Preclinical Evaluation and Phase I

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ChnicalTrual ofa
..
Tc-Labeled S 99m

Synthetic Polymer Used in Blood

Pool Imaging

RonaldJ. Callahan1 OBJECTIVE.Toobtaininitialdataonthesafetyandefficacyof a novelpolymeric,

synthetic
Alexei Bogdanov,Jr. blood pool contrast agent {O-monomethoxypoly(ethylene glycol)-O@succinyl)poly(N-E-L-ly
Alan J. Fischman syl{99mTc)diethylenetriamine pentaacetate monoamide, we performed a preclinical evaluation
andphase1clinical trial under an investigator-sponsoredinvestigationalnew drug application.
ThomasJ. Brady
MATERIALS AND METHODS. Methoxypoly(ethylene
glycol)ethylenetriaminopen
RalphWeissleder
taaceticacidwasformulatedinto a kit containingthepolymer,stannouschloride,anda buffer.
Kits were stored in frozen form for subsequentlabeling with technetium-99m. Acute and sub
acute toxicity studies were carried out in rats and rabbits. Healthy human volunteers (n = 6)
were then enrolled in a prospective, open-label phase 1 clinical study.
RESULTS. Animalstudiesshowedno signsof acuteor subacute
toxicityat doses280
timesthe proposeddosefor humans.In the clinical trial with humans,no significantabnor
malities of laboratory values, ECG findings, or hemodynamic parameters were seen. One vol
unteerexperiencedfacial flushingandpalpitations.Fourvolunteersshowedtypicalbloodpool
biodistnibution, with a blood half-life of 20.6 ±2.3 hr. At 24 hr after administration, 22.1% ±
2.5% of the injected dose had been excretedthrough the kidneys.Two other volunteers
showed a different biodistribution (primarily to liver and spleen), presumably associatedwith
labeling instability.
CONCLUSION. Syntheticmethoxypoly(ethylene
glycol)-graftedpolymerscan have
long circulation times in humans. Pharmaceuticals based on such polymers are expected to
have clinical applications in cardiovascular imaging, gastrointestinal bleeding studies, and
capillary leak imaging.

D iagnostic pharmaceuticals with a currently in clinical trials or in clinical prac

predominant distribution to the in
travascular space (blood pool
agents) are desirable for clinical imaging appli
cationssuchascardiovascular imagingandim
aging of vascular integrity. Cardiovascular
mapping, including gated cardiac blood pool
studies [I], perfusion imaging, functional im
aging, and MR angiography [2], have a vast
potentialin noninvasivelyobtainingphysio
logic information.Likewise, imaging of vascu
lar integrity or permeability is clinically
importantin imagingof tumorneovasculanity
ReceivedJune25,1997;acceptedafterrevision
January 22, 1998.
(angiogenesis)and in detectingsitesof inflam
mation or occult bleeding. The latter applica
All authors:Centerfor MolecularImagingResearchand
Divisionof NuclearMedicine,Departmentof Radiology, tions, involving imaging of the lossof vascular
MassachusettsGeneralHospitalandHarvardMedical integrity, require ultralong-circulating blood
School,Bldg.149,13thSt.,5403,Charlestown,MA02129. pool agents because vascular leaks may be
Address
correspondence
toR.Weissleder.
slowor intermittent[3, 4].
AJR1998;171:137—143
Several ultralong-circulatingagents, in
0361—803X/98/171
1—137 cluding RBCs [5—9],liposomes [10], or Ia
©AmericanRoentgenRaySociety beled human serum albumin (HSA), are
tice [I 1, 12]. Although effective in certain
clinical settings [12, 13], using RBCs has
some significant disadvantages: the need to
handlebloodproductswith associatedrisksto
operator and to patients, the relatively long
time necessaryfor the preparationof each
dose(20—30mm per patient),and the inabil
ity to prepare a multiple-dose batch for all pa
tients in a given day. An alternativelong
circulatingagenttestedin vivo is a liposome
based preparation labeled with a lipophilic
complex, 99mTc@hexamethylpropyleneamine
oxime [10]. Although this product clearly
showed extended blood half-life periods in
preclinical trials, the labeling procedure is
complicatedbecauseof theneedto preparethe
99mTchexamethylpropylene@neoxime be
fore liposomal labeling and the need to use
size-exclusion chromatography to separatefree
technetium-99mcomplex. Another blood pool
agentis @Tc-labeled HSA. Although label

AJR:171,July 1998 137

 Callahan et aI.

, J__,,__.cJ__o.,
@ @1.
,,.
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro
Backbone (poly-L-lyslne) chloride(Aldrich,Milwaukee,WI).The solutionwas
incubatedat room temperaturefor 10mm. Activated
monomethoxypoly(ethylene glycol)succinate was

,,, J.,. ProtectIve chain transferredto the poly-L-lysinesolutionand incubated

,@ is. (MPEG-succinate) for 4 hr atroomtemperature whilebeingmixed.1\vo
@ ,.I ,Loc,,,c.,,cocHc,sc,.,o$,..c,4 grams of DTPA cyclic anhydride (Pierce, Rockford,
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IL) was dissolved in 10 ml of dimethylsulfoxide and

added to the reactionmixturedropwise.The pH was
kept at 8 by the additionof I ml of 50% NaOH.The

@
:;i3Chelate
“¿OOC (DTPA)
scribed[17].
reaction mixture was stirred for 10 hr at room temper
ature. Intermediate products were analyzed as de

The solution was passed through a sterile apy

Fig.1.—Scheme
representing
methoxypoly(ethyleneglycol)ethylenetriaminopentaacetic Active rogenic cellulose nitrate membrane using a filter
acidstructure.
weight,35.5kDa;degreeofpoly (Nalgene, Milwaukee, WI) with 0.2-jim open
drugsubstanceofpolymerkitconsistsof backboneofpoly-i-lysine(molecular
merization,279)to which are covalently attached residues of methoxy(polyethylene)glycol-O-succinate(32%of ings. The solution was then diluted with 100 ml
total N-c-aminogroups).Remainderof aminogroupsare covalentlymodifiedwith DTPA.Drawingalsoshowsfor of sterile apyrogenic water and transferred into a
mulaandsimplifiedthree-dimensional
modelofbasicelementcontaininglysine—lysine
dimerbearingDTPAand 300-mi diafiltration cell equipped with a YMIOO
methoxypoly(ethyleneglycol)(MPEG)
elements(onetenthofactualMPEGlength).
membrane (Amicon, Beverly, MA). The cell was
pressurized using a nitrogen source and concen
ing kits for the preparation of 99mTc@HSA Materials and Methods trated to 30 ml at 25 psig (172,367 Pa). The con
are commercially available in the United Synthesis tent was then diluted with sterile apyrogenic
States,they are seldom used as a blood pool water and concentrated again. This concentra
imaging agent becauseof poor retentionin The drugsubstance is theradiopharmaceutical tion-and-dilution procedure was performed for
M@O-PL-{@@Fc}-UI'PA,((O-monon@thoxypoly(eth five cycles. An aliquot was removed for high-per
plasma[14, 15]. In one clinical trial, 99mTc@
—¿ @y@-@adnyl)poy(N-e-L-lysyl(@fc}dieth formanceliquid chromatography (HPLC) analysis.
HSA showed considerable enhancement of yleneiziamine pmta@ate monoamide).Thiscompound Thirty milliliters ofconcentrated methoxypoly(eth
the liver (presumably because of extrava is a m@remolecular chelate prepared as a graft ylene glycol)ethylenetriaminopentaacetic acid was
sation of labeled HSA) and was therefore copolymer of poly-L-lysine and monomethoxy transferred to an autoclaved lyophilization flask,
used for first-pass cardiac imaging per poly(ethyleneglycol)succinate.The copolymer was frozen in liquid nitrogen, and lyophilized.The dry,
formedwithin 5—10 mm after administration @uced in our laboratory by a multistep synthesis solid mass obtained after lyophilization was sub
[11]. The fast removal of an agentfrom cir that involved acylation of rnonomethoxypoly(ethylene jected to elementalanalysisat GalbraithLaboratories
culation may decreaseits diagnosticuseful glycol)in thepiesence ofa nucleophilic CatalySt conju (Knoxville,TN).The net formulaof methoxypoly(eth
ness for applications such as detection of _on ofthe acylated purified product, monomethoxy ylene glycol)ethylenetiiaminopentaacetic acid detived
poly(ethyleneglycol)succinate,to poly-L-lysine in the fromtheresultsofTNBS assayandelementalanalysis
slow gastrointestinal bleeding. Chemically
@ presenceof water-solublecarbodiimide and sullosuc
was which yielded a for
modifiedHSA (e.g., diethylenetriaminepen
cinimide; and acylation of monomethoxypoly(ethylene mula of MPEG@-poly(L-Lys)279-diethyleneuiamine
taacetic acetamide—HSA) [1 1] or mercaptoa
glycol)poly-L-lysine withcyclicanhydiideof DTPA. pentaacetic acetamide187.The formula was calcu
cyl-HSA [16j showedhigher radiochemical Production of monomethorypoly(ethylene gly lated assuming that the poly-L-lysineused for the
purity and better blood retention than parent col)succinate.—Methoxy[poly(ethylene)glycol]5000 synthesis had a mean of 279 lysine residues (poly
HSA in comparative technetium-99m label (Polyscience, Warrington, PA) was succinylated in dispersity. 1.1); that the degree of modificationof
ing studies. thepresence ofdimethylaminopytidine andpuiified poly-L-Iysinewith monomethoxypoly(ethylenegly
With regard and concern for the potential as described [19] with the use ofpyrogen-free glass col) was 32%, as determinedby TNBS titrationand
pitfalls and disadvantagesof existing agents, ware throughout the synthesis. Final purification of monomethoxypoly(ethylene glycol) quantitative
we havedevelopedan entirelysyntheticgraft the product was achieved using ethanol-washed determination; and that 96% of the remaining
copolymerand have formulatedit into a kit AG5O W-X8 resin (Bio-Rad, Hercules, CA). The amino groups were substituted with diethylenetri
yield of purified monomethoxypoly(ethylenegly aminepentaaceticacetamide.Theaveragemolec
for scintigraphic applications [17] and MR
col)succinatewas 57% oftheoretic yield. ular weight was calculated to be 550 kDa.
imaging [18]. This graft copolymerconsists Synthesisof MPEG@PL@-DTPA.—Stenie 0.1-
of a central poly-L-lysine chain containing rnolll carbonatebuffer was preparedby dissolving 4.2 KitFormulation
multiple diethylenetriamine pentaacetic ace g of sodiumbicarbonatein sterileapyrogenicwater. The polymer was formulated into a kit contain
tamide chelating groups (for labeling with 1\venty micmliters of 50% NaOH solution was ing stannous(II) chloride and sodium acetate for
gadolinium,technetium,or indium) (Fig. 1). added,and the solution was passedthmugh a sterile subsequent radiolabeling with technetium-99m.
The central chain is sterically protectedby filterwith0.4-janopenings.A solutionof I g of poly Each kit contained methoxypoly(ethylene gly
polyethyleneglycol sidechains,whicheffec L-lysine(Sigma, St. LOUIS,MO; molecularweight, col)ethylenetriaminopentaacetic acid, (1.765mg);
lively increase the hydrodynamic diameter of 35.5kDa;degreeofpolymerization,279)in 175ml of stannous(II)chloride (0.1 mg); sodium acetate (6.8
the molecule,decreaseimmunogenicity[18], 0.1-mol/lcarbonatebuffer was then prepared,and a mg); and sterile water for injection,USP (1 ml, pH
50-@da1@was vmthdrawnforaniinogmupdetennina5.3). The kit was packagedby filling6-mi syringes
and prolong blood half-life when compared
tiorL Monomethoxypoly(ethylene glycol)hydmxysulfo with a stock solutionof methoxypoly(ethylenegly
with the backbone alone. After early efficacy succinimidyl succinate was prepared using a modified col)ethylenetriaminopentaacetic acidstannate.Evacu
studies in animals [17], we present here mi method @ouslydesciibed[20]: We dissolved 9.6 g ated sterile empty vials were purged with argon to
tial data from a phase 1 clinical trial in ofmonomethoxypoly(ethylene glycol)succinatein25 remove oxygen, and I-ml aliquots of stock solution
healthy human volunteersto assessthe effi ml ofsterileapyrogenic waterandthenadded500mg werethentransferred intoeachvialusinga Micro
cacy and safety of the kit formulation. of N-hydroxysulfosuccinimide followedby 1 g of 1- Minispike assembly (International Medical Indus

138 AJR:171,
July1998
 Use of Labeled Synthetic Polymer in Blood Pool Imaging

tries, Watertown, MA) followed by additional
purging with argon. The lot was then transferred to
a —¿70°C
freezer and stored until further use.
PreclinicalLabeling EfticiencyTesting by HPLC
Five- to 100-mCi (185- to 3700-MBq) aliquots
of @@Tc-pertechnetate (Syncor, Woburn, MA)
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collection.The analysisincludedRBC,WBC,hemo
globin,hematocrit.platelets,glucose,blood urea ni
trogen, creatinine, total protein, albumin, globulin,
total bilirubin, alanine aminotransferase, aspartate
aminotransferase,and alkalinephosphatase.
Subacute toxicity—Subacute toxicity testing was
performed in nine experimental and five control
of-interest analysis was performed on all images
using an Icon computer (Siemens). Heart:liver and
heart:lungratioswerecalculatedas a measureof
blood pool activity.Totalurine was collectedduring
the0- to 2-hrand2- to 24-hrintervalsafterinjection.
For safetyevaluation,subjectsunderwentadditional
blood and urine tests 2 weeks after injection.

were added to kits in a volume of 0.5—2ml and in maleSprague-Dawleyrats (bodyweightrange, 100-

cubated for 30 mm at room temperature. Aliquots 125g) and four experimentaland three controlNew Dosimetry Calculation
of the kits (20—100@.ilcontaining 100—500@tCi Zealand white rabbits (body weight range, 2.5-3.0
[3700-18,500 kBq]) were then removed to deter kg). Each experimentalanimalreceivedsevenIV in Pharmacokinetic data obtained from region-of
mine the activity using a dose calibrator (Capintec, jections of methoxypoly(ethyleneglycol)ethylene interestanalysiswere usedto calculateradiation
Ramsey, NJ). The aliquots were then analyzed by triaminopentaacetic acid over 2 weeks. Control absorbed dose estimates for the compound. Data
HPLC (Hydropore SEC-S. 0.4 x 25 cm; Rainin animals were injected with saline only. Animals were expressed as a fraction of the administered
were dosed at a rate of 1400 pg of polymer per kilo dose by assuming that total counts measured in the
Varian Instrument, Wobum, MA), eluting them
with 0.0S-molIl sodium phosphate buffer with a grain of body weight (corresponding to approxi whole-body region of interest at the initial time
mately 280 times the human dose). Blood samples point were equal to the total counts in the adminis
pH of 6.8. The fractionswerepooled,andradioac
were collected from each animal for hematologic tered dose. The fractions of these counts contained
tivity was determined. The elution time of the ra
dioactivity peak was 4 mm. and serum chemistryevaluationbefore and after the in organsandwhole-body
regionsovertimewere
polymer injection. Blood analysis was the same as converted to percentage of administered dose by
for the acutetoxicitystudy. dividing the organ region counts by the initial
Labeling
whole-body counts and multiplying by 100.
The kit for clinical studies was radiolabeledby Forradiationdosimetrypurposes,one approach
addingto eachkit vial 100mCiof@mTc@pe@hfle@Human Subjects for an agent that is distributed primarily within the
tate diluted in 4 ml ofsaline obtained from a molyb
This study was approved by our Institutional Hu intravascular space is to assume that the activity is
denum-99--technetium-99m generator (Techneite;
man Research Committee and by the United States uniformly distributed within the total body, referred
Dupont, North Billerica,MA). After injection,kits Food and Dreg Administration under an investigator to as the “¿remainder
organs.―
The followingfive as
were incubated for 30 mm at room temperature.
sponsoredinvestigationalnew drug application.All sumptions based on the pharmacokinetic data ac
volunteers gave informed consent. Three male and quired in this study from workup ofblood and urine
Determination ofRadiochemical Purity
three female volunteers (21-44 years old; body sampleswereused in preparingdosimetryestimates.
Radiochemical purity was tested with instant
weightrange,60—91 kg; mean body weight,77 ±Il First, 85% of the administered dose is instanta
thin-layer chromatography using silica gel—im kg)wererecruited forthisstudy. Thevolunteers had neously and uniformly distributed throughout the
pregnated paper (Gelman Laboratories, Ann Ar no preexistingmedical conditions as evidencedby entire body and is eliminated with a biologic half
bar,MI) as the stationaryphaseand two separate physical examination and chemistry, blood, and urine lifeof2O.6hr.Second, 10%ofthe administereddose
mobile phases: acetone and saline [21]. A radio screenings. Baseline vital signs were taken and ECG is instantaneouslyand uniformly disnibuted within
chemical purity of greater than 90% (acetone) was was performedbeforeinjectionof2O mCi (70 MBq) the heartandis eliminatedwitha biologichalf-life
initially required before release of the material for of MPEG-PL-f@Tc}-DTPA (corresponding to of 20.6 hr. This assumptionis based on “¿standard―
human use. Because a vial-to-vial variation in ra 0.35 mg ofpolymer) by slow bolus injection (1 mm). data on the fraction of the total blood volume con
diochemical purity was observed during the trial, Blood testing included determination ofRBC, hema tamed in the heart chambers. Third, 5% of the ad
only vials with a purity greater than 95% were tocrit, hemoglobin, mean corpuscular volume, mean ministered dose is instantaneously taken up by the
used for the latter part of the study. corpuscularhemoglobin,platelets,WBC, neutro liver and is not excreted. Fourth, activity cleared
phils, lymphocytes, monocytes, eosinophils, and ba from the body is eliminatedby the kidneys into the
AnimalTo.xicityTesting sophils.Chemistrytestingincludeddeterminationof urinal)f bladder at a constant rate of 1% per hour
The purpose of the following experiments was sodium@potassium. chloride, CO2. blood urea nitro (23% divided by 24 hr) and continues for 10 physi
to determine if use of MPEG@PL@{@mTc}@DTPA gen, creatimne, calcium, alanine aminotransferase, cal half-life periods of technetium-99m (60 hr).
intestanimalswouldcauseanydetectable
acuteor aspartate aminotransferase, alkaline phosphatase, to Therefore, 54% ofthe administered dose is excreted
subacute toxicologic or pharmacologic effects as ad bilirubin, protein, albumin, and globulin. Urine into the bladder during the complete decay of the
measured by classic hematologic and blood chem testing includeddeterminationof glucose,bilirubin, technetium-99m. Fifth, the input half-life into the
istry profiles and gross observation. ketones, specific gravity, occult blood, pH, albumin, bladder is equal to the rate of clearancefrom the in
Acute toxicity—Acute toxicity testing was per urobilinogen,nitrite,WBC, and RBC. travascular space (20.6 hr). On the basis of these as
formed in 18 experimentaland six control male Continuous monitoring of vital signs and ECG sumptions, we calculated radiation absorbed dose
Sprague-Dawley rats (Harlan, Indianapolis, IN) were performed during the acquisitionof simulta estimates using the Medical Internal RadiationDo
(body weight range, 100—125
g). Each animal re neous whole-body anterior and posterior images be simetiy methodology [22]. Calculations were per
ceived a single IV injection of unlabeled methoxy ginning immediately after injection and at 2 and 24 formed using MIRDOSE3 computer software
poly(ethylene glycol)ethylenetriaminopentaacetic hr after injection. All images were obtained on an (Medical Internal Dosimetry Center of Oak Ridge
acid at a rate of 1400 @ig
of polymer per kilogram MS 2 camera(Siemens,HoffmanEstates,IL)using NationalLaboratories,Oak Ridge,TN) [23].
of body weight (corresponding to approximately the whole-bodyscanningmode.The instrumentwas
280 times the human dose). energy-calibratedwith a 15% window around the lmmunochemicalAnatysis
Three groups of six rats each were sacrificed by l40-keV photopeak of technetium-99m. The imme Humanantibodiesto kit components weredeter
etheroverdoseat 1hr.1day,or7 daysafterinjection. diate and 2-hr imageswere obtainedat a scan speed mined by enzyme-linked immunoabsorbentassay.
At the timeofsacrifice,bloodsampleswerecollected of 15cm/mm,andthespeedwasreduced to5 cm! Antigens (methoxypoly(ethylene glycol)ethylene
from each animal for hematologic evaluation. All mm for the 24-hrimage. thaminopentaaceticacid. with bovine serumalbu
blood samples were analyzed by an accredited mdc Whole blood samples were collected at 5 and 30 mm as a negative control) were adsorbed on the
pendentlaboratory (Tufts University VeterinaryDiag mm and at 1,2, and 24 hr after injectionfor radioac surface of high-adsorbing Immulon plates (Fisher
nostic Laboratory, Grafton, MA) on the day of tivity measurements. Organ and whole-body region Scientific, Pittsburgh, PA). Blood was serially di

AJR:171,July 1998 139

 •¿Wi@@@Overvlew
RabbitsParameterControl
of Laboratory Values from Subacute Toxicity Study in
GroupBefore GroupExperimental
StudyHematologyRBC StudyAfter StudyBefore StudyAfter

1.1Hematocrit0.39
(1012/I)6.1 ± 0.65.8 ± 0.65.8 ± 0.15.0 ±
0.01Hemoglobin ± 0.030.36 ± 0.020.38 ± 0.010.39 ±
11Platelets (gil)133 ± 8121 ± 6128 ± 8136 ±
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116WBC (109/l)370 ±65360 ± 72335 ± 36393 ±

1.1ChemistryBlood
(109/l)5.7 ± 1.35.9 ± 1.16.4 ± 0.85.0 ±

0Creatinine
urea nitrogen (mmol/dl of urea)596 ±71525 ±107589 ±125643 ±
9Protein (@imol/l)97 ± 988 ± 988 ± 88106 ±
(gil)53
10Alanine ± 550 ± 350 ± 350 ±
34Aspartate
aminotransferase(U/I)47 ±3126 ± 438 ± 1551 ±
19Alkalinephosphatase(U/l)91
aminotransferase (U/I)43 ±3717 ± 729 ± 1826 ±
±78425 ±13091 ± 78192 ± 95
Note—Thecontrol group containedthree rabbits,and the experimentalgroup receivingthe polymercontainedfour rabbits.Data are presentedas mean± SD.Eachrabbit received a 10-fold
humandose(14.0@g of polymerper kilogramof bodyweight,0.11mI/kgof kit reconstitutedin 4 ml of salinel or saline(0.11ml/kgl as a control.IV injectionswere madethrougha marginalearvein every
other dayfor 16days(seveninjectionstotal).Bloodsampleswere withdrawn beforeandafterthe completionof injections.

luted with phosphate-buffered saline containing 10
mg/mI of bovine serum albumin. The pH of the sa
line was 7.4. It was incubated with adsorbed anti
gens for 2 hr at room temperature and washed, and
antigen-hound immunoglobulins were determined
using goat anti(human IgG)peroxidase conjugate
(No. 6()5475: Boehringer Mannheim. Indianapolis.
IN) as described by the manufacturer. The analysis
was performed in triplicate.
StatisticalAnalysis
Quantitative results were analyzed using a one
tailed Student'sI test.Statisticalsignificancewas
assigned for p values of less than .05.
Results
PreclinicalLabeling Efficiency Testing
HPLC and instant thin-layer chromatogra
phy were used for purity assessmentafter we
had previously established that the Hydro of the radiopharmaceutical, the volunteer expe
pore SEC-S column matrix efficiently ab rienceda “¿metallic
taste:'becameflushed,and
sorbs any radioactive colloidal impurities. had palpitations. The blood pressure was stable
99mTcpertechnetate_labeledpolymer was but increasedslightly from 110 over 70 mm
eluted from the HPLC column as a single Hg to 150 over 90 mm Hg; pulse was 79 beats
peak, with an elution time of 3.9 mm. The per minute. Approximately 2 mm later the
overall loss of radioactivityduring the col symptomshadpassed,andby 20 mm all vital
umn run did not exceed 1—4% of loaded ra signs were normal. An ECG obtained at that
dioactivity during multiple test runs. Instant time did not show any changesfrom the base
thin-layer chromatographydata showedthat line ECG. To determinea possiblereasonfor
the reaction and concomitant altered biodistri
96—98%of the labeled component was non
mobile both in acetone and in saline, indicat bution, an immunochemical analysis of the
ing a complete reduction of pertechnetateby volunteer's blood was performed using en
stannous(II) chloride. zyme-linked immunoabsorbent assay. This
analysis did not reveal any preexisting, titrat
Toxicity
able antibodyagainstkit components.More
In the animals tested in the subacute (rab over, the blood sample obtained from a
bit; Table 1) and acute (rat; data not shown) volunteer who never received the drug sub
toxicity studies, no significant serum or he stance showed no statistically significant dif
matologic abnormalities were seen when ferencein reactiveimmunoglobulin content.
compared with normal laboratory values for
each species.Likewise, no significant abnor Imaging Results
malities in laboratory values were identified Serial anterior and posterior whole-body
in any of the six volunteers participating in images were acquired immediately and at 2
the phase I clinical trial (Table 2). and 24 hr after IV injection of MPEG-PL

c.@ One volunteer reported subjective symp

toms. Approximately 1 mm after IV injection
{99mTc}-DTPA. Visual inspection of images
showed a typical blood pool distribution in

Fig. 3.—Blood
eliminationcurve.
Plot-and-whiskergraph shows blood
-;0.0160.004@::@0.000 clearanceof methoxypoly(ethylene
Fig.2.—Scintigraphic
whole-body
image.Anterior glycol@ethylenetriaminopentaacetic
whole-body scintigrams obtained at 5 mm,2 hr,and 24 acid{@mTc}-DTPA plotted as per
hr after IV injection of 20 mCi (740 MBq( of methoxy 0510152025Time centageof injected dose per milliliter
poly(ethyleneglycol)ethylenetriaminopentaaceticacid )hr) of whole blood (mean±SEM;n = 4).
{99m1C}DTpA ID = injected dose.

140 AJR:171,July 1998

 Use of Labeled Synthetic Polymer in Blood Pool Imaging

four volunteers (heart and major blood yes
selsclearly visible) who had been adminis
tered MpEG@pL@{99mTc}@DTpAwith an
initial and subsequent radiochemical purity
of greater than 95% as determined by instant
thin-layer chromatography. In all four volun
teers blood pool activity was still visible at
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within30 mm after injection and 0.9±0.2 Pharmacokinetic Data and Dosimetry
after 2 hr. At 24 hr after administration, 0.06% ±
A different biodistribution, appearingto be 0.01% of the injected dose per milliliter of
reticuloendothelial (liver, spleen), was seen in whole blood was still present in blood (Fig. 3).
two volunteers(oneman andone woman).The Assuming a total blood volume of 7% of body
initial radiochemical purity of MPEG-PL weight,thisvaluecorresponds to32% ofthe in
{@Tc)-DTPA in these two volunteers was jected dose. The blood clearance calculated

24 hr (Fig. 2). The heart:lung ratio was 2.2 ± greaterthan 90% at the time of injection (one from multiple blood drawings was 20.6 ±2.3
0. 1 within 30 mm after injection and 1.9 ±0.3 of the two volunteersexperiencedthe subjec hr. assumingmonoexponentialdecrease(n =4;
after 2 hr. The heart:liver ratio was 1.1 ±0.2 tive symptoms already described). Fig. 3). The calculated percentage of the ad
ministered dose in the total blood volume ex
trapolated to zero time was 70%. Urinary
lft.@:1U*Oye@yjew VolunteersParameterBefore
of Laboratory Values In Six Healthy excretion was 7.5% ±1.4% ofthe injected dose
StudyAfter Study for the first 2 hr after administrationand 22.1%
Hematology ± 2.5% within 24 hr after administration.
Residence times, as defined by Medical In
RBC(1012/I) 4.5 ± 0.4 4.7 ± 0.6
ternal Radiation Dosimetry methodology for
Hematocrit 41 ±4 43 ± 4
the source organs used in dosimetry estimates,
Hemoglobin(gil) 139 ±14 146 ± 13
were calculatedto be 0.55 hr for the heartcon
Meancorpuscularvolume(fI) 90 ±2 90 ± 2 tents, 0.44 hr for the liver, 0.12 hr for the blad
Meancorpuscularhemoglobin(pg) 31 ±1 31 ± der contents,and 5.68 hr for the remainder of
Platelets
(10@/l) 251 ±41 268 ±37 the body. The radiation absorbed dose to mdi
WBC(10@/l) 5.9 ± 1.2 6.3 ± 1.3 vidual organs and the effective dose to the
Neutrophils 0.61 ± 0.10 0.65 ± 0.05 body are given in Table 3. The organ receiving
Lymphocytes 0.29± 0.06 0.24± 0.02
Monocytes 0.07± 0.03 0.07± 0.04 @AbsorbedDose Estimates
Eosinophils 0.03± 0.02 0.03± 0.01 Dose
TargetOrganAbsorbed
Basophils 0.01 ± 0.01 0.01± 0.01 (mGy/MBq)Adrenal
Chemistry glands0.004Bonesurface0.006Brain0.003Breasts0.003Eff
Sodium(mmol/l) 141 ±2 140 ± 3
Potassium(mmol/l) 4.1 ± 0.5 4.2 ± 0.5
Chloride(mmol/l) 105 ±2 103 ±
Carbondioxide(mmol/l) 28 ±2 31 ± 0 dosea0.004Effective
Bloodureanitrogen(mmol/dlof urea) 536 ±121 493 ± 86 equivalenta0.005Gallbladder
dose
Creatinine(@.imol/l) 80 ±9 80 ± 9 wall0.005Heartwall0.014Kidneys0.004Liver0.007Lower
Calcium(mmol/l) 2.22 ± 0.17 2.30± 0.10
Alanineaminotransferase
(U/I) 17 ±6 18 ± 6
Aspartate aminotransferase (U/I) 21 ±3 21 ± 2
Alkalinephosphatase(U/I) 66 ± 10 66 ± 17 wall0.004Lungs0.004Muscle0.003Ovaries0.004Pancreas0
largeintestine
Bilirubin(jimol/l) 10 ±3 10 ± 3
Protein (g/l) 71 ±2 70 ± 2
Albumin (g/l) 40 ±3 39 ± 4
Globulin(g/l) 31 ±2 29 ± 3
Urine marrow0.003Skin0.002Small
bone
Glucose Negative Negative
Bilirubin Negative Negative intestine0.004Spleen0.004Stomach0.004Testes
Ketones Negative Negative
Specificgravity 1.02± 0.01 1.02± 0.00
Occultblood Negative Negative
pH 6.6 ± 0.5 6.8 ± 0.8
Albumin Negative Negative
Urobilinogen Negative Negative body0.003Upper
Nitrite Negative Negative wall0.004Urinary
largeintestine
WBC Negative Negative wall0.008Uterus0.004
bladder
RBC Negative Negative
Note—Data
arepresented
asmean
ÂS±D. aDO@in mSv/MBq.

AJR:171,July 1998 141

 Callahan et al.

the highest dose was the heart wall, at 0.014
mGy/mBq, and the effective dose was 0.016
rem/mCi. On the basis of these estimates, an
administered dose of 20 mCi (740 MBq) to an
adult would result in a maximum organdoseof
1.04 rad (0.01 Gy) and an effective dose of
0.32 rem.
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(DTPA) attached to poly-L-lysine, and shield
ing methoxypolyethyleneglycol side chains.
These components were chosen because of
their safety (all indiyidual components of the
drug have been tested in humans) and their effi
cacy. Poly(ethylene glycol) is approved by the
Food and Dmg Administration as a vehicle or
preparation ofmany patient doses from a single
kit. All these factors are expected to increase
safety and improve patient throughput within
an imaging department.
Inert imaging agents with long circulation
times may have other potential applications in
clinical imaging. One such application would be

Discussion
Our goal was to investigatethe feasibility of
using a synthetic polymer for imaging of car
diac function (blood pool imaging). The results
of this phase 1 clinical trial are consistent with
prior dataobtainedin animals [17] andconfirm
a long circulation time of the synthetic graft
copolymer. In four of six subjects, the blood
half-life of MPEG-PL-(@Tc}-DTPA was
20.6 ±2.3 hr (range, 17.8—23.
1 hr), with a slow
urinary excretion (22.1% ±2.5% at 24 hr). Un
nary excretion presumably occurs becauseof
slow degradation ofthe graft copolymer, as the
protective monomethoxypoly(ethylene glycol)
chains slowly detach from the backbone in
blood [24], a phenomenon described for
monomethoxypoly(ethylene glycol)esters[25].
Our results indicate that high radiochemical
purity of the kit formulation is of the utmost
importance to ensuring long circulation times.
In two of the volunteers (one man and one
woman) a distribution to liver and spleen rather
than blood pool was observed. Radiochemical
purity of MpEG@pL@{@mTc)@DTpAat the
time of injection in these volunteers was
greater than 90% but was lower than the radio
chemical purity of the compound administered
to the other four volunteers (>95%). The ob
served accumulation in the liver and spleen of
the two volunteers cannot be explained by an
immune responsebecauseno antibodiesto kit
components could be identified in blood sam
ples. Likewise, colloid formation in thesekits
is also unlikely becausethis possibilityhad
been excluded by extensive pretrial HPLC test
ing. Kits prepared for nonhuman use, which
were filled using an open system,exhibited in
creasedstability when comparedwith kits pre
paredfor clinical use,which were filled using a
closed system. This observation suggeststhat
the method of filling and packaging influences
the in vitro stability of the kit. Future batches
for human use will be filled using an aseptic
open filling process followed by lyophilization
under an inert atmosphere. This process should
result in a kit with optimum in vitro stability.
Drug Substance
The graft copolymer consists of three corn
ponentswith different functions (Fig. I): a cen
tral poly-L-lysine chain, multiple chelators
base for a number of pharmaceutical prepara the mapping of capillary integrity and perme
tions, including Ganimagard (Baxter Laborato ability, which is often altered in inflammatory
ties, Glendale,CA) andVePesid(Bristol Myers and neoplasticprocesses[32], resulting in inter
Squibb, Princeton, NJ), and has low toxicity stitial accumulation of a given long-circulating
when administered orally, parenterally,or epi probe [24, 33, 34]. Because of the efficiency of
dermally. Poly(ethylene glycol) with a molecu tumoral accumulation of rnethoxypoly(ethylene
lar weight of 1—6
kDa was found to be excreted glycol)ethylenetriaminopentaacetic acid (ap
by the kidneys in mammals after IV injections proximately 3—7%of injected dose per gram of
[26, 27]. Furthermore, poly(ethylene glycol) tumor), chemotherapeuticagentshavealsobeen
and monomethoxypoly(ethyleneglycol)-modi attachedto the samegraft copolymer for effi
fled compounds (enzymes, lymphokines, and cient slow drug release once the conjugate is lo
so forth) have been tested in humans. For ex cated in tumors [19].
ample, monomethoxypoly(ethylene glycol)-L
asparaginase has been used to treat malignant Alternative ImagingAgents
lymphomasin clinical trials [28]. Poly-L-lysine Currently, the most widely usedblood pool
hasbeenapprovedfor phase1andphase2 cm imaging agentsarebasedon autologousRBCs
ical trials of an interferonogenic drug formula radiolabeledwith technetium-99mby eitherin
tion containing a mixture of polyinosinic and vivo or in vitro methods. Although adequate
polycytidylic acid complexed with poly-L blood pool images are routinely obtained with
lysine and carboxymethyl cellulose [29, 30]. these agents, the images are less than ideal be
No polylysine-mediated toxic effects have been cause of the time required for radiolabeling,
found in these trials. the need to prepare patient-specific doses, the
Monomethoxypoly(ethyleneglycol) side risk of misadministration to patients, and the
chains in the graft copolymer have several hazards to the operator during blood handling.
critical functions. First, the side chains are HSA is the only alternativekit compound
well known to reduce the immunogenicity of availablefor bloodpoolimagingin theUnited
proteins [31]. These protective properties States. Although HSA does not suffer from the
arise from the ability of polymeric chains to samedisadvantagesas autologous RBCs, car
create a hydrated shell around the central diac image quality is comparablewith that ob
polypeptide chain, thus masking it from eec mined with Tc-RBCs only immediately after
ognition and minimizing removal from circu IV administration [11]. One likely explanation
lation. Second, the side chains efficiently for this observation is that albumin is not ex
increasethe molecularweight and hydrody clusively distributed to the intravascular space
namic diameter of the molecule, resulting in but to other spaces as well [14].
long half-life periods in blood. Third, the at
tachment of monomethoxypoly(ethylene gly Future Studies Needed
col) side chains results in an increase in the This clinical trial shows that high-quality
relaxivity of copolymer labeled with para blood pool images in humans can be obtained
magnetic compounds, an observation that is usinga syntheticpolymer—amajor stepfor
of relevance for MR imaging of gadolinium ward in the goal of eliminating autologous
labeled copolymer [18]. blood productsfrom the armamentanumof di
agnostic agents. Further development of this
lmagingApplications
of Ultralong-CirculatingAgents agent will include optimizing the drug product
The most immediate imaging application of formulation(lyophilized kit) and testing its
the synthetic polymer would be for blood pool usefulness in imaging disease states such as
imaging. Gated cardiac imaging studies [1, 12] gastrointestinal hemorrhage, in localizing tu
and vascular imaging studies are commonly rnors, and in imaging inflammatory and infec
performedfor a varietyof indications.Corn tious conditions.
pared with labeled@Fc-RBCs, a simple poly
merkit wouldhavetheadvantages of avoiding Acknowledgments
the handling of blood products, decreasing the We thank Robert A. Wilkinson and Kirt
time for labeling, and making possible the Poss for technical assistance with animal im

142 AJR:171,July 1998

 Use of Labeled Synthetic Polymer in Blood Pool Imaging
aging and toxicity testing, Sandra Barrow for
acquiringclinical studies,andLynn Gedremis
for monitoring patientsand sampling blood.
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