ANTICANCER RESEARCH 29: 3777-3784 (2009)
`
`In Vivo Imaging of Human Colorectal Cancer Using
`Radiolabeled Analogs of the Uroguanylin Peptide Hormone
`DIJIE uu1.s, DOUGLAS OVERBEY1, LISA D. WATKINSON1.5, SAID DATBES-FIOUEROA 1·-",
`TIMOTHY J. HOFFMAN1.4.5, LEONARD R. FOIITE 1•3.S, WYNN A. VOLKERT1•2•5 and MICHAEL F. GIBLIN1•2•5•*
`
`1Research Service, Harry S, Truman Memorial Veterans' Administration Hospital, Columbia, MO 65201;
`Departments of 2Radiology, 3Medical Pharmacology and Physiology, and 4Tnternal Medicine, and
`5The Radiopharmaceutical Sciences Institute, Univen·ity of Missouri-Columbia, Columbia, MO 65211, U.SA.
`
`Abstract. Background: Uroguanylin is an endogenous
`peptide agonisi that binds ra the guanylate cyclase C
`is overexpressed
`receptor (GC-C). GC-C
`in human
`colorectal cancer (CRC), and exposure of GC-C-expressing
`cells to GC·C agonists results in cell cycle arrest and/or
`apoptosis, highlighting the therapeutic potential of such
`compounds. This study describes the first use of radiolabeled
`uroguanylin analogs for in vivo detection of CRC. Materials
`and Methods: The peptides uroguanylin and IP-uroguanylin
`were N-terminally labeled with the DOTA chelating group
`via NHS ester activation and characterized by RP-HPLC,
`ESl-MS, and GC-C receptor binding assays. The purified
`conjugates were radiolabeled with ln-111 and uud for in
`vivo biodistribution and SPECT imaging studies. In vivo
`experiments were carried out using SC!D mice bearing T84
`human colorectal cancer
`tumor xenografts. Results:
`Alteration of the position 3 aspartate residue to glutamate
`resulted in increased affinity for GC-C, with IC50 values of
`5.0±03 and 9.6±2.9 nM for IP-uroguanylin and DOTA-E3-
`111 Jn-DOTA-E3-
`uroguanylin,
`respectively.
`In
`vivo,
`uroguanylin demonstrated tumor uptake of 1.17±0.23 and
`0.61 ±0.07%JD/g at 1 and 4 h post injection, respectively.
`The specificity of tumor localization was demonstrated by co(cid:173)
`injection of 3 mg/kg unlabeled E3-uroguanylin, which
`reduced tumor uptake by 69% . Uptake in kidney, however,
`was dramatically higher for the uroguanylin peptides than
`for previously characterized radiolabeled E. coli heat-stable
`enterotoxin ( STh) analogs targeting GC-C, and was also
`
`Correspondence to: Dr. Michael F. Giblin, Harry S. Truman
`Memorial VA Hospital, Research Service Room A004, 800 Hospital
`Drive, Columbia, MO 65201, U.S.A. Tel: +l 5738146000 ell!,
`53669, Fax: + I 5738821663, e-mail: gibllnm@health.missouri.edu
`
`Key Words: Uroguanylin, E.coli heat-stable enterotox!n, guanylyl
`cyclase C, single photon-emitting computed tomography (SPECT),
`colorectal cancer, in vivo imaging.
`
`inhibited by coinjection of unlabeled peptide in a fashion not
`previously observed. Conclusion: Use of uroguanylin(cid:173)
`targtting vectors for in vivo imaging of colorectal cancers
`expressing GC-C resulted in tumor uptake that paralleled
`that of higher affinity heat-stable enterotoxin peptides, but
`also resulted in increased kidney uptake in vivo.
`
`Guanylate cyclase C (GC-C) is a type I transmembrane
`glycoprotein expressed on brush border membranes of
`intestinal epithelial cells, as wen as on transformed human
`colon cancer cell lines such as the T-84 cell line (1, 2). In
`the normal intestinal mucosa, GC-C receptors are expressed
`within the apical (luminal) face of epithelial cell membranes,
`and are therefore isolated from the bloodstream by cell-cell
`tight junctions (3-6). GC-C expression is maintained in
`transformed cells throughout the process of colorectal
`cHrcinogenesis, while expression of the endogenous GC-C
`ligands guanylin and uroguanylin is typically lost (7-9).
`Normally expressed at high levels within the lumen of the
`gut, GC-C is expressed on virtually all histologically
`confirmed primary and metastatic colorectal
`tumors
`examined in human patients, while normal tissues and other
`types of cancer express minimal or no GC-C receptors (4-
`6). GC-C receptors on colorectal tumors retain their ligand(cid:173)
`binding capacity, and expression of GC-C receptors does not
`vary as a function of metastatic site or grade of these tumors
`(5). The unique expression of GC-C by metastatic cells of
`colorectal origin within lymph nodes of patients undergoing
`staging for colorectal cancer (CRC) forms the basis for a
`PCR-based diagnostic lest that is currently undergoing
`clinical trials (10). GC-C expression has also formed the
`basis of development of ligand-based molecular agents for
`in vivo detection and therapy of colorectal cancer (9, 11-20).
`Uroguanylin is a 16 amino acid peptide with two disulfide
`bonds, and has mmornolar affinity for the GC-C receptor (21,
`22). Secretion of the endogenous peptides guanylin and
`uroguanylin into the lumen of the gut by enterochromaffm
`cells plays a role in regulation of ion and fluid homeostasis
`
`0250-7005/2009 $2 .00+ .40
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`ANTICANCER RESEARCH 29; 3777-3784 (2009)
`
`by activation of the cystic
`transmembrane
`fibrosis
`conductance regulator ( CFTR), generating net efflux of
`sodium, chloride, bicarbonate, and water into the lumen of
`the intestine (23), In nature, the E. coli heat-stable
`enterotoxin (STh) is expressed by enterotoxigenic strains of
`to co-opt the endogenous
`E. coli bacteria in order
`uroguanylin/GC-C ligand-receptor system. Bacteria such as
`E. coli have evolved heat-stable peptides which differ
`st1ucturally from uroguanylin family peptides in that they
`third disulfide bond, which is presumably
`possess a
`responsible for both increased resistance to heat/enzymatic
`degradation as well as superagonist activity. These
`guanylin/uroguanylin mimics are also the highest affinity
`ligands known for the GC-C receptor (22, 23).
`We have developed numerous analogs of the E.coli heat(cid:173)
`stable enterotoxin for the purpose of developing an optimal
`molecular imaging vector for colorectal cancer ( 11, 13-1.5).
`Such imaging constructs could enable noninvasive imaging
`of CRC patients, and help to define patient groups that could
`benefit from treatment with GC-C agonists. Here, we
`examine the consequences of using the structurally less
`complex uroguanylin molecule on targeting efficiency. ln
`vivo targeting was assessed using peptides with N-terminal
`DOTA macrocyclic chelators, labeled with the radionuclide
`In-111. Two analogs of the uroguanylin peptide have been
`compared with respect to in vitro binding affinity and in vivo
`biodistribution patterns in SCID mice bearing T84 human
`co1orectal cancer tumor xenogrnfts.
`
`Materials and Methods
`
`All solvents were either ACS certified or HPLC grade solvents
`obtained from Fischer Scientific and used as received, DOfA-NHS
`ester was purchased from Macrocyclics (Dallas, TX, USA). 111InCl3
`was obtained from Mallinckrodt Medical, Inc (St. Louis, MO, USA)
`as a 0,05 N HCI solution. Wild-type human uroguanylin was
`obtained from the American Peptide Company, and E3-uroguanylin
`was kindly provided by Dr. Kunwar Shailubhai at Callisto
`Pharmaceuticals, All other reagents were purchased from Aldrich
`Chemical Company. Human colon carcinoma T-84 cells were
`obtained from the American Type Culture Collection (ATCC) and
`maintained and grown for use in these studies in the University of
`Missouri Cell and Immunology Core facilities. MALDI-TOF mass
`spectral analyses were performed by the proteornics core facility at
`the University of Missouri-Columbia,
`
`High performance liquid chromatography ( HPLC). High performance
`liquid chromatography (HPLC) analyses were performed on a
`Shimadzt, system equipped with an SPD-20A lN detector imd an in(cid:173)
`line sodium iodide crystal radiometric detector, HPLC solvents
`consisted of H2O containing 0 .1 % trifluoroacetic acid ( solvent A)
`and acetoni tril.e containing O. I % trifluoroacetic acid ( solvent B).
`Conditions: A Phenomenex Jupiter C-18 (5 µrn, 300 A, 4.6x250 mm)
`column was used with a flow rate of i .5 ml/min, Gradient purification
`of compounds is achieved during a linear 30 minute ramp from 23%
`B to 33% B, followed by colunm rinse and re-equilibration.
`
`Peptide synthesis and radiolabeling. Fl9-STh(l-19) was iodinated
`by a modified lactoperoxidase method. Briefly, 2 µg peptide was
`suspended in 50 µJ 100 mM sodium phosphate buffer, pH 7 .5,
`containing 2 µg lactopemxidase and 0.25-1.0 mCi Na12~I. The
`reaction was initiated by addition of 2 µI of a l: 10,000 dilution of
`30% H20 2• The reaction wru, incubated 30 min at room temperature
`with occasional mixing, then diluted with dH20 and purified to
`homogeneity by RP-HPLC.
`DOfA labeling of the folded urogmmylin peptides proceeded using
`a l 00-fold molar excess of !he DITTA-NHS ester. The reactions were
`incubated in 150 mM HEPES ut 4'C overnight, quenched with TRIS
`buffer and purified by Cl8 RP-HPLC. HPLC purified DOTA-peptides
`were subsequently characteri7,ed by ESI-MS and in vitro cell binding
`assay. For the synthesis of ll!Jn-labeled compounds, aliquots of
`1 l 1InCl3 (0.2-2.5 mCi, 4-50 µ!) were added to solutions of 50 µg of
`respective peptides in 0.2 M ammonium acetate (200 µl). The pH of
`reaction mixtures was adjusted lo 5.8, and reactions were incubated
`for 1 hour at 80'C. After 1 hour, 2 mM EDTA (50 µl) was added to
`complex unreacted 111In+3. The resulting conjugates were purified to
`homogeneity by RP-HPLC. The 111In-metallated conjugates eluted
`between 1.1-1.9 minutes before the associated non-metallated species
`enabling collection of high-specific activity, no cllITier-added I llfo(cid:173)
`peptide conjugates. All purified 111Jn-peptido conjugates were then
`concentrated by passing through a 3M Empore C-18 HD high
`pcrformruice extraction disk (7 mm/3 ml) cartridge and eluting with
`50% ethanol in 0.1 M NaH2P04 buffer (500 µ!). The concentrated
`fraction was then reduced in volume under a stream of N2, and finally
`diluted with 0.1 M NaH2P04 buffer, pH 7 .0, to a final activity of
`approximately 2 µCi/JOO pl.
`
`In vitro eel/ binding swdies, Peptide IC50s were determined by a
`competitive displacement cell binding assay using 12SI-F19-STh(l(cid:173)
`l9). Briefly 3xl06 cells suspended
`in DMEM/F-12 media
`containing 15 mM MES and 0.2% BSA, were incubated at 37'C for
`I hour in presence of approximately 20,000 cpm 125I-tracer and
`increasing concentration of the DITTA-peptide conjugates. After the
`incubation, the reaction medium was aspirated and cells were
`washed three times with media. The rndioaclivily bound to the cells
`was counted in a Packard Ria.star gamma counting system. The %
`12'I-F19-STh(1-19) bound to cells was plotted vs. increasing
`concentrations of DOTA-peptides to determine the respective IC50
`values. For statistical considerations, duplicate in vitro cell binding
`experiments with each analog were performed. IC50 calculations
`were performed using the 4 parameter logistic model within !he
`program grafil (Erithacus Software, Ltd),
`
`in vivo pharmacokinetic studies of 111 ln-peptide analogs in SCID
`mice. Four- to 5-wcck old female ICR SCID (severe combined
`immunodeficient) outbrnd mice were obtained from Taconic
`(Germantown, NY). The mice were housed four animals per cage
`in sterile micro isolator cages in a temperature- and hnmidity(cid:173)
`controlled room with a 12-hour ligh!/12-hour dark schedule. The
`animals were fed sterile rodent chow (Ralston Purina Company, St.
`Louis, MO, USA) and wuter ad libitum. Animals were housed one
`week prior to inoculation of tumor cells and anesthetized for
`injections with isoflurnne (Baxter Healthcare Corp., Deerfield, IL,
`USA) at II rate of 2.5% with 0.41 oxygen through a non-rebreathing
`anesthesia vaporizer.
`Human colon cancer T-84 cells were injected on the bilateral
`subcutaneous (s,c.) flank with N5x106 cells in a suspension of JOO
`
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`Liu el al: Radiolabeled Uroguanylin Analogs
`
`A
`
`B
`"'N-,-1'.)- S,-b.,.;E,-L-O-V;..,N ... V;_A~.6-i-G-O'-L
`I
`. ·1
`
`Figure I. Struciures of(A) a11d uroguanylin (BJ E3-uroguanylln,
`
`Table I. Calc11/a1ed and observed (M+lfJ+ ya/11es and ICso values(~
`SD) for characterized peptidu.
`
`lll'IO" 1x1dr . 1X1b" · i x1.tJ•
`,1x1·0-:~ 1x10-•• 1X!O'" 1~1.0~
`[peptidel(M}
`
`Figure 2. JC50 ,matyscs ofuroguanyl/n airaJog Ji,p/aceme,r/ of 125/-F19.
`STh( 1-19) from TS4 human colortc/111 cancer cel/1.
`
`Peptide
`
`(M+H)+ Cale.
`
`(M+H}·Obs. ICso (nM)
`
`Results
`
`Uroguonylin
`DOTA•uroguanylin
`El•urognanylin
`DOTA-E3-urog11any\in
`
`1667.6
`2053.6
`1681.6
`2067.6
`
`1667.7
`2053.9
`1681.6
`2067.9
`
`39.8:1:14.9
`34.5±3.3
`S.0±0.3
`9.6:t2.9
`
`µI 3:1 PBS:Matrigel (BO Biosciences, Bedford, MA, USA) per
`injection site. T-84 cells were allowed to grow In vivo four to si,c
`weeks post inoculation, developing tumors ranging in sizes from
`0.06·0.59 grams. The biodlstributioa and uptalce of lllJn-DOTA(cid:173)
`uroguanylin and 111Jn-DOTA-E3.uroguanylin in tumor bearing
`SCIO mice was studied following randomization of animnls such
`that no significant (p<0.05) differences existed with respect to
`tumor sizes between test groups. The mice (average weight, 25 g)
`were injected with nliquots (50- tOO µI) of the radiolabelcd peptide
`solution (1-3 µCi) in each animal via the tail v.ein. Tissues, organs
`and tumors were excised from animals sacrificed at 1 hour and 4
`hour post-iajectlon (p.l.), weighed. and counted. Radioactivity was
`measured in a NaI counler and tile percent-injected dose per organ
`and the percent-injected dose per gram tissue were calculated.
`Animal studies were conducted in accordance willl the highest
`standards of care as outlined in the NIH guide for Core and Use of
`Laboratory Animals and the Policy and Procedures for Animal
`Research at the Harry S. Truman Memorial Veterans' Hospital and
`according to approved protocols.
`
`SPECTICT imaging. A combined micro-SPECT/CT unit (microCAT
`IT, Siemens Medical Systems) was employed for Single Photon
`Emitting Computed Tomography/ Computed Tomography imaging
`studies. One SCID mouse bearing T84 human colorectal cancer
`tumor l\enografts was injected intravenously with 220 11Ci (100 µI)
`111In-DOTA-uroguanylin solution and sacrificed at I hour p.i.
`Micro-SPECT scans of 60 projections were performed using a
`symmetrical 20% photopenk discriminating window. Volumetric
`data from SPECT nnd CT was visualized and image fused using
`Amira 3.1 (TGS, San Diego, CA, USA).
`
`Two DOTA-Jabeled uroguanylin nnulogs were synthesized in
`this work. and their characteristics as agents targeting the
`GC-C receptor were compared to tl1ose of previously
`synthcsiz:ed analogs of the E. coli heat-stable enterotoxin
`(STh) ( 13, 14). Each of these peptides is structurally rel.ated,
`and the two urogttanylin analogs differ only in a conservative
`D3E substitution (Figure 1). Uroguanylin peptides share
`significant sequence homology with STh, including absolute
`conservation of four cysteine residues that form a conserved
`array of disu]fide bonds (Figure 1). Analogs of the E. coli
`heat-stable enterotoxin also possess a third disulfide bond,
`lending these bacterial uroguanylin mimics exceptional
`stability and affinity for the GC-C receptor.
`Peptides were purified by RP-HPLC and characterized by
`MALDI-TOP MS and by a competitive displacement
`receptor binding assay utilizing T84 human colorectal cancer
`cells and 125I- labeled F 19-STh(l-19) (Table I, Figure 2) .
`Observed JC50 values for uroguanylin and DOTA(cid:173)
`uroguanylin (39.8:t 14.9 and 34.5±3.3 oM, respectively) were
`significantly higher than those of previously characterized
`STh analogs. as expected from previous studies of this class
`of peptide agonists (13-15, 22). A conservative D3E
`substitution, however, significantly increased the binding
`affinities of the resultant uroguaoylin analogs, with measured
`JC50 values for E3- urogunnylin and DOTA- E3-uroguanylin
`of 5.0±0.3 and 9.6±2.9 nM. respectively.
`To determine the effects of affinity differences on in vivo
`tumor localization, DOTA peptides were labeled with 111In ,md
`purified by RP-HPLC (Figure 3). Addition of N-tennioal DOTA
`moieties resulted in a 0.9-1.1-minute shift to earlier retention
`times for each uroguanylin peptide. Further 1.1-1.9-minute shifts
`to earlier retention times were ob~erved upon coordination of
`lllin by DOTA peptides. lllJn.Jabeled uroguanylin analogs were
`
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`ANTICANCER REsEARCH 29: 3777-3784 (2009)
`
`l,,,._ ____ ,. ,: l _ _
`
`Figure 3. RP-HPLC chromarogrmns of pur/fi'-d m1n-DOTA-E3-
`urog110nyUn (top), 111/n-.DOTA· urogua,.ylin (bottom).
`
`Figure 4, BlodistrlbUJion analysis of /lJ /11-DOTA-l'J-uroguonylin with
`and without co-injection of 70 µg unlabeled E1,uroguanylin, and I llJn(cid:173)
`DaTA-uroguanylin at I hr pi (%/Dig, N=4).
`
`found to posse~ chromatographic properties cotTesponding to
`those of previously cha:acterized 111In-labeled STo peptides,
`requiring similar acetonitrile concentrations for elution from a
`Cl8 RP-HPLC column. RP-HPLC purification of each lllJn.
`labeled peptide resulted in high specific activity radiotmcers that
`were subsequently tested in animal models.
`ln vivo, tumor uptake of 111hi-DOTA-E3-uroguanylin at l
`hour p.l. trended higher than that of 111In-DOI'A-uroguanylh1
`(1.04±0.07 and 0.88±033 %ID/g, respectively), although the
`difference did not achieve the p<0.05 significance level (Figure
`4). 111In-DCTfA-E3-uroguanylin demonstrated significant
`(p<0.001) specific tumor uptake in T84 human colorecta\
`cancer tumor xenografts, as uptake of this compound was
`reduced to 0.32±0.06 % ID/g at 1 hour p.i. by co-injection of
`saturating concentrations of unlabeled E3-uroguanylio (Figure
`4). Uptake in tumor at 1 hour p.i. was higher than for all other
`tissues, with
`the exception of kidney. Tumor/blood,
`tumor/muscle, and tumor/liver ratios at this timepoint wete 4.7,
`20.8, and 6.1, respectively. At 4 hours p.i., tumor uptake of
`111In-DITTA-E3-uroguanylin decreased to 0 .61±0.07 % ID/g.
`However, activity also rapidly washed out of nontarget tissues,
`resulting in tumor/blood, tumor/muscle, and tumor/liver ratios
`of 6l, 61, and 4.4, respectively at 4 hours p.1. These
`target:nontarget ratios compare favorably in certain respects
`with those obtained previously at the same time p.i. using STh
`peptides such as 1 t!In-DOI'A-R l.4 ,F19-STh(l-19) (21, 55, and
`4.4, respectively) (13), although tumor uptake was generally
`higher for 111Io-DOTA-labeled STh peptides ( 1.64 % ID/g at
`4 hour p.i. for 11 1In-DOl'A-R1.4,F 19-STh(l-19)) (13, 14) .
`The most significant difference between the In vivo
`biodistribution of uroguanylin analogs and previously
`characterized STh peptides related to uptake in kidney. While
`the observed kidney uptake of lllJn. DOTA-labeled STh
`analogs at 4 hours p.i. has previously been observed in the
`(13-15), 111In-DOI'A-E3-
`range of 2.2-4.3 %ID/g
`
`3780
`
`1111'>-COT/\·8-
`01 hr
`Urog"""y)l'l
`• I hr '"il,00)'A•R1,◄•
`Ftfl.SThf1·11l)
`.,tfir"'il-OOTA-et,
`u-.,g..,,y:t,
`JH t,t ' "11,COTA,Rt,4-
`F1M!Th(MG)
`
`~,
`Figure 5. Kidney up1ake of 111Jn-DOTA-l'J-urog1Wnylin al I and 4
`hour., post injectio,1 compared with kld,1ey uptake of 111Jn-DOTA-
`11'·'.f19.STh( 1-19)11 at the Sll/M ti~ points.
`
`uroguanylin kidney uptake at this timepoint was greater than
`10-fold higher (Figure 5). This high kidney uptake was
`blocked by co-injection of excess unlabeled E3-uroguanylin,
`decreasing from 28.3±10.6 %ID/g to 14.9±3.0 %ID at 1
`hour p .l. Despite this higher kidney uptake however, in vivo
`SPECT/CT i.mGgiog clearly showed specific uptake of l 11In(cid:173)
`DOTA-uroguanylin in T84 tumor xenografts (Figure 6). Toe
`tumor:kidney ratio of 1111n-DOTA-E3-uroguanylin is lower
`than previously observed for l llJn-labeled STh analogs (13-
`15). However, uptake in other nontarget tissues is also low,
`demonstrating the utility of radiolabeled uroguanylin pepti<k
`analogs for the locali7..ation of GC-C-expres~ing cells in vivo.
`
`Discussion
`
`Research into the development of peptide ligands targeting
`the GC-C receptor has resulted in the generation of
`numerous analogs with a diverse array of incorporated
`structural alterations. The development of GC-C targeted
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`

`Liu et al: Radiolabeled Uroguanylin Analogs
`
`affinity for GC-C only two- to three-fold lower than that of
`full-length STh analogs (13-15).
`Differences in hydrophobicity between uroguanylin and
`STh peptides did little to alter the pharrnacokinetic behavior
`of the compounds in vivo with respect to paran_ieters such as
`hepatic excretion. In vivo, lllln-DOTA-labeled uroguanylin
`peptides cleared rapidly from the bloodstream via the
`renal/urinary route, with >83% of injected activity excreted
`into urine at I hour p.i. Both uroguanylin analogs however
`had lower urinary excretion and higher retention in kidney
`than 111In-labeled STh peptides. Although DOTA-E3-
`uroguanylin demonstrated significantly higher binding
`affinity than DOTA-uroguanylio In vitro, this change did not
`result in significantly (p<0.05) increased tumor-specific
`uptake (Figure 4, Table I). At l hour p.i., the tumor
`specificity of 111 In-DOTA-E3-uroguanylin was demonstrated
`by a 69% drop in tumor specific uptake upon coinjection of
`a blocku1g dose of unlabeled uroguanylin peptide (Figure 4).
`Surprisingly, 111In-D0fA-E3-uroguanylin also demonstwted
`a high degree of uptake in kidney (Figure 5) . The high
`kidney uptake observed at l hour p.i. (28.3±10.6 %ID)
`incrensed even further at 4 hours p.i. to 49.4±3.2 % ID, a
`value more thnn IO-fold higher than was observed previously
`for 111In-DOTA-STh peptides at 4 hours p.i. (13, 14).
`Comparable uptake in kidney has never previously been
`observed for labeled heat-stable enterotoxin analogs, and
`suggests the possibility of a novel uroguaoylin receptor existing
`in kidney that can discrimlnate between uroguanylin and heat(cid:173)
`stable enterotoxin ligands. Several experimental findings have
`contributed to the belief that a receptor for uroguanylin family
`peptides dislinct from the well-characterized GC-C receptor
`exists. First, induction of natriuresis and kaliuresis by peptides
`in the uroguanylin family is observed in kidneys of GC-C
`knockout mice (24). Second, cellular responses to uroguanylin
`in immortalized human kidney epithelial (IHKE-1) cells were
`detected io whole cell patch clamp experiments that were
`distinguishable from responses to an STh peptide (2S),
`suggesting involvement of an unknown pertussis toxin-sensitive
`a protein in binding of uroguanylin in kidney cells. Thi.rd,
`uroguanylin knockout mice demonstrated a decrease in Na+
`excretion following oral salt loads when compared to wild-type
`controls, while GC-C knockout mice demonstrated 110 such
`impairment (26). In this stody, kidney uptake of 111In-DarA(cid:173)
`E3-Urogaanylin at 1 hour p.i. was reduced 47% by colnjection
`of 70 µg unlabeled E3-Uroguanylin. However, In vitro binding
`studies utilizing partially purified kidney membranes
`ll1Jn-DOTA-E3-
`demonstrated no specific binding of
`urogunnylin (Data not shown). Therefore, the question of
`whe1her the increased kidney uptake of uroguaoylin peptides
`observed here is due to ao as yet u11characteri1.ed uroguanylin(cid:173)
`speeific receptor in kidney, or alternatively is due to a structural
`property of the uroguanylin peptide such as its ac.idic N(cid:173)
`terminus, remains to be determined.
`
`3781
`
`Figure 6. SPECTICT image of a SCJD mouse bearing blla1eral hind
`}lank T84 human colorectal cancer tumor xenografts I hour p .i. of 220
`µCi IIIJn-DOTA-11,-oguanylin. Arrows indicale locations of tumors.
`
`radiopharmaceutical agents has until now centered on the use
`of tri-disu1fide analogs of the heat-s1able enterotoxin molecule,
`due to their having the highest affinity known for the GC-C
`receptor. In this work, we have compared the properties of
`such a peptide with those of structurally related uroguanylin
`peptides. Uroguanylin peptides are the endogenous ligands for
`the GC-C receptor, have lower affinity for the GC-C receptc>r,
`and Jack one of the three disalfide bonds present in heat-stnble
`enterotoxin peptides, thereby simplifying their chemical
`synthesis. Comparison of such peptides in vitro and in vivo
`makes possible the analysis oft.he effects of small variations in
`receptor binding affinities on tumor localiz.ation, and could
`also lead to the imaging of GC-C in vivo \\-'.ith o structurally
`simpler class of peptide ligands.
`In this study, we have synthesized two modified analogs
`of uroguanylin, and compared their in vitro and In vivo
`properties with previously characterized E. coll beat-stable
`entcrotoxin analogs possessing an array of linker sequences
`to N-tenninal DarA moieties. Each uroguanylin peptide also
`possessed an N-terminal DOTA chelating moiety, which is
`capable of complexing a wide range of trivalent imaging and
`therapeutic radionuclides, including 90y+3, 111 Jn+J, l49pm+3,
`153sm+3 , 166Ho+3, and 177Lu+3 . Each DCYI'A-peptide was
`synthesized and purified by C 18 RP-HPLC. Both DOTA(cid:173)
`urogunnylio and DOTA-E3-uroguanylin were shown 10 have
`lower affinity for the GC-C receptor than previously
`characterized DOTA-STh peptides (13-15). For example,
`DOTA-uroguanylin showed approximately a 10-fold lower
`affinity than DOTA-R1•4 ,F19-STh(l-19) in an
`in vitro
`competitive receptor binding assay. A conservative D3B
`substitution in wild-type human uroguanylin was shown to
`increase the affinity of the peptide for GC-C three- to four(cid:173)
`fold, resulting in a uroguanylin-based imaging probe with
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`

`ANTICANCER RESEARCH 29: 3777-3784 (2009)
`
`Peptides in the uroguanylin family are currently under
`active investigation as phannacologicel agents with potential
`applications in the treatment of colorectal cancer (9, 16-20,
`27-30). Such therapeutic use of uroguanylin peptides relies
`on the function of uroguanylin analogs as GC-C agonists,
`eliciting intracellular production of cGMP (23). Early
`investigations into the function of uroguunylin peptides
`demonstrated that bacterial ST peptides functioned as
`superagonists for the GC-C receptor when compared with
`uroguanylin analogs, having higher binding affinity as well
`as increased potency in assays meaYuring both cGMP
`synthesis and stimulation of short circuit current resulting
`from c1- efflux from GC-C-expressing cells (23). Subsequent
`demonstrations by other researchers that expression of GC-C
`is maintained or even up-regulated in colorectal cancer
`metastases ( 4-6), while expression of the endogenous
`guanylin ligand is lost (7, 8), led to the hypothesis that the
`endogenous ligands could function in a tumor suppressor role.
`Experiments lo address this hypothesis have been carried out
`in a number of different laboratories, and have confirmed the
`growth-inhibitory signaling elicited by peptides in
`the
`uroguunylin family (9, 16-20, 27-29). Several studies have
`demonstrated either cell cycle arrest or apoptosis in response
`to exposure of GC-C-expressing cells to STh ligands (9, 19,
`27-29), and treatment of APCmin mice with therapeutic levels
`of uroguanylin resulted in marked reduction of intestinal
`polyp formation in this in vivo model (9). Together, these
`results point to a possible therapeutic role for uroguanylin
`peptides in and of themselves in the treatment of colorectal
`cancer, either alone or in combination with phosphodiesterase
`inhibitors such as exisulind.
`The results presented here have implications both for the
`molecular imaging and therapy of colorectal cancer as well
`as for the function of uroguanylin as a specific effector of
`natriuretic and kaliuretic responses in the kidney. With
`respect to molecular imaging of colorectal cancer in vivo, the
`DOIA-uroguanylin peptides described here have been used
`to successfully image GC-C-expressing human colorectal
`cancer xenografts in vivo, with rapid clearance from the
`blood pool and minimal uptake in nontarget tissues other
`than kidney (Figure 6). Given their ease of synthesis relative
`to STh analogs and similar tumor:nontarget tissue ratios,
`uroguanylin analogs labeled with In-l 11 or other imaging
`radionuclides (e.g. F-18, Tc-99m, Ga-68, Cu-64) are worthy
`of further study. The ability to image GC-C-expressing
`malignancies in vivo would provide a useful adjunct lo the
`development of GC-C ligands as cancer therapeutics, The far
`higher kidney retention of radiolabeled uroguanylin however
`necessarily reduces contrast between tumor tissue and this
`organ. Although potentially acceptable in the context of
`imaging studies, such high kidney uptake would preclude the
`use of labeled uroguanylin analogs as radiotherapeutic agents
`in favor of STh-based peptide-receptor radiotherapy (PRRT)
`
`constructs. With respect to use of uroguanylin peptides for
`the treatment of disseminated colorectal malignancies, work
`is currently underway to further elucidate the nature of the
`kidney uptake observed in this study.
`
`Acknowledgements
`
`This material is the result of work supported with resources and the
`use of facilities at the Harry S Truman Memorial Veterans' Hospital,
`Columbia, MO 65201, and the University of Missouri-Columbia
`School of Medicine Deparlmenl of Radiology, Columbia, MO
`65211. This work was funded -by a United States Department of
`Veterans' Affairs Biomedical Laboratory Research and Development
`Service VA Merit Award, and a National Cancer Institute center
`grant (l P50 CA103130-0l).
`
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`Liu et al: Radiolabeled Uroguanylin Analogs
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