NOTE Annals of Nuclear Medicine Vol. 6, No. 1, 63-67, 1992 Preparation and biodistribution in mice of [11C]carfentanil: a radiopharmaceutical for studying brain u-opioid receptors by positron emission tomography Hideo SAJI,* Daisuke TSUTSUMI,* Yasuhiro MAGATA,** Yasuhiko IIDA,* Junji KONISHI** and Akira YOKOYAMA* *Faculty of Pharmaceutical Sciences and **School of Medicine, Kyoto University A potent u-opioid agonist, [11C]carfentanil, was prepared by the methylation of carfentanil carboxylic acid with [11C]methyl iodide in order to study brain u-opioid receptors by positron emission tomography. Synthesis (including purification) was completed within 25 min and the radiochemical yield was approximately 40%. The radiochemical purity of the product was more than 99% and its specific activity was 3.7-7.4 GBq/umol. Biodistribution studies performed in mice after intravenous injection showed a high brain uptake and rapid blood clearance, so a high brain/blood ratio of 1.5-1.8 was found from 5 to 30 min. Regional cerebral distribution studies in the mouse showed a significantly higher uptake of [11C]carfentanil by the thalamus and striatum than by the cerebellum, with the radioactivity in the striatum disappearing more rapidly than that in the thalamus. Treatment with naloxone significantly reduced the uptake of [11C]carfentanil by the thalamus and striatum. These results indicate that [11C]carfentanil binds specifically to brain u-opioid receptors. Key words: [11C]carfentanil, synthesis, biodistribution, opioid receptor, positron emission tomography INTRODUCTION WITH THE DISCOVERY of specific opioid receptors and subsequently of endogenous opioid peptides, considerable interest has been developed in the relationship of opioid receptor systems to pain control, psychiatric disorders, and neurodegenerative diseases.1,2 Recently, positron emission tomography (PET) has been applied to the localization and quantification of opioid receptors in the central nervous system.3-5 PET studies of opioid receptors require the use of a radioligand labeled with a suitable positron-emitting radionuclide that possesses a very high affinity for the target receptors.5-7 Cafentanil is a u-subtype Received September 20, 1991, revision accepted December 4, 1991. For reprints contact: Hideo Saji, Department of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-01, JAPAN. selective opioid agonist which is over 7,000 times more potent than morphine3,8-10 Accordingly, [11C]carfentanil (Fig. 1) has been synthesized and used to study u-opioid receptors in the human brain by PET.3-5,11,12 However, in order to fully assess the usefulness of this radioligand for PET studies, more detailed information on its biodistribution is required, including the regional cerebral distribution. In the present study, [11C]carfentanil was prepared and its biodistribution, including the regional cerebral distribution as well as the effect of naloxone on the distribution pattern, was studied in mice because the regional cerebral localization of u-opioid receptors is similar in mouse and human brains.13,14 MATERIALS AND METHODS The sodium salt of carfentanil carboxylic acid was kindly provided by Dr. R. Dannals (The Johns Hopkins Medical Institutions, U.S.A.). Naloxone hydrochloride was purchased from Sigma. The other chemicals used were all of reagent grade. Male ddY mice were supplied by Japan SLC Co. Ltd. Synthesis of [11C]carfentanil We synthesized [11C]carfentanil according to the method of Dannals et al.15 [11C]Carbon dioxide was produced via proton bombardment of nitrogen gas and the 14N (p,a) 11C reaction using an ultra-compact cyclotron (Sumitomo, Model 325), and was trapped in a solution of lithium aluminum hydride in tetrahydrofuran (THF). After evaporation of the THF, 54% hydroiodic acid was added, and the [11C]methyl iodide produced was trapped in dimethylformamide (DMF) under a stream of nitrogen gas. 1 mg of cafentanil carboxylic acid sodium salt was then dissolved in 100 ul of DMF in a reaction vial, and 200 ul of DMF containing [11C]methyl iodide was added to the vial. The vial was sealed and then the mixture was stirred at 35deg.C. After 5 min, the reacted mixture was purified with preparative HPLC on a 7.6x300 mm Lichrosorb RP-18 column eluted at 2.5 ml/min with methanol/0.1 M ammonium formate (7/3) (Rt=8.1 min for methyl iodide, Rt=15 min for carfentanil, and Rt=6.5 min for carfentanil carboxylic acid). The fraction corresponding to carfentanil was collected and evaporated to dryness. The residue was then dissolved in 3 ml of saline and 3.6 ml of 7% sodium bicarbonate solution, and filtered through a 0.22-um filter. Radiochemical purity was determined by analytical HPLC on a 3.9x250 mm Lichrosorb RP-18 column eluted at 2 ml/min with methanol/0.1 M ammonium formate (7/3) (Rt=11 min for carfentamil). The specific activity was estimated from the U.V. absorbance at 254 nm. Biodistribution in mice A dose of 1.11 MBq (5 ug/kg) of [11C]carfentanil was injected intravenously into male ddY mice weighing about 30 g. At various times after the injection, the mice were killed by decapitation, their organs were excised, and blood samples were collected by cardiac puncture. All samples were weighed and the radioactivity was counted in a well-type NaI scintillation counter. Results are presented as the % dose/g organ weight. Regional cerebral distribution in the mouse Male mice weighing an average of 30 g were injected intravenously with 0.1 ml of a solution of [11C]carfentanil (1.11 MBq, 5 ug/kg). At various times afterwards, the mice were killed by decapitation, and the various brain regions were dissected out on an ice-cold plate according to the method of Glowinski and Iversen.16 The wet tissue samples were then weighed, and the radioactivity was determined in a well-type NaI scintillation counter. Results were calculated in terms of the % dose/g tissue weight. Effect of naloxone on regional cerebral distribution in the mouse Naloxone (1 mg/kg) was injected simultaneously with the radioligand into mice weighing an average of 30 g. The animals were killed 5 and 30 min after administration and the uptake of the radioligand by various brain regions was determined as described above. RESULTS AND DISCUSSION Preparation of [11C]carfentanil We synthesized [11C]carfentanil by the methylation of carfentanil carboxylic acid with [11C]methyl iodide. The labeling and purification procedures were completed within 25 min after the finish of [11C]methyl iodide trapping and the radiochemical yield was approximately 38% based on the initial radioactivity of [11C]methyl iodide. Radiochemical purity was comfirmed to be more than 98% by HPLC (Fig. 2). The specific activity of the product was 3.7-7.4 GBq/umol at the end of the synthesis. When in vivo PET studies of neuroreceptors are performed, high specific activity of the radioligand used is important both to achieve a specific distribution (low specific activity results in excessive nonspecific binding) and to avoid receptor occupancy levels high enough to cause pharmacological effects.6,17,18 The specific activity obtained in this study was somewhat lower than the theoretical value (3.4x 105 GBq/umol), and this may have been due to contamination with carrier carbon-12 from the carbon dioxide in the target gas and methanol formed from the THF solution containing lithium aluminum hydride, as has been reported by some other groups.13,18-20 However, it has also been reported that there exists a lower limit at which it is difficult to overcome the effect of the carrier containing the final 11C-labeled product synthesized via [11C]methyl iodide and that this limit is about 0.2 umol.19 Since the total amount of carrier contained by [11C]carfentanil produced in this study was about 0.25 umol, a shorter synthesis time and a higher initial 11C radioactivity may be necessary to achieve a similar specific activity to that reported in clinical studies (42 GBq/umol).3,15 In vivo studies Table 1 shows the distribution of [11C]carfentanil in various organs of the ddY mouse. Radioactivity was cleared rapidly from the blood after injection. The brain showed a relatively high uptake of 3.7% dose/g at the initial sampling time (5 min), after which it declined steadily. The brain/blood ratio was high, ranging from 1.5-1.8 throughout the period from 5 to 30 min. High initial uptake was also observed in the lungs, kidneys, and liver, with the radioactivity in the former two organs clearing rapidly and that in the latter increasing further with time. The distribution of [11C]carfentanil radioactivity in various brain regions is shown in Table 2. Radioactivity was high in the thalamus and striatum, intermediate in the cortex, and low in the cerebellum. Accordingly, the ratios of radioactivity in the thalamus, striatum, and cortex to that in the cerebellum were 2.2-3.2, 1.9-2.3, and 1.4-1.7, respectively, at 5 to 30 min after injection. This marked regional variation in the distribution of [11C]carfentanil agreed well with the results obtained by in vitro mapping of u-opioid receptors.13,14 Futhermore, treatment with naloxone, a u-opioid antagonist,20 significantly reduced the uptake of [11C]carfentanil in the thalamus and striatum (73-74% inhibition for the thalamus and 62-82% for the striatum), so that the radioactivity in these two regions became almost the same as that in the cerebellum (Fig. 3). The time course of changes in radioactivity was different in the thalamus from that in the striatum, with the disappearance of striatal radioactivity being about twice as fast as that in the thalamus (T1/2=11.7 min for the striatum and 23.8 min for the thalamus) (Table 2). Since two subtypes of u-receptors (u1 and u2) have recently been proposed to exist,1,2,14,22 the differences in the distribution pattern of [11C]carfentanil by the thalamus and striatum might be related to the differential regional localization of u-receptor subtypes. In conclusion, the results of these in vivo regional cerebral distribution and competition studies indicated that [11C]carfentanil binds specifically to brain u-opioid receptors, offering a good basis for the application of this agent to clinical studies of such receptors. ACKNOWLEDGMENTS The authors wish to thank Dr. R.F. Dannals, Division of Nuclear Medicine and Radiation Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, U.S.A., for supplying carfentanil carboxylic acid sodium salt and carfentanil. This study was supported in part by the Ministry of Welfare and Education of Japan. REFERENCES 1. Pasternak GW: Multiple morphine and enkephalin receptors and the relief of pain. J Am Med Assn 259: 1362-1367, 1988 2. Wollemann M: Recent developments in the research of opioid receptor subtype molecular characterization. J Neurochem 54: 1095-1101, 1990 3. Frost JJ, Wagner Jr HN, Dannals RF, et al: Imaging opiate receptors in the human brain by positron tomography. J Comput Assist Tomogr 9: 231-236, 1985 4. Frost JJ, Mayberg HS, Sadzot B, et al: Comparison of [11C]diprenorphine and [11C]carfentanil binding to opiate receptors in humans by positron emission tomography. J Cereb Blood Flow Metab 10: 484-492, 1990 5. Mayberg HS, Frost JJ: Opiate receptors. In Quantitative Imaging: Neuroreceptors, Neurotransmitters, and Enzymes, Frost JJ, Wagner HN, (eds.), New York, Raven Press, pp 81-95, 1990 6. Sedvall G, Farde L, Kopp J, et al: Visualization of neuroreceptor functions in the living human brain. In Visualization of Brain Functions, Ottoson D, Rostene W, (eds.), New York, Stockton Press, pp 159-165, 1989 7. Kung HF: Radiopharmaceuticals for CNS receptor imaging with SPECT. Nucl Med Biol 17: 85-92, 1990 8. Van Daele PGH, De Bruyn MFL, Boey JM, et al: Synthetic analgesics: N(1-[2-aryl ethyl]-4-substituted 4-piperidinyl)N-arylalkanamides. Arzneim Forsch (Drug Res) 26: 1521-1531, 1976 9. Stahl KD, Bever WV, Janseen P, et al: Receptor affinity and pharmacological potency of a series of narcotic analgesic, anti-diarrheal and neuroleptic drugs. Eur J Pharmacol 46: 199-205, 1977 10. Titeler M, Lyon RA, Kuhar MJ, et al: u opiate receptors are selectively labeled by [3H]carfentanil in human and rat brain. Eur J Pharmacol 167: 221-228 1989 11. Frost JJ, Mayberg HS, Fisher RS, et al: Mu-opiate receptors measured by positron emission tomography are increased in temporal lobe epilepsy. Ann Neurol 23: 231-237, 1988 12. Frost JJ, Douglass KH, Mayberg HS, et al: Multicompartmental analysis of [11C]-carfentanil binding to opiate receptors in humans measured by positron emission tomography. J Cereb Blood Flow Metab 9 : 398-409, 1989 13. Pfeiffer A, Pasi A, Mehraein P: Opiate receptor binding sites in human brain. Brain Res 248: 87-96, 1982 14. Moshowitz AS, Goodman RR: Autoradiographic distribution of Mu1 and Mu2 opioid binding in the mouse central nervous system. Brain Res 360: 117-129, 1985 15. Dannals RF, Ravert HT, Frost JJ, et al: Radiosynthesis of an opiate receptor binding radiotracer: [11C]carfentanil. Int J Appl Radiat Isot 36: 303-306, 1985 16. Glowinski J, Iversen LL: Regional studies of catecholamines in the rat brain-I. The disposition of [3H]norepinephirine, [3H]dopamine and [3H]dopa in various regions of the brain. J Neurochem 13: 655-669, 1966 17. Arnett CD, Shiue C-Y, Wolf AP, et al: Comparison of three 18F-labeled butyrophenone neuroleptic drugs in the baboon using positron emission tomography. J Neurochem 44: 835-844, 1985 18. Fowler JS, Wolf AP: Positron emitter labeled compounds: priorities and problems. In Positron Emission Tomography and Autoradiography: Principles and Applications for The Brain and Heart, Phelps M, Mazziotta J, Schelbert H, (eds.), New York, Raven Press, pp 391-450, 1986 19. Iwata R, Ido T, Ujiie A, et al: Comparative study of specific activity of [11C]methyl iodine: a search for the source of carrier carbon. Appl Radiat Isot 39: 1-7, 1988 20. Dannals RF, Ravert HT, Wilson AA: Radiochemistry of tracers for neurotransmitter receptor studies. In Quantitative Imaging: Neuroreceptors, Neurotransmitters, and Enzymes, Frost JJ, Wagner HN, (eds.), New York, Raven Press, pp 19-35, 1990 21. Parterson SJ, Robson LE, Kosterlitz HW: Opioid receptors. In The Peptides : Analysis, Synthesis, Biology, Vol 6, Vdenfriend S, Meienhofer J, (eds.), London, Academic Press, pp 147-189, 1984 22. Pasternak GW, Wood PJ: Minireview: multiple Mu opiate receptors. Life Sci 38 : 1889-1898, 1986