A simple method of preparation for [123I]-(S)-(−)-IBZM

Appl. Radiat. Isot. Vol. 49, No. 4, pp. 369-372, 1998 © 1998 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain P I I : S0969-8043(97)00290-X 0969-8043/98 $19.00 + 0.00

Pergamon

A Simple Method of Preparation for ['23I]-(S)-(-)-IBZM THEODORE

S. T. W A N G *~, D O L O R E S M A L A S P I N A 2 and R O N A L D L. V A N H E E R T U M '

~Division of Nuclear Medicine, Department of Radiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A. and 2Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A. (Received 22 April 1997; accepted 7 July 1997)

Sterile, apyrogenic [~23I]IBZMwas prepared in a sealed, capped 'V' vial, followed by SEP-PAK C-18 cartridge purification, and then was placed under sealed vial condensation. The quantity of BZM present in the final product ranged from 3.3-5.9 ~tg,as measured by a UV spectrophotometer at 254 and 308 nm, Animal biodistribution studies revealed that the [~3I]IBZM prepared by this method which contained 5.9 ~tg of BZM, compared to the standard preparation method containing 0 Bg of BZM, resulted in identical brain uptakes at 15, 30, 60, and 120 min post-injection. The in vitro and in vivo studies demonstrated that a small amount of BZM presence in the final product did not affect the radiochemical purity, nor the D~ receptor binding capacity in the rat brain of [~23I]IBZM.The preparation time can be shortened to 1.5 h compared with at least 2-4 h needed for the standard method of preparation. This factor may be important in routine clinical application. © 1998 Elsevier Science Ltd. All rights reserved

Introduction It has been reported that (S)-(-)-3-iodo-2-hydroxy6-methoxy-N-[(1-ethyl)-2-pyrrolidinyl)methyl]benzamide (IBZM) possesses a high binding affinity to the brain dopamine D2 receptor (Kd = 0.43 nM) both in vitro and in vivo (de Paulis et al., 1985). It has also been reported that radioactive iodine ('25I) labeled IBZM has been studied for D2 receptor imaging in animals (Kung et al., 1988a). In recent years, [~23I]IBZM has been investigated in humans for D2 receptor imaging for Huntington's disease (Ichise et al., 1993), Parkinson's disease (Brucke et al., 1990; Knable et al., 1995), and Schizophrenic patients (Vallabajosula et al., 1997). The preparation of [t23I]-(S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(l-ethyl-2-pyrrolidyl)methyl]benzamide ([':aI]IBZM) is generally initiated by an oxidative electrophillic radioiodination in an acidic condition of (S)-(-)-2-hydroxy-6-methoxyN-[(1-ethyl)-2-pyrrolidinyl)methyl]bezamide (BZM; the precursor) with ['23I]NaI, followed by organic solvent extraction, evaporation, HPLC purification and in vacuo condensation with a radiolabeling yield of 30-50°,/0 (Kung et al., 1988b; Seibyl and Woods, 1992; Knable et al., 1995), At least 2--4 h are needed for the preparation of a sterile and apyrogenic *To whom all correspondence should be addressed. 1"Supplied by Nrodion, Canada. ARI 49/4~-D

product. Taking all of these stringent requirements into consideration, we have developed a simplified procedure in order to save time in preparation of the sterile-apyrogenic compound, which may be important in its routine clinical application.

Materials and Methods All reagents were purchased from commercial suppliers and used a s received, unless otherwise specified. UV spectra were recorded on a Beckman DU-7 UV-Vis spectrophotometer. Radioactivity was measured in a PACE-1 Gamma scintillation counter, Picker International. All glassware was depyrogenated in an oven for 5-8 h at 185 + 2°C before use. All aqueous reagents (except peracetic acid and NaHCO3 solution), were prepared with 'sterile water for irrigation, USP', divided into a few sterile-apyrogenic vials (25 ml), seal and capped, and steam sterilized in an autoclave, for 20-30 min under 181b/psi, at a temperature of 125°C. Radiosynthesis was performed in a fume hood, with a charcoal filter, and a vertical flow Laminar flow hood. The guidelines of the Good Manufacturing Practices, The Procedures of Pyrogenicity and Sterility Tests (USP, 23rd., ) were closely complied with. A septum-sealed 'V' vial (4ml) containing 10-40 mCi of [u3I]NaI in 0.1 M NaOH (45-750 p.l), supplied by the manufacturert, was used as an

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original reaction vial. The following reagents were added by injection into this vial through a 1 ml syringe: BZM (20 lag/80 ~tl, ethanol, USP), 0.1 M NH4OAc-HOAC (100 ~tl, pH 4.0), and peracetic acid (100 lal, 0.64 wt%, prepared freshly every preparation). The syringes were changed, but the same needle was used throughout. The reaction mixture was kept in the seal-capped vial at room temperature for 10-15 min, then, NaHSO3 (120 ixl, 300 mg/ml), and NaHCO3* (0.5-1.5 ml, 7.5%, pH 7.8) were injected into the vial until effervesce ceased. (Total volume: 1.2-3.0 ml). The reaction mixture was then drawn into a 3 ml syringe and loaded on a pre-washed (ethanol, 3 ml; H20, 3 ml) SEP-PAK-C18 Cartridge (Long body, classic)l', and eluted slowly with H20 (3 ml); the eluent was collected (vial 1). Then, the column was eluted slowly with ethanol (3 ml), and the eluent was collected (vial 2). The radioactivities of vial 1 and 2 were measured in a dose calibrator. The ethanolic solution vial (2) was sealed, capped, and attached to a sterile charcoal trap. The solvent was evaporated at 55-60°C from the sealed vial, under a stream of Ar gas until totally dried. The residue was dissolved by injection of 300 lal of ethanol followed by 10 ml of normal saline. The product, [t23I]IBZM, was filtered through a 0.22 lam filter. The radioactivity of the final product was assayed in a dose calibrator.

Determination of Radiochemical Purity of ImlIIBZM The radiochemical purity of []23I]IBZM was determined by a thin layer chromatography (TLC) method, as directed under the Thin-layer Chromatography (USP, 23rd., {261}). Adequate volumes of the sample were spotted on a TLC plate of silica gel (60 F-254, Merck). The plate was developed with a mixture of CHCI3:C2HsOH:NH4OH (9:1:0.2; v/v/v) to a distance of about 10 cm, air dried. We recorded the radiochromatogram, and measured the Rf value (0.7-0.8) of the radioactivity peak of [t23I]IBZM, and the Rf value (0.00) of the radioactivity peak of [~23I]NaI (Kung and Kung, 1990).

known amounts of BZM (0-25 ~tg/ml) with a blank (known amount of IBZM in 300 ul of ethanol and 10 ml of normal saline solution. From the BZM standard cure, then the quantities of BZM present in the samples were calculated.

Pyrogenicity Test [Limulus Ameobocytes Lysate (LAL) Test] For each U23I]IBZM preparation seven test samples were prepared for testing as follows: Four volume dilution of [~2q]IBZM samples, and one each of negative, positive, and internal positive samples were prepared. 0.2 ml of solution was added into each LAL tube:~. The tubes were then incubated at 37°C for 60 min. The E. Coli endotoxin sample showed gel formation (positive control and internal positive), and the water sample showed no gel formation (negative control); the unknown samples were then considered positive or negative depending on whether or not they formed gel. The mean end point of Endotoxin titer was determined (USP, 23rd.,

{85}). Sterility Tests The sterility tests were performed according to the guidelines from USP, 23rd., {71} and are briefly described as follows: We aseptically transferred a negative control (Sterile Water for injection, USP), a positive control (saliva), and a small sample ([~23I]IBZM), (100 lal each) to a tube of culture mediumfl, using a sterile syringe needle, the liquid was mixed with the medium, but not aerated excessively. The text mixture was incubated for 14 days at 30 to 35°C (for Thioglycollate) and at 20 to 25°C (for Triptic Soy Broth). At prescribed intervals during and at the conclusion of the incubation period, contents of all of the tubes were examined for evidence of microbial growth, such as the development of turbidity and/or surface growth (positive control). If no growth was observed in both the negative control and the test sample, the sample tested met the requirements for sterility.

Determination of BZM Concentration The amount of BZM present in the final product, [~2q]IBZM, was determined by measurement of UV absorbance (A), on a spectrophotometer, and calculated from the BZM standard curve at 254 ( e = 8 . 3 7 x 106) and 308nm (e=4.03 x 10'), the characteristic wavelengths for BZM. The standard curve was constructed by measuring a series of *Supplied by Sigma, St. Louis, MO. tSupplied by Waters, Milford, MA. :~Supplied by Endosafe, Charleston, SC. IlSupplied by Remel, Lenexa, KA.

Biodistribution in Animals [~23I]IBZM (100 gCi), containing 5.9 lag of BZM, and [~23I]IBZM-C (120 I~Ci), containing 0 Ilg of BZM, were injected into the tail veins of male rats (Sprague-Dawley), for a comparative studies in vivo. The animals were sacrificed at 15, 30, 60, and 120 min post-injection. Tissues were removed, weighed, and the radioactivity was determined in a gamma scintillation counter. Statistical analyses were performed using an unpaired Student's test. Results are presented as mean + SD.

A simple method of preparation for [~23I-(S)-(-)-IBZM

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Table 1. The radiolabelingyield, radiocbemicalpurity, pyrogenicity,sterility and BZM concentrationin various [ml]IBZMpreparations(n = 3) (mean value) [mI]NaI/mCi 10 20 30 40 Labeling yield 51% 56% 39% 33% (mCi) (5.13) (11.26) (11.77) (13.20) Radiochemical purity >95% >95% >95% >95% BZM/lag 5.7 5.9 4.4 3.7 Endotoxin titer EU/ml < 0.03 < 0.03 < 0.03 < 0.03 Sterility . . . . - Negative

Results

The results revealed that the radiolabeling yield of [~2JI]IBZM, prepared by the simplified method, was slightly higher (39-56%) than the [~23I]IBZM-C, prepared by the standard method (33-50%). The radiochemicai purities of [~23I]IBZM and [~23I]IBZMC were identical ( > 9 6 % ) . The a m o u n t of BZM presence in the product calculated from the BZM standard curve was 3.3-5.9 lag. The various amounts of BZM presence in the product, pyrogenicity, and sterility tests results are summarized in Table 1. The animal biodistribution studies showed that the brain concentration of [~23I]IBZM and [~23I]IBZM-C at 15, 30, 60 and 120 min of post-injection were almost identical (Table 2), and similar to other results cited in the literature (Kung et al., 1988a). Discussion

Using the methodology, described in the literature, [~23I]IBZM can readily synthesized. The standard procedure begins with 50 lag of BZM. Following the oxidative radioiodination reaction, the product, unreacted BZM, and [~23I]NaI were separated by extraction with organic solvent, followed by HPLC purification. The major disadvantages of this method are (1) adequate time is needed for the preparation (2-4 h), (2) at least two steps of HPLC purification are necessary and (3), multiple transfers could increase contamination as well as and exposure of the volatile radioactive iodine to the chemists. These complicated separation processes may prevent the widespread application of this agent in routine clinical nuclear medicine application.

One major difference between the standard and the simplified method is the presence of a small a m o u n t of BZM in our method of preparation. However, the radiolabeling yields (Table 1), and the in vivo results (Table 2), demonstrated that the small a m o u n t of BZM present (5.9 Bg) in the product would not affect the radiochemical yield or purity, nor would it affect the affinity of the brain D2 receptor binding of [~23I]IBZM. Since the potency of competitive inhibition constants between IBZM (K~ = 0.63 nM) and BZM (K~ = 31.1 nM) is 50 to 1 (Kung et al., 1988b), theoretically, the BZM should not be capable of competing with the IBZM at the D2 receptor binding site. This simplified method of preparation, however, needs only 1.5 h to complete the preparation. A kit formulation for the preparation of [~23I]IBZM has been previously reported (Kung et al., 1991), by reacting 4 lag of BZM with unspecified amounts (presumably tracer quantity) of [~23I] or [~25I], and by using a Accubound-C4 column to separate the unreacted iodide. By omitting the performance of pyrogenicity and sterility tests, the entire radiochemical synthesis could be achieved in 20 min. The aim of our study however, was prepare doses of [~23I]IBZM suitable for clinic use. We started with large quantities of [~2q]NaI (10-40 mCi) in each preparation, and under sterile and apyrogenic processes, obtained a radiopharmaceutical grade product, which is in compliance with our current I N D clinical protocol guidelines. The difference between these two methods is primarily in timing; the pyrogenicity (LAL) and sterility tests, which were performed in this study required at least 1 h of additional time.

Table 2. Biodistributionof [ml]IBZM-Cand [~23I]IBZMin ratsa PO inj. 15 rain 30 rain 1h 2h Compound C S C S C S C S Blood 2.39 2,22 1.50 1.47 1.30 1.18 1.03 1.06 Heart 0.34 0,37 0.27 0.20 0.15 0.11 0.14 0.08 Lungs 4.15 5.14 3.93 4.16 2.67 2.83 0.90 0.94 Spleen 0.55 0A9 0.38 0.41 0.24 0.16 0.22 0.19 Kidneys 1.82 2.03 1.48 1.69 1.07 1.21 0.56 0.88 Liver 4.93 5.16 3.26 3.77 2.28 2.14 0.56 1.05 Thyroid 0.04 0.07 0.06 0.07 0.07 0.09 0.07 0.09 Eyes 0.04 0.06 0.06 0.07 0.06 0.08 0.07 0.07 Brain 2.59 2.52 1.97 2.06 1.01 0.83 0.33 0.36 Muscle 0.09 0.13 0.33 0.35 0.23 0.30 0.12 0.26 "%Dose/organ (mean value) (n = 3).

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Conclusion By using this simplified method, (1) a sterile, apyrogenic [n3I]IBZM can be successfully prepared without several steps of organic extraction, evaporation, HPLC purification, and in vacuo condensation, (2) the preparation was mainly carried out in a single sealed and capped vial, thus reducing the potential contamination of volatile radioactive iodine, and (3) the preparation time can be shortened to 1.5 h, which may be important in routine clinical application of this compound. Acknowledgement--This work is supported by a grant from the Mathers Charitable Trust Foundation.

References Ichise, M. I., Toyama, H. and Fornazzari, L. et al. (1993) Iodine-123-IBZM dopamine D-2 receptor and Tc-99mHMPAO brain perfusion SPECT in the evaluation of patients with and subjects at risk for Huntington's diseases. J. Nucl. Med. 34, 1274. Knable, M. B., Jones, D. W., Coppola, J. R., et al. (1995) Lateralized differences in I-123-IBZM uptake in the basal ganglia in asymmetric Parkinson's disease. J. Nucl. Med. 36, 1216. Kung, M. P. and Kung, H. F. (1990) Peracetic acid as a superior oxidant for preparation of [~23I]IBZM: A

potential dopamine D-2 receptor imaging. J. Label. Compds. Radiopharm. XXVII, 691. Kung, H. K., Billings, J. J., Guo, Y. Z. and Mach, R. H. (1988a) Comparison of in vivo D-2 dopamine receptor binding of IBZM and NMPS in rat brain. Nucl. Med. Biol. 15, 203. Kung, H. F., Kasliwal, R., Pan, S. et al. (1988b) Dopamine D-2 receptor imaging radiopharmaceuticals: Synthesis, radiolabeling, and in vitro binding of (R)-(+) and (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2pyrolidinyl)methyl]benzamide. J. Med. Chem. 31, 1039. Kung, M. P., Liu, B.-L., Yang, Y. Y., et al. (1991) A kit formulation for preparation of Iodine-123-IBZM: A new CNS D-2 dopamine receptor imaging agent. J. Nucl. Med. 32, 339. de Paulis, T., Kumer, Y. and Johansson, L. et al. (1985) Potential neuroleptic agents. Chemistry and antidopaminergic properties of substituted 6-methoxy-salicylamides. J. Med. Chem. 28, 1263. Seibyl, J. P., Woods, S. W., Zoghbi, S. S., et al. (1992) Dynamic SPECT imaging of dopamine D2 receptors in human subjects with iodine-123-IBZM. J. Nucl. Med. 33, 1964. Vallabajosula, S., Hirschowitz, J. and Machac, J. (1997) Effect of Haloperidol dose on I-123-IBZM brain SPECT imaging in schizophrenic patients. J. Nucl. Med. 38, 203. The United States Pharmacopeia (USP 23rd), The National Formulary (NF 18th) (1995) Sterilization and Sterility Assurance of Compendial Articles), Good Manufacturing Practices, United States Pharmacopeial Convention, {71}, {85}, {1077}, {1211}. 1907-1977, 1976-1980.