Comparison of Technetium-99m AminoalkylDiaminodithiol (DADT) Analogs as PotentialBrain Blood Flow Imaging AgentsUrsula Scheffel, Howard W. Goldfarb, Susan Z. Lever, Ramon L. Gungon,H. Donald Burns, and Henry N. Wagner, Jr.

Divisions of Nuclear Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland

N-ethyl piperidinyl diaminodithiol (NEP-DADT), complexed with "Te has been developed as

an agent for the measurement of brain blood flow using SPECT. Studies in patients haveshown that "Te NEP-DADT enters rapidly into the brain, but also clears rapidly (t1/2= 17min). In this study nine new aminoalkyl DADT derivatives were synthesized, labeled with 99mTc

and tested in mice with the aim of developing an agent with increased retention in the brain.In addition, relationships between chemical properties of the derivatives and their in vivolocalization were investigated. The results were as follows: (a) the R-group and its isomerieconfiguration has a profound influence on the biodistribution; (b) "Tc aminoalkyl DADT

derivatives with apparent p«avalues of >6.9 show poor brain uptake (<0.40 % dose at 5min); (e) lengthening of the chain between the DADT moiety and the amino-R group from ethyl

to hexyl generally increases the apparent pKa and consequently lowers brain uptake; (d) acorrelation (r = 0.71 ) exists between initial brain uptake and the octanol-buffer partitioncoefficient; (e) "Te--*'-methyl NEP-DADT has the highest partition coefficient, relatively high

uptake, and longest retention in the mouse brain. This complex has characteristics suited forbrain blood flow measurements.

J NucíMed 29: 73-82,1988

he technetium-99m (99Tc) complex of N-ethylpiperidinyl diaminodithiol ([99mTc]NEP-DADT)has re

cently been developed in our laboratory (/). This agenthas good qualities for single photon emission computedtomography (SPECT) imaging of brain blood flow inexperimental animals (7,2) and normal volunteers (3).However, because of its relatively fast clearance fromthe brain, it cannot be considered optimal. Present daySPECT imaging technology with rotating cameras callsfor prolonged brain retention, preferably up to 1 hr, aswell as a fixed distribution during the time of the study(4). In order to evaluate the possibility of increasinguptake and retention of the 99mTc-labeledcomplex inthe brain, several derivatives of [99mTc]DADT were

prepared and tested in mice.Five new analogs of the N-aminoethyl and four an

alogs of the N-aminohexyl DADT complex were syn-

Received Feb. 9, 1987; revision accepted Aug. 18, 1987.For reprints contact: Ursula Scheffel. ScD. Div. of Nuclear

Medicine, The Johns Hopkins Medical Institutions, 301 TraylorResearch, 1721 East Madison St., Baltimore. MD 21205.

thesized, labeled with "Tc and separated by high

performance liquid chromatography (HPLC) into tworespective components (A and B). Their biodistributionin mice was compared with that of [99mTc]NEP-DADT

(A and B).An attempt was made at correlating chemicalstructure against in vivo localization in order to aid inthe custom design of future brain blood flow imagingagents of similar structure.

MATERIALS AND METHODS

Preparation of the DADT Complex and its AnalogsThe following ligands as the tri-HCl salts were prepared in

an analogous fashion to the procedure for NEP-DADT (/):N-ethylpiperidinyl DADT (NEP-DADT, I, see Fig. 1); N-ethylN,N'-dimethylamino DADT (II); N-ethyl 4'-methylpiperidi-nyl DADT (III); N-ethyl 4'-propylpiperidinyl DADT (IV); N-

ethylmorpholinyl DADT (V); N-ethylisopropylamino DADT(VI); N-hexylpiperidinyl DADT( VII); N-hexyl N,N'-dimethyl-

amino DADT (VIII); N-hexyl morpholinyl DADT (IX) andN-hexyl isopropylamino DADT (X). Each ligand was char

acterized by IR, NMR, and elemental analysis. The analyticdata was consistent with the assigned structures.

Volume 29 •Number 1 •January 1988 73

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HNINA+B

R

FIGURE 1Labelingof aminoethyl (I-VI)and ami-nohexyl (VII-X) DADT ligands with["Tclpertechnetate in the presenceof SnCI2. In each case two majorcomplexes (A and B) were formed.

R>vn

mr\

IV

VI

AAA/N<

Vffl IX

AAA/N-<

X

Tc-99m Labeling and Isolation of ComplexesLabeling of the different ligands with "Tc was performed

as previously described in detail (/). In brief, to a solution ofI mg of ligand in 0.4 ml of pH 7.1 phosphate buffer was addeda solution of stannous chloride dihydrate in ethanol (0.1 ml,1.33 x Ifr4 M), followed by 5-10 mCi sodium ("Tc]per-

technetate in 0.1-0.3 ml isotonic saline. All reactions werecarried out at room temperature for 15 min, except for ligandsIII and IV that were heated at 50"C for 30 min.

Separation and purification procedures depended on thetype of ligand. The "Tc aminoethyl analogs (Complexes I-

VI) were first extracted into hexane then separated by normalphase HPLC using a silica gel column' and méthylènechlo-

ride:ethanol (98:2) as solvent system. The two main peaks (Aand B, where B eluted first) were collected in separate flasksand five milliliters of isotonic saline were added to eachfraction. Subsequently, the organic phase was removed byevaporation under vacuum without heat. The "Te amino-hexyl derivatives (Complexes VII-X), on the other hand, wereseparated on a reverse phase HPLC column1 with acetoni-

trile:0.01 N aqueous ammonium acetate (45:55) as the solventmixture. In this system peak A eluted before peak B. Afterisolation, the fractions were subjected to rotoevaporation under vacuum for removal of acetonitrile. The purity of eachcomplex was determined in the following manner. For complexes isolated by normal phase HPLC, the collected fractionswere re-analyzed by normal phase HPLC and silica gel thinlayer chromatography*(95:5 CH2Cl2:EtOH).Complexes IV-Aand IV-B were analyzed by HPLC only. For complexes isolated by reverse phase HPLC, the collected fractions were reanalyzed by reverse phase HPLC. The purity was found to be>95% for all complexes except the following: I-B (90%), IX-A (70%), and IX-B (86%). The contaminant in these caseseluted near the void volume on reverse phase HPLC andprobably consists of salts of ["Te] pertechnetate generated

by partial decomposition of the complexes during the isolation

procedure. For use in biodistribution studies all HPLC separated "Te complexes were diluted to a concentration of 10

with isotonic saline.

Determination of Apparent lonization ConstantThe apparent pK, of each of the "Tc complexes was

estimated by measuring their octanol-buffer distribution coefficient (O-B DC) at various pH values. For this purpose, 10-100 /A(~5 MCi)of the "Tc complex were introduced into a

100 mm x 10 mm screw capped tube containing 3 ml ofeither HC1/KC1(0.05-0.2A/) at pH 1.0-2.2, QAMcitric acid/citrate at pH 3.0-6.0, 0.2A/ phosphate buffer at pH 6.0-7.5,or 0. 1M TRIS at pH 7.0-9.9. For each of the complexes O-BD.C. determinations were done in triplicate at 12 to 14 different pH values. Three milliliters octanol were added and eachtube was gently shaken on an automatic mixing apparatus for5 min. After a 5-min centrifugation at 1,500 g, 1 ml aliquotsof the octanol and the buffer phase were transferred into aplastic tube and counted in an automatic gamma scintillationcounter.* The counting error was kept below 3%. O-B DCswere plotted against pH. The apparent pK, values were estimated from the curves by two methods: derivative analysis bylocating the pH of maximum difference in O-B DCs and theHenderson-Hasselbach equation using the O-B DC for pro-tonated and unprotonated species.

Determination of Octanol-Buffer Partition CoefficientOctanol-buffer partition coefficients were measured by in

troducing 0.2-0.5 ml of each of the "Tc complexes into

isotonic phosphate buffer (5) at pH 7.4 (total volume: 3.0 ml),and then adding 3 ml of 1-octanol. After shaking the mixturefor 5 min and centrifuging for 3 min, the supernatant octanolphase was transferred into another tube and reequilibratedwith 3 ml of fresh buffer four more times. After the fifthpartitioning, two milliliter samples of each phase were removed and the "Tc activity in buffer and octanol deter-

74 Scheffel, Goldfarb, Lever et al The Journal of Nuclear Medicine

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mined. The partition coefficient was obtained by calculatingthe ratio of net CPM/ml of octanol to that of buffer.

Determination of Protein BindingProtein binding was evaluated by Ultrafiltration. Thirty to

two hundred microliter aliquots of solutions of the """Tecomplexes (10-15 ßCi)were added to 3 ml of pooled humanserum and incubated for 15 min at room temperature. Onemilliliter serum samples were transferred to Centricon-30microconcentrators'with >95% membrane retention for mol

ecules above 30,000 D. Centrifugation was performed at 4,000g for 20 min at 4°Cusing a fixed 34°angle rotor. Adsorption

to the membrane was measured by counting the filter afterremoval of filtrate and serum concentrate. Less than 5% ofthe "Tc activity was found with the filter membrane. Tech-netium-99m diethylenetriaminepentaacetic acid (DTPA),known not to bind to serum proteins (6), was recovered to100% in the filtrate under similar incubation and filteringconditions.

Biodistribution StudiesMale CD-I mice" weighing 25-35 g were injected intrave

nously into the tail vein with 0.2 ml (~2 ^Ci) of each of the"Tc-labeled DADT analogs. At various times after injection,

the animals were killed. Immediately before death, a bloodsample (0.1 ml) was collected from the jugular vein into aglass capillary which was then placed into a counting tubefilled with 0.9 ml water. Organs of interest (brain, heart, lungs,liver, spleen, kidneys, intestines) were dissected, weighed, andprepared for counting. The "Tc radioactivity was measured

in an autogamma scintillation counter. The percent of radioactivity in the whole blood was estimated using a bloodvolume of 7% of the body weight. The average recovery of"Te radioactivity at 5 min after injection, i.e., the sum of

the percentages of the dose found in all organs including thecarcass was 99.5 ±3.1%(mean of 15determinations ±1s.d.).

RESULTS

Figure 1 shows six different aminoethyl and fouraminohexyl DADT ligands which were prepared in ourlaboratory and labeled with Sn2+ reduced ["Tc]per-technetate. Each of the formed "Te complexes was

separated on HPLC. The two main constituents of theHPLC separation designated A and B, represent, mostlikely, geometric isomers of the complexes as shown for["Tc]NEP-DADT (7,8). Confirmation for the com

plexes in this series is still in progress.The organ distribution in mice for the different "Tc

aminoethyl- and aminohexyl DADT analogs is presented in Tables 1A and IB. The analogs are orderedaccording to their brain uptake at 5 min after i.v.injection. Technetium-99m NEP-DADT (Complex I,peak A) and "Tc N-ethyl-N,N'-dimethylamino

DADT (Complex II, peak A) had the highest brainuptake (1.95 and 1.94% of the injected dose, respectively), followed by 99mTc 4'-methyl NEP-DADT

(Complex III, peak A) (1.48% dose). Brain/blood ratiosfor these three compounds were also appreciably higher

(4.28, 3.97 and 3.18, respectively) than all oth-ers.Lengthening the chain length from ethyl to hexylbetween the DADT moiety and the amino R-groupsignificantly lowered brain uptake as demonstrated bythree out of the four hexyl analogs tested. The exceptionwas the "Tc aminohexyl morpholino derivative that,

in the A form, showed similar uptake in the brain asthe aminoethyl morpholino derivative. In the B-form,it demonstrated increased uptake (1.14% vs. 0.61%, p< 0.005).

All ["Tc]DADT analogs showed considerable ac

cumulation in liver and gastrointestinal tract. Lunguptake was variable, ranging from 0.88% to 11.21% ofthe injected dose. However, no relationship between invitro lipophilicity, as determined by the octanol-bufferpartition coefficient at pH 7.4, and lung uptake wasobserved.

Technetium-99m activity in brain and other majororgans was followed for a 2-hr period for complexeswhich had demonstrated high initial brain uptake, i.e.,for ["Tc]NEP-DADT (Complex I, peaks A and B),"Tc-N-ethyl N,N'-dimethylamino DADT (ComplexII, peaks A and B) and "Tc 4'-methyl NEP-DADT

(Complex III, peaks A and B). Brain and blood curvesfor four of these complexes were selected to be presentedin Figure 2. Technetium-99m NEP-DADT (I A) and"Tc N-ethyl N,N'-dimethylamino DADT (II, A and

B) disappeared rather rapidly from the brain. In contrast, "Tc 4'-methyl NEP-DADT (HI A) brain activity

remained relatively high over the entire observationperiod, falling from 3.1 ±0.17% dose/g (mean of 12determinations ±1 s.e.m.) at 5 min after injection to1.74 ±0.08% dose/g at 60 min and to 1.19 ±0.11%dose/g at 120 min. Blood clearance of "Tc 4'-methylNEP-DADT (III A) was rapid, resulting in high brain/blood ratios (3.2 at 5 min to 4.6 at 2 hr after injection).

The biodistribution data for Complexes I, II, and III(peaks A and B) at 30 and 60 min after injection arelisted in Table 2. Most striking was the rapid accumulation of "Tc activity in liver and GI tract. Thecombined uptake in these two organs ranged from 60-70% of the injected dose at 30 min to 70-79% at 60min. The majority of the remaining dose was found inthe carcass (data not shown). The results indicate thatthese lipophilic "Tc complexes are excreted primarily

via the biliary system and the gut.Several physicochemical properties of the different

"Tc aminoethyl and aminohexyl DADT complexes

were examined in order to investigate their relationshipwith biological localization. Drug entry into the brainis governed, according to Rapoport et al. (9), by threemain factors: (a) its lipophilicity, (b) its degree of ioni-zation at the pH of blood, and (c) its binding to serumproteins. Accordingly, the octanol-water partition coefficient, apparent dissociation constant and proteinbinding of each of the "Tc complexes was determined.

Volume 29 •Number 1 •January 1988 75

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Volume 29 •Number 1 •January 1988 77

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FIGURE 2Brain and blood clearance curves of["TcJNEP-DADT (I A), Tc N-ethylN,N' dimethylamino DADI (II A andB), and Tc 4'-methyl NEP-DADT(IIIA). Technetium-99m complex IIIAshowed longest activity retention inthe brain and highest brain to bloodratios.

Tim« Att«r Injiclion

Iminutol•Brain«Blood

Octanol partition coefficients for the different DADTderivatives at pH 7.4 ranged between 11 and 1,671 (logP = 1.04 - 3.22) (Table 3). When the log P data wereplotted against percent brain uptake at 5 min after i.v.injection (Fig. 3), a correlation of r = 0.71 was observed,indicating at least to a certain extent, a linear relationship between entry of the 99mTccomplex into the brain

and its distribution between octanol and buffer at thepH of blood. Furthermore, it was interesting to notethat the complex with the highest lipophilicity (log P =3.22) was the 4'-methyl-NEP-DADT (III) complex

which had been identified as having the longest retention in the brain (Fig. 2). The apparent ionizationcoefficients were determined by measuring the octanol-buffer distribution of the "Te complexes various pH-

values and constructing a curve, similar to a titrationcurve. The apparent pKa's ranged between 4.1 and 7.5

(Table 3). Earlier experiments with the parent complexes had indicated that the dissociation curves didnot represent equilibria of the coordinated amines.Technetium-99m complexes with apparent pK^of >6.9showed poor brain uptake (<0.40% dose at 5 min afterinjection). This latter finding agreed with the postulatethat ionized species pass the blood-brain barrier eitherpoorly or not at all (70). In addition, it was observedthat peak A of the aminoethyl complexes (that alwayspredominates in our labeling procedure) exhibited ahigher apparent pKa than peak B. In the aminohexylcomplexes, on the other hand, where the electron density of the amine is insulated from the complex core,no difference was seen.

Protein binding, as determined by ultrafiltrationranged between 86% and 99% for all ["TcJDADT

complexes (Table 3). In spite of this high degree ofbinding, some of the complexes exhibited relatively highinitial brain uptake after intravenous injection (seeTable 1). This leads to the conclusion that the on andoff kinetics of binding must occur relatively fast; in

particular, that these complexes are readily dissociatedfrom serum proteins in order to be able to penetratethe blood-brain barrier immediately after intravenousinjection.

DISCUSSION

Several laboratories have directed research effortstowards the development of 99mTc-labeledagents suit

able for evaluation of cerebral blood flow by SPECTimaging, a goal first stated by Oldendorf (77,72).

One ligand system which forms neutral, lipid solublecomplexes with 99mTc is propylene-amine-oxime

(PAO). It was first described by Troutner et al. (73) andVolkert et al. (14) in 1983. Studies with the originalcomplex in human beings showed relatively low first-pass extraction and lack of retention in the brain (75)and, therefore, led to the search for a better agent.Amersham International synthesized and tested over100 analogs of the PAO ligand (76), of these d-1 HM-PAO was selected. Several groups of investigators havereported encouraging results using ["Tc]HM-PAO inpatients with various neurologic disorders (77-79).However, the [99mTc]HM-PAOcomplex is not without

problems: its in vitro stability is rather limited (seesupplier's instruction sheet) and, in vivo, it decomposes

extremely fast after intravenous injection, leading torelatively high levels of 99mTcactivity remaining in the

blood stream (2,20).Another of the ligands developed was the diamino-

dithiol (DADT) ligand that forms neutral, lipid soluble"Tc (V) complexes (27,22) and has been shown tocross the blood-brain barrier (23). Kinetic studies ofseveral unsubstituted ["Tc]DADT complexes have

shown rapid transport into the brain, but clearancefrom the brain occurred within a few minutes. Increasedbrain uptake as well as longer retention has been demonstrated when a piperidinylethyl side chain was at-

78 Scheffel,Goldfarb,Leveret al The Journal of Nuclear Medicine

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Volume 29 •Number 1 •January 1988 79

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FIGURE 3Relationship between in vitro lipo-philicity of the ["TcJDADT com

plexes and their initial uptake in themouse brain. The lipophilicity wasdetermined by measuring the octanol-buffer coefficients at pH 7.4.Brain uptake was determined as percent of the dose at 5 min after injection.

2.0

1.0

r = 0.707

1 2 3

log Partition Coefficient

tached to the DADT ligand (N-ethyl piperidinyl-DADT, (NEP-DADT)) (7).

Preliminary studies with [99mTc]NEP-DADT in hu-

man volunteers have shown that washout of the com-

plex from the brain occurs with a half-time of 17 min(J). Since comparatively long (5-10 min per rotation)imaging times with present-day SPECT cameras arerequired to accumulate adequate counts for measure-

TABLE 3APhysicochemical Properties of [99Tc]Aminoethyl-Diaminodithiol Complexes

RComplex'^AhTA

|A«X_yIBf~\iSIIAl>(\

IIBf~\J~\-

IIIAr\_y

"IB/

\ ..( V- |vA^^J^IVB/

\ ' \-k \iA/ \ ».§ r\ v"\_yVB^u-<

5ÃŽPartition

coefficient(logP)'2.78

2.522.21

2.413.22

3.142.38

2.082.40

2.471.82

2.23Apparent5.3

4.46.1

5.45.5

4.74.34.05.1

4.17.1«6.7Percent

serumbinding*96.8

97.493.8

92.897.7

98.399.5

99.092.2

89.291.9

96.8

' Octanol-buffer partition coefficient at pH 7.4.

f Measured by determination of octanol-buffer distribution coefficient at various pH values.* Determined by Ultrafiltration.§Partially ionized at pH 7.4.

80 Scheffel, Goldfarb, Lever et al The Journal of Nuclear Medicine

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TABLE 3BPhysicochemical Properties of [99mTc]Aminohexyl-Diaminodithiol Complexes

RComplexA

A A /N > V" A/WV1^ jvilB•.i/

Vili AAAA/^\ VIIIBr

^ |X^AAA/H

P ixBH

-AAA/N-< XBPartition

coefficient(logP)'2.04

2.031.53

1.502.79

2.841.04

1.04Apparent

plO7.0*

7.5s6.9s

6.9s5.25.36.9s

6.9sPercent

serumbinding*97.9

96.894.7

93.492.2

89.293.0

96.0

' Octanol-buffer partition coefficient at pH 7.4.

f Measured by determination of octanol-buffer distribution coefficient at various pH values.* Determined by ultrafiltration.§Partially ionized at pH 7.4.

ments of brain blood flow, a 99mTcagent with longer

brain residence time and little or no redistribution couldbe highly desirable. As advances in SPECT imagingtechnology continue, this requirement will become lessstringent. In fact, the use of agents with a short residencetime could be utilized for repetitive or perturbationstudies on the same day.

The purpose of our present investigation was (a) tostudy different [99nTc]DADT derivatives as to their

distribution and retention in the brain and (b) to gaininsight into the relationship between structure of thecomplex and its biological function with the goal todevelop a better agent for the measurement of brainblood flow by SPECT imaging.

All complexes studied were lipophilic with octanol-buffer (pH 7.4) coefficients log p > 1.0, thus, accordingto Oldendorf (24), freely diffusible through the blood-brain barrier. However, another criterion has to be metfor a lipophilic agent to diffuse through the blood-brainbarrier: it has to remain unionized at physiological pH(25). Some of the [99mTc]DADTcomplexes in our series

were found to be partially ionized and hence, showedpoor brain uptake (<0.4% dose in mouse brain at 5min after injection). The [99mTc]DADTcomplexes that

demonstrated the lowest brain uptake (<0.2% dose at

5 min postinjection), were the aminohexyl DADT derivatives; all had apparent pKa values of 6.9 or greater.Serum protein binding was essentially the same for all[99mTc]DADTcomplexes tested. Whether the relatively

high binding determined by ultrafiltration was due tospecific protein binding or to adsorption of the lipophilic agents to serum components was not evaluated.A direct relationship between the amount of 99mTc

complex taken up in the brain and serum proteinbinding could not be demonstrated.

Of the complexes tested in mice 99mTc4'-methyl

NEP-DADT showed the slowest clearance from thebrain. SPECT studies in dogs, comparing 99mTc4'-methyl NEP-DADT with [99mTc]NEP-DADTdemonstrated no significant difference between the two 99mTc

complexes (2). In contrast, recent SPECT studies in thesame baboon showed a clear advantage of the methylated complex: brain clearance was prolonged, on anaverage, about two times. Reasons for the interspeciesdifferences are presently under investigation.

NOTES'(Micro-Porasil, 10 n\ 8 mm I.D., Waters Z module car

tridge) Waters Co., Milford, MA.

Volume 29 •Number 1 •January 1988 81

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f(4 ti Nova Pak, 8 mm I.D. Waters Z module) Waters Co.,

Milford, MA.'(Machery-Nagel) Brinkmann Instruments, Westbury, NY.'Packard Instrument Co., Inc., Downers Grove, IL.' Amicon, Danvers, MA."Charles River Laboratories, Wilmington, MA.

ACKNOWLEDGMENTS

This work was supported by E.I. Dupont de Nemours andCo., Inc.—NEN Medical Products Division and by USPHSGrant; CA 32845.

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82 Scheffel, Goldfarb, Lever et al The Journal of Nuclear Medicine

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1988;29:73-82.J Nucl Med.   Ursula Scheffel, Howard W. Goldfarb, Susan Z. Lever, Ramon L. Gungon, H. Donald Burns and Henry N. Wagner, Jr.  Potential Brain Blood Flow Imaging AgentsComparison of Technetium-99m Aminoalkyl Diaminodithiol (DADT) Analogs as

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