Progress in Radiopharmacology

INIS-mf—11544

Proceedings of the Vth International Symposium on Radiopharmacology

PROGRESS IN RADIOPHARMACOLOGY

EDITORS A. E. A. MITTA R. A. CARO C. O. CANELLAS

986

BUENOS AIRES - REPUBLICA ARGENTINA 1987 & ,f

Proceedings of the Vth International Symposium on Radiopharmacology

PROGRESS IN RADIOPHARMACOLOGY

EDITORS A. E. A. MITTA R. A. CARO C. O. CANELLAS

986

BUENOS AIRES - REPUBLICA ARGENTINA 1987 Proceedings of the Vth International Symposium on Radiopharmacology

PROGRESS IN RADIOPHARMACOLOGY

EDITORS

A. E. A. MITTA Comision Nacional de Energia Atomica Buenos Aires, Republics Argentina

R. A. CARO Facultad de Farmacia y Bioquimica Universidad de Buenos Aires, Republics Argentina

C. O. CAIVIELLAS Comision Nacional de Energia Atomica Buenos Aires, Republica Argentina PREFACE

This book contains most of the papers presented at the V International Symposium on Radiopharmacology held at Buenos Aires, Argentina, from the 29th to the 31st October, 1986. The papers were put into the same order as they were presented at the symposium.

The V Simposium was sponsored by the Argentine Atomic Energy Commission, which allowed, among other things, the edition of this book.

I want to acknowledge specially the cordial assistance of Profs.DlSiRicardo.A.Caro and Carlos. O.Canellas in the publication of the present book. PREFACE

The Executive Committee of the V International Symposium on Radiopharmacology acknowledges deeply the participation of all those who presented their paper, discussed the results or simply assisted to the sympos ium.

The proceedings we are publishing herewith are the result of the efforts of the authors who sent us the full papers, as well as the excellent work done by the Printing Department of the Argentine Atomic Energy Commission.

We hope that this publication will steer the efforts towards an increasing knowledge and utilization of radiopharmacology, as a branch of science capable of a peaceful application of radioisotopes for the benefit of Mankind.

The Editors TABLE OF CONTENTS

BIOLOGICAL EVALUATION OF '9mTc cis-PLATINUM IMI NOD IACETIC ACID COMPLEXES AS TUMOR IMAGING AGENT 1 A . Awa1uddin, J.J.Jacobs, D.W.Bourne, D.D.Madda1ena, J.G. Wilson and R . E.Boyd.

COMPUTED FUNCTIONAL ANALYSIS OF ""TC EHIDA KINETICS IN PATIENTS 22 V.Blaha, I.Cihak, F.Nicek and J.Horak

SYNTHESIS AND BIODISTRIBUTION OF SOME RADIOIODINATED DIAMINES 32 A.V.Joshua, J.G.Sandford, J.R.Scott and S.N.Muddukrishna

RADIOPHARMACOLOGY OF IMINOD[ACETIC ACID N-DERIVAT IVES ANALYSIS IN BIOLOGICAL MODEL AND COMPARISON TO HUMAN BEINGS 4 1 C.O.Canellas, M.G.ArgiieLles and A.E.A.Mitta

THE INFLUENCE OF MONOFLUORMETHYLH1STIDINE (MFMH) ON THE MAMMARY ADENOCARCINOMA M-2 58 M.C.P.ubio, M.E.Landa, H.Targovnik, L.L.Colombo, A.J. Scolnik and R.A.Caro

1-131 METAIODOBENZYLGUANIDINE (MIBG.I-I31) KINETICS IN A CARCINOID TUMOR 64 R.Schiavo, G.Concolino, F.Fazi, P . Iannantuono , S.Li.Voti, A.Manzara and P.Pavoni. 9 9m A SIMPLE METHOD FOR THE PREPARATION OF DIFFICULT Tc COMPLEXES USING SURFACE ADSORBED STANNOUS IONS 75 D.J.Madda1ena, G.M.Snowdon, A.Awaluddin and P.M.Pojer

BLOOD CELLS RADIOLABELING ACHIEVEMENTS, CHALLENGES AND PROSPECTS 86 J.Weininger and J.Trumper THE SYNTHESIS AND PROPERTIES OF 1 -(4 -IODO-5-NITROIMIDA ZOLYD-2-HYDROXY-3-METHOXYPROPANE A NOVEL RAD IOSENS IT IZER FOR USE AS AN HYPOX1C CELL MARKER 107 L.I.Wiebe, D.C.Jette, J.R.Mercer, B.Samuel, R.J.Flanagan J.Lee, B.E.Meeker and J . D.Chapmann.

SPECIFIC ACTIVITY DETERMINATION OF 1-125 LABELED PROLACTIN BY RECEPTOR AND RADIOINMUNOLOGICAL SELF-DISPLACEMENT METHODS 129 E.S.Rivera, G.A.Coiuccia, A.Venturino, S.Malkischer and R.A.Caro.

STRUCTURE-ACTIVITY STUDIES ON Tc PHENOLIC AMINOCARB£ XYLLIC ACID HEPATOBILIARY AGENTS 135 D.J.Maddalena, J.G.Wilson and G.M.Snowdon

IN VIVO STABILITY AND INERTNESS OF VARIOUS DIRECT LABELED AND CHELATE-TARGET PROTEIN 157 Gy.A.Janoki, L.Korosi, G.KLievnyi and B.Spett ] 8 h BJODISTRIBUCION Y ESTIMACION DOSIMETR1CA DEL Re HEDP 165 M.G.Noto y A.Manzini

METODOLOGIA PARA LA EVALUACION DE FARMACOS DECONTAMINANTES EN SISTEMAS "IN VITRO" 170 C.Fernandez Degiorgi, D.Dubner y I.Gomez Parada

PREPARACION Y CONTROL DE CALIDAD DE COMPUESTOS DE i86Re 184 M.G.Noto, R.A.Amor, D.A.Cavig1ia, M.T.Ratner, A.M.Schroder, J.C.Rocco y A.Manzini

DIISOPROPIL-IDA, MEBROFENIN E IODOFENIN MARCADOS CON n Q Tc "SU EVALUACION EN VARIOS MODELOS BIOLOGICOS" 188 M.G.Argue 1les, A.S.Leon, E.S.Verdera, E.Leon, C.O.Caftellas y A.E.A.Mitta QQ PREPARACION Y CONTROL DE CALIDAD DE FIBRINOGENO Tc 202 M.G.Noto, G.Rabiller, C.Garrie Faget, C.Fisman y A.Manzini LIST OF PARTICIPANTS 2O7 BIOLOGICAL EVALUATION 0^ qpMTc cis-PLATINl!M IMINOFJIACETIC ACID AGENTS. A.AWALUDDIN*, J.J. JACOBS**, ^.'/.B O.J.MADDALENA***^ .I.fi.WILSON*** AND R.E.BOYD * ISOTOPE HEPT^ UNIT TENAPA "IUKLEAR^ COMPLEX PUSPATI, BANGI, MALAYSIA. ** PHARMACY DEPT,, UNIVERSITY OF QUEENSLAND^ ST. LUCIA^ OLD^ AUSTRALIA, *** ISOTOPE HIVISION^ AUSTRALIAN ATOMIC F.NERRY COMMISSION^ LUCAS HEIRHTS RESEARCH LABORATORIES^ PRIVATE ^AIL BAG NRO I, 'IENAI, »!S'.^ ?23'I

^EPUBLICA ARGENTINA BIOLOGICAL EVALUATION OP 99mTc cis-PLATINUM IMINODIACETIC ACID COMPLEXES AS TUMOUR IMAGING AGENTS

A. Awaluddin1, J.J. Jacobs2, D.W. Bourne2, D.J. Maddalena3,

J.G. Wilson3 and R.E. Boyd3

Isotope Dept,- Unit Tenaga Nuklear, Komplex Puspati, Bangi,

Malaysia

2 Pharmacy Dept, University of Queensland, St Lucia, Qld,

Australia

Isotope Division, Australian Atomic Energy Commission, Lucas Heights Research Laboratories, Private Mail Bag No. 1, Menai, NSW, 2234, Australia.

ABSTRACT The biodistributions of three new "mTc labelled cis-platinum bifunctional tumour imaging agents were examined in mice bearing the EMT6 sarcoma between 15 minutes and 24 hours post injection. The three complexes were excreted primarily via the renal pathway into the urine but at quite different rates. All complexes had some affinity for the tumour, but complex TTI had the greatest, with tumour to blood and tumour to muscle ratios at 24 hours in excess of 10:1 and 18:1.

Keywords Tumour Imaging agents; 99raTc; EMT6 sarcoma mice; cisplatin; cis-diammineplichloroplatinuin (II); iminodiacetic acid — 3 —

INTRODUCTION

In recent years, considerable attention has been focused on the

development of radiopharmaceuticals for diagnostic scintigraphic

imaging of tumours. In a survey of 12 specialist journals issued between 1976 and 1981,.Weibe (1983) found more than 160 papers describing studies in animals on potential tumour imaging agents utilising a wide variety of radionuclides. Many more have been published since, but very few have been shown to be clinically useful.

Gallium-67 citrate is considered the agent of choice, but its use in diagnosis and the staging of tumour types has proved unsatis- factory since not all tumour sites or types are detectable. Other limitations are that it only reliably detects tumours greater than 1 cm in diameter; it localises in a variety of non- cancerous inflammatory lesions and scintigraphic imaging must be carried out after 24 to 48 hours due to high body backgrounds. Gallium-67 citrate also has poor physical properties which make it ill-suited to scintigraphic imaging with a gamma camera (Lentle, 1986). Consequently, there is a great need for new and better tumour imaging agents preferably based on

Technetium, or some other radionuclide with near optimal physical characteristics for gamma imaging.

One approach is to label drugs such as cancer chemotherapy drugs, which localise in certain tumours, with radionuclides and hope that the resultant complex does not lose its tumour specificity. -4-

Bleomycin labelled with Co localises well in a number of tumour

types and in some comparisons has been shown to be more sensitive

than fa Gallium citrate (Poulose et al., 1974; Grove et al.,

1974), but owing to its long physical half-life and poor imaging

characteristics it has not gained widespread use. Attempts at

replacing the b7Co with 111In or "mTc have not been success-

ful (Lentle, 1986).

Even before its clinical studies had been completed (DeConti

et al. , 1973), Lange et al. (1972) had examined the possibility

of using cisplatin, the first of a widely used group of platinum

coordination complexes with anti-tumour activity, as a tumour

imaging agent, by replacing some of the platinum in the complex

with the radionuclide, 193mPt.

In subsequent studies, cisplatin has been labelled with othar platinum radionuclides, including Pt (Wolf et al., 1973; Smith et al., 1974), i9xPt (Baer et al., 1985) and its nitrogen has been replaced with X"N (Haber et al., 1985). Although these compounds localise in some tumours, the physical properties of the radionuclides are less than ideal and complex syntheses with radioactive materials are required in their preparation.

A less obvious but more useful approach would be to synthesise a bifunctional chelate where the cisplatin acting as a tumour binding moiety was joined to a ligand capable of forming a stable complex with a variety of radionuclides. -5-

This paper describes the initial biological studies on a series

of three new organo-platinum complexes (figure 1) containing the

cis-dichloroplatinvtm moiety for tumour binding with benzylimino-

diacetic acids as tha chelating groups for Tc.

MATERIALS AND METHODS

Formulation of the Complexes The syntheses of organo-platinum compounds I, II and III have been described elsewhere (Awaluddin et al. , 1987). The "mTc complexes of the three compounds were prepared using a sodium borohydride reduction method. Five milligrams of compound I, II or III was dissolved in isotonic saline (1.0 mL) and sodium hydroxide (0.1 mL, 1.0 M) in a 10 mL vial. The pH was adjusted to 7 with 1.0 M HC1, generator-derived sodium pertechnetate (20 MBq, 0.1 mL) was added and the resultant solution diluted to 3.0 mL with isotonic saline. Sodium borohydride solution (0.1 mL, 2.8 mg/mL) was added dropwise and the solutions incubated for ten minutes then passed through a 0.22 \im membrane filter into a fresh sterile vial for use. Radiochemical purity was examined chromatographically using Gelman ITLC-SG ministrips developed with acetone-water (19:1).

Animal Studies

The tumour specificity of the Tc labelled complexes was evaluated by quantitative biodistribution studies at time intervals between 15 min and 24 h in groups of 3-4 specific pathogen free balb/c mice (18-23 g), bearing solid EMT6 sarcomas

(0.2-0.8 g) implanted in the flanks. The animals were injected -6-

intravenously via a tail vein with 100 \xL of the labelled complex, then placed unrestrained in a metabolic cage for the collection of urine and faeces. At the end of the prescribed time interval the animals were sacrificed by overdose of ether for tissue samples. Biodistribution results were calculated using the TISCON program (Maddalena, 1983).

RESULTS AND DISCUSSION The major route of blood clearance of the three Tc

labelled compounds in mice was via the renal pathway into the

urine. However, their rates of clearance were different

(figure 2). Blood clearance was fastest for the complex of

compound I (complex I), with 8% injected dose in the blood at

15 min post injection dropping to less than 1% at 24 h. Complex

III, had the slowest rate with 14% injected dose in the blood after 30 min post injection, dropping to 1% at 24 h, whereas the clearance of complex II was mid way between the other two.

Initially, the pertechnetate control cleared more slowly than complex III from the blood, but after 24 h less than 1% was left.

Cisplatin has a two-component plasma half-life curve in rats, the first (alpha) component is 5.9 - 16.1 min and the second (beta) component is .15.6 h - 44.9 days, depending on the source of the data (Litterst et al., 1979).

Although not measured in the present study, the alpha components of the complexes, would probably have been similar to the rat results for cisplatin since only 14% was left at 30 min (after -7-

three, ten minute half-lives, 12.5% would be left) for the

slowest of the three complexes; on the other hand the beta

components for the three complexes; used in this study would probably have been much faster (hours) than for the rats (days), suggesting that these complexes behave differently to cisplatin.

Urinary accumulation was fastest for complex I with 53% found in the urine at 15 min post-injection and 86% after 24 h (figure 3), whereas with complexes II and III, which clear more slowly, only

50 and 51% was found in the urine at 5 and 24 h respectively.

The pertechnetate control accumulated in the urine at a rate mid way between that of complexes II and III.

A rapid urinary excretion of the 99mTc complex of compound I, which is basically a phenyl-substituted hydroxybenzyliminodiacetic acid (HBIDA), is not surprising since a variety of Tc labelled phenyl-substituted HBIDA complexes have been found to be good urinary excretors (Maddalena et al., 1987). Lange et al., (1972) have reported that cisplatin excretes at a rate similar to that of complex II in mice with 79% being found in the urine 24 h after intraperitoneal administration.

The activities of the three complexes found in the kidneys at the earliest time intervals varied from 5.5% injected dose for complex II to 1.9% for complex III, but by 24 h post injection the activity found in all of the kidneys had decreased to 1% or less (figure 4). In contrast, the activity of the pertechnetate control found in the kidneys was about 1% for the early time intervals decreasing to 0.3% at 24 h. Boven et al. (1985), found -8-

higher amounts of cisplatin (1.9%) in the kidneys of nude mice after 24 h, whereas Manaka and Wolf (1981) found 2.9% in rat kidneys after the same period post injection.

The maximum amounts found in the liver for complexes I (6.3%) and

III (7.3%), for the early time intervals (< 2 h), were a little

less than that found for the pertechnetate control (8.3%) at

2 h (figure 5). By 4 h, however, the pertechnetate activity

(2.0%) had cleared to approximately half the amount of complexes

I (4.5%) and III (3.7%), and by 24 h the difference was even greater. The amount of cisplaten found in mouse liver at 24 h

(5.1%), by Boven et al. (1985) was approximately twice the amount for complexes I (2.8%) and III (1.7%) found in the liver at 24 h.

The biodistribution of complex II in the liver was different from those of complexes I and III. The activity stayed relatively high and constant (16-28%) for the whole 24 h period, suggesting a partial in vivo breakdown of the complex with colloid formation.

The hepatobiliary excretion of the complexes, as judged by the activities found in the gastrointestinal tract, was relatively low at all time intervals. Complex III, the slowest urinary excretor, had the greatest hepatobiliary output with 22% in the gastrointestinal tract after 4 h, whilst a maximum of 10.3% was found after 4 h with Complex I, the fastest urinary excretor.

The hepatobiliary output of Complex II was the lowest of the three Complexes with a maximum of 7.3% found after 5 h. -9-

The activities of complexes I and II in the stomachs of the mice were relatively high (5%) for periods up to 2 h, suggesting partial breakdown in vivo of these two complexes whereas the high concentration of complex III in the stomach at 2 h (18%) suggests a massive in vivo breakdown. The markedly different biodistributions of the three complexes and the pertechnetate control suggests, however that the complexes did not break down immediately after injection but remained intact long enough to get to the target tissue.

All three complexes concentrated in the tumours, with an peak uptake between 1 and 2 h post injection, however, complex III had a relative affinity for the tumour approximately three times greater than the other two complexes (figure 6). Complexes I and III had tumour-to-blood ratios in excess of 2.5 as early as 15 min post injection, rising to greater than 8 after 24 h, suggesting that these complexes were actively binding within the tumours (figure 7). These high tumour-to-blood ratios and high tumour-to-muscle ratios (figure 8) indicate that the complexes should have excellent imaging properties.

Manaka and Wolf (1981), found that [19bmPt] cisplatin- bleomycin and [1 b PtJ cisplatin-adriamycin complexes had tumour to blood ratios in mice bearing Ehrlich's acites tumour of

3.7 and 4.3 compared to 0.9 for [i95 j cisplatin. From their results, they suggested that improvements in localisation of cisplatin in tumours might best be brought about by tropophore complexes made up of two parts - a pharmacophore or transport part and a ponophore or tumour binding part. The results of the - 10- present study, in which the benzyliminodiacetic acid moiety might be considered the phanxiacophore and the cis-platinum moiety the ponophore supports this hypothesis.

CONCLUSIONS

The biodistributions of three TC labelled platinum bifunctional chelates and a [ '""Tc] pertechnetate control have been examined it*;, an EMT6 sarcoma implanted in balb/c mice. The complexes had different biodistributions to the pertechnetate control and all three concentrated more in the tumour than in the blood or muscle, suggesting that they may be useful as tumour imaging agents.

REFERENCES Awaluddin, A.B., Jacobs, J.J., Bourne, D.W.A., Maddalena, D.J.,

Wilson, J.G. and Boyd, R.E. (1987), Synthesis and characterisation of potential tumour scintigraphic agents. Int. J. Nucl.

Appl. Instr. Part A (in press).

Baer, J., Harrison, R., McAuliffe, C.A., Zaki, A., Sharma, H.L. and Smith, A.G. (1985), Microscale syntheses of anti-tumour platinum compounds labelled with i91Pt. Int. J. Appl. Radiat.

Isot. , 36., 181.

Boven, E., Nauta, M.M., Schluper, H.M.M., Elferink, P., Van Der Vijgh, W.J.F. and Pinedo, H.M. (1985), Secondary screening of platinum compounds in human ovarian cancer xenografts in nude mice. Eur. J. Cancer Clin. Oncol., 21, 1253-60. Ueconti, R.C., Toftness, B.R., Lange, R.C. and Creasey, W.A. (1973), Clinical and pharmacological studies with cis- -diamminedichloroplatinum (II). Cancer Res., 33, 1310-15.

Grove, R.B., Reba, R.C. Eckelman, W.C. and Goodyear, M. (1974),

Clinical evaluation of radiolabelled bleomycin (Bleo) for tumor detection, J. Nucl. Med., 15, 386-90.

Haber, M.T., Cooper, A.J.L., Rosenspire, K.C., Ginos, J.Z. and

Rottenberg, D.A. (1985), Synthesis of l3N cisplatin. J. Label.

Cpds_._ R?_di_opha£m. , 2^2, 509-16.

Lange, R.C., Spencer, R.C. and Harder, H.C. (1972), Synthesis and distribution of a radiolabeled antitumor agent: cis- diamminedichloroplatinum (II). J. Nucl. Med., 13, 328-30.

Lentle, B.C. (1986), Tumor detection with radiolabeled agents, iri Current Applications in Radiopharmacology, Ed. M.W.

Billinghurst, Pub. Pergamon, Sydney, pp.48-61.

Litterst, C.L., LeRoy, A.F. and Guarino, A.M. (1979), Disposition and distribution of platinum following parenteral administration of cis-dichlorodiammineplatinum (II) to animals. Cancer Treat Rep. ,

Maddalena, D.J. (1983), TISCON, a BASIC computer program for the calculation of the biodistribution of radionuclide labeled drugs in rats and mice. AAEC/E572. -12-

Maddalena, D.J. Snowdon, G.M. and Wilson, J.G. (1987), Structure-distribution studies on some potential hepatobiliary agents: 99rnTc-o-Hydroxybenzyliminodiacetic Acid Complexes. Int. J. Radiat. Appl. Instrum. Part B (in press).

Manaka, R.C. and Wolf, W. (1981), Cisplatinum compounds and analogues. j_ri RADIOPHARMACEUTICALS Structure-Activity Relation- ships, Ed. R.P. Spencer, Pub. Grune and Stratton, Sydney, pp.183-1'33.

Poulose, K.P., Watkins, A.E., Reba, R.C., Eckelman, W.C. and

Goodyear, M. (1975), Cobalt labelled bleomycin - a new radiopharmaceutical for tumor localisation. A comparative clinical evaluation with gallium citrate. J. Nucl. Med., 16, 839-41.

Smith, P.H.S. and Taylor, D.M. (1974), Distribution and retention of the antitumor agent Pt-cis-dichlorodiammine platinum

(II) in man. J. Nucl. Med., 15, 349-51.

Weibe, L.I. (1983), Small animal oncological models for screening diagnostic radiotracers. iri Animal Models in Radiotracer

Design. Ed. R.M. Lambrecht and W.C. Eckelman, W.C., pp.107-47.

Wolf, W., Manaka, R.C. and Ingalls, R.B. (1973), Radiopharma- ceuticals in clinical pharmacology: 195 Pt-cis-diammine dichloroplatinum (II). _in New Developments in Radiopharmaceuti- cals and Labelled Compounds, Vienna, IAEA, pp.205-20. - 1 3-

Captions of Figures

Figure 1 Structure of Platinum Compounds I, II and HI.

Figure 2 Blood Clearance of WmTc Platinum Complexes in Mice, (vertical bars represent standard deviations)

Figure 3 Urinary Clearance of 99mTc Platinum Complexes in Mice, (vertical bars represent standard deviations)

Figure 4 Kidney Uptake of 99mTc Platinum Complexes in Mice, (vertical bars represent standard deviations)

Figure 5 Liver Uptake of 99mTc Platinum Complexes in Mice, (vertical bars represent standard deviations)

Figure 6 Tumour Concentrations of 99inTc Platinum Complexes in Mice, (vertical bars represent standard deviations)

Figure 7 Tumour to Blood Ratios of 99mTc Platinum Complexes in Mice, (vertical bars represent standard deviations)

Figure 8 Tumour to Muscle Ratios of 99mTc Platinum Complexes in Mice, (vertical bars represent standard deviations) - 14-

9 J v I.R1OH R2-.CH2N(CH2COOH)2 CH2 -CH -C -NH

V *Pr* III.R1:CH2N(CH2COOH)2 R2=H Cl Cl

figure I 20 COMPOUNDS I © IU III

TcO4" o 15 UJ in o a o 10

figure 2 100 • COMPOUND I A CDMPOUND II • COMPOUND III 80 °TcO4- O Q 260

3 4 24 TIME (HR)

figure 3 8 COMPOUND KEY {• II A III- O" o

I/) o a

TIME (HR)

figure 4 COMPOUNDS I • If A

30 1/1 o Q

Q LLJ 20 -z.

3 4 24 TIME(HR)

figure 5 COMPOUNDS i • ii * in TCO4- o

2 3 4 TIME(HR)

figure 6 COMPOUNDS •i An •m 10 0TCO4- o

< o o o _j C I CQ a 5 o

0 3 4 TIME{HR) figure 7 TUMOR / MUSCLE RATIO in o o o

TO C

r«o

-1 ( - COMPUTED RF-ICTIONAL ANALYSIS OF 9QMTC EMin/\ KINETICS H PATIENTS

V. BLAHA, I, ClHAK, F. 'llCEK AND !. lloRAK

HEP. OF "IUCLEAR PEDICINE^ FACULTY OP -IEDICINE

AND HYRIENEJ, CHARLFS "NIVF.R^ITY^

CZECHOSLAVAKIA.

PUENOS ^IRES

REPUBLICA ARGENTINA 1987 23

COMPUTED FUNCTIONAL ANALYSIS OP "mTo EHIDA KINETICS IN PATIENTS

V. Blaha, I. Sihak, F. Nicek, J. Horat

Bep. of Nuol. Medioine, Faculty of Medioine and Hygiene, Charles University, Prague, Czechoslovakia

The differential diagnosis of the hepatobiliary disorders often oalls for a detailed funotional analysis of the kinetics of labelled compounds in this system, including the kinetics in the liver parenchyma. The prolongation of the transport by the hepatic parenchyma leads to delayed entrance of these substances into biliary ways. However, besides this, it also decreases the concentration of labelled compound in the bile with very negative oon- sequenoes for the interpretation of the study. Thus, our method of analysing the EHIDA (dietyl-imino-aoetanilido- diaoetio acid) kinetics is aimed particularly at the kinetics in the hepatic parenchyma*

Material and methods A group of 367 patients with different hepatobiliary or other gastrointestinal diseases was examined and eaoh study was quantified either in whole extent or at least partially. The cholesointigraphy is made with several small modifications of the commonly known methods: a) the patients were in a fasting state, buJ the biliary ways were routinelly evacuated by ingesting of 50 g oho- oolade 3 hours before the examination; -24-

b) the equipment used was LPOV Searle gamma oamera with the CUTE 250 Interteohnique oomputer; o) the administered aotivity in adults was oca 100 MBq, in icteric patients up to 500 MBq; d) the sointigrams, five minutes each, were taken on X ray film and 15 s data were stored into the memmory of the computer. The time of baslo study was 40 up to 60 min. One 5 min delayed sointigram was taken two hours after the beginning of the examination, additional delayed photos were taken if necessary; e) the following ROIs were selected: the heart with some part of lungs (not closely to the liver !), right and possible also left liver lobes (beyond the region of large biliary ways), oommon hepatio duot - whenever possible, common bile duot, gallbladder and duodenum. A simplified model of the SHIDA kinetios has been considered (fig. 1). The principle of the analysis is in determining the hepatio parenchyma kinetics. The hepatio ourre is analysed by a oomputer programme aooording to a two oompartement model (fig. 2). Both prinoiple liver femotions, the uptake and the exoretion of the tracer were determined and they were expressed as the half times - T. and Tax aooording to the equations 1, 2 (Pig. 3)*

A _ u J^** 4. k ak4«* (1)

ex -2 5-

With the help of the oomputer programme the overlap- ping liver activity was subtracted from the curves of the gallbladder (Pig. 4) and also from other biliary ways, The blood aotivity baokground was subtracted from the ourve of duodenum (Pig. 5)* Prom the liver curves the blood aotivity was not subtracted. In our study of 27 patients the liver ourve parameters either with and without blood background aotivity subtraction did not proofed significant differences. In posthepatic organs, the times of the first aotivity appearance in the gallbladder, oommon hepatic duct, common bile duot and duodenum were determined as well as the time of the maximum aotivity in both biliary duots. The rate of the initial aooumulation of the aotivity in the gallbladder is expressed by a linear constant (k). Besides these quantitative parameters, of course, each study was submitted to a visual analysis on the X ray film and particularly to repeated replay of the whole study on the oomputer soreen. In this way, it is often possible to observe changes, which are not observed on individual sointigrams, as, transitory spasm of the sphinter of Oddi, duo- d»nogastrioal reflux£ antiperistaltio motions of the duodenum etc.

Results We had not possibility to examine the control group. Thus, it was neoessary to derive normal values of the EHIDA kinetios from the patients, where a complex of oli- nical and laboratory examinations demonstrated no patholo- gical changes of the hepatobiliary system and where similar •26- disordere were also absent in the anamnesis* Tbe "normal" values demonstrates Fig. 6. The results obtained in the whole group of patients were submitted to a statistioal evaluation to obtain oer- tain general conclusions.

Conclusions Liver parenchyma kinetios: a) There is nigh statistioal correlation between the parameters in the right and left liver lobes; b) all the time parameters (3?4_ , T__ , Tm._) of the right lobe are mostly a little shorter; o) the statistioal correlation between -

the uptake (T*-) and exoretion (Tex) is absent, the uptake (Tj^J and liver curve peak time ( does exist (ooef. of oorr. » 21),

the exoretion (Tex) and liver ourve peak time is very high (ooef. of oorr. s 29) - it means, in prao-

tioe, that the prolongation of the Imax equals to chole- stasisj d) the subtraction of blood activity from the liver ourves does not significantly ohange none of the liver parameters.

Posthepatio kinetios: a) The activity appears in the gallbladder twioe more as the first comparing to the common bile duot; b) the statistioal correlation between - the first appearanoe of the posthepatio activity and the liver uptake (T^,) does exist, but it is low (ooef. of oorr. « 9); -2 7- the first app«aranoe of the posthepatlo activity and

the liver ourve peak time (Tmax) does exist (ooef• of oorr. c 18); the first appearanoe of the posthepatlo activity and

the liver excretion (?ex) is high (ooef. of oorr. = 26). Gallbladder filling:

a) Both parameters of the filling of the gallbladder ^Tin gallbl • ^ correlate with the liver uptake C^jjj)* ooef. of corr. = 9, reap. 6, but do not oorrelate with the liver exoretlon (1L,,); b) there is the statlstioal correlation between the first appearanoe of the activity in the gallbladder and the const, of the gallbladder filling, ooef. of oorr. = 10; o) the statistical correlation between both gallbladder k and ne filling parameters (T^ ga2ibi • ) * filling of the common bile duot does not exist. -.18-

Computed functional analysis of ^mTo EHIDA kinetics in patiente V. Blaha, I. Sihak, F, M5ek, J. Horak

List of figures: 1. The simplified model of the "mTo EHEXA kinetics in man 2. The model of the liver ourve analysis 3« Analysis of the liver curves by a computer programme 4. The subtraction of the liver parenchyma aotivity from the gallbladder curve 5. The subtraction of blood background aotivity from the ourve of duodenum 6. The "normal" values of the ^mTo EHIDA kinetios in man -29-

Intravascular space - disappearance constant **" tl2'

dilution clearance

extravascular hepatocytes space kidney Liver Tin Tmax Tex > dilution | _! *_ intracellular ' 1 Urinary space , Intrahepatic bile ducts ways

,,k in" r Extrahepatic bile ducts Gallbladder Tin 4 Choledochus T in ,. k ex "•

Duodenum Tin

figure 1

A3- recorded curve

A, - liver uptake (Tjn)

Aj- liver excretion (Tex)

60 . min)

f i. g u r e 2 -30-

DX :T(IM)« 3.7HIN T(EX)* 12.9HIH S!H: 4.6 13.7

f i gura 3

FUNKTIO HOIK.

AREA VES. FEL. /*

. DX.

f igura 4 -3 I-

DUOD. •

figure 5

imp. Norma (?nrox.) :

Tin 3 min

Tex 15 min

(Tmax 7-8 min)

Tcholed 8 m'n

Tvcs. fei. 8 min

> Kves. fe|. •••• 5

Tduod •• 8-2°

0 5 10 15 20 25 30 cas (min.)

figure 6 SYNTHESIS AND BIODISTRIBUTION OF SOME RADIOIODINATEn DIAMINES

A.V,JOSHUA, .I.G.SANDFORD, J.1?. SCOTT AND S.N.^UDDUKRISHNA

EDMONTON ^ADIOPHARMACE'JTICAL CENTRE, EDMONTON, ALBERTA, CANADA

BUENOS AIRES REPUBLICA ARGENTINA Synthesis and BiodistrLbution of Some Radioiodinated Diamines

A.V. Joshua, J.G. Sandford, J.R. Scott, S.N. Muddukrishna Edmonton Radiophurmaceutical Centre Edmonton, Alberta, Canada

Several years ago, Kung and coworkers showed the radioiodinated diamine, HIPDM, to be of value as a brain blood flow agent (1). While the emphasis has since shifted to potential agents labeled with Tc-99m, it was felt that investigation of some side chain analogues of HIPDM may help elucidate some of the factors necessary for the uptake and retention of radiolabeled compounds by the brain.

HIPDM and the analogues described in this study were prepared by the general method reported in 1983 by Tramposch and associates (3) (Figure 1).

The iodoaldehyde was condensed with the diamines in reflux ing benzene to form the imines which were reduced with sodium borohydride in ethanol. The products were purified by conversion to the dihydrochlorides and crystallization from methanol-acetone. The N-alkyl derivatives were then prepared by reductive alkylation with the appropriate aldehyde or ketone (formaldehyde for methyl and acetone for isopropyl) in methanol. and purified as the dihydrochlorides.

Compounds that were evaluated included HIPDM and its N'- d e me t h y I derivative, both of which were reported by Kung's group,and the N'-isopropyl derivative. In addition, compounds in which the dimethylaminopropy 1 side chain was replaced with the dimethyl aminoethyl, the piperidinoethyl, the rnorpholinopropyl, morphol inoethyl and the pyrrolodino- ethyl groups were investigated (Figure 2).

Radio labeling of all these derivatives by exchange proved to be relatively straight forward. The compound of interest was heated with 1—12 5 sodium iodide in the presence of acetic acid in a boiling water hath for '30 minutes followed by neutralization with sodium hydroxide. Radiochemical purity was always above 95% without further purification.

For the animal studies, the labeled compounds were diluted to 200 KBq (0.07 mg) per ml, and 0.1 mL was injected via the tail vein. At 5, 30 and 60 minutes after injection the animals were killed by C0_ asphyxiation and the uptake of radioiodine was determined in various tissues.

All the compounds investigated showed significant initial brain uptake in mice. The N'-demethyl derivative (Cmp II) had somewhat lower brain uptake but the retention was relatively good. The N'-isopropyl derivative (Cmp III) had -34-

iniLial upLake almost equivalent. Lo HIP DM but the retention was considerably poorer. Similarly, when the side chain is shortened by one carbon (Cmp IV), there is high initial brain activity but this falls very rapidly. The heterocyclic derivatives (Cmps V-VIII), had somewhat variable initial uptake but the retention of all these compounds tended to be poor (Table I).

Table _I

Brain Uptake in ICR Mice*

Percent Dose per Organ

Compound 5 Min . 30 Min. 60 Min.

1 6.70 6.44 5.30 (5.41-7.64) (5.90-6.96) (4.45-6.92) I I 4.17 4.52 3.93 (3.88-4.46) (3.74-5.64) (3.63-4.40) III 5.69 3.23 2.43 (5. 58-5.88) (2.95-3.53) (2.28-2.53) IV 5.35 2.47 0.89 (3.77-6.19) (2.38-2.57) (0.77-1.03) V 3.78 1 .75 0.77 (3.16-4.24) (1.70-1.78) (0.77-0.86) VI 5.94 3. 19 1.40 (5.47-6.24) (2. 57-3.65) (1 .22-1.55) VI 1 2.42 0.44 0.23 (1 .96-2.95) (0.31-0.57) (0.17-0.29) VI I I 3.72 3.40 ] .90 (2.09-4.93) (2. 51-4.86) (1.60-2.49)

*Mean of 3 Animals (Range)

These results indicate that the particular side chain seen in HIPDM is important for brain retention of the compound.

Yamamoto and associates have reported that HIPDM localizes in the pancreas of mice and rats (3) and more recently this group has reported initial clinical pancreatic studies with th1s compound (4). Several of the compounds we are reporting on here also have pancreatic uptake. In particular, compounds VI and VIII, the morpholinopropyl and pyrrolidinoethyl derivatives, had considerably higher pancreatic uptake in mice at 60 minutes as compared to HIPDM (Table II). -35-

Table II Pancreas Uptake in ICR Mice*

Percent Dose per Organ Compound 60 Minutes

I 2.66 (2.61-2.74) VI 6.17 (5.35-6.86)

VIII 3.44 (2.97-4.17) * Mean of Three Animals (Range)

These compounds were further evaluated in Sprague-Dawley rats at 60 minutes and 24 hours after injection. As can be seen HIPDM appears to have the best retention and pancreas to liver ratio at 24 hours. There was loss of most of the activity of Compound VIII from the pancreas by 24 hours (Table III).

Table III Pancreas Uptake in Rats

6^' 0V^ 4Minute Jt U. WA 1-1 V- V st-J MII 2•>*4 "^ Hour11 W V*. 1.s I*J # Compound ^Dose/Pancreas* P/L ^Dose/Pancreas* P/L 2.18 2.7 3.65 13.0 I (1.95-2.30) (3.31-4.21) 4.01 2.9 2.62 7.9 VI (2.92-4.64) (2.52-2.70) 3.89 1.6 0.07 0.2 VIII (3.18-4.41) (0.07-0.07)

| Mean of Three Animals (Range) % Dose / Gram Pancreas / %Dose / Gram Liver

HIPDM and Compound VI were labeled with 1-123 and images were obtained in rabbits at various times up to 24 hours after injection. With HIPDM the 24 hour images still showed considerable liver uptake and it was not possible to differentiate pancreas from liver activity, In addition there was kidney and intestinal activity (Figure 3). The images with Compound VI were considerably different than were expected from the studies in rats. By 24 hours, in rabbits, virtually, all the activity had cleared from the -3b-

liver. There still remained considerable activity in the intestine (Figure 4). By using background subtraction, it can be seen that the highest concentration of activity was in the pancreas (Figure 5).

In conclusion, the side chain configuration of HIPDM showed the best brain uptake and, especially, retention of those compounds investigated. There is an indication that other related compounds may be superior to HIPDM as potential pancreas imaging agents. The evaluation of these compounds may be complicated by differences in biodistribution among species. These differences appear to be related primarily to the rates of hepatobiliary clearance and intestinal transit. However, it appears that these or similar compounds may have potential value especially if they should prove to be indicators of pancreatic function.

References

1) Kung, H.F., Tramposch, K.M., Blua, M., J. Nucl. Med. 24, 66-72, 1983

2) Tramposch, K.M., Kung, H.F., Blau, M., J. Med. Chem. 26, 12 1, 1983

3) Yamamoto, K., Som, P., Srivastava, S.C., et. al., J. Nucl. Med. 26, 765-769, 1985

4) Yamamoto, K., Kuge, Y . , Saji, H., et:. al., J. Nucl. Med. 27(6), 1013, 1986 (Abstr.) FIGURE 1

CH CH=N R CH, CHO NH2-R 3\/V - "> //

1) NaBH4/ EtOH 2) HCl/EtOH

OHCHO, CH?-N-R ru CH -NH-R *• i CH, NaBH3CN,MeOH 3^ 2HC1 2HC1 2) HCl/EtOH -38-

FIGURE 2.

OH CH , 3 -N R-

R1 i i

CMPD B: -(CH 1 -N(CH ) I -CH3 2 3 3 2 D -H m -CH(CH IV -CH

-CH

VI -CH, O vn -CH- O

-CH -(C1 H ) -H vm 2 2 -39-

Figure 3

Rabbit Image 2k Hours After Injection of 1-123 HIPDM

Figure k

Rabbit Image 2k Hours After Injection of I-123 Compound VI -40-

Figure 5 Rabbit Image 2-^ Hours After Injection of 1-123 Compound VI (Background Subtracted) CO.ÜANELLAS, M.G.ARGUELLES AND A.E.^.MlTTA

COMISION NACIONAL DE ENER6IA AlOMICA DE LA REPUBLtCA ARGENTINA.

BUENOS ß.IRES REPU3HCA ARGENTINA 1"".7 RAD10PHARMAC0L0GY Ob' IMINODI ACETIC AGIO N-DttMVATIVES ANALYSIS IN BIOLOGICAL MODELS AND COMPARISON TO HUMAN BEINGS

Cane]las.0.0; ArgUelles.M.G and Mitta A.E.A National Atomic Energy Commission Research Subprogram, Application and Development in Nuclear Medicine. Buenos Aires, Argentina.

INTRODUCTION

h'rorn the time Harvey, Burke and Halko published their first- reports on images or" the liver- and biliary tract using "Bengal Rose" labeled with 1-1.31, the diagnostic file on liver and gal I bladder disorders has grown considerably. In addition to the wealth of information obtained over the years from contrastive radiology with iodates, new methodology has provided us with more than just functional information. The body processes involved in the conveyance of these chelates with hepatobi I i ary .affinity can be summed up as follows: 1.- the radiopharmaceutioa.) rapidly binds with the plasmatic carrier and reaches the hepatoeyte's bloodstream. 2.- its passage through the hepatoeyte and elimination through the biliary ducts. 3.- passage through intrahepatic ducts to gallbladder. 4.- passage through the cholodochus to the duodenum. Keeping in mind the above and that a pharmaceutical that goes through these steps, in order to prove useful, must have the properties of rapid plasmatic clearing, high hepatic uptake with an effective flow to the gal 1.bladder regardless of the tenor of the nonconjuqateu billirubin present, and very low kidney excretion, we decidod to make a complete radiopharmacological study of a series of iminodiacetic acid N-deri vat ives. employing not. only novel met'r .-; of synthesis but also reliable chroma togrotph i c systems as well as a biological model that would allow us to extrapolate from the experimental results to apply thorn to human;;. On the basis of -4 3-

the data obtained from the four derivatives used in the experiment, we were able to design a new derivative whose biological behavior in human beings we were able to predict and which we verified on normal volunteers and carriers of different clinicopathologic diseases.

MATERIALS AND METHODS

The method published by Harvey ot a1 was followed to obtain the N-derivatives. Modifications were introduced to improve the efficiency of the intermediate as well as end product reactions. The following four compounds were synthesized using the synthetic pathway as seen in figure 1: N-(2,6 dimethylphenylcarbamoylmethy1)iminodiacetic acid, N-(2,6 diethylphenylcarbamoylmethyl)iminodiacetic acid, N-(2.6 diisopropyIphenyIcarbamoyjmethyl)iminodiacetic acid and N-(4, butyIphenyicarbamoy1methy I)iminodiacetic acid. Also, the previously unknown N-derivative, N-(2,6 diisopro pyIphenyIcarbamoyiethyi 1)imi nodiacetic acid was obtained by way of the synthetic pathway seon in figure 2. The quality control methodology chosen was the determination of melting point, with a Kofler device, spectra of nuclear magnetic resonance at 100 mHz a Varian XL-110-15 spectrophotometer in a solution of DMS(H-3) with tetramethylsilene as an internal pattern, mass spectrometry at 70 eV on a Varian Mat-CH 7A spectrometer run by a Varian-Mat-Data system 166 computer with direct sample insertion and determination of the centesimal formula. We used Cl~Sn.2 H^O as a reducing agent and sodium pertechnetate (Tc-99m) from a "Gentec" generator. Each vial contained 20.0 mg of the N-derivative and 0.20 mg of the reducing agent, kept in a nitrogen atmosphere at a volume of 1.0 ml, to which 20 mCi (740 MBq) sodium pertechnetate (Tc-99m) was added resulting in a final volume of 2.0 ml. Given the fact that there are no reliable chromatographic systems to correlate the ohelate's radiochemical purity and -44-

figure

NH-C-CHjCl 6

NH7 * Cl-C-CH-C! 0 R

H-C-CH^CI n Z 0

NH-C-CH^N^CH^-COOH). 6 R%

R Y /( ) ) NH-C-CHrCl • HN(CI-L-COOH) «. 2 0 iT

-SY-NTHESIS OF N-DER1VAT-1VES - .'i 5 -

figure 2

C3H?

NH2 4- Br-C-CH-Sr H-C-CH-Br ii I

I 0 CM, C3H7 -3

NH. o cm

3 7 /"A NH" H CH-0r+HN(CH2COOH)2 \O) ^9HN(CH-COOHj 3 2 7

>NH-C-CHN(CHH:CCI- -4 6-

biod istr ibut i on, we found it. necessary to develop thorn. With ITLC(SO) as a base and methanol 8b% we differentiated the colloid from the bis-compound as the former remained in the bas° white the latter ran with the solvent front, the same as the free perteehnetate. By altering the polarity of the solvent mixture, we were able to inhibit- the migration of the N-deri.vati.ves while the: i'rtie perteehnetate remained Rf 1.0; tho composition of the solvent was methy 1 ethylketone (MftK):benzene;methanol (1.0: H.b:0.5). We also employed molecular screen (sephadex G-25 medium) stacked in glass tubes. After- preparation, the dead volume was eluted with a physiological solution and the distribution of the activity in the tube was determined with a Berthold Lb 2723 radiochromatograph at- a speed of 3000 mm/h. The lethal dose bO (LD-50) was determined for each one of the compounds to deduce the mass to be injected without causing toxic reactions, and the safety factor, calculated. Acute toxieity and sterility tests were run in accordance with Argentine National Pharmacopoeia (6th edition) methodology. To determine the presence of pyrogonie substances, not only was the standard technique of basal temperature variation on rabbits used but also the "limulus test", recently accepted by the US Pharmacopoeia. Since the speed of the distribution in the tissues of each organ will be determined by the bloodstream and by the ease with which the molecules can pass through the bloodstream's capillary network and penetrate the cells, it was necessary to employ a technique that would enable us to evaluate plasmatic kinetics and thus determine the "plasmatic half life" indicated by the duration and intesity of the desired effect of each one of the radiopharmaceuticaIs, being able to infer its behavior. We used Wistar nils for this and performed a carol i dea-carot idoana bypass on them by inserting a piast. ic cannula with mandrel, after" divuIsion and be cutting the right jugular vein (figure 3), ana a 1.0 m ioi.-j caihofer with a 0.7 mm internal diameter in (.he ioft primitive carotid artery, in order to attain OXUMCVIIPI^.M: .: i r ou , a';, i on (figure -47-

figur.- 3 -48-

figure 4 We fj I iced I he animal ne

•!. i Him. • .spec I. j nine I.( :r W i I h .') (Ictccl ui (•< >n I . i i n i l m .1 ''.-1 i 1 ''•' i ; crystal, I J/'V wide by /." hi^h, and inserted t h<- caroti.i < Mthetor . ' >0 _/(.(.: i of radiopharmaorufieaI w.is administered vi. I.IK; jugular pi asmat i e caiinuln and Liu- v,iri.il. ion in oireulai tn rad 1 oaet i v i ty was recorded up to }?.() miniU.es, by rocor d i nq the rcKul l.s on semi I o'jar i t.hni i e npr;i wo arrived at. I he v/i> I ue of the compononl.s, I.ho lunclioii, which q.ivt- us

I'he b i t>d i .'•; Lr i bu t i on of I IK; I ivi; r ad 1 opliarmaeeut ic.ils was estirrifited in mice (NIH strain) adm i n i sl.or i nq t.ho?m 0.1 ml ol I he oho la to vin the; (ail vein, then distributing thorn in metabolic boxes and saerif icing them at dii I'orf-nt 1. i mes by cervical traction. The abdominal cavity was opened (Mq. '•>). the eho I odochus pinched, and (.no gallbladder romoved withoul s;>i I i i nq its contents, t.h<--n 1 he I iver, spleen, duodenum, intestines, kidneys, bladder and urine (f iq (•>) . isy usinq

Wo considered two o i iminauion routes, th<; h i I iary and the renal, and by reinjectinq the biliary contents and urine, we wen.' able to evaluate the metabolic influence of each oi those paths. The gallbladder were removed from mice that had boon injected with different rad i opharmnoeu t i ea 1 s ond tiie ur'ine was collected in a riietat)olic box. Tho.se were measured in an automatic; gamma counter and they were administered to the same biological model as was used in the biodistribut ion. Afterwards, the animals were sacrificed at different times and the residual, radioactivity dose in eaen organ was determined. The results obtained were compared to trie standard ones by the match inn "t" test.

Since one of the objectives of i.his pr. • ect was the synthesis and biological evaluation of a new :\i-der i vat. ive -50-

f igu re 5 -b 1 -

figure 6 -52- and the subsequent extrapoJation from the results in the biological model use on human beings, tests were carried out in different branches of nuclear medicine, on normal volunteers and carriers of different clinicopathologic disorders. We used the new compound and, given its structural likeness to N-(2,6 diisopropylphenylcarbamoylmethyl)iminodiacetic acid, matching tests were performed. Eleven normal volunteers were compared who were administered the drug a woek apart. Twenty nine adult patients were divided into two clinicopathologic groups: acute and chronic cholecystitis, and wore given the N-(2,6 di isopropylphenylcarbamoylethy1 1) iminodiacetic acid. The new derivative was tested on 30 patients divided into four cLinicopathologic groups: acute cholecystitis, chronic cholecystitis, cirrhosis and jaundice. The takes were done uninterruptedly using a gamma camera, an image every minute for 35 minutes, followed by one every IS minutes. By creating areas of interest in the heart and the liver, we obtained the liver/heart activity radio at 5 and 60 minutes. Also, we estimated the appearance times of the intrahepatic ducts (IAT), extrahepatic ducts (EAT), gallbladder (GAT), duodenum (DAT), and renal persistence (RP).

RESULTS

Using the methodology described above, the following recjction efficiencies were obtained: N-(2,6 dimethylphenyl- carbamoylmethyl) iminodiacetic acid (7'3%), N-(2,6 diethylphenyl carbamoylmethyl)iminodiacetic acid (80%), N-(2,6 diisopropylphenylcarbamoylmethyl)iminodLacetic acid (75%), N-(4 butyl- phenylcarbamoylmethyl)iminodiacetic acid (90%) and N-(2,6 diisopropylphenylcarbamoylethyl 1)iminodiacetic acid (75%). The mass spectra, nuclear magnetic resonance and centesimal composition corresponded to the proposed structures. The LD-50 arrived at were 130j_5.8 mg/Kg for dimethyl derivative, 160j_4.2 mg/Kg for the dlethyl dor , vative, 150_»_6.3 mg/Kg for the diisopropyl derivative, 72j_2.4 mg/Kq for the 53-

p-butyl derivative, and 140j^6. 2 mg/Kg for the new derivative. With respect to these data, the following safety factors were obtained: 456 (dimethyl derivative), 526 (diethyl derivative), 561 (diisopropyl derivative), 252 (p-butyl derivative) and 491 (new derivative). No pyrogenous substances were found and the limulus test proves to be not only totally compatible with these radiopharmaceutical s but also homologous with the Argentine National Pharmacopoeia. Using the carotidea-carotideana bypass technique, we determined that the blood mixing time for ail compounds is 20 seconds, with no significant di fferences; the TJ$ short (hepatic uptake) and T^ long (renal excretion) were 2.5 and 92 minutes for the dimethyl derivative, 3.0 and 116 minutes for the diethyl derivative, 2.8 and 130 minutes for the diisopropyl derivative, 2.0 and 238 minutes for the p-butyl derivative and 2.0 and 136 minutes for the new derivative. With the f tgures obtainod in the biodistribution, variation graphs in function of time were drawn up of the injected dose for renal excretion, intestinal passage, hepatic clearing and biliary uptake. On the basis of these data, we determined the maximum uptake time in the bladder to be 5 minutes for the dimethyl derivative, 30 minutes for the diethyl derivative, 15 minutes for the diisopropyl derivative, 30 minutes for the p-butyl derivative and 15 minutes for the new derivative. In using the reinjection technique, we found that when biio was administered, a similar- uptake by the bladder occurred on the whole, staying above normal values of renal elimination. When urine was administered, we observed a completely different biological behavior: bladder uptake was practically nil and renal elimination, much higher than normal levels. In normal volunteers, treated with the dit:;opropyl derivative, we observed an IAT of 7.4+_1.4 minutes, an EAT of -54-

12.7^2.7 minutes, a GAT of 12.9^2.7 mJnutes, a DAT of 24 . 2j_4 . 6 minutes arid RP oi 8.8_»_3.8 minutes. For the new derivative, these values were 7.3^2.1 minutes, 11. 6j^3. 4 minutes, 13.7_«_4.7 minutes, 21.3j_7.0 minutes and 10.5j|_6.8 minutes, respectively. Among the acute cholecystitis cases, the results were and IAT of 11.4^3.9 minutes and 10.50.3 minutes, an EAT of 2?..0_>_A.8 minutes and 16.5_»_6.5 minutes for de di isopropyl derivative and for the new radiopharmaceutical, respectively. It was not possible to observe the gallbladder in any of the tests run.

In the cases of chronic cholecystitis, the results were an tAT of 9.4j|_2.6 minutes and 6.7_«_2.5 minutes, an EAT of 16.7_»_7.3 minutes and 12.4_*_4.J minutes for the diisopropyl derivative and the new derivative, respectively. In the tests run with diisopropyl derivative, the gallbladder did not appear, while with the new derivative, it was detected in throe of the patients.

DISCUSSION

Since the appearance of the dimethyl derivative, many more compounds have appeared. In this study, we have used only some of them, not because Lhey could not be synthesized, but because they did not seem relevant to our objectives. Our first objective was to achieve a greater reaction efficiency in tho methods proposed by Harvey et al. The changes introduced improved the reaction by 75-

began to work with dimethyl, diethyl, diisopropyl and p-butyl derivatives, which we had wide experience with as well as plenty of jibiography. The selection of the biological model was of fundamental importance! since we thought it should reflect the hepatic passage and the bladder uptake at the same time it would allow us to extrapoLate from these results for use in humans. The use of mice allowed us to infer both passages. Since there is no information whatsoever on the use of this model in evaluating ohelaLes with hepatobiliary affinity, we began by determining the value of the L.D-50 for the first four derivatives in order to compare them with the new compound and thus be able Lo calculate the mass needed to reach a safety factor acceptable to pharmacopoeia and guarantee its future use in humans. The use of the carotidea-carotineana bypass allowed us to estimate the degree of renal excretion in the radiopharmaceutical and the speed of hepatic uptake. Although the latter showed no significant differences among the different N-derivatives, the renal excretion did. This leads us to affirm that the variation in molecular weight and lipophiiic power bring about a variation in the movement and degree of metabolization in the hepatocyte which is reflected, precisely in different renal elimination. If we consider that the TJ& long was 92 minutes for the dimethyl derivative, 116 minutes for the diethyl derivative and 130 minutes for the diisopropyl derivative, we can relate the importance of the increase in molecular weight in renal excretion, and when we analyze that for the p-butyl derivative, this value is 238 minutes,we see the importance of the position of the substitute in the ring. By introducing a variation in the hydrocarbonate chain, we maintained the type and position of the phenylic substitute constant, in this way, we obtained a T%, of 136 minutes which, compared to that obtained for the diisopropyl derivative, showed no significant differences. When we compared the results of the lipophilic power. -56-

wo found that the dimethyl derivative showed -2.43^0.2; the di.et.hyl derivative -0.'70*0.2: the diisopropyl derivative 1.69j_0.19 and the p-butyl 2.79^0.1 whereas the new derivative showed .1 . 90+_0. 19. Note that the lowest degree of 1 ipophilieity appears in dimethyl derivative which, consequently, shows the fastest flow through the hepatobiliary structures and, as was proved by reinjection techniques, the lowest rate of metabolization. When analyzing the results obtained with the reinjections of the various radiopharmaceuticals, it was seen that when administering urine, renal elimination of radioac tivity was rapid, which shows an important decrease in the degree of 1ipophi1icity and accordingly, an increase in hydrosolubi1ity. The reinjection of the biliary contents produced a bladder uptake; equal to the normal product and lesser increase in renal excretion than that detected for uri ne. The parameter we decided to extrapolate from the biological mode I lor use in human beings was the radiopharmaceuticals' transit times for the different structures. The results tell us that the maximum uptake time in the gallbladder was 15 minutes for diisopropyl derivative and for the new derivative, when these are compared with those obtained in normal human volunteers, we see that there are no significant differences since the diisopropyl derivative shows a VAT of 12.9+_2.7 minutes and the new derivative 13.7_*_4.7 minutes. This demostrates the validity of our biological model, allowing us to affirm that the mouse is the most adequate animal for experimenting radiopharmacology of hepatobi1iary agents. As for the new derivative, we found that it is potentially useful in the diagnosis of hepatobi1iary diseases, showing rapid bloodstream transit and good hepatic uptake, a T4 of 2.0 minutes, high biliary accumulation, 8.8_^3.6 percent of the dose administered in 1 i> minutes, and low renal excretion. Finally, the reason for such biological behavior of the -57-

diisopropyl and the new derivative should be explained. The results from the reinjtjction techniquejs of urine and gallbladder show that the modification introduced in the molecular weight did not produce changes in biological behavior since it did not lead to modifications in lipophilic power, 1.69_»_0.19 for diisopropyl and 1.90jt_0.]9 for the new derivative, and this parameter is really the one that regulates cellular flow of these radiopharmaceuticals.

ACKNOWLEDGEMENTS

We would like to thank the nuclear medicine branches that tested these radiopharmaceuticals enabling us to realize the extrapolation from our experimental results.

REFERENCES Synthesis and radiolabeling of technetium radiopharmaceuticals based on N-subst ituted iminodiacet ic acid.'effect of radiolabeling conditions on radiochemical purity. Fields.A.T, Porter.D.W, Harvey.E.B and loberg M.D J.Lab.Comps.RadLopharm. 15 (387) 1978 A mathematical model for the liver uptake and excretion of d if thy 1 -IDA . Gelius.L, Skretting.A and Aas.M. Eur.J.Nuc.Med.6 (139) 1 98 I Structure-activity relationships for Tc-99m benziraidaiolyl methy1imino-diacetic acid he patobi1iary radiopharmaceuticals. J .Lab.Com.Rdaiopharm. 18 Nro I (191) 198 I Correlation analysis of substituents efects on the pharmacy kinetics of hepatobi1iary agents. Jansholt . A . L , Scheibe.P.O, Vera.D.R, Kron.K.A and Stadalnik.R.C J.Lab.Comp.Rdaiopharm. 18 (198) 198 1 Radiopharmaceuticals for he patobi1iary imaging. Chervu.L.R, Nunn.A.D and Loberg.M.D. Sem.Nuc1.Med. 7 Nro 1 1982 Comparative in vivo kinetics of some enw Tc-99m labelled acetanilido iminodiacetates. Chiotellis.E, Varvarigou.A ;i n d Koutoulidis.C Eur.J.Nucl.Med. 6 (24 1 ) 198 1 SI)

THE INFLUENCE OF MOMOFUlORMETHYUlISTimiE ON THE MAMMARY ADEW?CTWA M-2.

M.C.RUEIO* "I.E.LANDA* , H.TARGOVNIK** J L.L. COLOMBO*** , A.J.SCOLNIK**** AND R.A.CAPO****

ININFA, CONICET, BUENOS ÎPES, ÊP.^RPFNTINA ** CATEDRA DE QUIMICA BIOLOGICA^ FACULTAD DE MEDICINA, UNIVERSIDAP DE BUENOS AIRES^ BUENOS AIRES> REPUBLICA APPENTINA. *** INSTITUTO DE ONCOLOPIA ANREL M POFFO, FACULTAD DE HEDICINA, UNIVERSIDAD DE BUENOS AIRES^ REP. ARGENTINA. **** CATEDRA DE FISICA, FACULTAD DE FARMACIA Y BIOQUIMICA^ UNIVERSIDAD DF BUENOS ÎRES, DEP. ARGENTINA BUENOS AIRES REPURLICA ARGENTINA THE INFLUENCE OF MONOFLUORMETHYLHISTIDINE (MFMH) ON THE MAMMARY ADENOCAKCINOMA M-2

M.C. Rubio, M.E. Landa (ININFA, CONICET), H. Targovnik (Catedra de Qufmici Biologica, Facultad de Medicina, U.B.A.), L.L. Colombo (Instituto de Oncolo- gia "Angel H. Roffo", Facultad de Medicina, U.B.A), A.J. Scolnik and R.A. Caro (Catedra de Ffsica, Facultaci de Farmacia y Bioquimica, U.B.A.). Buenos Aires, Argentina.

It has been shown in previous papers, that the endogenous histamine content is high in animal and human tumors (1,2). It has also been demonstrated (3) that the H-110 Lymphoma has a high histamine biosynthetic c.-ipacity. Evidence was found that the administration of 3-H- histamine shows an increase of the radioactivity concentration in the tumor nuclear fraction. Similar results were obtained in the Balb/c mice bearing M-2 mammary adenocarcinoma (4); in this tumor we found also a higher histamine endogenous content. The high levels of biosynthetic activity of histamine in the M-2 tumor cannot be adscribed to a dissimilar nature of the histidine decarboxylase of the tumor or the intestine (5); the DNA and protein concentration of the nuclear fraction were not significantly different from that of normal tissues. We demonstrated also a non-covalent specific binding of histamine to certain DNAs (5). In a N- nitroso-N-methyl- urea induced mammary adenocarcinoma, a correlation between histamine biosynthesis and tumor growth was observed (6). The administration of Levamisole during more than 20 days decreased the histidine decarboxylase activity (6).

In the present paper we study the influence of monofluormethylhistidine (MFMH), a specific irreversible inhibitor of histidine decarboxylase (7), on the survival of M-2 bearing mice and its correlation with the histamine biosynthetic activity. Some preliminary results concerning the binding of 3-H-histamine to purified tumor DNA will also be reported.

MATERIALS AND METHODS

Animals and tumor.

Male and female Balb/c mice form the Instituto de On- cologia "Angel H. Roffo", aged between 2 and 3 months, were used. The animals were maintained in cages in groups of 5, with standard food and water "ad-libitum". The M-2 mammary adenocarcinoma was transplanted into these mice as described before (4). -60-

tlistidine decarboxylase activity.

The histamine biosynthesis was determined using 14-C histamine as substrate, measuring the liberated 14-C-carbon dioxide, as previously reported (4).

Lung and tumor DNA purification. Lung and tumor DNA was isolated and purified by phenol extraction and ethanol precipitation. DNA was previously preincubated with RNAase and pronase according with Maniatis et al. (8).

3-H-histamine binding to DNA.

Binding studies were carried out by dialysis equilibrium techniques in a semimicro cell with a semipsrmeable membrane (cut off of 12000 - 14000). The DNA solution (1 ml) of 1 ng/ml in buffer TRIS- 10 mM, pH = 7.5, with 1 ml EDTA was dialyzed against an equal buffer volume (1 ml). The 3-H-histamine concentration was 0.15 MM (S.A. 8 Ci/ mmol New England Nuclear). The equilibrium time was 6 hours. After dialysis was completed, 0.1 ml aliquots of the DNA solution (lung or tumor) and buffer, were analyzed for radioactivity, adding 5 ml of a scintillation solution having the following composition: PPO 5.5 g; POPOP 1 g; toluene 600 ml; ethanol 100 ml; triton X-100 300 ml. The samples were counted in a liquid scintillation spectrometer Packard 3255. Absolute activities were determined by the external standard channels ratio method.

Statistical analysis and drugs.

All the pertinent data were analyzed by the two-tailed unpaired Student's t-test.

A dose of 25 mg per kg body of MFMH was i.p. injected two times per day during the animal's life. The control group received similar volumes of saline solution twice a day.

RESULTS

The survival curves with MFMH and the control group are shown in Figure 1. In our experimental conditions, the survival half-times after transplantation is 22 and 8 days respectively. -•6 1-

100 n=11

oc 60 I 40

^ 20

0 I I * 10 K 18 22 26 Days

Figure 1. Survival curve after M-2 transplantation of control mice (••-•• ) and mice injected with MFMH ( A~A ).

The histidine-decarboxylase activity of the control group and the mice injected with MFMH can be observed in the Table 1.

TABLE I: Effect of monofluoromethylhistidine (MFMH) on histidine decarboxylase activity (HDC)

Tissue HDC (dpm.g .h ) Control animals MFMH treated animals

Intestine 4350 + 780 2528 + 850

Tumor 8240 + 1520 4297 + 1052

MFMH: 25 mg/kg i.p. 2 x per day. HDC: assayed 8 hours after last dose.

As shown in previous papers (4) the tumor histidine- decarboxylase activity was again high as compared to that of intestine. The fractional enzymatic activity remaining 8 hours after the last MFMH dose, was 0.42 and 0.48 respectively. These results were obtained at the 3rd. day after the tumor transplantation.

The results of the binding experiments of 3-H-histamine with DNA are summaryzed in the Table II. -62-

TABLE II: Binding experiments in equilibrium dialysis cell

3 Per cent H-Histamine bound

Tumor DNA Tumor DNA Lung DNA + 100 Histamine

35.5 + 1.5 5.1 + 2.4 21.2 + 2.3

3 Ligand concentration: 0.15 MM ll-histamine

DNA concentration: 1 mg/ml

It can be ooserved that the specific binding to the tumor DNA is approximately 30 per cent.

DISCUSSION

According to our previouis hypothesis (9) concerning the involvement of histamine metabolism in tumor growth, endogenous histamino content and the biosynthesis of this amine, are high in the studied tumor. It is interesting that the pattern of the decrease of survival in the curves on Figure 1 show a similar slope, with a delay of approximately 14 days. This seems to indicate that the inhibitory action of MFMH is time-limited. This might be due to a change of the enzyme conformation or a significant increase of the biosynthesis of the enzyme. An alternative explanation might be that the tumor change the regulatory growth factor. Future research will be carried out in order to analyze further these results.

Concerning the binding of 3-H-histamine to tumor and other DNA, it should be borne in mind previous results shown vith this tumor, showing that the nuclear fraction has a higher proportion of radioactivity after incubation with 3-H-histamine, than the intestine of the same animal (4); the 3-H-histamine was bound to certain but not to all DNAs (5). Our - 6 3 ••

present results, obtained with purified tumor DNA, may imply that histamirif interacts with DNA in a way similar to that proposed for polyamines (10) and that the binding to tumor DNA is higher that to lunp, DNA. In this sense it may be taken into account that a single point mutation in the DNA is sufficient to confer transforming properties to an oncogene (11). We consider that our results indicate a possible relationship between histamine, its binding to DNA and tumor growth.

REFERENCES

1. Zieher L.M., Scolnik A.J.: Medicina (Buenos Aires) 1970, 2_9j_ 1953.

2. Zieher L.M., Botto H.G., Botto I.S. de: Rev. argent. Cancerol. 1966, 8^ 78.

3. Scolnik A.J., Rubio M.C., Caro R.A., Ciscato V.A.: Biomedicine 1978, 28, 274.

4. Scolnik A.J., Rubio M.C., Comolli R.R., Colombo L.L., Caro R.A.: Biome- dicine 1981, 35, 84.

5. Scolnik A.J., Rubio M.C., Colombo L.L., Comolli R.R., Caro R.A.: Biomed. Pharmacother. 1984, 38, 465.

6. Scolnik A.J., Bergoc R.M., Rivera E.S., Venturino A., Comolli R.R., Rubio M.C., Caro R.A.: Acta Bioquimica Clinica Latinoamericana 1986, 20, 469.

7. Garbarg M., Barbin G., Rodergas F.., Schwartz J.C.: J. Neurochem 1980, 35, 1095.

8. Maniatis T., Frilsch E.F., Sambrook J.: Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (USA) 1982.

9. Scolnik, A.J., Rubio, M.C., Caro, R.A. TIPS b± 35G-357, 1985.

10. Abraham A.K.: Medical Biology 1981, 59^ 368.

11. Reddy E.P., Reynolds R.K., Santos F.., Barbacid M.: Nature (London) 1982, 300, 149. 131-1 TTAinflO^ZYLGUAMITW (131-1 MI3G) KINETICS IN A CARCnniJ) TMW.

R.SCHIAVO, n.CoNCOLIMO^ ^.RAZI, n.lANMANTUONO, S.LI* VOTI, A.MANZARA AND P,PAVONI

V.CLINICA MEDICA, "MIVER^ITA ni DOMA "|.A SAPIENZA"

REPURLICA ARGENTINA 1H87 5

131I-METAIODOBENZYLGUANIDINE (i31I-MIBG) KINETICS IN A CARCINOID TUMOR.

R. Schiavo, G. Concolino, F. Fazi, P. Iannantuono, S.Li. Voti, A. Manzara, P. Pavoni. V. Clinica Medica, Universita di ROMA "La Sapienza".

131I-MIBG is a radioiodinated adrenergic neuron-blocking agent (Fig. 1). As an adrenomedul1ary imaging agent was first employed by Sisson (1981) who demostrated marked uptake in eight patients with known pheochromocytoma. Since then 131I-MIBG has shown to be very useful - for diagnosis and radiometabolic therapy of pheochromocytoma and neurobiastoma (Me. Ewan 1985, Munkner 1985). During the last two years the utilization of 131 I—MIBG was extended to the localization - of paragangl ioma (Smit 198*0, carcinoid tumor (Fischer 198*0, metastatic medullary thyroid cancer (Sone 1985), melanoma and oat-cell carcinoma (Hoefnagel 1985). Therefore many different APUD neoplasms may be considered as a potential target for 131I-MIBG (Tab. 1) although the uptake and the kinetic of this tracer are still unclear A possible kinetic compartmental (mammi11ary) model is shown in Fig. 2 where compartment 1 represents blood pool and compartment 2 the APUD neoplasia. A third compartment, representing the neuroadrenergic — system with a much slower dynamic pattern, may also be hypothesized. We cannot exclude that the MIBG is partially metabolized in compartments 2 and 3, but most evidence is that the tracer is almost completely excreted by kidneys as unaltered compound (Mangner 1986). According to this model, theoretical "in vivo" time-activity curve obtained on compartment 2 is shown in Fig. 3, not taking on account - compartment 3- We report the dynamic-functional 131I-MIBG study performed in a — case of carcinoid tumor with liver metastases, comparing the kinetic parameters measured with those obtained in another pheochromocytoma. - 6 h -

MATERIALS AND METHODS A 35-year-old woman came to our observation with a history of pituitary adenoma (GH and prolactin producing) surgically treated 3 years before. One year after the surgery she began to experience — recurrent flushes with headache, diarrhea and vomiting. Urinary excretions of 5-hydroxytryptamine, 5-hydroxytryptophane and 5- hydroxyindolacetic acid were 3-4 fold more than normal. A CT abdominal scan revealed multiple hypodense lesions in the liver, confirmes by a radionuclide liver scan (99m Tc-Albucoll. 55,5 MBq i.v., Fig. 4). After the administration of 55,5 MBq 131I-MIBG i.v. serial scintiscans of the hepatic region were obtained for a period of 6-8 hours (a scan an hour). Serial counting was also performed on the spinal lumbar — column, in order to obtain a parameter for blood background correction (see appendix), on the liver and on every site we suspected a metastatic localization of the neoplasm (i.e. where an even limited — increase of activity was observed). Scintiscans and counting were then daily performed until the activity ratio: neoplasia/background approached to 1. Time-activity curve of the neoplasm was obtained with a best-fit procedure after background correction, and kinetic parameters (Ti/2, Tmax) were then evaluated.

RESULTS In the 1311—MIBG scan performed after 7 hours we observed a significant increase of activity in those liver regions that were hypoactive at the 99mTc-Albucol1 scan because of the carcinoid tumor metastases. This activity became more evident during the following days (Fig. 5). Time-activity curve analysis (Fig. 6) showed that maximal 1311—MIBG uptake by carcinoid tumor occurred at 48 Hours, while its biological half-time was 8 days and half. -6 7-

DISCUSSION Most data on the kinetic of 1311—MIBG, are derived from "in vitro" kinetic studies of neuroblastoma and pheochromocytoma cultured cells (Tobes 1985, Kimming 1986) or mice-transplanted neoplasms (Senekowitsch 1985), Few authors nave performed "In vivo" kinetic studies (Lindberg 1985, Nakajo 1985)- Our findings appear to be not in agreement with those elsewhere reported on carcinoid tumors (Hoefnagel 1986), on account of the very slow kinetic (Tl/2 = 8d, 12h) we have observed. Nevertheless biological 131I-MIBG — half-times values measured by the already mentioned Authors and ranging between 1 and k days are in agreement with our results in a patient with a pheochromocytoma. There are two possible explanations for understanding such a difference; a) We observed a particular carcionoid tumor with a very slow metabolic rate. b) the 1311—MIBG kinetic in carcinoid tumors is really slow according to their well known slow growth rate, so that the short half-time reported by Hoefnagel cou1H depenH upon the influence of blood activity that Author does not mention to have subtracted. In fact limiting the observation to the first 2k hours and without background correction we too measured a biological half-time of 3 days about. This different technique employed may explain also the disagreement between the results obtained by LinWberg (which in his paper stresses the importance of prolonging the study after k days) and those reported by — other authors. The difference: between this rsrcino id and pheochromocytoma regarding 131I-MIBG kinetic is confirmed by the analysis of the neoplasia/liver activity ratio curve (Fig. 7-8). Carcinoid/iiver and pheo/liver share a similar curve but in our study the latter reaches a plateau after 9 days while the former after 2 days only. Although we did not perform the quantitative compartmentai study the analysis of the time-activity curves, allowes us to suggest that the model hypothesized is correct on account of the pattern of the curve obtained on compartment 2 similar to the theoretical curve showed on Fig. 3. Analytical quantitative evaluation of turn-over rates was not performed. In fact it is not possible to define a physiological compound "traced" by 131I-MIBG, and there is not correlation between 131I-MIBG uptake and amine production by the tumor (Smit 198**, Cabal lero 1986). This tracer more probably mimicks the normal behaviour of many different amines by crestal neuron origin cells, -68-

CONCLUSIONS Although more data are necessary to understand if there is a significant variation in 131I-MIBG kinetics between the different kinds of APUD neoplasms we think that a dynamic-functional study, allowing the evaluation of the different biological half-times, may be helpful for the selection of these neoplasms which could be treated with 131I-MIBG and for the estimate of radiation dose required for the treatment.

APPENDIX (Magrini-Pavoni 1985) Total counts obtained on the neopiasia N _ should be considered as the sum of two terms;

%(t) = Nft(t) + N'B(t) (1) where N. is the activity present in the neoplastic cells and N'_ is the activity in blood perfusing the neoplasia and the surrounding tissues. As far as complete mixing of the tracer can be assumed N' counts are — proportional to the counts obtained over the spinal lumbar column (aortic activity) NQ

e N'B(t) - B NB(t) (2)

The constant £„ accounts for the different detection efficiency of blood activity in the two counting sites. As _ varies from subject to subject and for earn counting area in the same subject a normalization must be performed to counts recorded at time t=0. At this time:

NAB(0) = N'B(0) (3)

NAB(0) = Therefore E g (Q) and by sostitutions in (2) and then in (l) the net neoplasia counts can be obtained :

N.(t) = N._(t) - A" N (t) A AB N (0) -69-

REFERENCES CABALLERO 0., FERRIS J., VERDEGUR A., ESQUEMBRE C, CASTEL V.. Iodine-131 Metaiodobenzylguanidfne. J.Nucl.Med. 27:868,1986 (letter) FISCHER M., KAMANABROO D., SANDERKAMP H., PROSKE T., Scintigraphic imaging of carcinoid tumors with 1311—MIBG. Lancet 2 :165, 1984 (letter). HOEFNAGEL C. A., DE KRAKER J., MARCUSE H. R., VOUTEP A.,: Detection and treatment of neural crest tumors using I-13l-metaiodobenzylguanidine. Eur. J.Nucl.Med. 11: A 17*1985 (abstr.). HOEFNAGEL C.A., DEN HARTOG JAGER F. C. A., VAN GENNIP A. H., MARCUSE H. R., TAAL B. G. : Diagnosis and treatment of a carcinoid tumor using iodine-131 meta-iodobenzylguanidine. Clin.Nucl.Med. 11:150- 152,1986. KIMMIG B.. REICHARDT P., EISENHUT M., BRANDEIS W.E.: Kinetik von meta-jo-benzylguanidin in neuroblastom-zeliku1turen. NucCompact 1 7 :8it-86,1 986. LINDERBERG S., ERNEST I., FJALLING M., JACOBSSON L.,: The value of late images in 131I-metaiodobenzylguanidine (1311—MIBG) scanning of the adrenal medulla. Eur.J.Nucl. Med. 11:A 28,1985 (abstr.). MAGRINI A., PAVONI P..: Teoria dei traccianti, Roma, EMSI, 230-232, 1985. MANGNER T.J., TOBES M. C, WIELAND D. W., SISSON J. C. SHAPIRO B.. Metabolism of iodine-131 metaiodobenzylguanidine in patientes with metastatic pheochromocytoma. J.Nucl. Med. 2 7 :37-*+i+, 1 986 .

MC EWAN A. J., SHAPIRO B., SISSON J.C., BEIERWALTES W.H., ACKERY 0. M., : Radio-iodobenzylguanidine for the scintigraphic location and therapy of adrenergic tumors. Sem.Nuc.Med. 15=132-153,1985. MUNKNER T.: 1311- Metaiodobenzylguanidine scintigraphy of neuroblastomas. Sem.Nuc.Med. 15:15^-160,1985. NAKAJO M., SHIMABUKURO K., MIYAKI N., SHIMADA J., SHIRONO K., YOSHIMURA K., YONEKURA R., SHINOHARA S.,: Rapid clearance of iodine- 131 MIBG from the heart and Ifver of patients with adrenergic dysfunction and pheochromocytoma. J.Nucl.Med. 26 1357 — 365,1985. -7 0-

SENEKOWITSCH R., BODE W., MOLLENSTADT S., KRIEGEL H., BRUCHELT G. . BUCK J., TREUNER J., Tumor uptake and biokinetics of 131I-MIBG in nude mice hosting a human neuroblastoma, Nuc.Med. Comm. 6:555,1985 (abstr.).

SISSON J. C, FRANGER M. S., VALK T. W., GROSS M. D,, SWANSON 0. P., WIELAND D, M. . TOBES M. C, BEIERWALTES W. H., THOMPSON N. W. , : Sci nt i graphic localization of pheochromocytoma N.Engi. J.Med 305:12- 17, 1981.

SMIT A. J., VAN HESSEN L. H., HOLLEMA H., MUSKIET F. A. J., PIERS 0. A.: Meta- (1-131) Iodobenzylguanidine uptake in a non secreting paraganglioma. J.Nucl. Med. 2 5:98^-986,1984.

SONE T., FUKUNAGA M., OTSUKA N., MORITA R., MURUMAKA A., YANAGIMOTO S., TOMOMITSU T., NAKAYAMA H., HARADA T.,: Metastatic medullary thyroid canrer : localization with iodine-131 metaiodobenzylguanidine. J.Nucl.Med. 26:604-608, 1985.

TOBES M. C, JACQUES S., LLOYD R. V., SHAPIRO B., SISSON J. C: comparison of the in vitro pharmacodynamics of meta-iodobenzylguanidine (MIBG) to the in vivo scintigraphy, Nuc,Med.Comm. 6:585, 1985 (abstr.).

SUMMARY

131I-METAI0D0BENZYLGUANIDINE (131I-MIBG) KINETICS IN A CARCINOID TUMOR.

R. Schiavo, G. Concolino, F. Fazi P. Iannantuono, S.Li. Voti, A. Manzara, P. Pavoni (V. Clinira a, Universita di Roma "La Sapienza" Italy).

The 1311—MIBG kinetic was "in vivo" studied in a patient with liver metastases in case of carcinoid tumor. Serial scintiscans (55-5 MBq i.v.) and counting on spinal lumbar column, on liver parenchyma and on those suspected sites, on account of the activity measured, allowed us to validate a two-compartment model. Biological neoplasia half-time was 8 days and half, while maximal tracer uptake occurred at kS hours.. Our findings, showing a very slow 1311—MIBG kinetic, raise the possibility that exists a difference among different APUD neoplasms in 1311—MIBG turnover rates. Therefore a dynamic-functional study, with the evaluation of the diference biological half-times may be helpful for a more correct estimate of the radiation dose required for a radiometabolic 1311—MIBG treatment. -7 I-

LEGENDA

•Fig. 1 : 1311-Metaiodobenzylguanidine . Fig. 2: 13U-MIBG kinetic model. Fig. 3 •' 13H-MIBG time-activity theoretical curve. Fig. k: Liver colloid scan. Fig. 5: Liver 7 3TI-MIBG scan. Fig. 6: Time-activity 13H-MIBG curve. Fig. f: Carcinoid/liver activity ratio curve. Fig. 8: Pheo/liver activity ratio curve.

Tab. 1 : APUD cells, apudomas and their main endocrine production.

TAB. 1

APUD CELLS APUOOMAS ENDOCRINE PRODUCTION

Adrenergic neuron Neuroblastoma Catecolamines

Chromaffin eel 1 Pheochromocytoma Catecolami nes

Paragangliar eel 1 Pa? aganglioma

Thyroid "C" eel 1 Medullary thyroid Calci toni ne ca rci noma

Endocrine gastroenteric Intestinal carcinoid 5-HT, Gastrine eel 1

Kulchitsky bronchial Bronchial carcinoid 5-HT cell Mi rrocytoma ACTH

Melanocyte Melanoma -72-

N-H II CH2NHCNH2

figure I

1/ 3 J X)3 X3O

g _ neuroadrettergk; f _ /,/oorf 2 •= neoplamla

figure 2

1 I if

figure 3 -73-

S. * **'

figure 4 and 5 figure 8 "IS

\ SI'-TLF VETHO^ FOR "P!F. PT^PATW! OF c COMPLEXES "spin SURFACE AnSO^E* STArinnr,

D.J.'VDDALENA*, H ,.M . oNOWDON*, *\. AWALHDDIN** AND

0 Ml OA .(.(, i i .'OJER

* ISOTOPE DIVISION, ^IJGTPALIAN ATOMIC EKEPRY COMMISSION, LUCAS ''EIGHTS ^ESRARCH °RIVATE "AILBAP MPO .1, ^NAI, MSW, AUSTRALIA. ** ISOTOPE PEPAPTMENT, "NIT TRNARA 'IUKLEAP^ COMPLEX pUSPATI, D.ANGI, ^FLANHOR, MALAYSIA. *** AUSTRALIAN RADIATION LABORATORY,, LOWER PLENTY YALLAMBIE, VICTORIA, 3H85, AUSTRALIA.

AIRES "EPUBLICA ARGENTINA A SIMPLE METHOD FOR THE PREPARATION OF DIFFICULT 99mTc COMPLEXES USING SURFACE ADSORBED STANNOUS IONS.

Maddalena, DJ.1, Snowdon, CM.1, Avvaluddin, A.2 and Pojer, P.M.3

; Isotope Division, Australian Atomic Energy Commission, Lucas Heights Research Laboratories, Private Mailbag No.l.Menai, NSW, 2234, Australia.

2 Isotope Department, Unit Tenaga Nuklear, Komplex PUSPATI, Bangi, Seiangor, Malaysia.

3 Australian Radiation Laboratory, Lower Plenty Road, Yallambie, Victoria, 3085, Australia.

ABSTRACT

A simple new technique where stannous tin is adsorbed on the inner surface of plastic tubing and used to reduce [WmTc] pertechnetate prior to labelling radiopharmaceuticals, has been evaluated, using some lipophillic and metal containing ligands. Complexes formed using the technique had good labelling efficiencies and behaved the same in rat biodistribution studies as those prepared using conventional labelling methods.

KEYWORDS

Radiopharmaceuticals, Stannous ions, tin,99mTc , pertechnetate, adsorption. -II-

INTRODUCTION

The most widely used radionuclide for production of diagnostic imaging radiopharmaceuticals in nuclear medicine is Technetium-99m, normally supplied in solution as sodium [99mTc] pertechnetate. It is popular as it has excellent properties for medical imaging and can exist in multiple oxidation states allowing it to form a wide range of coordination complexes with both inorganic and organic ligands (Pinkerton et a/.,l985).

The preparation of 99mTc labelled radiopharmaceuticals using sodium pertechnetate usually requires that the pertechnetate be reduced, prior to its complexation, with the ligand of choice. A stannous salt, usually the chloride, is the reducing agent of choice since it is water soluble, effective at room temperature, relatively stable and non-toxic at the concenLations required.

Stannous ions in solution, however, have a number of disadvantages, in that they can cause precipitation or colloid formation with organic ligands especially tho^e that are highly lipophillic (Pojer and Baldas, 1981; van Wyk et ai., 1986) or those that contain other metals such as platinum (Awaluddin et al., 1987). The quantity of stannous ions may also influence the biodistribution of radiopharmaceuticals as in the case of bone agents (Srivastava et al., 1982). These complications have led to a variety of methods of immobilising the stannous ions on various supports to overcome some of its deficiencies Pe Schrijver, 1978; Camin et al., 1979; Horiuchi et al., 1981).

Recently, Salehi et cz/.,(l987), found that many common radiopharmaceuticals could be labelled with 99mTc by passing a solution containing a mixture of the ligand and sodium ["""Tc1 ^er^chnetate through an infusion catheter with stannous ions immobilised on the inside. In this study we have evaluated the technique using two vpes of plastic tube for labelling a platinum iminodiacetic acid complex which normally^recipitates out of -78-

solution with even small amounts of added stannous ions, a series of lipophillic hydroxybenzylglycine (HBG) derivatives and hexamethyl propylene amine oxime a highly lipophillic compound proposed for brain imaging studies (Volkert, et al., 1984).

MATERIALS AND METHODS

Thin wall poly vinyl chloride (PVC) and polyethylene (PE) medical grade tubing (1.0 mm internal diameter, Gosford Plastics, Sydney) cut into 300 mm lengths were used in all studies. The tubes were internally coated by a technique similar to Salehi et al. (1987). Solutions of stannous chloride dihydrate (25 mg in 10 mL isotonic saline), were passed through a 0.22 Jim membrane filter, and used immediately. Tubes were filled with the stannous solution and allowed to stand for 5 minutes before being emptied, then washed with 10 mL isotonic saline, dried with a stream of oxygen free nitrogen and clamped at both ends until use

To evaluate the efficiency of the tin coated tubes for preparation of 99mTc labelled complexes, 1 mL solutions of chloro hydroxybenzyliminodiacetic acid (CI-HBG) adjusted to pH 7.5, and hexamethylpropyl amine oxime (HMPnAO) in 0.02 M carbonate buffer plus generator-derived pertechnetate were aspirated and ejected slowly through 'tinned' tubes, several times. After each pass through the tube, 5 (iL samples were taken to examine labelling efficiency.

To examine whether the lipophilicity of a 99mTc labelled complex affected its labelling efficiency, a series of hydroxybenzylglycine (HBG) ligands, known to form neutral complexes with 99mTc over a wide range of lipophilicities, were used (Maddalena et al., 1987). One mL solutions (5 mg ligand / mL), pH 7.5, were passed through a 'tinned' tube four times then 5 \\L samples taken for estimation of labelling efficiency. -79-

The labelling efficiency of the complexes was studied by paper electrophoresis using Beckman paper strips at 300V for 1 hour in 0.05 M hepes buffer at pH 7.4. The strips were cut into 5 mm sections and counted in a Packard model 5650 automatic gamma counter at windows appropriate for"mTc.

To determine whether the 'tinned tube' method of labelling gave the same results as more conventional methods, three ligands HMPnAO, (Volleert et al., 1984), Cl-HGB, (Maddalena et al., 1987) and platinum compound II, (Awaluddin et al., 1987), were each labelled with 99mTc by their published methods and the tube method. The HMPnAO was labelled in 0.02 M carbonate buffer at pH 8.3 using stannous tartrate; the Cl-HBG in isotonic saline at pH 7.5 using stannous chloride in 1 M HC1 and the platinum compound II in 2% sodium chloride solution at pH 7.5 using sodium borohydride. The pairs of complexes were then compared by rat biodistribution studies at 1, 2 and 3 hours post injection.

The biodistribution studies on all complexes were carried out using female specific pathogen free Australian Albino Wistar (AAW) rats 8-10 weeks old. Groups of animals were injected intravenously via the tail vein with 100 |JL of the"mTc labelled complex using a glass micro-syringe (SGE), whilst under light ether anaesthesia, then placed in a metabolic cage for collection of urine and faeces. At the duration of the prescribed time "interval, the rats were sacrificed by an overdose of anaesthetic, for collection of tissue samples. The biodistribution of the complexes was calculated using the TISCON program (Maddalena, 1983).

RESULTS

Effect of type of plastic and number of passes

When a solution containing Cl-KBG and pertechnetate was passed through either a polyethylene (PE) or polyvinyl chloride (PVC) 'tinned' tube the labelling efficiencies of -80-

the complexes formed were the same. The labelling efficiency of the complexes after one pass was 82%, rising rapidly to a maximum of 96% after four passes. In the case of the HMPnAO / pertechnetate mixture the labelling efficiency was >99% after the first pass with tubes made of either type of plastic. These results suggested that either PVC or PE could be used, so for the remainder of the studies only the PVC tubes were used.

Effect of lipophilicity on labelling efficiency

The "mTc labelling efficiency of six derivatives of HBG, whose lipophilicity spanned nearly two orders of magnitude, (-0.61 to 1.28), after four passes through a 'tinned' tube is shown in table 1. The labelling efficiency was not found to be related to the lipophilicity of the complexes.

Effect of Tube Labelling Method on Biodistribution

The results of the rat biodistribution studies are shown in table 2. No significant differences were found between the biodistributioi.s of the HMPnAO labelled by either the stannous tartrate or the 'tinned' tube methods at 1 hour post injection.

When the biodistributions of the 99mTc labelled platinum complexes made by the two different methods were compared, one third more was found in the kidneys and a little le^s in the urine of the platinum complex made with the tube technique, suggesting that the.v; might be a difference between the complexes.

The complex prepared with the tube technique had a labelling efficiency of 98%, and only 1% of the injected activity was found in the stomach, an indicator of in vivo stability, suggesting that the preparation v/as stable both in vitro and in vivo. However, the other complex, prepared with sodium borohydride had a lower labelling efficiency of 90% and more than 2% in the stomach suggesting a less stable preparation. Consequently, it is likely that the differences between the the amounts of activity found -8 I- in the kidneys and urine of the complexes prepared by the two different methods were due to the variation in amounts of free pertechnetate in the two preparations.

When the biodistribution data from the 99mTc Cl-HBG complex prepared by the tube method was compared with the data from the conventional method using a t-test, significantly more activity was found in the kidneys (p<0.05) and urine (p<0.05) and significantly less in the gastrointestinal tract (p<0.05), suggesting that the complexes were different.

In both preparations the labelling efficiency was > 95% and the activity found in the stomach, was < 1%, suggesting that the complexes were stable both in vitro and in vivo. However, in the preparation made by the tube technique there was a change in the pH of the solution after it had passed though the tube four times. Since the 99mTc Cl-HBG complex is very sensitive to pH, (Maddalena et a/.,1987), it is likely that the differences in the biodistribution between the two methods are due to the pH change and might be eliminated by buffering the solution prior to passing it through the tube.

CONCLUSIONS

The use of 'tinned' polyethylene or poly vinyl chloride plastic tubing was found to be a rapid, and convenient method for labelling Iigands having a wide variety of lipophilicities, with 99mTc. The labelling efficiency of the ligands was not related to their lipophilicity suggesting that this technique may be useful for labelling lipophillic and other difficult ligands such as those containing metals, which are incompatable with free stannous ions in solution. -82-

REFEREiNCES

Awaluddin, A., Jacobs, J.J., Boume, D.W.A., Maddalena, D.J., Wilson, J.G. and Boyd, R.E. (1987) Synthesis and characterisation of potential tumour scintigraphic agents. Int J Nucl Appl Instr Part A (in press).

Camin, L.L., Liteplo, M.P., Pratt, F.P. (1979) Solid phase reductants for the preparation of Tc-99m radiopharmaceuticals. J Lab Compds Radipharm, 16, 24-5.

De Schrijver, M., (1978) Method and kit for the preparation of radiopharmaceuticals. Australian Patent Specification 39680.

Horiuchi, K., Yokoyama, A., Fujibayashi, Y. et al (1981) Tin-adsorbed resin for the preparation of 99mTc-radiopharmaceuticals: Stable complex of "Tc-bleomycin. Int J App Radiat hot, 32, 47-52.

Maddalena, D.J. (1983) Tiscon, a BASIC computer program for the calculation of the biodistribution of radionuclide labelled drugs in rats and mice. AAEC/E572.

Maddalena, D.J., Snowdon, G.M. and Wilson, J.G. (198/) Structure-activity studies on some neutral lipophillic "mTc hydroxybenzylglycine (HBG) derivatives. Submitted for publication.

Pinkerton, T.C., Desilets, C.P., Hoch, D.J., Mikelsons, M.V., Wilson, G.M. (1985) Bioinorganic Activity of Technetium Radiopharmaceuticals. J Chem Ed, 62,965-73.

Pojer, P.M. and Baldas, J. (1981) Technetium-99m-labelled N,N- diethyl- dithiocarbamate - a non-polar complex with slow hepatic clearance. Int J Nucl MedBiol, 8, 112,14. -8 3-

Salehi, N., Lichtcnstein, M. and Pojer, P.M. (1987) Technetium-99m radio/%- armaceutical preparation by surface adsorbed stannous ions. J Nucl Med (in press).

Srivastava, S.C., Richards, P., Yonekura, Y. et a/.,(1982) Long-term retention of tin following in-vivo RBC labeling. J Nucl Med, 23, 91-2.

van Wyk, A.J., Fourie, P.J., Bekker, P., Wood, S., Smith, K., Gerber, T. and du Preez, J. (1986) Comparison of new 99mTc -adamantadine-IDA compounds with WmTc BAT brain agents, in Current Applications in Radiopharmacology Ed. M.W. Billinghurst, Pergamon, Sydney, p 27-34.

Volkert, W.A., Hoffman, T.J., Seger, R.M., Troutner, D.E. and Holmes, R.A. (1984) "mTc-propylene amine oxime (99mTc-PnAO); A potential brain radiopharmaceutical. EurJNuclMed,9,5U-l6. -84-

Table 1.

Tube Labelling Efficiency of some 99mTc Complexes

Complex Lipophilicity Labelling Efficiency

HBG -0.61 88 NO2HBG -0.13 96 Cl-HBG 0.39 96 Br-HBG 0.71 94 DiCl-HBG 1.06 96 DiBr-HBG 1.28 92

Lipophilicity measured as log octanol-water partition coefficient.

Table 2

Biodis tribution of 99mTc Complexes in Rats HMPAO Cl-HBG Pt Complex II

Tissue SnTart. Tube SnCl2 Tube NaBH4 Tube

liver 16.8(0.4) 15.8(1.1) 15.2(0.2) 4.2(0.1) 1.8(0.2) 1.8(0.3) kidney 2.7(0.2) 2.7(0.1) 11.9(0.9) 15.0(0.7) 9.4(0.5) 13.4(1.5) muscle 16.8(0.6) 16.6(1.0) 1.6(0.3) 1.8(0.2) 3.6(0.8) 5.2(1.1) blood 6.5(0.4) 6.3(0.1) 3.2(0.3) 2.9(0.5) 3.9(0.1) 5.0(0.5) urine 11.8(0.8) 14.0(0.7) 20.9(1.9) 29.1(2.6) 62.2(2.4) 56.9(5.6) gut 26.5(1.2) 26.7(0.7) 43.0(1.4) 34.6(1.7) 4.9(0.7) 4.2(0.5) stomach 1.8(0.2) 2.2(0.4) 0.2(0.0) 0.3(0.0) 2.3(0.0) 1.0(0.5) brain 0.8(0.1) 0.6(0.1) - - - -

%LE 99.0 99.0 98.0 96.0 90.0 98.0

* means (standard deviation) of % injected dose of groups of 3 rats at 1,2 and 3 hours post injection respectively.

% LE - percentage labelling efficiency. CELLS RADIOLABELri^ ACHIEVEMENTS, CHALLENGES AW PROSPECTS.

IOLIE WEININGER AND JACOB TRUMPER

SOREQ NRC, 70600-YAVNE, ISRAEL

REPURLICA -87-

BLOOD CELLS RADIOLABELING: ACHIEVEMENTS, CHALLENGES AND PROSPECTS

Jolie Weininger and Jacob Trumper, Soreq NRC, 70600-Yavne, ISRAEL

1. INTRODUCTION

The potential value of the use of radiolabeled cellular blood elements for noninvasive medical diagnosis has long been recognized. A radionuclide is useful for diagnostic nuclear medicine if it is a high intensity photon emitter with suitable energy for imaging , if it emits no or minimal particulate radiation and if its physical half-life is of the order of magnitude of duration of the study being performed. Ideally, for blood cell labeling, one would like to have available a number of radiopharmaceuticals, each of wich could be administered to the patient either in vivo or, if this were not possible, after being mixed with patient's blood in vitro in or^er to label a specific cell fraction. One would like a raHionuclidic label wich assures reduced radiation dose to the cells and possesses physical properties favorable for imaging. The labeling of the blood cells must be stable both in vitro anH in vivo. The physiral anH biochemical Droperties, as well as the in vivo function and behavior of the labeled cells, must be preserved. The label, once incorporated into the cells, should neither be released during the study, nor reused. All the above mentioned conditions must be achieved by simple, fast and yet very reliable techniques in order for the imaging agent to be accepted. Plasma is used as the labeling medium in order to preserve ceil viability. Since the labeling yields were only 50-60% at first, it was necessary to separate the piasrm-biund activity before injection. Together with the separation procedures come -88-

problems of maintaining the sterility and apyrogenicity of the preparation, and assuring the viability and normal physiologic function of the labeled cells. During the last ten years many of these problems have either been overcome or reduced in magnitude, A variety of different cell fractions can now be labeled with reasonable reliability and are widely used for imaging in specific disease states. These examinations have proved their usefulness in the more accurate diagnosis of various specific pathologic conditions. It is known that many pathologic processes tend to be characterized by infiltration or aggregation of one or another cell type, which is relatively specific to the process. The ability to label a particular cell type which will seek out a diseased area of the body should greatly expand the diagnostic capabilities of nuclear medicine by giving it specificity, the lack of which has generally been its major limitation.

2. RED BLOOD CELL LABELING.

Of all the nuclear medicine procedures that have come into use over the years, none has had as significant an impact as the widespread use of technetium-99m labeled red blood cells ( Tc-RBCs) as an imaging agent. Tc-RBCs have revolutionized the field of cardiovascular nuclear medicine, in particular, by making possible non-invasive evaluation of various heart parameters. Tc-RBCs are also widely used as a blood pool imaging agent for the visualization of different vascular abnormalities: vascular malformations, hemangiomas and G. I. — bleeding. The mass and life span of red blood cells may also be determined with ^mTc -RBCs. Intentionally demaged "mTc-RBCs permit the evaluation of splenic function in an optimal manner. The labeling of RBCs, compared with that of other blood cells, is facilitated by several factors such as: - 8 <) -

a) RBCs are the most abundant of all cellular blood elements b) They are relatively easy to separate and manipulate in vitro and are not especially susceptible to physical or chemical damage. c) In vitro they are less dependent on energy and nutritional requirements than the other blood cells. d) RBCc are relatively easy to label due to the presence of a variety of cellular transport mechanisms and of hemoglobin wi-^h its active metal binding sites. Radionuclidic 1abelin^sof blood cells can be divided into two main categories: cohort (or pulse labels) and random labels. Cohort labels bind to bone marrow precursors that appear after a few days as labeled cells of uniform age in the — circulation. These labels are useful for the study of cell production rates and survival. Most of the RBC labeling procedures developed so far and those in predominant use today involve random labeling (i.e. labeling of circulating cells of all ages)\ and use 99m-Tcr , 51 rCr , 111TI n or 68Gar , as radiolabels,. , , . .

3. "mTc-RED BLOOD CELL LABELING

A) In vitro Labeling Methods After Tc was recognized as the "ideal" radioisotope and became the radioisotooe of choice for nuclear medicine imaging, multiple efforts were begun to label RBCs with it. The solution of technical problems involved in the improvement of labeling yields of Tc-RBCs was closely related to progress achieved in understanding Tc-chemistry and labeling mechanisms. Very early it was recognized that Tc, as pertechnetate, moves in and out of the red cells almost freely and only reduced Tc species could bind irreversibly with the cell components. Labeling methods which did not make use of reducing agents had, therefore, only minimal success. It was suggested that the pertechnetate could be reduced efficiently in the cell by pretreatment of the RBCs with a reducing agent This method gave -9(1-

the first dependable results. Sn(11) compounds were amonq the •first used to reduce pertechnetate and are still the most widely used reducing agents of all those available. Complexes like Sn-glucoheptonate (l), Sn-citrate (2), Sn-pyrophosphte (3). or Sn-DTPA were used to pretreat. or to "tin" the blood before labeling with TC - pertechnetate. Recently, by using radioactive stannous, it was shown that the plasma competes with the red cells for the available stannous-tin. The effect cf the complexing agent on the percent distribution of Sn (11) in whole blood was reported (4). It was shown that, of the available stannous-tin, RBC-bound Sn (11) amounts there are about 50% for glucoheptonate, about 35% for citrate, about k% for DTPA and about \% for pyroDhosphate. The rest, the extracellular Sn(ll). is almost entirely olasma-bound. The pretreatment of the whole blood with microgram amounts of stannous ion, as Droposed by Smith and Richards (2) and confirmed later by Gutkowski et al. (1) and Weininger cc al . (5) made possible the development of very simplified and reproducible high labeling yield procedures. Small samples of the patients blood are generally incubated for a few minutes with the — Sn-complex. In different methods one or more consecutive washing steps follow. The blood is centrifuged and the olasma that contains tin not taken up by the RBC is removed before adding the Tc - pertechnetate, in order to assure constant high labeling efficiency. Several kit and non-kit preparation procedures which consistently provide labeling yields of 95-98/ have been published some 10 years ago (1,2,5). The Brookhaven National Laboratory (BNL.) procedure, later kit method, using Sn-citrate (2/kg tin) as reducing agent, is widely used clinically as well as in the study of mechanisms involved in in vitro labeling methods (2). The removal of the plasma before the "tinning" step, proved - to be a very efficient way of improving the labeling efficiency. -9 1-

A Sn-glucoheptonate complex was used in two Soreq kit preparation procedures: a) usin

In all the preparation methods several ^onserutive washings with saline solution and the removal of tin-olasma before labeling with pertechnetate, increases the standard labeling yield to almost 100%. Several centrifugations to obtain better separation between RBCs and plasma before incubation with the reducing agent, however do not significantly increase the subsequent labeling yield. Srivastava and co-workers(6) recently deve'opeH a new in vitro technique, based on the BNL kit. This technique selectively labels RBCs in whole blood without outside handling of the blood sample and without centrifugations or washing steps. This method yields quantitative (> 95'/.) labeling of red blood cells in 1 ml whole blood means of the following closed, one-vial system: To the vial containing the lyophilized stannous citrate mixture (50^g tin) are added successively the heparinized blood and 5 min later 0.6 ml 1'/. sodium hypochloritc and 1 ml k,k/ disodium EDTA. Finally, tho desired activity of Tc pertechnetate in 0,5 to 3 ml saline is added. Aftei \r, min of incubation at room temperature a labelinq yield of >957 is obtained. The addition of EDTA effectively reducer, to a minimum the stannous tin content of plasma trapped between the packed red cells, thus assuring good labeling yields. It was t coortod that when samples of very low hematocrit blood were labeled without the use of EDTA. poor labeling yields were obtained due to the plasma effect. This new one-vial "whole-blood" method utilizes chemical inactivation (oxidation) of thf e vt race 1 1 u 1 ar , plasma-bound staonous tin, an<~t thus obviates the need for cell seoaration prior to the addition of per t echnct a t c . Sin'-e the RBCs remain in their native ol asms environment and there is almost no handling or manipulation, the RBCs do not suffer damage associated with the label ing orocedure.

Since there is no transfer involved, a'-.ootic condii ionr. are easy yo maintain. Ii war, shown c^ner imenta 1 1 y, that when reducing aqenf co-if.ai ni nq 50 to 100/^q Sn (ll) arc- used, the -i<:'u'ar tin uptake i'-. measured in mi croqranii, i.e. aboLt ten time., n, ,hcr tr.jn in -93-

previous techniques. Thus, the drawbark of limited stannous tin capacity for handling pertechnetate samples with high 99-Tc content, is virtually eliminated. The Soreq optimized one-vial labeling method (7) for 2 ml whole blood uses as the reducing agent a Iyophilized Sn-glueOheptonate complex with a formulated tin content of 50—65 /l/g Sn(11). This is the same formulation which has been widely used as a kidney and brain imaging agent and has been proved to have a shelf-life of at least one year. A very dilute sodium hypochlorite solution (0.26'/.) is added to oxidize the extracellular tin, thereby avoiding the plasma effect, without the use of EDTA. By adding to the RBCs the wanted Tc activity, in a volume not exceeding 0,5 ml, and incubating for 15 min, the red cells are labeled with an efficiency of 98-99^;. Studies performed on the RBCs labeled by this method showed that the labeling yield is almost unaffected by the presence of OKidizi'n g agents in the generator eluate and also that the timing of the elutions does not have to be controlled. In cases when a bolus injection is needed, the whole activity may be obtained in about I ml volume by centrifugation of the vial in an inverted position an^ withdrawing the packed RBCs with a syr i nge . Testing in humans showed that high intravascular activity and prolonged stability of the label in the blood pool were obtained. The experimental results are in good agreement with a biexponential decay, where k0% of the activity is cleared with a half-life of 3 hours and 60'/ with a half-life of 60 hours. As almost no free pertechnetate was observed in vivo during the first six hours after injection, this preparation was proved to be suitable for the diagnosis of patients with acute or intermittent upper gastrointestinal bleeding.

Mechanisms: Srivastava and co-workers (k) undertook exhaustive studies to define the mechanisms involved in the in vitro red cell labeling methods. They studied factors such as: a) uptake and distribution of Sn (11) in blood components b) role of oxidation and chelation in the chemical inactivation of the extracellular Sn(11). c) uptake kinetics and distribution of Tc in tinned RBCs d) effect cf carrier "mTc e) effect of plasma and other suspending media f) sites of binding of Sn(ll) and "mTc within RBCs h) long-term retention of tinned RBCs following in vivo 1abeling. B ) In Vivo Label ing

Ir. vivo reri blood cell labeling, as first proposed by Zimmer 9m anrt Pavel (8), was crucial for the wi"He use of ^ Tc-RBCs,,, especially in nuclear cardiology. Studies were performed which determined the optimum time lag between injection of the Sn (11) and the Tc-pertechnetate (30 min), the optimal stannous ion concentration (10-30 ji/q Sn(ll)/kg body weight) and the ootimal stannous preparation. A} t hou eft no difference was found between pyrophosphate, diphosphonate and citrate in influencing the — efficiency of red cell labeling is pyrophosphate has become the most widely used ligand. Routinely performed examination have shown that about 70 - 90'/, of the injected dose is in the circulation during the first two hours after injection, and at least 95/> of the activity in the blood is RBC-bound. The biologic half-life of RBCs labeled in vivo was found to be similar to that of RBCs labeled in vitro (5). In vivo labeling is widely usert, despite variable labeling efficiency and urinary excretion which were reported. Also, the eventual uptake by the gastric mucosa and thyroid of free pertechnetate, reduces the quality of the imaging. Attempts have been made to understand the source of extravascular activity and to reduce it. Especially high extravascular radioactivity was observed after bolus injection of the pertechnetate. One possible explanation of this undesirable effect could be that the amount of technetium injected exceeds the binding capacity of the Sn-RBCs, permitting the uptake of the excess b the thyroid and - 9 IS -

the gastric mucosa. The quality of the gated cardiac images was improved by injecting the pertechnetate slowly, thus increasing the number of Sn-RBCs that come in contact initially with the Tc pertechnetate. Improvement of the quality of the image was seen when 1-2 ml blood, containing the Sn-RBCs, was withdrawn for about 30 sec into the syringe containing th".pertechnetate and the reinjected slowly. The image was also improved when the stannous content of the pyrophosphate kit used for pretreating the red cells is increased to exceed 1 mg Sn (11) (9).

C) In Vivo/In Vitro Labeling

A modified in vivo/ in vitro method for labeling erythrocytes was reported by Callahan et al . (10). Twenty minutes after the •injection of Sn pyrophosphate (Sn-PYP), 3 ml of blood is withdrawn, incubated with 99m-Tc pertechnetate, and reinjected after 10 minutes. The labeling efficiency of the method was shown to be around 95%, so that only 5% of the activity, injected as free pertechnetate, is available for extravasru1ar distribution. This method seems to combine the superior binding efficiency of in vitro labeling with the convenience of the in vivo labeling method. Effect of anticoagulants on RBC labeling efficiency. It was found that the use of ACD as an anticoagulant provides more efficient binding efficiency than heparin in all labeling methods, in vitro and in vivo/in vitro. The lower labeling efficiency obtained when heparin is used may be explained by the formation of Tc heparin complex in the presence of Sn (11). This complex undergoes renal clearance and raises the urinary bladder activity -96-

D) Summary of Similarities and Differences between In Vitro and In vivo Labeling of RBCs with Tc Pertechnetate

Red cells are pretreated with stannous tin in both in vitro and in vivo labeling. The amounts of stannous ion that provide optimal Tc-labeling are almost the same in both cases: 1 /tg Sn(ll) for the in vitro labeling of 3 to 10 ml blood and 0,7-2,1 fl$ Sn (11) for in vivo labeling of the apDroximately 5 liters of circulating blood. Very soeeial precautions must be taken in order to keep the stannous tin suitable for in vitro Tc-labeling even for very short periods. In contrast, for in vivo labeling, after the stannous is injected into the blood stream, it remains suitable for red blood cell labeling fc; a period of months. In in vivo labeling only a fraction of the available tin is taken up by the RCBs : the remaining extracellular Sn(ll) in the plasma competes with the red cells for the Tc, depressing the label in yield. Therefore it becomes necessary either to remove physically or to oxidize the non-eel 1-bound tin before the addition of pert<^chnetate. On the other hand the non-eel 1-bound tin does not affect the in vivo labeling efficiency. Any time during the first 24h after Sn-PYP injection, in vivo labeling yields about 98% of the activity bound to the RBCs. The same ligands are used for the Sn-preparations in both methods, and in both cases do not influence the labeling efficiency: the determining factor is the amount of available atannous-t in. The choice of anticoagulant, the carrier Tc and other oxidants possibly present in the Tc pertechnetate seem to influence the labeling efficiency. The potential effect of patient medication on the quality of blood DOOI studies was recently reDorted. but further studies are nc-ccssary to pinpoint such interference more accurately. -97-

Patient radiation dose. Average radiation doses to the whole body, as well as to various organs, have been calculated The whole body dose, 20 mrad/mCi, is lower than the radiation dose to many other organs (30-70 mrad/mCi). This difference is due to the lower average blood content of the whole body compared to the listed organs.

Table 2.

Patient radiation doses for normal and for heat damaged RBCs(ll)

Organ Dose (mrad/mCi) Normal RBCs Heat damaged RBCs Whole body 20 20 Heart 78 Spleen 50 2.870 Liver 70 11 Blood 55 27 Lungs 56 Kidneys Sk Red marrow 33 -98-

.k. LABELING OF PLATELETS AND LLUKOCYTES

LipophMic cheiates of In with oxine , aretyl acetone, tropolone and mercapr.opyr idine N-oxide (Merc) have been used successfully for labeling platelets and leukocytes. These methods require prior cell separation from relatively large samples of autologous blood. In vitro agqrogaticn end in vivo viability of cells are better preserved when the labeling procedure is performed in autologous plasma. However,due to the cheiation of In with plasma transferin and other 1ipoproteins,the labeling in plasma is generally inefficient. Efficient labeling can be achieved in nonplasma media, but only at the expense of eel 1 viability. Various investigators have modified nonplasma labeling techniques so as to allow efficient incorporation of radioactivity, while minimizing adverse effects on cell viability and making the procedure convenient for routine handling. Different salt balanced media and different proportions of various anticoagulants are used for cell separation with different centrifugal forces. All these factors affect cell viability. Recently Thakur and colleagues (12,13) published kit procedures for the labeling of both platelets and leukocytes with In with negligible alteration of in vitro aggregabi1ity, A water soluble sodium salt of Me-rc is used to chelate In, permitting relatively high incorporation of the radioisotope in both platelets and leukocytes in plasma. The labeling yield is dependent on pH, Merc concentration and cell concentration. The blood cells were labeled using either preformed In-Mere or first incubating the cells with dry Merc and then with In. The latter method provided simple kit procedures that label both platelets and leukocytes with better efficiency than methods using either oxine or tropolone. In this procedure platelets were first incubated with 2 .5 ytl9 dry Mere and then labaled with In. The same dry Merc concentration can be used for optimal labeling -99-

of leukocytes when ACD is the anticoagulant. When instead of ACD, heparin is used, 20-^g Merc is necessary for optimal labeling. The reason for the need for this larger amount of Merc is not clearly understood. One explanation may be the formation of disulfides which cannot bind metal. Not more than 10 IU heparin/ml blood are used and generally a labeling efficiency of >75/ is achieved. The use of heparin as an anticoagulant for the labeling of leukocytes is imDortant because it reduces the contamination of the preparation with radioactive Dlatelets from about 30% to <3/. The radioactivity associated with erythrocytes remains <2'/,. The use of dry Merc has several advantages: the need to prepare In-Mere before proceeding to the cell labeling itself is eliminated thereby avoiding problems related to the quality control of this comdex. The labeling method as described is very simple and fast. The use of Merc instead of cither oxine or tropolone results in a much higher labeling efficiency in the In labeling of both platelets and leukocytes (Fig. 2, Table. 3) In dogs, platelets labeled with Merc in autologous PPP (platelet-poor-plasma) had a normal survival time (7«5 days). Twenty four hour old vascular thrombi were detected *+0 min after injection of labeled platelets, the thrombi/blood ratio being about 60. Forty-eight hour old pulmonary emboli (PE) could be detected by scintigraphy 90 min after injection, the PE/blood ratio being about kS. Leukocytes labeled by this procedures clear rapidly from normal lungs and migrate to abscesses providing abscess/blood rations of about 75,2*+ hours after injection. The liver uptake in this case is much reduced as comoared to cells, labeled with In-oxine, possibly due to better preservation of cell viability, i.e., Merc does not alter the phogocybi1ity of neu t rophi1os . The availability of kit procedures which nllow efficient labeling of human platelets and leukocytes in . >1 Tuna by a simple I 00-

and convenient method increases the potential value of radioiabeled platelets and leukocytes for noninvasive diagnosis,

5. SPECIFIC CELL LABELING USING MONOCLONAL ANTIBODIES

Labeling of a specific cell population in whole blood samples would eliminate the possible damage from cell separation procedures and keep the cells in their native plasma environment throughout the labeling procedure. A very promising approach is the labeling of cells with monoclonal antibodies directed against specific cell antigens. Various investigators have already started to search for cell specific antibodies (MAb) and to develop optimized methods 123 for in vitro labeling with various radionuciides such as lT« 12 5 131 111, 99m IT , IT , In anHd TTc. Factors such as the nature and amount of oxidant, reaction time substitution levels and specific activity affect not only the chemical labeling yields, but also the specific binding of the labeled MAb to cells in the whole blood, and the blood clearance of the label. Radiolabeling frequently alters the biological behavior of MAb and compromises the specificity of binding to the in vivo antigens. Sensitivity of the labeling chemistry is quite variable for different antibodies. In order to achieve maximum efficiency in imaging and/or in therapeutic applications, individual antibodies may require careful specific optimization of the labeling procedure using different radionuclides. Srivastava and co-workers (14,15) investigated Coller's 7E3 (16), an anti-platelet monoclonal antibody, which belongs to the IgG subclass and is directed against the fibrinogen receptor on the platelet surface. This antibody inhibits ADP-induced platelet aggregation and the ADP-induced binding of fibrinogen to platelets. The antibody was chosen for a pre-c!im"cal study in dogs because it cross react? with dog platelets. Studies were carried out to check various factors affecting the binding to platelets of 7E3 following labeling with I, ' Ji and I, as -10 1-

ii ri. ui^« iui- • .L '''in and Tc. New well as after chelation labeling witJh modified methods for labeling and characterization of the Mao were recently reported and their usefulness for general antibody labeling was disrussed (1?). Both iodinated 7E3 and indium labeled 7E3 displayed qreater binding soecificity in fibrinogen coated bead assay. The binding of radiolabeled JE1 to dog platelets at various levels of saturation of the binding sites showed that when 0,1-1 yu. g was incubated per ml dog blood, 75- 5% °f the activity became bound to the cellular fraction, of which >95% was associated with platelets. Twenty to 30% of the label remained in the plasma with >90% of it being found to be associated with the IgG fraction. Experiments with human whole blood showed that >90% of the activity was platelet-bound. When blood was clotted by adding thrombin, 80-90% of the activity was incorporated in the clot despite repeated washing. Fifty percent of the maximum binding took pla<~e at 6 - 8 min: the maximum was observed at kS - 60 min. When blood was incubated in - vitro with In-7E3 (100-150ml blood, 100/tg antibody) and the separated resuspended cells were reinjected, clots were visualized despite overlying herat blood pool activity- Three hours after injection the clot/blood ratio was about 15. Thus it was demostrated that the 7E3 antiplatelet monoclonal antibody binds hicjhly selectively to human and to dog platelets in whole blood samples and that it can be radiolabeled with high specific activity without loss of binding efficiency to platelets. For this reason radiolabeled 7^3 is considered as a promising agent for the in vivo localization of thrombi and vascular lesions. Several advantages of the antibody technique over conventional cell labeling are obvious: the antibody can be iodinated or labeled with metallic nucl ides and imaging is possible quite sooon after injection (5-30 min for venous thrombi with I-7E3-D1atelets as compared to 60 min for In-oxine platelets) and at the same time the clot-to-blood ratios were approximately twice that from conventional labeling. - 102-

Recently Som and co-workers (16) published a study about MAb 50H. 19, which reacts with human platelets. This MAb was converted to fragments, pretinned anrl made into kits for subsequent radiolabelinq with Tr. The antibody, which cross-reacts with dog platelets, was used to evaluate in vftro binding to blooc clots and in vivo experimental thrombi in dogs. The results of this study are summarized in Tables k and 5. ExDerimental thrombi in dogs could be visualized consistently within 2-3 hours post injection in peripheral veins and arteries, pulmonary arteries and the rigth ventricle. The advantages of this method may be summarized as follows: short and simple pre-imaging preoaration and rapid visualization of thrombi with no need for blood-pool subtraction or delayed imaging. The clinical effectiveness of this procedure still remains to be established. - I 0 3 -

REFERENCES

1. Gutkowski R.F., Dworking H.J., Porter W.C. et al., J Nucl. Med. 17 (1976), 1113. 2. Smith T.D. and Richards P. J. Nucl. Med. 17 (1976) 126-132 3. Stiegman J. and Richards P.. Setn in Nu<~l . Med. k (197*0 269- k. Srivastava S.C., Straub R.F., Meineken G.E. and Gil M.C., Current Applications in Radiopharmacology, Pergamon Press (1986) 166-183. 5. Weininger J., Trumper J., Lubin E. and Abrashkin S., Int. J. of Nucl. Med. Biol . 5 (1976) 190-195. 6. Srivastava S.C., Babich J.W. and Richards P., J Nucl. Med. ?-h (1983) P 128. 7. Trumper J., Weininger j. and Lubin E., Proc. .IV World Congress, Buenos Aires, 1986, i+79-P. 8. Pavel, D.G., Zimmer, A.M. and Patterson V.N., J. NuH. Med. 18 (1977) 305-308. 9. Lubin E. Unpublished results (1977). 10. Callahan R., Froeiich J.M., McKusick K.A. ei al., J. Nucl. Med. 23 (1982) 315-318. 11. Srivastava S.C. and Chervu L.R., Sem. in Nurl. MeH. 1 k (1 98^4) 68-82. 12. Thakur M.I., McKenney S.L. and Park C.H., J. Nucl. Med. 26 (1986) 510-517. 13. Thakur M.I., McKenney S.L. and Park C.H., J, Nucl. Med. 26 (1986) 518-523. Ik. Srivastava S.C., Straub R., Meineken G.E. et al., J. Nucl. Med. 2 5 (1986) P65. 15. Srivastava S.C., Meineken G.E., Scudder L.E. and Coller B.S. J. Nucl . Med., 26 (1985) P*+5. 16. Coller B.S., J. Clin. Invest., 76 (1985) 101-108. 17. Som P., Oster. H., Zamora P.O. et al., J. Nucl. Med. 27 (1986) 1315-1329. - I 04 -

Fig 1 The effect on the label ing yield of number of Tc atoms. On the upper abscissa, the number of Tc atoms is exoressed in terms of ingrowth period calculated for 10 mCi activity (5).

g Fig. 2 -v9xl0 Platelets suspended in 0,5 ml PPP were incubated either with Merc, oxine; or tropolone (dry), and labeled with In. Results indicate that Merc was agent of choice (11).

Table 1 Tc-RBCs labeling yield.1; as a function of oxidant quantities present in the pertechnetate solutions (5).

Table 3 Leukocytes labeling yields with In using dry Merc, oxine, and troDolone (12).

Table k In vitro performances of mTc 50H. 19 (16). - I 0 5 -

Ingrowth per 100, f»'

.'0 24 10 1 1 1 1 1 1 1 * *=±:

\ + • \

• • .„„,. -• KIM- A -Iwe R 1

Tc utomi

In. i I IK tiled > if i (tic- labeling yield «f Ihc number of Tc aioms. On the upper abscissa ilk- mimrvr •.>f (c. .Hums is expressed in terms of ingrowth period calculated for lOmC'i activity

101! p Fig. 2

~9 X )09 Platelets suspended in 0.5 ml PPP A were men bated either with (•) Merc; (B) oxme; or (•) tropolone (ri:v| and labeled with 1 "In by lollowing Method II. Results indicate that Merc was agent of choice I Ub-

I Mil i ' '' " f i KH( •. I ,!•• 11 :• \ ,-U •, .; . , ,,.. :r. •' I.\I l.irl . •! ...Jilii-

pri .en in ilic (H-I'L'I 'IIK'1.it. •.».nil i.in .

On n.v * l'i.K.\liiir \ I'I.K\.!I.I. ii

( t VMJI/itli- .i^\ u[ I in'l ( • • 1

\1n<), MIU 'i< ; '» ' i>

il (i \)w '."ii '/ IK> >i 11) •;'• ii

( I •()•-' \\\\ 'y.-v *•

DM. 4711 '\<)

•One niKTi>>;r.iin .>! Sir' i. iqii!\.ilc.-nt li' .' .•> ;IJ: Mud, U'sMnnmi: a wilcm-y ch.ui>.'..- ol M..' ' i,. M,.' i. 0 2S\ ,/L- H .'<).,. I I: ne « r,O. •'

TABLF: 5

LoukocyiBs Labelino Yields with '"In Using Dry Merc. Oxbrte, and Tropotone fiQ More Ox.ne T'opolcne

10 <6 ± 4.5 6.9 i 2 3 6 7 i .1 : 2b 96 ± 2.3 10.4 i. 2 ." ?3i i ? SO 55 dt 5 7 5.6 t 2 I 8 2 * .I 9

* 60 million lw*ocytee in 0 5 ml plasma were first xx iXate'S wrth the agent and tt<«n wilh dry " 'In

TABLE: 4

Technetium-99m 50H 19 Binding to Cellular Blood

Frc-iction

!:) viUo In vivo

OiiM"oi Miasma ('-) GPI-S ("••.) PUT.ma Co]

Dogs' 57 G » ? 9 -V :> ' ? '.' 3b.2 .' 5 -4 43.8 >: ->4 Humans' <•, 1 ." » ? 1 .iH H • ?i

' n =r^ 4

' n •••• 8 Faoh point n-pf." i'Mt' m.^.-ir. • -..1

TABLKj 5

Technetium-99m 50H. 19—Ciot

lo vi': o

Percnnt binding to clot i-'ercent fcn

Dogs' 57.2 + 1.9 69.9 - ^ 7 Humans' 74.G x 5.

0 = 4.

Each point represents mo an ± S (1 THE SYNTHESIS WH PROPERTIES OF 1-(4-

A NOVEL RADIOSENSITIZER FOR USE AS V! HYDOYJC CELL MARKER,

L.I.WIEBE*, D.C.JETTE*, .1.^.MERCER*, ^.SAMUEL*, 7..I.PLANARAN*. '.LEE**, n .E.MEEKER** AND J.T1, CHAPMAN**

* FACULTY OF PHARMACY AND PHARMACEUTICAL SCIFNCES, MNIVEPSITY OF ALPFPTA^ FDMONTON,, CANADAJ TBH ?y% ** DEPARTMENT OF RADIATION ONCOLOGY, CROSS CANCER INSTITUTE, AND DEPARTMENT OF RADIOLOGY AND

fllAHNOSTIC iMAniNP "NIV^ITY OF AL^ERTA, EDMONTON CANADA, T6H 1Z2.

BUFMOS AIRES REPURLICA ARGENTINA The Synthesis and Properties of i-(4-Iodo-5-nitroimidazolyl)-2-hydroxy-3-methoxypropane: A Novel Radiosensitizer for use as an Hypoxic Cell Markor

L.I. Wiebe1'2, D.C. Jette1, J.R. Mercer1, B. Samuel1, R.J. Flanagan1, J. Lee3, B.E. Meeker3 and J.D. Chapman3

1. faculty of Pharmacy and Pharmaceutical Sciences, University of Liberia, Fdmonton, Canada, T6H 2N8

2. Correspondence

3. Department of Radiation Oncology, Cross Cancer Institute, and Department of Radiology and Diagnostic Imaging University of Alberta, Edmonton, Canada, T6H 1Z2 - 109-

ABSTRACT l-(4-Iodo-5-nitroimidazolyl)-2-hydroxy-3-methoxypropane (4-I-5-NHMP) has been synthesized and radiolabelled with I for use as an j_n vivo marker of tumor hypoxia. 4-I-5-NHMP was found to be a potent radiosensitizer of hypoxic EMT-6 cells in culture, being 5-10 times more potent than misonidazole (MISO). The oxygen enhancement ratio (O.E.R.) for these cells at 10% survival was 2.85. Cytotoxicity studies with cultured EMT-6 cells produced 50% growth inhibition at 0.07 mM, a toxicity about 50 x greater than MISO toxicity under identical conditions. However, studies of the uptake of ^M-labelled 4-I-5-NHMP in both aerobic and hypoxic EMT-6 cells showed no metabolic dependent or hypoxia-specific uptake of the radiolabel. HPLC examination of the culture medium indicated that rapid metabolic deiodination took place in both oxygenated and hypoxic cell cultures, and that nit'o-reduction occured under hypoxic conditions. These data suggest that a deiodinated metabolite of 4-I-5-NHMP, rather than the parent compound, is the radiosensitizing species. In scintigraphic studies, uptake of radioiodide by the thyroid was consistent with rapid in vivo deiodination after i.v. injection of 4- I-5-NHMP into a normal Spraque-Dawloy rat or into f^DgFj mice baaring sub-cutaneous implanted Lewis lung carcinomas. - I 10-

INTR0DUCTI0N The effect of cellular oxygen concentration at the time of radiotherapy on the curability of human cancers was first reported by Gray et al1. Thorn!inson and Gray^ showed that specific histologic features, and in particular, necrosis, may correlate with the presence of treatment-resistant hypoxic cells. Several techniques for overcoming the radioresistance of hypoxic tumor cells to low LET irradiation have been investigated, one of which is the use of oxygen-mimetic, electron-affinic chemicals such as nitroimidazoles in combination with radiotherapy^'^'^. Misonidazole (MISO), a 2-nitroimidazole, has been widely studied in experimental radiation oncology as an hypoxic cell radiosensitizer, but the total drug dosage which can be tolerated (12 gm c) is limited by neurotoxicity . These clinical investigations were based on both tissue culture and in vivo experiments which address mechanisms of radiosensitization, mechanisms of drug cytotoxicity, drug metabolism, pharmacokinetics, and the selection of superior compounds for clinical application7'8'9-10. The hypoxic cell radiosensiti/er, MISO, becomes bound to components of hypoxic cells when metabolized by mammalian cells*1'-' jl3,14 Preliminary distribution studies in tumor-bearing mice have indicated that except for liver, the amount of C from C-M1S0 retained in tumor tissue after clearance of the unmetabolized drug was greater than that retained in other tissues . It was consequently suggested that the tumorrtissue ratios of MISO adducts might be large enough for detection of hypoxic regions within neoplasms by non-invasive procedures of nuclear medicine* after the drug was labelled with an appropriate gamma-emitting radionuclide . Several studies describing the synthesis, toxicity, sensitizing potenrv specificity of uptake - ] I I -

by hypoxic cells, biological activity and whole-body distribution of 2-nitroimidazoles labelled with radioisotopes of bromine and iodine have been sported17'18'19'20. The radioiodinated nitroimidazoles tested to date have had undesirably high 1ipophilicity or have b^en susceptible to j_n vivo dehalogenation. In an effort to circumvent these properties we have synthesized l-(4-iodo-5-nitroimidazolyl)-2-hydroxy-3-methoxypropane (4-I-5NHMP) and have tested it as a marker for hypoxic cells in vitro and vn vivo.

MATERIALS AND METHODS All reagents and solvents were; reagent grade and used without further purification. Thin-layer chromatocjraphic (TLC) analysis was carried out on silica gel micro plates (Whatman MK6F) using chloroform:methanol=4:1. Preparative thin-layer chromatocjraphy (PTLC) was performed using Whatman siln.a

li(jiii(i i liroma i (xjr'tiph ic (HPI C) analysis was carried out using a Waters HPLC system with a <. 18 reverse phase column eluted with 20% methanol in water at a flow rate of 2.0 ml min . Radioactivity in HPLC eluants was measured using a Nal(Tl) crystal flow detector in series with a UV detector. Silica gel for flash chromatography (Merck 9385; 20-40 microns) was eluted under pressure with methanol:chloroform-5:95. The H nuclear magnetic resonance (PMR) spectra were measured in DMS0-D6, on a Brucker AM300 Spectrometer. Ultraviolet absorption spectra were recorded on a Unicam SP 1800 spectrophotometer usinq water as solvent. Mass spectra were obtained with a Hewlett-Packard 5995A gas chromatograph -mass spectrometer using the DIP mode. All melting points are uncorrected. - 1 1 2 -

Synthesis 4,5-Di-iodoimidazole was prepared using a modification of the method of Pauley . Imidazole (20g) was dissolved in water (900 mL) which contained concentrated NH4OH (2.0 mL). Iodine (40 g) was added in 10 g portions, with 24h between additions. The precipitate which formed was filtered and washed with a saturated solution of potasium iodide, then redissolved in ethanol (200 mL). The crude iodoimidazole was reprecipitated from the ethanol by adding cold water. After filtering and washing with water the precipitate was air dried. This produced 29 g of a crude mixture of iodinated imidazoles. Di-iodoimidazole was separated from the crude mixture using flash column chromatography. 4(5)-Iodo-5(4)-nitroimidazole was prepared using the method of Hoffer e_t a_l . Di-iodoimidazole (5 g) was dissolved in a mixture of concentrated sulfuric acid (12 mL) and concentrated nitric acid (12 mL) at 0°C. The mixture was allowed to warm to room temperature and was stirred overnight. A precipitate was formed by pouring t.he nitrating solution over 1.5 volumas of ice/water. After washing the filtrate with saturated aqueous potassium iodide the iodo-nitroimidazole was recrystalized from ethanol and water. Yield 45% and mp. 280°C compared with another published report of 281°C23. 1 (5-lodo-4-nitroimidazOlyl)-2-hydroxy-3-methoxypropane (5-I-4-NHMP) was synthesized by reacting 1,2-epoxy-3-methoxypropane with 4(5)-iodo-5(4)- nit.roimidazole in the presence of potassium carbonate in refluxing ethanol, using the coupling method reported by Beaman et al . The product was recrystallzed from ethanol after purification by flash column chromatography. Yield 70%; MS(DIP, El, 70eV) 327.10 (M-t,8.3). This me! hod produced two isomers which were determined to be l-(4- iodo-5-nitroimidazoly: ' 7-!i.ydroxy~3~ 3-

methoxypropane (4-I-5-NHMP) and l-(5-iodo-4-nitroimidazolyl)-2-hydroxy-3- methoxypropane (5-1-4-NHMPj by PMR 25,26 j^ese isomers were separated using flash column chromatography, and purified by fractional crystallization and were investigated separately. PMR(DMSO-D6) 4-I-5-NHPH: 8.00 (s,lH, ring

proton), 5.28 (d,lH,-OH), 4.56 (dd,lH,NCH2), 4.21 (dd,lH,NCH2), 3.89

(m,lH,CH-0H), 3.33 (m,2H, CH2-0-), 3.28 (s,3H, 0CH3); mp 121-122°C, lit26 mp 121-122°C; UV max 324 nm. PMR(DMS0-D6) 5-I-4-NHPH: 8.05 (s,lH,

ring proton), 5.38 (d,lH,-OH), 4.22 (dd,lH,NCH2), 4.03 (dd,lH,NCH2), 3.97 26 (m,lH,CH-0H), 3.35 (m,2H, CH2O), 3.32 (s,3H, OCH3); mp 146-148°C, lit mp 143-144°C.

Radioiabelling Procedure The "melt method"2' was used to introduce radioiodine by halogen exchange. Na12^I (30 MBq; no-carrier-added) in ethanol (20 uL) was dried in a Reacti-vial (Pierce) and unlabelled 4-I-5-NHMP (1 mg) and pivalic acid (10 mg) were added to the vial, which was then sealed and heated (155 °C) for 30 minutes. After cooling, the residue was dissolved in ethanol (0.1 mL) and the final radiolabelled product was purified on PTLC. Radiolabelling yields were determined by HPLC. Elution times for the isomers were 14.7 min and 20 min at 2 mL min" . The radiochemical purity of the final product, as determined by HPLC, was greater than 95% and did not change with time (48 hours).

Partition Coefficient (P) Test compounds (50 ug mL"^) were dissolved in 0.05 M phosphate buffer (pH 7.4) and shaken for three hours with equal volumes of water and octanol. The concentration of sensitizer in the aqueous phase before and after octanol - I 14-

partitioning was determined by UV analysis at 324 nm, with reference to a calibration curve (Beer's plot) for the compounds over a concentration rangi* of 5-50 ug mL~*. Partition coefficients were calculated as ratios of concentration in octanol to concentration in aqueous phase.

Protein Binding (%PB) Test compounds (50 ug mL"^) were incubated under aerobic conditions for 1 h at 37°C with normal human serum albumin (Connaught) diluted to 5% w/v with tris-buffered saline. Ultrafiltration (Amicon.YMT filters) at 2000 x g for 20 minutes was used to separate free drug from protein-bound drug. The absorbance at 324 nm was used to determine drug concentration in the filtrate. Radiolabelled compound wa^ aiso used to determine protein binding. In this case the radioactivity in the filtrate was used to calculate drug binding.

Measurement of Tumor Cell Adduct Formation Murine EMT-6 fibrosarcoma cells were cultured as monolayers and transferred twice weekly in Waymouth's medium complete with 10% fetal calf serum. Cells grown to near confluence on 150 cm^ flask were trypsinized, centrifuged and resuspended in Spinner minimal essential medium (MEM) (Mg6 and Ca*- free) which contained 7% fetal calf serum. Cells (5 x 10° cells mL~*) were transferred to glass chambers designed to allow for control of cell culture temperature and oxygen tension in the gas phase . The cell suspensions were gassed (1L min ) with humidified mixtures of 95% air plus 5% CO2 or with 95% ultra-pure nitrogen (oxygen content less than 5 ppm) plus 5% CC>2. Solutions of sensitizer in sterile water were prepared at 20-50 x the final desired concentration and were added in small volume, ... the cell suspensions -\\b-

in the chambers. At various times, samples of the cells were removed through the sampling port and added to 10 volumes of cold 5% trichloroacetic acid to precipitate the macromolecular fraction. Samples were maintained on ice until they were filtered through cellulose acetate filters (Sartorius; pore size 0.45 um), held in a Millipore sampling manifold, to collect the macromolecular fraction. Filters were washed twice with cold 5% TCA, placed in scintillation vials and the I activity was determined with a gamma-counter (Beckman Model 8000). Samples of medium were analyzed by TLC (at various times during the incubation) for intact radioiodinated sensitiser. HPLC analyses were performed on the media after the incubation was complete. Samples of media were passed through ultrafilters (Amicon, YMT filters) to remove cells and protein prior to HPLC analysis.

Hypoxic Cell Radiosensitization The radiosensitizing activity of 1 -(4-iodo-5-nitroimidazolyl)-2- hydroxy-3-methoxypropane (4-I-5-NHMP) was assayed using EMT-6 mouse fibrosarcoma cells which were cultured and maintained as monolayers in HEM supplemented with 10.5% fetal calf serum and antibiotics. Cells in logarithmic growth in culture flasks were trypsinized and resuspended in Spinner MEM (Ca2+ and Mg^+ free) to which 6.2% fetal calf serum and antibiotics were added. Cell suspensions (10 cells mL ) were transferred to glass po _ i irradiation chambers'10 and made hypoxic by gassing at a rate of 1 L min l for 60 minutes with ultra-pure nitrogen plus 5% CO2 or aerobic by gassing with air and 5% C02- A 4-I-5-NHMP solution was prepared at 20-50 x the desired final concentration in the culture medium and added to the cell suspension 10 minutes prior to irradiation. The cell suspensions were 16-

irradiated at both 22°C and 37°C, with or without drug present, with gamma-rays from a Cs irradiator. The average dose rate in the liquid volume of the irradiation chamber was 1.4 Gy min , determined by Fricke dosimetry. An aliquot of the cell suspension was removed through the vessel sampling port after each dose fraction, diluted in complete MEM and plated in MEM (5 mL) in 60 xI5 mm plastic petri dishes. Cultures were incubated at 37°C for 6 days in a humidified air plus 5% CO2 atmosphere and then stained with methylene blue prior to enumeration of colonies. Radiation survival curves were constructed from surviving fractions determined at several doses of radiation.

Measurement of Drug Toxicity Cytostatic toxicity of 4-I-5-NHMP towards murine EMT-6 fibrosarcoma cells was measured using replicate culture samples containing approximately 10 cells. These were plated in 60 x 15 mm plastic dishes and incubated overnight, a procedure which resulted in exponentially proliferating cultures. The medium from these cultures was aspirated and replaced with warmed medium containing differing concentrations of test drug. After 24 hours incubation in the presence of the drug, the medium was removed from the cultures, cells were trypsinized to remove them from the plastic dishes and total cell number per petri dish was determined by Coulter counter measurements. Cell proliferation data in the presence of sensitizer were expressed AS the fraction of the cell increase observed in the control (absence of drug) .

RESULTS AND DISCUSSION The procedure used for nitration of di-iodoimidazole was first reported to 9 9 or produce 2- iodo-4(5) -ni troimidazole , but. subsequent woc/v has indicated - I I 7-

that the correct structural assignment is 4(5)-iodo-5(4)-nitroimidazole. Careful analysis of our PMR data support the assignment made by Dickens et al . No 2-iodo-4-(5)-nitro-imidazole derivative was formed using this synthesis. The coupling procedure for introducing the N-l side chain produced two structural isomers. The structure of these two compounds was assigned on the basis of mass spectrometry and PMR data. These data and the reported PMR spectra for iodinated nitroimidazoles"' support the assignment of two isomeric structures, l-(4-iodo-5-nitroimidazolyl)-2-hydroxy-3-methoxypropane (4-I-5-NHMP) and l-(5-iodo-4-nitroimidazolyl)-2-hydroxy-3-methoxypropane (5-I-4-NHMP), but are not in total agreement with PMR spectra reported for these compounds .

In our analysis the N-CH2 protons appear as two separate well resolved signals demonstrating both geminal coupling (J=13.7 Hz) and coupling to the adjacent methine proton. In addtion, the N-CH2 protons are somewhat deshielded by the 5-nitro group in 4-I-5-NMPH leading to a downfield shift of these signals relative to the equivalent protons in 5-I-4-NMPH. l-(4-iodo-5-nitroimidazoyl-2-hydroxy-3-methoxypropane (4-I-5-NHMP) was selected for further study. The partition coefficient of 4-I-5-NHMP was foun'd to be 1.97 +0.1 (n=12) compared to a value of 2.6 reported by Gupta et al^° and the plasma protein binding was determined to be 17.2 + 5.7 (n=12) percent. These values are higher than those for MISO but very similar to those for IAZR19 (Table 1), and fall within the range thought to be necessary for effective radiosensitizers.

The radiosensitization of EMT-6 cells by 0.1, 0.? and 0.5 mM 4-I-5-NHMP is shown in Figure 1. Sensitizing enhancement ratio studies (at 10% survival level; O.E.R. 2.85) show 4-I-5-NHMP to be 5 to 10 timrs :;ore potent as a - I 18-

hypoxic cell radiosensitizer than misonidazole (MISO). The single electron reduction potential of 441 mV reported for this compound would suggest thai 4-I-5-NHMP should be a less potent sensitizer than MISO; this unexpectedly high potency may be due to the relatively high toxicity of 4-I-5-NHMP. The concentration of 4-I-5-NHMP required to inhibit EMT-6 cell proliferation in vitro to 50% of untreated controls was 0.07 mM (lit26 0.48 mM). 4-I-5-NHMP is therefore approximately 50 times more toxic than MISO (3.9 mM for 50% inhibition) and 1.4 times more toxic than IAZR19 (Table 1). incubation studies using a mixture of 4-131I-5-NHMP and 5-131I-4-NHMP in aerobic and hypoxic EMI-6 cultures showed a small uptake of radioactivity after 15 and 30 minutes of incubation. At longer incubation times the amount of radioactivity remained low and eventually decreased slightly. TLC analysis of the culture medium revealed that these compounds were being de-iodinated by EMi-6 cells (Figure 2) under both aerobic and hypoxic conditions. Little dehalogenation was seen under either aerobic or hypoxic conditions when the incubation medium contained no cells, confirming the occurence of metabolic deiodination. The radiochemical purity remained greater than 88% during these control studies. Similar results were obtained using 4- M-5-NHMP alone. Analysis of the medium by HPLC failed to detect either 1-(2-hydroxy-3-methoxy) 5-nitroimidazole (4-NHMP) or l-(2,3-dihydroxypropyl)-5-nitroimidazole (desmethyl-5-NHMP), the compounds expected to be formed by metabolic de-iodination or de-iodination with 0-demethylation. 0-Demethylation is a major biotransformation reaction for Br-KlSO in this system . lhe chemical and radiochemical stability, low 1ipophilicity (partion coefficient) and moderate protein binding of 4-J- 5-NHMP indicated that this compound might be useful as a marker of hypoxic cell' 'ory little - I 19-

radioactivity was incorporated into the cells, indicating that the radiosensitizing properties of 4-I-5-NHMP might be due to a de-iodinated metabolite which is produced in the cell. Surprisingly, the simple dehalogenated products l-(2-hydroxy-3-methoxypropyl)-5-nitroimidazole (4-NHMP) and l-(2,3-dihydroxypropyl)-5-nitroimidazole (demethyl-4-NHMP) were not detected in the culture medium. This suggests that either dehalogenation takes place immediately after or during adduct formation, or that the metabolic pathway is more complex. Such a complex pathway might involve elimination or reduction of the nitro group simultaneously with dehalogenation. Although a reduction pathway would be plausible in hypoxic cultures, it is unlikley to exist under aerobic conditions, because the first reduction intermediate (nitro radical anion) is known to be stripped of its electron by molecular oxygen-* . Stratford et al c studied the sensitizing properties of an isomeric pair of imidazoles with similar substitution patterns. In their studies 4-iodo-5-nitro-1-methylimidazole and 5-iodo-4-nit.ro-l-methyl imidazole had Cjg values (concentration to produce sensitizing enhanced ratio of 1.6) values of 2.8 xlO"^ mol dm""* and 1.0 xlO'4 mol dm respectively, indicating that the 4-nitro compounds are more effective sensitizers than 5-nitro compounds, but both are less effective than 2-r,itro compounds. Gupta et al" report almost equal radiosensitizing potency for the two isomers (5-I-4-NHMP and 4-I-5-NHMP) despite a 2.5 x greater cytotoxicity, a 3-fold higher partition coefficient and a 56 mV greater single electron reduction potential for 5-I-*-NHMP. Very few in. vivo scintigraphic studies of the whole-body biodistribution and tumor uptake of radiolabelled nitroimidazole radiosensitizers have been reported to date. Tubis et al^'"^ demonstrated the uptake of a - I 20-

iOiI-labe11ed derivative of metronidazole by amebic liver abscesses using j_n vivo scintigraphy, without reference to the affinity of these compounds for hypoxic tissues. 4-Bromomisomdazole (4-Br-MISO) has been radiolabelled with cBr, and its radiosensitizing properties, metabolic debromination and metabolic O-demethylation i_n vivo in tumor-bearing mice have been demonstrated 1/j . Highly 1ipophilie iodinated derivatives [l-(4-iodophenoxypropyl)- 2-nitroimidazole; IPENI] of the radiosensitizer l-(2-phenoxyethyl)- 2-nitro- imadazole have also been labelled with * *I and studied in tumor-bearing mice. IPENI was resistant to dehalogenation i_n \QVO, but because of its high 1ipophi1icity it could not be adequately evaluated as a radiosensitizer in vitro . The synthesis of * F-labelled l-(2-nitro-l-imidazolyl)-3-fluoro- 2-propanol, which has potential as a hypoxic cell marker using positron emission tomography (PET), has been reported . Recently, 1-(5'-iodo-5'-deoxyribofuranosyl)-2-nitroimidazole has been synthesized and radiolabelled with 131^9,20^ j^e radiosensitizing properties and uptake in hypoxic cells in vitro, and the in vivo biodistribution of this compound have been investigated. Although it is a potent radiosensitizer with only moderate cytotoxicity, dehalogenation «f the sugar moity precluded sucessful in vivo application. The j_n vivo whole-body biodistribution of an i.v. injection (tail vein) of 4-[xJiI)-5-NHMP in a male Sprague-Dawley rat showed a general, almost uniform soft tissue distribution, with a gradual redistribution of actvity to the stomach and thyroid (within three hours). No other tissue was highlighted in thf- scintigrams, suggesting that deiodination was ra;.-id and complete. Similar distribution patterns were observed in B2O5F1 mice bearing subcutaneous, implanted lewis i ung carcinomas. Further in vivo st •.•-:• t.;. evaluate tn.i 2 1-

compound as a scintigraphic indicator of tissue hypoxia were not warranted because of this deiodination. Symptoms of neurotoxicity were apparent in this preliminary investigation. Rapid deiodination of 4-I-5-NHMP is in keeping with earlier observations with 4-8^Br-MIS0^ which also underwent extensive metabolic debromination of the imidazole ring. This rapid de(radio)halogenation is an important factor which limits the usefulness of these compounds as in vivo markers for hypoxic cells. An examination of the relative reactivities of the three carbon atoms in the imidazole ring30 suggests that halogenation at the 2-position v/ould provide compounds which would be more stable chemically and perhaps more resistant to biological degradation. However, their sensitizing properties and efficiency as hypoxic cell markers would have to be established.

ACKNOWLEDGEMENTS We wish to thank Carolyn Johnson for assistance in preparation of the manuscript. This work was supported by Alberta Cancer Board-Applied Research-Cancer Grant number H-161. - 1 22-

REFERENCES 1. L.H. Gray, A.D. Conger, M. Ebert, S. Hornsey and O.C.A. Scott. Concentration of oxygen dissolved in tissues at time of irradiation as a factor in radiotherapy. Br. J. Radiol. 26: 638-648 (1953). 2. R.J. Thomlinson and L.H. Gray. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer 9: 539-549 (1955). 3. G.E. Adams. Chemical radiosensitization of hypoxic cells. Br. Med. Bull. .29: 48-53 (1973). 4. J.D. Chapman, A.P. Reuvers, J. Borsa, J.S. Henderson and R.D. Miglior^ Nitroheterocyclic drugs as selective radiosensitizers of hypoxic mammal: cells. Cancer Chemotherapy Reports Part 1. 58: 559-570 (1974). 5. T.L. Phillips, T.H. Wasserman, J. Stetz and L.W. Brady. Clinical trials of hypoxic cell sensitizers. Int. J. Radiat. Oncol. Biol. Phys. 8: 327-334 (1982). 6. R.C. Urtasun, J.D. Chapman, M.L. Feldctein, R.P. Bond, H.R. Rabin, A.F. Wilson, B. Maynowski, E. Starreveld and T. Shnitka. Peripheral neuropathy related to misonidazole: Incidence and pathology. Brit. J. Cancer 3_7: Suppl. III. 271-275 (1978). 7. J.B. Chin and A.M. Rauth. The metabolism and pharmacokinetics of the hypoxic cell radiosensitizer and cytotoxic agent misonidazole, in C3H mice. Radiat. Res. 86: 341-357 (1981). 8. R.A.S. White, P. Workman and J.M. Brown. The pharmacokinetics and tumor and neural tissue penetration properties of SR-2508 and SR-2555 in the dog. An hydrophilic radiosensitizer potentially less toxic than misonidazole. Radiat.Res. 84: 542-561 (1980). 9. J.M. Brown, Y.N. Yu, D.M. Brown and W.W. Lee. SR-2508: A 2-nitroimidazole amide which should be superior to misonidazole as a radiosensitiser for clinical use. Int. J. Radiat. Oncol. Biol. Phys. I: 695-703 (1981). 10. I.J. Stratford. Mechanism of hypoxic cell radiosensitization and the development of new sensitizers. Int. J. Radiat. Oncol. Biol. Phys. 8:391-398 (1982). 11. J.D. Chapman, A.J. Franko and J. Sharplin. A marker for hypoxic cells in tumors with potential clinical applicability. Brit. J. Cancer 43: 546-550 (1981). 12. J.D. Chapman, K. Baer and J. Lee. Characteristics of the metabolism induced binding of misonidazole to hypoxic mammalian cells. Cancer Res. 43: 1523-1528 (1983). - 12 3-

13. G.G. Miller, J. Ngan-Lee and J.D. Chapman. Intracellular localization of radioactively-labelled misonidazole in EMT-6 tumor cells in vitro. Int. J. Radiat. Oncol. Biol. Phys. 8: 737-739 (1982). 14. A.J. Franko, J.D. Chapman and C.J. Koch. Binding of misonidazole to EMT-6 and V79 spheroids. Int J. Radiat. Onco]. Biol. Phys. 8: 737-739 (1982). 15. B.M. Garrecht and J.D. Chapman. The labelling of EMT-6 tumors in BALB/c mice with 14C-misonidazole. Brit. J. Radiol. 56: 745-753 (1983). 16. J.D. Chapman. The detection and measurement of hypoxic cells in solid tumors. Cancer 54: 2441-2449 (1984). 17. J.S. Rasey, K.S. Krohn and S. Freauff. Bromomisonidazole: Synthesis and characterization of a new radiosensitizer. Radiat. Res. 91: 542-554 (1982). 18. D.C. Jette, L.I. Wiebe and J.D. Chapman. Synthesis and in vivo distribution and metabolism of the radiosensitizer 4-( Br)bromomisonidazole in tumor-bearing and normal mice. Int. J. Nucl. Med. Biol. K): 205-210 (1983). 19. D.C. Jette, L.I. Wiebe, R.J. Flanagan, J. Lee and J.D. Chapman. Iodoazomycin riboside (l-(5'-iodo-5'-deoxyribofuranosyl)-2-nitroimidazole), A hypoxic cell marker. I. Synthesis and in vitro characterization. Rad. Res. 105: 169-179 (1986). 20. L.I. Wiebe, D.C. Jette, J.D. Chapman, R.J. Flanagan and B.E. Meeker. Iodoazomycin riboside (l-(5'-iodo-5'-deoxyribofuranosyl )-2-nitroimidazole, a hypoxic cell marker. II. In vivo evaluation in experimental tumors. Nuclear Medicine in Clinical Oncology (C. Winkler, Ed.) Springer-Verlag, Berlin, 402-7 (1986). 21. H. Pauly and E. Arauner. Uber den Gegensatz zwischen Jod und Brom bei der Imidazolsubstitution. J. Prakt. Chem.(2)118: 33 (1928). 22. M. Hoffer, V. Toome and A. Brossi. Nitroimidazoles II. Synthesis and Reactions of Iodo-nitroimidazoles (1). J. Heterocycl. Chem. 3(4): 454-458 (1966). 23. K.C. Agrawal, B.C. Millar and P. Neta Radiat. Res. 78, 532 (1979). 24. A.G. Beaman, W. Tautly, and R. Duschinsky. Studies in the Nitroimidazole Series III. 2-Nitroimidazole Derivatives Substituted in the 1-Position. Antimicrobial Agents and Chemotherapy. 520-530 (1967). 25. J.P. Dickens, R.L. Dyer, B.J. Hami 11 and T..A. Harrow. Reassignment of the structures of iodonitroimidazoles, its N-methyl derivatives and related compounds. J. Org. Chem. 46: 1781-1786 (1981). 26. R.P. Gupta, C.A. Lerroquette, K.C. Agrawal, J. Grodkowski and P. Neta. Potential radiosensitizing agents 7.4(5)-Iodo-5(4)nitroimidazole derivatives. J. Med. Chem. 28, 987-91 (1985). - 1 24-

27. J.P. Weichert, M.E. Van Dort, M.P. Groziak, and R.E. Counsel!. Radioiodination via isotope exchange in pivalic acid. Proceedings of the International Symposium on Radiohalogens. Banff (1985). 28. J.D. Chapman, E.A. Blakeley, K.C. Smith and R.C. Urtasun. Radiobiological characterization of the inactive events produced in mammalian cells by heliun and heavy ions. Int. J. Radiat. Oncol. Biol. Phys. 3: 97-102 (1977). 29. J.D. Chapman, J.A. Raleigh, J. Borsa, R.G. Webb and R. Whitehouse. Radiosensitization of mammalian cells by p-nitroacetophenone II. Effectiveness of analogues. Int J. Radiat. Siol. 21: 475-482 (1972). 30. Y. Takeuchi, H.J.C. Yeh, K.L. Kirk and L.A. Cohen. Adjacent lone pair (ALP) effects in heteraromatic systems. 1. Isotope exchange of ring hydrogens in alkylimidazoles. J. Org. Chem. 43: 3565-3578 (1978). 31. A.J. Varghese, S. Gulyas, and J.K. Mohindra. Hypoxia-dependant reduction of l-(2-nitro-l-imidazole)-3-methoxy-2-propanol by Chinese hamster ovary cells and KHT tumor cells in vitro and in vivo. Cancer Res. 36: 3761-3765 (1976). 32. I.J. Stratford, S. Hoe, G.E. Adams, C. Hardy and C. Williamson. Abnormal radiosensitizing and cytotoxic properties of ortho-substituted nitroimidazoles. Int J. Radiat. Biol. 43: 31-43 (1983). 33. M. Tubis, G.T. Krishnamurthy, J.S. Endow, R.A. Stein, R. Suwanik and W.H. Bland. Labelled metronidazoles as potential new agents for amebic hepatic abscess imaging. J. Nucl. Hed. 14: 163-171 (1975). 34. M. Tubis, A.F.C. daRocha, W.P. Souza, B.A. Meguerian and G.T. Krishnamurthy. Scintigraphic demonstration of ambebic liver abcesses with 131-iodine labelled bromometronidazole. J. Nucl. Med. 15: 36-38 (1976). 35. L.I. Wiebe, D.C. Jette and J.D. Chapman. Electron affinic compounds for labelling hypoxic cells: The synthesis and characterization of l-(2-(2-iodophenoxy)-ethyl)-2-nitroimidazole. Nuklearmedizin 23: 63-67 (1984). 36. P.A. Jerabek, T.A. Patrick, M.R. Kilbourn, D.D. Dischino and M.J. Welsh. Synthesis and biodistribution of i8F-labeled fluoronitroimidazoles: Potential in vivo markers of hypoxic tissue. Appl. Radiat. Isotopes 3J7, (;99-605 (1986). 25-

TABLE 1 Properties of radiosensitizers 5-I-NHMP MISO19 IAZR19

P value 1.97 + 0.1 0.43 2.1 ± 0.3 %PB 17.2 ± 5.7 <1 20.5 ± 1.6 50% toxicity 0.071 mM 3.9 mM 0.17mM SER (10% survival) 5 to 10* 1.0 5 to 10

relative to MISO SER - sensitization enhancement ratio - 126-

Figure 1. Radiosensitization of hypoxic murine E"MT-6 fibrosarcoma cells in tissue culture by addition of 0.1, 0.2, or 0.5 mM 5-I-4-NHMP.

Figure 2. Metabolic dehalogenation of 5-I-4-NHMP by murine EMT-6 fibrosarcoma cells in vitro. -127-

A AIR

• N2 4 0-5mM 4-l-NHMP a N2 «0-2mM 4-l-NHMP N2 «0.imW 4-l-NHMP NITROGEN

2 O I- o

2 >

CH,-CH-CH,-O-CH, OH

I i I 10 20 30 40 RADIATION DOSE (Gy)

figure - I 2 H -

100-

T 4- I-NHMP In medium • RADIOIODIDE In medium A 4-l-NHMP In cell culture O RADIOIODIOE in cell culture

CHrCH-CH,-O-CH, OH

0 15 30 60 90 120 TIME OF INCUBATION (MIN.)

figure 2 SPECIFIC ACTIVITY ^TERMINATION OF 125-1 LABELED PROLACTIN n»Y Dc.CEPTnP Af!D RAD 10 ITl IVO LAICAL SELF-DISPLACEHENT METHODS.

E.S.RIVERA, R.A.COLUCCIA^ A.VENTURINO, ^.^ALKISCHER

AND R.A.CARO

ir CATEDRA DE CISICA, ACULTAD DE FAPMACIA Y

BLOQUIMICA, (INIVEPSIDAD DE ^UFNOS '

BUENOS ^IRFS> ARPF.NTINA.

REPUBLICA ARGENTINA

1987 CbO

SPECIFIC ACTIVITY DETERMINATION OF 125-1 LABELED PJRDLACTIN BY RECEP1U. AND RADIOIMMUNOLOGICAL SELF-DISPIACEMENT METHODS. E.S. Rivera, G.A. Coluccia, A. Venturino, S. Malkischer and R.A. Caro. (Ca"tedra de Ffsica, Facultad de Farmacia y BioquJJnica, Universidad de Buenos Aires, Buenos Aires, Argentina).

It is now well known tJhat 125-1 lateled compounds maintain radiochenvLcal purity for a relatively short time after the labeling. In fact, once the labeling and the purification have been completed a steady increase of damaged molecules is observed which decreases the radiochamLcal and chemical purities of the solution. Several factors are involved in this phenomenon, such as primary and secondary autorradiolytic effects, the used labeling method, the time of contact with the oxydizing reagents, etc. It is therefore clear that the specific activity (S.A.), defined here as the absolute activity divided by the mass of the labeled compound maintaining its biological and/or inrnunological properties, decreases as a function of the tima elapsed after the labeling and the purification of the labeled compound. The S.A. immediately after the labeling and before the purification is relatively easily obtained fran tlie labeling yield if it is assumed that all the mass put into the reaction is biologically and/or jurmunologically active. Seme years ago (1) , it was demonstrated that the yield of the labeling process is overestimated if determined radioelec trophoretically; a simple and rapid paper radiochranatographic method gives correct values. However, the specific activities calculated fran those yields give values higher than the real ones, since even iitmediately after the labeling, not all the labeled molecules maintain their capacity to be bound specifically to a receptor site and/or to compete at the equilibrium. In the special case of competitive analysis, where affinity constants and nunber of receptor sites have to be calculated, such as the Scatchard plots in receptor and radioimmune analysis, it is essential to know accurately the true S.A. in order to calculate the correct values for the above mentioned parameters. In the present paper we study the specific activity of 125-1 labeled Prolactin by means of self-displacement of the hormone in binding experiments, using RIA or receptor techniques, according to a method similar to that proposed by Calvo et al. (2). The results obtained immediately after the labeling were compared to those calculated frcm yield measurements. The S.A. was determined by self-displacement in order to study its modification with the time elapsed after labeling. The change of S.A. with different labeling conditions and using RIA or receptor techniques for its determination was also studied.

MMERIALS AND METHODS. Rat Prolactin (r-Prl), kindly supplied by the National Institutes of Health (U.S.A.)* was labeled with 125-1**, using the Chloramine-T method (1) and tlie lactoperoxydase-hydrogen peroxyde method (3) . Both labeling teclmiques

* National Hormone and Pituitary Program, Baltimore, Maryland, USA. ** New England Nuclear, NEZ 033H. -1 j I -

were performed in PBS at pH - 7.4. The radiochemical purity of the used 125-1- iodide was electrophoretically checked prior to its use; in all the cases, we found less than 1 per cent 12S-I-iodate. The physicocheinical S.A. determination was carried out as described before (1). The 125-1 labeled r-Prl was purified by filtration through a Sephadex G-75 column, whenever the non-specific binding was higher than 10 per cent, the labeled hormone was repurified by the same method. For the receptor binding experiments, we used crude membrane preparations, obtained from female Sprague-Dawley rat livers. The tissue was homoge- nized in 1/5 (w:v) Dulbecco buffer and centrifuged at 20,000 x g during 30 minutes; then, the pellet was resuspended in PBS (1/5; w:v). The protein concentration of the membrane preparation was 3 mgAnl, as determined by the method proposed by Lowry et al. (4) . The preparations were fractioned and maintained at - 20 °C. 100 ul were used for the assays. The incubations were carried out at roam temperature during 16 to 20 hours. For the RIA experiments we used rat Prolactin antiserun, also kindly supplied by the National Institutes of Health (U.S.A.)*. The reagents were preincubated during 5 days at 4 °C; then, the second antibody was added and the preparations were incubated for 2 days more at 4 °C. The S.A. determinations by self-displacement were carried out by a method essentially similar to that described by Calvo et al. (2). In order to obtain the values of the affinity constants and the number of binding sites, the experimental results were analyzed by ocmputer through a direct linear plot method, as proposed by Cressie and Keightley (5).

RESULTS. The mean values of the specific activities immediately after the labeling, determined physicochemically, were compared to those obtained by RIA or receptor self-displacement methods. The results are suimaryzed in the Table I for labelings performed with Chloramine-T and Lactoperoxydase-hydrogen peroxyde (LPO). TABLE I

Specific Activity (uCi/ug) Labeling Ratio Physiccchem. RIA Receptor

Chloramine-T 28 - 17 0.61 LPO 40 - 24 0.60 Chloramine-T 81 29 - 0.36 LPO 63 17 - 0.27

* National Hormone and Pituitary Program, Baltimore, Maryland, USA. - 1 32-

Tlw ra*;io of the last rolunn of Table I. is defined as tho S.A. determined by receptor or RIA self-displacement divided by the S.A. determined pliysicochemical ly. The study of the r-Prl S.A. as a function of tint demonstrates Hint the beliaviour of tie labeled molecules analyzed by IUA or receptor technique?; in different. For receptor analysis, a clearly b.i phasic pattern of the curves is ob served; figure 1 is an example. No significant differences are observed if initial S.A. values smaller than 50 jCi/pg are compared. With higher S.A. values, the non-specific binding was so high Uiat tie experiemntal results demonstrated tlie non-feasibility of the assays under theee conditions.

TIME

Figure 1.- Specific Activity of 125-1 r-Prl determined by I«T|V- tor self-displacement (vCi/ug) as a function of tin*? (days).

In order to confirm that our S.A. values are correct, WP stulied the affinity constant ard the number of binding sites of tie sane preparation as a function of time, calculating the S.A. from selMisplacement PxperJ"10"' s- As it can be observed in Figure 2, tie affinity constant and the mmher of binding sites remained essentially constant for more than 20 days. These results confirm the validity of the employed methodology. The S.A. values as a function of time obtained with niA se.lf-displacement, clearly show an influence of tie initial specific activity, as oin br» observed in Figure 3. For S.A. values smaller than 50 vCi/jig a relatively stable behaviour can be observed. However, for S.A. valties well above 100 ,uCi/)ig, an initial rapid decrease of the specific activity is observed. Tlvis enn bn attri- buted, at least in part, to a significant proportion of di-iotlinated tyrosilie residues, as can be deduced from the data reported by Bolton (6) . - 133-

0 2

10 30 TIME 20 30 TIME

Figure 2.- Affinity Constant (Ka) given in nM and number of binding sites (B) given in nananolar concentration as a function of time (days).

TIME

Figure 3.- Specific Activity of 125-1 r-Prl determined by R.LA self-displacement (uCi/ug) as a function of time (days). - 1 '3 4 -

DISCUSSION. The results of Table I show thai the ratio between specific activities determined by self-displacement to those obtained physicochemLcally ir; higher for receptor than for RIA tediniques. Tliis may be due to the fact that tormones even with an important proportion of post-labeling damage, can te used for RIA; this explains the low values of the ratio of Table I for these experiments. In receptor analysis, these highly damaged labeled hormones are useless, since no specific binding can be obtained with them; thus, only labeled tiormones with low damage can be used. For these hormones the ratio of Table I will be ot>- viously high. Our results also show that, even immediately after the labeling,, the S.A. determined lay radiochromatography is always higher than that obtained by self-displacement. This is due to the fact that damaged molecules are always present in any labeling process, even immediately after its completion. Tlie proportion of damaged molecules increases significantly as a function of time. The amount of this increase depends on the labeling conditions and the analytical system used to determine the real specific activity. Our results also show that the decrease of S.A., for equivalent initial values, is more important for receptor thian for RIA tecliniques. These results may be explained taking into account that, antibodies are generally poly- valent and that the prolactin molecule lias many antigenic sites. On tl« oilier hand, prolactin has one active site to be bound to the receptor (7). If this active site is danaged, the specific binding is not possible. The results reported here show that whenever specific activities or parameters derived frcm it, such as the mass of the labeled hormone, are to be used, the greatest caution must be observed in order to avoid misleading results .

REFERENCES. (1) R.A. CARD, V.A. CISCATO, S.M.V. de GIAOOMINI, S. QUIROGA and R. RAIXICELIA: Int. J. appl. Radiat. Isotopes: 2^6, 527, 1975. (2) J.C. CALVD, J.P. RADICELLA and E.IJ. C1IARREAU: Biochem. J. : .212, 259, .1 Of?3. (3) J.J. TIDRELL and B.G. JOHANSSON: Biochem. Biophys. Act a: .251, 363, 197.L. (4) O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL: J. Bio]. Chem. : 193, 265, 1951. (5) N.A.C. CRESSIE and D.D. KEIGHTLEY: J. Steroid Biochem. ; U_, 1173, 1979. (6) A.E. BOLTON: Radioiodination Techniques. Review 18, Arrorsham, U.K., p. 18-20, 1985. (?) M.D. IDLLENBERG: Receptor Madels and tte Action of Neuro transmit, tors a:id Hor- mones, in: H.I. YAMAMUR* et al. (Editors): NeurotranJsnittcr Receptor Binding. Raven Press, New York, J 978. Mb

STRUCTURE-ACTIVITY STUDIES ON qqf/|Tc PHENOLIC AHIMOCAR^OXYLLIC ACIH HFPATO^ILIARY B

D.J P"ADDALENA, J,G.WILSON AND G.M.SNOWDON ISOTOPE DIVISION,, AUSTRALIAN ATOMIC FNERPY COMMISSION, LUCAS HEIGHTS RESEARCH LABORATORIES^ M PRIVATE ^AIL BAP *-!RO 1, ENAI, ^L ?.?3Ht AUSTRALIA

P-U^NOS AIRF.S REPURLICA A STRUCTURE-ACTIVITY STUDIES ON 99mTc PHENOLIC AMINO- CARBOXYLLIC ACID HEPATOBILIARY AGENTS.

D. j. Maddalena, J.G. Wilson and G.M. Snowdon.

Isotope Division, Australian Atomic Energy Commission, Lucas Heights Research Laboratories, Private Mail Bag No 1, Menai, NSW, 2234, Australia.

ABSTRACT

Biodistributions of a series of eight 99mTc hydroxybenzylsarcosine (HBS) complexes were carried out in rats and their urinary and hepatobiliary excretion compared with their lipophilicities, the influence of substituents on the phenyl ring and plasma protein binding ability. The charge on the complexes was determined by electrophoresis at varying pH values.

The HBS derivatives formed anionic complexes with 99mTc that excreted mainly via the urinary route. An increase in the lipophilicity of the complexes by substitution of halogens onto the phenyl ring led to an increase in serum protein binding and a decrease in the urinary output but had no direct effect on hepatobiliary output.

KEYWORDS

Structure-activity, 99mTc , hepatobiliary, iminodiacetic acid, sarcosine, biodistribution, rats. - 1 3 7 -

INTRODUCTION

Since the successful introduction in 1976 of 99mTc labelled 2,6-dimethylphenyl- carbamoylmethyliminodiacetic acid (dimethyl-HIDA) as a replacement for 131I Rose Bengal for hepatobiliary imaging (Loberg et al. ,1976), several studies have been carried out on a wide variety of phenyl-substituted variants of dimethyl HIDA in an attempt to find the hepatobiliary agent that would have the optimum imaging efficiency (Wistow et al.,1911; van Wyk et al.,\979; Chiotellis and Varvarigou,1980; Molter and Kloss,198l).

A wide variety of other compounds containing the iminodiacetic acid (IDA) moiety have been studied as possible alternatives to the phenylcarbamoylmethyl IDA (HIDA) structure (Subramanian et al., 1977; Fields et al., 1978; DeJuliis et al., 1980; Karube et al.,1981; Fritzberg et al., 1982; Hunt et al., 1984), but few have been studied in as much detail as the derivatives of HIDA.

Structure-distribution studies which relate some of the physico-chemical properties of HIDA derivatives to their biological activity were particularly useful for optimising their imaging properties (Nunn et al., 1983). Similar studies have been used to examine the hepatobiliary output of 99mTc complexes of o-hydroxybenzyliminodiacetic acids (Maddalena et al., 1987). This approach has not been widely applied to other potential hepatobiliary imaging compounds.

In this investigation, structure-distribution studies were carried out on "mTc complexes of eight hydroxybenzylsarcosine derivatives (HBS), each having various substituents in the phenyl ring, in an attempt to optimise the biological activity of the complexes for hepatobiliary excretion and to gain a greater understanding of factors affecting hepatobiliary affinity of "mTc chelates. The compounds (table 1) were selected because their structures are half those of the N,N' -bis[2-hydroxybenzylj ethylenediamine N,N' diacetic acids (HBED) which have good hepatobiliary imaging properties (Hunt et al., 1986). - ! 38-

MATERIALS AND METHODS

Formulation of Complexes

The synthesis of the compounds will be reported elsewhere (Wilson, 1987). The complexes were prepared by the stannous chloride reduction method. Twenty milligrams of the compound was dissolved in 3 mL of 0.1 M NaOH in a 10 mL vial; 0.1 mL of freshly prepared stannous chloride solution (20 mg of SnCljJHjO in 5 mL of 1 M HC1) was added and the pH adjusted as required with 1 M HC1. The resultant solution was diluted to 5 mL passed through a 0.22 nm membrane filter into a sterile presealed vial and purged with nitrogen gas before the addition of 0.1 mL (200 MBq) of generator- derived sodium f"mTc] pertechnetate solution. Preparations were incubated for a minimum of 10 minutes before use.

Radiochemical Purity

The radiochemical purity, complex stability and charge were determined on Beckman paper strips, in a Gelman electrophoresis chamber containing 0.05 M hepes buffer at pH 6.5 and 7.4, 0.05M tris buffer at pH 8.5 and 0.05 M carbonate buffer at pH 9.5. Analyses were undertaken at various times after preparation, using [99mTc] pertechnetate as a control, each determination being carried out for one hour at 300 V in a nitrogen atmosphere. The distributions were determined by cutting the paper strips into 5 mm sections and counting them in a Packard model 5650 automatic gamma counter.

Plasma Protein Binding and Partition Coefficients

The amount of plasma protein binding was measured with fresh hcparinised rat plasma, using disposable ultrafiltration units (Amicon Centrifree) as described elsewhere (Maddalcna ft ai, 1987). The plasma protein binding value was corrected by subtracting the amount bound to the filter using the same procecure for plasn.u p.\>iein binding but - 139-

substituting nitrogen-purged, 0.05 M phosphate-buffered saline at pH 7.4 for the rat plasma. The percentage of the complex bound to the filter was always less than 5%.

The lipophilicity of the complexes was measured by determining their partition coefficients between n-octanol and 0.05 M phosphate-buffered saline at room temperature (20°C) in a nitrogen atmosphere, as described by Maddalena et

The theoretical lipophilicity of the ligands was calculated as the sum of the fragmentary K values of the substituents on the phenyl ring of the ligand, using the data of Hansch and Leo (1979). The lipophilicity of the remainder of the ligand was assumed to be constant and assigned a value of zero.

Biodistribution Studies

Biodistribution studies in groups of three female specific pathogen free AAW (Australian Albino Wistar) rats of closely controlled body mass (220 +/- 20 g), were used to determine the hepatobiliary and urinary outputs of the 99mTc labelled HBS derivatives and 99mTc Cl-HBED. The animals were injected intravenously via the tail vein with 90 (iL of complex, using glass gas chromatography micro-syringes (SGE), while under light ether anaesthesia and then placed unrestrained in a metabolic cage for collection of urine and faeces. After 3 hours they were sacrificed for tissue samples and the results calculated using the TISCON computer program (Maddalena, 1983). The biodistribution of one complex, 99nTc Cl-HBS was examined as described above but at time intervals between 5 minutes and 24 hours.

To examine the effect of pH on the biodistribution of a complex, 99mTc Cl-HBS was examined as described above, however, for this study groups of rats were injected with complexes prepared at pH values between 6.5 and 9.5, then sacrificed at 3 hours post injection. - 14 0-

RESULTS AND DISCUSSION.

Electrophoresis

The three mono-halogenated ligands formed single negatively charged complexes with "mTc at pH 6.5 and two to three negatively charged species between pH 7.5 and 9.5, whereas the three di-halogenated ligands formed single negatively charged complexes up to pH 7.5 and two or three negatively charged species between pH 7.5 and 9.5 (figure 1). All the halogenated complexes were stable for at least four hours after preparation. The methoxy and methyl-HBS complexes were, however, unstable in vitro at all pH values. In contrast, 99mTc labelled chloro-HBED formed a single stable neutral peak at pH 6.5 and one or two negatively charged peaks between pH 7.5 and 9.5 (figure 2).

Biodistributions

The biodistributions of the WmTc HBS complexes in rats at three hours post injection arc listed in table 2. At this time interval, the hepatic excretion of the complexes was relatively complete, as is shown by the biodistribution study of 99mTc-chloro-HBS over 24 hours (figure 3).

Low free pertechnetate levels in the stomach ( <1% ) indicated that the two ligands which formed unstable complexes in vitro, methoxy-HBS and methyl-HBS, gave acceptable biodistribution results in rats when injected immediately after preparation. However, if allowed to stand for more than 10-15 minutes the complexes became unstable, their biodistributions resembling those of sodium [99mTc], pertechnetate (stomach > 15%).

Altering the pH of the 99mTc Cl-HBS complex between pH 6.5 and 8.5 was found to have no effect on its biodistribution (figure 4) even though the electrophoresis of the complex ai the different pH values indicated the presence of a mixture of labelled species. At pH - 1 4 I -

9.5. however, a larger proportion of the activity was found in the urine and less in the gastrointestinal tract suggesting that at this pH a different complex was being formed that had different biological characteristics.

Effects of Lipophilicity on Biodistribution

The physicochemica1 properties of the ligands and their 99mTc complexes are shown in table 1. In the octanol/buffer partition studies, a good linear correlation (r=0.99) was found between the theoretical lipophilicities (Liz) of the ligands and the log of the octanol/buffer partition coefficients (log P) of the complexes. This good correlation confirms that the substituents on the phenyl ring are the main causes of differences in the relative lipophilicity of the ligands and their 99mTc complexes and allows the prediction of the lipophilicity from published 7t values. Similar linear relationships have been found between HIDA and HBIDA derivatives and their "mTc complexes (Nunn, 1983; Maddalena et al., 1987).

The complexes were mainly excreted via the urinary pathway. When the activities of complexes in the urine were plotted against their lipophilicities, an inverse linear relationship with significant correlation (p<0.05) was found (figure 5). Similar relationships have been reported for"mTc HIDA complexes (Nunn, 1983),99mTc HBIDA complexes (Maddalena et al., 1987) and 99mTc pyridoxylidene-phenylalanine complexes (Kato-Azuma, 1982).

When the activities of the complexes found in the blood were plotted against their lipophilicities a significant linear correlation (p<0.05) was found (figure 5). This suggested that there might be a correlation between the amount bound to the plasma proteins and the lipophilicity of the complexes. When these variables were plotted, a highly significant (p<0.0l) linear correlation was found (figure 6). A similar relationship was found for"mTc HBIDA complexes (Maddalena et al., 1987). -142-

These relationships indicate that the activity of complex in the urine is dependent upon the activity of free complex in the plasma which, in turn, is dependent upon the lipo~hilicity of the complex, which can be modified by suitable substitution of halogen groups within the phenyl ring.

Although there was good correlation between the activities in the urine or blood and lipophilicity, no significant correlation was found between lipophilicity and the activity in the kidneys or gastrointestinal tract (figures 5 and 7).

One reason for examining the HBS derivatives as possible hepatobiliary agents was that in structural terms (except for a hydrogen atom) they were half of the structure of a series of HBED derivatives which perform quite well as hepatobiliary agents (Hunt et a/., 1986). It was possible that the HBS derivatives would form 2:1 ligand to"mTc complexes which would perform similarly to the HBED derivatives which form 1:1 complexes with a number of metals (L'Eplattenier et at., 1967). However, an examination of the biodistributions of related derivatives of each series such as the 99mTc chloro-HBS and WmTc chloro-HBED (table 2) quickly reveals that there is no similarity in their biological behaviour.

The Influence of Substituents in the Phenyl Ring

Structural alterations, involving changes in the lipophilicity of substituent groups on the phenyl ring and of th^j location relative to the carbamoylmethyl IDA moiety, have the greatest influence on the hepatobiliary clearance of 99mTc HIDA derivatives. Subramanian et ah, (1977) found that as the lipophilicity of the substituent group on the benzene rings of HIDA derivatives increased, the biliary excretion increased, and the urinary excretion decreased. Similar results have been found for a variety of HIDA derivatives (Chiotellis and Varvarigou, 1980; Mouer and Kioss, i 98 i; Nunn et a/.,Iv83) and also for benzimidazoly! IDA derivative.^ (Hunt et al., 1984). - 14 3-

In a comparison of the 99mTc chelates of six dimethyl-HIDA isomers, van Wyk et al.,(1979), found that the position of the methyl groups substantially influenced the hepatic and urinary excretions. The compounds substituted in the meta positions gave the best results. Chiotellis and Varvarigou (1980) also found that of the n-butoxy derivatives of HIDA, those substituted in the meta position had better characteristics than those substituted at the ortho or para positions.

In the present study, all the substituents were placed in the meta position with respect to the mcthylene iminodiacetic acid moiety. The halogen substituents increased the lipophilicity (log P) of the complexes by three orders of magnitude, from an estimated -2.54 for the 99mTc methoxy-HBS complex to 0.60 for the "mTc dibromo HBS complex, however, a linear change of hepatobiliary output was not found. After a rapid increase in hepatobiliary output from the methoxy-HBS complex (16.1%) to the chloro-HBS complex (31.5%), there was no further change in the hepatobiliary output even though the lipophilicity had changed by nearly two orders of magnitude (tablel).

Maddalena et a/.,(1987) found that alkyl substituens had a much greater effect on hepatobiliary output than halogens with 99mTc HBIDA complexes. The tertbutyl substituent, which had a similar lipophilicity (log P) to two chloro groups, more than doubled the hepatobiliary output (15.6 to 35.6%) and increased the activity in the liver by almost a factor of three (4.9 to 14.1%), suggesting that substituent type was more important than direct increases in lipophilicity.

Nunn et al.,(1983) found that the high hepatic outputs and fast hepatocellular transit times could be achieved with 99mTc HIDA complexes by designing HIDA derivatives with small alkyl substituents in the ortho position and additional substituents in the meta and para positions to produce the required lipophilicity. Kato-Azuma (1982) achieved similar results with "mTc pyridoxylidenephenylalarune complexes. It is possible that similar changes to the HBS derivatives might lead to improved hepatic excretion - 144-

CONCLUSIONS

The hydroxybenzylsarcosine (HBS) derivatives form anionic complexes with 99mTc which excrete mainly via the kidneys into the urine rather than by the hepatobiliary route, indicating that these complexes are unsuitable as hepatobiliary agents.

Increase of the lipophilicity of the complexes by substitution of halogens onto the phenyl ring increases the plasma protein binding and decreases the urinary output but has no direct effect on hepatobiliary output.

REFERENCES

Chiotellis, E. and Varvarigou, A. (1980) 99mTc labelled N-substituted carbamoyl iminodiacetates: relationship between structure and biodistribution. IntJ Nucl MedBiol, 7,1-7.

DeJuliis, CM., Graham, B.W., Mattson, R.J., and Sowell, J.W. (1980) Evaluation of technetium-99m-labeled iminodiacetic acid derivatives of 2-aminopyrroles as hepatobiliary imaging agents. J Pharm Sci, 69,731.

Fields, A.T., Porter, D.W., Callery, P.S., Harvey, E.B. and Loberg, M.D. (1978) Synthesis and radiolabeling of technetium radiopharmaceuticals based on N-substituted iminodiacetic acid: Effect of radiolabeling conditions on radiochemical purity. J Lab Compd Radiopharm, 15, 387-99.

Fritzberg, A.R., Bloedow, D.C., Klingensmith III, W.C. and Whitney, W.p. (1982) Comparative study of 99mTc labeled hepatobiliary agents based on napnthaler.e and similar ring systems, hit J Nucl Med Biol, 9,1 - i 1. 5 -

Hansch, C. and Leo, A. (1979) Substituent constants for correlation analysis in chemistry and biology. New York. Wiley.

Hunt, F.C., Maddalena, DJ. and Wilson, J.G. (1984) Technetium-99m benzimidazolyl iminodiacetic acid hepatobiiiary radiopharmaceuticals: structure biodistribution studies. Int J Nucl Med Biol, 11,219-22.

Hunt, F.C., Maddalena, D.J., Wilson, J.G. and Bautovich, GJ. (1986) Phenolic aminocarboxylate chelates of 99mTc as hepatobiiiary agents. Nucl Med Biol, Int J Radiat Appi Instrum Part B 13,289-93.

Karube, Y., Kono, A., Maeda, T., Ohya, M. and Matsushima, Y. (1981) A new series of Tc-99m-labeled hepatobiiiary tracers N'-Acyl- and N'-sulfonyl ethylenediamine-N,N- diacetic acids. J Nucl Med, 22,619-22.

Kato-Azuma, M. (1982) Lipophilic derivatives of 99mTc (Sn) pyridoxylidene- phenylalar.ine: a structure distribution relationship (SDR) study on technetium-99m complexes. Int J Appl Radioat hot, 33,937-44.

L'Eppiattenier, F., Murase, I., Martell, A.E. (1967) New multidentate ligands. VI. Chelating tendancies of N,N'-di(2-hydroxybenzyl) ethylenediamine N,N'-diacetic acid. / Amer Chem Soc, 89, 837-43.

Loberg, M.D., Cooper, M., Harvey, E., Cailery, P. and Faith, W. (1976) Development of new radiopharmaceuticals based on N-substitution of iminodiacetic acid. J Nucl Med, 17, 633-38.

Maddalena, DJ. (1983) Tiscon, a BASIC computer program for the calculation of the biodistribution of radionuclide-labeled drugs in rats and mice. AAEC/E572. - 146-

Maddalena, D.J., Snowdon, G.M. and Wilson, J.G. (1987) Structure-distribution studies on some potential hepatobiliary agents: 99mTc o-hydroxybenzyliminodiacetic acid complexes. Nucl MedBiol Int J Radiat Appl Instrum Part B (in press).

Molter, M. and Kloss, G. (1981) Properties of various IDA derivatives. J Lab Compd Radiopharm, 18, 56-58.

Nunn, A.D. (1983) Structure-distribution relationships of radiopharmaceuticals. Correlation between the reversed-phase capacity factors for Tc-99m phenyl- carbamoylmethyliminodiacetic acids and their renal elimination. / Chromatog, 255, 91- 100.

Subramanian, G., McAfee, J.G., Henderson, R.W., Rosenstreich, M., and Krokenberger, L. (1977) The influence of structural changes on biodistribution of Tc-99m labeled N- substituted IDA derivatives. J NuclMed, 18, 455-61. van Wyk, A.J., Fourie, P.J., van Zyl, W.H., Lotter, M.G. and Minnaar, P.C. (1979) Synthesis of five new 99mTc -HID A isomers and comparison with 99mTc- HID A. Eur J Nucl Med, 4, 445-48.

Wiison, J.G. (1987) Personal communication.

Wistow, B.W., Subramanian, G., Van Heertum, R.L., Henderson, R.W., Gagne, G.M., Hall, R.C. and McAfee, J.G. (1977) An evaluation of 99mTc -labeled hepatobiliary agents. J Nucl Med, IS, 455-61. - 14 7-

Table 1 Biodistribution* of ""Tc Complexes in Rats

Compound Blood Liver GIT Kidneys Urine

a methoxy 1.8(0.2) 2.1(0.2) 16.1(1.7) 16.8(0.5) 51.6(3.7) b methyl 3.2(0.8) 6.0(1.6) 18.0(2.3) 10.5(2.9) 45.0(6.9) c chloro 1.8(0.1) 3.9(0.5) 31.5(5.0) 13.1(1.3) 32.9(7.1) d bromo 2.6(0.2) 5.5(0.5) 31.2(1.3) 11.8(1.5) 38.3(2.5) e dimethyl 4.3(0.3) 4.7(0.4) 30.8(2.2) 8.9(0.5) 43.1(4.6) f iodo 3.4(0.3) 7.5(0.4) 27.4(1.2) 11.0(0.5) 38.8(2.8) g dichloro 3.2(0.5) 5.7(0.2) 26.4(2.1) 9.4(0.6) 42.2(1.8) h dibromo 3.0(0.4) 6.2(0.3) 30.2(2.7) 9.9(0.1) 33.9(0.2) Cl-HBED 2.8(0.2) 7.5(1.5) 66.2(3.0) 1.7(0.1) 13.8(1.1)

* means (standard deviations) of % injected dose (groups 3-5 rats) 3h post injection. - 14 8-

Tablc 2 Physicochemical Parameters of HBS Ligands

COOH

Compound Rl R2 MW In logP % bound

HBS a MeO H 225 -0.02 u u b Me H 209 0.56 u u c Cl H 230 0.71 -1.13 34 d Br H 274 0.86 -0 94 35 e Me Me 223 1.12 -0.49 39 f I H 321 1.12 -0.55 47 g Cl CL 264 1.42 0.36 57 h Br Br 353 1.72 0.60 71

Notes

1. MW = molecular weight of ligand. u = unstable. 2. values from Hansch and Leo (1979) 3. log P = log (octanol/buffer partition coefficient) of 99mTc complexes. 4. % bound = percent of 99mTc complexes bound to plasma proteins. - 149-

Captions of Figures

Figure 1 Electrophoresis of "mTc Cl-HBS versus pH.

Figure 2 Electrophoresis of "mTc Cl-HBED versus pH.

Figure 3 Biodistribution of 99nTc Cl-HBS in Rats.

Figure 4 Effect of pH of 99nTc Cl-HBS Complexes on Biodistribution in Rats.

Figure 5 Effect of Lipophilicity on the Biodistribution of 99mTc HBS Complexes in

Blood, Kidney and Urine in Rats.

Figure 6 Variation of Plasma Protein Binding with Lipophilicity of 99mTc HBS

Complexes.

Figure 7 Effect of Lipophilicity on the Biodistribution of 99nTc HBS Complexes in

Liver and GIT of Rats. - 150-

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RADIOHYGIENE, BUDAPEST,, HUNGARY.

HUENOS AIRES

^EPU?»LICA ARGENTINA IN VIVO STABILITY AND INERTNESS OF VARIOUS DIRECT LABELED AND CHELATE-TAGGEO PROTEIN

Gy.A.Janoki, L.Korrisi, G.Klivenyi and B.Spett ("FJC" Natl. Res. Inst. for Radiobiology and Radiohygiene, Budapest, Hungary) INTRODUCTION

Biologically important proteins, polypeptide hormones, cells etc. labeled with different isotopes are widely used both in nuclear medicine and in several fields of biological research. Their widescope use is limited, however, by the .L'act that in vivo stability of proteins labeled in different ways Cdirect labeling, chelating or conjugation) with 99mTc, 123-1, 111-In, 67-Ga etc. is not satisfactory. Dilution following intraveneous injection and the presence of competitive proteins (e.g. transferrin) in the serum may induce the detachment of the isotope from the protein molecule. In case of labeling with 99mTc the detached Tc may be reoxidized and transformed into pertechnetate or undergo hydrolysis. Biological distribution and pharmacokinetics of l:ho product:} of both processes differ from those of the original labeled protein and thus a distorted background may arise. With 111-In labeling, transchelation of the label to transferrin significantly enhances the proportion of nontarget activity (1. 2. 3. 4. 5.). In this study we were looking for methods giving precise information about composition and activity distribution of protein components both in the initial samples and serum samples after intraveneous administration. We tested the applicability of electroimmunoassay (EIA), polyacrilamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC) for the assessment of in vivo stability of labeled proteins. The model compound was human serum albumin labeled with 99mTc and 125-1, respectively. Bifunctional chelate labeling was done with desferrioxamine in this case protein was labeled with 67-Ga (6.). Bindib-tribution of the labeled compounds and their elimination from the blood were studied in rabbits.

METHOD'S Polyacrylamide gel Electrophoresis (PAGE) Samples were analysed as detailed published elswhere (7.). Shortly, the gel concentration was 7.5 % current 4 mA per column, electrode buffer 0.2 mol phosphate buffer of pH:7.2. Running time was 24 hrs at +4 C. High Performance Liquid Chromatography (Hi'LC) HPLC analysis cf protein was performed using Bio~Rad HPLC .system. Separation was performed using 300 x 7.5 mm Bio-Sil l!)K-250 molecular weight sizing column in series by a 75x7.5 iiim Bio-Sil TSK guard column. Samples (20/iil) were injected into the unit and elutcd using 0.02 mol NaH?P0, /0.0!> mol Ha2504 buffer (pH 6.8) at flow rate of 1.0 ml/min. - I 59-

Protein absorbance was monitored at 280 nm, gamma radiation was measured simultaneously using flow-through gamma-ray detector.

Electroimmunoassay (EIA) Human serum albumin content of rabbit serum sample was determined by EIA. Method published elswhere (8.). Elc-ctrophoresis was made under conditions as follows:0.1 mol barbital buffer pH:8.4, 6 mA/plate and 2 hrs. Anti HSA goat IgG (Phylaxia Vet.Biol.Co,) was used (120,ul antibidies in 12 ml agarose gel solution). The Standard used were 10, 15, 20, 25/ug/ml HSA concentration.

Biodistribution and pharmacokinetics The biodistribution of the various labeled protein was studied in male New Zeland rabbits using a scintillation car.ere. The blood clearence of the compounds was compared to that of the following reference compounds using standard blood sampling method: 67-Ga-citrate, 67-Ga-desferrioxamine (DF), 99mTc-RBC (labeled in vivo).

RESULTS and DISCUSSION

The PAGE studies in Fig. 1. well demonstrate the amounts and activity distribution of radiolabeled proteins. Activity recowered in protein bound Tc-Sn-HSA; monomer 37 %, dimer 27 %, trimer 13 %, higher polymers about 20 %. 125-I-HSA: monomer 78 %, dimer 10 %, polymer 10 %. The results of HPLC analysis of 99mTc-(Sn)-HSA is shown in Figure 2. Radiochromatograms presented in Fig. 2.A show that approximately 95 % of the radioactivity is protein bound. The results of serum sample analysis indicated that after 1 hr (Fig. 2.B) resulting from oxidation of reduced 99mTc, pertechnetate appears at the elution volumes of 13 ml. After 3 hrs (Fig. 2.C) label eluted as the high molecular weight species. The electroimmunoassay pattern of standard HSA series and serum samples taken at different points of time after injection of 99mTc-(Sn)-HSA ?.ve see- in Fig. 3.A. Control rabbits serum gives no precipitation peak. Result of in vivo stability of the three labeled proteins tested are summarised in Fig. 3B. The demonstrated values clearly show that HSA has been eliminated from the blood stream with identical rate in all the three cases. Differences in the activity of serum samples are unequivocally due to differences in labeling and type of binding. Sixty minutes after injection the circulating HGA carried 13 to 53 % less activity than at the time of injection. Radionuclide detachment of the three-te:jt compounds are as follow 99mTc-(Bn)-IISA 53 %, 67-Ga-OF-HSA 27%, 125-I-HSA 13%. !io i n t. I graphic pictures in Fig. 4. demon';' •;; ;c3 in vivo distribution. Signilicant liver a renal activity can see in c:a:;e of 99mTc- (fin )-HSA . The results of comparative blood cicarence examinations are illustrated in Fig. 5. 125-I-HSA :ir>ed at- a control show the - 160-

niost favourable blood activity rate. The disappearance of •67-Ga-DF from the blood Is caused by the secretion of the kidneys, 67-Ga-citrate excreted faster from the blood also. Significant differences are found among clearences of compounds No. 1-5.

Our experience with various labeled protein, especially with Tc-Sn-HSA system indicate that in vivo stability of these compounds are generally low. Following intravenous injection of proteins labeled with metal isotopes, due to dilution and to the presence of considerable amount of competitive proteins in the serum (e.g.transferrin), part of the label is being detached from the carrier protein. Distribution of the detached metal isotopes is different from the original distribution of the protein. This problem arises also with radiophamaceuticals based on monoclonal antibodies.

Acknowledgements This work was supported by the Scientific Council of the Ministry of Health. We thank Mrs.M.Lakner und R.Kaldi for their technical assistance, and Mrs.Cs.Paldcz for the typing of the manuscript. REFERENCES

1. Hnatowich D.S.: Label stability in serum of four radionuclides on DTPA-Coupled Antibodies - An Evaluation Nucl. Med. Biol. vol 13. pp 35-358, 1986.

2. DeNardo S.J, Peng J.B, DeNardo G.L, Mills S, Epstein A.L: Immunochemical aspects of monoclonal antibodies important for radiopharmaceutical development Nucl. Med. Biol. vol 13. pp 303-310, 1986

3. Paik C.H , Eckelmann W.C. and Reba R. C. .- Transchelation of 99mTc from low affinity sites to high affinity sites nf antibody. Nucl. Med. Biol. vol 13. pp 359-362, 1986

Goodwin D.A., Meares C.F : Bifunctional chelates for radiopharmaceutical labeling. In Radiopharmaceuticals. Structure Activity Relationships Edit. Spencer RP, Grune and Stratton. 1980: 14:281-306.

5. Janoki Gy.A., Korbsi L, Spett B.: Factors influencing the biological properties of labeled protein. In: Current Application in Radiopharmacclogy Edit. Billinghurst M. W. orgamon Press 1985; 271-276.

6. Jnnoki Gy.A., Harwig F.J., Chnnachni W. and Wolf W.: 67~Ga-Desferrioxamin;;-HSA: Synthesis of Chelon Protein Conjugates Using Carbodiimirie as a Coupling Agent. ..'nt. J. Appl . Radiat. Isot. 1983; "34:871-1)77. -16 1-

7. Janoki Gy.A., Korbsi L., Spett B. and Kocsar L.T.:Protein based radiopharmaceuticals: Applicability of polyacrilamide gel electrophoresis in the quality contor of labeled protein preparation. Progress in Radiopharmacy Ed. Cox P.H, Mather S.J, Sampson C.B. and Lazarus C.R: Nijhoff Publishers 1986. pp 570-576

8. Janoki Gy.A, Korbsi L.: Use of electroimmunoassay for the determination of in vivo stability of labeled (submitted to Nucl.Med.Communications) - 162-

FIGURE CAPTIONS

Fig. 1. Electrophoretic patterns and activity distribution and 99inTc-Sn-HSA of 125-I-HSA. Activity recovered in protein bound listed in the text. Fig. 2. HPLC radiochromatograms obtained by the analysis and serum stability of 99mTc-Sn-HSA. A. Analysis of 99mTc-Sn-HSA prior injection. B. Analysis of serum samples fron rabbits 99mTc-Sn-HSA was injected 60 minutes previously. C. 3 hours previously.

Fig.3 A. Electroimmunoassay pattern of standard HSA series and serum samples taken at different points of time after injection of 99mTc-Sn-HSA. B. Serum stability values of 99mTc-Sn-HSA, 67-Ga-DF-HSA and 125-I-HSA. Fig. 4. Gamms camera images of 99mTc-Sn-HSA (I.) and 67-Ga-DF-HSA (II.) in rabbits after 60 minutes of i . v . injection. Fig. 5. Blood clearence curves of various radiolabeled HSA preparation in rabbits. Control 125-I-HSA, 1.2.3. 67-Ga-DF-HSA (Ref.6), 4. 99mTc-RBC (in vivo labeled 5. 99mTc-Sn-HSA, 6. 67-Ga-citrate, 7. 67-Ga-DF (desferrioxamine) -163-

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CONTROL BTODTSTRIBI'CIOM Y ESTPWCWN TC MET RICA 1% RE HEDP,

Y n.^ANZINI

COMISION NACIONAL DE HNERGIA ^TOMICA DE LA ''EPUBLICA APR

BUENOS AIRES REPURLICA ARGENTINA BIODISTFIBUCION Y ESTEKACICN DOSIMETRICA DEL 186R5 HEDP.

W.G.NOTO y A.JVRNZINI (Comisión Nacional de Energía Atómica,Buenos Ai res,Argentina).

En 1979 fue propuesto el HEDP-186Fe para terapia paliativa del dolor en pacientes con metástasis óseas (1). En 1982 se propuso el metilendifosfonato(IOP) mar- cado con 186Re para el mismo fin(2). Se demostró(3) por imágenes centellográficas en animales de experimentación la mayor concentración de los fosfonatos-186Re en lesión ósea con respecto a tejido óseo normal. En un trabajo preliminar(4) se buscaron las condiciones óptimas de mar- cación con Re-186 de varios agentes óseos y a partir de los estudios realizados se seleccionó al HEDP-186Re para evaluar su probable utilización en humanos. El motivo de éste trabajo es estudiar la farmacocinética y estimar la dosis de radiación absor bida en diversos tejidos del organismo por la administración de éste radiofármaco.

Preparación del 186Re 0 NH,, .

El 186Re se obtuvo por irradiación neutrónica del ReO.NH. lográndose una actividad específica del 31 mCÍ/mg de Pe y una relación 188Re/186Re de 0,7 a las 24 horas post irradiación).

Preparación y control de calidad del HEDP-Re 186 A 1 mi de la solución de HEDP sal disódica(40 mg/ml)se le agregan 0,5 mi de solución dp C12Sn-2H20(50mg/ml en C1H 1 N). Se le adiciona luego un volumen de solución de ReO^NHj conteniendo como máximo 1,2 mg de Re. El tiempo necesario para comple- tar la reacción está relacionado en forma directa con el pH. En las condiciones des_ criptas la reacción se completa en 30 minutos y puede controlarse por cromatografía ascendente utilizando como soporte ITLC-SG y como solante netil etil cetona(el compuesto queda en el punto de siembra y el ReO4 migra con el frente del solvente). Corpletada la reacción el preparado se lleva a pH 6 y se controla la ausencia de Re reducido y/o coloide utilizando cromatografía ascendente, soporte ITLC-SG y solvente solución fisiológica(el compuesto migra con el frente del solvente y en el punto de siembra quedan las iirpurezas investigadas).

Ensayos de biodistribución Se inyectó el HEDP-Re 186 en ratas Wistar de aproximadamente 300 g de peso; los animales fueron anestesiados con éter en el momento de la administración intravenosa y con uretano (1 g/kg) 15 minutos previo al sacrificio, las cantidades administradas de HEDP por Kg de peso del animal oscilaron entre 2 y 5 iro/Kg. Los animales se sacrificaron a distintos tiempos post administración. La biodistribución en ratas 2 horas post administración se comparó con el compuesto de 99mTc de igual relación HEDP/Sn++.

Ensayos de Toxicidad Se determinó la DL50 del HEDP-Re en ratones adultos de 25g de peso por administra- ción lentra intravenosa. - 167-

Resultados y discusión

La Tabla N°l muestra el porcentaje de dosis inyectada de HEDP-186Re en los distintos órganos de rata a distintos tiempos post administración; se consideró la vía urina- ria como única vía de eliminación. :?1 porciento de dosis inyectada en fémur dos horas post administración para HEDP- 186Re y HEDP-99nric, de igual relación HEDP/Sn+4- es de 0,58+ 0,13* y 0,93* 0,0G res- petivamente . La dosis letal(DL50) determinada en ratones para el HEDP-Re es de 100mg/Kg coincidiendo con la del HEDP sal disódica. Los datos registrados en la bibliografía(5) dan para inyección rápida del HFDP en ratas y conejos valores de DL50 entre 40 y 70 mg/Kg y para inyección lenta 70-100 mg/Kg, no evidenciándose reacciones adversas por inyecciones repetidas de 20mg/Kg en perros, cerdos, ratas y conejos. En ratones (6) se determinaron valores de DL50 entre 40 y 50 mg/Kg, para soluciones de HEDP en su forma acida. Para el cálculo de dosis de radiación se tomó en consideración que el 20% de la dosis inyectada de HEDP-186Re se retiene en hueso y la eliminación sigue el decaimien- to radiactivo. La dosis de radiación en rad/mCÍ para el HEDP-186Re y HEDP-188Re para hueso y médula roja se incluyen en el Cuadro N°2. El valor de la dosis de radiación para riñon del 186 Be es de 5 rad/mCí; se incluyen también las dosis de radiación para el 89 Sr(8) y 32 P, agentes utilizados actualmen te para el mismo fin que el propuesto para el compuesto de 186Re estudiado. Conclusiones La ventaja del 186Re-HEDP con respecto al 89Sr y 32P(9) está dada en la mayor rela- ción que se obtiene entre lesión ósea y tejido óseo normal para el primero.Weber(10) utilizando 85 Sr en ratas da para esta relación los valores de 1,5 y 1,1 para tiempos post administración entre ly 24 horas y 120 horas respectivamente. Para el 99mTc HEDP(11) el valor de esta relación determinada en humanos es de 2,5 y en ratas(3) para el 186Re-HEDP de 4,5-5,4. Por lo tanto de las consideraciones anteriores se infiere que el 186Rs-HEDP es un fármaco adecuado para terapia paliativa del dolor en pacientes con metástasis óseas que no respondan a los calmantes convencionales.

BIBLIOGRAFÍA - l)Mathieu,L et al.Inst.y Apll.Radiat.Isot.30,725-727,1979. 2) Eisenhut,RInt. y Apll.Radiat.Isot. 33,99-103,1982. 3) Weininger,J. et al Nuklearmedizin 23,81-82,1984. 4) Noto,M.G et al;V Simp.Int.Radiofarmacología.Bs.As.Argentina, 29-31 Oct.1986 5) Dunson,G.et. al. Doig. Intell. Clin. Pharm. 7,470-474,1973. 6) Subrumanian G.et al. J.Nucl.Nted. 16,744-755,1975. 7) Jowsey,J. et. al. J.Lab.Clin. Med. 78,574-584,1971. 8) Silberstein.E.B. et. al.J. Nucí.Med. 26:345-348,19Rr> 9) Wallace. J.C. et al. Brit. J. Radiol. 50,664-661,1977. 10)Weber,et. al. Radiology 120,1976. ll)Fogelman,I. et. al. J. Nucí.Med. 20,98-101, 1979. l^Kaul, A. et al. Radiopharmac?euticals-Biokinetics Data and Results of Radiation Dose Calculations( Informationsdienst fur Nuklearmedi- zin, Berlin) DISTRIBUCIÓN BIOLÓGICA DE 186 RE HEDP EN RATAS WI STAR TIEMPOiHS. 1 2 4 6 15 20 24 48 70 90 141 190 ÓRGANO % DT + SD SANGRE 7,35 3,48 2,02 2,21 1,34 1,47 0,77 0,30 0,17 0,16 0,10 0,50 0,16 0,42 0,50 0,41 0,51 0,05 0,02 0,09 0,03

MSCLL0 6,83 3,72 3,31 1,36 1,53 2,03 0,70 0,91 0,68 0.32 0.57 0,35- 1,93 2,20 0,28 0,49 0,67 0,45 0,13 0,32 0,38 0,15 0,07 0,13

HUESO 33.15 24,82 33,61 20,23 20.23 15,32 17,40 10.86 9,05 8,35 9,11 7,61 6,21 1,95 0,07 1,90 5,43 0,32 0,20 0,65 2-82 2.23 1,70 2,17 HÍGADO 1.90 0,78 0,57 0,70 0.85 0,40 0,37 0,18 0,20 0.19 0,14 0,21 1,28 0,11 0,07 0,17 0,15 0,11 0,01 0,02 0,06 0,11 0.08 0,18 ESTOMAGO 0,60 0,53 0,73 0.93 5,25 8,91 2,21 0.26 0.57 0.11 0.09 0,31 0,01 0,60 0,34 1,58 1.5b 0,14 0-02 0,51 0.07 0.02 INTESTINO 2,87 1,86 1,81 3.65 2,15 2,88 1,24 0.88 1,21 0,25 0,25 0,03 0,69 0,26 0,95 0,86 0,47 0,03 0,43 0.14 1,12 0,02 .,07 0,01 R1Ñ0N 8'69 4.65 2,46 6,05 5,65 2.48 1,16 0,74 0,40 0,40 0.15 0,07 5,14 0,17 0,54 1,47 2,84 0,17 0,09 0,20 0,24 0,19 0,02 0,01 ORINA 37,44 52,38 55,22 64,58 62,61 63,22 75,95 85.52 87.39 89,79 89.59 91,82 11.83 1,38 0,38 5,14 9,16 7,43 0,10 0,14 5,85 2,02 1-28 2,62

FEMUR 0,67 0-58 0.66 0,40 0,45 0.40 0.35 0,20 0,21 0,22 0,20 0,16 0,10 0,18 0,00 0,03 0,07 0,11 0,02 0,01 0,05 0,08 0,03 0,05 - 169-

DOSIS DE RADIACIÓN DEBIDA A IA ADMINISTRACIÓN INTRAVENOSA DE AGENTES UTILIZADOS

EN TEFiAPIA PALIATIVA FN PACIENTES ODN METASTASIS OSEA (RAD/MCÍ) . (CUADRO N° 2)

AUTOR NUCLEIDO FORMA HUESO MEDUIA

SILBERSTEIN et al SR89 CIORURO 59 39

1,5% SR90 78 35

32 KAUL, et al (12) FOSFATO 56 57 P POLIFOSFATO 57 57

COMPARACIÓN CON EL COMPUESTO PROPUESTO

Re186 HFDP 3 3 -j QO Re HEDP 1 1 Y'iO

CM c «T"J

F.nio^ri, n,r)ljnfJr;p Y

'APADA,

' 11 r' i o ^

«i A METODOLOGÍA PARA LA EVALUACIÓN DE FÁRMACOS

DECONTAMINANTES EN SISTEMAS "IN VITRO"

C- Fernandez Degiorgi , D. Dubner, I. Gomez Parada (C .N . E .A.-Buenos

Ai res-Argent i na)

Debido a la generalización de) uso de la energía nuclear

para fines pacíficos, ha aumentado la posibilidad de incorporación de elementos radiotóxicos en trabajadores expuestos. Hay entonces un interés creciente en el desarrollo de métodos decontaminantes efectivos, principalmente para aquellos radionucleidos que tienen una vida media efectiva larga.

Teniendo en cuenta que los actínidos y lantánidos de im- portancia para la protección radiológica tienen alta afinidad por el hígado, se desarrolló un sistema experimental destinado a fa_ cilitar el estudio de la acción de los fármacos decontaminantes eri el tejido hepático.

En dicho sistema se distinguen tres etaoas :

1.- La aislación y cultivo de hepatocitos en presencia de un a- gente quelante.

2.- La obtención de la fracción citoplasmática soluble y su aná- lisis cromatográfico.

3.- La comparación del comportamiento del agente quelante en dicha fracción "in vitro" e "in vivo". - I 7 2 -

Se utilizó como r adionuc 1 eí do Cl Ce-144 y como agente

decontaminante DTPA Ca Na_

MATERIALES Y MÉTODOS

Se contaminaron ratas Wistar (250 g) con 0,3 mi de Cl_ Ce-

144 vía i nt rapen" toneal . A las 48 horas, la cavidad abdominal fue a-

bierta y una cánula fue insertada en la vena porta para realizar la -

perfusión "in situ" del hígado siguiendo la técnira quirúrgica emple^

da por Bhattacharyya (l).

Se realizaron dos perfusiones sucesivas, la primera con

40 mi de solución de Hanks modificada (libre de Ca y Mg con 25 mM de CO. HNa) y la segunda con 50 mi de la misma solución s¿ 2 + lina balanceada adicionada con 5 mM de Ca 0,05% de colagenasa (Sia^ nna tipo IV), 0,05'/. albúmina bovina (Sigma) y 0,01 '/., de estreptami ci na.

Esta última solución fue preoarada una hora antes de la perfusión,

termostat izada a 37 C y con burbujeo constante de una mezcla gaseosa

constituida por 5 J, de C0, 75 % dp ¡^ y 20 % de 0,, . El pH fue así mantenido igual a 7.5.

Despúe'j ie 5 minutos de oerfusión sin reciclaje el hígado fue inmediatamente removido, lavado, finalmente desmenuzado y dige-

rido con ]a misma solución de colagcnasa, durante 20 minutos a 37 C con agitación y gaseado.

La susoensión celular obtenida se filtró a través de una

ITMII.Í úo acaro de 350 me'.h y el filtrado r.o .entrifugó a 50 xg -173-

durante 2 mi ñutos.

El pellet fue lavado dos ve^es con medio de Waymouth MB 752/

1 con glutamina y sin bí carbonato, suplementario con 2,2í+ g/l de CO

HNa, 50 U de penicilina, 50 ug/rnl de estreptomicina y 10 % (v/v) de

suero fetal bovino.

El pellet final fue resuspendido en el mismo medio para dar

una concentración de 2 E + 05 células por mi .

El rendimiento celular y el índice de viabilidad fueron

calculados por duplicado usando una cámara de conteo de Neubauer.

El ensayo de viabilidad se basó en la capacidad de los hepatocitos

para excluir el colorante Trypan Blue.

Se procedió luego a la incubación de los hepatocitos aisla_ dos con el agente decontaminante.

Para ello se sembró 2 mi de dicha suspensión celular en cápsulas Falcon de plástico, se agregó DTPA CaNa, a una concentración final de 5 E-03 M y se 1 a mantuvo a 37 C durante 6 horas en un incu- bador humidificado y gaseado.

Toda la técnica se realizó bajo condiciones de asepsia.

La suspensión celular fue removida de la cámara de cultivo, cc-ntr i fugada a 100 xg durante 2 minutos y el Del let lavado dos veces con CINa (0,9 %).

Se determinó la actividad de Ce-l^ en el pellet y en el

"pool" de sobrenadantes con un cristal de pozo de INa, complementán- doT.c anfla primera etapa del diseño experimental.

El pellet fue resuspendido en sacarosa 0.25 M en Tris - C1H

(pH - .',-••), homogenei Hado en un disruptor tipo Dounce con diez golpes y cent r i f ugrdo a 'lU^ xg durante 10 minutos. 7 4 -

La secuencia se repitió nuevamente y el "pool" de sobrenadar!

tes que representa el extracto citoplasmático se centrifugó a 10$000xg

durante 80 minutos. El Del let fue descartarlo y el sobrenadante que

ahora es la fracción citoplasmatica soluble fue óromatografi ado en co_

lumnas de Ultrogel AoA 22 (de 70 x 1,6 cm), usándose como buffer de -

elución Trisoloridrico pH= 8, CiNa 0,1 M, azida sódica 0,02 %.

El eluato fue monitoreado espectrofotométricamente a 2%U nrn y se recogieron automáticamente fracciones de h mi. En cada una se determinó la actividad de Ce-l't^.

Las columnas fueron calibradas con transferrina humana

PM - 69000 y ferritina de bazo de caballo PM = 700000 - 800000 y e'i volumen vacío fue determinado con Blue Dextran.

Para la tercera y ultima etapa se inyectaron ratas intraperitonealmente con 0TPA Ca Na en dosis equivalente a la utilizada on terapéutica humana (30 uM/kg Deso) 2k horas después de la <-ont arni nación .

Los hepatocitos fueron aislados con la misma técnica e inmediatamente homogeneizados omitiéndose la incubación "in vitro" durante 6 horas

RESULTADOS

La distribución de la actividad de Ce - ]kh {% de actividad total ) de hepatocitos aislados in¡ubados con el - 175-

quolante y sus respectivos controles se muestra en la Tabla 1. El

valor ríe la relación pel 1et/sobrenadante es de 0,8^ y 1,71 res-

pect í vamente.

E] valor de la relación pellet - sobrenadante es un índi- ce de la actividad de Ce-1^4 retenida por las células.

En el gráfico 1 se muestra el perfil de absorbancia de]

(itosol de hepatocitos aislados o incubaHos sin DTPA Ca Na y i a distribución de la actividad de Ce-l^ (como '/ de actividad recu- perada) entre las fracciones del eluato después de la cromatografía en Ultrogel AoA 22. Se observa un pico de actividad de Ce-lit'+ que coincide con el volumen de elución de la ferritina y otro que se encuentra on los volúmenes corresponaientes a la región del límite cíe

•xr lusión inferior del ultrogoi AoA 22 (rango de fraccionamiento

1200000 - 100000).

En el gráfico 2 se muestra el perfil de absorbancia y el h ir, t nq r ,-irun de a<-¡ -i viciad de Ce—IM+ n^ra hepatocitos incubados con —

DTPA Ca Na,..

fn la Tabla 2 se muesiia la relación entre la actividad de Cr' - 1 L\h qu'1 anare^e en las fracciones de alto y bajo PM corres- pondiente ,i 1 citosol de hepatocito; in ubados con el quelante. r.omp¿ r

En el gráfico 3 se observa el perfil de absorbancia y el hi--tograrm de actividad do Ce - 1 h¡ de pues de la cromatografía en

Ulrr-igel AoA 22 de") citosol obtenido de ratas tratadas con 0TPA - i 76-

Ca Na a las 2k horas de la contaminan' ón. Los picos de actividad

de Ce - ]kU aparecen al icjuiit que en la experiencia "in vitro'1

coincidiendo con el volumen dfí elución de la ferritina y con los

volúmenes de la región del límite de exclusión inferior del

Ultrogel AcA 22.

En el gráfico h te observa el perfil de absorbencia y el

nistograma de actividad para las ratas controles.

En la tabla 3 se munstra la relación entre la actividad

He Ce - 1^4*4 liqado a las fracciones de alto y bajo PM correspondiera

te al citosol de ratas cortí decontaminante y sin tratar. El valor -

de dicha relación resulta igual a 0,59 y 6,05 para muestra y control

res pert i vamente.

CONCLUSIONES

La incubación durante 6 horas de la célula aislada en presencia del quelante es un método adecuado para usar en una primera evaluación de un potencial agente decontaminante.

La validez de este método está sostenida por el hecho de nue con un decontaminante de efecto ya conocido como el DTPA Ca Na se observó una Hisminución de la retención de Ce - 1^*4 en las célu- las en cultivo.

Si bien en este sistema experiment a 1 se tienen en cuenta factores que involucran Dernieabi) idad de membrana y constante de - 17 7-

estabilidad de los complejos formados, no considera factores espe-

ciales involucrados en experimentos con animales tales como dist.ri_ burión de la droga y su metabolismo.

Los resultados preliminares arerra del posible mecanismo de la acción del decontaminante obtenido por el análisis cromato- gráfico, muestran una distribución diferente del raiionucleido entre los compuestos de alto y bajo PM en los hepatocitos incubados en pre_ sencia del quelante respecto ele los controles.

Para dilucidar si tal efecto no es debido a un artefacto de la técnira "in vitro", el decontaminante se administró en forma sistémica y los resultados obtenidos muestran un modelo de dis- tribución similar aunque de menor magnitud.

BIBLIOGRAFÍA

1.- BHATTACHARYYA, M. H. and LINDENBAUM, A. Association of Plutonium with Isolated Liver Parenchymal Cells Following Injection of

Monomeric Plutonium into Mice. Radiat. Res. 66,552-565 (1976).

2.- POPPLEWELL, D. S. Plutonium Uptake by Cell Cultures in Presence of Some Chelating Agents. Health Phys. 2S.i+1?-i+20 (1973). - I /8-

TAliLA

1 S T R 1 B U C 1 0 N I) E LA ACT IV 1 DAP 0 E C E ' '*'* (%) DESPUÉS

DE LA INCUBACIÓN DE HEPATOC1TOS ATSLADOS

SIN DTPA.Na Ca ( 1 ) CON DTPA.Na ,Ca (2) J

pel let sobrenadante pellet sobrenadante

62 . 5 + 3 .4 37.1 + 3.4 46. 1 + 2. 7 53.9^2.7

P/S =1 .7 1+0.28(3) P/S = O.8 5 + 0 . 09( i)

1) valor medio de 5 experiencias + D.S

2) valor emdio de 4 experiencias +_ D.S

3 ) valor emdio obtenido de las relaciones determinadas separadamente para cada experiencia + D.S -179-

TABI.A 2

HKPATOC1TUS INCUBADOS

CON DTPA. Na Ca SIN DTPA.Na Ca

Alto PM/Bajo PM=0.08 Alto PM/Bajo PM=2.6

TABLA '3

RATAS TRATADAS RATAS CONTROL CON DTPA. N;) Ca

Alto PM/Kajo PM=0.59 Alto PM/Bajo PM=6.05 HEPATOCI TOS INCUBADOS SIN DTPA.Na Ca

o. i

o *

-3 v/1

PERFIL DE ABSORBANCIA (254 nm) OBTENIDO POR CROMATOGRAFÍA EN ULTROGEL AcA 22 DE LA FRACCIÓN SOLUBLE DE

HEPATOCITOS AISLADOS DE RATAS CONTAMINADAS CON Cl,, Ce-144 E INCUBADOS SIN DTPA Na3 Ce.

GRA FICO HEPATOCITOS INCUBADOS CON DTPA.Na3.Ca

3 4 5 í * % 9 tó i i. U 15 i4 U U

r T C PFRFIL IE Aí." -R8A!¡::f (254 nir) 0E FNIDP PO? CROWTOFRAr If- EÍU-'LT^"-r,EL :c= 2? DE L-" RACC1OÍ! SOLUBLE •EPÍTO1" T5 iJSLÍDns Di SATÍS COMAWI'IADAS CCN Cl, Ce-l

CR AFICO RATAS TRATADAS CON DTPA.Na3.Ca

ierre ±t¡iL4iiii±p±rri±n±rtrn mmm^.

m .-i—~._J—i.i , ii py - .- áfe^iEitia; mm^ 13

PERFIL D£ ABS0R8ANCIA (254 nm) OBTENIDO POR CROMATOGRAFÍA EN ÜLTROGEL AcA 22 DE LA FRACCIÓN SOLUBLE DE HEPATOCITCS AISLADOS DE RATAS CONTAMINADAS CON Cl3 Ce-144 Y TRATADAS CON DTPA Na3 Ca GRAFICO3 PrRFIl OF ABSORBANCIA (254 nm) OGTENIDC POR CROMATOnRAF!/1 EM ULTRHREL AcA 2? DE LA FRACCIOU SOLUBLF OE HEPÄTOCITOS AISLADOS DE RAWS CPNTAMINAOAS CON Clj Ce-]44 CRAFICO 4 )'I Y rof!TPO|_ v\F CALTW

DE 136 RE

M.n.'ioTO, R./\.' *\.M.SCHRODER, !.C.Pocen Y ^.^AMZINI

COMISIÓN ''ACION^L HF TNER^I^. ATÓMICA DE LA nEPURLicA ARGENTINA.

BUENOS \t J

PREPARACIÓN Y CONTROL DE CALIDAD DE COMPUFSTOS DE 186 Re. M.G.Noto; R. A. Amor;D.A.Caviglia;M.J.Ratner; A.N!.Schroder; J.C.Rocco; A.Manzini (Comisión Nacional de Energía Atómica,Buenos Aires,Argentina). En 1979 Mathiew y col.(1) realizaron centaellogramas óseos en ratas con 186Re-HEDP (hidroxietilidendifosfonato) y 99irtTc-HFDP comprobando que la concentración en tejidos óseos era similar para los dos compuestos. Por lo tanto, y dadas las caracterís_ ticas físicas del 186Re y la conocida propiedad del 99mTc-HEDP de concentrar prefe- rentemente en lesión ósea, propusieron el 186íte-HEDP para tratamiento paliativo del dolor en pacientes con metástasis óseas. En 1983 Eisenhut (2) realizando centello- gramas en conejos demuestra que el 186Re-MDP(metilendifosfonato) y el 99mTc-KDP concentran en forma similar en tejido oseo. En 1984(3) Weninger y col. administran 186Re-HEDP a ratas y ratones con lesión ósea y obtienen una relación entre lesión ósea y tejido óseo normal de 4-5,4. El motivo de éste trabajo ha sido buscar las condiciones óptimas de marcación de MDP, HEDP,PYP(pirofosfato) y FDIMPfetiendiaminotetrametilenfosfórico) con 186Re y la bio- distribución de estos compuestos en animales de experimentación a fin de deteminar el agente terapéutico más apropiado para su eventual utilización como paliativo del dolor en pacientes con metástasis óseas. Materiales y método Producción de 186ReO NH v 13 2 f-,1 186Re se obtuvo por irradiación neutrónica ^9:3x10 n/cm seg) durante 5 días del Re metálico o bien directamente del perrenato de amonio. En el primer caso luego de la irradiación se trató el Re metálico con agua oxigena- da al 10% durante dos horas y posterior neutralización con NH.OH al 25% (2). Al fin de irradiación la actividad específica fue de 37 mCí/mg de Re. Cuando r.e utilizó Re metálico la masa de Re se determinó por espectrofotcmetría en base a la formación de un complejo amarillo con sulfocianuro de potasio en medio clorhídrico 6N y posterior extracción con éter etílico midiendo la absorbancia de la fase orgánica a 430 nm. (4) El Cuadro N°l muestra los períodos de semidesintegración, principales radiaciones emitidas, energía y abundancia de los principales radionucleídos del Re obtenidos por irradiación neutrónica. La pureza radionuclídica se determinó desde 24 horas hasta varios meses posteriores a la irradiación. A las 24 horas la relación 188Re/ 186Re fue de 0,7. La pureza radioquímica de la solución de perrenato (186Re) se determinó por cromato- grafía ascendente en papel utilizando como solvente C1H 0,3 N fRf ReO 4:0,65). Marcación de compuestos con Rel86 "1 Cuadro N°2 muestra las condiciones de marcación do los compuestos ce 186Re estudiados. La velocidad de marcación aumenta ai disminuir ol pH;sin einbarao la baja so lubjlidad de alguno de los compuestos en su forma acida limita los valores de pH de marcación a los señalados. La solución toma una colorac ±1 arribar aue so intensifica a medida quo se forma el complejo de Re. i o marcación se controló por cromatografía ascendente en sn¡*,rbe "¡TIC-PC utilizando solvente metílotiIcetona (Rf compuesto-Re. <),0; Pf Ro04 : 1,0). -18 6-

Una vez completada la marcación el pH se llevó a 6,0 y se controló la ausencia de coloide por cromatografía en ITLC-SG y solvente solución fisiológica (Rf conpuesto Re: 1,0; Rf Re reducido y/o coloide:0,0). Los compuestos permanecieron estables por lo menos durante 8 horas post marcación aún después de diluirlos 10 veces. Ensayos de biodistribución Se realizaron estudios de biodistribución en ratas Wistar de 250-300 g de peso; se sacrificaron a las 2 horas post administración in'_-avenosa. La concentración en tejido óseo de los diversos compuestos de 186Pe fue la siguien- te: MDP > HEDP > PYP > EDIMP

La Tabla N°3 muestra la distribución biológica del MDP y HEDP marcados con Re-186 en diversos órganos.

Conclusiones Se concluye que el HFDP-186Re es el aaente que presenta mejores características dado que: 1) Los valores de la concentración ósea son comparables a los del MDP-186Re. 2) El MDP tiende a precipitar a pH bajos(2). 3) El MDP-Re 186 tiende a aumentar su concentración en el sistema retículo endote- lial cuando la dosis es mayor de 1,2 mg MDP/Kg de peso corporal. 4) Teniendo en cuenta la similitud entre los compuestos de Re y fe y en base a los datos bibliográficos (5) sería de esperar una mayor relación metástasis ósea/tejido óseo normal para el HFDP-186 Re comparado con el MDP-186 Re.

Bibliografía 1)-Mathieu, L. et al.Int.J.Appl.Radiat.Isot.30,725-727,1979 2)-Eisenhut,M. Int.J.Appl.Radiat.Isot. 33,99-103,1982. 3)-Weininger,J et al.Nuklearmadizin.23,81-82-,1984. 4)-Beeston,J.M. et al.Analytical Chem.Vol. 25 N°651,1953. 5)-Fogelman,I et al. J. Nucl.Med. 20,98-101,1979.

Cuadro N"l Tl/2 Principal Emisión Energía-MEV Abundancia 1,071 76,6 f3 0,933 23,4 i b6 Re 90,64 / 0,137 9,4 0,12J 0,7 i H7 -

Cuadro Nül

Tl/2 Principal Energía Abundancia h Emisión MFV I8 2,116 79,0 1 1,965 20,0

188 Re 16,98 a 0,155 15,2 0,633 1,3

Cuadro N°2 -Condiciones de marcación de compuestos de Re. t i Moles Ata Sn Vol PH Tiempo de PH X 10~4 x 10~4 mi marcación marcación (final) (min.) (min.)

PYP 1,5 1,0 2 3,0 90 6 HEDP 1,5 1,0 2 1,5 30 6 MDP 1,5 1,0 2 3,0 90 6 EDTMP 4,16 0,3 2 2,0 60 6

(•f- ) Átomos gramo de Re: 7 xlO

Cuadro N°3 -Distribución biológica en ratas Wistar 2 horas post administración.

HET>P-186Re MDP-186Re % DI ? DI Sangre 3,48 í 0,16 3,97 í 0,53 Músculo 3 -i? ± 2,20 3,02 + 0,53 Fueso 24,82 - 1,95 27,56 + 0,83 + Hígado 0,78 - 0,11 1,11 0,12 Bazo 0,08 - 0,03 0,03 + 0,03 Pulmón 0,13 í 0,01 0,22 + 0,09 Intosti no 1 ,86 - 0,26 3,26 + 0,07 Estómago 0,53 - 0,01 0,58 + 0,52 Testículos 0,38 - 0,11 0,18 + 0,08 Riñon 4,65 - 0,17 ?.,?,( 0,60 Orina 54,08 - ] ,38 5 ñ ,JLi 4,40 CON "^Tc "Sil EVALUACIÓN' EN VARIOS "OTÓLOS W

\ F.LFON**, C.O.CANELLAS* Y A.E.A.^ITTA*

COMISIÓN NACIONAL DE :A DE

CENTRO DE INVESTIHACIONR ?ns. MONTEVIDEO, IJ i I SOPKÜH I !.- I DA , MEBROFEN [_N E IQPOFENIN MARCADOS CON Tc-99m

" S 11 EVALUACIÓN K N V A R 1_O S M O I) K U) S JiJjOU) GJ COS"

M . G . A r g ü e ] les*, A . S . 1, o ó n * * , l¿ . S . V t> r d e r a * * , E.León**,

C.O.Ciiiu'l l;is* y A . K . A . M i L t a "•• " Sub prog rama de Investigación, Desarrollo y Ap Li cae ion en Mi'd icin;i Nuclear. Comisión Nacional de Energía Atómica. Buenos Aires. República Argentina. * * Centro de Investigaciones Nucleares. Montevideo. II r u g u a y .

I NT R 01)1) CC I ON

El origen del uso d e a gentes de visual izacion h e p a to biliar, dentro de la medicina nuclear, lo debemos referir .1 I empleo del "Rosa de Bengala" y de la "Bromo sulftaleína", ambos ma reacios con 1-131. Debido a la alta dosis de radia ción a que se veían expuestos los pacientes tratados con estos agen!es es que fueron reemplazados rápidamente ante la aparición de los qucl .itos del T c - 9 9 m . Esta nueva etapa i'iinu'iizu cuando i. o b e r g presento su primer derivado del acido ¡mi nod iaci't ico, el ampliamente conocido "dimetil-IDA". A p.irl i r de ese momento se sintetizaron varios derivados del IDA con distintos sustituyen tes en el anillo bencénico.Den tro de esta 1 í n e a se encuentra el " c¡ i i s o p r o p i 1 - I I) A " , q u e a poco de su aparición se convirtió en el radio fármaco de m a y o r utilidad en el estudio de las vías hepatobiliares. I' c r o las tareas de investigación de los distintos grupos no se detuvieron y es asi que lio y día se dispone de una nue va línea de agentes para la visual ilación di1 los distintos i r a s t 'i i" n ii •- h e p á ticos y " e s i c vi I a res, se t r a t a de los d e r í v a do 11 ,i 1 o ;,(• n a d (i s del acido i m i n od i a c o t ico. Dent r o de nuestros propósitos se e n e i: e n ! r a , no s o 1 o la i- v a 1 ii .i c i o n r a d i o f a r m a c o I o g i c a , h i n o a d em a : ¡ estudio de p :> : a ni <• t r • • s que ¡ios p«' r ni i ' a n inferir i

• si'.1, producios i' n seres ¡i u nía n o .s .

<• " ni' > r e p i e -i <• n ( .i t : v i • .i (I " ¿í ni I) a s ;; e ,¡t .r a i i o n e s de derivados - 19 0-

d c I IDA hiunos o I e g i d o al " I) i i s i d ;i " , de la p r i m e i a , y .1 1

"Moltrol i'ii i n " e " lodofonin" d e a 1 s c: gunda .

MATERJ_AUiS Y MKT0D0S

r.i r ,i 1 .i obtención d e ) 'i c i do N- ( 2 , f> d i ¡snprnpi 1 i'cnl i

i'¿irb¡ini() i I met i ]) ¡miiiod i acct ico ( d i i s i d i ) se siguió el ni c

lodo publicado por Harvey y col al cual lo incorporamos mod i I i c a c ion es que no s permitieron elevar el rendimiento

il c los intermediarios, así c orno de los productos finales.

Del mismo modo se procedió ron la síntesis del ácido N--(

) -- i odo - 2 , 4 , 6 t r i me t i 1 f e n ¡ I o a r b amo i 1 me t i 1 ) i m i n od i a c u L ico

(ioddfenin) y el /u: ido N- ( 3 -b r orno- 2 , 4 , 6 t r i me t i 1 f e n i 1 c a r b a m o i I m e t i 1 ) i m i n o d i a c é t i c o (mebrofen ¡n) ( I i K I I r a 1 )

Tara los tres derivados se optó como metodología de control de calidad la determinación del punto de fusion utilizando un equipo Ko f1 e r, además se realizaron los es perl r o .s de resonancia magnética nuclear a 100 MHz, en Milu

«ion de d i me t i I suI I o x i d o d e nt e r ad o y pjl ron interno de le

I r.-mict i I s i 1 ano . La espectrometría de masa se realizo a 70

>• V en un cspcrl rómcl ro comandado por computadora v p i o v i s I o de inserción directa de la muestra. Se realizo un m i c r o a n a

1 i s i s para la obtención de la composición centesimal.

1' a r a la marcación se prepararon viales conteniendo

2 0 m >; del N - d e r i v a d o , 0.2 m ;> del agente erductor, C I S n .

.'Mil, mantenido en un ambiente de nitrógeno y en un volumen d i • 1.0 ral . A esta solución se le incorporaron 20 m ('. i de solución de p e r t e c n e c i a t o de sodio (T c- 9 9 m ) provenientes ti e un generador " G e n t e c " , resultando un volumen tina] de

2.0 ni 1 , a un pH de 5 . '> .

V a ; a la determinación de la pureza r a fi i o q u í ni i c a s e u I i i izo cromatografía! ascendente, empleando ionio soporte ¡T¡.C(SG) y i-1 un o •,nlv('iU"S de en r i i il,i inri .mu I H '•> % y I ,i ni e :' c 1 . i m e i i 1 i í i 1 i'i'liin.i ( * I u e n o ( I . 0 : I . ') I . C. • n el primero c! . • > s «ol ven! CÍ el c o ni pue •• t o m i g r a con e I ¡rene al i gu ,i 1 que el pe i Í e c n e c i a t o libre quedando el c i • I << i el e • n <- I iiri; •«. I a r a la p; e . c 1 a •' i g,¡ 19 1-

NH- NH- CK H3C HCKc) Br CH CK

OC-CH-Cl 2 NH- Í H3C CH. Cl-CO-CHrCl Br Chi

OC-CKCl Of-CHrN(CH,COOH HN 2 2

H3C CH, HN(CHfCOOHT \*\J\Ji )ijy Br CH. CH.

SYNTHESIS OF MEBR0FEN1N - 192-

IO,K,AcOH (85%)

HN(CHrCOOH);

CH2 CH2 COOh COO'i-

OF IO:X)FEN!N

finir."1 1 19 3-

n i c a los Kl son 0.0 p ¿j r ¡i el complejo y el coloide y 0.5 pa r .i el porte c n c c ¡ a t o libre. I. a I) ¡ o d i s t r i b u r i ó n se e s L i m ó en ratones, cepa N 1 H , deja dos en ayunas por 2 4 horas , a los cuales se les administro 0.1 ml del quelato, vía vena de la cola. A continuación se los distribuyó en jaulas metabolicas y a distintos tiempos se los sacrificó por tracción cervical. Mediante la apertura de la cavidad abdominal se pin. • el colédoco y se extirpó la vesícula biliar, sin perder su contenido. A continuación se t1 x L r a | c r o n el hígado, bazo, duodeno, intestinos, r i ñ u n >? s , ve jiga y orina. Utilizando una cámara de ionización se determinó 1 ,-j actividad remanente en cada uno de ellos; con los valores obtenidos se construyeron las curavs de porciento de dosis inyectada en función del tiempo post inyección.

S iond» ¡ ;i renal un ;i de las v í a s de c 1 i m i n ación de estos productos y resultando de interés su evaluación, utilizamos perros para su estudio. Los animales fueron sedados por admi n i st rac i ó n i n t r amu s c u1 a r de c1 o r p r orna z¡na (M.R C omb e1e n) y posteriormente cateterizados. Se les administró el radiofárma co vía vena safena y se colecto la orina a diferentes interva los de tiempo durante 120 minutos. Con los valores obtenidos se construyeron las curvas de actividad por mililitro en fun c" ion del volumen excretado. Teniendo en cuenta la relación existente entre el com p o r t ;i m ¡ e n I o biológico de una droga y el coeficiente de partición oi'liinol/aguii es que utilizamos este parámetro para predecir 1 ¡i posible vía de e 1 i m i n a c i <í n de los compuestos estudiados. Para estudiarlo se marco cada derivado en la forma li a b i t u al, luego se tomó 1.0 mi de la solución y se mezcló con igual volumen de octanol, manteniendo la mezcla en agitación a 37 ¡;rados centígrados durante 120 minutos. Transcurrido ese tiempo se separaron las dos capas y se tn i d i ó ] ,¡ r a d i o a c t i v i d a á e n cad a u 11. i de ellas, calculándose el coeficiente de partición, expresa ti o ¡. orno )n a c I i v ] (1 .i el o n o c | iinol / J c t i v i d a d <.• n j u u a . IM-

H •cH c f • l_o: 0 UJ )-i •H O •H 0 C4 a ^ CM CO

. 1

OJ rH O

I .Mebrofenir ?•; ?• Disida

22 i 3 Q lodof er.in

?0 . mm

j 18 i

16 .

14 \

12 1

6 1

30 )6-

c •H f CO () I o mm!••••i PI c •fe n i r0 ^^ u H O en TÍ F Q •H 0 c: M m B D -o Í §

U-I. i o Q

M \A M -\ "0 ) ._> CM an S "^''} •-/.• 1 (T j ft < T tí ^ y h-l

'->

u- .n -A ! o o "ei-.rof er.in

3 [U Xodofenin

« 1 20 30 f igura _6 19 9-

En [odas las preparaciones 1 a pureza radioquímica lúe superior al 9 5% En la figura 2 se observa la variación del porciento de la dosis administrada en función del tiempo en vesícula biliar; para simplificar para cada uno de los derivados se g r a f i c a n tres tiempos, 5 , 1 r> y K) minutos, los que fueron si'h'ci-inniídiis luego de construir la curva de porciento de dosis en (unción del tiempo desde los 5 a los 180 minutos u I i ; i /. a i) d o s e s e i s a n i m a 1 e s en I .i dclcrui n a c ion d e c a d a u n o de los puntos. De este gráfico es evidenti' que la máxima captación no concuerda entre los derivados ha 1 o g ena d o s y el d i i s i d a ; es así que mientras que el pico máximo para este último se en ruentra a los 1 r) minutos, para los h a 1 o g e n a d o s se halla en los 5 minutos. Ten ¡ elido e n cuanta los valores encontrados, a idénticos tiempos, para la c a p t a c i ó n h e p a I. i c a (figura i ) se calcularon las reí a c i o n e s e n t r e I a s c a |> I a c i o n e s e n vesícula e h i g a d o (figura 4) encontrándose el mayor cociente a los 15 minutos. Respecto de al excreción renal, luego de construir la curva de porciento de la dosis i n y e c t a d ¿t versus tiempo, como en los casos anteriores, se eligieron para la comparación los tiempos que se detallan en la figura 5 (15, 30 y 60 minutos). Cuando se realizo la co r r o b o r a c i ón de estos resultados en otro modelo biológico, perros, se obtuvo la gráfica de la figura 6.

CONCI.US I ONKS

Lo primero que nos planteamos al comenzar este trabajo luc I o j', r ,i r s ¡ni et i/.ir los derivados li a I o j; e n a d u s , que y .i habían prcsenl adn o t r o s a u t o re s , el objetivo lúe cumplido o b te n i e nd o ii n i e n d i m i e n t o d e r e a c c ion :; a I i ¡ .i c t o r i o . [!n,i • e /. 1 o g r ado es- to, y en b a s e a I a rxper i e ncía r e c o g .d a c on 1 o s d e mas d e r i v a (los, lue f( ii e ! raíamos de e n <" << n ¡ r ,, c p .,• r . i me í ros que también nos pe rmi t iesen i n I e r i r el c orr, port am . e n I o d e a q u e I 1 o s : tiempo de I'Q[)I;:K L.I I>OK 1 LIOO

DKR I VAIJO in '-> Ai

disid.-i 1 . b9j_O. IV mobrof'onin 1 . 46_t_0./6 ¡odofenin 1.b»•0.01 •20 I -

m a x i m a captación vesicular, excreción renal y poder 1 i p o f í

I ico. Anal izando los valores de los coeficientes de partición

( I i g u r a 7 ) sería lógico suponer que el comportamiento b i o 1 ó g i

ce- de ellos tendría que ser similar; esto no ocurre y es p r e c i s a mcnLe t a excreción renal el rol que mas los diferencia, sef.ún este análisis.

Surge de esto que no solo el grado de 1 i p o f i 1 i c i d a d de un compuesto es import ante como elemento d o co n t r o I sino que también lo es la evaluación del comportamiento biológico en un mude lo adecuado, ratones y/o porros, dado que existen otros factores que entran en juego durante el tránsito citoplasma tico de los que latos, como son los átomos de halógeno incur p o r a d o s .

A G K A I) E C_J_M 1_E_NTOS_

.- Parcialmente auspiciado por los contratos del Organismo

Internacional de Energía Atómica (01E A) 3 3 3 7 / R B y 3338/RH.

.- Al grupo de Aplicaciones Biológicas y Veterinarias del

dentro de Investigaciones Nucleares de la Rcpúb lira Orion

tal del Uruguay.

In vitro properties and clinical use of T c- 3 bromo-2,4,6 trimethyl-IDA in sequential hepatobiliary scint igraphy. G . F . (Í a m u z z i , B.D'Angelini, P.Tortore, A . B i g g i and G . P I a s s s i o The J.NucI.Med and Allied Sciences Vol 28 Nro 3 (16 7-179)

An structure-distribution-relationship approach leading to the development of Tc-99m "mebrofenin": An improved chole.scint igra phic agent. A.D.Nunn, M.D.Loberg and K.A.Ciiiley J.Nuc.Med. Vol 24 Nro 5 (423-430) _[_ 9_8_ 3 Tc-mfbrofenin: 1 n c o r p o r a t i on as a rouI i n e a g e n t i or h epato biliary studies. S . V e r d era, A . 1. e ó n , C . 0 . C a nol 1 a s , K . I, e ó n , if . i'> a i t o i , .1 . .! . I. ó p e /. a nd li . A . K . M i t t a . New perspectives in nuclear medicino. Fart ; . edited .) y i' . C o x and K. Tonya. Gordon and Breach Science P u b L i s h e ." ;-. . I 9 8_'> . Y CO'ITPni nc CALIDAD IF FPTIW'II q0"Tc.

)j ^.RAMLLFR., C.^ARRIF FACET, C.FlS^AN Y

A.MANZINI

COMISIÓN NACIONAL DE F^ERHIA ATÓMICA DE LA REPÚBLICA ARHFNTINA.

REPÚBLICA VGENTINA PREPARACIÓN Y CONTROL DE CALIDAD DE FIBRINOGENO 99m Te.

M.G.Noto;G.Rabí 11er;C Garrié" Faget;C.Pisman;A.Manzini-Comisión Nació nal de Energía Atómica,Buenos Aires,Argentina.

Dada la limitación de los agentes iodados utilizados actualmente en la detección de trombosis agudas, debido a las características físicas del 125 I y 131 I, se ha desarrollado un método de preparación de fibrinógeno para marcar con 99m Te utilizando coito reductor Sn-H- en medio alcalino. (1) , (2) , (3).

Materiales y Método Se utilizó fibrinógeno humano liofilizado (Inmuno A.G.,Viena, Austria) cuyo conteni_ do es:

o. Fibrinógeno Humano 24,39 dtrato Trisódico dihidratado 14,63 Glicina 12,20 Cloruro sódico 12,20 Glucosa 36,53

Se preparó una solución de fibrinógeno de 5 mq/ml en solución acuosa de C^Sn^EUO de 0,2 mg/ml; se le adicionó igual voliaren de solución alcalina de Sn-H- (0,2 mg/ml de Cl2Sn-2H20 en glicina 0,05 M pH 12-12,2). Este preparado se dejó durante tiempos variables en un baño termostatizado a 20 °C, se fraccionó 2 ml/vial a través de filtro de rrembrana de 0,22 micrones y líofilizó. Todas las operaciones se realizaron en forma aséptica y en ambiente de nitrógeno. Al liofilizado se le agregó 2-3 mi de solución de 99m TCO4 proveniente de un generador GENTEC. Los controles de pureza radioquímica fueron realizados entre 20 y 120 minutos post marcación por cromatografía en soporte ITLC-SG y solvente metanol 85% (Rf fibrinó- geno -99mTc: 0,0; Rf 99mTcO^ :lf0) y en columna de sephadex G-25 eluída con solu- ción fisiológica: el 99MTc hidrolizado queda en el punto de sieitfora, el 99mTcO4 a 1/3 de la columna y al final de la misma el fibrinógeno - 99mTc. Los controles de coagulabilidad se llevaron a cabo con sangre de conejo: a 0,1 mi de la solución de fibrinógeno-99mTc en tubo de centrífuga se le agregó directamente de la oreja del conejo 3-4 mi de sangre; se incubó a 37° C durante 2 horas y posteriormente se rompió el coágulo con varilla de vidrio, centrifugó y midió la actividad en coágulo y suero. Los ensayos de biodistribución se llevaron a cabo por inyección intravenosa en rato HOP que se sacrificaron a los 60 minutos post administración. El animal entero y los órganos disecados se midieron en cámara de ionización.

Resultados En el Cuadro N°l se muestra el rendimiento de marcación en función del tiempo de in cubación a 20°C del fibrinógeno (Sn) a los 20 minutos post marcación con 99Tc, de-~ terminado por cromatografía (ITLC-SG). El Cuadro N°2 muestra la distribución biológica en ratones I hora post administra- ción intravenosa de fibrinógeno-Sn-99mTc; los valores representan el valor promedio de 8 preparaciones incubadas durante 22 horas r. 20°C e inyectadas 20 minutos post marcación. El Cuadro N°3 muestra el rendimiento de marcación y distribución biológica en ratones 1 hora post administración intravenosa de preparados con distintos tiempos de incubación, a los 80 minutos post marcación con 99 mTc. El valor de la relación entre el porcentaje de dosis inyectada en sangre e hígado se correlaciona directamente con la calidad del fibrinógeno-Sn-Tc 99m, de tal manera qvs el tiempo de incubación puede reducirse siempre que se aumenten los tiempos post marcación del producto; sin embargo por debajo de 18 horas de incubación los tiempos post marcación deberían au- mentarse considerablemente para obtener porcentajes superiores al 95%. Para preparados con tiempos de incubación entre 22 y 23 horas y tiempos post marca- ción de 20 minutos, el ensayo de coagulabilidad dio el valor de 73,8 - 1,03% (promedio de 4 preparaciones) y el 99mTc hidrolizado determinado por cromatografía en gel fue manor del 5%; en todos los casos el pH del fibrinógeno-Sn-99mTc estuvo entre 10,0 - 0,1. De los datos suministrados en los ensayos se concluye que el tiempo, óptimo de incu_ bación está entre 22 y 23 horas; por lo tanto se adopta como método de preparación 22 horas de incubación a 20°C, pasaje por filtro de membrana de 0,22 micrones y frac cionamiento de 2 rKL/vial inirediatanente antes de realizar la liofilizacióh.

Bibliografía l)-Jeghers,O;et al;-A chemical method for the labelinc? of fibrinogen with Tc99m. -Eur.J.Nucl.Med.,3:95-100,1978. 2)-Arguelles,M.;Palcos,M.C.;-Estudio de las condiciones de marcación de fibrinógeno humano con Te 99iru- 6"Congreso Argentino de Biología y Nfedicina Nuclear,Buenos Aires,24-28 de noviembre de 1980. 3)-Lavie, L et al.-Factors affecting the labeling of human fibrinogen with Tc99m in vitro.Int. J.Appl.Radiat.Isot.Vol. 35 N°2 pp 99-102,1984. CtJADRO N°l - Rondimionto -.i- a-.-.r: KM-.,; do i f • liri.-,6>.nü.^-(Sn) con ^rríic en función del tiempo de inciKicióii -i 20'"'(.' y ?.() mi rrutos r;ost maroacíom.

LJBACKJN "ION :^\CTON MARCACIÓN US %

S f.6 ,0- 8/) ' 1 82, íi 1 10,0

11 ' 7 ' 1¿ 94,1 ± 2,7 17 74 •i í •' f • 94,7 ± 3,3 18:30 Vb •••,! t,. \ >A 86,0 ± 1,5 78,3* S,7 íí 5: .30 87,8 -- 7,5

CUADRO N"2 - BicxHstrjl'Ucióo o.v, ratory-f; 1 h'jro MDSI; adininistiación intravenosa de fihrinóqei,o - f;cji-YI\-:.

()]

Hf q; ido ' ' -' * ' Riñ6n Orina s.n.i. '•i RENDIMIENTO DE MARCACIÓN 80 MIN. POST ADICIÓN DEL 99MTC Y DISTRIBUCIÓN BIOLÓGICA EN RATONES

1 H POST ADMINISTRACIÓN EN FUNCIÓN DEL TIEMPO DE INCUBACIÓN DEL FIBRINGGENO(SN) A 20 °í

INCUBACIÓN RENDIMIENTO DISTRIBUCIÓN BIOLÓGICA % DI

8 92, 1*2, i

1 10 30 91, 5*1.

14 91, 9±2, 1 ¡46, 2*0 ,7 18,7-0,1 11,4*2,3 8,3*1,9 ¡14,8*3,5 2,5-0,0 I ,8 ¡45 .7*1 ,5 8,1*1,6 9,0*0,4118,2*3,7 2,5*0,3 18 93 7±1 i i 20 96 ,9*2 2 ;47 ,3*2 ,0 19,8-0,8 7,9r0,5 6,5*1,7!17,9*3,4 2,4*0,3

22 95,5*1 ,4 ¡48,8*1 ,5 19.9-0,1 8,0-0,1 7,6-1,1)15,2-2,8

+ 2". 95 .4*1 ,5 ,1 21.9*1,3: 8,0*0,2 7,2*l.5¡13,2*0,3 ; 2,3*0,2 207

LIST OF PARTICIPANTS

AMIGO. Viviana BLAHA. Vaclav Hospital A.Ro f fo De p.Nucl.Med. Av.San Martin 5481 Faculty of Medicine and Capital Federal Hygiene. Charles University Buenos Aires Prague. Srobárova 50, República Argentina Prague 10 10034 Chekoslovaquia ANGU[LERI. Leopoldo Service Med. Nucl. BERG0C. Rosa Universite Nancy Facultad de Farmacia y 18 R u e Lionnois Bioquímica. Dpto de Física Nancy 54000 Cátedra de Radioisótopos F rani' i a Cap.Federal. Rep.Argentina

ARC1PRETE.Carlos CAMIN. Leopoldo Comisión Nacional de E.I.DuPont de Neumours Knergía Atómica 331 Treble Cove Rd . Av.Libertador 8250 No rth Billerica (1429) Capital Fede Massachuset t s 0 1862 r ;J 1 . Buenos Aires. USA República Argentina CAMPOS. Estrella ARGUELLES. María Facultad de Química Comisión Nacional de Av.Gral .Flores 2 1 24 Energía Atómica Montevideo. Av.Libertador 8 2 50 República Oriental de (1429) Buenos Aires Uruguay. República Argentina CAMELLAS. Carlos Osear BARMASCH. Martha Comisión Nacional de Comisión Nacional de Energía Atómica Knergía Atómica. Av.Libertador 8250 (1429) Av . 1. ibert ador 8250 Capital Federal. Buenos (14 29) Buenos Aires Aires. República Argentina República Argentina CARO. Ricardo BARRIO. Jorge Facultad de Farmacia y Bio Lab. of Nuclear química. Universidad de Medicine. Division Buenos Aires. Cátedra de o I Bio-Physics. UCLA Física. Radioisótopos. School of Medicine. Buenos Aires. República Los Angeles, CA 90024 Argentina. USA CASTAGNINO. Juan BARBOZA. Marice) Facultad de Ciencias Exactas instituto de Pesquisas y Naturales. Universidad de K n practicas. Buenos Aires. Ciudad Univor Ciudad Universitaria •sitaría. ;' ;i h e i 1 ó n \ \ . S .i o I' .i u 1 o . Brasil C a p i í. a I i' c .i <• r .i I . Buen o .s A i r c i República A r ¡\ o n t i n a . B K N IT E 7. . Mari a l'royrd o I5 <.• r u CAUL. S ,i n t i .i j-,o Comisión Nacional de Comisión N.io idnal úc Energía Energía Atomic;-! Atómica. Av.Libertador Av . Lil)l

COLOMBKTT I . Li> I iu DALESS10. Gilberto 8 13 N.Gleanda le Avenue Facultad de Farmacia y Glendale, CA 9 1206 Bioquímica . Capital Pede USA ral. Buenos Aires. República Argent ina COLUCCIA. Graciela Facultad de Farmacia y GAMBINO . Nora Bioquímica. Universidad Facultad de Ciencias Exactas de Buenos Aires. Dto. y Naturales. Ciudad Univejr de Física. Cátedra de sitaria. Pabellón II. Buenos Radioisótopos. Capital Aires. República Argentina Federal. Buenos Aires. Re pública Argentina DUBNER. Diana Comisión Nacional de Energía COUTO. Susana Atómica. Av.Libertador 8250 ('Omisión Nacional de (1429) Capital Federal. Bue Energía Atómica. nos Aires. República Argcn Av .Libertador 8250 (1429) tina. Buenos Aires. República Argent ina FERNANDEZ DEGIORGI.Cristiaa Comisión Nacional de Enej: CHACON. María gía Atómica. Av.Libertados Proyecto Perú. 8250 (1429) Capital federal Comisión Nacional de Buenos Aires. República Energía Atómica. Argent ina. Av-Libertador 8250 (1429) Buenos Aires. República FONTANA. Luís Argentina. Facultad de Farmacia y BÍ£ química. Universidad de B ue_ CROOK. James nos Aires. República Argentina Oak Ridge Associated Universities. Medical and FRA --A DE SUAREZ. Amanda Health Sciences Division Comisión Nacional de Energía Tennessee Atómica. Av.Libertador 8250 USA ( 1429) Capital Federal . República Argentina CISCATO. Vicente Facultad de Farmacia y BÍ£ SILVERA. Franco química. Universidad de BUJ? Centro de Medicina Nuclear nos Aires. Dto. de Física. Hospital de Clínicas. Cátedra de Radioisótopos. Montevideo. República Orien Buenos Aires. República tal del Uruguay. Argent ina FURNARI. Liliana CYNBERKNOP. Dora Comisión Nacional de Energía Academia Nacional de MedicJ^ Atómica. Av.Libert ador 8250 na. Servicio de Medicina (1429). Capital Federal. Nuclear. Buenos Aires, re pja • Buenos Aires. República blica Argentina. Argent ina.

D'AMORE. Beatriz GAVILÁN. Moisés. instituto Nacional de l'ar m a Instituto de invest i (¡aciones cología. Av. Caseros 2161. y Ciencias de la Salud-Rio Capital Federal Buenos Aires do ! ;i Plata y G e r e s a . República Argentina. P a r a g u a y -209-

GARCJA. Eloy KRAMAIZ. Emilia U. F.R.G.S Comisión Nacional de Ener Biofísica gía Atómica. Av.Libertador 9 0 049 Porto Alegre R. D 8 2 5 0 (14 2 9). Capüal Federal. Brasil Buenos Aires, república A rge n t i n a. GOMKZ DE CASTIGL1A. Silvia Comisión Nacional de Ene£ LANÜABURO. Juan Carlos gía Atómica. Av.Libertador Centro de Altos Estudios 8250 (1429). Capital Federal en Ciencias Exactas. Av . Buenos Aires. República de Mayo 1396. Dto de Biol£ Argent ina gía. Buenos Aires. RepúbH ca Argentina. GODOY SANCHEZ. Nelson Comisión Chilena de Energía LAGE. Marcelino Nuclear. Av. Nueva Bilbao Comisión Nacional de Energía 12301. Santiago de Chile Atómica. Av.Libertador 8250 Cl] i 1 e (1429). Capital federal. República Argentina. GOMEZ PARADA Inés Comisión Nacional de Ener LARCHER. Ana gía Atómica. Av.Libertador Comisión Nacional de Energía 8250 (1429). Capital Federal Atómica. Av. Libertador 8250 Buenos Aires. República (1429). Capital federal. Argent ina República Argentina

GAI.BAR INI . Alicia LARUMBE. Fernando F ¡i <• u 1 t a d d c Q u í m i c ;i Comisión Nacional de Energía Marcelino Sosa 2779. Gral Atómica. Av.Libertador 82 5 0 Flores y Yatai. Montevideo (1429). Capital Federal. U r u guay Buenos Aires. Rep. Argentina

HE I,US. Frank LEGUIZAM0N. Carlos Geman Cancer Research Center Comisión Nacional de Energía Institute of Nuclear Medicine Atómica. Av.Libertador 8250 Fm Meildelberg Feld 280 (1429) Capital Federal. Bue 69 0 0 Neiideberg . Federal nos Aires. Rep. Argentina. Republ ic LEON. Alba HNATOWICH. D.J Centro de Investigaciones Department of Nuclear Medicine Nucleares. Av.Mataojo S/N l¡ n i v . Massachusetts Medical Montevideo. Rep. Oriental Center. del Uruguay. USA MADDALENA. Demon HERNANDEZ. Leopoldo Australian Atomic Energy ('omisión Nacional do Energía Commission-Private Mail-Bag Atómica. A v.L i b e r t a d o r 8250 Sutherland (14 2 9). Capital. Federal. Bue Australia nos Aires. Rep. Argentina MALKISCHER. Susana 1R í BARNK. Nestor Facultad de Farmacia y Facultad de Farmacia y B i o Bioquímica. Universidad de química. Universidad de Bue Buenos Aires. República nos Aires. Rep. Argentina Argent ina. - 2 10-

MAROTTE. María PARRA. Lucia C o misión Nacional de Clínica Marly. Energía Atómica. Av. Carrera 13 4940. Entrepiso Libertador 8250 (1429) 101. Bogotá. Colombia Capital Federal. Bue nos Aires. R e p. Aregnt ina ORCE. Luis Comisión Nacional de Energía M I C H E L 1 N . S c v e r i n o Atómica. Av.Libertador 8250 Comisión Nacional do (1429). Capital Federal. Energía Atómica. Av. Buenos Aires. Rep. Argentina Libertador 8250 (1429) Capital Federal. Buenos OTAVIANO. Graciela Aires. R i> p . Argentina Instituto de Investiagciones Cardiológicas. Marcelo T de MITTA. Aldo Emilio Antonio Alvear 2270. Capital Federal Comip i ón Nacional de Buenos Aires. Rep. Argentina Energía Atómica. Av. Libertador 8250 ( 1429) . PALAU . Virginia Capital Federal. Buenos Centro de Altos Estudios en Aires. Rep. Argentina. Ciencias Exactas. Av. de Mayo 1396. Dto. Biología. MITTA. Marisa Capital Federa]. Buenos Comisión Nacional de Aires. Rep. Argentina. Energía Atómica. A v . Libertador 8250 (1429) PARERA . Victoria Capital Federal. Buenos Facultad de Ciencias Exactas Aires. Rep. Argentina. y Naturales de la Univers^ dad de Buenos Aires. Ciudad MORAN. Alberto Universitaria. Pabellón II. Centro de Altos Estudios 4 to piso. Rep. Argentina. en Ciencias Exactas Av.de Mayo 1428. Capital PALCOS. María Cristina Federal. Dto de Biología Comisión Nacional de Energía Buenos Aires. Rep. Argentina Atómica. Av . Libertador 8250 (1429). Capital Federal. Bue MORENO. Iris nos Aires. Rep. Argentina. Hsopital Ramos Mejia Urquiz a 6 7 3. Buenos Aires PAVONI. Pietro República Argentina Univ.Policliníco Umberto i Clínica Medica. 00100 Roma NOTO. María Italia Comisión Nacional de Energía Atómica. f. v. Libertadro 825 0 RAYMOND. Paulin (14 2 9) Capital Federal. Faculté de Medicine Buenos Aires. Rep.Argentina. 27 Bd. Jean Moulin i 3385 Marseile Ceedx 5. N0UJAIM. A.A F r ancia University of Alberta Div.of Bíonucleonic & PEARSON David Radiopharmacy. Faculty o 1 Unit for Metabolic. Medicine l'hcirmacy . Ed moni on. Al bcrl.i Guy H o :, j) i t a I Medical School T6C 2G3 Canada London. S L .T homa s Street. London Bridge -2 11-

PEC0RIN1. Viet o rio ROBLES. Ana María Comisión Nacional do Ener Centro de Investigaciones gía Atómica. Av. Libertador Nucleares. Av.Mataojo S/N 82 50 ( 14 2 9) . Capital Fed£ Montevideo. República Orieii ral. Buenos Aires. Republi tal del U ruguay. ca Argén Lina. RAMOS. Miguel PEREZ. A Servicio de Medicina Nuclear Comisión Nacional de Enejr Hospital de la Fuerza Aerea gía Atómica. Av.Libertador Ventura de Argentina la Vega 8 2 50 ( 1429 ) . Capital l'ede 3 697. Capital Federal. ral. Buenos Aires. KepúbM Buenos Aires. República Argejí ca Argentina tina.

PISA REV. Mario RAYUDU. Gariemlla Comisión Nacional de Ener Rush University Medical Center gía Atómica. Av. Libertador 17 5 3 W. Congress Parway. 8 2 5 0 (14 2 9) Capital Federal Chicago. Illinois 60612 Buenos Aires. República USA . A r e g n t i n ¿i SCOTT. John i'QZZ I . Osear Edmonton Radi opharmaceutics] C OLÍ i s 11' n N .i c i o ii a 1 el e E n e r ('. entre. 1 15 6 0 University gía Atómica. A v . 1. i b e r t a d o r Avenue . Edmonton. Alberta 8 2 5 0 (14 2 9). Capital Federal T6G 1Z2 Buenos Aires. República A r Canada g e n L i n a . SOUTO . Be a I. r í v. RABILI.ER. Graciela Centro de Inevst igaciones Comisión Nacional de Ene_r_ Nucleares. Av.Mataojo S/N gía Atómica. Av.Libertador Montevideo. República Oriental 8250 (1429). Capita] Federa] del Urugu ay . Buenos Aires. República Argejí t i n a . ROCCO. Juan Carmelo Comisión Nacional de Eenrgía SANTOS. Regina Atómica. Av. Libertador 8250 I ti s 1 iluto l'otlugii e s d e O n c o (14 2 9) Capital federal. Buenos logia de Franco Gentil Aires. República Argentina. Rúa Proe Lima Bastos Palhava Lisboa. Portugal. SCHRODER. Ana María Comisión Nacional de Energía JACQUES. Robert Atómica. Av. Libertador 8250 Service Medicine Nucleaire (1429) Capital federal. Buenos Francia. University Nancy Aires. República Argentina. 18 Rue Lionnois 54.000 Nancy. Francia RIVERA. Elena Facultad de Farmacia y Bioquí STL INGARDI .Claudio mica. Universidad de Buenos I. ,i p 1 e x S . A . Aires. República Aregntina. Lisandro d e i a Torre 2161- Buenos Aires. Rep. Argentina RUBIO. C a• i o s Facultad de F .i rni:\ c i a y Bioquí S U K E S H . M . R mica. Universidad de Buenos B i o m i c a [N C. Edmondon Research Aires. Uto de Farmacología. 4 411A-20 Avenue Buenos Aires. República Argén C a nada t i n a . -2 I 2-

RUTTY SOLA. Gi sel le VALDEZ. Isidora Comisión Nacional de Er.er Comisión Nacional de Energía gía Atómica. Av.Libertador Atómica. Av.Libertador 8250 8250 (1429) Capital Federal (1429). Capital Federal. Bu¿ Buenos Aires. República Argén nos Aires. República Argentj^ tina. na .

SCHESINGEK. Tuvia VINIEGRA. Mi r ian Soreq Nuclear Research Center Servicio de Medicina Nuclear Yavneh . Israel. Centro Gallego de Bueno' Aires. Belgrano 2199 S Y KED . Tom República Aregntina Research Fellow University of Alberta WEIN1NGER. Joliette Department of Inmunology Soreq-NCR Edmonton. Alberta Rcidiopharmacie Dept. Canada 70600-Yaune Israel TAJA.M.R Comisión Nacional de Energía W1EBE.Leonard Atómica. Av. Libertador Div.of Bionucleonics & 8 2 5 0 (14 2 9) Capiuil Federal radio pharmacy Buenos Aires. República Argén Faculty of Pharmacy tina University of Alberta Edmonton, Alberta T6G 2N8 TURNER . Conn ie Canada Faculty of Pharmacy University of Alberta ZAN1N0VICH Angel Edmonton, Alberta T 6 G Servicio de Medicina Nuclear 2N8. Canada Hospital de Clínicas José de San Martín. Buenos Aires. TROPAREVKY. Maria República Aregntina. C om i H i ó n Nacional de Energía Atómica. Av.Libertador 8250 ZOLLE. Use (1429) Capital Federal. Bue Zud Medical Clinic. Div. of nos Aires. República Argentina Nuclear Medicine Faouisongasse 13. Vienna TRUMPER. Jacobo A u s t r i a Sorcq-Israel 2 S h k o 1n i k St. Rehovot 76209. Israel

van WYK. A.J Atomic Energy Corporation Isotope Production Centre Private Bag X256 Pretonia. South Africa

VEKDKRA . Silvia C entro de Investí t* ;t rione s Nucleares. Av.Mat. aojo S / N Montevideo. Uruguay.