- Email: [email protected]
PET study of the distribution of [11C]fluoxetine in a monkey brain
Nucl. Med. Biol. Vol. 22, No. 5, pp. 613-616, 1995
Pergamon
0969-8051(94)00146-4
Copyright 0 1995ElsevierScienceLtd Printedin Great Britain. All rights reserved 0969~8051,'95$9.50+ 0.00
PET Study of the Distribution of [“C]Fluoxetine in a Monkey Brain CHYNG-YANN
SHIUE’*, GRACE G. SHIUE’, KURTIS and MARIA F. O’ROURKE3
G. CORNISH*
‘Department of Radiology, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, U.S.A. and ‘Department of Physiology and 3Department of Pharmacology, University of Nebraska Medical Center, Omaha, NE 68198, U.S.A. (Accepted 16 December 1994)
No-carrier-added [“C]fluoxetine (2) was synthesized by methylation of norfluoxetine (1) with [“C]H,I in 20% radiochemical yield in a synthesis time of 40 min from EOB with a specific activity of 0.48 Ci/pM (EOB). In oiuo study in mouse indicated that the uptake of 2 in mouse tissues was high and the radioactivity remained constant throughout the study. The uptake of 2 in mouse brain was 4%/g. PET study in a Rhesus monkey also showed that the uptakes of 2 in different brain regions were similar and the retention of radioactivity in these regions remained constant throughout the study (80 min). Analysis of arterial plasma by HPLC showed that only 20% of radioactivity in the plasma remained as 2 at 30 min post-injection. These results suggestthat the uptake of fluoxetine in monkey brain is probably not receptor mediated. Rather, blood flow, lipophilicity or other transport mechanisms may play a role in its uptake.
Introduction Positron Emission Tomography (PET) has been used to study the human physiology non-invasively. Recently, it is emerging as an important tool for drug research and development. With PET and appropriate radiotracers, one can study drug distribution, pharmacokinetics and pharmacodynamics (London, 1993). Fluoxetine (Prozac), (+)-N-methyl3-phenyl-3-[(a,a,a-trifluoro-p-tolyl)oxy]propylamine, is a potent clinically used antidepressant with potential applications in the treatment of other psychiatric disorders (Murphy et al., 1986; Fuller et al., 1991). Like other antidepressants, the mechanism of action of fluoxetine has been proposed as a selective inhibitor of serotonin reuptake in presynaptic neurons (Fuller, 1987; Schmidt et al., 1988;Wong et al., 1975). Consequently, carbon-l 1 and fluorine-l 8 labeled fluoxetine have been synthesized and evaluated in
rodents as the serotonin reuptake marker (Kilbourn et al., 1989; Schaffel et al., 1990; Das and Mukherjee, 1993; Hammadi and Crouzel, 1993). Unfortunately, lipophilicity of the molecule has been suggested as one of the contributers to its poor specific binding in rodent brain, therefore, it is not a suitable ligand for studying serotonin reuptake sites with PET. In spite
of this, as fluoxetine is such a potent antipsychotic drug, it is of interest to study its uptake, distribution and metabolism in primate or human brain such that
it will provide useful information for psychiatrists in the treatment of patients. In a continuing effort to study drug distribution and pharmacokinetics with PET (Schlyer et al., 1992; Shiue et nl., 1989, 1993a), WC have also synthesized [“Clfluoxetine (2) and studied its regional distribution
in a monkey brain.
Part of this study has already appeared (Shiue et al., 1993b).
Materials
and Methods
Fluoxetine-HCl and nortluoxetine-maleate were provided by Eli Lilly and Co., Indianapolis, IN. Radioactivity was determined using a calibrated chamber (Capintec CRC-12, Capintec Inc.) and a sodium iodide well counter (Packard Gamma Counter 5000 Series, Packard Instrument Company, IL). Thin layer chromatographic
(TLC) analyses were
performed on plastic-backed silica gel TLC plates (Merck, U.S.A.). High performance liquid chromatography (HPLC) analyses were carried out with a Sonntek liquid chromatograph equipped with both UV and radioactivity monitors. For the preparative separations, a semi-preparative C,, column (10 x 250mm, Sphrisorb 50DS, Phenomenex) was used with CH,CN : 0.1 M HC02NH, (80 : 20) as the solvent with a flow rate of 6 mL/min. For the specific activity and plasma metabolites determinations, an analytical C,s column (4.6 x 250 mm, Sphrisorb 50DS) was
used and eluted with CH,CN:O. 1 M HCO,NH,
*Author for correspondence. 613
Chyng-Yam
614
(70: 30) as the solvent with a flow rate of 1.5 ml/min. The retention time of 2 in this system was 10.5 min. Prepurcrtiori of norfluoxetine
,frw
he
(I)
A mixture of norfluoxetine maleate (2 mg) and sodium hydride (2 mg) in tributyl phosphate (0.3 mL) was stirred at room temperature for 30 min and then stood until all of sodium hydride was precipitated. The norfluoxetine free base in tributyl phosphate was transferred to a reaction vessel for subsequent methylation with [“Cliodomethane. Production c?f’no-carrier -a&led (NCA) [” Cjodomethane No-carrier-added [“Cliodomethane was prepared from [“C]O, in an automated system (NKK, Japan). [“C]Oz was produced by the 14N (p, a)“C nuclear reaction on the CT1 RDS 112/00 cyclotron with 1% 0, (99.993%) in N, (99.999%) as the target gas. The [“C]Oz was reduced with LAH and then reacted with HI to give [“Cliodomethane. No -carrier -added (NCA) [” Cfluoxetine
(2)
NCA compound 2 was synthesized by methylation of norfluoxetine (1) with [“C]H,I followed by purification with HPLC (Scheme 1). Thus, NCA [‘*C]H31 prepared as described above was dispensed into a small reaction vessel containing the freshly prepared compound 1 in 0.3 mL of tributyl phosphate at ice-bath temperature. The solution was heated at 120°C for 7 min. The mixture was injected into a semi-preparative HPLC [C,,, 10 x 250mm, Sphrisorb SODS, Phenomenex; CH,CN: 0.1 M HCO,NH, (80:20) as the solvent with a flow rate of 6mL/min]. The effluent of the HPLC column was passed through an UV detector at 254 nm and into a radioactivity detector. The fraction containing [“Clfluoxetine (2) was collected from 12-14.5 min after injection. The HPLC eluate was evaporated, the residue co-evaporated with 1 mL of 2% HCl-ethanol and finally with 5 mL of ethanol to dryness. To the residue was added 3 mL of normal saline and the resulting solution was filtered through a 0.22 pm cellulose acetate membrane filter (Millipore) into a multi-injection vial. The radiochemical yield of 2 was 20% (EOB) in a synthesis time of 40 min from EOB. The radiochemical purity of 2 was 98% with a specific activity of 0.48 Ci/pM (EOB) as determined by the UV absorbance of the radioactive peak compared Ch
CF, [“C]H31 /(BuO)+O 12O”C,7 min
Scheme 1. Synthesis of carbon-l 1 labeled fluoxetine.
Shiur 6’1rd.
with a standard solution of fluoxetinc Thus. from 500mCi of [“C]O:, 25 mCi of compound 2 wab obtained 40 min from EOB. The K, of compound 2 was 0.38 [silica gel, CH,CN : MeOH (4: 1) as solvent].
Tissue distribution of’ NCA (“Cfluo.wtbw
(2)
in miw
Female CF-1 mice, 20-25 g, were rcstramed in a plastic holder and injected in a lateral tail vein without anesthetic with 0.2.-0.5 mCi of compound 2 in 100 p L of isotonic saline solution. The mice were killed by cervical dislocation and decapitation at 5. 30 and 60 min after injection. The dissected tissues were blotted to remove adhering blood and placed in tared counting vials. A sample of blood was obtained by heart puncture after cervical dislocation. The radioactivity in the entire tail was measured in a NaI detector (Packard) to verify the potency of the tail vein injection. If the ratio of percent uptake in the tail over the percent uptake in the muscle was greater than 3, it was considered a mis-injection. The radioactivity of each sample was also measured in a NaI detector, the sample weighed and the activity expressed as percent of injected dose/g of tissue decay corrected to time of injection. PET stud-v
of’
[“Cfluoxetine
(2) in a monke.)
A male Rhesus monkey (Macaccr mulrtta) weighing 12 kg was initially anesthesized with 17 mg/kg of ketamine administered i.m. approx 2.5 h prior to isotope injection. Once the monkey was anesthesized pentobarbital (20 mg/kg) was administered i.v. followed by insertion of an endotracheal tube. The monkey was maintained under pentobarbital anesthesia for the remainder of the study. A cannula for blood sampling was placed by percutaneous insertion into a femoral artery. A second cannula was inserted into the opposite femoral vein for isotope injection. After the study the monkey was returned to its home cage and allowed to fully recover from anesthesia. The positron emission tomography used for this study was a Computer Technology and Imaging (CTI/Siemens) whole body ECAT 93 l/08-16 PET scanner. The monkey’s head was placed in a plastic head holder and a foam mold (Alpha Cradle Imaging Head Immobilizer, Smithers Medical Products Inc., Tallmadge, Ohio) was used to stabilize the head. Before the radioligand injection, a 20min transmission scan was performed using eight 68Ge filled ring sources to measure regional attenuation. The monkey was injected (i.v. bolus) with 12.6 mCi of [“Clfluoxetine (2). Regions of interest corresponding to cortex, temporal lobe and cerebellum were selected as described previously (Shiue et al., 1993a, b). These regions are sufficiently large structures in the monkey brain that quantitative assessment of radioligand concentrations in these structures are only minimally affected by partial volume effects.
Distribution of [“C]fluoxetine in monkey brain 0.025
Monkey plasma metabolite analysis After injection of [“Clfluoxetine (2), blood samples were collected from the previously described arterial catheters. During the first 2 min, 1 mL of blood samples were drawn manually at approx. 10 s intervals. More blood samples were collected at 5, 10, 30, 60 and 80 min post-injection. Blood samples were placed in heparinized tubes and centrifuged. Duplicate aliquots (0.3 mL) of plasma sample were counted in the gamma counter and the counts corrected for background and decay. Selected plasma samples (1, 10, 30 and 60 min) were analyzed for radioactive metabolites and unchanged compound 2. An aliquot of plasma (0.2-0.4mL) was added to 2 mL of CH,OH, vortexed and centrifuged. The supernatant, with the addition of an authentic fluoxetine, was injected into the HPLC system (4.6 x 250 mm, Sphrisorb 50DS, Phenomenex); CH,CN:O.I M HCO,NH,, 70: 30, 1.5 mL/min). The retention time of compound 2 in this system was 10.5 min.
Results and Discussion Fluoxetine has been labeled with both carbon-11 and fluorine- 18 and evaluated as a serotonin reuptake sites marker (Kilbourn et al., 1989; Scheffel et al., 1990; Das and Mukherjee, 1993; Hammadi and Crouzel, 1993). Due to its high lipophilicity, this compound has been proven not to be a suitable serotonin reuptake sites tracer. However, fluoxetine is such a potent antidepressant, it is of interest to study its uptake, distribution and metabolism in the primate or human brain with PET. been [“ClFluoxetine has synthesized by norfluoxetine with either methylation of [“C]formaldehyde (Kilbourn et al., 1989) or [“Cliodomethane (Scheffel et al., 1990). We have synthesized [“Clfluoxetine (2) by methylation of norfluoxetine (1) with [“C]HJ in (BuO),PO at 120°C for 7 min followed by purification with HPLC in 20% yield in a synthesis time of 40min from EOB. Table 1 shows that the relative tissue uptake of compound 2 in mouse after i.v. administration is
Table 1. Tissue distribution
of I’ClAuoxetine
615
in mice*
% Dose/g (mean + SD)
9::
0.020
4 P g ‘r’
0.015
000 gas
Cerebellum Cortex A Temporal lobe
0 0
0.010
&F 0.005
0
il
20
40
60
80
Scan midpoint (min) Fig. I. [“ClFluoxetine uptake in monkey brain
lungs > kidneys > heart > spleen > liver > brain > blood. The uptake of compound 2 in these tissues is high and the retention of the radioactivity remains constant throughout the study (60 min). The uptake of compound 2 in mouse brain was high (approx. 4%/g) which was in agreement with the results of Scheffel (1990). The distribution of radioactivity in the monkey brain following injection of [“Clfluoxetine (2) is depicted in Fig. 1. The uptakes of radioactivity in different monkey brain regions (cerebellum, cortex and temporal lobe) were similar and the retention of the radioactivity in these regions remained constant throughout the study (80min). The uptake of compound 2 in different mouse brain regions was shown not to be proportional to serotonin uptake site densities and also not to be inhibited by pretreatment with paroxetine (Scheffel et al., 1990). A structurally similar compound, sertraline, was also shown not to bind specifically to serotonin uptake sites (Hume et al., 1989). These results suggest that the uptakes of fluoxetine and sertraline in mouse brain or monkey brain are probably not receptor mediated. Rather, blood flow, lipophilicity or other transport mechanisms may play a role in their uptakes. The appearance of metabolites in the monkey plasma following injection of compound 2 was relatively rapid. Analysis of arterial plasma by HPLC showed that only 20% of radioactivity in the plasma remained as parent compound 2 at 30 min post-injection (Table 2). Fluoxetine has also been shown to undergo rapid demethylation to norfluoxetine in the rat brain. At 60 min post i.p. injection, 60&70% of compound remained as fluoxetine (Fuller et al., 1978; Schmidt et al., 1988).
Time after injection Orgall Blood Brain Heart LUIl@ Liver SplWl Kidneys Small intestines *n =3
5 min 0.50 + 0.02 3.91 F 1.26 9.54 i 2.37 28.52 F 3.96 4.88 f 0.83 3.90 f 0.25 9.19*0.09 3.73 + 0.38
30 min 0.56 i 0.07 3.88 i 0.72 2.81 f 0.52 22.66 f I S7 5.74 f 0.84 6.18 + 0.88 5.80 f 0.61 4.13 f 1.04
60 min 0.67 i 0.04 3.96 f 0.76 2.86 + I.31 22.97 f I .50 6.21 f 1.40 5.86 k 0.88 4.42 + I .29 3.63 k 0.61
Table 2. Analysis of “C radioactivity in monkey plasma following iniection of I”Clfluoxetine (2)* Min after iniection
30 60
% of total radioactivity extracted
% of unchanged 12) in olasma
85 82
*Plasma samples were extracted with 2 mL of MeOH
22 22
Chyng-Yann Shiue et (II.
616
Conclusion [“ClFluoxetine (2) has been synthesized by methylation of norfluoxetine (1) with [“C]HJ in 20% yield in a synthesis time of 40 min from EOB. In uiuo studies in mouse revealed that the relative tissue uptake of compound 2 in mouse after iv. administration was lungs > kidneys > heart > spleen > liver > brain > blood. The uptake of compound 2 in these tissues was high and the retention of the radioactivity remained constant throughout the study. PET study in a Rhesus monkey also
showed that the uptakes of compound 2 in different brain regions were similar and the retention of the radioactivity in these regions remained constant throughout the study (80min). Analysis of arterial plasma by HPLC showed that only 20% of radioactivity in the plasma remained as compound 2 at 30 min post-injection. These results suggest that the uptake of fluoxetine in monkey brain is probably not receptor mediated. Rather, blood flow, lipophilicity or other transport mechanisms may play a role in its uptake. research was carried out at the Creighton Center for Metabolic Imaging and supported by a grant from Health Future Foundation. The authors thank MS Julie Prellwitz and Dr John D. Sunderland for technical assistance and NKK (Japan) for providing us with the automated [“C]H,I system.
Acknowledgemenfs-This
Hammadi A. and Crouzel C. (1993) Synthesis of [“F]-(s)fluoxetine, a selective serotonin uptake inhibitor. J. Label. Compds. Radiopharm. 32, 703-10. Hume S., Myers R., Manjil L. and Dolan R. (1989) Sertraline and paroxetine fail tests in ~ivo as 5-HT rcuptake site ligands for PET. J. Cereb. Blood Flow Metab. 9 (Suppl. l), s117. Kilbourn M. R., Hara M. S., Mulholland G. K., Jewett D. M. and Kuhl D. E. (1989) Synthesis of radiolabeled inhibitors of pre-synaptic monoamine uptake systems: [“F]GBR13119 (DA). [“Clnisoxetine (NE) and [“Clfluoxetine (5HT). J. Label. Compds. Radipharm. 26, 412414. London E. D. (1993)Zmaging Drug Action in the Brain. CRC Press, Boca Raton. Murphy D. L., Mueller E. A., Garrick N. A. and Aulakh C. S. (1986) Use of serotonergic agents in the clinical assessmentof central serotonin function. J. Clin. Psych&. 47 (Suppl. 4), 9-15. Scheffel U., Dannals R. F., Suehiro M.. Wilson A. A., Ravert H. T., Stathis M., Wagner H. N., Jr. and Ricaurte G. A. (1990) Evaluation of “C-fluoxetine as in ZX~O ligands for the serotonin uptake site. J. Nucl. Med. 31,883 (abstract). Schmidt M. J., Fuller R. W. and Wong D. T. (1988) Fluoxetine, a highly selective serotonin reuptake inhibitor: a review of preclinical studies. Br. J. Psychiat. 153 (Suppl. 3), 4046. Schlyer D. J., Volkow N. D., Fowler J. S., Wolf A. P., Shiue C. Y., Dewey S. L., Bendriem B., Logan J., Raulli R., Hitzemann R., Brodie J., Alavi A. A. and MacGregor R. R. (1992) Regional distribution and kinetics of haloperidol binding in human brain: a PET study with [‘*F]haloperidol. Synapse 11, 10-19. Shiue C. Y., Vallabhajosula S., Fowler J. S., Dewey S. L., Zhou Y.-G. and Wolf A. P. (1989) Carbon-11 (+)- and (-)-ketamine: synthesis and PET studies in a baboon. J. Label. Cmpds. Radiopharm. XXVI,
References Das M. K. and Mukherjee J. (1993) Radiosynthesis of [F-lS]fluoxetine as a potential radiotracer for serotonin reuptake sites. Appl. kadiat. Isot. 44, 835-842. Fuller R. W. (1987) Pharmacoloaic DroDerties of serotonergic agents and antidepressant drugs. J. Clin. Psychiat. 48 (Suppl. 3), 5-l 1. Fuller R. W., Snoddy H. D., Perry K. W., Bymaster F. P. and Wong D. T. (1978) Importance of duration of drug action in the antagonism of p-chloroamphetamine depletion of brain serotonin-comparison of fluoxetine and chlorimipramine. Biochem. Pharmacol. 27, 193-198. Fuller R. W., Wong D. T. and Robertson D. W. (1991) Fluoxetine, a selective inhibitor of serotonin uptake. Med. .
,
Rex Rev. 11, 17-34.
I
_
_
246-248.
Shiue C. Y., Shiue G. G., Rysavy J. A., Pleus R. C., Huang H., Bai L.-Q., Cornish K. G., Sunderland J. J. and Frick M. P. (1993a) Fluorine-18 and carbon-l 1 labeled amphetamine analogs-synthesis, distribution, binding characteristics in mice and rats and a PET study in monkey. Nucl. Med. Biol. 20, 973-981. Shiue C. Y., Shiue G. G., Cornish K. G., O’Rourke M. F. and Suderland J. J. (1993b) The pharmacokinetics of [“Clfluoxetine in a monkey brain. In Xfh International Symposium on Radiopharmaceutical Chemistry, Kyoto, Japan, 534-536. Wong D. T., Bymaster F. P., Horng J. S. and Molloy B. B. (1975) A selective inhibitor for uptake of serotonin into synaptosomes of rat brain: 3-(‘p-trifluoromethylphenoxy)N-methyl-3-phenylpropylamine. J. Pharmacol. Exp. Ther. 193, 804-8 11.
Pergamon
0969-8051(94)00146-4
Copyright 0 1995ElsevierScienceLtd Printedin Great Britain. All rights reserved 0969~8051,'95$9.50+ 0.00
PET Study of the Distribution of [“C]Fluoxetine in a Monkey Brain CHYNG-YANN
SHIUE’*, GRACE G. SHIUE’, KURTIS and MARIA F. O’ROURKE3
G. CORNISH*
‘Department of Radiology, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, U.S.A. and ‘Department of Physiology and 3Department of Pharmacology, University of Nebraska Medical Center, Omaha, NE 68198, U.S.A. (Accepted 16 December 1994)
No-carrier-added [“C]fluoxetine (2) was synthesized by methylation of norfluoxetine (1) with [“C]H,I in 20% radiochemical yield in a synthesis time of 40 min from EOB with a specific activity of 0.48 Ci/pM (EOB). In oiuo study in mouse indicated that the uptake of 2 in mouse tissues was high and the radioactivity remained constant throughout the study. The uptake of 2 in mouse brain was 4%/g. PET study in a Rhesus monkey also showed that the uptakes of 2 in different brain regions were similar and the retention of radioactivity in these regions remained constant throughout the study (80 min). Analysis of arterial plasma by HPLC showed that only 20% of radioactivity in the plasma remained as 2 at 30 min post-injection. These results suggestthat the uptake of fluoxetine in monkey brain is probably not receptor mediated. Rather, blood flow, lipophilicity or other transport mechanisms may play a role in its uptake.
Introduction Positron Emission Tomography (PET) has been used to study the human physiology non-invasively. Recently, it is emerging as an important tool for drug research and development. With PET and appropriate radiotracers, one can study drug distribution, pharmacokinetics and pharmacodynamics (London, 1993). Fluoxetine (Prozac), (+)-N-methyl3-phenyl-3-[(a,a,a-trifluoro-p-tolyl)oxy]propylamine, is a potent clinically used antidepressant with potential applications in the treatment of other psychiatric disorders (Murphy et al., 1986; Fuller et al., 1991). Like other antidepressants, the mechanism of action of fluoxetine has been proposed as a selective inhibitor of serotonin reuptake in presynaptic neurons (Fuller, 1987; Schmidt et al., 1988;Wong et al., 1975). Consequently, carbon-l 1 and fluorine-l 8 labeled fluoxetine have been synthesized and evaluated in
rodents as the serotonin reuptake marker (Kilbourn et al., 1989; Schaffel et al., 1990; Das and Mukherjee, 1993; Hammadi and Crouzel, 1993). Unfortunately, lipophilicity of the molecule has been suggested as one of the contributers to its poor specific binding in rodent brain, therefore, it is not a suitable ligand for studying serotonin reuptake sites with PET. In spite
of this, as fluoxetine is such a potent antipsychotic drug, it is of interest to study its uptake, distribution and metabolism in primate or human brain such that
it will provide useful information for psychiatrists in the treatment of patients. In a continuing effort to study drug distribution and pharmacokinetics with PET (Schlyer et al., 1992; Shiue et nl., 1989, 1993a), WC have also synthesized [“Clfluoxetine (2) and studied its regional distribution
in a monkey brain.
Part of this study has already appeared (Shiue et al., 1993b).
Materials
and Methods
Fluoxetine-HCl and nortluoxetine-maleate were provided by Eli Lilly and Co., Indianapolis, IN. Radioactivity was determined using a calibrated chamber (Capintec CRC-12, Capintec Inc.) and a sodium iodide well counter (Packard Gamma Counter 5000 Series, Packard Instrument Company, IL). Thin layer chromatographic
(TLC) analyses were
performed on plastic-backed silica gel TLC plates (Merck, U.S.A.). High performance liquid chromatography (HPLC) analyses were carried out with a Sonntek liquid chromatograph equipped with both UV and radioactivity monitors. For the preparative separations, a semi-preparative C,, column (10 x 250mm, Sphrisorb 50DS, Phenomenex) was used with CH,CN : 0.1 M HC02NH, (80 : 20) as the solvent with a flow rate of 6 mL/min. For the specific activity and plasma metabolites determinations, an analytical C,s column (4.6 x 250 mm, Sphrisorb 50DS) was
used and eluted with CH,CN:O. 1 M HCO,NH,
*Author for correspondence. 613
Chyng-Yam
614
(70: 30) as the solvent with a flow rate of 1.5 ml/min. The retention time of 2 in this system was 10.5 min. Prepurcrtiori of norfluoxetine
,frw
he
(I)
A mixture of norfluoxetine maleate (2 mg) and sodium hydride (2 mg) in tributyl phosphate (0.3 mL) was stirred at room temperature for 30 min and then stood until all of sodium hydride was precipitated. The norfluoxetine free base in tributyl phosphate was transferred to a reaction vessel for subsequent methylation with [“Cliodomethane. Production c?f’no-carrier -a&led (NCA) [” Cjodomethane No-carrier-added [“Cliodomethane was prepared from [“C]O, in an automated system (NKK, Japan). [“C]Oz was produced by the 14N (p, a)“C nuclear reaction on the CT1 RDS 112/00 cyclotron with 1% 0, (99.993%) in N, (99.999%) as the target gas. The [“C]Oz was reduced with LAH and then reacted with HI to give [“Cliodomethane. No -carrier -added (NCA) [” Cfluoxetine
(2)
NCA compound 2 was synthesized by methylation of norfluoxetine (1) with [“C]H,I followed by purification with HPLC (Scheme 1). Thus, NCA [‘*C]H31 prepared as described above was dispensed into a small reaction vessel containing the freshly prepared compound 1 in 0.3 mL of tributyl phosphate at ice-bath temperature. The solution was heated at 120°C for 7 min. The mixture was injected into a semi-preparative HPLC [C,,, 10 x 250mm, Sphrisorb SODS, Phenomenex; CH,CN: 0.1 M HCO,NH, (80:20) as the solvent with a flow rate of 6mL/min]. The effluent of the HPLC column was passed through an UV detector at 254 nm and into a radioactivity detector. The fraction containing [“Clfluoxetine (2) was collected from 12-14.5 min after injection. The HPLC eluate was evaporated, the residue co-evaporated with 1 mL of 2% HCl-ethanol and finally with 5 mL of ethanol to dryness. To the residue was added 3 mL of normal saline and the resulting solution was filtered through a 0.22 pm cellulose acetate membrane filter (Millipore) into a multi-injection vial. The radiochemical yield of 2 was 20% (EOB) in a synthesis time of 40 min from EOB. The radiochemical purity of 2 was 98% with a specific activity of 0.48 Ci/pM (EOB) as determined by the UV absorbance of the radioactive peak compared Ch
CF, [“C]H31 /(BuO)+O 12O”C,7 min
Scheme 1. Synthesis of carbon-l 1 labeled fluoxetine.
Shiur 6’1rd.
with a standard solution of fluoxetinc Thus. from 500mCi of [“C]O:, 25 mCi of compound 2 wab obtained 40 min from EOB. The K, of compound 2 was 0.38 [silica gel, CH,CN : MeOH (4: 1) as solvent].
Tissue distribution of’ NCA (“Cfluo.wtbw
(2)
in miw
Female CF-1 mice, 20-25 g, were rcstramed in a plastic holder and injected in a lateral tail vein without anesthetic with 0.2.-0.5 mCi of compound 2 in 100 p L of isotonic saline solution. The mice were killed by cervical dislocation and decapitation at 5. 30 and 60 min after injection. The dissected tissues were blotted to remove adhering blood and placed in tared counting vials. A sample of blood was obtained by heart puncture after cervical dislocation. The radioactivity in the entire tail was measured in a NaI detector (Packard) to verify the potency of the tail vein injection. If the ratio of percent uptake in the tail over the percent uptake in the muscle was greater than 3, it was considered a mis-injection. The radioactivity of each sample was also measured in a NaI detector, the sample weighed and the activity expressed as percent of injected dose/g of tissue decay corrected to time of injection. PET stud-v
of’
[“Cfluoxetine
(2) in a monke.)
A male Rhesus monkey (Macaccr mulrtta) weighing 12 kg was initially anesthesized with 17 mg/kg of ketamine administered i.m. approx 2.5 h prior to isotope injection. Once the monkey was anesthesized pentobarbital (20 mg/kg) was administered i.v. followed by insertion of an endotracheal tube. The monkey was maintained under pentobarbital anesthesia for the remainder of the study. A cannula for blood sampling was placed by percutaneous insertion into a femoral artery. A second cannula was inserted into the opposite femoral vein for isotope injection. After the study the monkey was returned to its home cage and allowed to fully recover from anesthesia. The positron emission tomography used for this study was a Computer Technology and Imaging (CTI/Siemens) whole body ECAT 93 l/08-16 PET scanner. The monkey’s head was placed in a plastic head holder and a foam mold (Alpha Cradle Imaging Head Immobilizer, Smithers Medical Products Inc., Tallmadge, Ohio) was used to stabilize the head. Before the radioligand injection, a 20min transmission scan was performed using eight 68Ge filled ring sources to measure regional attenuation. The monkey was injected (i.v. bolus) with 12.6 mCi of [“Clfluoxetine (2). Regions of interest corresponding to cortex, temporal lobe and cerebellum were selected as described previously (Shiue et al., 1993a, b). These regions are sufficiently large structures in the monkey brain that quantitative assessment of radioligand concentrations in these structures are only minimally affected by partial volume effects.
Distribution of [“C]fluoxetine in monkey brain 0.025
Monkey plasma metabolite analysis After injection of [“Clfluoxetine (2), blood samples were collected from the previously described arterial catheters. During the first 2 min, 1 mL of blood samples were drawn manually at approx. 10 s intervals. More blood samples were collected at 5, 10, 30, 60 and 80 min post-injection. Blood samples were placed in heparinized tubes and centrifuged. Duplicate aliquots (0.3 mL) of plasma sample were counted in the gamma counter and the counts corrected for background and decay. Selected plasma samples (1, 10, 30 and 60 min) were analyzed for radioactive metabolites and unchanged compound 2. An aliquot of plasma (0.2-0.4mL) was added to 2 mL of CH,OH, vortexed and centrifuged. The supernatant, with the addition of an authentic fluoxetine, was injected into the HPLC system (4.6 x 250 mm, Sphrisorb 50DS, Phenomenex); CH,CN:O.I M HCO,NH,, 70: 30, 1.5 mL/min). The retention time of compound 2 in this system was 10.5 min.
Results and Discussion Fluoxetine has been labeled with both carbon-11 and fluorine- 18 and evaluated as a serotonin reuptake sites marker (Kilbourn et al., 1989; Scheffel et al., 1990; Das and Mukherjee, 1993; Hammadi and Crouzel, 1993). Due to its high lipophilicity, this compound has been proven not to be a suitable serotonin reuptake sites tracer. However, fluoxetine is such a potent antidepressant, it is of interest to study its uptake, distribution and metabolism in the primate or human brain with PET. been [“ClFluoxetine has synthesized by norfluoxetine with either methylation of [“C]formaldehyde (Kilbourn et al., 1989) or [“Cliodomethane (Scheffel et al., 1990). We have synthesized [“Clfluoxetine (2) by methylation of norfluoxetine (1) with [“C]HJ in (BuO),PO at 120°C for 7 min followed by purification with HPLC in 20% yield in a synthesis time of 40min from EOB. Table 1 shows that the relative tissue uptake of compound 2 in mouse after i.v. administration is
Table 1. Tissue distribution
of I’ClAuoxetine
615
in mice*
% Dose/g (mean + SD)
9::
0.020
4 P g ‘r’
0.015
000 gas
Cerebellum Cortex A Temporal lobe
0 0
0.010
&F 0.005
0
il
20
40
60
80
Scan midpoint (min) Fig. I. [“ClFluoxetine uptake in monkey brain
lungs > kidneys > heart > spleen > liver > brain > blood. The uptake of compound 2 in these tissues is high and the retention of the radioactivity remains constant throughout the study (60 min). The uptake of compound 2 in mouse brain was high (approx. 4%/g) which was in agreement with the results of Scheffel (1990). The distribution of radioactivity in the monkey brain following injection of [“Clfluoxetine (2) is depicted in Fig. 1. The uptakes of radioactivity in different monkey brain regions (cerebellum, cortex and temporal lobe) were similar and the retention of the radioactivity in these regions remained constant throughout the study (80min). The uptake of compound 2 in different mouse brain regions was shown not to be proportional to serotonin uptake site densities and also not to be inhibited by pretreatment with paroxetine (Scheffel et al., 1990). A structurally similar compound, sertraline, was also shown not to bind specifically to serotonin uptake sites (Hume et al., 1989). These results suggest that the uptakes of fluoxetine and sertraline in mouse brain or monkey brain are probably not receptor mediated. Rather, blood flow, lipophilicity or other transport mechanisms may play a role in their uptakes. The appearance of metabolites in the monkey plasma following injection of compound 2 was relatively rapid. Analysis of arterial plasma by HPLC showed that only 20% of radioactivity in the plasma remained as parent compound 2 at 30 min post-injection (Table 2). Fluoxetine has also been shown to undergo rapid demethylation to norfluoxetine in the rat brain. At 60 min post i.p. injection, 60&70% of compound remained as fluoxetine (Fuller et al., 1978; Schmidt et al., 1988).
Time after injection Orgall Blood Brain Heart LUIl@ Liver SplWl Kidneys Small intestines *n =3
5 min 0.50 + 0.02 3.91 F 1.26 9.54 i 2.37 28.52 F 3.96 4.88 f 0.83 3.90 f 0.25 9.19*0.09 3.73 + 0.38
30 min 0.56 i 0.07 3.88 i 0.72 2.81 f 0.52 22.66 f I S7 5.74 f 0.84 6.18 + 0.88 5.80 f 0.61 4.13 f 1.04
60 min 0.67 i 0.04 3.96 f 0.76 2.86 + I.31 22.97 f I .50 6.21 f 1.40 5.86 k 0.88 4.42 + I .29 3.63 k 0.61
Table 2. Analysis of “C radioactivity in monkey plasma following iniection of I”Clfluoxetine (2)* Min after iniection
30 60
% of total radioactivity extracted
% of unchanged 12) in olasma
85 82
*Plasma samples were extracted with 2 mL of MeOH
22 22
Chyng-Yann Shiue et (II.
616
Conclusion [“ClFluoxetine (2) has been synthesized by methylation of norfluoxetine (1) with [“C]HJ in 20% yield in a synthesis time of 40 min from EOB. In uiuo studies in mouse revealed that the relative tissue uptake of compound 2 in mouse after iv. administration was lungs > kidneys > heart > spleen > liver > brain > blood. The uptake of compound 2 in these tissues was high and the retention of the radioactivity remained constant throughout the study. PET study in a Rhesus monkey also
showed that the uptakes of compound 2 in different brain regions were similar and the retention of the radioactivity in these regions remained constant throughout the study (80min). Analysis of arterial plasma by HPLC showed that only 20% of radioactivity in the plasma remained as compound 2 at 30 min post-injection. These results suggest that the uptake of fluoxetine in monkey brain is probably not receptor mediated. Rather, blood flow, lipophilicity or other transport mechanisms may play a role in its uptake. research was carried out at the Creighton Center for Metabolic Imaging and supported by a grant from Health Future Foundation. The authors thank MS Julie Prellwitz and Dr John D. Sunderland for technical assistance and NKK (Japan) for providing us with the automated [“C]H,I system.
Acknowledgemenfs-This
Hammadi A. and Crouzel C. (1993) Synthesis of [“F]-(s)fluoxetine, a selective serotonin uptake inhibitor. J. Label. Compds. Radiopharm. 32, 703-10. Hume S., Myers R., Manjil L. and Dolan R. (1989) Sertraline and paroxetine fail tests in ~ivo as 5-HT rcuptake site ligands for PET. J. Cereb. Blood Flow Metab. 9 (Suppl. l), s117. Kilbourn M. R., Hara M. S., Mulholland G. K., Jewett D. M. and Kuhl D. E. (1989) Synthesis of radiolabeled inhibitors of pre-synaptic monoamine uptake systems: [“F]GBR13119 (DA). [“Clnisoxetine (NE) and [“Clfluoxetine (5HT). J. Label. Compds. Radipharm. 26, 412414. London E. D. (1993)Zmaging Drug Action in the Brain. CRC Press, Boca Raton. Murphy D. L., Mueller E. A., Garrick N. A. and Aulakh C. S. (1986) Use of serotonergic agents in the clinical assessmentof central serotonin function. J. Clin. Psych&. 47 (Suppl. 4), 9-15. Scheffel U., Dannals R. F., Suehiro M.. Wilson A. A., Ravert H. T., Stathis M., Wagner H. N., Jr. and Ricaurte G. A. (1990) Evaluation of “C-fluoxetine as in ZX~O ligands for the serotonin uptake site. J. Nucl. Med. 31,883 (abstract). Schmidt M. J., Fuller R. W. and Wong D. T. (1988) Fluoxetine, a highly selective serotonin reuptake inhibitor: a review of preclinical studies. Br. J. Psychiat. 153 (Suppl. 3), 4046. Schlyer D. J., Volkow N. D., Fowler J. S., Wolf A. P., Shiue C. Y., Dewey S. L., Bendriem B., Logan J., Raulli R., Hitzemann R., Brodie J., Alavi A. A. and MacGregor R. R. (1992) Regional distribution and kinetics of haloperidol binding in human brain: a PET study with [‘*F]haloperidol. Synapse 11, 10-19. Shiue C. Y., Vallabhajosula S., Fowler J. S., Dewey S. L., Zhou Y.-G. and Wolf A. P. (1989) Carbon-11 (+)- and (-)-ketamine: synthesis and PET studies in a baboon. J. Label. Cmpds. Radiopharm. XXVI,
References Das M. K. and Mukherjee J. (1993) Radiosynthesis of [F-lS]fluoxetine as a potential radiotracer for serotonin reuptake sites. Appl. kadiat. Isot. 44, 835-842. Fuller R. W. (1987) Pharmacoloaic DroDerties of serotonergic agents and antidepressant drugs. J. Clin. Psychiat. 48 (Suppl. 3), 5-l 1. Fuller R. W., Snoddy H. D., Perry K. W., Bymaster F. P. and Wong D. T. (1978) Importance of duration of drug action in the antagonism of p-chloroamphetamine depletion of brain serotonin-comparison of fluoxetine and chlorimipramine. Biochem. Pharmacol. 27, 193-198. Fuller R. W., Wong D. T. and Robertson D. W. (1991) Fluoxetine, a selective inhibitor of serotonin uptake. Med. .
,
Rex Rev. 11, 17-34.
I
_
_
246-248.
Shiue C. Y., Shiue G. G., Rysavy J. A., Pleus R. C., Huang H., Bai L.-Q., Cornish K. G., Sunderland J. J. and Frick M. P. (1993a) Fluorine-18 and carbon-l 1 labeled amphetamine analogs-synthesis, distribution, binding characteristics in mice and rats and a PET study in monkey. Nucl. Med. Biol. 20, 973-981. Shiue C. Y., Shiue G. G., Cornish K. G., O’Rourke M. F. and Suderland J. J. (1993b) The pharmacokinetics of [“Clfluoxetine in a monkey brain. In Xfh International Symposium on Radiopharmaceutical Chemistry, Kyoto, Japan, 534-536. Wong D. T., Bymaster F. P., Horng J. S. and Molloy B. B. (1975) A selective inhibitor for uptake of serotonin into synaptosomes of rat brain: 3-(‘p-trifluoromethylphenoxy)N-methyl-3-phenylpropylamine. J. Pharmacol. Exp. Ther. 193, 804-8 11.