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A rapid and simple Sep Pak method for purification of radioiodinated IQNP, a high affinity ligand for the muscarinic receptor
Nuclear Medicine & Biology, Vol. 26, pp. 859 – 863, 1999 Copyright © 1999 Elsevier Science Inc. All rights reserved.
ISSN 0969-8051/99/$–see front matter PII S0969-8051(99)00050-5
TECHNICAL NOTE
A Rapid and Simple Sep Pak Method for Purification of Radioiodinated IQNP, a High Affinity Ligand for the Muscarinic Receptor D. W. McPherson and F. F. (Russ) Knapp, Jr. NUCLEAR MEDICINE GROUP, LIFE SCIENCES DIVISION, OAK RIDGE NATIONAL LABORATORY (ORNL), OAK RIDGE, TENNESSEE, USA
ABSTRACT. A simplified procedure for the purification of 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1iodo-1-propen-3-yl)-␣-phenylacetate (IQNP) stereoisomers utilizing a silica Sep Pak (SSP) is described. Iodine-131-E- and iodine-125-Z-(R,R)-IQNP were isolated after SSP purification in 80% and 75% radiochemical yields, respectively. The biodistribution of iodine-131-E-/iodine-125-Z-(R,R)-IQNP, purified either by SSP or high performance liquid chromatography (HPLC), was evaluated in female rats and demonstrated no significant differences in the uptake in various organs and cerebral regions. The utilization of SSP thus affords a simple and rapid method for the purification of IQNP for use in a variety of animal studies. NUCL MED BIOL 26;7:859 – 863, 1999. © 1999 Elsevier Science Inc. All rights reserved. KEY WORDS. IQNP, Sep Pak purification, Muscarinic receptor
INTRODUCTION The muscarinic acetylcholinergic receptor complex (mAChR) has been observed to exhibit five distinct subtypes (m1–m5) by cloning methodology (3). Changes in the density or function of the various mAChR subtypes have been postulated to play an important role in various dementias such as Alzheimer’s and Huntington’s diseases (4, 5, 7, 11). The expected clinical importance has spurred the development of new mAChR subtype selective drugs and ligands for the potential treatment and diagnosis of Alzheimer’s disease. One of these new ligands, 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1-iodo1-propen-3-yl)-␣-phenylacetate (IQNP, Fig. 1) demonstrates high in vitro and in vivo binding affinity (1, 2, 9, 12, 14). The E-(R,R)isomer of IQNP demonstrates high binding to m1 subtype after the modest binding to the m2 subtype washes out and the Z-(R,R)isomer displays non-subtype in vivo selective binding in both rats and baboon studies. In addition, it was observed that utilization of a dual-labeled “E/Z-(R,R)-IQNP cocktail” allowed for the in vivo cerebral quantification of the m2 mAChR subtype in female Fischer rats (10). Use of this technique is being evaluated for the determination of the mAChR subtype selectivity for new and established ligands and drugs in vivo. Our method for the radioiodination of IQNP involves the no-carrier-added iodination of a tributylstannyl intermediate with a 3% hydrogen peroxide solution as the oxidizing agent. Following high performance liquid chromatography (HPLC) purification, the radioiodinated IQNP isomers are obtained with high specific activity in a total reaction time of 3 h. To expedite the preparation of IQNP for use in animal experiments, a simplified procedure of the final purification of IQNP was investigated. The use of a C-18 Sep Pak (CSP) for the purification of radioiodinated IBZM, a high
affinity dopamine D2 ligand, has been reported recently (6, 13). We now report the development of an improved purification procedure of IQNP utilizing a silica Sep Pak (SSP) to afford the radioiodinated IQNP isomers with sufficient specific activity for use in various animal studies. MATERIALS AND METHODS
General E- and Z-(R)-1-azabicyclo[2.2.2]oct-3-yl (R)-␣-hydroxy-␣-phenyl␣-(1-tributylstanyl-1-propen-3-yl)acetate (TBTQNP) were prepared as described previously (8, 9). All reagents were of reagent grade and used without further purification. Sodium iodine-125 (specific activity 17 Ci/mg) and sodium iodine-131 (specific activity 9 Ci/mg) were purchased from New England Nuclear Life Sciences Division (Roxbury, MA USA). CSP and SSP cartridges were purchased from Waters Corporation (Cary, NC USA). HPLC was performed with a Waters model 510 HPLC pump and model 454 variable ultraviolet detector (254 nm) (Cary, NC USA) utilizing a Beckman Model 170 radioisotope flow detector (Fullerton, CA USA). The HPLC conditions for the purification of the stereoisomers of IQNP utilized a mobile phase of methylene chloride: (ethanol ⫹ 1% triethylamine) [98:2], flow rate of 2.5 mL/min, and a Waters semipreparative Porasil column (3.9 mm ⫻ 30 cm). Radiochemical thin layer chromatographic (RTLC) analysis was performed using Analtech glass-coated silica GF plates (250 m) (Newark, DE USA) and a mobile phase of chloroform:methanol (8.5:1.5, Rf ⫽ 0.6). The plates were analyzed using a Bioscan System 200 Imaging Scanner with an Autochanger 1000 (Washington, DC USA).
Preparation of Iodine-125 Z-(R,R)-IQNP Address correspondence to: D. W. McPherson, Ph.D., Oak Ridge National Laboratory, Post Office Box 2008, Building 4501, Oak Ridge, TN 378316229, USA; e-mail [email protected] Received 1 May 1999. Accepted 5 July 1999.
An ethanolic solution of Z-(R,R)-TBTQNP (1 mg/mL, 100 L), 0.1 N HCl (200 L), and an ethanolic sodium iodine-125 solution (7.8 mCi in 100 L of 0.1 M NaOH and 100 L of ethanol) were added to a 3-mL vial. A 3% hydrogen peroxide solution (50 L) was
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Preparation of Iodine-131 E-(R,R)-IQNP E-(R,R)-TBTQNP was prepared as described above with 4.1 mCi sodium iodine-131 to afford 3.3 mCi iodine-131-E-(R,R)-IQNP (80%). An aliquot of the radioligand was subjected to HPLC purification as described above to afford iodine-131-E-(R,R)-IQNP with specific activity greater than 1,000 mCi/mol. The radioligand, purified by SSP and HPLC, was stored in ethanol in a freezer until used.
Biodistribution Studies
FIG. 1. Structure of 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣(1-iodo-1-propen-3-yl)-␣-phenylacetate (IQNP), 1.
added, the vial sealed, and the solution stirred at room temperature for 30 min. A 10% sodium bisulfite solution (500 L) was added, the solution stirred 1 min, and a saturated sodium bicarbonate solution (1.0 mL) added slowly. The resultant solution was transferred to a syringe attached to a CSP. The reaction vial was rinsed with acetonitrile (100 L) and water (1.0 mL) and these rinses were added to the syringe. The volume in the syringe was brought up to 10 mL with water and the solution passed through the CSP. The CSP was washed with water (2 ⫻ 4 mL) and acetonitrile (0.25 mL). The crude radioiodinated product was removed from the CSP with acetonitrile (2 mL) followed by chloroform (5 mL). The crude radioiodinated ligand solution was evaporated to dryness under a stream of nitrogen, the vial rinsed with chloroform (3 ⫻ 1 mL), and these washes transferred to a syringe containing an SSP. The SSP was washed sequentially with chloroform:methanol (v/v) solutions (99:1, 4 mL; 95:5, 4 mL; and 90:10, 8 mL) and each wash was collected separately. The chloroform:methanol (90:10) fraction was evaporated to dryness under a stream of nitrogen to afford 5.9 mCi of iodine-125 Z-(R,R)-IQNP (75% yield) with a radiochemical purity greater than 97% as determined by RTLC analysis. An aliquot of the radioligand was subjected to HPLC purification as described previously to afford iodine-125-Z-(R,R)-IQNP with specific activity greater than 1,000 mCi/mol. The radioligand, purified by SSP and HPLC, was stored in ethanol in a freezer until used. Following the initial in vivo animal experiments, the remaining iodine-125-Z-(R,R)-IQNP ethanol solution was stored in a freezer for 1 month. This solution was evaporated to dryness, dissolved in chloroform (3 mL), and subjected to SSP purification as described above. Iodine-125-Z-(R,R)-IQNP was obtained in greater than 97% radiochemical purity as determined by RTLC analysis.
Biodistribution studies were performed using female VAF Fischer rats (⬃150 g). The animal care and use procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and were reviewed and approved by the Oak Ridge National Laboratory Animal Care and Use Committee. The animals were allowed food and water ad libitum before and during the course of the experiments. Ethanolic solutions of iodine-125-Z-(R,R)-IQNP (100 L) and iodine-131-E(R,R)-IQNP (100 L), both of which were purified either by SSP or HPLC, were combined and acidified by the addition of a 0.1-N HCl solution (100 L). The resultant solution was diluted with saline (10 mL) and filtered (Millipore) into a sterile injection vial. Iodine-125-Z-(R,R)-IQNP (10 Ci/animal) and iodine-131-E(R,R)-IQNP (4 Ci/animal) were administered by intravenous injection (0.5 mL) into a lateral tail vein of Metophane-anesthetized rats (N ⫽ 5). The rats were euthanized by cervical fracture following Metophane anesthesia 3 h postinjection of the radioligand. The various organs were removed, rinsed with saline, blotted dry, and weighed in tared vials. Blood samples were obtained from the heart cavity after removal of the heart. Brains were dissected on ice into the regions of interest immediately upon removal. Tissue samples were counted in a Packard Minaxi 5000 sodium iodide gamma counter and the calculated injected dose/gram for iodine125 was corrected for iodine-131 spill over into the iodine-125 window. An analogous procedure was used for the injection of the iodine-125-Z-(R,R)-IQNP solution (10 Ci/animal), which had been stored in the freezer for 1 month before SSP purification. RESULTS AND DISCUSSION A dual-labeled E- and Z-(R,R)-IQNP solution, when co-injected into the same animal, demonstrates the potential for the determination of the concentration of cerebral m2 mAChR subtype in vivo (10). In addition, this dual-labeled “cocktail” demonstrates the potential for the evaluation of mAChR subtype selectivity of various drugs and ligands in vivo. To facilitate the preparation of the radioiodinated IQNP isomers, a simplified purification of this ligand
TABLE 1. Elution Profile of E- and Z-(R,R)-IQNP from a Silica Sep Pak Utilizing Increasing Concentrations of Methanol Iodine-131-E-(R,R)-IQNP Eluent CHCl3:CH3OH (v/v, mL) 1:0 (3) 99:1 (4) 95:5 (4) 90:10 (8)
Iodine-125-Z-(R,R)-IQNP
Percent of activity eluted
% IQNP (by RTLC)
Percent of activity eluted
% IQNP (by RTLC)
0.5 0.4 7.3 91.7
0 12 97 97
0.8 0.6 6.4 92.3
0 14 75 97
IQNP ⫽ 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1-iodo-1-propene-3-yl)-␣-phenylacetate; TLC ⫽ thin layer chromatography.
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FIG. 2. Comparison of the cerebral biodistribution of iodine-131-E-(R,R)IQNP via silica Sep Pak or high performance liquid chromatography (HPLC) purification at 3 h postinjection.
via SSP was investigated. E-(R,R)-IQNP labeled with sodium iodide-131 and Z-(R,R)-IQNP labeled with sodium iodide-125 were prepared as reported elsewhere (9). Upon elution from a CSP, the respective solutions were evaporated to dryness under a stream of nitrogen. Each radioactive isomer was dissolved in chloroform and
loaded onto individual SSPs. Each SSP was subsequently eluted with increasing concentration of methanol in chloroform (Table 1). Elution with chloroform:methanol solutions increasing from 0% to 5% methanol concentration by volume effectively removed a radioactive impurity (free iodide) from the crude reaction mixture.
FIG. 3. Comparison of the cerebral biodistribution of iodine-125-Z-(R,R)-IQNP via silica Sep Pak or high performance liquid chromatography (HPLC) purification at 3 h postinjection.
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FIG. 4. Comparison of whole body biodistribution of iodine-131-E-(R,R)-IQNP via silica Sep Pak or high performance liquid chromatography (HPLC) purification at 3 h postinjection.
Elution of the SSP with a chloroform:methanol solution (90:10) afforded greater than 90% recovery of the applied radioactivity as the various radiodinated IQNP isomers. Iodine-131-E-(R,R)-IQNP and iodine-125-Z-(R,R)-IQNP migrated with a cold standard for each isomer and were observed to be greater than 97% radiochemical pure by RTLC analysis. To evaluate if the radiolabeled ligand obtained via SSP purification was suitable for use in animal studies, an aliquot of each radioiodinated isomer obtained from purification via SSP was subjected to HPLC purification. A solution containing iodine131-E- and iodine-125-Z-(R,R)-IQNP, both purified either by an SSP or HPLC, was then administered to groups of rats (n ⫽ 5) and the cerebral distribution was determined (Figs. 2 and 3, respectively). It was observed that the cerebral biodistributions of the E or Z isomer were not significantly different (p ⬎ 0.05) when the isomers were purified either by SPP or HPLC. In addition, the biodistribution in selected tissues was similar (Figs. 4 and 5, respectively).
FIG. 5. Comparison of whole body biodistribution of iodine-125-Z-(R,R)-IQNP via silica Sep Pak or high performance liquid chromatography (HPLC) purification at 3 h postinjection. ND ⴝ not determined.
When labeled with iodine-125 (t1/2 ⫽ 60 days), IQNP is relatively stable in ethanol storage over extended periods (2 months) in a freezer with minimal decomposition (⬍10%) as determined by RTLC analysis. The use of an SSP was then investigated as a rapid method for the purification of radioiodinated IQNP stored under these conditions. From this study it was observed, after SSP purification, the biodistribution in various tissues of iodine-125-Z-(R,R)-IQNP that had been stored in a freezer for 1 month was analogous to that observed above (Fig. 5). CONCLUSION The utilization of a SSP was investigated as an alternative method for the rapid purification of the radioiodinated isomers of IQNP to be used in animal studies. Iodine-125-Z-(R,R)-IQNP and iodine131-E-(R,R)-IQNP were obtained in radiochemical yields of 75% and 80%, respectively, after SPP purification in approximately
A Rapid and Simple Sep Pak Purification of IQNP
1 hour. In vivo biodistribution studies in female rats using a solution containing iodine-131-E- and iodine-125-Z-(R,R)-IQNP demonstrated the ligands purified either by SSP or HPLC displayed similar cerebral and whole body biodistribution. The use of an SSP thus affords a simple and rapid method for the purification of radioiodinated IQNP affording the ligand with sufficient specific activity and radiochemical purity for use in various animal studies. Work was supported at the Oak Ridge National Laboratory (ORNL) by the United States Department of Energy, Office of Science under contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corporation. The authors also thank Dr. S. Kennel and Mr. A. Beets for assistance with the in vivo biodistribution studies.
References 1. Bergstrom K. A., Halldin C., Hiltunen J., Swahn C. G., Ito H., Ginovert N., Hall H., McPherson D. W., Knapp F. F. Jr., Larsson S., Schnell P. O. and Farde L. (1998) 125I- and 123I-labeled E-(⫺,⫺)IQNP: Potential radioligands for visualization of M1 acetylcholine receptors in brain. Determination of metabolites using HPLC. Nucl. Med. Biol. 25, 209 –214. 2. Bergstrom K. A., Halldin C., Savonen A., Okubo Y., Hiltunen J., Swahn C. G., McPherson D., Knapp F. F. Jr., Larsson S., Schnell P. O. and Farde L. (1998) Iodine-123 labelled Z-(R,R)-IQNP: A potential radioligand for visualization of m2 muscarinic acetylcholine receptors in brain with SPECT. Eur. J. Nucl. Med. 25, 843. 3. Buckley N. J., Bonner T. I., Buckley C. M. and Brann M. R. (1989) Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol. Pharmacol. 35, 469 – 476. 4. Flynn D. D., Ferrari-DiLeo G., Mash D. C. and Levey A. I. (1995) Differential regulation of molecular subtypes of muscarinic receptors in Alzheimer’s disease. J. Neurochem. 64, 1888 –1891. 5. Jenni-Eiermann S., Von Hahn H. P., Honegger G. C. and Ulrich J.
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6. 7. 8.
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10. 11. 12.
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(1984) Studies on neurotransmitter binding in senile dementia. Gerontology 30, 350 –358. Kung M.-I., Liu B.-L., Yang Y.-Y., Billings J. J. and Kung H. F. (1991) A kit formulation for preparation of iodine-123-IBZM: A new CNS D-2 dopamine receptor imaging agent. J. Nucl. Med. 32, 339 –342. Levey A. I. (1996) Muscarinic acetylcholine receptor expression in memory circuits: Implications for treatment of Alzheimer disease. Proc. Natl. Acad. Sci. USA 93, 13541–13546. McPherson D. W. and Knapp F. F. Jr. (1996) Structure elucidation via stereoselective synthesis of the acetate center of labeled 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1-iodo-1-propen-3-yl)-␣phenylacetate (IQNP). A high affinity muscarinic imaging agent for SPECT. J. Org. Chem. 61, 8335– 8337. McPherson D. W., Lambert C. R., Jahn K., Sood V., McRee R. C., Zeeberg B. R., Reba R. C. and Knapp F. F. Jr. (1995) Resolution, in vitro and initial in vivo evaluation of isomers of iodine-125 labeled 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1-iodo-1-propen-3-yl)-␣phenylacetate (IQNP). J. Med. Chem. 38, 3908 –3917. McPherson D. W., Luo H., Beets A. L. and Knapp F. F. Jr. (1998) A novel in vivo method for the determination of muscarinic ligand subtype selectivity. Eur. J. Nucl. Med. 25, 843. Nordberg A., Alaguzoff I. and Winblad B. (1992) Nicotinic and muscarinic subtypes in the human brain: Changes with aging and dementia. J. Neurosci. Res. 31, 103–111. Rayeq M. R., Boulay S. F., Sood V. K., McPherson D. W., Knapp F. F. Jr., Zeeberg B. R. and Reba R. C. (1996) In vivo autoradiographic evaluation of isomers of 1-125-labeled 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1-iodo-1-propen-3-yl)-␣-phenylacetate (IQNP). A high affinity ligand for the muscarinic receptor. Recept. Signal Transduct. 6, 13–34. Wang T. S. T., Malaspina D. and Van Heertum R. L. (1998) A simple method of preparation for [I-123]-(S)-(⫺)-IBZM. Appl. Radiat. Isotopes 49, 369 –372. Zeeberg B. R., Boulay S. F., Sood V. K., Rayeq M. R., Danesh R. A., McPherson D. W. and Knapp F. F. Jr. (1997) In vivo autoradiographic competition studies of isomers of [iodine-125]IQNP against QNB demonstrating in vivo M2 muscarinic subtype selectivity for QNB. Recept. Signal Transduct. 7, 45–54.
ISSN 0969-8051/99/$–see front matter PII S0969-8051(99)00050-5
TECHNICAL NOTE
A Rapid and Simple Sep Pak Method for Purification of Radioiodinated IQNP, a High Affinity Ligand for the Muscarinic Receptor D. W. McPherson and F. F. (Russ) Knapp, Jr. NUCLEAR MEDICINE GROUP, LIFE SCIENCES DIVISION, OAK RIDGE NATIONAL LABORATORY (ORNL), OAK RIDGE, TENNESSEE, USA
ABSTRACT. A simplified procedure for the purification of 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1iodo-1-propen-3-yl)-␣-phenylacetate (IQNP) stereoisomers utilizing a silica Sep Pak (SSP) is described. Iodine-131-E- and iodine-125-Z-(R,R)-IQNP were isolated after SSP purification in 80% and 75% radiochemical yields, respectively. The biodistribution of iodine-131-E-/iodine-125-Z-(R,R)-IQNP, purified either by SSP or high performance liquid chromatography (HPLC), was evaluated in female rats and demonstrated no significant differences in the uptake in various organs and cerebral regions. The utilization of SSP thus affords a simple and rapid method for the purification of IQNP for use in a variety of animal studies. NUCL MED BIOL 26;7:859 – 863, 1999. © 1999 Elsevier Science Inc. All rights reserved. KEY WORDS. IQNP, Sep Pak purification, Muscarinic receptor
INTRODUCTION The muscarinic acetylcholinergic receptor complex (mAChR) has been observed to exhibit five distinct subtypes (m1–m5) by cloning methodology (3). Changes in the density or function of the various mAChR subtypes have been postulated to play an important role in various dementias such as Alzheimer’s and Huntington’s diseases (4, 5, 7, 11). The expected clinical importance has spurred the development of new mAChR subtype selective drugs and ligands for the potential treatment and diagnosis of Alzheimer’s disease. One of these new ligands, 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1-iodo1-propen-3-yl)-␣-phenylacetate (IQNP, Fig. 1) demonstrates high in vitro and in vivo binding affinity (1, 2, 9, 12, 14). The E-(R,R)isomer of IQNP demonstrates high binding to m1 subtype after the modest binding to the m2 subtype washes out and the Z-(R,R)isomer displays non-subtype in vivo selective binding in both rats and baboon studies. In addition, it was observed that utilization of a dual-labeled “E/Z-(R,R)-IQNP cocktail” allowed for the in vivo cerebral quantification of the m2 mAChR subtype in female Fischer rats (10). Use of this technique is being evaluated for the determination of the mAChR subtype selectivity for new and established ligands and drugs in vivo. Our method for the radioiodination of IQNP involves the no-carrier-added iodination of a tributylstannyl intermediate with a 3% hydrogen peroxide solution as the oxidizing agent. Following high performance liquid chromatography (HPLC) purification, the radioiodinated IQNP isomers are obtained with high specific activity in a total reaction time of 3 h. To expedite the preparation of IQNP for use in animal experiments, a simplified procedure of the final purification of IQNP was investigated. The use of a C-18 Sep Pak (CSP) for the purification of radioiodinated IBZM, a high
affinity dopamine D2 ligand, has been reported recently (6, 13). We now report the development of an improved purification procedure of IQNP utilizing a silica Sep Pak (SSP) to afford the radioiodinated IQNP isomers with sufficient specific activity for use in various animal studies. MATERIALS AND METHODS
General E- and Z-(R)-1-azabicyclo[2.2.2]oct-3-yl (R)-␣-hydroxy-␣-phenyl␣-(1-tributylstanyl-1-propen-3-yl)acetate (TBTQNP) were prepared as described previously (8, 9). All reagents were of reagent grade and used without further purification. Sodium iodine-125 (specific activity 17 Ci/mg) and sodium iodine-131 (specific activity 9 Ci/mg) were purchased from New England Nuclear Life Sciences Division (Roxbury, MA USA). CSP and SSP cartridges were purchased from Waters Corporation (Cary, NC USA). HPLC was performed with a Waters model 510 HPLC pump and model 454 variable ultraviolet detector (254 nm) (Cary, NC USA) utilizing a Beckman Model 170 radioisotope flow detector (Fullerton, CA USA). The HPLC conditions for the purification of the stereoisomers of IQNP utilized a mobile phase of methylene chloride: (ethanol ⫹ 1% triethylamine) [98:2], flow rate of 2.5 mL/min, and a Waters semipreparative Porasil column (3.9 mm ⫻ 30 cm). Radiochemical thin layer chromatographic (RTLC) analysis was performed using Analtech glass-coated silica GF plates (250 m) (Newark, DE USA) and a mobile phase of chloroform:methanol (8.5:1.5, Rf ⫽ 0.6). The plates were analyzed using a Bioscan System 200 Imaging Scanner with an Autochanger 1000 (Washington, DC USA).
Preparation of Iodine-125 Z-(R,R)-IQNP Address correspondence to: D. W. McPherson, Ph.D., Oak Ridge National Laboratory, Post Office Box 2008, Building 4501, Oak Ridge, TN 378316229, USA; e-mail [email protected] Received 1 May 1999. Accepted 5 July 1999.
An ethanolic solution of Z-(R,R)-TBTQNP (1 mg/mL, 100 L), 0.1 N HCl (200 L), and an ethanolic sodium iodine-125 solution (7.8 mCi in 100 L of 0.1 M NaOH and 100 L of ethanol) were added to a 3-mL vial. A 3% hydrogen peroxide solution (50 L) was
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Preparation of Iodine-131 E-(R,R)-IQNP E-(R,R)-TBTQNP was prepared as described above with 4.1 mCi sodium iodine-131 to afford 3.3 mCi iodine-131-E-(R,R)-IQNP (80%). An aliquot of the radioligand was subjected to HPLC purification as described above to afford iodine-131-E-(R,R)-IQNP with specific activity greater than 1,000 mCi/mol. The radioligand, purified by SSP and HPLC, was stored in ethanol in a freezer until used.
Biodistribution Studies
FIG. 1. Structure of 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣(1-iodo-1-propen-3-yl)-␣-phenylacetate (IQNP), 1.
added, the vial sealed, and the solution stirred at room temperature for 30 min. A 10% sodium bisulfite solution (500 L) was added, the solution stirred 1 min, and a saturated sodium bicarbonate solution (1.0 mL) added slowly. The resultant solution was transferred to a syringe attached to a CSP. The reaction vial was rinsed with acetonitrile (100 L) and water (1.0 mL) and these rinses were added to the syringe. The volume in the syringe was brought up to 10 mL with water and the solution passed through the CSP. The CSP was washed with water (2 ⫻ 4 mL) and acetonitrile (0.25 mL). The crude radioiodinated product was removed from the CSP with acetonitrile (2 mL) followed by chloroform (5 mL). The crude radioiodinated ligand solution was evaporated to dryness under a stream of nitrogen, the vial rinsed with chloroform (3 ⫻ 1 mL), and these washes transferred to a syringe containing an SSP. The SSP was washed sequentially with chloroform:methanol (v/v) solutions (99:1, 4 mL; 95:5, 4 mL; and 90:10, 8 mL) and each wash was collected separately. The chloroform:methanol (90:10) fraction was evaporated to dryness under a stream of nitrogen to afford 5.9 mCi of iodine-125 Z-(R,R)-IQNP (75% yield) with a radiochemical purity greater than 97% as determined by RTLC analysis. An aliquot of the radioligand was subjected to HPLC purification as described previously to afford iodine-125-Z-(R,R)-IQNP with specific activity greater than 1,000 mCi/mol. The radioligand, purified by SSP and HPLC, was stored in ethanol in a freezer until used. Following the initial in vivo animal experiments, the remaining iodine-125-Z-(R,R)-IQNP ethanol solution was stored in a freezer for 1 month. This solution was evaporated to dryness, dissolved in chloroform (3 mL), and subjected to SSP purification as described above. Iodine-125-Z-(R,R)-IQNP was obtained in greater than 97% radiochemical purity as determined by RTLC analysis.
Biodistribution studies were performed using female VAF Fischer rats (⬃150 g). The animal care and use procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and were reviewed and approved by the Oak Ridge National Laboratory Animal Care and Use Committee. The animals were allowed food and water ad libitum before and during the course of the experiments. Ethanolic solutions of iodine-125-Z-(R,R)-IQNP (100 L) and iodine-131-E(R,R)-IQNP (100 L), both of which were purified either by SSP or HPLC, were combined and acidified by the addition of a 0.1-N HCl solution (100 L). The resultant solution was diluted with saline (10 mL) and filtered (Millipore) into a sterile injection vial. Iodine-125-Z-(R,R)-IQNP (10 Ci/animal) and iodine-131-E(R,R)-IQNP (4 Ci/animal) were administered by intravenous injection (0.5 mL) into a lateral tail vein of Metophane-anesthetized rats (N ⫽ 5). The rats were euthanized by cervical fracture following Metophane anesthesia 3 h postinjection of the radioligand. The various organs were removed, rinsed with saline, blotted dry, and weighed in tared vials. Blood samples were obtained from the heart cavity after removal of the heart. Brains were dissected on ice into the regions of interest immediately upon removal. Tissue samples were counted in a Packard Minaxi 5000 sodium iodide gamma counter and the calculated injected dose/gram for iodine125 was corrected for iodine-131 spill over into the iodine-125 window. An analogous procedure was used for the injection of the iodine-125-Z-(R,R)-IQNP solution (10 Ci/animal), which had been stored in the freezer for 1 month before SSP purification. RESULTS AND DISCUSSION A dual-labeled E- and Z-(R,R)-IQNP solution, when co-injected into the same animal, demonstrates the potential for the determination of the concentration of cerebral m2 mAChR subtype in vivo (10). In addition, this dual-labeled “cocktail” demonstrates the potential for the evaluation of mAChR subtype selectivity of various drugs and ligands in vivo. To facilitate the preparation of the radioiodinated IQNP isomers, a simplified purification of this ligand
TABLE 1. Elution Profile of E- and Z-(R,R)-IQNP from a Silica Sep Pak Utilizing Increasing Concentrations of Methanol Iodine-131-E-(R,R)-IQNP Eluent CHCl3:CH3OH (v/v, mL) 1:0 (3) 99:1 (4) 95:5 (4) 90:10 (8)
Iodine-125-Z-(R,R)-IQNP
Percent of activity eluted
% IQNP (by RTLC)
Percent of activity eluted
% IQNP (by RTLC)
0.5 0.4 7.3 91.7
0 12 97 97
0.8 0.6 6.4 92.3
0 14 75 97
IQNP ⫽ 1-azabicyclo[2.2.2]oct-3-yl ␣-hydroxy-␣-(1-iodo-1-propene-3-yl)-␣-phenylacetate; TLC ⫽ thin layer chromatography.
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FIG. 2. Comparison of the cerebral biodistribution of iodine-131-E-(R,R)IQNP via silica Sep Pak or high performance liquid chromatography (HPLC) purification at 3 h postinjection.
via SSP was investigated. E-(R,R)-IQNP labeled with sodium iodide-131 and Z-(R,R)-IQNP labeled with sodium iodide-125 were prepared as reported elsewhere (9). Upon elution from a CSP, the respective solutions were evaporated to dryness under a stream of nitrogen. Each radioactive isomer was dissolved in chloroform and
loaded onto individual SSPs. Each SSP was subsequently eluted with increasing concentration of methanol in chloroform (Table 1). Elution with chloroform:methanol solutions increasing from 0% to 5% methanol concentration by volume effectively removed a radioactive impurity (free iodide) from the crude reaction mixture.
FIG. 3. Comparison of the cerebral biodistribution of iodine-125-Z-(R,R)-IQNP via silica Sep Pak or high performance liquid chromatography (HPLC) purification at 3 h postinjection.
D. W. McPherson and F. F. Knapp, Jr.
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FIG. 4. Comparison of whole body biodistribution of iodine-131-E-(R,R)-IQNP via silica Sep Pak or high performance liquid chromatography (HPLC) purification at 3 h postinjection.
Elution of the SSP with a chloroform:methanol solution (90:10) afforded greater than 90% recovery of the applied radioactivity as the various radiodinated IQNP isomers. Iodine-131-E-(R,R)-IQNP and iodine-125-Z-(R,R)-IQNP migrated with a cold standard for each isomer and were observed to be greater than 97% radiochemical pure by RTLC analysis. To evaluate if the radiolabeled ligand obtained via SSP purification was suitable for use in animal studies, an aliquot of each radioiodinated isomer obtained from purification via SSP was subjected to HPLC purification. A solution containing iodine131-E- and iodine-125-Z-(R,R)-IQNP, both purified either by an SSP or HPLC, was then administered to groups of rats (n ⫽ 5) and the cerebral distribution was determined (Figs. 2 and 3, respectively). It was observed that the cerebral biodistributions of the E or Z isomer were not significantly different (p ⬎ 0.05) when the isomers were purified either by SPP or HPLC. In addition, the biodistribution in selected tissues was similar (Figs. 4 and 5, respectively).
FIG. 5. Comparison of whole body biodistribution of iodine-125-Z-(R,R)-IQNP via silica Sep Pak or high performance liquid chromatography (HPLC) purification at 3 h postinjection. ND ⴝ not determined.
When labeled with iodine-125 (t1/2 ⫽ 60 days), IQNP is relatively stable in ethanol storage over extended periods (2 months) in a freezer with minimal decomposition (⬍10%) as determined by RTLC analysis. The use of an SSP was then investigated as a rapid method for the purification of radioiodinated IQNP stored under these conditions. From this study it was observed, after SSP purification, the biodistribution in various tissues of iodine-125-Z-(R,R)-IQNP that had been stored in a freezer for 1 month was analogous to that observed above (Fig. 5). CONCLUSION The utilization of a SSP was investigated as an alternative method for the rapid purification of the radioiodinated isomers of IQNP to be used in animal studies. Iodine-125-Z-(R,R)-IQNP and iodine131-E-(R,R)-IQNP were obtained in radiochemical yields of 75% and 80%, respectively, after SPP purification in approximately
A Rapid and Simple Sep Pak Purification of IQNP
1 hour. In vivo biodistribution studies in female rats using a solution containing iodine-131-E- and iodine-125-Z-(R,R)-IQNP demonstrated the ligands purified either by SSP or HPLC displayed similar cerebral and whole body biodistribution. The use of an SSP thus affords a simple and rapid method for the purification of radioiodinated IQNP affording the ligand with sufficient specific activity and radiochemical purity for use in various animal studies. Work was supported at the Oak Ridge National Laboratory (ORNL) by the United States Department of Energy, Office of Science under contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corporation. The authors also thank Dr. S. Kennel and Mr. A. Beets for assistance with the in vivo biodistribution studies.
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