tomography (PET) imaging

N-Me-Phe--Ty~,~ D-Trp

A

I

Lys

Lys-Cys-Lys-~Ala-Hcys~Val ~ B

His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys GIn'Mct'Ala'VaI'Lys'Lys-Tyr-Lcu" Asn"Scr'llr

C

Hz

DTPA-Leu-GIu-Glu-Glu-Glu-GIu-Ala-Tyr-Gly-Trp-Met-AspPheNH=

D

pGlu-Gln-Arg-Lcu-Gly-Asn-Gln-Trp-Ala-VaI-Gly-His-LeuMet-NH 2

Fig 5. Schemstlc presentstlon of (A) NeoTect (P829), (B) vasoactlve Intestinal peptlde, (C) OTPA-mlnlgastrln, and (D) bombesln. Amino acids in bold are directly Involved In receptor binding. Amino acids are all L-Isomers except where notad. Unusual amino acids: H-cys, homo-Lcye; aaAla, acetylamlnoAla; N-Me-Phe, N-msthyl-Phe. s,s,~,l~

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range of other effects (Table 1). VIP promotes both the growth and proliferation of normal and malignant cells. More importantly, VIP action is receptor-mediated, and the receptors (VIPR) are upregulated in a variety of diseases (Table 1). Two receptor subsets have been identified. In addition, VIPR has a wide distribution in normal lung, GI tract, peripheral blood cells, and kidney. VIP was synthesized by using solid-phase synthesis and was then labeled with 1231by using the iodogen method. 98 The product was purified by passage through a high-pressure liquid chromatography reverse-phase C18 column to remove the unlabeled VIP and unreacted I231. The usual dose was 150 to 200 MBq, containing less than 1 lag of peptide. The normal distribution showed mostly lung activity, which constituted 40% of ID. Liver, kidneys, and bladder showed similar amounts of uptake (4% to 7%). All tissue activity declined as a function of time. Lung activity declined to 8% at 24 hours. The kidneys were the main excretion route. About 5% of the 123Iwas present in the feces at 24 hours, le3I-VIP was rapidly cleared from the blood, with less than 5% of the injected activity present at 5 minutes. The only side effect was a modest (5% to 10%) and transient (10 minutes) drop in blood pressure. The thyroid (blocked) received the highest absorbed dose (0.104 mGy/MBq), with the bladder and lungs next at 0.077 and 0.067 mGy/MBq, respectively. 123I-VIP was initially studied in patients with colorectal cancer, pancreatic and gastric cancer, and carcinoid tumors. 1~ Although the number of patients in each category who had identified lesions was small, the results were promising. Both primary and metastatic 123I- Vasoactive Intestinal Peptide lesions in liver lung and lymph nodes were visualized. In a larger Virgiolini et al have promoted the use of 123I-vasoactive series of patients with carcinoid tumors. OctreoScan was found to intestinal peptide (VIP), but it still remains investigational.98-1~176 be superior to 123I-VIP.1~ 123I-VIPhad a slightly lower sensitivity VIP is a 28-amino acid peptide that was first isolated from porcine (-80%) compared with OctreoScan (-90%) in detecting both primary and recurrent tumors. Also. mIn-OctreoScan identified duodenum (Fig 5B). As it name implies, VIP is a potent vasodilator and has effects on both peripheral and pulmonary blood pressures. more lesions 141% v 25%) not detected by other imaging modalities. The combination of the 2 agents did not improve the It is effective even at pM (1 ktg/3,000 mL) quantities. It has a wide

is very useful for determining the malignant character of these masses. However, this technology is expensive and its availability is currently limite&95 99mTc-NeoTectis easy to prepare. 96 Blood half-life is short (t,~= 4 minutes), and imaging is performed at 2 to 4 hours PI. In healthy individuals, the agent localizes in the kidney, spleen, bladder, thyroid, and bone. The kidney is the critical organ, with an absorbed dose of 0.089 mGy/MBq (0.33 rad/mCi). At 4 hours PI, 70% to 80% of the activity in blood and 61% o f excreted radioactivity in urine still bound to NeoTect. Protein binding is small, only 10% to 20%. NeoTect, recently approved, 96 has a modest sensitivity, specificity, and accuracy (70%, 79% to 86%, and 72% to 74%, respectively) for determining the malignant character of SPN. However, there was a high percentage of both false-negatives (29%, 1- to 7-cm lesion size on CT) and false-positives (17%, 7/42, inflammations). Figure 6 shows 99mTc-NeoTect images of a patient with chronic obstructive pulmonary disease (COPD) with SPN detected in the left lung on CT. Focal activity seen in the right lung had no corresponding density on CT. NeoTect has high affinity (Kd - 2 nmol/L) for SSTR subtypes 2, 3, and 5, 97 Even though the presence of SSTR has not yet been shown on nSCLC, it has been argued that the high sensitivity in this disease is caused by lymphocytic cells or other SSTR-containing cells at the site. Adverse reactions to NeoTect were minor (4% to 5%).

Fig 6. 99mTc-NeoTect image, 4 hours PI (28 mCi), of a patient with COPD who presented with suspicious-looking lesions in the left lung on CT. (A} Anterior, (B) posterior planar chest, (C) transverse, (D) coronal, and (E) sagittal SPECT Images show focal area of uptake in right lung but not left. Subsequent CT confirmed presence of lesions.

D im

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RADIOLABELED PEPTIDES

305

diagnostic information. Thus, 123I-VIP would not be a costeffective replacement or competitor for Hlln-OctreoScan in detecting neuroendocrine tumors. In contrast, ]23I-VIP could fill a diagnostic gap in detecting pancreatic cancer.l~176 For this disease, surgery is currently the only therapeutic option. Even after resection, there is a very high recurrence rate. CT is the most reliable imaging technique, but because of the lack of specific symptoms, pancreatic cancer is usually diagnosed at an advanced stage of the disease. The 5-year survival rate is very low (3%). In a study of 60 patients, ]23I-VIP had sensitivity in detecting patients with only a primary lesion (90%). This agent was not as good in patients with distant metastases and local disease. It could detect only 32% of the primary lesions but had high sensitivity with liver disease (90%). Where this agent might have a big advantage was in 5 patients for whom disease was detected by ]23I-VIP before CT became positive. In a direct comparison, ]23I-VIP was superior to ]]]InOncoScint for identifying the lesions in colorectal carcinoma. ]~ Two groups of researchers have developed 99mTc-labeled VIP analogues. 1o4.]o5 99mTc_P1666 has been administered to 15 patients with GI cancerJ ~ Lung activity was lower (22% v 40%) than ]23I-VIP and declined sharply to less than 0.4% at 24 hours PI. Dosimetry was comparable with ]23I-VIP, with the bladder getting the highest dose (0.020 mGy/MBq). However, side effects were more severe, and more than 50% of patients experienced mild to moderate effects. In contrast, Thakur et al ]~ observed no side effects with their analogue, 99mTc-TP-3654, in 16 patients. This compound is VIP with a spacer and an added peptidic chelator. Blood clearance was rapid, with most activity excreted in the urine (70%) and the remainder (30%) through the liver. In healthy volunteers, lung uptake was only 4.5% ID. Activity was present in the bladder, kidneys, and gallbladder. In the patients, 99mTc-TP3654 was positive where it was supposed to be positive. Figure 7 shows a patient diagnosed with an osteosarcoma and injected with 99mTc-TP-3654. More studies are necessary to determine if either of these agents has an advantage over 123I-VIP, but their results were encouraging.

Cholecystokinin-B/Gastrin, Bombesin, and Epidermal Growth Factor The receptors for these 3 compounds are under active investigation as targets, but only very preliminary results have been

reported. In each case, investigators are still trying to determine which peptide analogue is the best. Compounds directed at the cholecystokinin-B (CCK)-B/gastrin receptor are the furthest along in development.l~176 CCK and gastrin are involved in regulating various functions in the GI tract (Table 1). Similar to the other peptides already discussed, CCK can act as a growth factor for normal and malignant cells. 1]~ The actions of CCK are mediated by 2 receptors, CCK-A and CCK-B, which have, respectively, a low and high affinity for gastrin. Using receptor autoradiography, Reubi et al have shown a high incidence (90%) of CCK-B receptors in medullary thyroid cancers but not in differentiated thyroid cancers. Behr et al have used a variety of gastrin derivatives conjugated with 1311, 1HIn, and 9oy in pilot experiments to detect and treat medullary thyroid cancers. 1~176 In the most recent study, an ]]]In-labeled DTPA derivative of minigastrin (Fig 5C) was studied in 35 patients (111 to 185 MBq).]06 There was expected uptake in the stomach, and the main route of excretion was the kidney. No uptake was observed in the liver or spleen. Sensitivity appeared to be high (88%) compared with other imaging modalities, but there was no explanation to identify which lesions were missed or why. Nonetheless, the CCK-B/gastrin receptor agent seems to be potentially useful. Bombesin, a 14-amino acid peptide (Fig 6D), originally isolated from frog skin, has a high affinity for gastrin-releasing peptide receptor (GRPR). Bombesin belongs to a peptide family that includes GRP, a 27-amino acid and neuromedin B, a 10amino acid peptide derived from pig tissue. GRPRs are overexpressed in a variety of cancers, and their activation stimulates both cell growth and proliferation (Table 1). Studies with these receptors have only been performed in cultured cells or with animal models. However, a number of groups are examining different analogues. 8"]]~'H2 These investigators have been able to produce analogues that retain their affinity for the receptor after labeling with either 99roTe or ~]lln. Epidermal growth factor (EGF) is a rather large peptide (5 kilodaltons) that is involved in cell growth, as its name implies. In 30% to 60% of breast cancer biopsies, its receptor (EGFR) is overexpressed 100-fold compared with normal cells. I]3 This receptor is an attractive target for both diagnostic and therapeutic probes because its up-regulation is inversely correlated with estrogen receptor expression. Up-regulation is also directly correlated with poor response to hormonal therapy. Patients identified with widespread hormone-resistant disease may be candidates for systemic chemotherapy. Early studies were performed in cultured cells and animals. HaH7 Labeling with ]|~In or 99mTc did not reduce the receptor affinity,~a't~5.~~7 and in animal models, tumor uptake was specific.

Therapeutic Agents L Bone

Background MIBI

VIP

Fig 7. VIP receptors are expressed on adenocarclnoma. A 50-year-old man with adenocarclnoma of the right shoulder was given 10 mCI of 99mTc-TP-3654 (VIP). The shoulder mass (VIP) was detectable within 15 minutes PI (anterior). Sestamlbl scan was also positive (MIBI) with less Intense uptake, and bone scan (bone) shows Increased bone uptake (anterior). (Reprinted with permlselon. 1~

A number of factors influence the development of therapeutic agents and their clinical use. Radionuclide therapy is usually proposed for patients with widespread disease that is not amenable to focused radiation therapy or is refractory to chemotherapy. This is particularly true of neuroendocrine disease. Whereas various somatostatin analogues have been successful at alleviating syrup-

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toms, these peptides have failed in advanced disease. H8 With the success of Zevalin H9 (IDEC Pharmaceuticals, San Diego, CA) and Bexxar ~2~ (Corixa, Seattle, WA) for the radioimmunotherapy for B-cell lymphoma, commercial interests are now more likely to support research for developing a radionuclide-labeled therapeutic agent. For therapeutic purposes, variations in tumor physiology are important. If the tumor mass is well vascularized, the small peptide can easily diffuse into the tumor mass. It can then bind uniformly via the receptor to cells that are distributed throughout the mass. If a peptide-radionuclide complex is bound to the cell's surface, it may or may not be internalized. The tumor need not be welt vascularized, and the pepfide receptor complex need not be internalized if the radionuclide is a high-energy [3-emitter, eg, 9Oy (maximum 2.3 MeV; particle range, 1,000 pm; - 1 0 0 cells). Cell killing could take place with the nuclide only on the cell's surface or at the tumor's periphery. A nuclide with this range allows for the possibility of "crossfire." Cells can be irradiated in the nearby vicinity or near a necrotic central zone. However, toxicity could increase by irradiating normal cells. Another downside of the 9Oy label is that this nuclide does not have a ",/-photon for imaging. Alternatively, highdose I~In therapy with its Auger electrons is a possibility. Auger electrons have a very short penetration range (0.02 to 10 larn) in tissue (cell diameter ~ 10 pm).~2~ Nuclear localization is essential for this therapeutic effect because the Auger electrons have short penetration distance, and a double-strand DNA break is the most lethal lesion to the cell. 122 This means that Hlln can potentially be used for therapeutic applications if the complex containing the nuclide could be internalized in the target cell. This would allow the Auger electrons to interact with critical cellular components, particularly the DNA, while causing minimal toxicity to normal cells.

A

B

1]]I n - O c t r e o S c a n

Krenning et a172 first showed that the biologic half-life of mIn-OctreoScan in tumors was more than 700 hours. Then it was shown in vitro that the mIn-OctreoScan was internalized into cells and localized in the cytoplasm and near the nucleus. 123 More recent data support the nuclear localization of 1~1inOctreoScan. a24'lz5 Janson et al124 used tumor tissue from patients injected with mln-OctreoScan and showed that it was first localized on the tumor membrane, cytoplasm, and then finally inside the nucleus. In another cell culture study, mln-OctreoScan was incubated with receptor-positive and -negative cells, and then the nuclear cell fraction was isolated, a2s The presence of 111In in the nuclear fraction increased as a function incubation time only for the receptor-positive cells. A number of groups are using lalIn-OctreoScan for the treatment of patients with refractory neuroendocrine disease. 126-129 These studies are in the phase UII stage. More than 85 patients have been treated, with a 62% to 69% response, either stabilizing the disease or reducing tumor size. Figure 8 shows a patient treated with a high dose of mln-OctreoScan. A diagnostic dose was first used to identify SSTR-positive patients. In 1 study, patients were treated with 1 high dose (6.6 GBq). 13~ In other studies, patients were treated with doses ranging from 6 to 11 GBq with a slow 4to 6-hour infusion every 3 to 4 weeks, up to 6 times. 127'129A31 The cumulative dose for responding patients ranged from 20 to 74 GBq. The estimated absorbed dose for 6 to 7 GBq was 300 to 1,400 cGy for the kidney, 200 cGy for the spleen, and 13 cGy for the bone marrow. 127 The kidney was the dose-limiting organ, and the main toxicity appeared to be hemopoietic. Most patients

C

Fig 8. Effects of radionuclide therapy on a patient with hepatic metastasis from a neuroendocrine tumor. Patient received 7 doses, for a total of 1.8 GBq mln-OctreoScan. Panel A shows the planar image OctreoScsn (anterior [left] and posterior [right]) In a patient treated on the therapeutic protocol. Multiple areas of increased uptake of the radioisotope, including the liver, are evident. CT images from the pre- (B) and early posttreatment (C) scans show no significant change In the size of the tumors, but show extensive central necrosis (dark areas). Dosimetry of a large representative lesion in the left lobe indicates that the estimated dose of radiation to this lesion is 1,828 cGy. (Reprinted with permission from Weiner RE, Thakur ML: Handbook of Radiopharmaceuticals: Radiochemistry and Applications. John Wiley Publishers (in press). ~4s

RADIOLABELED PEPTIDES

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experienced a transient decline in the number of blood cells, grade 3/4 (National Institutes of Health grading). Platelet number was most affected and hemoglobin the least) 27"13~ Even though the kidney received the highest absorbed dose, the irradiation had little effect on kidney function. 127"13~

90y_DOTA_Octreotide As an alternative therapeutic approach, modified octreotide, D-PheLTyr3, has been labeled with another chelate, 1,4,7,10tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) (Fig 9). This chelate, D-PheLTyr3-octreotide-DOTA (DOTATOC), also identified as SMT487 (Novartis Pharma, Basle, Switzerland), has a high affinity for both Hlln and 9Oy (log KML = 29), and SSTR binding affinity remains high (K d -- nmol/L).~32 In contrast, 9Oy is not tightly bound to DTPA. In vivo, it is eluted from the DTPA and is then deposited in the bone marrow. The advantage of DOTA is that the H ~In-labeled derivative can be used to estimate the biodistribution (and therefore the dosimetry) of the 9~ 86y, a positron emitter, can be used for this purpose in laboratories equipped with PET. Otte et a1133'134 were first to treat 10 patients. Initially, mlnDOTATOC was directly compared with H]In-OctreoScan. H~InDOTATOC had the same diagnostic precision but was cleared faster from the blood and other tissues) 33 Six of the 10 patients received multiple treatments of 9~ with cumulative doses ranging from 6 to 12 GBq) 34 Two patients had tumor regression, 2 had stable disease, and 2 had partial remission. For a single dose, 2 had tumor regression and 2 had stable disease. There was minimal renal or bone marrow toxicity (9/10 < grade 1), consistent with the relatively low organ-absorbed doses estimated from baboons injected with 86y-DOTATOC)35 The critical organ

was the kidney, with 2 to 3 mGy/MBq. The liver received 0.32 to 0.53 mGy/MBq, and bone marrow received 0.03 to 0.07 mGy/ MBq. Infusion of amino acids reduced the kidney dose by 20% to 40%. In a dose escalation study, cohorts of 5 to 7 patients (28 patients total) were treated, starting with 1.1 GBq of 9~ and increasing in 0.4-GBq increments) 36 Patients received 3 equal doses 2 months apart. No major toxicity was observed up to 2.6 GBq per cycle, except that 1 patient developed delayed renal toxicity (grade 2) at 4.4-GBq total dose. The majority of patients responded with either some tumor reduction (25%) or stabilized disease (55%). Only 20% had progressive disease. Subsequently, this group has reported a continuation of the escalation with added amino acid infusion) 37 Most patients (60%) had GI toxicity after the amino acid infusion, but few had adverse reactions to up to 4.8 GBq per cycle of 9~ One patient had grade-3 hematologic toxicity, and 2 had grade-1 renal toxicity. Sixty percent of patients had objective responses. Phase II trials are planned, supported by Novartis to treat breast cancer and SCLC.138

9~ Virgiolini et al have developed this octreotide analogue as an alternative treatment. 139 DOTA-lanreotide (DOTALAN) binds SSTR subtypes 2, 3, 4, and 5 with high affinity (Kd -- 4 to 10 nmol/L). A few studies in patients have been performed) 4~ So far in these 8 patients, only 1 response has been observed. The biodistribution and uptake of 1]qn-DOTALAN was quite different from those of ~l~In-OctreoScan. Compared with 9~ 9~ yielded a much higher (20- to 30-fold) bone marrow dose, 0.94 to 1.67 mGy/MBq, ~a~ leading to more hematologic toxicity. This may limit the use of the analogue. ~=~In_DOTALAN must be used for biodistribution studies before therapy.

64Cu.TETA-Octreotide

A

"~176162 "- N.

coo.

/ \ COOH

B

DOTA-CO-NH-D-Phe-Cys ~ T y r \ SI D-Trp i I S i Lys Thr(ol)-Cys --- T h r ~ Fig 9. Schematic drawing of the structures of (A) DOTA and (B) DOTA coupled to 9 modified octreotlde (tyr replaces phe In position 3). Amino acids In bold are directly Involved In somatostatln receptor binding. Amino acids are all L-Isomers except where noted or an amino alcohol, Thr(ol). DOTA Is coupled to the peptldes via an amlde bond, and the methylene groups (CH=) are omitted for clarity, la4

Anderson et al developed a chelate, 1,4,8,1 l-tetraazacycllotetradecane-N, N',N",N'"-tetraacetic acid (TETA), to attach 64Cu to octreotide) 42 The advantage of this nuclide is that it has both a g" (0.579 MeV, 39%) and 13+ (0.653 MeV, 17%), and it can be made by a reactor or medical cyclotron. Thus, it could be imaged with a PET camera and provide therapy. However, the short half-life (12.7 hours) makes transport difficult. Eight patients with neuroendocrine disease were studied with both 64Cu-TETAOctreotide (111 MBq) and ~' ~In-OctreoScan. In 5 patients, lesion identification was identical. In 2 patients, ~Cu-TETA-octreotide identified more lesions, and it was the reverse in 1 patient. ~Cu rapidly cleared from blood, with only 8% of the ID remaining in circulation at 4 hours. The absorbed doses were low compared with other therapeutic analogues. The bladder wall received the highest dose (0.062 mGy/MBq), and the liver and bone marrow received 0.091 and 0.013 mGy/MBq, respectively.

mln-EGF I~'In-EGF has been tested in breast cancer cultured ceils and normal mice) 13 HIln-EGF is rapidly internalized in receptorpositive cells, and 15% was translocated to the nucleus. High-dose treatment reduced the growth rate of receptor-positive cells and decreased the surviving fraction by 100-fold. There was no effect

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on receptor-negative cells and no evidence o f hepatic or renal toxicity. This approach s e e m s promising because it parallels the application o f high-dose 111In-OctreoScan therapy.

ACKNOWLEDGMENT

The typing assistance of Priscilla Paretta is gratefully acknowledged.

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