THE UNIVERSITY OF NEW MEXICOCOLLEGE OF PHARMACY

ALBUQUERQUE, NEW MEXICO

The University of New Mexico

Correspondence Continuing Education Coursesfor

Nuclear Pharmacists and Nuclear Medicine Professionals

VOLUME IV, NUMBER 4

The Current Status of Radiolabeled Peptidesf or

Imaging and Therapy

by:

John W. Babich, Ph,D,, R. Ph.George H. Hinkle, M. S,, R.Ph., BCNP

Co-sponsored by: ➤Amersham HEALTHCARE

H ~c IJnivcrsity of New Mexico College of Wtirmacy k approval by the Am.ri.~ Council on Pharmaceutical Wucation w

t9 a provider of continuing pharmaceutical education, Program No. 039-000 -95-003-Hw. 2,5 Contnct Houm or .25 Cm’s

Eddor

Director of Pharmucy Cotiinuing ~ucation

William B. Hladik III, M.S., R.Ph.College of Pharmacy

University of New Mexico

Assoctie Edtior

aizd

Production Spectiist

Sharon I. Ramirez, Staff AssistantCollege of Pharmacy

University of New Mexico

While the advice and information in this publication are believed to bc true and accurate at press time, neither the author(s) northe tiitor nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publishermakes no warranty, express or implied, with respect to the material conkined herein.

Copyright 1995University of New Mexico

Pharmacy Continuing EducationAlbuquerque, New Mexico

THE CURRENT STATUS OF RADIOLABELED PEPTIDES FOR IMAGING

STATEMENT OF OBJECTIVES

The purpose of this correspondence lesson is to increase the reader’s knowledge of current research efforts in thedevelopment and utilization of radiolabeled peptides for nuclear medicine imaging. In addition, the reader will reviewthe clinical use of the first radiolabeled peptide approved for use by the U .S.F.D.A., In-11 1 pentetreotide.

Upon successfd completion of this tied, the reader shotid be able to:

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2.

3.

4.

5.

0 6.

7.

8.

9.

10.

11.

12.

describe the general chemical structure of peptides.

compare and contrast the structural differences of peptides with that of monoclinal antibodies.

)ist the radionuclides that have been used for radiolabeling peptides for both pre-clinical and clinical use.

summarize the chemical bond formation that allows peptides to be radiolabeled with radionuclides useful fornuclear medicine imaging.

compare and contrast the radionuclides that have been used for radiolabeling peptides.

describe the biological role of naturally occurring peptides.

describe the different classes of peptides useful for nuclear medicine imaging.

discuss the development, characteristics, preparation, quality assessment procdure, pharmacology,pharmacokinetics, indications and efficacy of In- 111 pentetreotide.

discuss the potential role of vasoactive intestinal peptide in nuclear medicine imaging.

discuss the potential role of chemotactic peptides in nuclear medicine imaging,

discuss the potential role of RGD and RYD peptides in nuclear medicine imaging.

discuss the potential role of atrial natriuretic peptide in nucledr medicine imaging.

1

COURSE OUTLINE THE CURRENT STATUS OF RAD1OLABELEDPEPTIDES FOR IMAGING

1,

II.

III.

IV,

v.

VI.

VII.

VIII.

Ix.

x.

INTRODUCTION

RADIOLABELING TECHNIQUES

A. IodinesB. Technetium-99mc. Iridium-l 11 and Gallium-68D. Flourine-18

BIOLOGICAL ROLE OF PEPTIDES

CLASSES OF PEPTIDES FOR IMAGING

A. Natural biologically active peptidesB. Single chain antigen binding proteinsc. Hypervariable region peptide analogs

CURRENT PEPTIDE RADIOPHARMA-CEUTICALS

TUMOR IMAGING

A. Somatostatin receptor imaging with In-111 pentetreotide

1. Development2. Product description3. Clinical pharmacology and

pharmacokinetics4. Approved indications and

potential uses5. Clinical efficacy

B, Vasoactive Intestinal Peptide (VIP)

INFECTION IMAGING

A. Chemotactic peptides

THROMBUS IMAGING

A. RGD and RYD peptides

OTHER PEPTIDES

A. Atria] Natriuretic Peptide

SUMMARY

(ANP)

by

John W. Babich, Ph. D., R.Ph.Massachusetts General Hospital

Harvard Medical SchoolBoston, Massachusetts

and

Gmrge H. Hinkle, M. S., R.Ph., BCNPThe Ohio State University Medical Center

Columbus, Ohio

INTRODUCTION

Peptides play an important role in such fundamentalbodily functions as horneostasis and cell signaling (l).Recently they have been the focus of much interest asdiagnostic radiopharmaceuticals(2). Peptides representa logical next step in radiopharmaceutical developmentas they can be made to possess some of thecharacteristics which make monoclinal antibody (MAb)technology so attractive as targeting agents. T’hepromise of MAb technology, in relation to diagnosisand therapy, has not been fulfilled (3). The large ●molecular weight of these proteins is significantlyresponsible for the limitations of this approach.Barriers to diffusion into diseased tissue, as well as thenatural long-term blood residence time of these largeproteins, results in low target-tissue accumulationcompounded with high background activity, therebylimiting their utility. The promise of MAb technology,however, resides in its unique characteristic ofgenerating proteins which recognize particularmolecular sequences of other proteins or carbohydratesand which bind to these sequences with relatively highafinity (10-8 - 1010 M). Peptides may provide anavenue which allows us to exploit the specific bindingpotential of such compounds without the disadvantagesassociated with large molecular size.

It appears likely that high affinity binding to suchantigens can be accomplished using smaller fragmentsof MAbs or synthetic peptides. In addition tomimicking MAbs in application, peptides foundendogenously also have diagnostic utility, as naturally.-occuring peptides play important roles in cell signalin~and control. As more is learnd about the role ofpeptides in tissue regulation and disease, it is likelythat the abnormal expression of peptide receptors or ●overproduction of peptide messenger may be a criticalkey in the evolution and control of disease (4-6).

2

Exploration of such phenomena with radiolabeledprobes may result in useful diagnostic information withpotential for therapeutic application.

Radiolabeled peptides represent a new class of

@agents for exploitation of radiopharmaceuticals.Comprised of polymers of amino acids, peptidesdisplay a variety of sizes and shapes as well as biologicactivity. Peptides are formed through thepolymerization of amino acids. Of the 20 amino acidsusually found in nature, 19 of them have the generalstructure HZN-CHR-COOH. They differ only in thechemical structure of the side chain R. The 20thamino acid, proline, is similar but its side chain isbonded to the nitrogen atom to give the imino acid.Except in the case of glycine, the central carbon atomis asymmetric and is always the L-isomer. Unlessindicated, all the amino acids discussed herewith willbe assumed to be the L-isomers. More than half of theamino acids commordy encountered in peptides andproteins contain functional side chains. These includeacidic carboxy-groups (aspartic, glutamic acids); basicresidues (lysine, arginine, histidine); hydroxy-aminoacids (serine, threonine, tyrosine); sulfur-containingamino acids (cysteine, methionine); and, finally, thehetero-cyclic indole ring of tryptophan. In addition,there are amino acids with aliphatic residues (alanine,valine, Ieucine and iso-leucine) as well as the aromatic

o

amino acid phenylalanine and, glycine, the simplestamino acid with only a hydrogen atom for a side chain.

Amino acids are linked into peptides and proteinsby the peptide bond. The polypeptide backboneconsists of a repeated sequence of three atoms of eachresidue in the chain, These include the amidenitrogen, the alpha-carbon and the carbonyl-carbon.

-NH-’CHR-CO-NH-’CHCOCNHNCHRCCO-CO-

The peptide bond is formed when the amino group ofone amino acid condenses with the carboxylic acidgroup of a second amino acid, liberating water (7).The peptide bond confers a particular three-dimensional structure on polypeptides. The hydroxylgroup is nearly always trans to the carbonyl oxygen.The peptide bond unit is rigid and planar and nofreedom of rotation occurs around the peptide bonddue to the partial double-bond nature of the peptidelinkage. There is no such character between thealpha-carbon and the amino group or the alpha-carbon and the carboxylic acid group. Therefore, alarge degree of freedom is found on either side of thepeptide bond, allowing the peptides to take a wide

evariety of conformations.

Polymerization of amino acids through peptidebond formation results in linear polypeptide chains.All proteins and polypeptides have this basic

structure. Proteins differ only in the number of aminoacids linked together and the sequence in which thevarious amino acids occur. The sequence of aminoacids in a polypeptide chain generally identify a proteinunambiguously. Peptides are often referral to aspolymerized amino acids with a relatively smallnumber of residues and a defined sequence. There isno maximum number of residues in a peptide. Theterm peptide is appropriate to a chain or polymer ofamino acids if its physical properties are those expectdfrom the sum of its amino acid residues and there is nofixed three-dimensional conformation.

Polypeptides are longer chains usually of definedsequence and length, and proteins are generally thosepolypeptides that occur naturally and have a definitethree-dimensional structure under physiologicalconditions. For the sake of our discussion we will usethe term peptide throughout, referring to a number ofamino acid polymers with biological activity or thosethat are analogs of biologically-active peptides.

Since each of the 20 amino acids is chemicallydistinct and each can, in principal, occur at anyposition in a polypeptide chain, there are 20 to the nthpower different possible polypeptide chains, n aminoacids long (e.g., 205 = 3,200,000 different polypeptidechains possible for five amino acids). Hence, therepertoire of potential peptides that can be described isvast and provides an opportunity for exploitation inradiopharmaceutical development, as well as dmgdevelopment (8).

When assembled into small peptides, amino acidshave generated a large number of hormone-releasingfactors, neurotransmitters and neuromodulators. Largerpolypeptides comprise molecular recognition systemssuch as the immunoglobulins, receptors and enzymes.

RADIOLABELING TECHNIQ~

As discussed above, peptides are comprised of thesame basic building blocks as proteins, thereforetechniques which have been developed forradiolabel ing proteins can be applied to peptides.However, one must consider that since peptides possessfewer amino acid residues than proteins there arefewer sites available for labeling. The likelihood ofmodifying an amino acid residue crucial for biologicalacitivity is therefore gredter. Consequently,optimization of labeling must be determinedempirically. In addition, due to the high potency ofmany peptides and the low tissue concentrations oftheir target receptors, specific activity becomes acritical element in the preparation of radiolabeledpeptides. Efforts to prepare peptides with extremelyhigh specific activity are required to avoid potentialflooding of the receptor with cold or unlabeled peptide.

3

For more than a decade, a variety of methods forlabeling proteins with radioisotopes of iodine,technetium, iridium, gallium, carbon, and fluorine havebeen developed. These methods can be applied to thelabeling of peptides. What follows is a discussion ofradiolabeling methods that have been, or could be,applied to peptide radiopharmaceuticals.

IodineIodination is probably the most widely utilized

method for radiolabeling peptides. Although originallydevelopd for the in-vitro applications of radioabeledpeptides, such as in RIA assays, radioiodination canalso be applied for imaging purposes. This requires theuse of isotopes such as Iodine-123 and Iodine-131, aswell as Iodine-124. Peptides maybe directl y iodinatedon reactive aromatic acid res’iduessuch as tryrosine (9).To accomplish this, oxidizing agents are used togenerate electrophilic iodine species from NaI*.Reagen@ available for direct iodination include thecommordy used agents, Chlorarnine T, iodinemonochloride, N-bromosuccinimide, and Iodogen (10).

Quantitative labeling can be achieved with mostpeptides through the optimization of reaction conditionsand the removal of unwanted radioactive by-products.In general, purification of the radiolabeled peptiderequires chromatographic techniques such as ionexchange, reverse-phase or size-exclusionchromatography. In many cases, direct labeling hasproved to be such a reliable and straightforwardtechnique that many peptide analogs have beenprepared with tyrosine substituted for phenylalanine oradded to the peptide sequence in positions which areunlikely to affect biological activity or receptor binding(e,g., [Tyr’]-octreotide),

Alternative approaches to direct labeling also existand these include using pre-labeled reagents such as theBolton-Hunter reagent [N-succinimydyl-3 -(4-hydroxyphenyl) proprionate], In this case, an activatedaromatic compound, a phenol residue, isradioiodinated, purified, and attached to the peptidesequence by acylation of a free amino group, such asthe N-terminal amine of the peptide sequence or theepsilon mino group of a 1ysine residue (11).

One potential drawback of using iodine as aradiolabel for peptides, is the observation in vivo ofrapid dehalogenation. This has been documented,particularly in the case of radioiodinated antibodies,and has led to the use and investigation of non-phenolicaromatic compounds as radiolabeling reagents [(i.e.,N-succinimidyl-5 -Iodo-3-pyridine carboxylates (12), N-succinimidyl-3-Iodobenzoate (13) and N-succinimidyl-4-Iodobenzoate (14,15)]. In general, these reagents areprepared from trialkyl stannyl precursors and demon-strate greater in vivo stability than directly labeled

polypeptides. The improvement in stability is thoughtto be due to the structural differences of thesecompounds from iodo-tyrosines and thyroxine whichare substrates for dehalogenases in a number of tissues.It has also been found that the Bolton-Hunter reagent ●is resistant to dehalogenation (16). While still havinga phenol group, the Bolton-Hunter reagent possesses analtered aliphatic chain and lacks the amino group oftyrosine. These structural differences impart greaterstability to dehalogenation, over direct labeling oftyrosine residues.

The structural requirements for minimizing in vivodehalogenation has been reported to include (a) the ringsubstituents (b) alkyl chain length, and (c) the nature ofthe linkage to the polypeptide (17). For example, ithas been shown that an octratide analogradioiodinated with N-succinimyd yl-5-Iodo-3-p yridinecarboxylate has greater s~dbility than directly iodinated[Tyr3]-Octreotide (18).

The application of these iodinatable conjugates topeptide radiolabeling should hold promise for futuredevelopment of stable radiopharmaceuticals with regardto therapy. While Iodine-123 and Iodine-13 1 havebeen used for scintigraphic imaging of peptides (19,20)the limited availability and the high cost of Iodine-123,and the high radiation dose and poor imaging qualitiesassociated with the use of Iodine 131 have promptedinvestigations into other radionuclides.

T~hnetium-99mTechnetium-99m is an ideal radionuclide for peptide

labeling due to its low cost, widespread availability,excellent imaging properties, and favorable dosimetry.In addition, Tc-99m can also be obtained at highspecific activity. There are a number of reports in theliterature which show a variety of approaches toradiolabeling proteins with Tc-99m. These includeboth direct and indirect methods.

In direct labeling methods, technetium is reduced inthe presence of the protein or peptide and binds toamino acid residues or functional groups. Thisapproach assumes the protein or peptide will providea stable reproducible coordination environment for Tc-99m. In general, TcO~ is reduced in the presence ofthe protein or peptide and unbound Tc-99m is removalby chromatographic methods (21). A more recentapproach for labeling immunoglobulins with Tc-99mhas been to utilize the thiol groups present in theprotein as Tc-99m affme coordinating atoms. Thesupposition is that disulfide reduction liberates freesulfiydryls which preferentially bind Tc-99m whenpresented as a weak complex (e.g., Tc-99mglucoheptonate) (22). In general, direct labeling is ●complicated by the tendency of technetium to formunstable complexes, and there is poor control over the

4

labeling sites (23), Despite its limitations, directlabeling has proved populm for the preparation of Tc-99m antibodies.

o

The direct approach is unlikely to be broadlyapplicable to the labeling of peptides for severalreasons. The need for Tc-99m to stabilize its bondingwith multiple coordination sites would result in atomsfrom several amino acids being bound to the metal.This would restrict the three-dimensional flexibility ofthe peptide and possibly inhibit the optimal receptor-binding conformation. In addition, most small peptidesdo not contain disulfides and, although the secondaryand tertiary structures of proteins and antibodies ares~dbilizedby multiple non-covalent interactions, this isnot the case with peptides. In many peptides thatcontain disulfide bridges slight alterations in ringstructure can result in dramatic alterations in biologicalactivity. Small cyclic peptides, such as oxytocin andvasopressin, exhibit a marked decrease in biologicalactivity with increasing ring size. In an effort to

prepare Tc-99m octreotide it has been shown thatdirect labeling via disulfide reduction reduces receptoraffinity by four orders of magnitude (24) therebyconfirming that alterations of the cyclic nature of thispeptide dramatically reduces biological activity.Irrespective of these considerations, direct labeling ofpeptides has been reported for several peptides,

●including thyrotropin-releasing hormone (TRH) (25)and, more recently, for a nineteen-residue analog ofIaminin (PA22-2) (26). Jn neither case were receptorbinding studies reported indicating the effect oflabeling on affinity.

In order to overcome the problems associated withdirect labeling, methods have been developed whichutilize high affinity bifinctional chelates to bindtechnetium. The itiirecr or bifunctional methodsemploy metal chelating compounds which are able tobe covalently attached to the peptide through a reactiveside group and bind technetium with availablecoordinating groups. These chelates can be used in oneof two ways. Jn the first method, the bifunctionalreagent is pre-labeled with Tc-99m and then conjugatedto the peptide in a manner similar to the Bolton-Hunteriodinating reagent. The second method utilizes thechelating agent as part of the overall peptide sequenceand radiolabeling VdkeS place after peptide purification.Both methods have been highly successful forradiolabeling antibodies and could be directly appliedto large peptides. To achieve high specific activity insmall peptides, both the unreacted labeling reagent andunlabeled peptide must be removed from the reaction

@mixture when the first method is used. Since thesespecies can be similar in size to the labeled peptide,chromatographic purification may not bestraightforward. In addition, if more than one reactive

group is present on the peptide, multiple speciw canresult. Theoretically, the itiirect method should yieldonly a single radiolabeled species, and chromatographicseparation is usually simpler.

Such bifunctional chelating agents include DTPA(27), N,S,(28), N,S (29), BATO (30) and 6-hydrazinonicotinate (3 1). Radiolabeling is achieved byincubating the chelate-peptide conjugate with a lessstable complex of Tc-99m such a Tc-99mglucoheptonate. The more stable complex is favoredand the Tc-99m is transferred to the chelating group ofthe peptide-conjugate. Recently, Tc-99m labeling ofseveral peptides has been reported using a variety ofmethods, including HYNIC (32) and diaminedithiol(33) derivatized chemotactic peptides for infectionimaging, The HYNJC and diamindithiol derivatkedchemotactic peptides demonstrated biological activityand receptor binding characteristics similar to theunmodified peptide. The use of N,S, and N,S forgenerating analogs of the apolipoprotein SP-4 forimaging atherosclerosis has also been reported (34).

Theoretically, itiirect labeling of the peptide usingbifunctional chelators affords a preferable product overthat achieved with direct labeling. The chemistry ofTc-99m labeling is better defined and more predictableusing the itiirect or bifunctional chelate method.

Indium-111 and Gallium-68Iridium-l 11 is a Group III metal which possesses a

chemistry and half-life which makes it ideal forradiolabeling intact immunoglobulin. This is due tothe prolonged pharmacokinetics of these proteins andthe ease of achieving rapid, stable labeling usingbifunctionalized derivatives of the polyamino-polycarboxylate chelates, EDTA (35) and DTPA (36).DTPA has been used as a bifunctional chelating agentfor radiolabeling alpha-melanocyte stimulating hormone(alpha-MSH) with Iridium-l 11 for imaging melanoma(37), labeling formulated chemotactic peptides forinfection imaging (38), labeling laminin fragments fortargeting tumor-associated laminin-receptors (39), andlabeling atriaJ natriuretic peptide (ANP) for imagingANP receptors in the kidney (40). The positron-emitting isotope, Gallium-68 has been used forradiolabeling chemo~~ctic peptides (41) and asomatos~dtin analog (42), using DTPA anddesferrioxamine as bifunctional chelates, respectively.

Fluorin&lSJn an effort to utilize positron emission tomography

(PET) for peptide imaging, a number of reagents havebeen described. Proteins have been radiolabeled with“F using N-succinimidyl-4-[ ’gF]fluorobenzoate (43),methyl 3-[18F]-5-nitrobenzimidate and 4-[18F]fluoroacylbromide (44) and more recently, N-succinimidyl-4-

5

(fluorobenzylamino) suberate (45) and[“F]fluoropropionic acid and [’*F]fluoroacetic acidderivative (46). Reports of “F labeled peptides includelEFinsulin labeled via a 4-(bromomethyl)benzoyl amineintermediate (47), lnF-fluoroproprionyl octrmtide (48),and most recently, 4-[18F]fluorobenzoate conjugatedchemotactic peptide (49).

BIOMGICAL ROLE OF PEPTIDES

Naturally-occuring peptides function primarily incell-signaling. The need for signaling orcommunication between cells comes from the need toregulate development and organize tissuw, as well asto control growth and division and coordinate function.This communication can occur in three ways. One is

remote signaling by secreted molecules, the second iscontact signaling between plasma membranes ofadjacent cells, and the third, is synaptic signaling,again between adjacent cells.

Types of remote signaling differ in the distance inwhich they operate. The first, endocrine signaling,occurs when hormones are secretd from a tissue andtravel throughout the body allowing their messengermolecules to target cells in distant areas of the body.The second, paracrine signaling, occurs when cellssecrete local chemical mediators which are so rapidlytaken up and metabolized or immobilized that theiractions affect cells ordy within a few millimeters ofwhere they are secreted. The third, synaptic signaling,is confined to the nervous system. Cells secreteneurotransmitters--the chemical messengers whichdiffuse across a synaptic cleft and act only on theadjacent cells. Again, rapid immobilization ordegradation of the neurotransmitter limits thesecompounds from acting in distant cells. In all cases,the chemical signal interacts with the receptor protein,which initiates the response.

In generaJ, hormones are greatly diluted in thebloodstream and therefore must be able to act at verylow concentrations (less than 10-8 M).Neurotransmitters are much less diluted and canachieve local concentrations approaching 5 x 10-4M.Synaptic receptors have relatively low affinity, and asa result do not respond significantly to the very lowconcentrations of transmitter that may reach them viadiffusion from remote sites. Peptide hormones act ina paracrine fashion as neurotransmitters. Mosthormones and chemical mediators are water soluble.

CLASSES OF PE~IDES FOR IMAGING

For the sake of discusson we can broadly classifypeptides in two ways based on the origin of the initialamino acid sequence of interest. The first group would

include naturally occuring proteins and peptides actingin a cell signaling capacity (i.e., hormone, growthfactors, neurotransrnitters, etc.). These peptides actupon naturally-occurring target molecules such asreceptors and would also include analogs of naturally- ●occurring peptides such as the somatostatin analogoctrmtide. The second group would be comprised ofpeptide analogs of immunoglobulins or MAbs. Thesewould include low molecular weight fragments ofMAbs, peptide analogs of MAb antigen-bindingregions, as well as synthetical y-derived peptides ofepitope-binding region or hypervariable region peptidesof MAbs.

Radiolabeled peptides targeted to the variousreceptors or cell membrane target proteins may allowfor:

1.

2.

3.

4.

5.

In

characterization of tumor biochemistry(receptor status)tumor detection and staging (location andextent)screening (at presentation for extent of diseaseand follow up)receptor-guided delivery of cytotoxins (e.g.,radionuclides, drugs, toxins)therapeutic monitoring (of conventional orbiological therapies)

addition, therapeutic approaches involvinp the ●inhibition or disruption of autocrine growth factorscould be studid in detail with radiolabeled peptideprobes.

Natural Biologically Active PeptidmIn contrast to antibodies, low and intermediate

molecular weight biologic peptides may be moresuitable starting points for radiopharmaceuticaldevelopment. Peptides are necessary elements in avariety of biologic processes, functioning as hormones,neurotransmitters, neuromodulators, growth andgrowth inhibition factors, and cytokines. In manycases, peptides display affinities for their receptors thatare significantly greater than that of monovalentantibody fragments.

The molecular weights of biologically-activepeptides are extremely diverse, ranging from 3-5residues in TRH, enkephalins and bacterialchemoattractant peptides to over 200 residues ingrowth hormone. However, in many cases molecularrecognition sites are restricted to specific areas of thesequence. For example in PTH, and ACTH, biologicalactivity is conferred by the N-terminal sequence.Likewise, for very small peptides like TRH and ●bacterial chemoattractant peptides the C-terminus canbe extended without significant alterations in receptor

6

binding or biological activity. A particular advantageof this focality of binding domains is the ability tomodify sequence regions that do not participate inreceptor interaction for radiolabeling or optimization ofbiodistribution. For the smaller peptides, thesemodifications can be performed by the direct chemicalsynthesis of analogs by methods of solid-phase orsolution synthesis. For larger peptides (> 50 residues),analogs are more efficiently prepared by molecularcloning, followed by chemical modification in somecases.

Single Chain Antigen Elnding ProteinsThe large three-dimensional structure of

immunoglobulins allows for a variety of functions, inaddition to antigen recognition or binding. Thevariable region of the immunoglobulin’s heavy andlight chains make up the antigen recognition portion ofthe protein, whereas, the constant region(predominantly the Fc region) of the immunoglogulinconfers its effecter function. It should be noted thatthe molecular sequences recognized by antibodies arerelatively small sequences (5-6 mer) of carbohydratesor amino acids. Hence, stripping down the antibody toits antigen binding site only would greatly reduce itsmolecular bulk as well as other biological functions notnecessarily related to antigen binding. Suchapproaches to improving MAb applications to imaginghave been reported with some encouraging results.

Recently, it has been suggested that imaging withradiolabeled Fv fragments which are about 50%smaller than Fab’s, may minimize many of theproblems associated with the use of intact antibodiesand conventional fragments. Although these reagentscan be produced by fragmentation of intact antibodies(50), most single-chain binding proteins (sFv’s) havebeen prepared by recombinant DNA techniques (51-55). In this approach, the gene sequences coding forthe variable regions of the light and heavy chains ofan antibody are linked with a specifically designedoligonucleotide sequence and the construct is expressedin E. coli. In the final product (25-30 kDa), thecarboxyl terminus of variable region of the light chainis linked to the amino terminus of variable region ofthe heavy chain by a 12-20 residue peptide. Theseproteins display similar antigen specificity as the parentantibody. Recent studies with these SFV’S showedrapid tumor penetration (53, 56,57). For example,with the pancarcinoma antibody CC49, the time formaximal penetration of SFVis 30 min while invdctIgGrequired 48-95 hr to attain similar concentrations.Autoradiographic studies comparing the penetration ofinrdct IgG with F(ab)z, Fab and SFV’Srevealed thatSFV’S distribute uniformly in tumors while intactantibodies concentrate in the region of, or immediately

adjacent to, the blood vessel. The distributions ofF(ab)z and Fab fragments showed intermediatepenetration in a size related manner (56,57). Whenapplied to in vivo imaging, SFV’Sclear rapidly from thecirculation (tl,z alpha - 2-5 min and tl,2 beta - 2-5hr), have low levels of accumulation in backgroundorgans, and localize rapidly in target tissues(53,58,59). Although in some cases absolute levels ofaccumulation in target tissues were lower than withlarger fragments, target-to-background ratios werecomparable or greater.

Hypervariahle Region Peptide AnalogsSince antigen binding is largely imparted by the

hypervariable portions of the variable region ofantibodies, synthetic peptide analogs of these regionsmight have even more favorable imaging propertiesthan SFV’S. Unfortunately, although peptides derivedfrom hypervariable region sequences can bind antigenswith similar specificity to the native antibody, affinityis usually significantly reduced. However, by usingsophisticated methods of molecular design, thisproblem may be surmountable. For example, it hasbeen demonstrated that conformationally-constrainedand dimeric peptides derived from hypervariable loopsequences can bind antigen with affinities that are up to40-fold higher than linear sequences (60). Similarly,by using nuclear magnetic resonance analysis inconjunction with molecular modeling, it has beenpossible to determine the effects of specific amino acidresidues on antigen binding (61). Based on this type ofinformation, peptides with improved binding propertia~have been designed and synthesized. With thesetechniques, it may be possible to modify the peptidesto optimize biodistributi(m and pharmacokinetics.Unfortunately, these strategies are complex and requiredetailed knowledge of the three-dimensional structureof the antibodies and peptides. One importantapplication of hypervariable region peptides to in vivoimaging has already been reported for thrombusimaging (62).

CURRE~ PE~IDE RADIOPHARMACEUTICALS

Numerous biologically -activepeptides have potentialas imaging agents. Although imaging applications ofthese reagents are still relatively new, many importantimaging agents are currently under development. megrowing interest in peptides may be due in large partto the successful clinical application of theneuropeptide, somatos~dtin and its analogs. In thefollowing sections, we will review the current status ofradionuclide imaging with biologically-active peptides.

7

TUMOR IMAGING

Somatostatin Receptor Imaging with In-IllPentetreotide

Development. Although In-1 11 pentetrmtide wasordy recently approved by the USFDA for clinical use(6/2/94), research which ld to the compound’sdevelopment began over twenty years ago with theisolation of a polypeptide from the hypothalamus ofseveral animals (64). That same year, the peptide,named somatostatin because it inhibited the secretion ofsomatotropin from the pituitary gland, was structurallyidentified and syntbwized (65,66). Years of researchhas shown the endogenous hormone somatostatin toprocess multiple functions in the endocrine hommstasisof the human body. Table 1 illustrates the variousorgans and body functions influenced by the naturally-occurring neuropeptide.

Table 1. Effects of Somatoshtin on Organ Systems

Organ System Effect

Anterior Pituitary Gland Ifiibition of secretion(growth hormone,thyrotropin)

Gastrointestinal Tract Inhibition of guthormone secretion,abso~tion, motility

Pancreas Inhibition of secretion(insulin, glucagon,enzymes)

Liver Decreased bloodflow, inhibition ofsecretion of bile acid

Kidney Fluid-electrolytebalance

Adapted from O’Dorisio, Wood, O’Dorisio(Reference81)

The promise of inhibiting growth hormonetherapeutically led to the characterization and synthesisof the 14 amino acid structure seen in Figure 1A.Somatostatin had limited therapeutic value because ofa short (2-3 minute) biological half-life. A long-actinganalog of somatostatin was synthesized to use as atherapeutic agent to inhibit hypersecretion of hormonesfrom a variety of neuroendocrine tumors (67). The

octreotide analog, shown in Figure lB, has ordy eightamino acids but maintains the sequence of Phe-Trp-Lys-Thr found essential for function. In addition,alteration of the terminal ends of the peptide led tolonger circulation times and prolonged biologic ●activity.

In vitro binding studies were conducted byradiolabeling somatostatin and octrmtide with iodine-125 (68). The feasibility of nuclear medicine imagingof somatostatin receptor-containing neuroendocrinetumors was first shown using Iodine- 123 Tyr3-Octrwtide (see Figure lC), in which the third aminoacid was changd from Phe to Tyr for subsequentiodination (63). Although 1-123 provides excellentnuclear properties for external imaging, accelerator-production with its associated limited availability ofhigh purity 1-123 necessary for optimal radioiodinationof Tyr3-Octreotide, cre~ted difficulties in production.Clinical studies showed 1-123 Tyr3-Octrmtide wascleared rapidly by the hepatobiliary system leading toexcretion and accumulation in the intestines whichcaused difficulty interpreting abdominal and pelvicimages (63). To overcome the production difficultiesand problematic biodistribution patterns of 1-123 Tyr3-Octrmtide, radioactive metal labeling of octreotide viachelation was introduced (70) Starting with octreotide,the DTPA moiety is attached to the d-phenylalanylgroup at the N-terminus (Figure lD). Four -COOHgroups from DTPA are available for forming a metal- ●binding complex with the radioactive metal, indium-111. Attaching the large chelate group at the farthestdistance from the receptor binding portion of thecompound allowed the iridium-111 radiolabeled speciesto retain the ability to bind somatostatin receptors.Indiurn-111 DTPA-D-Phel -Octreotide (In-1 11pentetrmtide) has a longer physiologic half-life thanthe 1-123 Tyr3-Octreotide allowing more time for tumorlocalization. The longer physical half-life of In-111allows delayed imaging. In- 111 pentetreotide notbound to receptor sites is rapidly cleared through thekidneys reducing non-specific uptake and increasing thetarget to background ratios (T/S).

Product Description. In-1 11 pentetrwtide(OctrwScan@-Mallinckrodt) is a radiolabeled analog ofsomatostatin supplied as a single dose kit. Each kitincludes a 10ml reaction vial containing a Iyophilizedmixture of 10~g pentetreotide, 2 mg gentisic acid, 4.9mg anhydrous trisodium citrate, O.37mg anhydrouscitric acid, and 10 mg inositol. A second 10 ml vialcontains In- 111 chloride sterile solution in 1.1 ml ofdilute (0.02N) hydrochloric acid (3mCi/ml In-111chloride at calibration). The vial containing the In-111also has 3.5pg/ml of ferric chloride. To complete ●the radiolabeling step, the contents of the In-111 vialis added to the reaction vial using the stainless

8

—

A Somatostatin I

B I Octreotide i cl ’231labeled Tvr3-Oareotide~— L ,H

D Ilndium ‘“In Pentetreotide ~

Figure 1. Somatostatin and its analogs. A. Native, human somatostatin. B. Synthetic analog ofsomatostatin with prolonged biologic activity used for control of symptoms associated with neuroendocrine

—

tumors, C, Iodine-123 labeled somatostatin derivative first usd for nuclear medicine imaging. D. IridiumIn- 111 pentetratide (OctrmScan@-Mallinckrodt Medical, Inc.).

9

Table 2. Radiolabeling of OctrmScanm @ndium In-Ill Pentetrmtide).

Note: Read complete directions thoroughly before starting prepa~”on.

The Kit Containa:

One 10-ml reaction vial containing 10~g pentetrmtide, 2mg gentisic acid, 4.9mgtrisodium citrate, 0.37mg citric acid and 10mg inositol.

One 10-ml vial containing 1. lml of 3.0 mCi/ml In-111 chloride in 0.02N HCI.

One 25G x 5/8” stainless steel nedle.

One package insert and label.

Procedure Precautions and Notes

1. All transfers and penetrations of the vial stoppers by a needle must use aseptic technique.2. Wear waterproof gloves during the entire procedure and while withdrawing the patient dosage from the

OctreoScan@reaction Vial+3. Transfer In-1 11 chloride sterile solution with an adequately-shielded, sterile syringe using the transfer needle

in the kit.4. Adequate shielding should be maintained at all times until the preparation is administered to the patient,disposed of in an approved manner, or allowed to decay to safe levels of radioactivity. A shielded, sterile syringeshould be used for withdrawing and injecting the preparation.5. Do not inject into TPN administration bags or associated intravenous lines.

Procedure for the Preparation of Iridium In-Ill Pentetreotide

1.2,3.

4,5.6.

7.

8.

9.

10.

11.

Place the OctrwScan@ reaction vial in a lead dispensing shield.Swab the rubber stopper of the reaction vial with an appropriate antiseptic and allow the vial to dry.Aseptically remove the contents of the In-111 chloride sterile solution vial using the needle provided and ashielded, sterile syringe.Inject the In-1 11 chloride sterile solution into the OctrwScan@ reaction vial.Gently swirl the OctrmScan” reaction vial until the Iyophilized pellet is complete]y dissolved.Incubate the In-1 11 pentetrwtide solution at or below 25°C (77”F) for a minimum of 30 minutes.Note: A 30-minut e incubation time is reauired. Shorter incubation periods may result in inadequate labeling.Using proper shielding, visually inspect the vial contents. The solution should be clear, colorless, and freeof particulate matter. If not, the solution should not be used. It should be disposed of in a safe and approvedmanner.Assay the In-1 11 pentetreotide solution using a suitably calibrated ionization chamber. Record the date, time,total activity, and patient identifier (e.g., patient name and number) on the radioassay information label andaff~xthe label to the lead dispensing shield.The labeling yield of the reconstituted solution should be checkd before administration to the patient,according to the instructions given in Table 3. If the radiochemical purity is less than 90%, the productshould not be used.Store the reaction vial containing the In-1 11 pentetreotide solution at or below 25 ‘C (77 ‘F) until use. TheIn-1 11 pentetreotide must be used within six hours of preparation.If desired, the preparation can be dilutd to a maximum volume of 3mL with 0.9% Sodium Chloride Injection,U .S .P. immd-iatel y prior to injection. The sample should be drawn up into a shielded, sterile syringe andadministered to the patient.

10

Table 3. Recommended Method for Determination of hbeling Yield of Iridium In-Ill Pentetreotide

● Reauired Materials

1. Waters (Division of Millipore) Sep-P@ C18 Cartridge, Part No, 51910.2. Methanol, 15rnL (Caution: toxic and flammable. Exercise due caution)3. Distilled water, 20mL4. Disposable syringes:

2-10mL, no needle requird2-5mL, no needled requiredl-lmL, with needle

5. Three disposable culture tubw or vials, minimum 10mL capacity6. Ion Chamber

Preparation of the Sep-Pak Cartridge

1. Rinse the Sep-Pak cartridge with 10mL methanol as follows:Fill a 10mL syringe with 10mL methanol, attach the syringe to the longer end of the Sep-Pak cartridge,and push the methanol through the cartridge. Discard the eluate in a safe and approved manner.

2. Similarly, rinse the cartridge with 10mL water. Ensure that the cartridge is kept wet and that there is noair bubble present, If an air bubble is present, rinse the cartridge with additional 5mL of water. Discardthe elute.

Sample Analysis

●1.

2.

3.

4.

Assay

1.

2.

3.4.

Using a lmL syringe with n~le, withdraw 0.05 -0.1 rnL In-111 pentetreotide from the OctreoScan@reaction vial. Apply the preparation to the Sep-Pak cartridge through the longer end of the cartridge. Makesure that the sample is migrating on the column of the cartridge. Note: After this step, the cartridge andall solutions eluted from it will be radioactive.With a disposable 5rnL syringe, slowly (in dropwise manner) push 5mL water through the longer end ofthe cartridge, collecting the eluate in a counting vial or tube. Label this eluate as “Fraction 1.”Similarly, elute the cartridge with 5mL methanol. Be sure that this solution is pushed slowly through thelonger end of the catiridge so that the elution occurs in a dropwise manner. Collwt this fraction in a secondculture tube or vial for counting. Label it as “Fraction 2.” Push two 5mL portions of air through thelonger end of the cartridge and collect the eluate with Fraction 2.Place the Sep-Pak cartridge in a third culture tube or vial for assay.

Assay the activity of Fraction 1 in a suitably-calibrated ionization chamber. This fraction contains thehydrophilic impurities (e.g., unbound In-1 11).Assay the activity of Fraction 2. This fraction contains the In-111 pentetreotide,Assay the activity of the Sep-Pak cwridge. This component contains the remaining non-elutable impurititi,Dispose of all of the materials used in the preparation, the sample analysis, and the assay in a safe andapproved manner.

Calculations

1. Percent In-1 11 pentetratide = (Fraction 2 activity/Total activity) X 100%

@Where Total activity = Fraction 1 + Fraction 2 + activity remaining in Sep-Pak

Note: If this value is less than 90%. do not use the ~ret)aration. Discard it in a safe and au~roved manner,2. Percent hydrophilic impurities = (Fraction 1 activity/Total activity) X 100%3. Percent non-elutable impurities = (Activity remaining in Sep-Pak cartridge /Total activity) X 100%

11

steel needle provided in the kit (See Table 2). Afier anincubation period of 30 minutes at room temperature,the finished In-1 11 pentetrwtide can be dilutd to amaximum volume of 3 ml with 0.9% Sodium Chloridefor Injection USP prior to quality asswsment (seeTable 3) and patient infusion which must be donewithin six hours of preparation.

In addition to the radiolabeled somatostatin receptorbinding peptide, the finished product contains citrate asa buffer and inositol and gentisic acid as stabilizers.

Clinical Pharmacology and Pharmacokinetics.Pentetreotide is a long-acting, cyclic octapeptideanalog of the human hormone, somatostatin, that hasbeen coupled with a DTPA moiety. The DTPA chelateenables radiolabeling with In-111 while the octapeptideanalog confers somatostatin receptor recognition andbinding.

In-1 11 pentetreotide binds to somatostatin receptorson cell surfaces throughout the body, concentrating intumors that contain a high density of somatostatinreceptors such as those listed in Table 4. Within anhour of injection, the radiopharmaceutical primarilydistributes from plasma to extravascular body tissues.Visualization of somatostatin receptor-rich tissuesincluding normal pituitary, thyroid, liver, spleen andurinary bladder is seen (71). Lymphocytes are alsothought to possess sornatostatin receptors which led toclinical investigation of the usefulness of the radiophar-maceutical to detect malignant lymphomas (71). The10~g of pentetreotide found in the radiopharmaceuticalis 5-20 times less than that of therapeutic dosages ofoctrwtide and is not expected to exact clinical]y-significant somatostatin-like effects (72).

Table 4. Neuroendocrine tumors known to havesomatostatin receptors.

GastrinomaInsulinomaCarcinoidSmall Cell Lung CancerParagangliomaGlucagonomaNeurohlastomaPhmchromocytomaPituitary TumorsMedullary Thyroid CarcinomaLymphomasMeningiomasAstrocytomasMerkel Cell Tumors

After intravenous injection, In-1 11 pentetreotide

radioactivity leaves the plasma rapidly with only one-third of the administered dosage remaining in thecirculation at ten minutes post-infusion. Blood levelscontinue to drop with less than 1% of the radioactivedose found in the blood pool twenty hours post- ●infusion (72). Radioactivity levels in the liver andspleen remain low (2-3 %) over a 48-hour period.Despite this, the spleen is the organ that receives thehighest radiation absorbed dose (2.46 rads/mCi).Imaging of intestinal tumors is aided by the use oflaxatives to diminish abdominal backgroundradioactivity from the small amount of hepatobiliaryclearance of the drug.

in-l 11 pentetreotide clearance is primarily by renal 4

excretion with 25% of the injected dosage recovered inurine at three hours, 50% at six hours, 85% at 24hours and over 90% by two days (72). The biologicalhalf-life of In-111 pentetreotide is approximately sixhours. This rapid biodistribution and systemicclearance coupled with urinary excretion lead to highT/B ratios for imaging. Hepatobiliary excretionaccounts for less than 2% of the injected dosage overthree days. In vivo stability studies indicate thatplasma and urine radioactivity is predominantly in theparent form (72).

Approved Indications and Potetiial Uses. In-111pentetreotide is indicated for the scintigraphiclocalization of primary and metastatic neuroendocrinetumors bearing somatostatin receptors (72). Table 4 ●lists those neuroendocrine tumors that have beenlocalizd in clinical investigations. In addition, breastcancer, non-Hodgkin’s lymphomas, Hodgkin’s disease,sarcoidosis, tuberculosis, and gliomas have beenvisualized with In-1 11 pentetreotide scintigraphy(71,74)

High dose octreotide therapy may lead to tumorregression in acromegaly, islet cell tumors, andmetastatic carcinoids (75). In- 111 pentetreotidescintigraphy has been used to identify those patientswith uptake at somatostatin receptors, and therefore,more likely to respond to octrmtide therapy for tumorregression and control of hormonal hypersecretionsyndromes such as secretory diarrhea. Patients withnegative studies would be spared the expense andbothersome daily injections of octrmtide.

ClinicaZ Efficacy. In-111 pentetreotide is the firstdrug approved by the FDA based entirely on Europeantrial data (76). Clinical studies totaling 365 patientsindicated gredter than 86% success in detecting neuro-endocrine tumors. A subgroup of 39 patients evaluatedwith tissue confirmation provided a sensitivity andspecificity rates of 85.7% and 50%, respective y, forIn-1 11 pentetreotide imaging compared with 68%

●sensitivity and 12% specificity for CT/MRI.

12

Fiure 2. Whole body anterior (on Icft) and posterior (on right)images of a 43 y. o. male with metastatic VIPoma with unknownprimary cancer taken 24 hours after infusion with 6.6mCi of In-111pcntetreotide, Patient was receiving 150pgm of octreotide thrwtimes daily for symptomatic control. This study was conducted todetermine if all lesions were somatostatin-rweptor positive. Alongwith normal uptake in the kidneys, liver, spleen, and thyroid, there

are numerous lesions seen in the thoracic, lumbar, and cervicalspinal skeleton, as well as ribs, pelvis, and right side of the htid.In tiddition, there are numerous lesions seen in the liver and thesubhepatic region.

Vasoactive Inttitinal Peptide (VIP)

The neuropeptide vasoactive intestinal peptide (VIP)has recently been shown to be a promising agent fortumor imaging (77). VIP is a 28 amino acid peptide(3326 daltons) of the glucagon-secretin family, firstcharacterized from porcine duodenum (78). VIP hasa range of biological activities depending on the targettissue. First characterized as a vasodilatory agent withpotent effects on peripheral and pulmonary bloodpressures (75), this peptide also stimulates secretion ofvarious hormones (79,80). VIP also acts as animmunomodulator (81) and promotes growth andproliferation of normal and malignant cells (82).

VIP receptors are found on cell surface membranes

throughout theg~strointestinalRecently, VIP

body, peripheral blood cells (83), thetract (84) lungs (85) and kidneys (86).receptors were identified on various

human tumors in amounts exceeding that of peripheralblood cells (87). This finding led to an initial study of1-123 labeled VIP in 79 patients with various tumors.1-123 VIP showed good sensitivity for primary andrecurrent colorectal, pancreatic and gastricadenocarcinoma as well as detecting liver and lymphnode metastasis. This study also compared 1-125octreotide in 38 patients. VIP was superior to 1-125octreotide in detecting adenocarcinomas and wasequivalent in detecting intestinal endocrine tumors.

Subsequent to these findings, Reubi evaluated thepresence of VIP receptors on a variety of humancancers (88). Over three hundred tumors were studiedfor VIP receptor expression by in vitro receptorautoradiograph y. For comparison somatostatinreceptors were also studied in the same tissue samplesusing I-125-[Tyr3]-octreotide. The results of this studyshowed a high incidence of VIP receptors present onnumerous tumor types as described in Table 5.However, VIP receptors were found on only half ofundifferentiated phachromocytomas, small-cell lungcancers, neuroblastomas and gastroentero-pancreatictumors.

Table 5, TUMORS FOUND TO EXPRESS VIPRECE~ORS BY AUTORADIOGRAPHICMEANS.

Tumor Type

Carcinomasbreastendomtitriumprostate (and prostate cancer metastasis)bladder

Adenocarcinomasovariancolonicpancreatic

Othergastrointestinal squamous cell carcinomanon small cell lung cancersIymphomasastrocytomasglioblastomasmeningiomas

All differentiate neuroendocrine tumors.

When compared with the results obtained with 1-125[Tyr3] octrmtide, significantly more tumors were

13

found to express VIP receptors than somatostatinreceptors. These data suggwt a strong likelihood thatVIP receptor targeting may be useful for tumorimaging in vivo.

INFECTION IMAGING

Despite the succws of several agents for imaginginfection, in most cases localization requires 24 hoursbefore l-ions can be visualized. This is a seriousclinical deficiency. The problems of tie prolongdinterval between in vitro cell labeling, injection, andlesion detection could be reduced by imaging with anagent capable of binding in vivo to both circulatingwhite blood cells (WBCS) as well as WBCS alreadypresent at the site of inflammation, Candidatemolecules with these characteristics include:Interleukin-8 (89), platelet factor-4 (90) and thepeptide, N-formyl-methionyl-leucyl-phenylalanine(Formyl-MLF) (91-93). Of these potential agents,Formyl-MLF is particularly promising. The followingis a review of the development of chemotactic peptidesas new agents for imaging infection.

Chemotactic peptides

Leukocytes follow a chemoattractant signal tomigrate to focal sites of inflammation. Formyl-MLF isa bacterial product that initiates leukocyte chemotaxisby binding to high-affinity receptors on the white bloodcell membranes. These receptors are present on bothpolymorphonuclear leukocytes and mononuclearphagocytes. As granulocytes respond to a chemoat-tractant gradient, the aff~nityof the receptors decreasesas additional receptors are expressed, until the site ofinflammation is reached (94-96). It has been previousl ydemonstrated that many synthetic analogs of Formyl-MLF bind to neutrophils and macrophagw with equalor greater affinity comparti to the native peptide(92,97,98). Due to the very small molecular size ofFormyl-MLF (M.W. 437), its molecular structure canbe readily manipulated to d~ign an optimal imagingagent.

The first use of a radiolabeled chemotactic peptidefor abscess localization was reported in 1982 (99,100).Unfortunately, the radiolabeling methods available atthe time of these studies yielded agents of relativelylow specific activity, requiring pharmacologicalamounts of peptide for imaging. These doses ofpeptide were shown to produce profound transientreductions in peripheral leukocyte levels in rabbits anddogs (100-104).

Chemotactic peptides of the class formyl-MLF aresomewhat ided for developing into radiodiagnostics.It is known that C-terminal modification can be donewithout significantly altering the binding properties of

the peptide, This has led investigators to derivative thesequence by adding C-terminal groups that contain afree amino group (e.g., Lys or alkyldiamine) (105). Asmall series of such peptides have been prepared andderivatized with DTPA for labeling with In-111.Thae peptides maintained their ability to bind to theFormyl-MLF receptor (7-50 nM affinity) and maintainbiological activity (3-150 nM for Of production).Upon Iabeling, the agents were shown to localize inintramuscular injections in a rat model. Accumulationof the radiolabel was rapid as was blood clearance.Such properti~s suggestd that Tc-99m would be bettersuited for this application than In- 111.

Recently, the preparation of hydrazino nicotinamide(HYNIC) derivatives of a series of chemotacticpeptides has been reported, based on Formyl-MLFK(106). HYNIC had been previously shown to be afacile and stable bifunctional chelating agent for Tc-99m (107). Peptides with HYNIC covalently linkedthrough a C-terminal amide bond maintained biologicalactivity (ECw ‘s: 12-500 nM for 0~ production) andreceptor binding (ECW,, 0.12 - 40 nM bindingafftnity). Labeling was achieved by incubation of thepeptide in acetate buffer with equal volume of Tc-99mglucoheptonate. Using reverse phase HPLC, labeledpeptide can be separated from unlabeled peptides andresulting specific activities > 10,000 mCi/pMole couldbe obtained. The Tc-labeled peptid~ retained receptorbinding with ECW <10 nM. Biodistribution of theindividual peptides were similar with low levels ofaccumulation in most normal tissues. In rats, all of thepeptides concentrated at the site of infections (T/B =2.5-3.0: 1) by one hour. In rabbits outsr~nding imagesof infection were obtained with T/B ratios > 20:1 at16 hr. after injection.

In an effort to more clearly define the mechanismsof localization and the relative ability of Tc-labeledchemotactic peptides to image infection, thebiodistribution and infection imaging properties of Tc-99m-labeld-Formyl-Met-Leu-Phe-Lys-HYNIC (Tc-99m HP) were compared with In-11 l-labeledleukocytes (In-l 11 WBC’S) in rabbits withintramuscular E. coli infections (108). After co-injection, the distributions of Tc-99m HP and In-111WBCS were similar and the sites of infection were wellvisualized with both radiopharmaceuticals. The T/Bratios of the labeled peptides were approximate] y threetimes greater on average than that of In-111 WBC.Absolute accumulation at the site of infection wasresponsible for the significantly higher T/B calculatedfrom direct tissue sampling (33: 1 vs. 8:1 for Tcpeptide and In-WBC, respective y). Activity in normaltissue (skeletal muscle) was similar for both agents.

Blood fractionation studies demonstrated that, only- 25% of the circulating Tc-99m radioactivity was

14

associatd with WBC’S, whereas, greater than 85% ofthe circulating In-1 11 radioactivity was cell associated,For both radionuclides binding to r~ blood cells wasminimal (<270). In contrast, fractionation of the pusdemonstrated that for both Tc-99m and In-111,approximate y 90 % of the radioactivity was associatdwith WBC’S, Column chromatography of the plasmademonstrated that >95 % of the plasma associated Tc-99m radioactivity eluted at an apparent molecularweight of -150,000 (IgG fraction) with noradioactivity detected at the elution position of the freepeptide.

Further studies (109) comparing Tc-99m peptideand In-111 DTPA-IgG in rabbits with E. coli infectionindicated that Tc-99m-labeled chemotactic peptideslocalize to a significantly greater extent than In-111IgG and the correlation between the degree oflocalization of these two tracers is not statisticallysignificant, suggesting that non-specific mechanismsdo not play a major role in accumulation of Tc-99mpeptide at sites of infection. At all imaging times, theT/B for the peptide was significantly higher than forIgG and much lower levels of peptide accumulate innon-target tissues.

As chemotactic peptides are known to producetransient neutropenia in rabbits and dogs, the dose-response of Formyl-NleLFYK-DTPA in monkeys hasbeen evaluatd (110). The peptide induced a cleardose-dependent reduction in peripheral leukocyte levelsin the animals. The reduction in leukocyte numberoccurred almost immediate y after injection and rapidl yreturned to baseline. No significant effects ondifferential white blood count, blood pressure, pulserate, or respiration rate were detected. Despite theseresults, the fact that HYNIC-conjugated chemotacticpeptides can be labeled at high specific activities(> 10,000 mCi/pMole) allows imaging experiments tobe perfomed at doses of cold peptide that are morethan 1,000-fold lower than the dose that elicits asignificant but transient reduction in white blood cellcount . This finding supports the safety of thesecompounds for use in humans when labeled at highspecific activities.

The data presented to date indicate hat Tc-99mlabeled chemotactic peptides possess severalcharacteristics which make them attractive for infectionimaging. Recent data has also shown that Tc-99mlabeled chemotactic peptides are capable of high qualityvisualization of infection in both dogs (111) andprimatw (112). Further efforts in this area should leadto clinically-usable infection imaging agents.

THROMBUS IMAGING

Vascular thrombosis occurs in about 2.5 million

pmple annually in the United States alone.Thrombosis formation is frequently associatd withpatients who undergo hip replacement or suffer fromhip fracture, Vascular thrombosis can Itid topulmonary embolism, a potentially lethal condition.New ways of detecting vascular thrombosis or deepvenous thrombosis (DVT) are needd. Two methodsnow widely used are contrast venography and B-mode(compression) ultrasonography. These techniques havethe limitation of not being able to determine if thethrombus is hematologically active and therefore likelyto continue to grow or embolize.

The current radionuclide test for DVT isradionuclide venography. Several tracers such as Xe-133, Kr-81m, Tc-99m labeled colloids,macroaggregated albumin or erythrocytes have beenused for this application. Advantages to radionuclidevenography are that it makes use of readily-availableradiopharmaceuticals and results can be obtainedquickly. The test, however, is not specific forthrombi. The images only show cold spots distal tosites of obstruction and not specific binding to thrombi.Also the technique cannot distinguish between acuteand chronic thrombi.

Newer agents have been develop~ based on thechemical nature, mode of formation and fate ofthrombi. The current understanding of thrombusformation begins with platelet deposition behind avenous valve followed by platelet aggregation andrelease of thrombin (113), Fibrin deposits form on theplatelets and retract. The thrombus may resolve ormay continue to recruit more platelefi and more fibrinor the thrombus may dislodge where it will find itsway to other vessels (i. e., pulmonary vessels). Agentshave therefore been directed against fibrin or platelettargets,

Fibrin directed targeting includes radiolabeledfibrinogen (the precursor to fibrin) (114). Fibrinogenis converted to fibrin monomer by action of thrombin.Advantages to radiolabeled fibrinogen includesignificant accumulation on fresh thrombi and lack ofantigenicity of this protein. However, fibrinogen isinsensitive to pre-existing thrombi, anticoagulants areknown to interfere with binding and blood clearance isslow .

Monoclinal antibodies (MAbs) have also been usedin thrombus imaging as well (115). MAbs have theadvantage of being producd to recognize specificcomponents of the thrombus complex. Once raisedagainst a target epitope, the MAbs can beenzymatically cleaved to form smaller fragments withdifferent biological clearance rates. Several MAbsagainst fibrin have been developed (116) which bindthe central chain of fibrin (anti- u or anti- B MAbs),which bind the carboxy terminal domains of

I

I

15

fibrin/fibrinogen (Anti-recogntie fibrin dimers.

D antibodies) and which

RGD and RYD peptidwGlycoprotein-IIb/IIIa(GP-IIb/IIIa) are

transmembrane proteins on the surface of platelets.They comprise a heterodimer complex which changesconformation in response to platelet activation. Thecomplex does not bind fibrinogen until after activation,a necessary phenomena for cell-cell attachment duringplatelet aggregation. The GP IIb/IIIa complex belongsto the family of cell adhesion molecules calledintegrins. The subfamily of compounds that recognizethe GP IIb/IIIa complex generally contain the tripeptidesequence Arg-Gl y-Asp (RGD). Fibrinogen con~ainsthis peptide sequence in two locations of the alphachain.

The IgM, PAC1, binds stimulatd platelets with anaffinity of 5 nM but does not bind unstimulatedplatelets (117) (similar to fibrinogen). The epitopebinding region of this IgM has been mapped and the21amino acid sequence contains the tripeptide sequence,Arg-Tyr-Asp (RYD). The synthetic 21 amino acidpeptide was shown to inhibit fibrinogen-dependentplatelet aggregation as well as PAC 1 binding toplatelets. Substitution of G (glycine) with Tyr in theRYD sequence of the peptide resulted in a peptide withincreased anti-platelet potent y over the originalsequence (118).

Knight and co-workers have evaluated a series ofRGD and RYD containing peptides as thrombusimaging agents (1 19). In this study 16 to 31 residueanalogs of the hypervariable sequence of tie IgM,PAC 1, which binds stimulated platelets with an affinityof 5 nM (but not unstimulated platelets) were prepared.All of the peptides contained a tripeptide binding group(RYD or RGD) and a metallothionein-derived sequence(KCTCCA) for Tc-99m labeling. These peptidescleared rapidly and images of fresh thrombi in thejugular veins of rabbits and day old thrombi in thefemoral veins of dogs were obtained within 1-2 hoursafter injection. In control experiments, a Tc-99mlabeled nonspecific peptide did not image the thrombi.These investigators concluded that the use of RGDpeptides is technically feasible but that further improve-ments are necessary for reliable thrombus imaging.

OTHER PEPTIDES

Atrial Natriuretic Peptide (ANP)ANP is a 28 amino acid peptide which is produced

in the cardiac atrium. Receptors for ANP are found inthe kidneys, adrenals, and lungs (120). In the kidneythese receptors are most numerous in the glomeruli andplay an important role in fluid, electrolyte, and blood

pressure homeostasis (121). Various types of bindingsites have been repotied for ANP (122,123) and thesedifferent ANP receptors have now been cloned (i,e.,A, B and C).

The ability to image ANP receptors has recently ●been demonstrated in monkeys (124) and rabbits (125).In the monkey, 1-123 labeled ANP was rapidly boundto ANP receptors in the kidneys and lungs. Theobserved uptake was demonstrated to be receptormediated by competition studies using simultaneousinjection of unlabeled ANP. In a more recent report(126), after intravenous administration to monkeys, I-123 ANP was shown to localize primarily in the kidneyand lungs, and blood clearance was rapid. This studydemonstrated that localization was receptor mediatedbased on displacement studies using cold ANP and C-ANP. Biodistribution in the monkey correlated wellwith the presence of ANP receptors determined by exvivo receptor binding assays.

The diagnostic implication of ANP receptor imagingare not firmly established as yet but likely will allowassessment of renal glomerular diseases. Since ANPreceptor density and aff~nity are influenced by variousphysiological and pathological conditions, clinical anddiagnostic applications seem possible.

SUMMARY

Over the past several years there has been a large ●body of data accumulated on the use of somatostatinanalogs for tumor imaging (73). FDA approval of In-111 pentetreotide may signal a turning point inradiopharmaceutical design and development. Inaddition to In-111 pentetreotide, other peptides havefound their way into preclinical and clinical studies,such as ANP (126) and VIP (77), insulin (47),captopril (127), platelet factor 4 (128), interleukin- 1(129), aM2, a synthetic peptide mimicking thecomplementarily determining region of an anti-tumormonoclinal antibody (130). Also a significant amountof preclinical data is available on the the use ofchemotactic peptides for infection imaging (131).Clearly, the scope for futurebased radiopharrnaceuticalsbioactive peptides continue toprocesses, greater interest inprobes will no doubt follow,

REFERENCES

applications- of-peptide-is vast. As naturalbe implicated in diseaseusing them as imaging

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Fischman AJ, Babich JW. Rubin RH. Infectionimaging with technetium-99 m-labeled chemotactic

peptide analogs. Semirl Nucl Med 1994;24(2): 154-68.

QUESTIONS

1. Radiolabelti peptides are composed ofwhich of the following?

a. fatty acidsb. sugarsc. lipidsd, amino acids

2. Which of the following radionuclides hasb~rt used for radiolabeling peptides fordiagnostic imaging?

a. iodine-123b. technetium-99mc. iridium- 111d, all the above

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3. Which of the following structures has b=nshown to provide the greatest penetrationinto tumors?

a. single-chain antigen bindingproteins (sFv’s)

b. Fab antibody fragmentsc. F(ab)2 antibody fragmentsd. whole, intact antibodies

4. Which of the following fractions from theSEP-PAK quality assessment of iridium In-111 pentetratide contains the hydrophilicimpurities from the product?

a.b.c.

d.

5, Which

SEP-PAK cartridgefraction number 1 eluted in waterfraction number 2 eluted inmethanolall the above

of the following amino acidresidues must be present in a peptidestructure before dirmt radioiodinationlabeling can occur?

a. tyrosineb. aspartic acidc. serined. valine

6. Somatostatin is involved in which of thefollowing types of cell signaling?

paracrine:: endocrinec. synapticd. all the above

7. Which of the following components of theIn- 111 pentetreotide kit acts as a stabilizer?

a. citric acid

b. ferric chloride

c. gentisic acid

d. hydrochloric acid

8, Which of the following blood cells arethought to possess somatostatin receptors?

erythrocytes;: plateletsc. stem cellsd. lymphocytes

9. Radiolabeled atrial natriuretic peptider~eptor imaging should allow diagnosis ofwhich of the following?

a, renal glomerular diseaseb. myocardial infarctionc. small cell lung carcinomad. Hodgkin’s and non-Hodgkin’s

lymphoma

10. Which of the following chemical classesdoes HYNIC (hydrazino nicotinamide)come under?

a. biologically active polypeptideb. monoclinal antibodyc. bifunctional chelated. amino acid

11. Technetium-99m labeled chemotacticpeptides have been shown to possess highbinding affinity to which of the followingtypes of blood cells?

erythrocytes;: lymphocytesc. plateletsd. none of the above

12. Both RGD and RYD peptides for thrombusimaging contain how many amino acids intheir sequence?

a. 14b. 8c. 5d. 3

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13. Which of the following tumor types areknown to have sornatostatin r~ptors onthe tumor cells?

a. small cell lung cancerb. carcinoidc. ph~hromocytomad. all the above

14. Which of the following is the primaryroute of clearance of radioactive In-111pentetreotide?

hepatobiliary system:: reticuloendothelial systemc. renal excretiond. none of the above

15. Which of the following tumor types areknown to have vasoactive intestinal peptide(VIP) receptors on the tumor cells?

a. non-small cell lung cancerb. breast carcinomac. colon adenocarcinomad. all the above

16. Technetium-99rn labeled chemotacticpeptides have been indicated for nuclear‘m~icine imaging of whichfollowing disorders?

infection;: myocardial infarctionc. colorecti cancerd. stroke

17. Which of theincluded in the

following aminostructure of RGD

a. tyrosineb. glycinec. glutamic acidd. serine

of the

acids ispeptide?

18. Radiolabeled peptides may allow for whichof the following clinical indications?

a. tumor detwtion and stagingb. therapeutic monitoring ●c. characterization of tumor

biochemistryd. all the above

19. Which of the followingtime for preparedpentetreotide?

30 minutes:: 120 minutesc, 6 hoursd. 18 hours

20. Which of the following

is the expirationiridium In-ill

has b=n studiedfor its potential as a thrombus imagingagent in nuclear medicine?

a. radiolabeled anti-fibrin antibodiesb. radiolabeled fibrinogenc. radiolabeled colloidsd. all the above

21, The quantity of pentetreotide in the In-111pentetreotide kit is times lessthan that of therapeutic dosages ofoctratide used to control the symptoms ofneuroendocrine tumors.

a. 1-4b. 5-20c. 20-50d, 50-80

22. Which of the following adjunctindications is suggested for use in patientsundergoing nuclear mdicine imaging withIn- 111 pentetreotide in order to improvethe images?

dexamethasone:: morphinec. furosemided. laxatives

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23.

@

24,

25.

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Which of the following is considered thecritical organ (receives the highestradiation absorbd dose) for In-111pentetreotide?

urinary bladder;: kidneys

spl~n:: liver

Which of the following radioiodinationmethods provides greater stability whenradiolabeling octreotide analog with iodineradionuclides?

Chloramine-T:: N-succinimydyl-5 -iodo-3-pyridine

carboxylateiodine monochloride

;: Iodogen

According to the literature, which of thefollowing peptide compounds has beenradiolabeled with the positron emittingradionuclide, fluorine-18?

chemotactic peptide:: octreotidec. insulind. all the above

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