Receptor-mediated radiopharmaceuticals

T I P S - Februa~ 1984

57

from nerves) than against prolonged applications which permit equilibrium to be reached, a-antagonists do not even reduce the amplitude of e.j.ps, either in our laboratory or in any others where e.j.ps have been recorded, We agree entirely with Dr Bryan on one point; we have not produced enough evidence to prove the existence of the ~receptor. It is a hypothetical receptor whose existence is needed to explain the data, Before rejecting it, Dr Bryan needs to explain (I) why a-antagonists do not block the e,j.p.,(2) why noradrenaline applied by iontophoresis to restricted regions mimics e.j.ps whereas nomdrenaline in the superfusing solution does not. We repeat that experiments in which only muscle tension or contraction are measured are unlikely to provide clear challenges to the idea of a second pool of receptors, because a long chain of intermediate events separates the e.j.p, from activation of the contractile apparatus. The e.j.p, is the closest event to the neurotransmitter-receptor interaction that we can observe. However, the idea that the receptors responsible for e.j.ps are distinct from c~-receptors should only receive full acceptance if a selective antagonist for "v-receptors is found.

Rmliag Ust 1 C-'heung, D . W .

(1982) J

Physiol. (London)

328, 461-468

2 Suzuki, H. (1983) J. Physiol. fLondon) 336. 47-59 3 Bevan, J. A., Bevan, R. D. and Duckies, S. P. (1980) in Handbook of Physiology Secdon 2. Vol. 2, pp. 515--566, American Physiological Society, Bethesda 4 Hirst. G. D. S. and Neild. T. O. {1981) J. Physiol. fLondon~ 313, 34.',-3511 5 Kuffler, S. W. and Yoshikami. D. (1975~ J. PhyxioL ¢London) 252, 46.%482 6 Hhst. G. D. S. and Neild, T. O. (1981) Br. J. Phannacol, 74, 189P 7 Holman, M. E, and Surprcnam, A. t lqgO) Br.

J. PhammcoL 74. 189P 8 Surprcnant. A., Neild, IF. (). and tlolman, M. E. ( 19&:qPfluge~ An h. 39~. 342-349 9 gadiner, J, S,, Bamos, P., Hamilton, C, A.

and Dollcry, C. T, 11979) Biochem. Biophys. Res. Commun. 90, 142-149 T, O, Neild is a National Health and Mediml Re~arch Council Research Fellow m the Depan. merit of Physiology at Monmh Uni~rsiO'. He graduated in physiology from Bmtol Uni~say and workedon molluscanphamua'oioD, m Bristol and Paris before moving Io Monash, His main research inten'~tis in the physiology of the circulatory system. G, D, S. H i m is a Fellow m the John Cunin in p~ma,t~ ~,, ~ u,~ ~ ~., worked in the DeOannmu of Pkamlacology,

School of Medk~ Rm,an.h. He ~ Ed~urS~ ~

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Receptor-mediated rad io p h a r m a c e ut ica I s Ann M. Carroll and Michael R. Zalutskv lh'partm,r;tl " I R+.l,b<,do~t. I l o r l u r d Boston. ,~A 02115. USA

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The development o f radiotracers with defined affinities for specific receptor syswms is a powntial!v ttst'[ui +qVwc~a'h t,, lit," dt,St+qOi Of ruchut.,h,mna~eut, als h,r ,::,, h',~r medicine. Radiolabelled ligands, such as hormones and neuemeansmitters, are being synthesized and evaluated as diagnostic tools for diseases winch can be characterized either by changes in receptor coneenlration or by novel receptor expression. Since the utility o f a radiopharmaceutical is dependent upon the achievemem o f adequate ratios o f target ~o nontarget accumulation o f radioactivio', design o f radid~'acers via the receptor approach attempts to exploit those syswms in which receptor is present in significantly higher concemration at potential target si,e~ such as tumors. Receptor-mediated radiotracers are also curremly being used in animal models to investigate the normal tissue distribution o f drug and hormone receptors and the in-vivo pharmacokinetics o f receptor binding. The successfuA derelopmera o f receptor-avid radiopharmaceuticals will depend on the abili~.. "to synthesize Iigands o f high specific radioactivily which retain their high affmil)- and binding specificitt." for the receptor after radiolabeiling. Nuclear medicine i~rspective The use of radioactive compounds for the noninvasive study of organ anatomy and function is the traditional domain of nuclear medicine. When combined with insmma:ntation capable of measuring the distribution of radioactivity in the body, these tracers represent valuable tools in diverse areas of medical d~agnosis. Isotopes which decay by y-emission are most suited for this application because the range of their radiation permits their detection external to the body. The suitability of a y-eminer for routine use in nuclear medical imaging is determined by several criteria. The isotope should be easily produced and should decay with an energy and photon yield that maximizes spatial resolution and detection effb ciency. Decay characteristics should be such that the radiation absorbed dose received by the patient is minimal. Finally, the half.life of the isotope should be com. patible with the imaging schedule follow. ing its administration to the patient. The isotope which is used most extensively in clinical nuclear medicine is technetium99m (mmTc). It has a half-life of 6 h and is conveniemly produced via a ~ l o J a ' T c radionuclide generator. The y-rays emitted in the decay of W"Tc have an energy of about 140 keV, which is well suited for use with most conventional nuclear medicine devices. Various organic and Lrsaganic

molecules and colloids have been labelled with ~'~Tc and are currenti~ used in a ~lde variety of diagnostic applications. Most radiophannaceuacals in routine u~ hax¢ bi~Mt..mbut,m propcrhc, x~h~ch are biochemicali) nonspecific. Fc,r example. ~m'~Tc-sulfur colloid, which is used for hepatic imaging, accumulates in the hver as a result of phagoq, tic uplake b) Kupffer cells: lung tmagtng glth radi,,L, bctlcd micTt~phcrt~ exph~ts particle ,~.'qtJcqra:lon m capillaH l~'d~. In&'ed. the chnlc.d apl+hcation,, ol m:,n.~ ,l~cnP, such a,, g,dhum-~ +

citrate and ~'Tc-HIDA were disco,,ercd serendipitousi). Gallium,67 citrate, while being tested as a bone imaging aeent, ~as discovered m localize in certain soft tissue tumors'. J°"Tc-HIDA. a technetium label led analog of the ant,-arrhxthmtc drug lidoc.6ne, was mitiall) investigated as a myocardial imaging, agent. Due to its liver u p ~ e and excretion via the bile, n is currentb used as a hepatobiliar) agenP. The information provided by these bit~'hcPllca~l~

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eeuticals is primaril~ anatomic. %~tth the development of newer imaging modalities, the strength of nuclear medicine will be m its ability to provide functional inform& tion. External visualization of function wi~! depend on the development of biochemically specific radioffactrs, mat the next generation of radiophannaceaticals will

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58 rely on the development of more biologically relevant compounds. One such approach is the use of radiolabelled ligands targeted to physiologically relevant recep. tor molecules. Receptor-targeted localization of

radiopharmaceuticats Radiolabelled IL~ands which show specific binding affinity for receptor molecules have been termed 'substrate specific'3. and these differ from substrate nonspecific radiopharmaceuticals in that they do no~.localize on the basis of gross physinchemical properties, it is hoped that selective radioligand uptake at the target site can be achieved by exploiting the ligand's specific, high affinity stereochemical interaction with selectively expressed receptor components. Radioligands currently under investigation include steroid and polypeptide hormones. neurotransmitters and. in a broader specificity range, desialylated glycoproteins which bind to galactose-recognizing recep. tots on mammalian hepatocytes. They also include antibody molecules which recognize and bind with high affinity to discrete antigenic determinants. The potential usefulness of this approach to radiopharmaceutical design is based on the selective target tissue expression of functionally associated receptors. In addition to anatomical imaging of specific target tissues, it may be possible that such radiopharmaceuticals will permit the detection of quantitative changes in receptor expression, which for certain systems has been correlated with particular pathological conditions4. For example, cardiac adrenergic and muscarinic receptors have been reported to undergo quantitative shifts in expression as a result of thyroid disease, diabetes and myocardial ischemia, and brain neumtransmitter receptors show changes which can be correlated with many neurological disorders. Increased expression of steroid receptors in breast, endometrial and prostatic carcinomas is well estab. fished. Also, many tumor-associated anti. liens have been described. Since the,affinity constant for receptor--ligand interactions is generally not altered in pathological states, it may be lX~ssibleto measure quantitative changes in n:~ceptorexpression and to relate d~se changes to specific disease states4. Selection ofradioUpnd derivatives In the .design of receptor-mediated radiophamm,~euticals, it is necessary to screen ligard analogs carefully to ensure that they bi~d to the receptor wi~h sufficient a~Tmity ard that they retain this affinity after radiol~d3elling. In-vitro competitive binding assays measure the ability of lignnd

analogs to displace binding of the parent compound front the relevant receptor, in initial screening, unlabelled derivatives compete with a labelled parent compound in order to weed out compounds with low affinity. Estradiol derivatives, for example, compete with [*Hlestradioi for binding to receptor in cytosolic preparations of imma. ture rat uterus. Those compounds with affinity sufficient to displace the parent ligand are then labelled with the isotope of interest, and the displacement study is repeated with the labelled analog. This is an important step, as the labelling procedure may cause changes in both ligand affinity and specificity. For example, introduction of a bulky halogen label at the phenolic A ring of estradiol interferes with the stereoselective interaction between ligand and receptor, and its affinity is significantly decreased, Labelling with halogens often increases the lipophilicity of the ligand, which could result in nonspecific partitioning into lipidrich components, Also, changes resulting from the introduction of the radionuclide can increase nonspecific binding of ligand to certain serum components'. The extent of nonspecific binding can be quantified by addition of serum or cellular components to the binding assay. This information can then be utilized in future strategies for radioligand design. In vitro testing can also determine the specific activity (radioactivity per mole) of the radioligand. This should be high enough to ensure that satura. tion of receptor by unlabelled ligand molecules cannot occur. It is also important to establish that the radioisotope is firmly finked to the ligand so that it will remain stable/n vivo. Once the affinity and specificity of the iigand is demonstrated /n vitro, the receptor concentration in the target organ required to achieve an acceptable target to nontarget (T:NT) ratio can he calculated. A simple expressiou of ligand--receptor interactions useful for preliminary estimation of T:NT ratios is the standard Scatchard binding isotherm: B/F = [R]. K.(I - ~ , )

for target tissue detection. Katzenellenbogen has synthesized a variety of estradiol derivatives and using this approach has calculated the K, values required for successful imaging of breast tumor masses with these compounds over a variable range of receptor expressions. However, the above extrapolations from :r~-vitro binding describe the optimal situs. tion of equilibrium binding which is rarely attainable/n vivo. It is necessary to consider variables influencing nonspecific binding in vivo as well as other factors which will detract from the theoretical estimate These factors include the presence of endogenous ligand or ligand-like structures and the effect of physiological para~.~eters which alter the rate of transport of ligand to the target site. Also, the imaging device is unable to distinguish between the radioactivity from intact and metaboi. ized ligand; thus, it is important to know the rate of ligand metabolism in vivo. Two current substrate-specific radiopharmaceutical mt~lels exe.mplif.v different approaches in ligand-receptor imaging. The use of radiolabelled estradiol derivatives for imaging breast tumors is an example of attempted exploitation of high afl'mity iigand-receptor interaction. The second, the binding of asialoglycoproteins to the liver cell surface molecule hepatic binding protein (HBP), uses a different approach. In this system subsaturating doses of moderate affinity ligand are administered in order to obtain information about changes in several physiological parameters in the target organ as a function of time. Breast tumor imaging with high affinity

Upnd The selective localization of a labelled hormone to its functionally responsive target tissue/n vivo was first shown in 1962 when Jensen and Jacobsen demonstrated uptake of [SH]estradioi by immature rat uterus6, a tissue which is estradiol growth. responsive. When high specific activity [q.l]estradiol was injected, the uterus showed a higher and more persistent accumulation of label than estradiol non. responsive tL~sues, One hour after injection, uterine activity was I(~-20 times greater than that in the blood and other nontarget t'.'~.~,s, with the liver receiving the highest nontaxget dose. Biochemical analysis oftis. sue extracts showed that while the majority of the radioactivity associated with the liver was due to the presence of metabolizc~d

in which B:F, the bound to free ratio, is equivalent to T:NT, R is the receptor concentration, K, is the binding association constant, and ~ is the fractional saturation of the receptor. Binding increases, there. fore, with an increase in either affinity or receptor concentration, and the degree to which this expression is less than optimal is ligand fragn~ents, uterine activity was a function of fractional saturation. Given a radiolignnd of known aWmity receptor associated. This system illustrates how a ligand of and with a selected minimum T:NT value, this equation can be used to calculate bow suffi~.ntly Idgh affinity, specificity and much unoccupied receptor must be present activity can accumulate selectively in

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were also studied. The lesions in one were successfully imaged whereas axillao' metastases in the other, a patien~ with a known receptor positive primary, did not show signifgant radiolabel uput~. The results suggest that radiolabelled ~radiol derivatives m y prove useful as a guide to surgery and in follow.up evaluation of bream canc~ ~ , bm ~m n~dts mu~ be inteq~-ted can=tully. For ¢xample, tumor cell l~temgem:ity m y msuh in metastatic lesions which differ from the primary in receptor expression, a dmafion which could lead to a false negative result.

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/n~ffon. Prom Kmtzeneilenbogena a/,', with per. mission.

receptor-bearing target tissue. The rat uterus model has become an important one due to the discover, a decade after Jensen and Jacobsen's experiment, that breast tumors express estradiol receptors. Normal breast tissue is estradiol receptor poor, whereas aproximately two-thirds of breast carcinomas contain variable but higher amounts (0.3-3 nmoles mg-* of the receptor"s. Several lahomtories have been pursuing the development of'x-emitting estradiol analogs for use in imaging breast tumors. The measurement of binding of these labelled derivatives to rat uterine estradiol receptors both in vitro and in vivo ixovides a convenient system for testing them for clinical use. One compound developed by KalzeneHenbogen et al., 16o-['Br] bromoestradiol-lT~,yieldedgood uterusto blood and tumor to blood ratios when injected into dimethylbenzanthracene (DMBA) mammary tumor-bearing rats (Fig. i, Ref. 7). Uterus and tumor showed the highest cow cenlration of label; furthermore, the activity in these tissues could be displaced by cold estradiol, which is indicative of the specificity of binding. Liver activity, however, could not be displaced, which sup. ports the belief that activity in this organ is due to the wesence of labelled metabolites. Contrast between tumor and other tissues was sufficient to allow tumor detection with a gamma csmeras. Based on these results, a small number of cancer patients were also imaged*, The lX~magy tumors in ~ of four patients were detectable after injection of bromine. 77-16.a-estradiol. After the study, the tumor which was not imaged was determined to be estradiol.mceptor negative. Two late-stage metastatic disease patients who were not r~eiving endocrine therapy

The approach outlined for radindiagno~ tic imaging of breast ttmm~ was based on attainingmaximal mget to nomarget ratios using a high affinity ligand for anmomgal delineation of receptor positive tissue. A different approach is that of KJrohnet aL who are using a radioligand of moderate affinity in order to achiev= a dynamic, functional analysis of liver physiology*. They an= studying the binding of asialoglyco. proteins to HBP. a comlx)oent e x p r ~ d on liver hepatocytes. In mammak, ck.~,ialvlated serum glxo~:n'otcins bind irrcversibh to ImP. By loss of terminal stalk: acid. a ph~.',iological p ~ that targets proteins for catabolism, penultimate galactose residues are exposed, and binding to HBP can occur. Once binding has occurred, the ligand-receptor complex is end(x'~t¢~d and transferred to I)~osomes. The substrate is metabolized ~4th the subsequent release of catabolites into the bkx~d folio~'ed bx excretion via glomerular fihratkm. The ligand in this study is galactosecoupled albumin, desigmated neogalactalbumin, labelled with the "~mitter ~=Tc. ~ T c - N G A is a good compound for this study because it does not interact with the reticuloendothelial system and shows no bili~y accumulation. Since the binding strength of this l i g ~ p t o r interaction is correlated with the number of galactose

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rcsaktt.~, cxp~v~-d, and a,, N(~:X ~ln Ix" c~J~'d ~lth xanabk' amount,, ~,fgaLh.l(v,c. it i*~po~l~" It) pnvJucc radKdig.,nd~ u~-ith:t ~id¢- r;mge of affin|tk-,, hn ttBP The admini~aUon of medcra~ affinity radioligand in a subsa, m~ing dose msuhs in binding c~mtndk:d bx ,~s.l-~rck.'r kmetK~, m ~oh~.'ht~" ra~',:of b0ndangv, omtroiled by the number of functional rece~ors as well as by ;he binding rate constant, equal to k. [Rece~or] [Ligand]. The model is sensitive, therefore, to both hepatic blood flow and fmgtionM (n:celmw-bearing) hepatic mass. Binding, not flow, becomes the ram-limiting step. Thus, by designing a ligand of moaermc rather than high affinity, and by giving a selected sub~urmmg dose of radiolil~l, Krohn eta/. have esta~. lished an experimental model which ma) be appropnate for a chnam~:, pharma~.~k=met_~anahm,, ~I hcpaltc lur,¢t,m In a ~ of the liver uptake of m"TcNGA in rabbits,regions of m t e t ~ were selected over the liver and blood pool. and t~vity" curves were genc~r-aU~. Data were analysed to delblgate ~ separate comets: the extrahepatic blood pool, the liver blood pool and the recelptorbeating b e p m o c ~ populanon (Rg. 2). R m h ~ than justprovide informationabout the l~ese~e or absence of nx'emors, this model is designed to stud) muhiple physiological processes including organ blood flow. iigand binding and iysosornal metabolism. Alterations m t,hese distinct oarameters m ) be identifi~le b) perfonnnga muhiparameter pharmacokinenc malysis. Th~ ultimate goal ot the, approach t~, to Jiagnose particular disease states based on observed changes in these physiochemical varameters. For exampk,. Incr Im~th,4, ~ . S~.'h ;~', hcl:~Itltls,chronic akx~holl~m. t~patoma and met;L~tat,: dt~a.,~', max -~+~ distin,.lne ~ltterr~ of alterat=on in r¢.~.'cptor concentration, h h ~ l tidy,. ~a~'ulanI,~. :igand mctaboh.~m and exerctton Some dl.~a.,¢." :t.~.~.'tated alterattom, haxc alread~ occn delineated ~=th " ]c-N£;A and t~ork

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TIPS- February 1984

~0 is in progress to elucidate how these variables change in normal, tumor-bearing and chemically-induced cirrhotic animals. Krobn et al. have postulated that this type of study, in conjunction with information provided by serum protein and enzyme analysis, will be useful in the diagnosis and follow-up of patients with these diseases.

compounds without the steric conforma. tionai changes resulting from the introduction of nonisotopic labels such as halogens. Spatial resolution attainable by poaltmn tomography is also superior; deep as well as superficial structures are detectable, and T/NT ratios of as low as two can be detected. These isotopes have short half. lives (less than I h), resulting in lower radiation exposure to the patient. Short halflives, however, also present an obstacle tq the use of positron-emitters in that they require on-site cyclotron production and the development of rapid synthetic techniques. Special positron detector devices are also required, and provision of the required apparatus for the production and detection of positron.emitters is costly. However, some investigators are currently designing and testing positron labelled radioligands with the hope that establishment of superior diagnostic agents may result in efforts to increase the practicality of the apggoach. The potential for development of clink cally useful mbstrate specific radioligends, whether met by positron.emitting or y-emitting analogs, will depend on their ability to meet the criteria which have been outlined. From this merger of nuclear medicine and pharmacology, high specific activity radiotracers with high specificity and the appropriate binding affinity for particulat target tissue associated receptors may become important diagnostic tools for noninvasive study of both organ anatomy and function.

Future outlook The identification of other tissue specific ligand-receptor systems will most probably lead to the investigation of their utility in the development of substrate specific radio. pharmaceuticals. Brain and heart have been analysed extensively for neurotransmitter receptor content; these tissues show both regional variation in receptor expression and quantitative shifts in expression associated with metabolic and other disorders 4'm'". For example, quantitative changes in the expression of nemou'ansmittcr receptors in selective regions oftbe brain have been observed in Huntington's disease, Parkinson's disease, Alzheimer's dementia and schizophrenia. Also, levels of cardiac adrenergic and acetylcholinergic receptors change characteristically as a result of ischemia and certain metabolic disorders. Other nonreceptor cell surface components can be identified in a stereoselective manner by antibodies. The extreme specificity and ease of production of monoclonal antibodies suggests that they will be useful as radioligands for the detection of tumor-associated antigens in vivo. In some cases, compounds labelled with ReadUlg l~. i Callmy, P. S., Faith, W. C., Loberg, M. D., positron.emitting isotopes rather than Fields, A. T., Harvey, E. B. and Cooper, M. D. y-emitters may be necessary, for the produc(1976)J. Med. Chem. 19, 962--964 tion of clinically acceptable radioligands. 2 Edwards, C. L. and Hayes, R. L. (1969)J. Nuci. Positron-emitting nuclides, such as ~q~, Meal. IO, 103-105 ~h:), and nN, would enable isotopic sub. 3 Gibson, R. E., Eckelman, W. C., ~ i , stitution of radioactive atoms into parent W. J.,Jiaag, V., Mazaitis,A., Paik,C., Komm,

T. and Reba, R. C. {1979) in Principlesof Radiopharmacoh)gv (('olumhclll. l... cd.I. pp. 17-3~, ('R(" Prc~. ('1...roland 4 Gibson. R. E. (1982) m Receptor.Binding Rad/otracen (Eckelman, W. C., ed. 1, Vol. II, pp. 186-212,C R C Peas, Cleveland 5 Katzenellenbr~n, J A., Carlson, K. E., Heiman, D. F. and Lloyd, J. E. (1980) in Rad~ophar. maceutk'als: Strucmre-Activily Relationships ISwnccL R, I'.. cd.I. pp. 2~,~. (;nmc ,rod SIl~,llon. New ~l'ork

6 Jemen, E. V. and Jacobsen, H. I. (1962) Recent Pro&. Horm, Res. 18, 387--414 7 Katzcncllcnlmgcn. J. A.. Scndcmfl. N t;,. McEIwmcy. K. D., O'Bricn, H. A. and Welch. M. J. ! 1~811J. Nucl. Med. 22.42-47 8 McEIvaney, K. D., Katzenellenbogen, J. A.o Sharer, K. E.: Siegal, B. A., Senderoff, S. G. and Welch, M. J. (1982)J. N~cl. Med. 23, 425-4.~ 9 Krohn, K. A., Stnlminik, R. C. and Vent, D. (1982l Diagn. Imaging, 24-31 IO Eckelnum, W. C., Gibson, R. E., R z e ~ i . W. J,. Jian8, V., Mazaitis,J. K,, Paik,C., Komai, T. and Reba, R. C. (t979) in Pzmc~ples of Radiopharma('ologv (Columlx'tti. I... cd.). pp. 251-273, CRC Pros& CIcvchmd II Kulm, M. J. (1982) in Receptor.Binding Ro~lio~cers (Eckelnum. w. C., ed.), Vol. H, pp. 37-50, CRC Press,Cleveland

Ann M. Carndl completed her Ph. D. m mmmnology at the Sloan.Kettering Division of Comell University Gmdume School ol Medical Sciences in 1982. Following a eme-year fellowship at the Departmem of Radiology at the Blgham and Women's Hospital, she is curremh." , Research Fellow it, the Department of Pathology at tlan.ard Medical School,

Michael Zalutsky received his Ph.D. in nuclear chemistry from Washington University in 1974. He completed a postdoctoralfellowshipat Argonv~, National Laboratory, and from 1977 to 1980 &: held a posMon as a mdiopharmaceutical chemist at the University of Chicago. He is currentlyan assistant pv~fessor in radiology and nuclear medicine at tlan,ard Medical School.