554
`
`Bioconjugate Chem. 2001, 12, 554−558
`
`Stabilization of 90Y-Labeled DOTA-Biomolecule Conjugates Using
`Gentisic Acid and Ascorbic Acid
`Shuang Liu* and D. Scott Edwards
`
`Medical Imaging Division, DuPont Pharmaceuticals Company, 331 Treble Cove Road,
`North Billerica, Massachusetts 01862. Received November 30, 2000; Revised Manuscript Received April 2, 2001
`
`Radiolytic degradation of radiolabeled compounds is a major challenge for the development of new
`therapeutic radiopharmaceuticals. The goal of this study is to explore the factors influencing the
`solution stability of a 90Y-labeled DOTA-peptide conjugate (RP697), including the amount of total
`activity, the activity concentration, the stabilizer concentration, and the storage temperature. In
`general, the rate of radiolytic decomposition of RP697 is much slower at the lower activity concentration
`(<4 mCi/mL) than that at the higher concentration (>10 mCi/mL). RP697 remains relatively stable
`at the 20 mCi level and room temperature while it decomposes rapidly at the 100 mCi level under the
`same storage conditions. Radical scavengers, such as gentisic acid (GA) and ascorbic acid (AA), were
`used in combination with the low temperature (-78 °C) to prevent the radiolytic decomposition of
`RP697. It was found that RP697 remains stable for at least 2 half-lives of 90Y when GA or AA (10 mg
`for 20 mCi of 90Y) is used as a stabilizer when the radiopharmaceutical composition is stored at -78
`°C. The stabilizer (GA and AA) can be added into the formulation either before or after radiolabeling.
`The post-labeling approach is particularly useful when the use of a large amount of the stabilizer
`interferes with the radiolabeling. The radiopharmaceutical composition developed in this study can
`also apply to other 90Y-labeled DOTA-biomolecule conjugates. The amount of the stabilizer used in
`the radiopharmaceutical composition and storage temperature should be adjusted according to the
`sensitivity of the radiolabeled DOTA-biomolecule conjugate toward radiolytic decomposition.
`
`INTRODUCTION
`
`We have been interested in the development of new
`diagnostic and therapeutic radiopharmaceuticals based
`on small-molecule receptor ligands (1-18). In our previ-
`ous contribution (19), we reported the synthesis of
`a DOTA-conjugated vitronectin receptor antagonist
`(SU015: 2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-
`1-cyclododecyl)-acetyl-Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-
`cyclo{Lys-Arg-Gly-Asp-D-Phe}) and its 90Y complex (RP697
`in Figure 1). Through a series of radiolabeling experi-
`ments, we developed a formulation for routine prepara-
`tions of RP697 in high yield with the radiochemical purity
`(RCP) of > 95%. However, RP697 prepared using this
`formulation was not stable over time at room tempera-
`ture, particularly at high activity levels.
`90Y is an (cid:2)-emitter with a 2.27 MeV (cid:2)-particle and a
`2.67 day half-life. Due to the high energy of the (cid:2)-particle,
`the radiolabeled cyclic peptide is very susceptible to
`radiolytic decomposition. Since the tumor uptake of
`RP697 is largely dependent on the receptor binding of
`the two cyclic RGD-containing peptide motifs, radiolytic
`decomposition may lead to the decreased therapeutic
`efficacy and unwanted radiation toxicity to other normal
`organs such as liver and bone marrow. The mechanism
`for radiolytic decomposition is thought to be mediated
`by the formation of free radicals, such as superoxide (•O2)
`and hydroxyl radicals (•OH), in the presence of a large
`amount of high-energy (cid:2)-particles (20). Radical scaven-
`gers such as human serum albumin (HAS), gentisic acid
`(GA), and ascorbic acid (AA) have been used as stabilizers
`
`* To whom correspondence should be addressed. Tel: 978-
`671-8696;
`FAX:
`978-436-7500;
`E-mail:
`shuang.liu@
`dupontpharma.com.
`
`Figure 1. Structure of RP697.
`for the radiolabeled antibodies (21-23). It was also found
`that freezing the antibody after radiolabeling up to the
`time of administration could considerably improve the
`immunoreactivity by decreasing the diffusion and inter-
`action of free radicals with the radiolabeled biomolecule
`(22).
`The purpose of this study is to explore the factors
`influencing the solution stability of RP697, including the
`activity concentration, the total activity level, the stabi-
`lizer concentration, and the storage temperature. We
`have chosen AA and GA as stabilizers since they have
`been approved for pharmaceutical or radiopharmaceuti-
`cal applications. The ultimate goal is to find the optimized
`radiopharmaceutical composition to maintain the solu-
`tion stability of RP697. In principle, the radiopharma-
`ceutical composition developed in this study should also
`apply to other 90Y-labeled DOTA-biomolecule conjugates.
`
`EXPERIMENTAL PROCEDURES
`Materials. Acetic acid (ultrapure), ammonium hydrox-
`ide (ultrapure), ascorbic acid (sodium salt), diethylene-
`
`© 2001 American Chemical Society
`10.1021/bc000145v CCC: $20.00
`Published on Web 06/12/2001
`
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`90Y-Labeled DOTA-Biomolecule Conjugates
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`Bioconjugate Chem., Vol. 12, No. 4, 2001 555
`
`triaminepentaacetic acid (DTPA), and gentisic acid (so-
`dium salt) were purchased from either Aldrich or Sigma
`Chemical Co., and were used as received. 90YCl3 (in 0.05
`N HCl) was purchased from New England Nuclear Life
`Sciences, North Billerica, MA. The cyclic pentapeptide
`cyclo(Arg-Gly-Asp-D-Phe-Lys), as its trifluoroacetic acid
`(TFA) salt, was prepared according to the literature
`method (24). Synthesis of SU015, 2-(1,4,7,10-tetraaza-
`4,7,10-tris(carboxymethyl)-1-cyclododecyl)-acetyl-Glu(cyclo-
`{Lys-Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-D-
`Phe}), as its trifluoroacetic acid (TFA) salt will be
`described in a companion paper (19).
`General Procedure for the Synthesis of RP697.
`To a shielded clean 5 mL vial containing 100 μg of SU015
`and 0-10 mg of sodium gentisate (GA) or sodium
`ascorbate (AA) dissolved in 0.5 mL of 0.5 M ammonium
`acetate buffer (pH 8.0) was added 20-40 μL of 90YCl3
`stock solution (∼20 mCi) in 0.05 N HCl. The reaction
`mixture was heated at 95-100 °C for 5 min. A sample of
`the resulting solution was diluted 20-fold with 2 mM
`DTPA solution (pH 5), and then analyzed by radio-HPLC
`and ITLC. Each condition was run twice, and the
`radiochemical purity (RCP) data are presented as an
`average of two independent measurements.
`Analytical Methods. The HPLC method used a HP-
`1100 HPLC system with a UV/visible detector (λ ) 220
`nm), an IN-US radio-detector, and a Zorbax C18 column
`(4.6 mm × 250 mm, 80 Å pore size). The flow rate was 1
`mL/min. The mobile phase was isocratic from 0 to 18 min
`using 87% solvent A (0.025 M ammonium acetate buffer,
`pH 6.8) and 13% solvent B (acetonitrile), followed by an
`isocratic wash using 40% solvent A and 60% solvent B
`from 19 to 25 min. The retention time of RP697 is 14-
`16 min. The ITLC method used Gelman Sciences silica
`gel ITLC paper strips and a 1:1 mixture of acetone and
`saline as eluant. By this method, RP697 migrates to the
`solvent front while [90Y]colloid and [90Y]acetate remain
`at the origin. The corrected RCP for RP697 was calcu-
`lated by subtracting the percentage of [90Y]colloid and
`[90Y]acetate obtained by ITLC from that obtained by
`radio-HPLC.
`
`RESULTS
`Room Temperature Stability of RP697 at Low
`Concentration (4 mCi/mL). In the first experiment, we
`prepared three RP697 vials according to the standard
`procedure. The first vial contains only 100 μg of SU015,
`the second vial contains 100 μg of SU015 and 5 mg of
`GA, and the third vial contains 100 μg of SU015 and 5
`mg of sodium ascorbate (AA). After addition of 20 mCi
`of 90YCl3 in each vial, the reaction mixtures were heated
`at 100 °C for 5 min. After radiolabeling, the resulting
`solution was diluted to a concentration of 4 mCi/mL with
`saline, and the solution stability of RP697 at room
`temperature was monitored by radio-HPLC over 6 days.
`Figure 2 shows a typical radio-HPLC chromatogram for
`RP697. The peak at 14 min is from RP697. The peak at
`∼3.5 min is probably from a combination of several
`hydrophilic 90Y-containing species while the peak at 24
`min is from more lipophilic 90Y-containing species. Since
`most of the radioimpurities are less than 1%, no efforts
`were made for further characterization. Figure 3 shows
`the RCP change over time for RP697. It is clear that
`RP697 remains relatively stable (RCP ) 96.5% at 0 h
`and 94% at 144 h post-labeling) in the presence of GA
`and AA while it decomposes rapidly in the absence of GA
`and RCP is only 86% at 6 days post-labeling.
`GA Concentration and Solution Stability. In this
`experiment, RP697 was prepared according to the general
`
`Figure 2. Typical radio-HPLC chromatogram of RP697.
`
`Figure 3. Effect of stabilizers on solution stability of RP697
`at room temperature. Each condition was run twice, and the
`radiochemical purity (RCP) data are presented as an average
`of two independent measurements.
`
`Figure 4. Effect of GA concentration on solution stability of
`RP697 at room temperature. Each condition was run twice, and
`the radiochemical purity (RCP) data are presented as an average
`of two independent measurements.
`
`procedure using two levels (2 and 10 mg) of GA and 100
`μg of SU015 for 20 mCi of 90YCl3. The total volume was
`0.5 mL, and the activity concentration was 40 mCi/mL.
`After radiolabeling, the resulting solution was kept at
`room temperature, and the solution stability of RP697
`was monitored by radio-HPLC over 6 days. Figure 4
`shows the RCP change over 6 days for RP697. The RCP
`of RP697 decreased from 96.5% at 0 h to 93% at 144 h
`post-labeling when 10 mg of GA was used for 20 mCi of
`activity. The RCP dropped much more rapidly from 97%
`at 0 h to 65% at 144 h when only 2 mg of GA was used
`to stabilize 20 mCi of RP697. This suggests that 2 mg of
`GA is not sufficient to maintain RCP above 90% and 10
`mg of GA is needed to stabilize RP697 over 2 half-lives
`of 90Y. Compared to the results from the previous experi-
`ment, it seems that the rate of decomposition of RP697
`is also dependent on the activity concentration.
`Activity Level and Solution Stability of RP697.
`In this experiment, we prepared two RP697 vials using
`two different levels of 90Y. In one vial, it contained 10
`mg of GA, 100 μg of SU015, and 20 mCi of 90YCl3 (total
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`Liu and Edwards
`
`renal clearance are particularly important to improve the
`tumor-to-background ratio and to reduce the radiation
`burden to other major organs (kidney, liver, and bone
`marrow). The new radiopharmaceutical must have high
`radiochemical purity (RCP g90%). Unlike diagnostic
`radiopharmaceuticals, therapeutic radiopharmaceuticals
`have to be manufactured and released under GMP (Good
`Manufacturing Practice) conditions, and delivered for
`clinic applications. Therefore, the new therapeutic ra-
`diopharmaceutical must retain its chemical and biological
`integrity during release and transportation.
`Therapeutic radiopharmaceuticals comprising (cid:2)-emit-
`ting radionuclides may undergo autoradiolysis during
`preparation, release, transportation, and storage. During
`radiolysis, emissions from the radionuclide attack the
`metal chelate, targeting the biomolecule, and other
`compounds in proximity, which results in inter- and
`intramolecular decomposition or destruction of the ra-
`diometal chelate or the biomolecule. Radioactivity, which
`is not linked to the targeting biomolecule, will accumulate
`in nontargeting tissues and lead to the unwanted radia-
`tion toxicity to nontargeting tissues. Thus, it is important
`that the radionuclide remains linked to the targeting
`moiety, and the specificity of the targeting biomolecule
`is preserved. As a matter of fact, a major challenge for
`the development of a therapeutic radiopharmaceutical is
`to stabilize the radiolabeled compound during radio-
`labeling, release, and transportation.
`Radiolysis is caused by the formation of free radicals
`such as hydroxyl and superoxide radicals. Free radicals
`are very reactive toward organic molecules such as
`peptides. To prevent radiolysis and stabilize the radio-
`labeled biomolecule, a radical scavenger or radiolytic
`stabilizer is often used either during or after the radio-
`labeling. A stabilizer is often an antioxidant, which
`readily reacts with hydroxyl and superoxide radicals. In
`general, the stabilizer for the therapeutic radiopharma-
`ceutical should have the following characteristics:
`low
`or no toxicity or immunoreactivity when it is used for
`human administration, no interference with the receptor
`binding of the radiolabeled compound to the target cells
`or tissue(s), and the ability to stabilize the therapeutic
`radiopharmaceutical for a reasonable period of time
`(preferably 2 half-lives of 90Y) for preparation, release,
`storage, and transportation.
`There are several commercially available antioxidants.
`Human serum albumin (HAS) has been used as a
`stabilizer for the radiolabeled antibodies (21-23). Ascor-
`bic acid is an antioxidant, and an FDA-approved phar-
`maceutical suitable for human injection. Gentisic acid has
`been used as a stabilizer in 99mTc radiopharmaceutical
`formulations (28-30). The stabilizer can be added into a
`therapeutic radiopharmaceutical formulation either be-
`fore or after the radiolabeling. For pre-labeling addition,
`the stabilizer is added before addition of 90YCl3 stock
`solution. During the radiolabeling process, the stabilizer
`is subjected to a certain degree of thermal decomposition,
`particularly at elevated temperatures. For the post-
`labeling addition, the stabilizer is not exposed to the
`heating process; thereby it may have longer stabilizing
`effect and higher stabilizing efficiency. The post-labeling
`approach is particularly useful when the addition of a
`large amount of stabilizer in the radiopharmaceutical
`formulation interferes with the radiolabeling.
`There are several factors influencing the solution
`stability of RP697. These include the amount of activity
`in each vial, the activity concentration, the relative
`amount of stabilizer, and the storage temperature. For
`example, the rate of radiolytic decomposition of RP697
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`3 of 5
`
`Figure 5. Effect of the activity concentration on solution
`stability of RP697 at room temperature. Each condition was run
`twice, and the radiochemical purity (RCP) data are presented
`as an average of two independent measurements.
`
`Figure 6. Solution stability of RP697 at -78 °C. Each condition
`was run twice, and the radiochemical purity (RCP) data are
`presented as an average of two independent measurements.
`
`volume ) 0.5 mL). In another vial, RP697 was prepared
`using 500 μg of SU015, 50 mg of GA, and 100 mCi of
`90YCl3 (total volume ) 2.5 mL). After radiolabeling, the
`resulting solution was kept at room temperature, and the
`solution stability of RP697 was monitored by radio-HPLC
`over 6 days, as shown in Figure 5.
`Solution Stability of RP697 at -78 °C. RP697 was
`prepared using the standard radiolabeling procedure (100
`μg of SU015, 10 mg of GA for 20 mCi of 90YCl3, and
`heating at 100 °C for 5 min). For the vials containing
`100 and 200 mCi of activity, all the component levels
`were increased proportionally. For the vial using AA as
`the stabilizer, AA (200 mg/mL) was dissolved in 0.5 N
`ammonium acetate buffer (pH 7.4) and added to the
`reaction mixture (10 mg of AA for 20 mCi of 90Y) after
`radiolabeling. After addition of AA, all the vials contain-
`ing RP697 were kept in a dry ice box (-78 °C). Samples
`of the reaction mixture were analyzed by HPLC at 0, 24,
`56, and 138 h. Figure 6 shows the RCP change over 6
`days for RP697. Apparently, RP697 can be stabilized by
`the combination use of a stabilizer and low-temperature
`storage at -78 °C.
`
`DISCUSSION
`There has been great current interest in radiolabeled
`small peptides as therapeutic radiopharmaceuticals (25-
`27). In developing a new receptor-based therapeutic
`radiopharmaceutical, several factors need to be consid-
`ered to satisfy the clinical need and to comply with FDA
`regulations. The therapeutic radiopharmaceutical must
`demonstrate therapeutic efficacy and favorable pharma-
`cokinetics, including high and fast tumor uptake, high
`tumor-to-background ratio, long tumor residence time,
`and fast renal clearance. High tumor uptake and fast
`
`

`

`90Y-Labeled DOTA-Biomolecule Conjugates
`
`Bioconjugate Chem., Vol. 12, No. 4, 2001 557
`
`is much slower at the activity concentration of 4 mCi/
`mL than that at a concentration of 40 mCi/mL (Figure
`3). It needs 10 mg of GA or AA to maintain the RCP of
`RP697 (20 mCi) above 90% over 2 half-lives of 90Y (Figure
`4). RP697 remains relatively stable at room temperature
`if 10 mg of GA is used for 20 mCi of activity. It
`decomposes much faster when 50 mg of GA is used for
`100 mCi of activity under the same storage conditions,
`even though the GA/activity ratio remains the same
`(Figure 5). Therefore, the solution stability data obtained
`from the low activity level (<20 mCi) cannot be simply
`extrapolated to high activity levels (>100 mCi).
`In addition to the use of an antioxidant in the radio-
`pharmaceutical formulation, storing the reaction mixture
`at low temperature (-78 °C/dry ice) also has a dramatic
`impact on the solution stability of RP697. For example,
`RP697 remains stable for at least 2 half-lives of 90Y at
`-78 °C (Figure 6) while it decomposes rapidly at room
`temperature. Freezing the reaction mixture containing
`RP697 offers two advantages: stopping the diffusion of
`free radicals and slowing down the reaction kinetics
`between free radicals and radiolabeled cyclic peptide.
`Thus, it is recommended that the radiopharmaceutical
`be stored at low temperatures to avoid extensive radi-
`olysis during release and transportation. The amount of
`antioxidant used in the radiopharmaceutical formulation
`and the storage temperature during release and trans-
`portation should be adjusted according to the sensitivity
`of a specific radiolabeled receptor ligand toward radiolytic
`decomposition.
`
`CONCLUSIONS
`In this study, we explored the factors influencing the
`solution stability of RP697, a 90Y-labeled DOTA-peptide
`conjugate, and found that both the amount of total
`activity and the activity concentration have significant
`impact on the stability of the 90Y-labeled cyclic peptide.
`The rate of radiolytic decomposition of RP697 is much
`slower at the lower activity concentration (<4 mCi/mL)
`than that at the higher concentration (>10 mCi/mL).
`RP697 remains relatively stable at room temperature
`when it is prepared at the 20 mCi level while it decom-
`poses rapidly at the 100 mCi level under the same storage
`conditions. Therefore, the solution stability data obtained
`from the low activity level (<20 mCi) cannot be simply
`extrapolated to high activity levels (>100 mCi).
`To prevent extensive radiolysis, we used both GA and
`AA as stabilizers. The stabilizer can be added into the
`formulation either before or after radiolabeling. For the
`post-labeling addition, the stabilizer is not exposed to the
`heating process; thereby it may have a longer stabilizing
`effect and higher stabilizing efficiency. The post-labeling
`approach is particularly useful when the addition of a
`large amount of stabilizer in the radiopharmaceutical
`formulation interferes with the radiolabeling of the
`DOTA-biomolecule conjugate.
`
`ACKNOWLEDGMENT
`
`Acknowledgment is made to Dr. M. Rajopadhye and
`Anothy R. Harris for the synthesis of DOTA-conjugated
`cyclic peptide (SU015).
`
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