position in vivo. This validation of the chemical integrityof a radiopharmaceutical in vivo becomes especially important with the less stable (4) or biologically reactivefunctionalities of the newer agents (5—10).

The first technetium heart agent to be successful inhumans, technetium (t-butyl isocyanide)6@ [99mTc-TBI](11), exhibited high myocardial extraction, but slow clearance from the lungs back into the blood as well as veryhigh uptake and prolonged retention in the liver (12). Thestructurally similar compound technetium (2-carbomethoxy-2-isocyanopropane)6@ [99mTc..CPI]is also a lipophilic cationic complex containing six identical functionalized isonitrile ligands (10). The terminal methyl esterfunctions on the ligands were included in the design ofthis molecule to introduce an unstable organic functionalgroup that could undergo metabolism or decompositionthus enhancing biologic excretion after initial myocardialextraction had occurred.

Preliminary screening of 99mTcCPI in rabbit demonstrated high initial accumulation of activity in the heart,liver and kidney with rapid clearancefrom blood and lung,followed by moderate hepatobiliary and renal clearanceand more gradual myocardial washout (13). During toxicologicand preclinicaldosimetryevaluationsof 99mTc@CPIkit formulations in other species, however, distinct differences in biodistribution were observed in that no myocardial accumulation was observed in rat or mouse (14).Further screening in other species along with pharmacologic evaluation ofthe compound's radiochemical integritywere performed to ascertain the nature ofthese differencesand to determine the ability of the guinea pig model topredict the behavior of an agent in human.

MATERIALS AND METHODS

The metastable radionuclide @mTc,as sodium pertechnetate(30-150 mCi/ml, 1.1—5.5GBq/ml), was obtained from a commercial @Mo/@mTcgenerator (DuPont/Biomedical Products, N.Billerica, MA). Synthesis and characterization of CPI, the

@Tc(CPI)6@complex and 99mTcTBI have been published elsewhere (10,15).

The cationiccomplex technetium(2-carbomethoxy-2-isocyanopropane)@(@Tc-CPl) contains terminal ester groups thatwere included to provide a pathway for in-vivometabolism ofthiscompound,therebyenhancingitsperformanceas a myocardial perfusion agent. Biodistnbution studies of the cornpound demonstrated rnyocardial accumulation in rabbit,guineapig,andchick,butnotinratandmouse.Radiochernicalanalysisby HPLC after in-vitro incubationof @“Tc-CPlinblood plasma from the various species confirmed enzymatichydrolysis to numerous new compounds. Rat and mouseserum produced complete hydrolysis of this agent after incubationfor less than 5 sec at 25°Cor rates greater than500 times those observed in human, rabbit, guinea pig andchickserum.Chemicalsynthesisandisolationof themonohydrolyzed species with subsequent biodistribution studies inguinea pig and rabbit confirmed that this neutral lipophiliccomplex did not accumulate in heart tissue. It is concludedthat varying rates of enzymatic in-vivo hydrolysis produce theinterspecies biodistnbution differences and may account forthe moderatemyocardialclearancerelativeto other isonitrilecomplexes.

J NucI Med 1992; 33:1357-1365

nterspecies variation in biodistribution has long plaguedthe development ofradiopharmaceuticals, especially in thediscovery of technetium heart (1,2) and brain perfusionagents (3). No single animal is an acceptable model forhumans; therefore, validating preclinical biodistributionstudies is an important step before proceeding to clinicaltrials. Evaluation of the biodistribution of a compoundalone, when the biochemical mechanisms for localizationor the pharmacokinetics are unknown, reduces nuclearmedicine to the simple visualization of anatomy. Empirically obtained biodistribution data should also includeradiochemical analysis as a test for metabolism or decom

ReceivedDec.10,1991; revisionacceptedFeb.27,1992.For reptintscontact:JamesF. Kronauge,PhD,Brighamand Women's

Hospital,DepartmentofRadiology,75FrancisSt.,Boston,MA02115.

Interspecies Variation of 99mTc-CPI•Kronauge et al 1357

Interspecies Variation in Biodistribution ofTechnetium (2-Carbomethoxy-2-Isocyanopropane)6@James F. Kronauge, Michael A. Noska, Alan Davison, B. Leonard Holman, and Alun G. Jones

The Joint Program in Nuclear Medicine, Harvard Medical School, and Brigham and Women ‘sHospital, Department ofRadiology, Boston, Massachusetts and Department ofChemistry, Massachusetts Institute of Technology,Cambridge, Massachusetts

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ChromatographyReversed-phase thin-layerchromatography (RP-TLC)was per

formedon WhatmanMK C-18platesdevelopedin methanol/acetonitrile/tetrahydrofuran/aqueous buffer (ammonium acetate, 0.5 M) (3:3:2:2, v:v:v:v). High-performance liquid chromatography (HPLC) was carried out on a sequential dual-detectorsystem,uv-vis (254 nm) and Nal-based radiometric detection(WatersAssociates,Woburn, MA) as describedpreviously(16).AnalyticalHPLC analysisof @mTc@CPIand its metaboliteswasaccomplished in a reversed-phase mode (RP-HPLC) with a C8bonded(5 tiM)sphericalsilicaparticlestationaryphase(BrownleeOS-MP cartridge, 100 mm x 4.1 mm) (Rainin Instruments,Woburn MA) and a gradient mobile phase of 100% aqueousbuffer (ammonium sulfate, 0.05 M, pH 5.5) to 95% methanol ina 5-mmlineargradientat a flowrate of 1ml/min.

Biological samples were made ready for RP-HPLC analysisusing a pre-wet (C-l 8) Sep-Pak Cartridge (Waters, Milford MA)prepared by first injecting,via a syringethrough the cartridge,absolute ethanol (5 ml) followed by distilled water (5 ml) andfinally air (10 ml).

Radiochemical PreparationsTechnetium-99m-CPI was synthesized by addition of@mTcO4_

(20—200mCi, 0.74—7.4GBq, 1—2ml 0. 15 M saline) to a vialcontaining sodium dithionite(5.O mg, 0.029 mmol), ethanol (0.25ml, 95%) and the liquid CPI ligand (5 @l,0.039 mmol) followedby heating at 65°Cfor 30 mm. Separation of the radiolabeledcomplex from reagents was performed by loading the cooledsolution (1.0 ml) onto a pre-wet C-l8 Sep-Pak and flushing thecartridge with saline (0.15 M, 10 ml) followed by ethanol/water(30%, 5 ml) and ethanol/saline(95%, 5 ml). Radiochemicalpurity was determined by both RP-HPLC and RP-ITLC to be>95% and stable for more than 6 hr. The identity of the @mTc@CPI specieswas confirmedby RP-HPLCwith the characterized

@Tc(CPI)6@and a retention time of9.0 mm (k' = 4.30); RP-TLCRf= 0.7.

Preparationof the FirstHydrolysisProductof @“TcCPI

The neutral monohydrolyzedproduct of the cationic @mTc@CPI complex [Tc(CNC(CH3)2COOCH3)5(CNC(CH3)@COO)]°,[Tc(CPI)5(H-CPI)]°, was synthesized by incubating the reconstituted 99mTcCpI preparation at pH 9.0 for 10 mm at 25°Cfollowed by neutralization to pH 6.0 with HC1 (0.5 M). Thesolution (100 mCi, 3.7 GBqJml) was loaded onto a C-18 SepPak,and the cartridgewaselutedwithethanol/saline(30%,5 ml)followedby ethanol/water (90%, 5 ml) to isolate the neutrallipophilic complex in an overall 5% yield. The RP-HPLC retention time was 8.2 mm (k' = 3.8); RP-TLC Rf = 0.6.

Animal BiodistributionAlbino rats (male CD, 400—650g) and albino mice (male CD

1, 25—35g) were obtained from Charles River Breeding Laboratories, Wilmington, MA; male Hartley guinea pigs (300—600g)from Elm Hill, Chelmsford, MA; and 4-day-old Leghorn chicks(40—60g) (SPF-utility chicks) from Spafas, Norwich, CT. NewZealand albino rabbits (3—4kg)were purchasedfrom Pine AcreRabbitry, Assonet, MA.

Dynamic gamma camera imaging studies were performed afteri.v. injection of 99mTc@pI (1.0 mCi, 37 MBciJO.2 ml) undersodium pentobarbital anesthesia. Images were collected on aLEAPcollimatedGE400Tgammacamera(GE MedicalSystems,

Milwaukee, WI) at 60-sec intervals for 1 hr in a 64 x 64 matrix.Time-activity curves for heart, lung, liver and kidney were obtamed without backgroundsubtraction.

Quantitative biodistribution studies were performed in mice,rats, guinea pigs and 5-day-old chicks. Unanesthetized mice wereinjected with 99mTcCpI (25 DCi,0.9 M&iJlOO @d)via a tail veinand killed by cervical dislocation. Rats and guinea pigs under i.p.sodium pentobarbital anesthesia (35—50mg/kg body weight) wereadministered 99mTCCPI(100 XCi,3.7 MBq/lOO gil) via an exposed femoral vein and killed by cardiac puncture. Unanesthetized chicks were injected with 99mTc@CPI(25 zCi, 0.9 MBq/l00@il)via a wingveinand killedby ether asphyxiation.

ln•VitroHydrolysis of @Tc-CPIIn-vitro hydrolysis of the radiolabeled compound was exam

med using plasma from freshly drawn heparinized blood. Humanblood wasobtainedfrom fasted(>8 hr), healthymaleand femalevolunteers, with normal HDL, LDL and triglyceride levels. Forinterspecies comparison, hepannized blood was taken by cardiacpuncture from ether-anesthetized male albino guinea pigs, miceand rats and from unanesthetized rabbits via an ear vein. Bloodsamples were centrifuged at 3000 x g for 5 mm to separate cells,and the plasma was kept cold (4°C)until use. In each hydrolysisexperiment, plasma (0.20 ml) was pipetted into a borosilicateculture tube and equilibrated to 37°Cin a water bath. The 99mTc@CPI complex (10—50mCi/ml, 0.37—1.85 GBq/ml, 20 @tl)wasadded, the contents were shaken and incubation was continuedfor various periods of time. Enzymatic hydrolysis was halted bythe addition of cold (4°C)absolute ethanol (1.0 ml) and coolingin an ice bath to precipitate serum proteins. The samples werecentrifuged (15 mm, 2500 x g, 4°C)and the supernatant wasanalyzed by RP-HPLC. Repeated RP-HPLC analysis on the samesampledemonstratedthat further hydrolysisdid not occur in theethanolic supernatant.

RESULTS

Biodistribution StudiesDynamic gamma camera studies in rabbits provided a

simple direct comparison ofpharmacokinetics for differenttechnetium compounds. Figure 1A shows a rabbit wholebody image obtained 60 mm postinjection of 99mTc@TBI,which served as our relative standard. Distinct visualization of the heart is made just above the persistently hotliver. Time-activity curves for this agent (Fig. lB) showthe high initial lung uptake with gradual clearance thatobscures visualization of the heart until 20 mm postinjection when the heart-to-lung ratio exceeds 2. Dynamicgamma camera imaging of 99mTcCpI in the same species(Fig. 2A) also reveals good visualization of the heart;however, as appreciated in the time-activity curves, resolution ofthe heart from background lung activity occurredmuch earlier in the study due to the lower initial pulmonary accumulation and more rapid clearance (Fig. 2B).Blood activity, measured by venous sampling from the earopposite that of injection, showed very rapid clearance to0.04% per gram by 5 mm postinjection. Thus, greater than95% of the heart-associated activity reflects retentionwithin the myocardium. The conspicuous donut can beobserved in the high-resolution image shown in Figure 2C,

1358 The Journal of Nuclear Medicine•Vol.33 •No. 7 •July 1992

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I C

B

.. LIL'ER

e 15 30 45

A

BLIL'ER

. .@ @IDNEY

.‘ ..--..--- -.- - - HEART

LUNG

60

FIGURE 1. (A) Representativewholebodyimageof rabbitat 60-mmpostinjection of @“Tc-TBl(1.0 mCi).(B)Time-activity curvesfor fasted,anesthetizedrabbitout to 60-mm postinjection. Regions ofinterestare normalizedto cpm/pixel.

15 30 45

which is an anterior view of the thoracic cavity of therabbit at 60 mm postinjection. Additionally, liver andkidney clearance, with half-times of 24 mm and 16 mm,respectively, were far more rapid than those of 99mTc@TBI.Consequently, the more rapid blood clearance and hepatobiliary excretion of 99mTc@CPIenhances the target-tobackground ratios earlier in the study.

Quantitative biodistribution studies performed in theguinea pig at increasing times after intravenous injectionof99mTc@CPIconfirmed 1.3% ±0.3% ofthe injected doselocalized in the heart at 5 mm postinjection (Table 1). Theheart activity gradually decreased over time to 0. 19% ±0.06% at 4 hr postinjection with a t½of 1.1 hr. The rapidclearance of background activity from blood and lungthrough high extraction by liver and kidney meets therequirements for a myocardial perfusion agent to assessinduced transient ischemic events. Also, both rapid hepatobiliary and renal excretion were observed, which was in

concordance with the rabbit pharmacokinetic data. Attimes greater than 15 mm after injection, the continueddecrease in liver and kidney activity, without increase inblood concentration, indicates that hepatobiiary and renalexcretion occurred without reabsorption.

A comparison of biodistribution data for 99mTc@CPIinfour different species demonstrates a distinct variation inmyocardial accumulation. Table 2 summarizes the biodistribution data for guinea pig, rat, mouse and chick at 5mm postinjection. For these early times after injection,the amount of 99mTcCpI retained in the heart of guineapig and chick was relatively high at 1.25% ±0.32% and1.33% ±0.26% ID, respectively, which gave heart-to-lungratios on a per gram basis of 2.4 and 1.8, also consistentwith the rabbit images. However, for rat and mouse, thenegligible activity in the heart (a factor of 20 less than thatin guinea pigs) resulted in heart-to-lung ratios less than 1.0

with no possibility of myocardial visualization.

A

FIGURE 2. (A) Representativewholebodyimageof rabbitat 15-mmpostinjection of @“Tc-CPl(1.0 mCi).(B)Time-activity curves for fasted, anesthetizedrabbitout to 60-mm postinjection. Regions-ofinterest are normalizedto cpm/pixel. (C)High-resolutionstatic 5-mmacquisitionofrabbit heart obtainedat 60-mmpostinjection.

1359Interspecies Variation of @mTc-CPl•Kronauge et al

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TABLE IBiodistributlonof @“Tc-CPIin GuineaPigsas%ID/OrganOrgan

5 min* 30 min*@ min*4hr*Heart

1.25±0.32 1.11±0.13 0.68±0.040.19 ±0.06Blood1.40±0.27 0.92±0.15 0.63±0.100.30 ±0.06Lungs0.91±0.09 0.36±0.04 0.10±0.020.08 ±0.02Uver13.3±2.20 2.24±0.82 1.21±0.200.30 ±0.04Kidneys10.4 ±2.55 8.20 ±1.81 5.12 ±1.430.54 ±0.22*Me@ofn>4@im@js±sd(@_

1).

GuineaPigRatMouseChick%ID/organ

%ID/g%ID/organ%lD/g%lD/organ%ID/g%lD/organ%lD/gHeartI

.25±0.32 0.90±0.200.066 ±0.0030.057 ±0.0020.046 ±0.0160.37 ±0.191 .33 ±0.264.46 ±1.28Blood1

.40±0.27 0.053±0.0083.82 ±0.330.16 ±0.0182.16 ±0.611 .14±0.42—1 .83±0.60Lungs0.91±0.09 0.38±0.110.25 ±0.0640.12 ±0.0150.23 ±0.0621 .09 ±0.451 .03 ±0.312.48 ±0.64Liver13.3±2.20 0.87±0.2920.0 ±2.151.27 ±0.1111.5 ±2.405.73± 1.1021.9 ±1.0412.3 ±1.40Gut34.4±4.29 —37.5 ±5.72—50.0 ±5.23—24.0 ±3.78—Kidneys10.4±2.55 3.04±0.782.71 ±0.880.91 ±0.271.37 ±0.752.63 ±1.185.79 ±1.4013.4 ±4.35Bladder0.51±0.35 —9.30 ±3.84—10.9 ±3.35—3.84 ±0.73—Spleen0.58±0.10 1.01±0.230.048 ±0.0110.06 ±0.0070.048 ±0.0260.47 ±0.150.30 ±0.125.81 ±2.92Brain0.029±0.004 0.008±0.0010.015 ±0.0030.008 ±0.0010.045 ±0.0090.1 1 ±0.0260.048 ±0.0070.051 ±0.004Muscle14.2±4.50 0.079±0.0207.78 ±1.630.049 ±0.0117.09 ±3.410.57 ±0.35—0.63 ±0.17*Me@ofn@5animals±s.d.(,—

1).

resulted in the eventual observation of all nine hydrolysisspecies predicted for a rigid octahedral hexacoordinatecomplex as identified in Table 3. All the newly generatedhydrolysis products were observed to have progressivelyshorter RP-HPLC retention times, indicating increasinghydrophilic character, as would be expected for the increasing number ofdeprotonated carboxylic acid groups on thenewly generated complexes. Even the neutral monohydrolyzed product [99mTc(CpI)5(H..Cpfl]Othe first new speciesobserved in the chromatograph, has a retention timeshorter than the cationic 99mTc@CPIcomplex. The intramolecular separation of the cationic technetium(I) centerfrom the terminal carboxylic acid, which is deprotonatedat physiologic pH, produces a zwitterionic species with anoverall neutral charge but yet more hydrophiic than theparent cation. Assignment of HPLC peak identities shownin Table 3 was made by comparison with characterized[99Tc(CPI)6]@hydrolysisproducts and measurementofpeak integral ratios for juxtaposed species in the chromatograph to determine cis/trans and fac/mer geometric isomers based on a random sequential process as shown inScheme 1 and described in detail elsewhere (10).

Incubation of 99mTcCpI in rabbit plasma for 2 mm at37°Cfollowedby HPLCanalysisrevealed84%of theactivity present as the original cationic agent with theremaining 16% consisting primarily of the monohydrolyzed neutral product. Similar low initial rates of in-vitrohydrolysis were observed after incubation in guinea pigand human serum with 93.3% ±0.4% and 90.9% ±1.0%,respectively, of 99mTcCpI remaining after 2 mm at 37°C.If incubationin theseplasmasamplesis allowedto continue for longer periods of time, increased hydrolysis isobserved with numerous species generated as exemplifiedin Figure 4 for the reaction in human serum after 40 mmat 37°C.Control studies on the rate of decomposition bysimple alkaline catalyzed hydrolysis in pH 7.4 bufferedsaline showed a significant but much slower rate of degradation. Table 4 contains the relative percent of eachtechnetium compound present after incubation at 37°C

For guinea pig and chick, extraction and elimination ofactivity by the two major excretion routes were differentfrom that in rat and mouse, with only 570% of the totalactivity excreted by 5 mm in the former two speciescompared with 71.6% in the latter. This difference ofbiologic clearance rate does not appear to be due to morerapid blood clearance but to lower uptake of the agent inthe muscle and fat of rat and mouse. The initial ratios ofactivity distributed between liver and kidney excretorypathways is not significantly different between the twogroups ofanimals. At 5 mm postinjection, 46.8% ±5.6%and 10.3% ±2.5% ofthe activity were in the hepatobiliaryand renal clearance pathways, respectively, for guinea pigand chick compared with 59.5% ±7.8% and 12.14% ±4.4%, respectively, for mouse and rat.

As predicted from the biodistribution results, dynamicimaging studies in the guinea pig revealed good visualization ofthe heart by 5 mm postinjection with heart-to-lungand heart-to-liver ratios of 2.0 and 1.1 after 30 mm asshown in the time-activity curves (Fig. 3). The gammacamera images for the rat are strikingly different in thatno delineation of the heart from the blood pool can bemade. The liver clearance rates for both species weresimilar with a t½of 14 mm.

Radiochemical Analysis of @Tc-CPlIn-Vitro Hydrolysis

Radiochromatographic analysis by HPLC of 99mTc@CPIafter incubation at pH 10 for varying lengths of time

TABLE 2Biodistribution of @Tc-CPlin Four Species of Animal at Five Minutes Postinjection

1360 The Journal of Nuclear Medicine •Vol. 33 •No. 7 •July1992

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0 7

coo coo-@ coo‘I,,.-I ,@ 5O%@ I ...@.‘@@@ ..,@.‘

0'Ncoo .OOC@―Ncoo. ooc@ @coo80% coo

@—,---@ C's @_@_%@ fac@@

20%coo- coo- coo

,,,@. I.,..@ 100 %@@ 33%

•“r@ ‘°‘Ncoo •“@Ncoocoo- coo- coo

trans mer trans

15 23

Peakelutionorderk'Identity12.23[Tc(H-CPl)@]522.61[Tc(CP1)

(H-CPI)5]432.81trans-[Tc(CPIMH-CPI)4]342.94cis-[rc(CPIMH-CPI)4]353.14mer-[Tc(CPl)@(H-CPl)@]263.25fac-[Tc(CPl)@(H-CPI)@]273.34trans-[Tc(CPi)4(H-CPl)@r183.46cis-[Tc(CPl).(H-CPl)@r193.68[Tc(CPI)5(H-CPI)]°104.20[Tc(CPI)@]@(CPI)

= CNC(CH3)@COOCH3,(H-CPI) = CNC(CH3)@COO.

I A1728

1298 ••LU/ER

860@ •• HEART

430 •‘•...... @•@•@

LUNG

. . 60e0 ;@@ 45 c@@j SCHEME I. Distributionof productsforsequentialhydrolysis of a rigid octahedralhexacoordinatecomplexwith ratios of geometric isomers predicted if hydrolysis occurs at random. Hydrolysis is shownas a continuationfrom the monohydrolyzedzwittenonicneutralcompound.

a time as 10 sec, virtually only the dihydrolyzed specieswas present. Measurements at shorter time intervalsshowed that even after 3 sec at 25°Ccomplete conversionof the cation of the neutral or anionic product had occurred (data not shown).

Not only does the rate of hydrolysis differ between thesespecies but so does the ratio ofgeometric isomers observedfor the metabolites of this octahedral complex. Figure 6shows a comparison of the products obtained after incubation at 37°Cfor 2 mm in rat plasma and 70 mm inhuman plasma. Although the resolution of peaks is not

FIGURE 4. RadiographicdetectionforHPLCanalysisof@mTcCPIbefore(A)and after (B) in-vitro incubationwith humanserumfor 40 mmat 37°C.

FIGURE3. (A)Timeactivity curves following intravenousinjection of @“TcCPI (0.5 mCi) intofasted, anesthetizedguineapig out to 60-mmpostinjection.(B)Time-activity curvesfollowingintravenousinjection of @“Tc-CPl(0.5 mCi)into fasted,anesthetizedrat outto 30-mm postinjection.

for 30 mm in plasma from human, rabbit, guinea pig, ratand an aqueous solution buffered to pH 10. After theseprolonged in-vitro incubation times, in no case did asignificant amount of the parent cation remain.

A striking contrast was observed in the initial rate ofhydrolysis and the ratio of products obtained after incubation of 99mTcCpI with plasma from rat or mouse compared with the other species tested. Figure 5 compares thechromatographs obtained at 37°Cafter a 2-mm incubationin human plasma and a 10-secincubation in rat plasma.In human plasma, the radiochemical purity was similar tothe control analysis. In rat plasma, however, after as short

TABLE 3Reversed-PhaseHPLC Analysis of @“Tc-CPlHydrolysis

Products

B

A

“Tc(CNC(CH,)fOOCH3),

8

1361Interspecies Variation of @mTc-CPl•Kronauge et aI

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GuineaPeak#pH10PigRabbitHumanRat1<0.5<0.5<0.5<0.5<0.522.42.8<0.51

.8<0.532.611.21.92.1<0.5411.56.37.06.21.0518.833.117.814.924.8613.217.97.34.32.578.211.49.88.455.8827.74.627.424.515.1913.59.626.136.1<0.5102.13.12.71.7<0.5*

Expressedas percentof activitypresent.

TABLE 4Metabolites of @Tc-CPlAfter Incubation in Plasma of

DifferentSpeciesat 37°Cfor 30 Minutes*

large, it can be seen that the first dihydrolyzed specieseluted on this system (trans-[Tc(CPI)4(H-CPI)2]') is present in much greater concentration in the rat plasma thanthe longer retained geometric isomer (cis-[Tc(CPI)4(H-CPI)2]'). By contrast,the longerretainedcis isomerispresent at four times the concentration of the trans-dihydrolyzed species following extended incubation in human plasma. The more rapid rate of metabolism and thealternate products produced indicate a completely different enzyme system in the blood ofrat or mouse comparedto the other species tested.

Biodistributlon of [@“Tc(CPI)5(H-CPl)]°Dynamic camera imaging of a rabbit following intrave

nous injection of[@mTc(CPI)5(H@CPI)]0gave images strikingly different from those ofthe parent cationic agent (Fig.7A). No heart activity is detectablein this 10-mm image,

FIGURE 5. RadiographicdetectionforHPLCanalysisof @‘TcCPI following in-vitro incubationat 37°Cin human plasmafor 2mmShOWingthemajorftyof radiolabelpresentasthecaflonandin rat plasmafor 10 sec containingprimarilythe dihydrolyzedproduct.

1362 The Journal of Nuclear Medicine •Vol. 33 •No. 7 •July1992

. Hydrolysis of @TC(cpi)6'@inRatPlasma

. Hydrolysis of @“Tc(cpi)@inHumanPlasma

--@@ —*.-- _________

FIGURE 6. HPLC analysisof @“Tc-CPIfollowingin-vitroincubationat 37°Cfor 70 mmin humanplasmaand2 mmin ratplasma demonstratingformation of different ratios for cis andtrans geometric isomers of the dihydrolyzed metabolite.

which shows only liver, intestine and kidney distribution.The time-activity curves (Fig. 7B) indicate a consistentheart-to-lung ratio of 1.0 and a very rapid biiary clearance.

A comparison of biodistribution for the monohydrolyzed neutral species [99mTc(CPI)s(H@CPI)]owith the parentcationic agent 99mTcCpI in guinea pig at 5 mm postinjection is presented in Table 5. The most significant differencebetween the two structurally similar compounds is thatthe percent of injected [99mTc(CpI)s(HQpI)]Oactivity retamed in the heart is one-tenth that observed for thecationic 99mTcCpI species. Also apparent is that the totalactivity excreted through the liver is slightly higher for themore hydrophilic monohydrolyzed species (57.7% ±14.8%) than for the original 99mTc@CPI(47.7% ±6.5%)with the concurrent inverse relationship observed for renalexcretion, 2.3% ±0.7% for the neutral compound versus10.9% ±2.9% for the cation. These mean values are thereverse of what one would expect on the basis of lipophilicity alone; consequently, overall charge must play a significant role on the rate of liver versus kidney extractionfrom the blood for these two compounds.

DISCUSSION

Biodistributlon

Initial biologic screening of the technetium ester isonitrue complexes was performed in the rabbit because of itsfavorable predictive value with other potential heart agents(1 7). The 4—5kg size of these animals is convenient forwhole-body imaging on a gamma camera to obtain dynamic distribution with maximal resolution. In addition,the possibility of re-injecting the same animal, via the earveins, minimizes intraspecies variation. Evaluation of biodistribution by dynamic gamma camera imaging, despitethe drawback of limited quantitative data, allowed directcomparison of pharmacokinetics for different technetium

Tc-CPI

c-CPI in Human Plasma2 ml, at 37―C

—Trans

-1@ “‘Tc(CPI)4(H-CPI),I2—C/s -‘

@--@ tl@ios@atTc-CPI in Rat Plasma

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0 15

[Tc(CN(CH3)@COOCH3)@]@[Tc(CN(CH3)@COOCH3MCN(CH3)@COO)J°%lD/organ%lD/gram%lD/organ%ID/gramHeart1.25

±0.320.90 ±0.200.10 ±0.020.065 ±0.13Blood1.40±0.270.053 ±0.0082.16 ±0.930.074 ±0.019Lungs0.91±0.090.38 ±0.110.252 ±0.0350.076 ±0.014Liver13.3±2.200.87 ±0.299.11 ±1.940.358 ±0.086Gut34.4±4.29—48.6±12.9—Kidneys10.4

±2.553.04 ±0.782.12 ±0.480.494 ±0.146Bladder0.51±0.35—0.176 ±0.220—Spleen0.58±0.101.01 ±0.230.056 ±0.0190.078 ±0.031*Meanofn6animals±s.d.(i—

1).

30 45

AB

LUNG@ ‘@ . . . . .@,

FIGURE 7. (A) Representativewholebody image of rabbit at 15-mm postinjection of [@“Tc(CPl)@(H-CPI)]°(1.0 mCi), amonohydrolyzedmetaboliteof parent cationic agent. (B) Time-activity curves forfasted, anesthetized rabbit out to 60-mmpostinjectionof [@“Tc(CPl)@(H-CPI)1°.Regions of interest are normalized to cpm/pixel.

S

complexes. The more rapid clearance of activity frombackground organs to enhance target-to-nontarget ratioswas the criterion for a second generation myocardial perfusion agent.

The guinea pig was chosen for initial quantitative biodistribution studies because of its critical predictive valuein screening other cationic myocardial imaging agents (2).Heart accumulation of@mTc@CPIhas also been confirmedby imaging and modeling studies in dogs, pigs and primates (13) and by imaging studies in humans (18,19). Thesomewhat unusual inclusion of the chick in the interspecies comparison of biodistribution was to confirm thevalidity of using cultured chick myocytes for cellular kinetic studies (20). Thus far, the only species that have notshown myocardial uptake oP@mTc@CPIare rat and mouse.These two species do, however, show heart uptake forother technetium isonitrile compounds, including 99mTcTBI and 99mTcMIBI (11,21,22) and other catiomc agents(23).

The choice of an “ideal―animal model depends uponthe particular distribution property for which one is screen

ing, with the limitation that it be cost-effective. For thenext generation heart agent, the debate centers aroundwhether a higher extraction efficiency is critical for analyzing disease states and, if so, can it be obtained withoutincreasing transient extraction by background organs, i.e.,blood cells, lungs, systemic muscle or fat, that may gradually release the agent back into the bloodstream andinterfere with acquiring the stress image. There appear tobe two compartments to the uptake equation, a nonspecific component proportional to the lipophilicity of thecomplex and a membrane-specific component, which ismore dependent upon molecular shape and charge distribution (24). For making comparisons of myocardial extraction ofdifferent compounds, a cell culture model offersthe greatest control of conditions and variables. The cxamination of other properties that are prerequisites for asuccessful myocardial agent requires testing for blood interactions, preferably in human blood, and performingpharmacokinetic studies by dynamic imaging of mediumsized animals, i.e., rabbit and guinea pig, and toxicitystudies in the least expensive species available. In short,

TABLE 5Biodistribution of @‘Tc-CPland [@‘Tc(CPl)@(H-CPl)]°in Guinea Pigs at Five Minutes Postinjection

1363Interspecies Variation of @“Tc-CPl•Kronauge et al

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there is no “ideal―model for the human and it is better tobegin with the most optimistic system that gives the greatest number of potentially successful compounds.

As part of our initial screening for biologic properties,gamma camera images of rabbits injected with @mTc@CPIwere promising due to the early clear visualization of theheart. These images correlated with uptake studies incultured avian myocytes (20) indicating a high extractionefficiency of 99mTCCPIand agreed with the model of afreely diffusible cationic compound that was retained intracellularly by the high negative membrane potentials ofmyocytes and mitochondria (25). The absence of heartuptake in the biodistribution studies with rat and mousewas potentially contradictory to this general model ofaccumulation and raised doubts whether to proceed tohuman trials. Had initial screening of @mTc@CPIbeenperformed by biodistribution in the mouse, by far the leastexpensive mammalian species, its potential as a myocardial perfusion agent would have been missed. By contrast,the biodistribution and pharmacokinetics of 99mTc@CPIinrabbit and guinea pig indicate that this compound hasoptimal biologic behavior for a myocardial perfusionagent.

RadiopharmacologyThe @mTc-CPIcomplex can be easily produced in ra

diochemical yields 95% and is stable in solution at pH 4to 7 for more than 24 hr. However, when diluted inphysiological buffer at pH 7.4 or greater, significant hydrolysis is observed, as much as 38% in 1 hr. This rate ofchemical decomposition or alkaline catalyzed hydrolysisis still much slower than that observed in human, rabbitor guinea pig serum with rates that are themselves almost500 times slower than those observed in rat and mouseserum. The exceptionally rapid hydrolysis of @mTc@CPIthat occurs in rat and mouse serum and the absence ofheart uptake of the monohydrolyzed species in eitherguinea pig or rabbit indicate that metabolism ofthe parentcompound in the blood is the origin of the interspeciesbiodistribution differences. Additionally, an appreciationof how drastically the rate of esterase activity can varybetween species suggests an explanation for the interspeciesdistribution differences observed with other compounds,such as the brain perfusion agent technetium N,N'-l,2-ethylenediyl-bis-L-cysteine diethyl ester (@mTc@ECD)(26).

The firstproductof hydrolysis,althoughstilllipophiicwith a net overall neutral charge, unlike other neutralcomplexes (27-29) does not accumulate significantly inthe heart of guinea pig or rabbit and is not taken up inisolated cultured chick heart cells (30). If, as has beenproposed, cationic isonitnile complexes are retained inmyocytes in proportion to the plasma and mitochondrialmembrane potentials (25), then a neutral compoundwould not be expected to be retained. Uptake ofthe neutralmyocardial perfusion agent Cardiotec®by contrast appearsto occur by a different mechanism, namely nonspecific

lipid partitioning (29). It should be noted that the lipophiic character of the neutral Cardiotec®agent is considerably greater than that of either 99mTc@CPIor its neutralmonohydrolyzed product. This much greater lipophilicityis required for a neutral compound to be extracted efficiently by the heart and the absence of charge allows itsrapid washout from myocardial tissue.

The fact that the neutral [99mTc(CPI)s(H@CPI)oJ,monohydrolyzed specie is not accumulated in guinea pig heartalso suggests an explanation for the moderate myocardialwashout of@mTc@CPIobserved in animals and in humans(31) in comparison to other isonitrile complexes (12,21,32). Hydrolysis of a terminal ester group of 99mTc@CPI,after localization in guinea pig myocardial tissue, removesthe catiomc charge and allows diffusion out of the heartwith the concentration gradient. This makes for an optimalagent because hydrolysis produces a radiolabel that clearsfrom the heart and does not re-accumulate. It is not knownwhether the rate of hydrolysis after the cationic agent is inthe heart is dominated by enzymatic or chemical hydrolysis.

CONCLUSION

Addition of the reactive methyl ester group to an isonitnle ligand followed by coordination to a reduced technetium central atom produces a lipophilic cationic complexthat undergoes both base-catalyzed hydrolysis and enzymatic hydrolysis in vitro. The parent cationic 99mTc@CPIagent exhibits high initial uptake and retention in themyocardial tissue of rabbit, guinea pig and chick butvirtually no accumulation in the heart of rat or mouse.The sequential metabolites ofthis agent are of increasinglyhydrophilic character and negative charge, features whichproduce quite different biodistribution properties. Consequently, we conclude that rapid enzymatic hydrolysis of99mTcCpI in the blood of rat and mouse produces metabolites possessing a peripheral negative charge and lowerlipophilicity, characteristics that inhibit their diffusionacross the plasma membrane of heart tissue and retentionbased on a negative myocardial membrane potential.

ACKNOWLEDGMENTS

Financial support provided by USPHS grant 5-ROl-CA43970and DOE grant DE-F902-87ER60526. The authors wish to thankRebekah A. Taube for assistance in editing this manuscript.

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1365Interspecies Variation of @‘@Tc-CPl•Kronauge et aI

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1992;33:1357-1365.J Nucl Med.   James F. Kronauge, Michael A. Noska, Alan Davison, B. Leonard Holman and Alun G. Jones 

+6(2-Carbomethoxy-2-Isocyanopropane)

Interspecies Variation in Biodistribution of Technetium

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