BASIC SCIENCES

Technetium-99m d,l-HM-PAO: A NewRathopharmaceutical for SPECT Imagingof Regional CerebralBlood PerfusionRudi D. Neirinckx, Lewis R. Canning, Ian M. Piper, David P. Nowotnik,Roger D. Pickett, Richard A. Holmes, Wynn A. Volkert, Alan M. Forster,Peter S. Weisner, Janet A. Marriou, and Sue B. Chaplin

Pharmaceuticals Development Department, Amersham International pk, Amersham,Buckinghamshire, England; and Department ofRadiology, University of Missouri,Harry S Truman Memorial Veterans Hospital, Columbia, Missouri

Following investigation of a large number of new ligands based upon propylene amine oxime(PnAO)the d,l-diastereoisomer of hexamethyl propyleneamine oxime (HM-PAO)was selectedas the preferred ligand for @9―Tcas a tracer for cerebral perfusion imaging. The neutral,lipophilic 99mTccomplex of d,l-HM-PAO was formed in high yield by stannous reduction of

@Mofl@―Tcgeneratoreluateusinga kit formulationof the ligand.Two minutesfollowingi.v.administration of this complex in rats, 2.25% of the injected dose appears in the brain. Lithewashout of the tracer is observed up to 24 hr postinjection. By qualitative autoradiographiccomparison with iodoantipyrine this new radiopharmaceutical displays blood flow dependentbrainuptakewith little redistributionof the tracerovertime.Thelipophilic @“Tccomplexconverts slowly in vitro to a secondary complex. This conversion process may account for theability of [@“Tc]d,l-HM-PAOto be retained within the brain without redistribution.

J NucI Med 28:191—202,1987

he discoveries of new radiopharmaceuticals suchas selenium-75-labeled di-(piperidinoethyl)selenide(PIPSE) and di-(morpholinoethyl)selenide (MOSE) (1),iodine-123- (1231) labeled p-iodo-N-isopropylamphetamine (IMP) (2) and N,N-dimethyl-N'-(2-hydroxy-5-iodo-3-methylbenzyl)- 1,3-propanediamine (HIPDM)(3). and thallium-20l (20Tl) diethyldithiocarbamate(DDC) (4) have generated wide interest in possibilitiesfor routine imaging of regional cerebral blood flow(rCBF) using rotating head single photon emission computed tomography (SPECT). In particular, [‘23I]IMPhas attracted much attention (5,6), and has been theagent of choice for SPECT studies of rCBF ( 7). However. none of these radiopharmaceuticals is suitable forroutine rCBF studies. Selenium-75 and 201T1 displaypoor physical characteristics, while ‘231suffers from highproduction costs, and limited availability.

As technetium-99m (@mTc)has none of these disadvantages. several investigators have sought to develop

ReceivedMar. 18, 1986:revisionaccepted Oct. 1. 1986.For reprints contact: R. D. Neirinckx, PhD. Amersham Inter

national plc. Pharmaceuticals Researchand Development. WhiteLion Road. Amersham. Buckinghamshire,England.

new rCBF tracers based upon this radionuclide (8—11).The main biologic requirements for such a radiopharmaceutical are the abilities to cross the intact bloodbrain bamer (BBB) and to distribute in the brain proportionally to blood flow. Once in the brain, the tracershould retain a fixed regional distribution for a timesufficient to permit image acquisition. For a rotatinggamma camera system, this is typically 20—30mm.

To cross the BBB, the technetium complex has to berelatively small (<500 daltons), lipophilic, and possessa net charge of zero. While the new tracers have therequired chemical characteristics, and are capable ofcrossing the BBB, all display rapid efflux from braintissue (8—11). Recently, a diamine dithiol (DADT)derivative with an amine side chain was reported toprovide a 99mTccomplex with improved brain retention(12).

Following the discovery that the 99mTccomplex ofpropyleneamine oxime (PnAO) (Fig. I) is neutral andlipophilic ( 11) and demonstrates transient flow-relatedbrain uptake in rats (13), dogs (14), and humans (15),a large number ofderivatives ofPnAO were synthesizedat the Amersham International Laboratories (16). Theaim of this work was to obtain a ligand which not only

191Volume 28 •Number 2 •February 1987

MATERIALS AND METHODS

Chemistry1. Synthesis of d,l-HM-PAO. Organic compounds were

characterized by melting point, 1H NMR,t and IR spectroscopy, and elemental analysis@.In addition, the stereoconfiguration of d,l-HM-PAO was confirmed by an x-ray crystalstructure analysis'.

(a). Preparation of 4,8-dia:a-3,6,6,9-tetra-methylundecane3,8-diene-2, JO-dione bisoxime. 2,3-Butanedione monoxime'(1 1.66 g, 115.4 mmol) was dissolved in benzene (50 ml)containing acetic acid (75 ml), and the solution was broughtto reflux in an apparatus fitted with a Dean and Stark trap,and under a nitrogen atmosphere. To this was added a solutionof 2,2-dimethyl-l,3-propanediamine (5.0 g, 5.88 ml, 49mmol) in benzene(l00 ml) over a period of5 hr. The resultingyellow brown solution was refluxed for a further 16 hr undernitrogen, then allowed to cool to room temperature. The

precipitated solid was removed under suction, and washed

with a little cold (—40°C) acetonitrile, giving the product as awhite powder. Drying under high vacuum for 2 hr gave theproduct: 7.9 g (60%). Recrystallization from benzene gavecrystals for analysis. (m.p. 132—134°C): IR (KBr) 1,640 cm'(C=N. C=NOH) ‘HNMR (d@-DMSO)Ã3́.2 (4H, s, CH2N),1.9 ( I2H. S. MeC). 1.0 (6H. s, CMe2). Anal. calc. forC13H24N402:C, 58.2: H. 9.0: N, 20.9: Found: C, 58.1: 8.9: N.20.9.

(b). Preparation of@RR,SS@-4,8-dia:a-3,6,6,9-tetra-methy/-undecane-2, JO-dionebisoxime (d,/-HM-PAO). The above bisimine (75 g, 287 mmol) was slurried in 95% aqueous ethanol(690 ml) at 0°C. Sodium borohydride ( 10.9 g, 287 mmol) wasadded in portions over 30 mm, and the mixture stirred at 0°C for 2 hr. Water (230 ml) was added and the mixture wasstirred well for a further 2 hr. The ethanol was removed andmore water (140 ml) was added. The pH was adjusted to I 1,and the resulting precipitate was removed by filtration, washedwith a little water, dried giving the impure HM-PAO: 36 g(47%). Double recrystallization from acetonitrile provided thediastereoisomeric mixture of HM-PAO free from major impurities: 25 g (33%), (m.p. 121—125°C). Fractional crystallization from ethyl acetate provided the d,l-diastereoisomer(m.p. 128—130°C): IR (KBr) 3,310 cm@ (OH), 3,219-3,100

HNMe

Me

e

OHFIGURE 1Structure of PnAO.

transported 99mTc across the BBB, but allowed theradiotracer to be retained with a fixed distribution fora time sufficient to permit SPECT imaging. From thatstudy, the ligand which combined the best overall features of high brain uptake, fixed regional distributionwithin the brain. and ease ofradiopharmaceutical preparation was hexamethyl propyleneamine oxime (HMPAO). The potential of HM-PAO shown in laboratoryanimals (1 7) was confirmed by the initial clinical findings (18,19).

HM-PAO exists in two diastereoisomeric forms, d,land meso- (Fig. 2). While the original clinical studieswith HM-PAO were conducted with a mixture of thesetwo isomeric forms it was subsequently shown in rats(20) andhumans(21) thatoneofthe diastereoisomers,d,l-HM-PAO, provides a @mTccomplex with superiorbrain uptake and retention compared with the complexfrom the stereoisomeric mixture. We now report onfurther studies which examine the biologic and chemical characteristics of [@mTcJd,l-HM-PAO.

Me

FIGURE 2Diasteriosomers of HM-PAO. meso-HM-PAO d,1-HM-PAO

The Journal of Nuclear Medicine192 Neinnckx,Canning,Piperetal

(OH.NH): ‘HNMR (d6-DMSO)ô10.3(2H. s, OH). 3.3(2H.s.NH). 3. 12 (2H.q.CH), 2. 12(4H,q,CH2). 1.64 (6H,s,ME),1.07 (6H,d.Me), 0.78 (6H,s,Me). Anal. calc. for C,3H28N4O2:C, 57.3:H. 10.4:N. 20.6:Found: C. 57.5:H. 10.2:N, 20.6%.

Stereoisomeric purity was confirmed using normal phasehigh performance liquid chromatography (HPLC), employinguv detection of the HM-PAO diastereoisomers.

2. Technetium complexation. Sodium pertechnetate@@mTcwas obtained from a commercial @Mo/@mTcgenerator.@The99mTc99m complex ofd.l-HM-PAOwas formed usinga freezedried formulation (Ceretec@)of 0.5 mg of d,l-HM-PAO, 7.6@Lg of stannous chloride dihydrate, and 4.5 mg of sodium

chloride. in a sealed 10-mI glass vial under an atmosphere ofnitrogen. Five milliliters ofsodium pertechnetate (99mTc,2—7mCi/mI) was injected into the vial. The vial was shaken todissolve the solid contents. Complex radiochemical purity(RCP) was assayed by thin layer chromatography.

The influences ofgenerator eluate age, time since previousgenerator elution. and radioactive concentration on RCP werestudied. Eluate was taken from generators which had beeneluted 2. 24. and 70 hr previously. These eluates were used toreconstitute vials ofthe freeze-dried formulation at 0.5, 2. and4 hr postgenerator elution. These vials were analyzed forpercentage primary and secondary complexes, reduced hydrolyzed technetium and pertechnetate at 2. 30. and 60 mmpostreconstitution. A single vial was reconstituted with 256mCi of generator eluate. and the contents analyzed at 2. 10,and 30 mm postreconstitution.

3. Analtwis ()f99mTccomp/exes. The distribution of radioactivity on chromatograms was quantitatively analyzed usinga 256 channel gas flow energy-proportional counter interfacedto an HP 98 16t@computer system.

(a). Thin lm'er chromatography. A combination of threechromatographic systems was used for a complete characterization of the radiochemical composition of the solutionsprepared in 2. above.

Ten microliter test samples were applied 2.5 cm from thebase of three chromatographic strips, two were ITLC/SG@ (2x 20 cm) and the third was Whatman No. 1 (2 X 30 cm).Immediately after the application ofthe sample, the chromatograms were developed by ascending chromatography in tankscontaining fresh solvent to a depth of 1 cm. One ITLC/SGstrip was developed in 2-butanone (MEK) and the other in0.9% saline. The Whatman No. 1strip was developed in 50%aqueous acetonitrile. After development. the strips were dried,and the radioactivity distribution was determined.

(h). Electrophoresis. Ten microliter test samples ofthe @mTccomplex ofd.l-HM-PAO were applied to Whatman 541 strips,saturated with 50 mM pH 7.4 phosphate buffer, in an electrophoresis bath4@.300V was applied across 20 cm of the stripfor 1 hr. The strips were dried. and the distribution of radioactivity on the strip was determined.

(i.) High performance liquid chromatography. The complex

was analyzed using a divinylbenzene-styrene copolymer column―fitted to a radioactivity detector. At a flow rate of 2mI/mm. 20 mM phosphate buffer pH 7.4 was pumpedthrough the column. When the test sample was injected ontothe column, a 0—25%linear solvent gradient of tetrahydrofuran (THF) was commenced (25% THF in 6 mm). Theradioactivity detector was fitted to a ratemeter and microcomputer programmed for peak integration.

Biology1. Biodistrihution of 99mTc complexes. Biodistribution

studies were performed in male Sprague Dawley rats (130—I70 g). Under light ether anesthesiaeachanimal wasadministered 100 zl of the test preparation through the lateral tailvein. The RCP of all preparations was checked prior toinjection. and at all times. the purity of the @mTccomplexwas not less than 85%. At killing, samples of blood. muscle.bone. skin. and fat were collected in preweighed containers.Other organs were removed intact. as listed in Table 3. Allorgans and tissues were assayed for radioactivity in a twincrystal automatic gamma counter. The accumulated activityin each organ or tissue was calculated as a percentage of thetotal dose. For blood. bone. muscle, skin. and fat the calculation was based upon the measured activity and weight of thesample. and body composition data (blood. bone. muscle,skin. and fat are 5.8. 5.0, 43.0, 18.0. and 7.0%. respectively).

2. Whole-body autoradiography. Carbon-l4- (‘4C)labeledd,l-HM-PAO was prepared from [2,2-dimethyl-C'4]-2,2-dimethyl- I,3-propanediamine and butanedione monoxime bythe method outlined for the synthesis of the inactive ligand.The specific activity was 105 mCi/mmol. The material wasstored as a freeze-dried powder at —20°C, and when required.this was dissolved in sterile water to give a radioactive concentration of 370 @Ci/ml.

Four Sprague Dawley rats (95—105g) were injected with[‘4C]d,l-HM-PAO(37 @zCi)and four rats with [‘@mTc]d,l@HM@PAO(36 mCi in 600 @zl)througha lateraltail vein under lightether anesthesia. At 2 mm and at 1 hr postinjection twoanimals from each group were killed. Twenty micrometerwhole-body sagittal sections were prepared by standard procedures and exposed to a fast x-ray film°°for 99mTc,or aslower, fine grain filmttt, for ‘4C.

After exposure for an appropriate period, films were develo_ and, in each case, films of the midline section and alateral section were used as negatives to produce contact prints.On the latter, regions with high radioactive concentrationappear black, and low activity regions appear white.

3. Rat brain autoradiography. Four male Sprague Dawleyrats (250—400g) were anesthetized with sodium pentobarbital(50 mg/kg, i.p.) and their right jugular vein cannulated. Theanimals were killed at specific times following the i.v. injectionof 1ml of[@mTc]HM@PAO(40—50mCi of@mTc)over 20 sec.Killing times were 5 sec following peak brain uptake (determined by a collimated Nal (TI) crystal detector placed overthe head and interfaced to a multichannel analyzer), and 1. 5.30. and 60 mm postinjection. After killing, the brains wereremoved and frozen in liquid nitrogen. Twenty micrometerserial coronal sections were taken through the cerebral hemispheres and cerebellum at —24°Cin a refrigerated microtome.The sections were mounted on plastic cover strips, dried. andexposedto x-rayfilm.*U

For studies comparing the cerebral distribution of [99mTc]d,l HM-PAO and ‘4C-labelediodoantipyrine ([‘4C]IAP*).theabove procedure was used. One animal was injected with a0.75-ml solution containing 43 mCi of [99mTcJd,l..HM..pAOand 62 @Ciof [‘4CJIAP.Following killing at 5 sec postpeak,and slice preparation as described above, slices were placedon fast x-ray film***for 24 hr to provide the @mTcautoradiograph. Three days later, when @mTchad decayed to background levels, the slices were exposed to a slow filmm for 10

193Volume 28 •Number 2 •February 1987

Me

3 4

FIGURE 3Synthesis of HM-PAO.

days to provide the ‘4Cautoradiograph. Each section wasstained with 2% cresyl violet and then fixed in 70% ethanol.The cresyl violet stain provided a clear differentiation of greyand white matter. Images were stored in a microcomputer,using a system described previously (22). Images shown inFigure 8 were obtained from the monitor display.

4. Calculation ofradialion doses. Using the standard MIRDI 1 format (23). radiation doses to humans receiving intravenous [99mTc]dIHMpAO were estimated on the basis of therat biodistribution data. Account was taken of a number ofurinary bladder voiding patterns.

5. Toxicology.Toxicology studieshavebeenperformed inmale and female rats and rabbits following single and repeatedintravenous injections. For single dose studies, animals received 17 vials/kg (equivalent to 1.200 vials/70 kg); for repeatdose studies. animals received 14 consecutive daily doses of14.3 vials/kg (the equivalent to 1.000 vials/70 kg).

RESULTS

ChemistryThe route ofsynthesis ofHM-PAO is shown in Figure

3. Condensation of the propanediamine (Fig. 3, no. 2)

with two molecular equivalents of the keto-oxime (Fig.3, no. 1) provides the bisimine (Fig. 3, no. 3) in 60%yield. Reduction of the two imine groups with sodiumborohydride provides HM-PAO (Fig. 3, no. 4) as anequal mixture of the two diastereoisomers, meso- andd,l, as demonstrated by HPLC analysis (Fig. 4). Repeated crystallization from ethyl acetate permits theseparation of the two diastereoisomers (Fig. 4), butresults in considerable reduction in the overall yield ofthe ligand. An x-ray crystal structure analysis demonstrated that the stereoisomer with longest retention onHPLC analysis was d,l-HM-PAO (Fig. 5).

The freeze-dried formulation of d,l-HM-PAO allowsthe 99mTc complex to be prepared simply by addinggenerator eluate to the vial. Thin layer chromatographypermits the quantitative determination of radioactivecomponents following complex formation. In Table 1are listed the R1 values of the observed radioactivecomponents on the three chromatographic systemsused. Addition of [99mTc]pertechnetate to this freezedried formulation provides the primary lipophilic com

Hplc Traces of HM—PAOOiastereoisomers

Th,., HPLC t@ it f,-@. tt@iy.t. of tho ligood, oo@og UV dot.otxo. of ,iuted

f.@.ottot. . Tho ,otontto@ t@,* .o.o.hoo. .. .tooto@.

Mixture of meso—and d. 1—HM—PAO

meso—HM-PAO d. 1-HM-PAOFIGURE 4HPLC analysis of HM-PAO diastereoisomers.

194 Neirinckx,Canning,Piperetal TheJournalof Nudear Medicine

Me Me

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There is a slow conversion of the primary complex toa secondary complex. In biologic studies this conversionlimits the time postpreparation in which the materialcan be used. In all cases, the radiopharmaceutical wasused with proportion of the primary 99mTc complexnever < 85%.

The rate of loss of primary complex is increasednotably when using generator eluate of age >2 hr. oremploying a high radioactive concentration. In thesecases, higher pertechnetate levels are the primary causeof the reduced proportion of primary complex.

The time since previous elution of the generator haslittle effect on the rate of loss of primary complex withthe commercial 99Mop9mTc generatortt used in thisstudy. However, evaluation ofa number of commercialgenerators revealed that, in some cases, the rate of lossof primary complex increased when using eluate froma generator, not previously eluted within 24 hr (TyrrellDA et al. unpublished data).

HPLC analysis also demonstrates the formation oftwo 99mTccomplexes of d,l-HM-PAO, as shown inFigure 6. The analysis shown was performed 30 mmafter complex formation.

We have demonstrated previously (24) that HPLCretention on this reverse phase system can provide anestimate of the lipophilicity of 99mTc complexes. Tra

ditionally, lipophilicity is determined by the concentration ratio (P) of a compound partitioned between twoimmiscible solvents (usually n-octanol and water). Thismethod is only suitable for pure compounds, but acalibrated HPLC system can provide estimates oflog Pfrom the retention times of components in a mixture.The estimated log P value for the primary complex is1.2 and the value for the secondary complex is <0. Byelectrophoresis, both the primary and secondary cornplexes of d,l-HM-PAO remain at the point of application, suggesting that both are neutral.

BiologyThe biodistribution of the primary complex of d,l

HM-PAO is shown in Table 3. The primary complexdisplays 2.25% (% i.d. in whole organ) brain uptakesoon after injection. Retention of activity is high, with84% of the initial activity (decay corrected) remainingin the brain 1 hr postinjection, and 73% at 24 hrpostinjection. Background activity clears by both thehepatobiliary and urinary pathways.

An initial period of rapid blood clearance to 12% ofinjected dose is followed by a phase of very slow clearance. Examination ofthe distribution ofthis radioactivity in blood reveals that >80% of that activity is in thered blood cells.

Whole-body autoradiography (Fig. 7) provides aqualitative assessment of regional distribution of theprimary 99mTccomplex ofd,l-HM-PAO. Uptake of thetracer within the brain is clearly visible. However, the

Oh

FIGURE 5Structure of d,I-HM-PAO from x-ray crystal analysis.

plex of d,l-HM-PAO in >90% yield immediately aftercomplex formation. Other observed components are aless lipophilic 99mTc complex of d,l-HM-PAO (termedthe secondary complex), and very small amounts ofreduced hydrolyzed technetium and pertechnetate.

Combined use of the three chromatographic systemsallows the full quantitative assessment of the radiochemical composition ofthe mixture using the following relationships:

% reduced, hydrolyzed Tc = % activity remainingat the origin in the Whatman No. 1/50% aq.acetonitrile system;

% pertechnetate = % activity at the solvent frontin the ITLC/saline system;

% secondary complex = % activity at R@0-0.3 inthe ITLC/MEK system minus % reduced hydrolyzedTc:

% primary complex = % activity at R10.3-l in theITLC/MEK system minus % pertechnetate.

Table 2 lists the observed proportion of each radioactive component over a period following the additionof [99mTc]pertechnetate to the freeze-dried formulation.

TABLE 1R1Values of the @“TcComponents on TLC

[@‘°TcJd,l-HM-PAOprimary0.9—1.000.9—1.0[@°TcJd,l-HM-PAOsecondary000.9—1.099mTc

reduced,hydrolyzed000[@“Tc]pertechnetate0.9—1.00.9—1.00.9—1.0

. System 1 : ITLC/SG—MEK.

t System 2: ITLC/SG—saline.

4 System 3: Whatman No. 1 —50% aqueous acetonitnle.

195Volume 28 •Number 2 •February 1987

Generator eluate/kit reconstitution detailsACP at timesafter reconstitution(%)2

mm30 mm60mmTimesince Ageof.previous

generator Totalactivityelutionof eluate on kit added tothegenerator

reconstitution vial (mCi in5(hr)(mm) ml) No. fNo. 2@ Tc04RHT@No. 1No. 2 Tc04RHTNo. 1No. 2Tc04RHT5

27.3 960 04923 15869142240 28.1 951 14895 427881225

31.3 932 15889 0384110424120 33.5 953 12925 1287742240

27.8 951 13884 5377101125

32.2 953 02934 0388100270120 30.0 962 02933 1388722240

30.0 953 01934 12838912mm10mm30mmNo.

1No. 2 TcO@RHTNo. 1No. 2 Tc04RHTNo. 1No. 2Tc04RHT18

30 256 963 11943 21767171No.

1 = Primarycomplex of@‘°‘°Tc)dJ-HM-PAO.tNo. 2 = Secondary complex of [‘“TcJd,lHM-PAO.S

RHT = RedUced, hydrolyzed technetium.

TABLE 2Determination of RCP, Examining the Influences of Generator Eluate Age, Time Since Previous Elution, and

Radioactive Concentration

uncomplexed ligand does not cross the BBB; the autoradiographs following administration of ‘4C-labeledd,lHM-PAO demonstrate the absence ofthe free ligand inthe brain.

Results of studies to determine the regional distribution of the primary [99mTc]d,l@HM@PAOcomplexwithin the rat brain are shown in Figure 8. Qualitatively,the regional distribution of[99mTc]d,l@HM@PAOat 5 secpostpeak brain uptake is similar to that observed for‘4C-labelediodoantipyrine (lAP), a reference tracer forrCBF (25). The prominent uptake of both tracers inregions rich in gray matter is highlighted by comparisonof the autoradiographs with the cresyl violet stained

section. Figure 9 displays the results of temporal measurement of the regional distribution of [99mTc]d,l@HM@PAO in the rat brain. Over the period of study (1 hr)there appears to be little redistribution of the tracer asthe image ofthe blood flow distribution pattern remainsunaltered.

Results oforgan radiation dose calculations are summarized in Table 4. The highest doses are received bythe urinary bladder and upper large intestines.

In all toxicity testing, no treatment-related effectswere evident. Observations were made ofbody weights,clinical signs, food and water consumption, hematology, blood chemistry, ophthalmology, and urinalysis.

Th-9@ d ,1 HM-PAOpri@ry co@1ex

@ft-99. d ,1 HM-PAOaecondary coqlex

FIGURE 6HPLC analysis of [@Tc]d,l-HMPAO. Retention U@ in ainute@

TheJournalof NuclearMedicine196 Neinnckx,Canning,Piperetal

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Volume 28 •Number 2 •February 1987 197

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FIGURE 7Whole-body autoradiographs of [@‘°Tc]d,I-HM-PAOand [14C]d,l-HM-PAO. Left column: [@“Tc]d,l-HM-PA0.Rightcolumn: [14C]d,I-HM-PAO.Top row: lateral section, 2 mm. Second row: midline section, 2 mm.Third row: lateral section,60 mm. Bottom row: midline section, 60 mm.

At termination complete necropsy and histopathologyrevealed no abnormalities.

DISCUSSION

The ligand d,l-HM-PAO readily forms a neutral,lipophilic complex with @mTcfrom a freeze-dried ‘kit'to provide a new radiopharmaceutical for imaging cerebral perfusion. The quality (RCP) of the radiopharmaceutical is dependent upon several factors concerning generator eluate age. time since previous elution ofthe generator, and radioactive concentration. It is therefore recommended to (a) use eluate from a @mTcgenerator which was previously eluted within 24 hr; (b) use

generator eluate within 2 hr ofelution; (c) add no morethan 30 mCi of 99mTc to the vial; and (d) use theradiopharmaceutical within 30 mm of reconstitution.

High brain uptake was demonstrated in rats, and upto 48 hr postinjection little washout occured. A qualitative assessment of regional distribution of the tracerwithin rat brain indicates a blood flow-dependent uptake with minimal redistribution of the tracer up to atleast 1 hr postinjection. The background activity levelsclear, principally by the urinary system, but alsothrough the hepatobiliary system. In humans, the higherproportion of cardiac output to the brain gives rise tohigher brain uptake (21).

The rat biodistribution data were used to estimate

The Journal of Nuclear Medicine198 Neirinckx,Canning,Piperetal

lung spleen kidney gut lining

\‘@@:@‘@

liver

stomach mucosa

0spleen

lung

liver gut contents

411

blood

@ (@ .,li_,@___ur ne

pancreas

FIGURE 8Comparison of [@TcJd,l-HM-PAOand [14Cjiodoantipyrmnecerebral distnbution.(Rat brainautoradiographswith stained sections).

w

the radiation dosimetry in humans. These estimatesindicate that no problems due to radiation exposurecan be anticipated at dose levels of up to 15 mCi inhumans, even with delayed bladder voiding. These datacompare favorably with the corresponding dose estimates for [‘23I]IMP,even discounting the contaminantsof 241and 125j in the iodinated radiopharmaceutical(26). Similarly, the acute toxicity study employing thefreeze-dried formulation as test article would indicatethat no toxicological effects can be expected at normalhuman dose levels.

The structure of the primary technetium complex ofd,l-HM-PAO has been determined (27) and shown tobe very similar to the complex obtained with PnAO(28). While both ligands can transport technetiumacross the BBB [99mTcJpnAO behaves like a freely diffusable tracer, whereas [99mTc}d,l-HM-PAO is retainedin the brain and displays a fixed regional distributionwithin that organ. This is essential for SPECT imagingusing the rotating head gamma camera. It has beenproposed that retention in the brain of [@mTc]d,l@HM@PAO results from in vivo conversion of the primarycomplex to the more hydrophilic species (29). It hasbeen shown previously (30) that neutral compoundswith log P values in the range of 0.9—3.5can cross theintact BBB. The log P value for the primary complexfalls within this range, but the value for the secondary

complex is below the range. Studies to confirm themechanism of retention are underway.

A high proportion of the radioactivity remaining inthe blood appears to be trapped within red blood cells;it is possible that the mechanism for entrapment withinthese cells is similar to that ofbrain retention. The highblood levels (10—12%of injected dose) of this radiopharmaceutical should not adversely affect SPECT image quality. In humans. the volume ofblood within thebrain is 3 1 ml, or 0.6% of total blood volume (31).Technetium-99m-d,l-HM-PAO displays 4. 1% i.d. uptake in the brain in humans, with blood levels at 1 hrpostinjection averaging 12.0% of injected dose (21).Thus, the contribution of blood activity to total countsfrom the brain is only 1.7% (1 hr postinjection).

The ability of the primary complex to cross the BBBis highlighted further by the whole-body autoradiographs comparing the biodistribution of [99mTcJd,l@HM-PAO, and the ‘4C-labeledligand. While uptake ofthe complex is clearly visible, the ligand itself is nottransported across the BBB. Hence, d,l-HM-PAO onlyaccumulated in the brain when complexed to 99mTcasthe primary complex. These findings are similar to thoseobserved for [99mTcJpflAO and ‘4C-labeledPnAO (32).Thus, the primary 99mTccomplex of d,l-HM-PAO canbe classified as a “technetiumessential―radiopharmaceutical (33).

Volume 28 •Number 2 •February 1987 199

,.*@t_'@ “?frSt@@i::@:@@‘A'

FIGURE9Regional distribution of d,I HM-PAO in the rat brain as a function of time. From left to right: thalamus, midbrain, andcerebellum. From top to bottom: 1 mm, 5 mm, 30 mm, and 60 mm.

detail provided by [@mTc]d,l@HM@PAO.Recent studiesin rabbits (34) and dogs (35), which quantitativelycompare the regional distribution of [@mTc]d,lHMPAO in the brain with labeled microspheres, confirmthat [@mTc]d,l@HM@PAOhas a blood flow-related braindistribution.

Extensive clinical studies have now been conductedat a large number of centers (21,36—42)and have confirmed the promise shown by the preliminary data

Technetium-99m d,l-HM-PAO displays a regionaldistribution in the brain which, from the qualitativeautoradiographic comparison with lAP, appears to beblood flow dependent. The 5-sec [99mTc]d,l@HM@PAOautoradiograph displays far better substructural detailthan the corresponding lAP autoradiograph. The lowerquality image provided by lAP could be attributed tosome diffusion of this tracer within the brain aftersacrifice, but served to highlight the excellent structural

200 Neinnckx,Canning,Piperetal The Journal of Nudear Medicine

Absorbedradiationdoserads/mCimGyfMBq2-hourly4-hourlyNo2-hourly4-hourlyNoOrganvoidsvoidsvoidsvoidsvoidsvoidsBone

(total)0.0140.0140.0160.0040.0040.004Redbonemarrow0.0240.0240.0270.0070.0070.007Testes0.0080.0090.0160.0020.0020.004Ovaries0.0400.0420.0520.01

10.0110.014Lungs0.0090.0090.0090.0020.0020.002Thyroid0.0050.0050.0050.0010.0010.001Total

body0.0150.0160.0190.0040.0040.005Breast0.0150.0160.0190.0040.0040.005Liver0.0420.0420.0420.01

10.0110.011Smallintest.wall0.1000.1000.1050.0270.0270.028Upperlarge intest.wall0.1800.1800.1830.0490.0490.050Lowerlargeintest.wall0.0780.0800.0910.0210.0220.025Kidneys0.0970.0970.0980.0260.0260.027Urinary

bladderwall0.0520.0910.3280.0140.0250.089Brain0.0150.0150.0150.0040.0040.004

TABLE 4Technetium-99m d,I-HM-PAO Summary of Organ RadIatiOnDoses

presented here. Technetium-99m d,l-HM-PAO has provided high quality SPECT images in patients with avariety of cerebrovascular and neurological disorders.

NOTES

0Gallenkamp.t R24B. Perkin-Elmer, Norwalk. CT.

t 684. Perkin Elmer, Norwalk. CT.

* Elemental Micro-Analysis Ltd.

C North London Polytechnic.

•0Aldrich Chemical Co., Milwaukee. WI.tt Amersham International plc. Buckinghamshire, England.

*1 Hewlett Packard Co.. Andover, MA.

k* Gelman Sciences. Inc.. Ann Arbor. MI.

“PRP-l , Hamilton Co., Reno. NV.

•0•Osray M3. Agfa-Gevaert Rex, Inc., White Plains, NY.tyt Industrex A. Eastman Kodak Co., Rochester, NY.

*1* MRF 3 1 CT film, DuPont Co.. No. Billerica, MA.

nt* 5p@.5 Kodak film, Eastman Kodak Co.. Rochester, NY.

ACKNOWLEDGMENTS

The authors thank Mr. W. Gibbs(Amersham International)for the determination of IR and NMR spectra, and Drs. P.Tasker and K. Hendrick (North London Polytechnic) for thex-ray crystal structure determination. The development of d,lHM-PAOinvolvedthe effort of a large number of chemistsand physiology staff in the Pharmaceuticals Research andDevelopment Department of Amersham International, andresearchers at the University of Missouri, Columbia. In particular. the authors thank the following for their contributions:Dr. C.D.R. Hewat. Dr. D.A. Tyrrell. Dr. R.C. Harrison, Dr.B. Higley.Dr. J.F. Burke. Mrs. V.J. Bayne.Mr. R.P. Pettitt,Professor D.E. Troutner, Dr. S. Jurisson, Mr. T. Hoffmann,and Mrs. E. McKenzie.

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202 Neinnckx,Canning,Piperetal The Journal of Nuclear Medicine