The Distribution of the Muscarinic Acetyichoiine Receptor Antagonists, Quinuciidinyi Benziiate and Qulnuciidinyi Benziiate Methiodide (both tritiated), in Rat, Guinea Pig, and Rabbit

Raymond E. Gibson, William C. Eckelman, Frank Vieras*, and Richard C. Reba

School of Medicine, George Washington University Medical Center, Washington, D.C. and *Armed Forces Radiobiology Research institute , Bethesda, Maryland

The distribution of [3Hjquinuclidinyl benzilate and its methiodide salt was deter mined in rat, guinea pig, and rabbit. Accumulation in the myocardium of up to 2% of the injected dose per gram of tissue was obtained with both compounds, providing heart-to-blood ratios of @—‘3Oand heart-to-lung ratios of @4.The accumulation in the heart was blocked (89%) by preinjection of atropine. The distribution of tritium in rabbit heart corresponds to the muscarinic receptor densities determined in vitro. Cakulation of the theoretical maximum for the bound-to-free ratio, based on in-vitro equilibrium binding isotherms, resulted in ratios in reasonable agreement with the experimental results. Because of the high accumulation in the heart with low serum concentration, we conclude that the methiodide salt of quinuclidinyl benzilate repre sents an ideal parent structure for the design of a receptor-binding gamma-emitting radiopharmaceuticalfor imaging of the myocardium. J NuciMed20:865—870,1979

Current methods for external imaging of the myo gand concentrations in the target tissue and work cardium are based on the metabolic requirements able. target-to-blood ratios. of the heart [Tl-201 (1), (11C)palmitic acid (2)], or Two receptors are present in the myocardium in metabolic trapping [2-('8F)fluoro-2-deoxy-D-glu sufficiently high concentration to make localization cose (3)], or the deposition of technetium [Tc-99m feasible: a) the .beta-adrenoceptor (8), and b) the Ppm (4)] and certain lipophilic drugs [1-131 tetracy muscarinic acetylcholine receptor (9). Both are cell dine (5)]. An alternate mechanism that may prove surface receptors that are present in concentrations useful is the localization of a radiolabeled ligand of 10 to 20 pmole/gram of heart tissue. For these exhibiting a specific, high-affinity interaction with receptors there are a limited number of drugs that a receptor present in the myocardium (6). The the have sufficient affinity (K,, of 10@to 10'°M1) to oretical requirements have been described (7) for deliver the desired radioactivity to the target and to both the quantity of receptor needed in the tissue provide sufficiently large target-to-blood ratios. The and the affinity required to give adequate radioli investigation of beta-adrenoceptor antagonist local ization in the myocardium has been the subject of Received Dec 28, 1978; revision accepted March 19, 1979. several previous communications (10,11). This re For reprints contact: Raymond E. Oibson, George Washing ton University Medical Center, School of Medicine, 2300 Eye port describes results on the myocardial localiza St., N.W., Washington, D.C. 20037 tion of two high-affinity, tritiated antagonists of the

Volume 20, Number 8 865 GIBSON, ECKELMAN, VIERAS, AND REBA

H suesfrom the heart, lungs,and liver wereexcised, blotted to remove excess blood, solubilized in NCS tissue solubilizer in capped vials, and counted in a liquid-scintillation counter. Blood samples were similarly solubilized and counted. Samples were dark-adapted for at least 4 hr and counted through two cycles to ensure reproducible values. Quench ing corrections were made with an external stand ard and calibrated curve. The results are expressed as %dose/g of tissue and were obtained from at least five animals. The affinity of [3H]QNB and [3H]MQNB for guinea pig heart muscarinic receptor was deter mined using the heavy-membrane fraction of the

* ventricular muscle. H In two rabbits, 50 @Ciof [3H]MQNB was injected through an ear vein and the animals were killed at 30 mm by a blow to the neck. Blood was taken by R=H@ UNB cardiac puncture, and the heart, lungs, and liver R = CH3; MQNB were removed. The heart was dissected into the left and right atrial and ventricular muscle (VM), yen FIG. 1. Structures of quinuclidinyl benzylate (QNB) and its tricular septum, and papillary muscle. In one rabbit methyl quaternary derivative (MONB). Counter ion (x) for MONB is iodide. the left ventricular muscle was cut into 16 sections. Three 20-mg samples of each tissue were solubi lized and counted as above. muscarinic acetylcholine receptor: [3H]quinucidinyl In-vivo drug displacement studies. Various con benzilate ([3H]QNB) and its methiodide salt centrations of unlabeled muscarinic agents were in ([3H]MQNB) (Fig. 1). jected 15 to 30 mm before the injection of [3HJMQNB. Quantities ofunlabeled drug were cho MATERIALS AND METHODS sen to provide initial serum concentrations of I @M, [3HIQNB (32 Cilmmole) was purchased commer 10 @M,or 100 @M,assuming that the volume of ciallyt. The molecule is nominally labeled in the distribution is 3.5% of the animal's weight (see 4' ,4'-positions of the benzilate moiety (Fig. 1). The Table 3). radiochemical purity is specified as greater than 98%. [3H]MQNB, the N-methyl derivative of RESULTS [3H]QNB, was prepared by mixing 1 mCi of Distribution in rat. Distribution of tritium in rat [3H]QNB with 1 ml CH3I at room temperature for and guinea pig VM, lung, and blood, following in 2 hr in the dark. The excess CH3I was removed jection of [3H]QNB and [3HIMQNB, is summarized under a stream of dry N2, and the methiodide salt in Table 1. There is no significant difference in the was solubilized in 95% EtOH. The product exhib accumulation of tritium in the rat VM between 15 ited one radioactive peak on TLC in butanol, water, mm and 2 hr with [3H]QNB, but the methyl qua acetic acid (60:20: 10; ml ofeach) and ethanol, acetic ternary salt suffers a reduction in activity of ½to acid, water (60:30: 10), with R1 values of 0.32 and ‘/4between 15 mm and 2 hr. The heart-to-blood 0.36, respectively. In the same two systems, ratio (H/B) for [3H]MQNB is somewhat higher than E3HIQNB exhibits Rf values of 0.65 and 0.73. The that for [3HJQNB at 15 mm, whereas the reverse is [3H]MQNB was stored at —25°C and was used the case at 2 hr. The quaternary salt exhibits a large within 5 days of preparation. Unlabeled atropine, heart-to-lung (H/L) ratio at 15 mm (12.7), whereas pilocarpine, oxotremorine, and nicotine were pur that of [3HIQNB is modest (1.6). We therefore used chased commercially. [3H]MQNB for the remainder of our studies. Distribution studies used male Charles River Distribution in guinea pigs. During studies on the guinea pigs (400 g), Sprague-Dawley rats (350 g), tissue distribution of a radioiodinated tyramine de and New Zealand Albino rabbits (6 kg). In rats and rivative of practolol (9), large species-related dif guinea pigs anesthetized with sodium pentobarbital, ferences were observed in the accumulation of the 4 @Ciof E3HIQNB or [3H]MQNB was injected via radiolabel in the VM of rat and guinea pig. We have the femoral vein and the animals were killed by therefore used guinea pigs as a second animal model exsanguination at various times after injection. Tis in this study. The %dose/g in the guinea pig VM

866 THE JOURNAL OF NUCLEAR MEDICINE BASIC SCIENCES RADIOCHEMISTRY AND RADLOPHARMACEUTICALS

TABLE 1. DISTRIBUTION OF TRITIUM RADIOACTIVITY AFTER INJECTION OF 4 @Cl(0.13 pmol) OF [3H]QNBAND [‘H}MQNBIN RATS AND GUINEA PIGS*

. % dose/g s.d.t Ti me Animal Compound (hr) Heart Blood Lung H/B H/L Rat QNB ¼ 1.89±0.24 0.103 ±0.008 1.25 ±0.37 18.4 01.6 2 1.88±0.57 0.076 ±0.014 0.34 ±0.13 24.6 6.0 MQNB ¼ 2.35 ±0.49 0.075 ±0.015 0.184 ±0.017 31.5 12.7 2@ 0.60±0.06 0.064 ±0.005 0.103 ±0.018 9.3 6.0 MQNB 2@ 0.88±0.16 0.072 ±0.007 0.17 ±0.02 12.2 5.3 4 0.46 ±0.20 0.075 ±0.006 0.14 ±0.04 6.3 3.7

Guinea pig MQNB ¼ 1.61 ±0.24 0.050 ±0.004 0.55 ±0.14 32.2 3.0 ½ 1.57±0.18 0.057 ±0.005 0.51 ±0.17 27.3 3.3 ¾ 2.74±0.83 0.070 ±0.005 0.86 ±0.09 38.7 3.2 1 1.38±0.25 0.058 ±0.009 0.49 ±0.08 23.9 2.8 2@ 0.82±0.24 0.036 ±0.015 0.32±0.15 28.4 3.0 2@ 0.80±0.15 0.026 ±0.003 0.30 ±0.14 31.6 2.7

* ONB = quinuclidinyl benzilate, MONB = N-methyl quinuclidinyl benzilate. t Each group contained at least five animals. 1@Determinedduringdifferentweeksto demonstratereplicability. following injection of [3H]MQNB compares favor distribution of tritium in the blood and lungs of two ably with the results for rat VM at both 15 mm and rabbits, the %dose/g in various regions of the heart 2 hr (Table 1). The accumulation in the lung is was determined (Table 2). The concentration of trit higher at 15 mm, resulting in a relatively low H/L ium in the atrial muscle is 1.5 to two times that in value, but at 2 hr the HIL ratio is not significantly the ventricular muscle. The differences in the different from that obtained in rat. The H/B values %dose/g in the left and right ventricular muscle, the obtained at five different times remain reasonably ventricular septum, and papillary muscle are not constant and at high values (range from 24 to 39). significant. The H/B ratio for rabbit is 17.2, whereas This is in striking contrast to the results for the rat the H/L ratio is 1.8. The distribution of tritium in and may reflect a difference in metabolic behavior the left VM is more or less uniform over 16 sec between the two species. tions, ranging from 0.24 to 0.39 %dose/g tissue. Distribution in rabbit heart. In addition to the Higher values appear to be obtained in the region of the apex, but the differences may not be signif icant (average and standard deviation: 0.32 ±0.04 TABLE 2. DISTRIBUTION OF TRITIUM %dose/g). RADIOACTIVITY AFTER INJECTION OF [‘HJMQNB In-vivo drug competition. The accumulation of (50 @iCl)IN RABBIT, AND COMPARISON WITH tritium in the rat heart is blocked by prior injection RECEPTOR CONCENTRATION DETERMINED IN VITRO of the muscarinic antagonist atropine (Table 3). An intramuscular injection of 1 mg/kg atropine, 15 to ofTissue!(pmole/total*serumgramcardiacTissue .Density*% 30 mm before injection of [3H]MQNB, reduced the concentration of tritium in the heart, thereby re %dose/g ratioprotein)receptors ducing the H/B ratio. Preinjected atropine impaired Serum 0.019 — — — the accumulation of [3H]MQNB in the rat heart: Left ventricular uptake was reduced to 50% of control with i.v. muscle 0.33 17.2 37.1 28.9 injection of 4.9 ;@gof atropine, and to 11% with 49 Right ventricular @g.The muscarinic agonist, oxotremorine, and the muscle 0.34 18.3 52.7 15.2 mixed agonist-antagonist, pilocarpine, were not ef Ventricular fective in blocking tritium localization at the test septum 0.31 16.5 58.1 21.7 Papillary muscle 0.43 23.1 — — dosages. In addition, the nicotinic acetylcholine re Left atrial ceptor agonist, nicotine, was also ineffective in muscle 0.48 23.1 302 16.4 blocking tritium accumulation in the heart. Right atrial muscle 0.68 36.6 200 17.8 In vitro determination of KA. The affinities of Lung 0.10. 9.4 — — [3H]QNB and [3H]MQNB for the muscarinic ace Liver 0.23 12.5 — — tylcholine receptor in the guinea-pig heart were de * Data from ref. 14. termined from Scatchard plots (12) (Fig. 2). The results obtained for the methyl quaternary salt vary

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TABLE 3. DISTRIBUTION OF TRITIUM RADIOACTIVITY AFTER INJECTION OF [‘H]MQNBIN RATS IN THE PRESENCE OF OTHER CHOLINERGIC DRUGS %Dose/g ±s.d. at 2 hrt Blocking Agent [@M]* Heart Blood Lung H/B H/L Atropine5.6Controli.m.-49 ,.&g 1 0.76 ±0.26 0.098 ±0.021 0.13 ±0.03 7.7 6.7Atropine 0.74 ±0.10 0.077 ±0.001 0.11 ±0.16 9.5 3.0Controli.v.-4.9 /L9 1 0.50 ±0.09 0.140 ±0.015 0.18 ±0.04 3.6 5.2Atropine 1.00 ±0.31 0.114 ±0.016 0.21 ±0.07 8.7 0.8Controli.v.-49 @Lgt 10 0.10 ±0.01 0.102 ±0.011 0.12 ±0.01 1.0 5.7Oxotremorine 0.91 ±0.19 0.082 ±0.010 0.16 ±0.02 11.1 4.8Control i.v.-35 @g 11.4 0.70 ±0.14 0.130 ±0.040 0.15 ±0.03 5.7 6.1Oxotremorine 0.89 ±0.12 0.057 ±0.018 0.145 ±0.01 17.4 6.2Control i.v.-350 @g 114.0 0.793 ±0.16 0.084 ±0.030 0.133 ±0.03 10.1 8.9Pilocarpine 1.13 ±0.33 0.057 ±0.011 0.131 ±0.02 21.0 6.0Control i.v.-41.6 @Lg 10 0.71 ±0.14 0.049 ±0.008 0.12 ±0.02 14.7 4.5Pilocarpine 0.73 ±0.21 0.064 ±0.014 0.163 ±0.03 11.8 6.0Control i.v.-416 IL9 100 0.71 ±0.14 0.049 ±0.008 0.12 ±0.02 14.7 4.5Nicotine 0.73 ±0.21 0.064 ±0.014 0.16 ±0.03 11.8 4.7Controli.v.-27.5 ,.L9 10 0.59 ±0.16 0.052 ±0.014 0.13 ±0.03 11.6 5.3Nicotine 0.81 ±0.29 0.107 ±0.032 0.15 ±0.03 7.8 6.9Control i.v.-275 @Lg 100 0.94 ±0.19 0.067 ±0.013 0.14 ±0.02 14.6 7.1* 0.98 ±0.24 0.060 ±0.008 0.13 ±0.02 16.2

wasinjectedMolarity in plasma assuming a 17-mI plasma value as the distribution space. Cholinergic blocking agent [3H]MQNB.t¼to @/2hr before 4 @Ci(0.13 pmol) of killedtwoControl experiments were carried out on same day. Each group contained at least five animals, which were [3H]MONB.Similarhr after injection of results were obtained in guinea pig following the i.m. injection of 500 @gof atropine.

according to different lots of [3HIQNB from the DISCUSSION supplier.t Initial studies using lot No. 1140-053 in dicated that the affinities of the tertiary and quater Distribution studies with [3H}QNB and its qua nary compounds were the same, but the maximum ternary derivative, [3H]MQNB, have shown that binding (RT) was different (solid triangles and cir these two muscarinic antagonists localize in the des, Fig. 2). Subsequent studies with a different lot heart in sufficient concentrations and with suffi of [3H]QNB (No. 1068-137) provided different re ciently large target-to-blood ratios to permit the ex sults. The maximum amounts bound were the same ternal imaging of the heart with a gamma-emitting for [3H]MQNB and [3H]QNB but the affinities for radiolabeled analog. The kinetic studies with the muscarinic receptor differed. Isotopic dilution [3H]MQNB in guinea pigs suggest that the peak studies with the methyl bromide salt of QNBI in concentration of tritium is obtained rapidly (be dicated that the specific activity of [3H]MQNB was tween 15 mm and 1 hr) and that the H/L ratio over reduced by 50% compared with the starting mate the entire time range is constant and large. rial. The [3H]QNB is nominally labeled and it is A quantitative, specific interaction of the ligand possible that some of the incorporated tritium is with the receptor is essential if external imaging is located on the aliphatic carbons of the quinuclidinyl to be used to monitor the change in receptor con moiety as well as on the phenyl rings of the benzi centration as a function of disease (13). It is there late moiety. We speculate that a contaminant in the fore important to prove receptor specificity in vivo. methyl iodide, which is stabilized by metallic cop In rats, an 1-125 derivative of a beta-adrenoceptor per, catalyzes the exchange of aliphatic H-3 with antagonist, practolol (11), gave an H/B ratio of 20:1. protons from moisture present in the methyl iodide. But this was subsequently proven to be species When the data obtained from the first lot of specific (rabbit and dog did not provide equivalent [3H]QNB were corrected for the change in specific results), and the localization in the rat myocardium activity, the binding curves obtained for the was shown to be the result of interactions with [3H]MQNB are the same as those obtained with the proteins other than the beta-adrenoceptor. The newer lot (open triangles, Fig. 2). Thus, the affini specificity of the interaction of the tritiated antag ties for [3H]QNB and [3H]MQNB are 7.0(±1.1) x onist, [3H]MQNB, is suggested by the results from 10@M1 and 2.1(±0.52)x 10@M1, respectively, two experiments. First, the distribution of the ra and the receptor concentration (RT) is 16.3(±2.0) dioligand in the heart (Table 2) closely parallels the pmole/g of ventricular muscle (wet weight). These demonstrated concentrations of the muscarinic re are the results of five determinations. ceptor (14). Secondly, the radioligand can be dis

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carmnic agonist, is lethal at higher concentrations. Pilocarpine, a mixed agonist—antagonist, exhibits an in-vitro affinity ‘/250of atropine (Table 4). At the highest concentration tested, 416 @g,we would not expect this compound to block [3H]MQNB binding. Pharmacologically, it may appear surprising that an estimated 1-MM injection of atropine is required to block 50% of the [3H]MQNB binding. This is 1000 times that predicted from the KA (Table 4). The 0

K radioligand concentration is very small relative to U. 0 the concentration of receptors. Under similar con ditions of receptor excess in vitro, a l0@ M con centration of atropine (100 times that predicted from KA) would be required to displace 50% of the [3H]MQNB binding [calculations based on the cubic equation for two ligands binding to one receptor (i5)]. Distribution and metabolism most likely ac count for the additional ten-fold concentration of atropine required. When the radioligand concentration is far smaller than the total receptor concentration (RT), the max imum bound-to-free ratio (indicative of target-to blood ratio) approximates the product of KA and B (pmol./g tissue) RT. Deviations from this ratio are likely in the pres FIG. 2. Scatchard plot of [‘H]QNB(•)and [3H]MONB (A) ence of nonspecific binding (7). For this calculation binding to the heavy-membrane fraction of guinea pig's yen tricular muscle. The association constants for this determi we assume that the muscarmnic receptors are uni nation are 5.7 x 10@M' for [‘HIQNBand 2.0 x 10@M' for formly distributed through the volume of the heart [3H]MQNB;RT = 7.4 pmol/g. muscle and that the density of the heart is close to 1, i.e., RT 16.3 nM. The products of RT and KA for [3H]QNB and [3H]MQNB are 114 and 34, re placed by the known muscarinic antagonist atro spectively. These values are in reasonable agree pine. ment with the experimentally obtained ratios and The in-vivo distribution of [3H]MQNB in the rab suggest little serum protein binding, particularly for bit heart provides the highest concentration of ra [3HJMQNB. dioligand (in %dose/g tissue) in the atrial muscle. The methyl quaternary salt of QNB (clinidinium The left VM has two-thirds the concentration of the bromide) is used clinically as an antispasmodic left atrial muscle and one half that of the right atnal agent in the drug Librax. Although Librax contains muscle. These results are in qualitative agreement with the density of receptors in those tissues (Table 2). The higher atrial concentration may be of no TABLE 4. APPARENT ASSOCIATION CONSTANTS consequence, however, because the atrial muscle FOR MUSCARINIC CHOLINERGIC AGONISTS AND represents only 10% of the weight of the myocar ANTAGONISTS dium. Thus the percentage of the total receptors Kapp@Guinea-pigK8@@tCompound present in the heart predicts that the left VM (with 29% of the total) and the ventricular septum (with ileumRabbit heart 22%) will accumulate the largest fraction of the QNB 3.3 x 10@ 27.8 x 10@ injected dose (Table 2). Images obtained with a Scopolamine 3.3 x 10@ Methylatropine 5.10 x 10@ gamma-emitting analog of QNB should provide ra Atropine 0.25x 10@ 1.0 x 10@ dioligand distributions similar to those obtained Isopropamide 0.17 x 10@ with 11-201, 2-['8F]fluoro-2-deoxyglucose, and Oxotremorine 0.001 x 10@ 0.007 x 10@ Pilocarpine 0.001 x 10@ [11Cjpalmitic acid in the normal heart. Arecoline 0.0003 x 10@ Accumulation of tritium in the heart is blocked Acetycholine 0.0003 x 10@ 0.002 x 10@ by 50% after a 4.9 @gi.v. preinjection of atropine, Carbachol 0.0003 x 10@ and can be almost completely blocked by 49 @g. * Data from ref. 9. The remaining drugs in Table 3 were not effective t Data from ref. 14. at the tested concentrations. Oxotremorine, a mus

Volume 20, Number 8 869 GIBSON, ECKELMAN, VIERAS, AND REBA

2.5 mg of clinidinium bromide, the drug has no as a radiopharmaceutical for measuring regional myocardial reported adverse effect on the heart. The tertiary glucose metabolism in vivo: tissue distribution and imaging base is a potent psychotomimetic agent that can studies in animals. J NucI Med 18: 990-996, 1977 4. HOLMAN BL, DAVIS MA, HANSON RN: Myocardial infarct produce unwanted central nervous system effects, imaging with technetium-labeled complexes. Semin NucI but the methyl quaternization prevents crossing of Med 7: 29-35, 1977 the blood—brain barrier. The high accumulation in 5. MALEK P, RATOSKY J, VAVR1JIN B, et al: Ischemia detecting the heart, low nonreceptor protein binding, and radioactive substances for scanning cardiac and skeletal specificity for the receptor suggest that MQNB is muscle.Nature 214: 1130—1131,1967 6. ECKELMAN WC, REBA RC, GIBSON RE, et al: Receptor the best parent structure for the design of a recep binding radiotracers: a new class of radiopharmaceuticals. tor-binding gamma-emitting radiopharmaceutical. J NucI Med 20: 350-357, 1979 7. GIBSON RE, ECKELMAN WC, RZESZOTARSKIWI, Ct al: Ra diotracer localization by receptor-ligand interactions. in FOOTNOTES Principles of Radiopharmacology. Colombetti L, ed. West t New England Nuclear Corp. , Boston, MA. Palm Beach, FL. CRC Press Inc., in press @ Clinidinum Bromide, Hoffman—LaRoche, Nutley, NJ. 8. HARDEN TK, WOLFE BB, M0LIN0FF PB: Binding of iodi nated beta adrenergic antagonists to proteins derived from ACKNOWLEDGMENTS rat heart. Mo! Pharmacol 12: 1- 15, 1976 9. YAMAMURAHI, SNYDERSH: Muscariniccholinergicrecep We thank Dr. W. I. Rzeszotarski for establishing the proce tor binding in the longitudinal muscle of the guinea pig ileum dure for the synthesis of [3H]MQNB. We thank E. Barron, N. with [3H]quinuclidinyl benzilate. Mo! Pharmacol 10: 861- Fleming, M. Flynn, J. Josza, and J. Warrenfeltz of the Armed 867, 1974 Forces Radiobiology Research Institute for their technical as 10. K0MAI T, ECKELMAN WC, RZESZOTARSKI Wi, Ct al: Prep sistance with the animal-distribution studies. Research was con aration and evaluation of beta adrenergic derivatives. J La @ ducted according to the principles enunciated in the @Guidefor belied Co@npounds and Radiopharmaceuticals 13: 217, 1977 the Care and Use of Laboratory Animals' ‘prepared by the (abst). Institute of Laboratory Animal Resources, National Research ii. JIANG VW, GIBSON RE, RZESZOTARSKIWI, Ct al: Radioiod Council. This study was supported in part by Grant No. HL mated derivatives off3-adrenoceptor blockers for myocardial 19127awarded by the National Heart, Lung and Blood institute, imaging. J Nuci Med 19: 918—924, 1978 DHEW. /2. SCATCHARD G: The attractions of proteins for small mole cules and ions. Ann NY Acad Sci 51: 660-672, 1949 REFERENCES /3. JACOBS 5, CUATRECASAS P: Cell receptors in disease. N I. BRADLEY-MOORE PR, LEB0wITz E, GREENE MW, Ct al: Engi J Med 297: 1383-1386,1977 Thallium-20l for medical use. 11: Biologic behavior. J NucI 14. FIELDS JZ, ROESKE WR, MORKIN E, YAMAMURA HI: Car Med 16:156-160,1975 diac muscarinic cholinergic receptors, biochemical identifi 2. HOFFMAN Ei, PHELPS ME, WEiss ES, et al: Transaxial cation and characterization. J Biol Chem 253: 3251—3258, tomographic imaging of canine myocardium with “C-pal 1978 mitic acid. J NucI Med 18: 57-61, 1977 /5. RODBARD D, LEWALD JE: Computer analysis of radioligand 3. GALLAGHER BM, ANSARI A, ATKINS H, et al: Radiophar assay and radioimmunoassay data. Acta Endocrinol (Suppi maceuticals XXVII. ‘8F-labeled 2-deoxy-2-fluoro-D-glucose 147) 64: 79-103, 1970

14th INTERNATIONAL SYMPOSIUM RADIOACTIVEISOTOPESIN CLINICALMEDICINEANDRESEARCH January 9- 12, 1980 Bad Gcistein, Austria

The 14th International Symposium on “RadioactiveIsotopes in Clinical Medicine and Research―will be held January 9—12,1980 in Bad Gastein, Austria.

TheSymposiumwill beopenedbythe“Gastein-lecture―whichwillbegivenbyaninvitedguestlecturer.Thereisnothematiclimitationfor theprogram.Deadlinefor paperregistrationisSeptember15,1979.

Opportunities for smaller expert discussionswill be available as well as the traditional “FreePaper―sessions.

For further information contact: Prof. Dr. R. Hofer Head, Dept. for Nuclear Medicine 2nd Medical Univ.Clinic Garnisongasse 13, A.1090 Vienna, Austria

870 THE JOURNAL OF NUCLEAR MEDICINE