Fluoro-norchloroepibatidine: Preclinical assessment of acute toxicity

Nuclear Copyright

Medicine &a Biology, Vol. 0 1997 Elsevier Science

24, pp. 743-747, Inc.

1997

ISSN

0969-8051/97/$17.00 + 0.00 PII SO969.8051(97)00120-O

ELSEVIER

Fluoro-norchloroepibatidine: of Acute

Preclinical Toxicity

Assessment

Patricia E. Molina,’ p2* Yu-Shin Ding,’ F. luy Carr~ll,~ F. Liang,3 Nom D. Volkow,’ Naomi Pappas, ’ Michael K~har,~ Naji Aburnrad,2 S. John Gatley’ and Joanna S. Fader’ ‘BROOKHAVEN HOSPITAL.

NATIONAL

MANHASSET.

LABORATORY,

NY 11070,

‘RESEARCH

UPTON,

NY 11973,

TRIANGLE UNIVERSITY,

‘DEPARTMENT

INSTITUTE, ATLANTA,

OF SURGERY,

NORTH

SHORE

TRIANGLE

PARK,

NC 27709,

RESEARCH GA

UNIVERSITY ANL> ‘+EMORY

30322

ABSTRACT. “Fluoro-norchloroepibatidine (exo-2-(6-fluoro-3-pyridyl)-7-azabicyclo-[2.2.l]heptane [NFEP]), a labeled derivative of epibatidine, has shown promise for imaging brain nicotinic acetylcholine receptors with in conscious rats. NFEP (1.5 (I.g/kg; administered PET. We determined the dose-dependent effects of NFEP intravenously) resulted in 30% mortality. Neither 0.5 pg/kg or 0.25 &kg NFEP resulted in any significant changes in cardiorespiratory parameters, but plasma catecholamines increased (2- to 3sfold). Further studies are needed to determine the safety of NFEP that are specifically designed to assess the catecholamine response. Our results suggest that it is not advisable to initiate human PET studies with [“F]*NFEP without further evidence supporting its safety. NUCL MED BIOL 24;8:743-747, 1997. 0 1997 Elsevier Science Inc. KEY WORDS. Positron emission

Fluoro-norchloroepibatidine, tomography

Nicotinic

acetylcholine

receptors,

Hemodynamics,

INTRODUCTION

MATERIALS

Epibatidine (exo-2-(6-chloro-3-pyridyl)-7-azabicyclo~[2.2.l]heptane [NFEP]), an alkaloid isolated from the skin of the poisonous frog Epipedobates tic&r, is an agonist at the nicotinic acetylcholine receptor (nAChR) with a potency about 20 times greater than that of (-)-nicotine (1). Recent studies with [3H]epibatidine and with [ ‘Hlnorchloroepibatidine demonstrated in uioo labeling of nAChRs in mice, suggesting that radiolabeled derivatives of epibatidine could be used in neuroimaging of nAChR in human subjects (6, 14). We and others recently synthesized a ‘sF-labeled derivative of epibatidine (exo-2-(6-[‘“F]fluoro-3-pyridyl)-7-azabicyclo-[2.2.l]heptane, [‘*FINFEP) and showed that it also binds with high specificity and affinity to nAChR in mouse brain and provides exquisite images of the thalamic nAChR in the baboon using positron emission tomography (PET) (6, 10, 11, 17). Though [‘“FINFEP would appear to be an ideal radiotracer for in viuo PET imaging of the nAChR in humans, the high toxicity of the parent compound, epibatidine (3, 4, 8, 16), raised the concern as to whether the structurally similar NFEP could be safely administered to humans. We therefore undertook this study to examine the toxicity of NFEP and to use this information to estimate the safety margin that would be expected in a typical PET study using [“F]NFEP. Studies were carried out in awake rats at three different doses (1.5, 0.5 and 0.25 p.g/kg intravenously) measuring hemodynamic, electrocardiograph (EKG) and respiratory parameters and circulating levels of catecholamines. In addition, in preliminary studies, the effects of NFEP (0.01-0.6 +g/kg) on hemodynamic parameters were determined in anesthetized baboons undergoing a PET study.

Because of the documented high toxicity of epibatidine in animals and the similarity between epibatidine and NFEP, these compounds were handled with great care (gloves, lab coat, safety glasses and a shield). It is recommended that concentrated solutions be handled in a hood and that all solutions and residues of the compounds be clearly labeled and disposed of following practices for highly toxic substances. All animals studies were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care Committee.

*AJdrcss for Correspondence: Dr. Patricia Molina, Medical Brookhaven National Laboratory, Upton, NY 11973-5000. Accepted 9 July 1997.

Department,

Studies

AND

Toxicity,

METHODS

in Rats

ANIMALS PREPARATION. Male Sprague-Dawley rats (325-350 g body weight, Taconic Farms, Germantown, NY) were housed in a controlled environment, exposed to a 12/12 h light/dark cycle and fed a standard rat diet (Purina Rat Chow, Ralston Purina, St. Louis, MO) for 1 week before being used in an experiment. On the day before the experiment, animals were anesthetized with an intramuscular injection of ketamine (9 mg/lOO g) and xylazine (0.9 mg/lOO g). Using aseptic surgical techniques, we implanted a catheter (PE-50) in the left carotid artery and advanced it to the arch of the aorta, and another catheter was placed in the right jugular vein as previously described by Delman et al. (5). EKG electrodes (insulated 26 AWG copper wire) were sutured to the anterior chest musculature over the superior aspect of the right hemithorax and over the superior and inferior aspects of the left hemithorax. Leads were then tunneled out through the intrascapular region. After surgery, the animals were returned to individual cages, fasted overnight and provided water ud libitum. Experiments were started between 0600 and 0800 h the following day, with rats conscious and unrestrained throughout the duration of the protocol. The carotid catheter was connected through a

744

I’. E. Molina

three-way stopcock to a pressure transducer for monitoring arterial blood pressure and the EKG leads were connected to an EKG monitor (detailed below). Basal arterial blood pressure, baseline EKG recordings, and baseline blood pH and blood gases were obtained prior to injecting the NFEP. Animals (six to eight per group) were injected intravenously with one of three doses of NFEP (manuscript describing the synthesis is in preparation): 1.5 p,g/kg = 7.8 nmol/kg, 0.5 kg/kg = 2.6 nmol/kg or 0.25 p,g/kg = 1.3 nmol/kg. Having determined that the high dose resulted in mortality, the medium and low doses were used for further characterization of the hemodynamic and respiratory effects of the drug. Additional blood samples for pH and catecholamine concentrations were taken 5, 15, 30 and 45 min after NFEP injection. CARDIAC, HEMODYNAMIC AND ACID-BASE STATUS. Rats were implanted with EKG leads 24 h prior to the protocol. The leads were attached to an EKG monitor (Sirecust 404, Siemens Medical Systems, Englewood, CO), and the EKG was monitored continuously on lead II from just prior to the NFEP injection up until 30 min after the drug administration. Blood pressure was measured using the same monitor (Sirecust 404) attached to the carotid catheter. Blood pressure was determined intermittently prior to drug administration and up to 30 min later, alternating use of the catheter between monitoring pressure and withdrawing blood via a three-way stopcock. Blood samples (125 FL) were obtained for the determination of pH and blood gases at baseline, 5, 15, 30 and 45 min after NFEP injection (170 pH/Blood Gas Analyzer, Corning Medical, Medford, MA).

Analytical Methods. Blood withdrawn for the determination of catecholamines was transferred to a tube containing 20 FL of 9% EDTA and 6% glutathione, and the plasma was analyzed for epinephrine and norepinephrine as previously described (13). In brief, 3, 4-dihydroxybenzylamine (DHBA) was added to plasma as an internal standard, and catecholamines were absorbed onto alumina at pH 8.5 and eluted with perchloric acid (PCA; 0.1 M). The processed samples were quantified by high performance liquid tomography (HPLC) using a chromatographic analyzer with a catecholamine column and an electrochemical detector (Bioanalytic Systems, West Lafayette, IN). Using this system, the detection limit for epinephrine, norepinephrine and DHBA was 10 pg. Statistical Analysis. Because the high dose of NFEP was associated with mortality in two of six rats studied, only the medium and low doses were used for the full characterization of the hemodynamic, hormonal and respiratory effects of NFEP. Differences in these parameters between basal and after drug administration were established using ANOVA followed by Student Newman-Keuls. Statistical significance was set at p < 0.05.

Preliminary

Studies

in Anesthetized

Baboons

Adult female baboons (Papio anubis) were initially anesthetized with ketamine hydrochloride (10 mg/kg), intubated with a cuffed endotracheal tube (Porex, Fariburn, GA) and transported to the PET facility. An esophageal stethoscope/core body temperature probe (SpaceLabs Bedside Monitor model 903038-1326, Redmond, WA) was inserted to automatically monitor core body temperature during the study. General anesthesia was started and maintained with oxygen (1500 mL/min), nitrous oxide (800 mL/min) and isoflurane (1.2-1.5%). A venous and an arterial catheter were then inserted into the antecubital vein and popliteal artery, respectively, using 20gauge catheters. The venous catheter was used for tracer

et al.

injection and the arterial catheter for blood sampling and for automatic blood pressure recording (SpaceLabs Bedside Monitor). EKG electrodes were attached to the chest of the baboon and connected to an EKG monitor (SpaceLabs Bedside Monitor). On average, it took approximately 60 min from the time of ketamine injection to the time of [‘sF]NFEP injection. The anesthesia was maintained between 3 and 6 h, depending on the experimental protocol. Continuous monitoring of blood pressure, EKG, heart rate and core body temperature was started at least 30 min prior to [‘*F]NFEP injection and recorded throughout the scanning period. Simultaneously, a capnograph (SpaceLabs) was employed using a side stream vacuum sampling technique that acquires respiratory data from the endotracheal tube. This allowed constant monitoring of ETCO,, minimum CO,, respiration rate, inspired and expired nitrous oxide percents and inspired 0, percent. A pulse oximeter was also attached, which allowed continuous monitoring of hemoglobin oxygen saturation (SpO,). Three different baboons were studied on multiple occasions. A total of 15 separate injections of [‘*F]NFEP of different specific activities (1.3-3.2 Ci/kmol at time of injection), alone or with other compounds, were made, allowing the assessment of the effect of chemical masses of NFEP ranging from 0.2 pg to 10 I.L~ (0.01-0.6 p,g/kg) on blood pressure, EKG, heart rate, core temperature, respiration rate, ETCO, and SpO,. Circulating levels of catecholamines were not determined in the baboon studies.

RESULTS Studies in Rats MORTALITY. In the six animals who (1.5 kg/kg), there was 30% mortality animals in the high-dose group recovered mental period.

received high-dose NFEP within 5 min. Surviving over the 45-min experi-

HEMODYNAMIC PARAMETERS. In the high-dose NFEP, mean arterial blood pressure (MABP) averaged 119 ? 2 mmHg during the basal period and reached an average of 151 ? 5 mmHg 30 set after drug injection (Fig. 1). MABP averaged 145 ? 6 mmHg 1 min after injection and had returned to 122 ? 3 mmHg by 3 min after NFEP injection. MABP was 113 -+ 2 mmHg 15 min after injection of NFEP and was not different from baseline at 30 and 45 min. Heart rate averaged 358 ? 24 beats/mm during basal and decreased to 190 ? 43 (30 set), 168 ? 11.96 (1 min) and 313 ? 24 (3 min), but had returned to 333 ? 49 by 5 min after drug injection (Fig. 2). The hemodynamic effects of NFEP at the medium and low dose were insignificant. In the medium-dose group, MABP averaged 123 ? 3 mmHg during basal, rose to 142 2 3 mmHg within 30 set of NFEP injection, but was not different from baseline at 1 min (128 -C 5 mmHg) and remained stable thereafter. No significant changes in heart rate were noted in this group (346 ? 19 beats/min at basal and 363 -C 18 beats/mm). Similarly in the low-dose group, NFEP did not produce any changes in either MABP (117 t- 2 mmHg at basal and 124 2 2 mmHg after 1 min) or in heart rate. CARDIAC ABNORMALITIES. The EKGs cant changes from the baseline rhythm. high-dose NFEP demonstrated varying mogenicity, as shown in Figure 3. None either the medium or low dose, however, in cardiac rhythm. BLOOD

an

GASES.

immediate

The injection alteration in

were monitored for signifiAnimals injected with the degrees of cardiac arrhythof the rats injected with manifested any alterations

of the high dose of NFEP resulted in breathing pattern, which initially

Acute

Toxicity

of Fluoro-norchloroepibatidine

745

160,

*a

100 i--,

0

7

1

2

3

~4

5

Time (min)

'--~.

'~-,~-

-~.

0

15

POST INJLClXON

3U WCONDS

POST

5 MINU’IW

_ I 45

30

Time (min)

FIG. 1. Mean arterial blood pressure (MABP) of animals injected with high ( 1.5 pg/kg) (f?lled circles), medium (0.5 @kg) (Riled squares) and low (0.25 pg/kg) (Ned triangles) doses of epibaticlme as a function of tune after its administration. For clarity, insert depicts values obtained during the first 5 min after epibatidine injection. Values are mean + SEM (n = 6-8 per group). Asterisk denotes p < 0.05 compared to basal values.

consisted of a significant suppression in respiratory rate (unfortunately, this was not possible to assess with the naked eye), followed by a compensatory increase in respiration from baseline value of 105 2 7 breaths/mm to 153 2 7 breaths/mm at 2 min. This remained elevated for the remainder of the observation period (45 min). No significant alterations were noted in the respiratory rate of the animals that were injected with either the medium or the low dose of NFEP (Table 1). Baseline arterial blood pH averaged 7.50 + 0.01 in all three groups. A transient decrease in blood pH was observed during the first 5 min after high-dose NFEP administra-

2

3

Time (min)

E‘400 g 350 g 300 L 5 250 f

200

!

150

I 0

r-15

,,rrr.

30

45

Time (mln)

FIG. 2. Heart rate (beats per minute [BPM]) of animals injected with high (1.5 pg/kg) (filled circles), medium 0.5 pg/kg (filled squares) and low 0.25 t.rg/kg (filled triangles) doses of epibatidine as a function of time after its administration. For clarity, insert depicts values obtained during the fast 5 min after epibatidine injection. Values are mean + SEM (n = 6-8 per group). Asterisk denotes p < 0.05 compared to basal values.

INJKTION

POST INJECIION

3U MINU’I’BS

FIG. 3. Representative ing and after epibatidine from an animal treated

tracing obtained at baseline and duradministration of an EKG recording with the high dose of epibatidine.

tion, with no changes noted in the animals injected with the medium and low doses of NFEP. Arterial blood PCO, showed a transient, statistically insignificant 10% increase in the high-dose NFEP group. No changes were noted in the mediumand low-dose groups. Arterial PO, decreased transiently during the first 3 min after NFEP injection in the high-dose group basal values of 83 ? 3% to 59 ? 28%, but had recovered at 5 min. No changes in any of the parameters assessed-pH, PCO,, PO, and HCO,-were noted in the animals administered the mediumor low-dose NFEP. CATECHOLAMINES. Plasma epinephrine and norepinephrine levels of saline controls did not change throughout the course of the experiment and averaged 277 + 47 pM and 284 i- 84 pM, respectively (Fig. 4). Intravenous administration of the high-dose NFEP resulted in a marked lo-fold increase in epinephrine levels 5 min after its administration, which owing to variability, did not reach statistical significance. Thereafter, epinephrine levels dropped, but were significantly higher than basal values at 15-45 min after NFEP injection. The medium dose of NFEP produced a delayed 2-fold increase in circulating epinephrine levels 45 min after its administration. While no immediate effect was noted in the low-dose group, epinephrine levels were 2- to 3-fold higher than basal at 30 and 45 min after NFEP injection. Norepinephrine levels were elevated lo-fold 5 min after the

746

TABLE

P. E. Molina

1. Effects

of Medium-

and

Low-Dose

NFEP

on Blood

Gas

and

AcidMBase

et al.

Parameters

Blood gas

Dose level

DH

Med Low

7.52 7.51

+ 0.02 i- 0.01

7.52 7.53

5 0.01 ? 0.02

7.51 7.52

+- 0.02 k 0.01

7.52 7.54

5 0.02 t 0.01

7.53 -+ 0.01 7.54 ? 0.02

PCO,

Med Low

36.2 38.9

k 1.6 -c 1.5

36.3 37.2

? 0.9 -t 2.4

36.3 36.1

2 1.1 ? 1.9

36.4 36.3

? 1.8 k 1.4

33.9 31.3

2 2.3 ? 2.2

PO,

Med Low

81.2 89.5

+ 2.5 ? 10.2

85.3 83.4

-+ 3.1 k 4.6

86.7 89.0

k 1.7 ? 6.1

88.2 91.8

? 0.1 ? 4.2

91.4 97.4

5 2.4 2 5.2

HCO,

Med Low

29.7 31.2

t 0.1 ? 1.1

30.0 31.0

2 1.1 + 1.1

29.4 29.4

k 1.7 ? 1.3

30.0 29.7

? 2.1 ? 0.9

27.4 26.6

-c 1.3 2 1.2

0 min

5 min

15 min

30 min

Values are mean ? SEM of animals injected with low- (0.25 pp/kg) and medium-dose and HCO, concentrations at hasal, 5, 15, 30 and 45 min after NFEP injection.

(0.5 j&kg)

administration of the high dose of NFEP. However, owing to the variability in the response, these values did not reach statistical significance. Norepinephrine levels were elevated 5-fold 15-45 min after NFEP injection. A delayed increase in circulating norepinephrine levels was observed in the animals that were administered the medium dose of NFEP. Values were significantly higher (2-fold) than basal levels at 30 and 45 min after NFEP injection. A similar late increase in norepinephrine levels was observed in the animals

with the low dose of NFEP. Values than basal levels at 30 and 45 min after

injected higher

NFEP. Blood samples were collected

45 min

for pH, PCO,,

were NFEP

PO,

significantly injection.

Studies in Baboons No significant change in heart rate, respiration rate, SpO, and ETO, after injection of [‘“FINFEP in doses ranging from 0.01-0.6 kg/kg were noted in baboons. The EKG tracings recorded at the end of each study were not different from tracings recorded at the baseline. However, occasionally a 20-55% increase in mean arterial blood pressure was documented within 20 min of the NFEP injection.

DISCUSSION

0

15

0

15

30

45

30

45

= 6000 , E 3

Time

(min)

FIG. 4. Arterial plasma levels of epinephrine (top panel) and norepinephrine (bottom panel) of animals injected with high (1.5 p,g/kg) (filled circles), medium (0.5 pg/kg) (filled squares) and low (0.25 pglkg) (fdled triangles) doses of epibatidine as a function of time after its administration. Values are means f SEM (n = 6-8 per group). Asterisk denotes p < 0.05 compared to basal values.

Acute toxicity studies in animals are usually required to obtain approval for administering any chemical compound to humans. The information obtained from such studies has been useful in estimating the margin of safety for administration of the specified chemical mass of positron emitter-labeled compounds for human PET studies. Though the LD,, has been used extensively in the past, recent guidance from the Food and Drug Administration generally supports the use of single-dose studies in animals as a basis for single-dose studies in humans (2). It also proposes that test compounds be administered to small numbers of animals to identify doses causing no adverse effect and doses causing major lifethreatening toxicity rather than calculating lethality parameters based on large numbers of animals (15). PET radiotracers are usually derived from chemical compounds that have fairly low toxicity in animals and/or those for which there is human safety data. They are typically administered in very small doses (e.g., microgram or submicrogram levels) that are usually devoid of pharmacological effects. The ratio of the toxic dose to the radiotracer dose (safety margin) is usually quite large, and for this reason, chemical toxicity is not usually a limiting factor in obtaining approval for their use in human studies. For example, two PET radiotracers, [“Clcocaine and [“C]L-deprenyl, have LDs,s of 17.5 and 81 mg/kg intravenously in rats (12) and in mice (16), respectively, and thus when a total mass of 10 pg (0.14 pg/kg for a 70 kg subject) is administered in a PET study, the safety margin relative to a toxic dose is 125,000 and 578,000. The present study in awake rats determined the effects of doses of NFEP ranging from significant toxicity to no measurahle effect. Our results indicate that 1.5 pg/kg NFEP administered intravenously resulted in 30% mortalitv, I while at 5 ..,.~ &kg no significant effects on .

Acute

Toxicity

of Fluoro-norchloroepibatidine

cardiorespiratory parameters were observed. These results are similar to epibatidine itself, which showed significant toxicity at minimally detectable cardiorespiratory effects in anesthetized rats at 0.5 kg/kg (9). While 0.5 and 0.25 kg/kg of NFEP had no cardiorespiratory parameters, these doses produced a 2- to 3-fold elevation of plasma catecholamines relative to baseline at 45 min. Although this was not manifested by either an increase in heart rate or in MABP, this increase in circulating catecholamines may reflect a delayed stress response and warrants further investigation. Having determined the relative safety of the compound at the 0.5-0.25 pg/kg dose level in rodents, we performed preliminary PET studies using [“F]NFEP in anesthetized baboons. Though we observed no cardiorespiratory effects nor alterations in their EKGs, a moderate rise in MABP was noted. Although the changes in pressure could be attributed to varying depths in anesthesia and such changes are common during PET studies with other tracers, our studies do not allow us to dissociate the observed fluctuations in pressure from the effects of the NFEP administration. Nevertheless, one could speculate that in the conscious animals, the rises in pressure could have been of greater magnitude than those observed under anesthesia. Thus, further studies are needed to elucidate the effects of NFEP on MABP, in particular in awake laboratory animals, before the evaluation of [‘“FINFEP in human subjects. In addressing the safety of performing PET studies with [ISFINFEP in humans, it is the specific activity of the radiotracer that determines the chemical mass of NFEP to be administered. NFEP is prepared in our laboratory by a nucleophilic aromatic substitution reaction of a t-Boc-protected derivative of epibatine in which no-carrier-added fluoride ion is substituted for a trimethyl ammonium group (7). This precursor has a 15,000-fold lower potency for the nAChR than bromo-epibatidine, which has also recently been used to synthesize [‘“FINFEP (F. I. Carroll, unpublished data) and which reduces concerns over precursor contamination of the product. In addition, the substitution reaction proceeds in unusually high yield (70%) and very high-specific activities are obtained (2-5 Ci/pmol at end of bombardment). With a synthesis time of 65 min, a specific activity of 1.3-3.2 Ci/kmol can be obtained at time of injection. Based on the high tracer uptake and specificity for the thalamus in hahoons, we estimate that a 5 mCi injection of NFEP would he sufficient for a human PET study. This would correspond to 0.74 pg or 0.01 pg/kg for a 70 kg individual, a factor of 50 lower than rhe dose that we determined to have no cardiorespiratory effects and a factor of 150 lower than the toxic dose. Nevertheless, in view of the delayed elevation of catecholamines in the awake rats as well as the occasional rise in MABP in the anesthetized baboon, we conclude that further study on the safety of NFEP is warranted prior to its recommendation for human PET studies.

CONCLUSION These studies established: (1) a dose of 1.5 kg/kg in awake rats results in significant mortality, with death occurring in two out of six rats; (2) a dose of 0.5 kg/kg produces no significant effects on cardiorespiratory parameters either in awake rats or in anesthetized hahoons undergoing a PET study with [‘“FINFEP; (3) doses of 0.5 and 0.25 kg/kg in awake rats are associated with a 2- to 3-fold elevation in plasma catecholamines at 45 min post-NFEP administration. Thus, even though the synthesis of [‘sF]NFEP in highspecific activity would permit human PET studies to he carried out with the administration of a total dose
747

taken together supporting the

our safety

results do not provide sufficient evidence of NFEP for use in human PET studies.

This research eras carried out, in part, at Brookhaven National Laboratory under contract DE-AC02-76CH00016 with the U.S. Department of Energy and supported by its Office of Health and Environmental Research. This research was also supported in part by the Office of Naval Research (N00014-95-l-0865), by the National Institutes of Health (NINDS NS-153800, and RR-00165), by the Council for Tobacco Research, by the National Institutes on Drug Abuse and by Fisons Pharmaceuticals (now AstraArcus USA). The authors are grateful to Payton King, Rebecca Naukam and Luping Qian for their excellent technical assistance.

References 1. Badio B. and Daly J. W. (1994) Epibatidine, a potent analgesic and nicotinic agonist. Mol. Pharmacol. 45, 563-569. 2. Choudary J., Contrera J. F., DeFelice A., DeGeorge J. J., Farrelly J. G., Fitzgerald G., Goheer M. A., Jacobs A., Jordan A., Meyers L., Osterherg R., Resmck C., Sun J. and Temple R. (1996) Response to Munro and Mehta ~rooosal for use of single-dose roxicoloev studies m sun~x)rr sinele-dose &es of new drugs in’humans. Clin. Pl;;?macol. Ther. 59, 265-2‘$7. 3. Damaj M. I. and Martin B. R. (1996) T o I-~rdnce to the antinociceptive effect of epihatidine after acute and chronic administrauon in mice. Eur. J. Pharmacol. 300, 51-57. 4. Damaj M. I., Creasy K. R., Grove A. D., Rosencrans J. A. and Martin B. R. (1994) Pharmacological effects of epiharidine optical enantiomers. Brain Res. 664, 34-40. 5. Delman K., M&k S. K., Abumrad N. N., Lang C. H. and Molina P. E. (1996) Resuscitation with lactated ringer’s solution after hemorrhage: Lack of cardiac toxicity. Shock 5, 298-303. 6. Ding Y.-S., Gatley S. J., Fowler J. S., V o Ik ow N. D., Aggarwal D., Logan J., Dewey S. L., Liang F., Carroll F. I. and Kuhar M. J. (1996) Mapping nicotinic acetylcholine with PET. Synapse 24, 403-407. 7. Ding Y.-S., Llang F., Fowler J. S., Carroll F. 1. and Kuhar M. J. (1997) Synthesis of [‘sF]norchlorofluoroepihatidine and its N-methyl derivative: New PET ligands for mapping nicotinic acetylcholine receptors. I. Label. Cmpnds. R&&arm. in press. 8. Fisher M., Huangfu, D., Shen T. Y. and Guyenet I’. G. (1994) Eplhatidine, m alkaloid from the poison frog Epipedobates tricolor, is a powerful ganglionic depolarizing agent. J. Phunwcol. Exp. Ther. 270, 702-707. 9. Horti A., Ravert H. T., London E. D. and Dannals R. F. (1996) Synthesis of a radio tracer for studying nicotinic acetylcholine receptors: (~)-exo-2-(2-[‘“F]fluoro-5-pyridyl)-7-azahicycl~~[2.2.l]heptan~. J. Label. Cmpnds. Radiopharm. 38, 355-365. 10. Horti A., Scheffel U., Dannals R. F., Stathis M., Finley P. A., Raven H. T. and London E. D. (1996) [‘sF](“)e~o-2-(2-[‘~F]fluoro-5-pyrldyl)-7-azahicyclo[2.2.l]heptane, a radioligand for m viva labeling and imaging of central nicotinic acetylcholme receptors [abstract]. J. Nucl. Med. 37, 1 I. 11. London E. D., Scheffel U., Kimes A. S. and Kellar K. J. (1995) In viva labeling of nicotinic acetylcholine receptors in hram with [‘Hlepihatidine. Eur. J. Pharmacol. 278, Rl-R2. 12. Merck (1989) Merck Index, 11th edn. (1989) Merck and Co. Inc., Rahway, NJ. 13. Molina P. E., Hashiguchi Y., Ajmal M. and Ahumrad N. N. (1994) Differential hemodynamic, metabolic and hormonal effects of morphine and morphine-6-glucuronide. Brain Res. 664, 126-132. 14. Scheffel U., Taylor G. F., Kepler J. A., C arm11 F. I. and Kuhar M. J. (1995) In viuo labeling of neuronal nicotinic acerylcholine receptors with radiolabeled isomers of norchloroepihatidine. Neurure@rt 6, 2483-2488. 15. United States Government (1996) Smgle dose acute toxicity testing for pharmaceuticals. Federal Register 61, 43934-43935. 16. Sullivan J. P., Decker M. W., Briom J. D., Donnelly-Roberts D., Anderson D. J., Bannon A. W., Kang C.-H., Adams P., Piattoni-Kaplan M., Buckley M. J., Gopalakrishnan M., Williams M. and Arneric S. P. (1994) (+)-epihatidine elicxs a diversity of in eutr~ and in uvo effects mediated by nicotinic acetylcholine receptors. 1. Pharmncol. Exp. Ther 271,624-631. 17. Villemagne V. L., Horti A., Scheffel U., Raven H. T., Finley P., London E. D. and Dannal R. F. (1996) Imaging nicotinic acetylcholine receptors in hahoon hrain hy PET [abstract]. J. i&cl. Med. 37, 1 lp.