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NEUROPHARMACOLOGY

Pharmacological and Toxicological Evaluation of 2-Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-F-A-85380), a Ligand for Imaging Cerebral Nicotinic Acetylcholine Receptors with Positron Emission Tomography

Neuroimaging Research Branch (D.B.V., A.G.M., S.I.C., A.G.H., A.S.K.), Intramural Research Program, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland; Department of Pharmacology (S.R.T.), Georgetown University Medical Center, Washington, D.C.; Department of Comparative Medicine (D.L.H.), Johns Hopkins University School of Medicine, Baltimore, Maryland; BioReliance, Inc. (V.O.W.), Rockville, Maryland; Departments of Psychiatry and Biobehavioral Sciences (E.D.L., A.O.K.), Molecular and Medical Pharmacology (E.D.L.), and the Brain Research Institute (E.D.L.), David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California

Received July 27, 2004; accepted August 26, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
2-[¹⁸F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[¹⁸F]F-A-85380), a positron emission tomography (PET) radioligand for neuronal 42^* nicotinic acetylcholine receptors, was evaluated for its pharmacology and safety. In the Ames test for mutagenicity, 2-F-A-85380 was without effect in five bacterial strains. No evidence of gross pathology or histopathological changes occurred in either 2-day acute (0.4-4000 nmol/kg i.v.) or 14-day expanded acute (40-4000 nmol/kg i.v.) toxicity studies in mice. Similarly, hematology and serum chemistry values in rhesus monkeys administered 60 nmol/kg i.v. were not affected over 14 days. Like nicotine, 2-F-A-85380 produced convulsions in mice at very high doses. The ED₅₀ value of 2-F-A-85380 for eliciting tonic-clonic convulsions (5.0 µmol/kg i.v.) was nearly 4 times greater than that of nicotine (ED₅₀ = 1.4 µmol/kg i.v.). Lower doses of 2-F-A-85380 (30-300 nmol/kg i.v.) and nicotine (20-400 nmol/kg i.v.) increased systolic and diastolic blood pressure, heart rate, and cardiac contractility in rats. Notably, the PR, QRS, or QTc intervals of the rat electrocardiogram were unaffected by either drug. Dosimetry studies indicated that the urinary bladder wall was the critical organ and total radiation exposure was within acceptable limits. Estimated doses of 2-F-A-85380 required to elevate blood pressure and heart rate by 10% ranged from 40 to 58 nmol/kg i.v. Nevertheless, the estimated radiopharmaceutically relevant dose of [¹⁸F]2-F-A-8380 required for initial PET imaging studies, 10 pmol/kg, is less than 1/4000th of the doses calculated (40-58 nmol/kg i.v.) to elevate blood pressure and heart rate by 10% in humans and should elicit no clinically significant effects and have acceptable dosimetry.

Developed by Abbott Laboratories (Abbott Park, IL), A-85380 exhibits selectivity for 42^* nAChRs. Its K_i values for different nAChR subtypes at 23°C and selectivity ratios (in parentheses) are as follows: 42^*, 0.017 nM (1); 7, 17 nM (1000); 34, 14 nM (800); and muscle, 320 nM (19,000) (Mukhin et al., 2000). Introduction of a fluorine atom at the 2-pyridyl position of A-85380 yielded 2-F-A-85380, which has a K_i value of approximately 50 pM for the 42^* nAChRs (Holladay et al., 1998; Koren et al., 1998), and retained high selectivity. The affinity of 2-F-A-85380 for the 42^* subtype is at least 2500 times greater than that for other major nAChR subtypes, 7, 34^* ganglionic receptors, and muscle receptors (Pavlova et al., 2000). Compared with the K_i values for 42^* nAChRs of 0.017 nM for A-85380 and 0.010 nM for 5-I-A-85380, the affinity of 2-F-A-85380 is slightly lower (0.050 nM) but not enough to limit the potential advantages it affords as a PET imaging radioligand.

In 1998, the successful syntheses of both unlabeled (Koren et al., 1998) and ¹⁸F-labeled 2-F-A-85380 (Dollé et al., 1998; Horti et al., 1998) were described. PET studies of 2-[¹⁸F]FA-85380 in nonhuman primates demonstrated a higher ratio of specific-to-nonspecific binding and more favorable kinetic parameters compared with ¹¹C-radiolabeled nicotine (Chefer et al., 2000). Furthermore, 2-[¹⁸F]F-A-85380 exhibits a binding pattern in the mouse brain that reflects the distribution of nAChRs, with saturable binding that is accompanied by relatively low nonspecific binding. Drug interaction studies using pretreatment with cytisine, (-)-nicotine, mecamylamine, or (-)-scopolamine followed by 2-[¹⁸F]F-A-85380 demonstrated that radioligand binding to nAChRs most likely occurs at the agonist binding site of the ion channel complex (Horti et al., 1998). Comprehensive characterization of 2-[¹⁸F]F-A-85380 suggested that it would be superior to [¹¹C]nicotine as an imaging agent. Additional preclinical pharmacological and toxicological studies providing evidence that 2-[¹⁸F]F-A-85380 could be used safely for human PET imaging studies have been briefly reported (Vaupel et al., 2002) and are described in detail herein.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Test and Control Materials. 2-F-A-85380 trifluoroacetate was synthesized in two batches following published procedures (Koren et al., 1998), and aliquots were stored in glass vials at 0°C prior to use in animal studies. Based upon elemental analysis of 2-F-A-85380 trifluoroacetate, the molecular weights were 433 (batch A, C₉H₁₁FN₂O · 2.2 C₂HF₃O₂) and 410 (batch B, C₉H₁₁FN₂O · 2.0 C₂HF₃O₂). Batch B was used for the mutagenicity study, and batch A was used for all other experiments. Purity exceeded 99% based on NMR and high-performance liquid chromatography analysis. 2-[¹⁸F]F-A-85380 used in the dosimetry study was prepared as described previously (Horti et al., 1998). Nicotine hydrogen tartrate, mecamylamine hydrochloride, and hexamethonium bromide were purchased from Sigma-Aldrich (St. Louis, MO). The vehicle for injection was 0.9% sodium chloride, USP.

Ames Test. The mutagenicity of 2-F-A-85380 was evaluated in the Ames test and performed by using four histidine-requiring strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, and TA 1537) and Escherichia coli strain WP2uvrA (contracted to BioReliance Corporation, Rockville, Maryland through MPI Research, Mattawan, Michigan). Strains TA 98 and TA 1537 are reverted by frameshift mutations, strain TA 1535 is reverted by base pair substitutions, and strain TA 100 is reverted by mutagens causing both frameshift and base pair substitution mutations. E. coli strain WP2uvrA is reverted by base pair substitution mutations rather than frameshift mutations. Dose levels of 2-F-A-85380 (50, 150, 500, 1500, and 5000 µg/plate), an ethanol (100%) vehicle control (negative control), and positive controls were plated in triplicate and tested in the presence and absence of a standard S9 metabolic activation mixture per the plate incorporation method (Maron and Ames, 1983). The dose levels were selected based on the results of a preliminary toxicity assay (data not reported). For positive controls, strain-specific reference substances were tested with and without the S9 mix.

Animal Studies. Protocols using mice were approved by the Animal Care and Use Committee of the National Institute on Drug Abuse Intramural Research Program, and the protocol for the cardiovascular study in rats was approved by the Georgetown University Medical Center Animal Care and Use Committee.

Physiological and Behavioral Measures Acquired in the Mouse. A battery of physiological and behavioral measures was used as a screen in mice. Parametric measures included body weight, changes in body temperature, locomotor activity counts, and development of the Straub tail position (scored on a 0 to 8 scale). Tremors, forepaw treading, transfer arousal, motor incoordination, changes in respiration, and convulsions constituted the most useful nonparametric measures. A detailed description of the battery has been published (Vaupel et al., 2003). To accommodate the rapid kinetics resulting from intravenous administration, the test battery was completed within 6 min. Rectal temperature was measured 30 min before drug administration and 66 min afterward. Immediately after the injection, the mouse was placed in an empty, clear plastic cage (28 x 17 x 13 cm) with the floor divided into four quadrants. Behaviors, particularly central nervous system effects, such as convulsions, tremors, forepaw treading, changes in the breathing pattern, and Straub tail, were observed for 0 to 2 min; locomotor activity was counted from 2 to 4 min; and transfer arousal and motor incoordination were assessed beginning at 4 min after injection. The battery was repeated 60 min later followed by measurement of rectal temperature. Mice were observed for any latent toxicity for a period of 1 week after testing.

Convulsive Dose ED₅₀ Study in the Mouse. The convulsant efficacy was evaluated in CD-1 mice using intravenously administered 2-F-A-85380, and the ED₅₀ value for producing convulsions was calculated using the method of Litchfield and Wilcoxin (1949). 2-F-A-85380 was tested at doses of 3.5, 6, and 10 µmol/kg; nicotine, whose toxicity includes convulsions, served as the standard and was tested at doses of 1, 1.25, 1.5, and 2 µmol/kg. Generally, 10 mice (five males and five females) were tested at each dose. Observations of additional physiological and behavioral effects were initiated immediately after drug administration into the tail vein and repeated 60 min later. The convulsions were primarily of the clonic or tonic type, or a combination of both, and the loss of the righting reflex for a minimum of 5 s was required to fulfill the criterion as a convulsion. Afterward, mice were observed daily for a 1-week period.

Acute 2-Day and Expanded Acute 14-Day Toxicity Studies in Mice. The acute and expanded acute toxicity of 2-F-A-85380 was evaluated following a single intravenous injection followed by either a 2- or 14-day observation period. Male and female CD-1 mice were used in both studies. Food and water were available ad libitum throughout the study, and a 12-h light/dark cycle was always in effect. Physiological and behavioral effects were measured immediately and 1 h after intravenous administration. Mice were subsequently housed singly throughout the observation period and then euthanized by cervical dislocation and prepared for necropsy. Gross pathological examinations were completed on all animals, necropsies were performed, and tissue samples were harvested for histopathology. At the time of necropsy, tissue samples of the following organs were preserved in 10% neutral buffered formalin: adrenal glands, bladder, brain, cecum, colon, heart, ileum, kidneys, liver, lungs, pancreas, spleen, stomach, testes/epididymides, and uterus/ovaries. Any tissue exhibiting visible lesions or apparent abnormality was examined histopathologically using standard procedures. All injections, observations, necropsies, and histopathology were performed with the investigators blinded to the treatments.

In the acute 2-day toxicity study, five male and five female mice were randomly assigned to each treatment condition using two blocks of experiments. In the first block, three doses of 2-F-A-85380 (0.4, 40, and 200 nmol/kg) and saline were evaluated. Because the 200-nmol/kg dose did not elicit pronounced pharmacological effects, a 4000-nmol/kg dose was added and tested with another saline control in the second block. Prior to treatment, body weights and rectal temperature were measured. Approximately 36 to 48 h after drug administration, each mouse was weighed, euthanized, and prepared for necropsy.

The expanded acute 14-day intravenous toxicity study used four randomized treatment groups (five male and five female mice per group). Each mouse received a single intravenous injection of 2-F-A-85380 [0 (saline), 40, 200, or 4000 nmol/kg] followed by a 14-day observation period. Body weights were recorded on day 1 before the first injection, day 8, and day 15 before euthanasia and necropsy.

Cardiovascular and Electrocardiogram Assessment in Rats by Telemetry. Adult Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) (three males and three females) were housed individually and fed based on a rate of 5 g per 100 g of body weight per day to maintain a stable body weight.

The test compound 2-F-A-85380, nicotine, and vehicle were administered intravenously to fully conscious rats that had been surgically prepared to permit data acquisition by telemetry (Transoma Medical, Inc., Arden Hills, MN) as previously described in detail (Tella, 1996; Vaupel et al., 2003). Systolic and diastolic blood pressure, heart rate, cardiac contractility, locomotor activity, and temperature were measured every 20 s, and electrocardiogram data were acquired every 2 min using data telemetry. On study mornings, animals were placed in the test chambers, and their catheters were flushed with heparinized saline and connected to an external catheter housed inside a flexible spring, which was attached to the plastic skull pedestal. This apparatus enabled data collection while the rats moved freely about the chamber. Catheters were preloaded with the drug (0.1-0.2 ml), and rats were habituated to the test chamber for 30 to 120 min to allow their physiological responses to stabilize. Data were monitored continuously during the habituation period. Once the physiological variables had stabilized, drug or vehicle was administered manually over a 2-s period by a 0.3-ml saline flush of the preloaded catheter; data acquisition continued for 2 h.

Using a within-subjects design and six rats, three compounds, 5-I-A-85380, 2-F-A-85380, and nicotine, were evaluated sequentially in three series of experiments, each consisting of four doses of the test compound and vehicle, which was administered twice. Preliminary experiments in a separate group of rats were used to establish the minimally-to-maximally effective dose range for the cardiovascular activity. The six rats were divided into two equal groups that were tested separately; however, due to a battery failure in the single female rat included in the first group of three rats, it became necessary to test four rats in the second group. Thus, for 2-F-A-85380, the groupings and dosing sequence were as follows: group 1, two males, 30, 100, 0 (saline vehicle), 10, 300, and 0 nmol/kg; and group 2, three females, one male, 100, 30, 0, 10, 0, and 300 nmol/kg. The dosing sequence and results for nicotine and 5-I-A-85380 have been published (Vaupel et al., 2003). On test days, all animals in a group were tested concurrently and received the same dose. The testing interval was either daily or every other day during the week. At the completion of these experiments, antagonism studies, using two ganglionic blockers, consisted of a single pretreatment of either mecamylamine hydrochloride or hexamethonium bromide followed 20 min later by 300 nmol/kg 2-F-A-85380 or 400 nmol/kg nicotine using the procedures as previously described.

2-F-A-85380 trifluoroacetate was tested at doses of 10, 30, 100, and 300 nmol/kg (corresponding to 4.3, 13, 43.3, and 129.9 µg/kg). Saline was the vehicle control. The doses of nicotine hydrogen tartrate (mol. wt. = 498) were 20, 60, 201, and 400 nmol/kg (corresponding to 10, 30, 100, and 200 µg/kg). Nicotine was prepared in a buffered vehicle (saline/Tris, pH 7.4) having a final pH value within the range of 3.3 to 4.3; its vehicle was a solution of 10 mM acetic acid in saline, pH 3.4. Antagonist doses were 3 mg/kg (14.7 µmol/kg) i.v. for the mecamylamine hydrochloride and 20 mg/kg (55.2 µmol/kg) i.v. for hexamethonium bromide.

Hematology and Serum Chemistry Tests in Nonhuman Primates. The expanded acute effects of 60 nmol/kg 2-F-A-85380, administered intravenously over 20 s, on hematology and serum chemistry were measured in four male rhesus monkeys, 5 to 6 years old, ranging in weight from 7 to 11 kg. All samples were acquired after the animals were anesthetized with Saffan. Baseline control blood samples were drawn prior to the intravenous administration of 2-F-A-85380, and subsequent samples were withdrawn 40 min afterward, the next day (ca. 24 h later), 5 or 6 days afterward, and 15 days after dosing. Determination of any abnormalities was based on a comparison to normal values provided by Animal Diagnostic Laboratory, L.L.C. (Baltimore, MD) and published reference values (Fuller et al., 1992; Buchl and Howard, 1997; Hom et al., 1999).

2-[¹⁸F]F-A-85380 Dosimetry in Mice. Radiation dosimetry for 2-[¹⁸F]F-A-85380 was assessed in male and female CD-1 mice following published procedures (Vaupel et al., 2003) with minor modifications. A solution of 2-[¹⁸F]F-A-85380 in saline (0.2 ml) was administered as a bolus injection into the lateral tail vein, and groups of mice (n = 3) were euthanized at 5, 15, 30, 60, 120, and 240 min after treatment with each time point replicated for each sex. Male mice (body weight, 30.9 ± 0.5 g) received 9.3 MBq/kg (0.25 mCi/kg) of 2-[¹⁸F]F-A-85380 with a specific activity of 59 GBq/µmol (1600 Ci/mmol) at the time of injection. The specific activity of the 2-[¹⁸F]FA-85380 prepared for the study with female mice (body weight, 28.4 ± 0.3 g) was 109 GBq/µmol (2950 Ci/mmol) at the time of injection. To ensure that both groups received the same mass per kilogram, female mice received 18.1 MBq/kg (0.49 mCi/kg), which adjusted for the differences in specific activity. As a result, animals of each sex received 0.16 nmol/kg 2-[¹⁸F]F-A-85380. Absorbed radiation doses were calculated based on a phantom model for a 70-kg adult human using the MIRDOSE program (v 3.1) and a dynamic bladder model.

Data Analysis and Statistics. For the convulsant efficacy study, ED₅₀ values were determined by log-probit analysis (Litchfield and Wilcoxin, 1949) using the PHARM/PCS program (Microcomputer Specialists, Valparaiso, IN). Cardiovascular, temperature, and locomotor activity data were checked for artifacts, corrected for missing data, and normalized for each individual by subtracting the baseline value from the respective data set. Baseline periods were determined for individual experiments and typically obtained within 10 to 20 min before drug administration. Vehicles were administered twice, and the replicates were pooled before analysis. Mean changes in blood pressure, heart rate, QA interval, temperature, and locomotor activity were calculated using all consecutive 0.33-min data points for 0 to 5-, 5 to 10-, 10 to 30-, 30 to 60-, and 60 to 120-min periods as well as for the entire experiment, 0 to 120 min. Mean changes were obtained by dividing baseline-corrected areas under the time-action curve for each time period by the number of minutes comprising the time period. For studies using the ganglionic blockers mecamylamine and hexamethonium, analysis of the time-course curves following pretreatment with these antagonists indicated the most effective blockade was present during the first 20 min after the administration of 2-F-A-85380. Therefore, the mean changes from baseline over the first 20 min were used to evaluate the effectiveness of the antagonism produced by each ganglionic blocker. Cardiac contractility was evaluated indirectly using the QA interval, defined as the time between the Q point of the ECG and the onset of the upstroke of the aortic blood pressure pulse wave (Cambridge and Whiting, 1986). Increases in contractility correspond to decreases in the QA interval. In addition, maximal decreases or increases in heart rate for individual rats occurring during the first 10 min of the study were acquired to address issues of nicotine toxicity. Peak pharmacological activity was obtained from the time-action curves.

PR, QRS, QTc, and RR intervals of the ECG were measured continuously throughout the experiment over 2-min epochs, and data points represented an average of 25 to 60 waveforms. The QTc interval uses Bazett's correction for heart rate. Time-period analysis of the ECG was similar to that described for the cardiovascular component and consisted of the following sequential periods of time: 0 to 10, 10 to 30, 30 to 60, 60 to 120, and 0 to 120 min. In addition, individual differences from baseline for the 2-, 4-, 6-, 8-, and 10-min time points were analyzed separately.

Cardiovascular and ECG statistical analyses consisted of two-way repeated measures ANOVA [main effects: treatment (i.e., doses) and sex] followed by a Dunnett's test to analyze the 2-F-A-85380 and nicotine dose-response data sets separately using SigmaStat (SPSS Inc., Chicago, IL). A Student-Newman-Keuls test was used to provide an analysis of simple effects for significant interactions (treatment x sex). Relative potency estimates and ED₁₀ values were obtained using the custom-designed program Bioassay 6.1 (D. B. Vaupel; programmed by MED Associates, St. Albans, Vermont). The ED₁₀ value, defined as the dose producing a 10% increase in blood pressure or heart rate within 5 min of drug administration, was calculated if the original, untransformed data exhibited linear regression. The data were subsequently expressed as a percentage of control values, the ED₁₀ value was obtained by regression analysis, and inverse prediction techniques were used to estimate the magnitude of the cardiovascular effect in response to the ED₁₀ dose. For antagonism experiments, a two-way repeated measures ANOVA [treatments (saline, 2-F-A-85380, mecamylamine + 2-F-A-85380, and hexamethonium + 2-F-A-85380) and sex] was followed by one-way paired t tests to evaluate effectiveness of the antagonists for different time periods following 2-F-A-85380 administration.

	Results

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Ames Test. All criteria for a valid study were fulfilled as described in the protocol. Neither precipitate nor appreciable toxicity was observed. In the mutagenicity assay, no positive responses were observed (Table 1). The highest dose of 2-F-A-85380 tested (5000 µg/plate, 50 mg/ml = 0.122 mol/l based on a mol. wt. of 410) is 7 x 10⁸ fold larger than the mass dose of 3 mCi of 2-[¹⁸F]F-A-85380 at a specific activity >5000 Ci/mmol, as proposed for PET imaging in human subjects. This conservative safety ratio assumes that the mass dose of the radioligand is initially distributed entirely within a 4-liter blood compartment [(70 kg b.wt. x 10 pmol/kg dose)/4 liters = 175 x 10^-12 mol/l (1 liter corresponds to 1 kg)].

Acute 2-Day and Expanded Acute 14-Day Toxicity Studies in Mice
Physiological and Behavioral Effects. Since physiological and behavioral responses were evaluated within the first 6 min after dosing for both acute and expanded acute studies, these data were pooled and combined with results from the convulsant study, which incorporated the same measurements during the identical time period. The Straub tail response, reduced locomotor activity, and a decrease in body temperature represented the parametric effects produced by 2-F-A-85380 over a dose range of 0.4 to 4000 nmol/kg (Fig. 1). Nonparametric measures included labored breathing, gasping, motor incoordination, forepaw treading, and depression of the transfer arousal response. The appearance of the Straub tail position was dose-dependent (p < 0.0001) and log-linear, with the effects of the 200- and 4000-nmol/kg doses differing significantly from the vehicle control value (Fig. 1). Temperature and locomotor activity were affected only by the 4000-nmol/kg dose, which appeared to represent near maximal changes. For temperature, 4000 nmol/kg produced a 7.9 ± 0.4°C (n = 20) decrease (p < 0.01) 60 min after treatment when compared with the 0.8 ± 0.1°C (n = 40) increase measured after the vehicle. Locomotor activity, measured as quadrant crossings, was almost absent 2 to 4 min after 4000 nmol/kg (1.4 ± 0.7, n = 7), whereas the mice averaged 14.4 ± 1.0 (n = 40) crossings after vehicle treatment. All effects appearing shortly after intravenous drug administration, except for temperature, were absent when mice were returned to the animal housing facility 2 to 3 h after testing. Analysis of pooled data from these studies and the convulsant study demonstrated that female mice were more sensitive to developing the Straub tail response (p < 0.001). Using the appearance of drug-induced behavior as a cutoff point, the projected imaging dose of 3 mCi of 2-[¹⁸F]FA-85380 (>5000 Ci/mmol) in a 70-kg human subject (dose, 10 pmol/kg) is less than 1/4000th of the highest dose that produced no noticeable behavioral effect (40 nmol/kg; Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Dose-response curves for the development of the Straub tail position, hypothermia, and depressed locomotor activity in mice were based upon the pooled effects of the acute 2-day and expanded acute 14-day toxicity studies and the convulsant efficacy study. Symbols and error bars represent means ± S.E.M. Open symbols, aligned with the V on the abscissas, represent the saline vehicle effect (0 mg/kg). Straub tail and motoric effects were measured within 6 min after intravenous drug administration, whereas the change in temperature was determined 60 min after treatment. Significant treatment (dose) effects were determined from a two-way ANOVA (main effects, treatment and sex). Differences from the pooled saline vehicle measurements (n = 40) were determined using a Dunnett's test and are indicated by a star (p < 0.01); n = 10 for the 0.40-, 3,500-, 6,000-, and 10,000-nmol/kg doses, and n = 20 for the 40-, 200-, and 4,000-nmol/kg doses. Sex was a significant (p < 0.001) main effect only for the Straub tail effect, with female mice having a higher mean score (5.1) than males (3.6).

Body Weight. Body weight was not affected in the acute 2-day toxicity study. For the expanded acute toxicity study, mean initial body weights ± S.E.M. for males and females were 29.7 ± 0.2 and 26.2 ± 0.2 g, respectively, and throughout the 14 days both sexes gained weight continuously, with males gaining more (4.2 g or 14%) than females (1.9 g or 7%) (p < 0.001). This difference represents normal weight gain and was not attributable to 2-F-A-85380 treatment.

Gross Pathology and Histopathology. Gross pathology associated with 2-F-A-85380 treatment was minor and infrequent. Furthermore, organ weights were not affected by treatment with 2-F-A-85380 in either study (Table 2). In the expanded acute 14-day study, organ weights (mean ± S.E.M.) for the kidneys and liver obtained from male mice (0.71 ± 0.02 and 2.48 ± 0.07 g, respectively) weighed significantly more than those from females (0.52 ± 0.02 and 1.82 ± 0.04 g, respectively) (all p < 0.001), an unremarkable finding that was consistent with their group differences in body weight. Histopathological results indicated a low incidence of lesions, which were characterized as mild in scope and observed commonly as background lesions in mice (Table 2). None of the findings was associated with 2-F-A-85380 treatment. Last, there was no evidence of pathology at the tail injection sites at the time of necropsy.

Convulsant Efficacy in the Mouse. 2-F-A-85380 produced dose-dependent clonic or tonic convulsions [ED₅₀ (95% CL) = 5.0 (4.0-6.4) µmol/kg]. The appearance of the Straub tail position (Fig. 1), reduced locomotor activity (Fig. 1), hypothermia (Fig. 1), motor incoordination, a diminished transfer arousal response, labored respiration that was sometimes accompanied by gasping (3 of 10 mice at 6 µmol/kg and 4 of 10 mice at 10 µmol/kg), and forepaw treading were commonly observed with high, nonlethal doses of 2-F-A-85380. All 10 animals convulsed at 10 µmol/kg, three of which died within 35 s. Hypothermia consisted of a marked 8 to 9°C decrease in rectal temperature that developed over a 1-h period in surviving mice. A safety ratio assessment was based on the ED₁₀ value of 2-F-A-85380 to elicit convulsions, which is 3.1 µmol/kg. This dose is 3 x 10⁵ times the proposed dose of 3 mCi of 2[¹⁸F]F-A-85380 in humans (10 pmol/kg), based upon a body weight of 70 kg and specific activity of >5000 mCi/mmol.

Nicotine likewise produced dose-dependent convulsions [ED₅₀ (95% CL) = 1.4 (1.2-1.7) µmol/kg. At a dose of 1.5 µmol/kg, convulsions (7 of 10) were most aptly described as "running fits" (extremely rapid running around the perimeter of the cage), and 1 of 10 mice died. After 2 µmol/kg nicotine, clonic seizures were followed by tonic convulsions and running fits; 4 of 10 mice died. Nicotine also reduced locomotor activity, elicited the Straub tail response, and produced motor incoordination and labored breathing (data not shown). Neither nicotine nor saline had an effect on temperature when measured 1 h after intravenous administration. There were no sex-related differences in convulsant activity linked to either drug, and no additional deaths occurred during following week.

Cardiovascular and Electrocardiogram Studies of 2-F-A-85380 in Rats
Systolic and Diastolic Pressure. 2-F-A-85380 dose-dependently increased systolic and diastolic blood pressure after intravenous administration (Fig. 2). The increases in pressure were rapid, occurring within 3 min. Based upon time-course curves, peak increases of 38 ± 2 mm Hg in systolic pressure (Fig. 3A) and 30 ± 1 mm Hg diastolic pressure appeared 0.33 min after the administration of 300 nmol/kg 2-F-A-85380. The lowest dose of 2-F-A-85380 tested, 10 nmol/kg, produced peak increases of 5 ± 4 mm Hg in systolic pressure and 4 ± 2 mm Hg in diastolic pressure.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Time-period analyses for the cardiovascular changes produced by 2-F-A-85380 following intravenous drug administration that demonstrate the dose-response relationships throughout a 2-h period. For all panels, the left ordinate for each set of bar graphs represents the baseline-corrected mean change in six rats, three female and three male; the right ordinate represents data expressed as a percentage of the mean baseline value. The mean baseline values ± S.E.M. based on all five treatments were as follows: systolic blood pressure, 119 ± 5 mm Hg; diastolic blood pressure, 87 ± 4 mm Hg; heart rate, 293 ± 16 beats/min; and QA interval, 44.5 ± 0.4 ms. Vertical bars represent the S.E.M. All rats received all treatments, and the vehicle was saline. A star indicates single-dose effects that differed significantly from the vehicle response (p < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 3. Vasopressor effects of 2-F-A-85380 on systolic and diastolic pressure in unanesthetized, freely moving rats are reduced by pretreatment with the ganglionic blockers mecamylamine and hexamethonium. A, time-course curves demonstrating both the hypotensive effects of mecamylamine (Mec) and hexamethonium (Hex) and their ability to reduce the increase in systolic blood pressure produced by 2-F-A-85380 (2FA). Changes were determined from baseline corrected data. Either Mec (3 mg/kg) or Hex (20 mg/kg) was administered intravenously as a bolus at the -20 min arrow; 300 nmol/kg (129.9 µg/kg) 2FA was administered at the time 0 arrow. For the saline and 2FA control curves, a single bolus injection was administered at time 0. Each curve is based on data acquired from the same six rats. B, increases in systolic and diastolic blood pressure produced by 300 nmol/kg 2-F-A-85380 were effectively antagonized by pretreatment with Mec or Hex during the first 20 min following 2FA administration. Bar graphs represent the mean changes ± S.E.M. (n = 6) from baseline corrected data that occurred throughout the first 20 min after 2FA or saline administration (0 min). Following a 2-way repeated measures ANOVA (treatments, sex), significance of specific comparisons was based upon a priori one-tailed paired t tests. , significantly greater than the saline vehicle effect (p < 0.001); *, significantly less than the corresponding 2FA treatment condition (p 0.01).

Significant treatment effects of 2-F-A-85380 were present for mean changes in pressure for all sequential time periods comprising the 2-h time course as well as for the entire 2-h experiment (Fig. 2). During the first 5 min after administration, the 30-, 100-, and 300-nmol/kg doses of 2-F-A-85380 significantly increased mean systolic and diastolic pressure. Thereafter, only the high, 300-nmol/kg dose continued to produce significant effects, characterized by prolonged elevations in the mean change in systolic pressure, which diminished from 20 to 6 mm Hg over 120 min, and in the mean change in diastolic pressure, which decreased from 17 to 5 mm Hg throughout the first 60 min. Regression analyses of the 0 to 5-min increases in mean systolic and diastolic pressure were significant (p < 0.00001) and log-linear for data, whether expressed in mm Hg or as a percentage of baseline values. ED₁₀ values, estimated to produce a 10% elevation in systolic and diastolic pressures for intravenously administered 2-F-A-85380, were 59 and 40 nmol/kg, respectively. Sex differences were absent except for one nonphysiologically relevant case.

Heart Rate. Heart rate was increased by 2-F-A-85380 throughout the testing period, most effectively at the 300-nmol/kg dose. For this dose, the onset of tachycardia was rapid, with an initial peak increase at 1 min (70 ± 19 beats/min). However, the early peak was followed by a more slowly developing second and somewhat larger peak (81 ± 17 beats/min) 45 min after treatment. The 100-nmol/kg dose exhibited a similar pattern, with a second peak also occurring at 45 min, but the mean increase in rate was not significant (Fig. 2). The late-developing peaks in heart rate are reflected in the mean changes illustrated in Fig. 2. Two hours after the 300-nmol/kg dose, heart rate had not returned to pretreatment levels, but based on the rate of decrease, the time at which heart rate would return to baseline levels was within 3 h.

Significant treatment effects were present for each period analyzed. However, neither the 10-, 30-, nor 100-nmol/kg dose of 2-F-A-85380 significantly increased heart rate above control levels at any time (Fig. 2). By comparison, the tachycardia produced by the 300-nmol/kg dose was significant for all time periods. Comparisons of the magnitudes of heart rate increases and linear regression probability levels indicated that the most robust linear dose-response relationships occurred during the 10 to 30-min period (p < 0.001). Using data from this time period, the ED₁₀ estimate for 2-F-A-85380 to elevate heart rate was 58 nmol/kg, which was predicted to produce an increase of 27 beats/min (95% CL: 12-42 beats/min).

Sex was a significant factor for the tachycardic response induced by 2-F-A-85380 only during the initial 5 min after drug administration. At this time, male rats were more sensitive than females. Mean changes in heart rate for the 0 to 5-min time-period were +32 beats/min for males and +9 beats/min for females.

Safety Ratios for Cardiovascular Effects. Safety ratios based upon predicted cardiovascular responses were derived on the assumption that a 10% increase in blood pressure or heart rate would be clinically acceptable. Dosages of 2-F-A-85380 calculated to produce 10% increases in systolic pressure, diastolic pressure, and heart rate were 59, 40, and 58 nmol/kg i.v., respectively. With the projected radiopharmaceutically-relevant dose of 2[¹⁸F]F-A-85380 estimated to be 10 pmol/kg, this dose is less than 1/4000th of the dose expected to produce a 10% increase in diastolic blood pressure, which appeared to be the most sensitive of the cardiovascular measures.

Cardiac Contractility and Electrocardiogram. The only change in the QA interval produced by 2-F-A-85380 was a significant reduction at 300 nmol/kg, indicating increased cardiac contractility. This effect was present for all time periods except for the 5- to 10-min period (Fig. 2). With respect to the electrocardiogram, baseline values (mean ± S.E.M., n = 6) of three critical ECG intervals, the PR (48 ± 1 ms), QRS (17 ± 1 ms), or QTc (164 ± 8 ms), were unaffected by any dose of 2-F-A-85380 tested, even during the first 10 min after treatment with 300 nmol/kg 2-F-A-85380, which effectively increased heart rate and blood pressure.

Effects of Nicotinic Ganglionic Blockers on Vasopressor Effects. In the 20-min period between the administration of the antagonists and 2-F-A-85380 injection, mecamylamine significantly reduced (p < 0.05) systolic and diastolic blood pressure (-17 ± 6 and -13 ± 5 mm Hg, respectively) compared with the effects of the saline vehicle values (4 ± 2 and 4 ± 2 mm Hg) (Fig. 3A). Similar decreases in pressure produced by hexamethonium (-11 ± 3 and -7 ± 6 mm Hg) were not significant (Fig. 3A). Heart rate was increased by hexamethonium (69 ± 17 beats/min; p < 0.05) and mecamylamine (46 ± 18 beats/min; N.S.). This combination of hypotension and increased heart rate is indicative of ganglionic blockade. Increases in systolic and diastolic pressure produced by 2-F-A-85380 were effectively antagonized by mecamylamine and hexamethonium throughout the first 20 min after 2-F-A-85380 administration (Fig. 3B), although the hexamethonium blockade exhibited a shorter duration of action (Fig. 3A). The combination of mecamylamine and 2-F-A-85380 produced smaller increases in systolic (p 0.04) and diastolic (p 0.01) blood pressures than 2-F-A-85380 alone throughout the 2-h observation period. There were no significant interactions between either of the ganglionic blockers and 2-F-A-85380 for heart rate.

Mecamylamine and hexamethonium similarly antagonized the increases in systolic and diastolic pressure produced by 400 nmol/kg nicotine (p < 0.05), except that the duration of the hexamethonium-induced antagonism was limited to the first 10 min after nicotine administration (data not shown). Nicotine-induced elevations in systolic and diastolic blood pressure remained significant for 120 and 60 min, respectively (Vaupel et al., 2003), and mecamylamine effectively reduced these increases (p = 0.01 and p = 0.004, respectively).

Locomotor Activity in Rats. Although there was a significant treatment effect for increased locomotor activity for all time periods (data not presented), the significance was driven by the stimulatory effects of the 300-nmol/kg dose, because no other dose increased activity. This stimulant effect had a rapid onset of action, remained stable for 45 min before diminishing, and was present over the course of the entire 2 h of the study.

Body Temperature in Rats. The effect of 2-F-A-85380 on temperature was both dose- and time-dependent. Temperature was unaffected by the 10- and 30-nmol/kg doses, whereas significant peak reductions of -0.64 ± 0.26°C and -0.53 ± 0.27°C (p < 0.05, n = 6) in core temperature developed 20 min after the 100- and 300-nmol/kg doses, respectively. The corresponding change produced by the vehicle was +0.01 ± 0.04°C. Interestingly, the hypothermic response to 300 nmol/kg was followed by a significant rise in temperature that peaked 90 min after treatment (+0.65 ± 0.19°C compared with the change after the vehicle, -0.23 ± 0.09°C; p < 0.05).

Hematology and Serum Chemistry Tests in Nonhuman Primates. All baseline hematology and serum chemistry values were within normal limits for the 18 parameters measured (Table 3). Following the administration of 60 nmol/kg 2-F-A-85380, hematology and serum chemistry values remained unchanged except for a subnormal hematocrit on day 5/6, which may be attributable to two missing values (Table 3).

Dosimetry Studies in Mice. Whole-body distribution studies with 2-[¹⁸F]F-A-85380 conducted in mice demonstrated that, after bolus administration of radioligand, most of the radioactivity was rapidly cleared via the urinary pathway regardless of sex. Fractions of administered radioactivity cleared via the urinary pathway were 89 and 90% for males and females, respectively. The corresponding t_1/2 values were 0.48 and 0.50 h. Consistent with these findings, the urinary bladder wall received the highest radiation dose, making it the critical organ. Radiation dose-equivalent values calculated using the dynamic bladder model and a 2.4-h voiding interval are listed by rank-order in Table 4. Radiation dose levels calculated to be absorbed by the bladder wall were at least 10 times greater than the exposure received by any of the next five individual organs.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The development of 2-[¹⁸F]F-A-85380 as a PET imaging agent for 42^* nAChRs yielded a compound that fulfilled safety requirements for a radioligand to be administered intravenously in tracer, nonpharmacologically active masses. At pharmacologically active doses, the effects of 2-F-A-85380 resemble those of nicotine. With respect to mutagenic activity, 2-F-A-85380 demonstrated no activity in the bacterial reverse mutation assay, confirming the results of Valette et al. (2002), except to note that the high dose in our study, 5000 µg/plate, was 22 times larger than the 200-µg/plate high dose used by Valette et al. (2002).

Results of acute and expanded acute toxicity studies in mice were negative. Gross pathology was unremarkable, and histological examinations indicated a low incidence of lesions, which were characterized as mild in scope, observed commonly in mice, and not associated with 2-F-A-85380 treatment. Doses of 2-F-A-85380 evaluated in mouse toxicity studies ranged from behaviorally inactive (0.04 nmol/kg) to marginally convulsant (3500 nmol/kg). Actions of higher doses were screened in a convulsant efficacy study. Together, these three studies covered a dosing continuum of 0.04 to 10,000 nmol/kg for 2-F-A-85380.

In a small cohort of monkeys, there were no overt acute effects of 60 nmol/kg i.v. 2-F-A-85380 on hematology and serum chemistry over a 15-day period, which was comparable to the length of the expanded acute toxicity in mice. The low hematocrit observed on day 5 (Table 3) was attributed to the small sample size, as two samples could not be analyzed; the missing samples belonged to animals having the two highest control hematocrit values among the total sample size of four. This 60-nmol/kg dose of 2-F-A-85380 is 6000 times higher than the imaging dose of 10 pmol/kg.

The conclusion that the urinary bladder wall was the critical organ with respect to radiation exposure was substantiated by results from studies in humans (Kimes et al., 2003). The fraction of radioactivity cleared with urine in humans (91%) was almost identical to that in mice (90%). Nonetheless, the renal clearance of radioactivity in human was much slower than that in mice (biological half-lives, 4.0 and 0.5 h, respectively). Consistent with this difference in half-life, the estimate of the radiation dose-equivalent value for the urinary bladder wall in humans was approximately one-third (180 µSv/MBq) of that predicted from the mouse studies (461 µSv/MBq) using a 2.4-h voiding interval.

At subconvulsant doses in mice, 2-F-A-85380 resembled nicotine and 5-I-A-85380 by producing the Straub tail response, depression of spontaneous locomotor activity, hypothermia, motor incoordination, and respiratory distress. Convulsions produced by 2-F-A-85380 as well as 5-I-A-85380 (Vaupel et al., 2003) differed from nicotine in that the latter produced a type of seizure characterized as running fits, which sometimes preceded the onset of other convulsant activity. At the highest doses tested, all three compounds elicited clonic and tonic convulsions and some lethality. Mechanistically, the central nervous system stimulatory effects of toxic nicotine doses leading to convulsions can cause death due to ensuing depressant actions that result in respiratory failure due to central paralysis and peripheral blockade of muscles required for breathing (Taylor, 2001). The labored, slow respirations and gasping movements of the mouth of mice receiving subconvulsant or convulsant doses of 2-F-A-85380, which at times were followed by death, were consistent with the lethal actions associated with nicotine.

2-F-A-85380 is clearly safer than epibatidine-based ligands developed to image nAChRs. For example, fluoro-norchloroepibatidine has an LD₅₀ value of 41 nmol/kg i.v. in mice (Horti et al., 1998). This dose is substantially lower than the ED₅₀ value of 5 µmol//kg i.v. for 2-F-A-85380 to produce nonlethal convulsions.

The positive and negative inotropic effects on cardiac contractility can be measured indirectly using the QA interval (Cambridge and Whiting, 1986; Gelzer and Ball, 1997; Takahara et al., 2001). There was no evidence of any cardiodepressive activity, and increased contractility was observed only with the high, 300-nmol/kg dose of 2-F-A-85380. By comparison, we previously reported that nicotine increases contractility in the rat over a range of 60 to 400 nmol/kg based on decreases in the QA interval (Vaupel et al., 2003).

Measurement of the PR, QRS, and QTc intervals of the ECG over a period of 2 h following the administration of 2-F-A-85380, in doses ranging from minimal-to-near maximal effectiveness with respect to increasing heart rate, demonstrated no changes in these intervals. Changes in these ECG intervals can result in pronounced alterations in cardiac rhythmicity, leading to potentially fatal arrhythmias. Torsade de points is a ventricular arrhythmia characterized by cyclic changes in amplitude and polarity of the QRS complex and is usually observed in the presence of a prolonged QT interval (Pourrias et al., 1999); however, there was no evidence that QTc prolongation posed a risk factor for 2-F-A-85380 with doses as high as 300 nmol/kg i.v. The absence of any effect on the ECG provides a margin of safety that is at least 30,000 times larger than the estimated 10-pmol/kg radiopharmaceutical dose to be used in humans. The lack of effect of 2-F-A-85380 on the ECG makes it superior in terms of safety to the epibatidine-related PET radioligand ¹⁸fluoronorchloroepibatidine, which produced deaths in rats that were associated with various cardiac arrhythmogenicities (Molina et al., 1997).

Nicotine characteristically increases heart rate and blood pressure in humans (Volle and Koelle, 1965), and these effects develop after either intravenous or inhaled nicotine (Henningfield et al., 1985). Similar cardiovascular actions occur in animals, with relatively high intravenous doses producing a rapidly developing transient bradycardia (Driscoll, 1976; Kubo and Misu, 1981; Henningfield et al., 1985). The vasopressor responses to nicotine are antagonized by the ganglionic blockers hexamethonium, a peripherally restricted antagonist, and mecamylamine, a centrally and peripherally active blocker, in animals (Tseng et al., 1993; Khan et al., 1994; Dhar et al., 2000) and humans (Rose et al., 2001). The dose-related pressor and positive chronotropic effects of 2-F-A-85380, as well as the antagonism of its pressor effects by mecamylamine and hexamethonium, are consistent with the effects of a nicotine-like agonist. In addition, no episodes of transient bradycardia, which may be indicative of cardiotoxicity (Pourrias et al., 1999), were observed in the present study using doses as high as 300 nmol/kg.

The mechanisms responsible for the cardiovascular effects of 2-F-A-85380 have not been studied and at present can only be postulated based on the actions of nicotine, which are complex, and its interactions with the antagonists hexamethonium, mecamylamine, and dihydro--erythroidine. Peripheral actions of nicotine include ganglionic stimulation, release of norepinephrine from sympathetic nerve terminals and epinephrine from the adrenal medulla and/or chromaffin cells, and stimulation of chemoreceptors (Taylor, 2001). Supraspinal central sites of action include the nucleus of the solitary tract and the C1 region of the rostral ventrolateral medulla (Tseng et al., 1993; Dhar et al., 2000). At the spinal cord level, putative targets are nicotinic receptors on preganglionic sympathetic neurons within the intermediolateral cell column of the thoracolumbar spinal cord, which innervate the preganglionic sympathetic chain (Khan et al., 1994a,b). Because the high dose of 2-F-A-85380 produced near maximal increases in systolic and diastolic blood pressure in the rat, it is possible that 42^*, 34^*, and 7 nAChR subtypes associated with peripheral and central loci involved with autonomic regulation may contribute to the pressor response of nicotine and possibly its positive chronotropic action.

In conclusion, the evaluation of the pharmacology and toxicology of 2-F-A-85380, as well as its radiation dosimetry of 2-[¹⁸F]F-A-85380 proposed for use with PET to image 42^* nAChRs in humans, fulfilled Investigational New Drug (IND) safety requirements for a radiolabeled compound to be administered intravenously at radiopharmaceutical-relevant doses. 2-F-A-85380 had no activity in the reverse mutation assay (Ames test) and in acute and expanded acute toxicity studies. Effects on serum chemistry and red and white cell counts were minimal in rhesus monkeys. Over a wide range of doses studied (0.4-4000 nmol/kg), the physiological pharmacology of 2-F-A-85380 resembled nicotine. In terms of safety, convulsant activity and cardiovascular effects were considered critical with respect to the appearance of possible nicotine-like actions. Intravenous 2-F-A-85380, like nicotine, produced convulsions that were sometimes lethal. Nevertheless, the ED₁₀ value for eliciting convulsions (3.1 µmol/kg) exceeded the 10-pmol/kg dose of 2-[¹⁸F]F-A-85380 needed for imaging nAChRs by a factor of 300,000. There was no evidence of cardiac depression or changes in cardiac rhythmicity. The occurrence of any increases in blood pressure or heart rate are expected to be within an acceptable 10% change from baseline based on our estimate that the imaging dose is 1/4000th of the dose estimated to elevate diastolic pressure by 10% (40 nmol/kg) and 1/5800th of the dose predicted to increase heart rate by 10%. Based on the assessment of functional alterations in vasopressor activity, contractility, and rhythmicity of the heart, 2-[¹⁸F]F-A-85380 was considered acceptable for use in humans as a PET imaging agent for neuronal nicotinic acetylcholine receptors. Initial human studies from our laboratory have substantiated our prediction that 10-pmol/kg radiopharmaceutical tracer doses of 2-[¹⁸F]F-A-85380 would not be associated with any overt pharmacological activity (Kimes et al., 2003).

	Footnotes

 
This study was supported by the Intramural Research Program of the National Institute on Drug Abuse (NIDA). Dr. David L. Huso was supported in part by National Institutes of Health Grants RR000171 and CA062924. We also thank Drs. David McCann and James Terrill of NIDA for providing financial resources to acquire the bacterial reverse mutation assay.

Preliminary reports of this work were presented previously: Vaupel DB, London ED, Horti AG, Koren AO, Mukhin AG, Chefer SI, Pavlova OA, Stratton M, Huso DL, Tella SR, et al. (2002) 2[F-18]F-A-85380 acquires phase I approval as a radiotracer for imaging alpha4beta2 nicotinic receptors in human PET studies (Abstract 704). Drug Alcohol Depend 66:S187, College of Problems on Drug Dependence, 2002; and Vaupel DB, Koren AO, and Tella S (2001) Radiotracers for imaging nicotinic acetylcholine receptors (nACHRs): electrocardiogram effects of 5-I-A-85380 (5IA), 2-F-A-85380 (2FA), and nicotine. Program 240.2, Abstract Viewer, Society for Neuroscience, 2001.

doi:10.1124/jpet.104.073999.

ABBREVIATIONS: 2-[¹⁸F]F-A-85380, 2-[¹⁸F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine; PET, positron emission tomography; nAChR, nicotinic acetylcholine receptor; 5-[¹²³I]I-A-85380, [¹²³I]5-iodo-3-(2(S)-azetidinylmethoxy)pyridine; A-85380, 3-(2(S)-azetidinylmethoxy)pyridine; ANOVA, analysis of variance; CL, confidence limit; HTC, hematocrit.

Address correspondence to. Dr. D. Bruce Vaupel, NIDA IRP, Neuroimaging Research Branch, 5500 Nathan Shock Drive, Baltimore, MD 21224. E-mail: bvaupel{at}intra.nida.nih.gov

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