A Strategyfor the Study of CerebralAmino Acid Transport Using Iodine-123-Labeled Amino Acid Radiopharmaceutical: 3-Iodo-alpha-methyl-L- tyrosine

Keiichi Kawai, Yasuhisa Fujibayashi, Hideo Saji, Yoshiharu Yonekura, Junji Konishi, Akiko Kubodera, and Akira Yokoyama

Faculty ofPharmaceutical Sciences, Science Universityof Tokyo, Tokyo, Japan and School ofMedicine and Faculty of Pharmaceutical Sciences, Kyolo University, Kyoto, Japan

In the present work, a search for radioiodinated tyrosine We examined the brain accumulation of iodine-i 23-iodo-al derivatives for the cerebral tyrosine transport is attempted. pha-methyl-L-tyrosine (123I-L-AMT)in mice and rats. l-L-AMT In the screening process, in vitro accumulation studies in showed high brain accumulation in mice, and in rats; rat brain rat brain slices, measurement ofbrain uptake index (BUI), uptake index exceeded that of 14C-L-tyrosine.The brain up and in vivo mouse biodistribution are followed by the take index and the brain slice studies indicated the affinity of analysis of metabolites. In a preliminary study, the radio l-L-AMT for earner-mediatedand stereoselective active trans iodinated monoiodotyrosine in its L- and D-form (L-MIT port systems, respectively; both operating across the blood and D-MIT) and its noniodinated counterpart (‘4C-L- brain barrier and cell membranes of the brain. The tissue tyrosine) are tested. homogenate analysis revealed that most of the accumulated radioactivity belonged to intact l-L-AMT, an indication of its The initial part of the work provided the basis for a stability. Thus, 1231-L-AMTappears to be a useful radiophar radioiodinated tyrosine derivative to be used for the meas maceutical for the selective measurement of cerebral amino urement of cerebral tyrosine transport. In particular, the acid transport. data on conformational isomerism and the enzymatic deiodination ofamino acids appeared as important factors J Nucl Med 1991; 32:819-824 to be considered.Thus, methylation of the L-tyrosine is estimated as the most plausible approach for providing the needed stability; and the alpha-methylated derivative, the 3-iodo-alpha-methyl-L-tyrosine (I-L-AMT, Fig. 1) is cho arious mental disorders have been associated with sen. Compatibility ofthis I-L-AMT is comparatively tested alterations in cerebral amino acid metabolism (1 ); in with its parent, L-MIT, molecule and its use as the radio schizophrenic patients, for example, a defect of tyrosine iodinated agent for the selective measurement of cerebral transport has been suggested (2). Although some studies amino acid transport is discussed. on the use of radiolabeled amino acids with cyclotron produced ultra short-lived radionuclides have been at MATERIALSAND METhODS tempted (3), they are limited to PET centers; a radioiodi nated amino acid, providing similar data, would offer Preparation of 125l-L-AMTand 1231-L-AMT wider applicability. !odine-l25-Na! was obtained from Amersham Japan and 1231 The multifunctional role of amino acids, participating Na! was provided by Nihon Medi-Physics, Japan. All other not only in protein synthesis but also as substrate for chemicals used were of reagent grade. Iodine-l25-L-AMT and various metabolic pathway, are well known (4). Our pre ‘23ILAMTwerepreparedby theconventionalchloramine-T method as follows. Chloramine-T (Aldrich) at a concentration of vious work indicated that the election of radioiodinated 2.0x lO_8molin 10MlofO.05M phosphatebuffer(pH6.2)was amino acid derivatives with metabolic selectivity or high added to a mixture of L-AMT (1.0 x l08 mol, Aldrich) and active membrane transport affinity was possible (5,6). carrier-free ‘25!-Na!(7.4-37 MBq) in 35 zl of 0.4 M phosphate buffer (pH 6.2). To prepare 123!-L-AMT,L-AMT (I .0 x l0_6 mol in 25 @tlof 1 N phosphoric acid) and chloramine-T (2.0 x lO_6 Received Oct. 31, 1989; revision accepted Oct. 19, 1990. mol in 20 Mlof 0.4 M phosphate buffer) were added to 500 @zlof For reprintscontact:KelichiKawal, PhD, Facultyof PharmaceuticalSd ences,ScienceUniversityofTokyo,12, lchigayaFunagawara-machi,Shinjuku carrier-free 123!-Na!(74-1 11 MBq) solution adjusted to pH 10. ku, Tokyo, 162, Japan. The resultant solution was allowed to stand for 2 mm, and 20 zl

CerebralAmino Acid Transport •Kawai et al 819 injected with 200 @dofsaline containing 3.7 kBq ofthe radioactive 1231 sample and 3H-H20 via the right common carotid artery. The CH3 rats weredecapitated 15secafter injection,and the radioactivity ofthe right halfofthe brain rostral to the midbrain was measured. HO@ CH2-@-COOH The BUI was calculated by the following equation: tissue 25!or ‘4C'tissue3H @ BUI = . . 12 4 3 x 100. NH2 inj. soln @!or ‘C/inj. soln H In competition studies, the injected solution contained 1 x FiGURE1. Structureof 123l-3-iodo-alpha-methyl-L-tyrosmneiO-@M cold L-tyrosine or L-AMT, a concentration that is about (1@l-L-AMfl. 14 times that of the endogenous tyrosine in normal rat plasma (10). of 10%saturated sodium metabisulfite solution were then added. In Vitro Accumulation Studies in Rat Brain Slices The radioiodinated L-AMT was purified by Sephadex LH-20 In vitro accumulation studies using rat brain slices were per (Pharmacia) column chromatography (10 x 200 mm, eluant, formed as previouslyreported (11). Male Wistar rats weighing ethylacetate:methanol:2 N ammonia = 40:lO:4)(7). The labeling 250—300g were killed by decapitation and the brains were quickly efficiency and radiochemical purity were studied by silica gel dissected. The brain tissue was washed with ice-cold Krebs-Ringer thin-layer chromatography (TLC, MERCK; Art. 5553) using two phosphate buffer (pH 7.4) and then sliced with a conventional solvent systems, i.e., methanol: acetic acid = 100: 1 (Rf value: I- Stadie-Riggs slicer (12). The slices (each weighing 100 ±5 mg) L-AMT, 0.50; I, 0.75) and methanol: 10% ammonium acetate were next placed into a vial containing 1.9 ml of Krebs-Ringer = 10: 1 (Rfvalue: I-L-AMT, 0.55; 1, 0.80). phosphate buffer as the incubation medium and preincubated for As references, we used [U-'4C]-L-tyrosine (NEN; NEC-289E) 10 mm for temperature equilibration. Then, 0.1 ml ofthe buffer as a labeled natural amino acid, ‘251-3-iodo-L-tyrosine(‘251-L-containing a radioactive amino acid was added and incubation MIT) and ‘251-3-iodo-D-tyrosine(‘25I-D-MIT)prepared by the was performed at 37°Cfor 30 mm. At the end of the incubation method mentioned above, and 123I-N-isopropyl-p-iodoamphe period, the medium was removed, and the slices were washed tamine ([‘23111MP,Nihon Medi-Physics). twice in 2 ml of ice-cold buffer. The radioactivity of the slices In Vivo MouseBiodistributionStudiesand Analysisof was then measured by the above-mentioned method. The per Metabolites centage of the dose accumulated per gram of slice was calculated DdY male mice weighing25 g received0.1 ml of radioactive as follows: amino acids (‘251-L-AMT,‘25I-L-MIT,or ‘251-D-MITat 1.6 x Accumulation % dose/g slice i0'@ mol and 11.1 kBq; ‘4C-L-tyrosineat 4.0 x l0@°mol and @@içe1251or 7.4 kBq) via the tail vein and were then killed at various time x 100. intervals. For the measurement of radioactivity, a well-type scm inj. soln 125!or ‘4Cx slice weight tillation counter(Aloka; ARC-300) was used for the 125J..and 123!.. Inhibition studies of the membrane active transport system labeled compounds. For ‘4C-L-tyrosine,100 mg of tissue was were performed using incubation at 4°Cand ouabain inhibition. dissolved in 1 ml of NCS tissue solubilizer (Amersham) and For the ouabain inhibition study, brain slices were preincubated incubated at 50°Cfor 3 hr, followed by the addition of 8 ml of at 37°Cfor 20 mm in medium containing 1 x l0@ M of ouabain toluene scintillator containing DPO and POPOP. The radioactiv before addition of the radioactive sample. In the competitive ity was measured using a liquid scintillation counter (Aloka; inhibition studies, cold L- or D-tyrosine solution at a final con ARC-900). centration of I x l0@ M and the radioactive samples were An aliquotoftissue washomogenizedand the 5%trichloroa simultaneously injected. The final radioactive amino acid con cetic acid-precipitated fraction was trapped on a glass filter (Toyo; centration was 2.7 x ‘M (1.85 kBq/ml, no-carrier-added) GC-50) to allow measurement of the radioactivity incorporated for ‘251-L-AMTand 1.0 x l0@ M (1.85 kBq/ml) for ‘4C-L- in the protein. Furthermore, the tissue supernatant was separated tyrosine. by TLC using the solvents mentioned above to examine the metabolites. RESULTS Measurement of Partition Coefficients Preparationof 125l-L-AMTand 123l-L-AMT The partition coefficients of ‘231-L-AMT,‘4C-L-tyrosine,and No-carrier-added ‘251-L-AMTand ‘23I-L-AMTwith ra [23!JIMP were measured using 2.0 ml ofn-octanol as the organic phaseand 2.0ml ofO.l M phosphatebufferasthe aqueousphase diochemical purities greater than 95% and radiochemical (pH 7.0 for brain tissueand pH 7.4 for plasma).N-octanoland yields of 50%—60%were obtained after purification. the bufferwerepre-mixedtwiceusinga mechanicalmixer for 1 In Vivo MouseBiodistributionStudiesand Analysisof mm at room temperature. Then, 20 @lofradioactive sample were Metabolites added and mixed twice using a mechanical mixer for 1 mm at The brain accumulation of ‘251-L-AMTin mice is shown room temperature. The radioactivity of 200 @tlofeach phase was in Figure 2 compared with ‘251-L-MIT,‘251-D-MIT,and measured after centrifugation. ‘4C-L-tyrosine.Comparison of ‘251-L-MITwith ‘251-D- Measurementof BUI MIT showed that the L-isomer exhibited higher brain BUIstudieswereconductedbasedon the method reportedby accumulation than the D-isomer, indicating that stereo Oldendorf (8,9). Male Wistar rats weighing 250—300g were selectivity for the L-isomer occurred in the brain. The

820 The Journal of Nuclear Medicine •Vol. 32 •No. 5 •May 1991 )O.O 100

J•__@@@@%4 0 @ o/@T @• I ‘@C-L-Tyr @ 1.0 T 50

o@o 125@LMIT@T 125I-L-A@ I j I 0

@‘9—.@------@251-D-MIT 0 I I 6'O 1251.LAMT 11@C-L-tyrosine 0 15 30 Time (mm) FIGURE 4. Fateof1251-L-AMTand“C-L-tyrosineinthemouse FIGURE 2. Brainaccumulationof 125l-L-AMT,1251-L-MIT,1251.. brain10 mmafter injection.Eachbar representsthe mean±s.d. D-MIT, and “C-L-tyrosinein mice. Each point represents the of three animals. Analysis for the amino acid and acid soluble mean±s.d. of four to five animals.(S, 125l-L-AMT;0, 125l-L-MIT; metabolitefractions of 14C-L-tyrosineare from only one animal. 0, 1251-D-MIT;A, “C-L-tyrosine). (Vertically striped bar, amino acid fraction; open bar, protein fraction; obliquely striped bar, acid soluble metabolite fraction; solid bar, free iodinefraction). alpha-methyl derivative, ‘25I-L-AMT,showed higher ac cumulation than ‘25I-L-MIT.Moreover, in the early stage up to 10 mm after injection, ‘251-L-AMTshowed superior 5% of the radioactivity in the brain as L-tyrosine, while uptake to its parent compound, ‘4C-L-tyrosine,although 37.4% and 56.8% was found in the protein precipitate and after that ‘25I-L-AMTlevels rapidly decreased in the brain. the acid soluble metabolites, respectively. In the case of As shown in Figure 3, rapid clearance of ‘25I-L-AMT ‘25I-L-AMT,more than 93% of the radioactivity in the from the blood was observed and the clearance curve was brain was found as free amino acid, and not as protein or found to be similar to that of ‘25I-D-MIT,which was stable as free iodine. In Table 1, the percentages of radioactivity against enzymatic deiodination (6). Redistribution in the in the amino acid and free iodine fractions of ‘25I-L-AMT blood, which was shown with ‘4C-L-tyrosine, was not in mouse tissues are shown compared with ‘25I-L-MIT.In observed. As a result, 30 mm after injection, ‘251-L-AMT the case of ‘251-L-AMT,free iodine was about 1% of the showed lower blood levels than ‘4C-L-tyrosine. radioactivity in the brain and less than 4% in the liver, The chemical forms of the radioactive compounds in kidney, and urine in contrast with 1251-L-MIT. the mouse brain 10 mm after injection are shown in Figure 4. Mice injected with ‘4C-L-tyrosineexhibited less than Partition Coefficients The partition coefficients of ‘231-L-AMT,‘4C-L-tyrosine, and [‘23I]IMPare shown in Table 2. lodination and meth 50.0 ylation ofthe tyrosine molecule increased the lipophilicity to some extent, but the partition coefficient of ‘231-L-AMT was lower than that of [‘231]IMP,which is a well known lipophilic brain-seeking amine (13,14). Little difference of E 10.0 V0 TABLE 1 5.0 In Vivo Stability of lodinated Amino Acids* > 1251-L S i-D-MIT V 1Brain93.02 AMT-AMTFreel1251-L MIT-MIT Free 0 1@@C-L-Tyr

1251LAMT (0.78)Liver55.25(2.65)1.08

0 15 30 60 .88 3.87 Time (mm) (5.39)Kidney (7.05)1 (1.34)1 (2.90)66.25 (1.42) (1.45) (4.55) (6.13) FIGURE 3. Clearanceof 125l-L-AMT,125l-L-MIT,1251-D-MIT, Urine94.93 93.09 3.81 4.79 94.21 and 14C-L-tyrosinefrom the bloodin mice.Eachpoint represents (1.19)* (2.45)2.21(2.00)14.07(1.52)75.07 the mean ±s.d. of four to five animals.Carbon-i4-L-tyrosine was determinedin plasmasample.(•,125I-L-AMT;0, 125l-L-MIT; Ten minutes analysis 0, 1251-D-MIT;A, 14C-L-tyrosine). (MeOH:AcOH = 1after00:1).intravenousinjection,TLC

CerebralAmino Acid Transport •Kawai et al 821 TABLE 2 200 Partition Coefflcients* 7.41@l-L-AMT—0.879pH 7.0pH

(0.004)14C-L-tyrosine—1(0.003)—0.911 .98 100 (0.05)[1@l]lMP0.722(0.02)—2.00

(0.008)* (0.026)0.940 E

log(n-Octanol/0.1 M phosphate buffer).

0 125k-L-AMT 14C-L-tyrosmne the partition coefficients between pH 7.0 and pH 7.4 was observed, in contrast with [‘231]IMP. FIGURE 6. Inhibitionof 125l-L-AMTand 14C-L-tyrosineaccu Brain Uptake Index mulationin rat brainslices.Eachbar representsthe mean±s.d. Iodine-l25-L-AMT showed a high BUI of 62. 1 ±4.3, of five experiments. (Vertically striped bar, samples incubated for in contrast with the 34.2 ±4.7 observed for ‘4C-L-tyrosine 30 mm at 37°C;horizontallystriped bar, samplesincubatedat (Fig. 5). In addition, the brain uptake of ‘25I-L-AMTwas 4°C;open bar, samples loaded with 1 x 10@M ouabain and preincubatedfor 20 mm; obliquely striped bar, L-tyrosine, and inhibited by the simultaneous injection ofcold L-AMT or dotted bar,D-tyrosine,at 1 x 10@M wereaddedsimultaneously). @ cold L-tyrosine, and a similar effect was detected with ‘4C- * = p < 0.005, = p < 0.001. L-tyrosine. In VftroAccumulationStudiesin Rat BrainSlices The results of the accumulation and inhibition studies DISCUSSION of 1251-L-AMTand ‘4C-L-tyrosineare shown in Figure 6. With incubation at 37°Cfor 30 mm, ‘251-L-AMT(158.4 ± Recent advances on PET brain studies have stimulated 9.4%/g) showed a similar accumulation to ‘4C-L-tyrosine the search for radiopharmaceuticals for SPECT studies. (15 1.0 ±l6.6%/g). With incubation at 4°C,the accumu Tyrosine or its derivatives have been radioiodinated or lation of both labeled amino acids was significantly de radiolabeled with positron-emitting radionuclides, but creased.Inhibition of accumulation was also detected in they have been used mostly for studies of protein synthesis the presence of ouabain. In addition, stereoselective inhi or elucidation ofbiochemical pathways in normal or path bition was detected only in the presence ofcold L-tyrosine. ologic state (15—17). These characteristics of ‘251-L-AMTwere shared by ‘4C-L- Studies of cerebral amino acid transport are rare and tyrosine. only basic research has been reported, particularly with tyrosine (8,18). At first, basic experimentation was carried out with the radioiodinated tyrosine, MIT. The availability of radioiodinated MIT in its L- and D-form provided the 80 preliminary basis for this work. In vivo biodistribution studies in mice showed high brain accumulation of the L MIT, but not that of the D-MIT, a good indication of the 60 a) well known stereospecificity of the blood-brain barrier, in V the transport of neutral amino acids (8,18). However, 0) tissue analysis revealed the metabolic unstability of L

a MIT, a metabolic intermediate of thyroid hormones, which is subjected to rapid enzymatic deiodination (19, 1.. 20). Use of a radioiodinated tyrosine derivative for amino 20 acid transport studies requires an enzymatically stable derivative of L-MIT. Methylation of L-MIT was considered since alpha 0 methylation has been reported to bring metabolic stability [email protected]. ‘4C-L-tyrosmne to the tyrosine molecule (21,22). Radioiodination of al pha-methyl substituted L-MIT was carried out and the FIGURE5. Brainuptakeindexof 125l-L-AMTand“C-L-tyrometabolic stability of radiolabeled I-L-AMT needed for its sinedeterminedin rats. Eachbar representsthe mean±s.d. of brain transport measurement was screened. four to six animals. L-AMT and L-tyrosine were loaded at 1 x io@M.(Verticallystripedbar,controlBUI;openbar,L-tyrosine, In the early biodistribution stage, I-L-AMT showed and obliquely striped bar, L-AMT, at 1 x 1O@M were added higher brain accumulation than its non-methylated L-MIT simultaneously).; p < 0.005, @;p < 0.001. and still higher, ifcompared with its parent ‘4C-L-tyrosine;

822 The Journalof NuclearMedicine•Vol. 32 •No. 5 •May 1991 this difference was assessed by the BUI. These results present study will provide an important basis for the indicated that not only methylation but the iodination of evaluation of I-L-AMT accumulation in human brain. the tyrosine molecule induced no interference in the trans port affinity of L-tyrosine into the brain. ACKNOWLEDGMENT The most interesting findings were obtained in the post The authors wish to thank Nihon Medi-Physics Co. Ltd. for injection analysis of tissue metabolites. As expected, the the generous supply of 123!-NaI. presence of the methyl group in the alpha-position con tributed to the stability of I-L-AMT against deiodination. TLC analysis revealed only 1%—2%of free iodine com REFERENCES pared with 65%—75%in the case of L-MIT. On the other 1. Bustany P, Henry iF, Rotrou J, et al. 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(continued from page 802) SELF-STUDY TEST Gastrointestinal Nuclear Medicine A. Thereisevidenceforobstructionoftheafferent(pro ximal) limb of the Billroth II reconstruction B. Onthebasisoftheimagesalone,itcanbecon cluded that enterogastric reflux is responsible for the 4 patient's symptoms. C. A calculatedpeakenterogastricrefluxindexof64% @t@: indicates that the patient's symptoms are likely to be relieved by surgical diversion of bile with a 45-cm @4;t:;.;@., Roux-en-Ylimb. D. Thepatient'ssymptomsarelikelyrelatedto post operative gastroesophageal reflux. E. Theabsenceofbiliousvomitingmakesitunlikely that the patient's symptoms are related to alkaline reflux gastritis.

Figure 2B

I-

ITEM 1: NeonatalJaundice In infants less than 3 months of age, assessing the degree of hepatic ANSWER:D extractionmayaid in differentialdiagnosis.Good extractionwith a high Theinitialangiographicphasewithrapidsequentialimagingisauseful liver-to-heartactivity ratio at 5 minutes is suggestiveof biliary atresia. component of hepatobiliary scintigraphy, in that unsuspected Visually,there is rapid clearance from the blood pool with prominent abnormalities,suchasan arteriovenousmalformationinthe liveror renal hepaticuptake Evaluationofthetime—activitycurveswillconfirmprompt inflammatory lesion, may occasionally be demonstrated. Avascular, uptakeof tracer by the liverwith either no washoutover 60 minutesor photon-deficientareaswithinthelivermaysuggestcyst@dilatedbileducts aslightdecreaseafterreachingpeakactivity.Thisisinkeepingwiththe asinCaroli'sdiseaseoracholedochalcyst.Thereisnospecificalteration relativelygood hepatocytefunction at this stage. Beyondthe age of 3 inhepaticperfusionusefulfordistinguishinghepatocytedysfunctionfrom months,theeffectofthe prolongedobstructionon liverfunctionbecomes biliary atresia, however. evident as hepatic extractiondecreases.If the initial hepatic uptake is The kidneys are the alternate route of excretion of the @Tc-lDApoor,then the patientis more likelyto have neonatalhepatitis(or some derivatives.Renal and bladder activity is prominent with either poor othercauseof hepatocellulardysfunction).Typically,thetime—activity hepaticuptakeand excretion(eg., hepatitis),or good hepaticextraction curve shows an early peak due to vascular activity.The shape of the butnoexcretion(biliaryatresia).Normally,somerenalandbladderactivity curve parallelsthat ofthe cardiac blood pool, becausethere is minimal is expectedbecaus@dependingon the specificiminodiaceticacid activeuptakebytheliver.Withlessseverehepatitistheextractionratio derivative5%-15% ofthe injecteddose will be excretedbythe kidneys. isdecreased.Thecurvesshowdelaysduptakebythe liverwitha washout The degree of renal excretioncannot be used to difterentiate rateequal to or slightlygreaterthan the rate of decrease in blood-pool hepatocellulardiseasefroman obstructiveprocess. activity.lnthe latterinstance,excretionintothe bowelmaybeanticipated Inthenewbornperiod,itisnotusualtoseegooddefinitionoftheintra inthe delayedimages.Thus,an evaluationofthe earlyhepaticextraction hepaticducts,andvisualizationofthe gallbladderisinfrequent.When and washoutduring the first 60 minutesmay suggest the presence of the gallbladderis seen,however,intrahepaticbiliaryatresiacan be biliary atresiaor neonatalhepatitis,provided the patient is less than 3 excluded.Extrahepaticobstruction is stillpossible,but dilatationof the months old. bileductswilloftenbeevidentbyultrasonography.Nonvisualizationof Rfrences thegallbladderisusualwithimpairedhepatocytefunctionandprolonged 1. GerholdJP@KlingensmithWCII, KuniCC.et al. Diagnosisof biliary atresiawith hepatictransitofthe radiopharmaceutical.Thus,nonvisualizationofthe radionuclide hepatobiliary imaging. Radiology 1983;146:499-504. intrahepatic bile ducts and gallbladder may be seen with both hepatitis 2. Majd M, Reba AC. Altman AP Effect of phenobarbital on @mTc-lDA and biliaryatresia,and thesefeaturescannot serveto distinguishthese scintigraphy in the evaluation of neonatal jaundice. Semin Nuci Med disorders. 1981;11:194-204. (continued on p. 829)

824 The Journal of Nuclear Medicine •Vol. 32 •No. 5 •May 1991