Stereoselective Potencies and Relative Toxicities of Coniine

Chem. Res. Toxicol. 2008, 21, 2061–2064

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Stereoselective Potencies and Relative Toxicities of Coniine Enantiomers Stephen T. Lee,* Benedict T. Green, Kevin D. Welch, James A. Pfister, and Kip E. Panter Poisonous Plant Research Laboratory, Agricultural Research SerVice, United States Department of Agriculture, 1150 E. 1400 N., Logan, Utah 84341 ReceiVed June 24, 2008

Coniine, one of the major toxic alkaloids present in poison hemlock (Conium maculatum), occurs in two optically active forms. A comparison of the relative potencies of (+)- and (-)-coniine enantiomers has not been previously reported. In this study, we separated the enantiomers of coniine and determined the biological activity of each enantiomer in Vitro and in ViVo. The relative potencies of these enantiomers on TE-671 cells expressing human fetal nicotinic neuromuscular receptors had the rank order of (-)coniine > (()-coniine > (+)-coniine. A mouse bioassay was used to determine the relative lethalities of (-)-, (()-, and (+)-coniine in ViVo. The LD50 values of the coniine enantiomers were 7.0, 7.7, and 12.1 mg/kg for the (-)-, (()-, and (+)- forms of coniine, respectively. The results from this study demonstrate that there is a stereoselective difference in the in Vitro potencies of the enantiomers of coniine that directly correlates with the relative toxicities of the enantiomers in ViVo. Introduction Conium maculatum L., commonly referred to as poison hemlock or spotted hemlock, is a member of the Umbelliferae family and is known worldwide for its acute toxicity to animals. The affected species include cattle, sheep, goats, horses, elk, pigs, poultry, range turkeys, quail, and humans (1). The principle toxins in C. maculatum have been identified as piperidine alkaloids of which coniine (1) and γ-coniceine are the most prevalent and account for most of the toxicity (1, 2). Coniine (1) is a nicotinic acetylcholine receptor (nAChR) agonist. Whole cell current recordings of Xenopus oocytes expressing fetal rat muscle type nAChR exhibited a dosedependent response to coniine (3). The IC50 values of coniine (1) for the displacement of [125I]-R-bungarotoxin or [3H]-cytisine from chick embryonic muscle and brain preparations have been shown to be in the micromolar range (4). In ViVo, it is documented that coniine (1) causes a biphasic response of first stimulation followed by blockade of nicotinic receptors in the central nervous system and periphery (1, 5). Clinical signs of poisoning include protrusion of the nictitating membrane, excessive salivation, and frequent urination and defecation, loss of coordination, muscle weakness, and tremors followed by collapse and death due to respiratory failure (5, 6). Coniine (1), as found in C. maculatum, is a mixture of (+)and (-)-enantiomeric forms, with the (+)-form as the predominant enantiomer (2). Stereochemical integrity is a significant factor in determining the specificity of biological effects of chiral compounds, both in natural products and synthetic compounds (7). For example, the (+)-form of ketamine is four times more potent in humans than the (-)-form, and in human neonates, the (-)-enantiomer of ibuprofen is more effective than the (+)enantiomer (8). No toxicity studies have been reported with both of the coniine (1) enantiomers measured separately. Single oral doses of enantiomeric mixtures of coniine (1) have been shown to be toxic to ewes, mares, cows, quails, chicks, and turkey * Corresponding author. Tel: (435) 752-2941. Fax: (435) 753-5681. E-mail: [email protected].

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chicks (1). The oral LD50 of an enantiomeric mixture of coniine in mice is reported to be 100 mg/kg (1). Little is known about the specific toxicities of the individual coniine enantiomers in ViVo and in Vitro. Previously, we reported the separation, isolation, and toxicity of anabasine (2) enantiomers from Nicotiana glauca and ammodendrine (3) enantiomers from Lupinus formosus plants (9, 10). In both cases, we observed differential toxicities between the individual ammodendrine enantiomers and individual anabasine enantiomers. These results led us to investigate the toxicity of the individual coniine (1) enantiomers. To separate coniine (1), we used preferential crystallization with (+)- and (-)-mandelic acid. Enantiomers of compounds often have very different biological activities; thus, it was important to measure the relative potencies of the (+)- and (-)-coniines (1) individually. In our study, the neuromuscular nicotinic receptor (nAChR) agonist potencies of coniine (1) enantiomers were assessed using a human tumor cell line (TE-671) expressing fetal muscle type nAChRs, and the toxicities of the enantiomers were measured in a mouse lethality bioassay.

Materials and Methods Materials. Ammonium hydroxide, N,N-dimethyl formamide (DMF), and sulfuric acid were obtained from Fisher Scientific (Pittsburgh, PA). (()-Epibatidine was obtained from SigmaAldrich (St Louis, MO) and ammonium acetate from VWR (Bristol, CT). Fetal bovine serum and penicillin/streptomycin were from Media Tech, Inc. (Herndon, VA), Dulbecco’s modified Eagle’s medium was from the American type Culture Collection (Manassas, VA), and the fluorescence dye kits were purchased from Molecular Devices (Sunnyvale, CA). Isolation of (+)- and (-)-Coniine. Coniine (1) was isolated using previously described methods (11). (()-Coniine (1.0102 g, 7.95 mmol) was added to a 50 mL screw top glass test tube. (()-Coniine-HCl was prepared by bubbling HCl gas through the 40 mL diethyl ether solution resulting in a fine white precipitate. The diethyl ether was filtered from the crystals, the crystals collected, and dried in Vacuo. The (()-conine-HCl was

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recrystallized (3×) by dissolving the (()-coniine-HCl in hot acetone and allowing it to crystallize at -20 °C for 16 h. Finally, the (()-coniine-HCl crystals were filtered from the acetone, crystals collected, and dried in Vacuo (0.813 mg, 4.96 mmol, 62% yield): fine cottony crystals, sublimes 155-167 °C; [R]25D ) -0.4° (c ) 0.50, MeOH). Optical rotations were recorded on an Autopol IV polarimeter (Rudolph Research Analytical, Flanders, NJ). (()-Coniine (502.6 mg, 3.95 mmol) and (-)-mandelic acid (605.5 mg, 3.98 mmol), were added to a 16 mL screw top glass test tube. To the (()-coniine and (-)-mandelic acid mixture was added 1.6 mL of MeOH and the mixture warmed until all (()-coniine and (-)-mandelic acid was dissolved, then anhydrous diethyl ether (4.00 mL) was added and the mixture put on ice. Crystals of (+)-coniine (-)-mandelate began to form within 30 min of adding the diethyl ether. After 16 h at -20 °C, the solvent was filtered from the crystals, the crystals collected, and dried in Vacuo. The crystals were recrystallized (3×) by dissolving the crystals in MeOH (1.6 mL), adding diethyl ether (4 mL), and then allowing them to crystallize at -20 °C. The (+)-coniine (-)-mandelate crystals were filtered for a final time, collected, and dried in Vacuo (314.7 mg, 1.13 mmol, 57% yield): feathery needles, mp 115 °C; [R]25.1D ) -47.8° (c ) 0.52, MeOH). This same procedure was used with (()-Coniine (513.7 mg, 4.04 mmol) and (+)-mandelic acid (635.1 mg, 4.17 mmol). The (-)-coniine (+)-mandelate crystals were collected and dried in Vacuo (364.0 mg, 1.30 mmol, 65% yield): feathery needles, mp 117-119 °C; [R]24.7D ) +49.0° (c ) 0.60, MeOH). (+)-Coniine (-)-mandelate (77.9 mg, 0.279 mmol) was weighed into a 16 mL screw top glass test tube and then dissolved in distilled deionized water (2 mL). The solution was basified to pH 9 by dropwise addition of ammonium hydroxide. The basic solution was extracted (2 mL, 2×) with diethyl ether. The diethyl ether extracts were combined and filtered through anhydrous Na2SO4 into clean 7 mL screw top glass vials and the solvent evaporated under N2 at 60 °C until approximately 1 mL of diethyl ether remained. (+)-Coniine HCl was prepared by bubbling HCl gas through the 1 mL diethyl ether solution resulting in a fine white precipitate. The diethyl ether was filtered from the crystals, the crystals collected, and dried in Vacuo. The (+)-conine-HCl was recrystallized (3×) by dissolving the (+)-coniine-HCl in hot acetone and allowing it to crystallize upon cooling. Finally the (+)-coniine-HCl crystals were filtered from the acetone, crystals collected, and dried in Vacuo (12.4 mg, 0.0975 mmol, 35% yield): fine cottony crystals, sublimes 160-196 °C; [R]26.1D ) +4.6° (c ) 0.49, MeOH). The same procedure was used to obtain (-)-coniine-HCl from the (-)coniine (+)-mandelate (82.6 mg, 0.300 mmol). The (-)-coniineHCl crystals were collected and dried in Vacuo (19.3 mg, 0.152 mmol, 51% yield): fine cottony crystals, sublimes 160-193 °C; [R]26.1D ) -5.2° (c ) 0.50, MeOH). Acute Toxicity Determinations. Known amounts of (()coniine-HCl, (+)-coniine-HCl, and (-)-coniine-HCl were dissolved in physiological buffered saline solution at concentrations of 1 mg/mL. The solutions were stored in sterile injection vials for toxicity testing. Seventy-five Swiss-Webster male mice, 15 to 20 g (Simonsen Laboratories, Gilroy, CA) were weighed after a 12 h fast and dosed intravenously via the tail vein. Prior to injection, the mice were randomly divided into 3 groups of 25 and maintained under a heat lamp for 15 min to dilate the tail vein. The tail vein was cleaned with 70% ethanol, and i.v. injections were accomplished with a tuberculin syringe equipped with a 1.27-cm-long 27-gauge needle. The volume injected

Lee et al.

(0.05-0.2 mL) varied depending on the dosage delivered. Time of injection, clinical effects, and time of death were noted and recorded. Mice were closely observed for 1 h after injection. The protocol for animal use in this research was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Utah State University, Logan, Utah. The LD50 for individual alkaloid toxicity was determined by a modified up-and-down method (12) and was calculated using the PROC PROBIT procedures of SAS (SAS Institute Inc., Cary, NC) on a logistic distribution of the survival data. Confidence (fiducial) intervals (95%) were also calculated using the same program. Nicotinic Agonist Actions on the Fetal Human nAChR. The rhabdomyosarcoma cell line TE-671 was obtained from ATCC (Manassas, VA, USA), and the membrane depolarization responses from the addition of nicotinic agonist toxins were measured by changes in fluorescence of a membrane potentialsensitive dye as previously described by Lee et al. (10), with the following modifications. The membrane potential dye solution was prepared by dissolving one vial of the Molecular Devices dye (Catalog number R8042) into 22 mL of Hanks’ balanced salt solution (HBSS) supplemented with 20 mM Hepes (pH 7.4). Ninety-six-well black walled cell culture plates were equilibrated to room temperature for 10 min, medium, aspirated, and replaced with 100 µL of the membrane potential dye solution into each well. The cells were incubated with the dye at room temperature for 30 min before experiments were initiated. Serial dilutions of a compound for concentrationresponse analysis were prepared in 96-well V-bottom plates by addition of the required volume of a methanolic stock solution. After evaporation of the methanol, the compound in each well was redissolved in membrane potential dye solution. Fluid (agonist or KCl) additions and membrane potential measurements were performed using a Flexstation II (Molecular Devices Corporation, Sunnyvale, CA, USA). Readings were taken every 1.12 s for 255 s, a total of 228 readings per well. The first 17 s were used as a basal reading. At 18 s, 50 µL of a test compound was added to assess agonist activity. At 180 s, 25 µL of KCl in saline was added to attain a final concentration of 40 mM KCl in the dye-HBSS solution bathing the cells. This served as a depolarizing calibrant and to correct for interwell differences in dye loading and cell count. Responses were calculated as equal to: (Fmax(compound) - FBasal)/(Fmax(calibrant) - FBasal). Depolarizing responses to agonists were normalized to the maximum response generated by (()-epibatidine and fitted to a sigmoidal dose-response equation and graphed with Prism version 4.03 (GraphPad Software, San Diego, CA, USA) to determine EC50 using a sigmoidal dose-response equation with variable slope, efficacy (maximal activation), and Hill coefficients. Ten duplicate wells with concentrations of epibatidine from 0.01 nM to 1 mM in log10 increments were included in every 96-well plate, and a stable baseline and maximal epibatidine response of 60% of the KCl calibrant was required for data from that plate to be included in analysis of individual compound responses. In all cases, the limit for statistical significance was set at P < 0.05.

Results and Discussion (+)- and (-)-coniine (1) (Figure 1) were isolated from (()coniine using preferential crystallization with (+)- and (-)mandelic acid. Racemic, (()-coniine, (+)-coniine, and (-)coniine were converted to HCl salts by reaction with HCl gas. The measured optical rotations for (+)-coniine ([R]26.1D )

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Figure 1. The chemical structures of coniine (1), anabasine (2), ammodendrine (3), and nicotine (4). (*) Denotes the asymmetric carbon.

Figure 2. Representative reponses of epibatidine and coniine enantiomers in TE-671 cells. The change in relative fluoresence over time of responses from one experiment with TE-671 cells for epibatidine, coniine, (+)-, and (-)-coniine. Agents were added at the times indicated by arrows. Responses represent the concentration of agonist that elicited the maximum membrane depolarization for each compound (10 µM, and 1 mM for epibatidine and the three forms of coniine).

+4.6°) and (-)-coniine ([R]26.1D ) -5.2°) were equal and opposite confirming that they were isolated enantiomers while the measured optical rotation for (()-coniine ([R]25D ) -0.4°) confirmed it was a racemic mixture. Pharmacological potencies of the coniine (1) enantiomers were determined with TE-671 cells, which express fetal human neuromuscular nAChRs. Representative tracings of responses from single wells of each compound from the same experiment are depicted in Figure 2. The TE-671 cells had a resting membrane potential as shown by the basal fluoresence during the first 17 s of the experiment. Upon the addition of an agonist, there was a rapid rise in fluorescence associated with changes in membrane potential from the activation of nAChR. After 180 s, a volume of KCl solution was added to the wells to achieve a final concentration of 40 mM KCl and to clamp the membrane potential at approximately -35 mV (13). The KCl induced fluorescence served as the calibrant for the calculation of the compound responses as previously described (10, 13). Epibatidine was the most effective at increasing fluorescence associated with membrane depolarization and was used for the comparison of the other agonist responses. The EC50 of epibatidine was calculated at 0.013 µM (95% confidence intervals ) 0.011-0.018 µM, from 3 plates of duplicate wells each). This EC50 was similar to the value previously reported by Lee et al. (10), who reported an EC50 value of 0.016 µM. The concentration-effect relationships for the two enantiomers and the racemic mixture on TE-671 cells are displayed in Figure 3, and the EC50 values and 95% confidence limits are listed in Table 1. As described above, the responses of the TE671 cells to the coniine enantiomers were normalized to the maximal epibatidine response at 10 µM. Epibatidine is the most potent known nAChR agonist and the response at 10 µM represents the maximal nAChR activation (10, 13). The effica-

Figure 3. Concentration-effect relationships with best-fit lines for the actions of coniine compounds on membrane potential sensing dye fluorescence in TE-671 cells. In each experiment with TE-671 cells, the membrane depolarization resulting from the addition of (-)-coniine, (()-coniine, or (+)-coniine log10 molar concentrations indicated was measured and displayed as a percentage of the maximal epibatidine response shown in Figure 2. Each data point represents three experiments of duplicate wells.

Table 1. LD50 and EC50 Values for the Enantiomers of Coniine coniine form (-) (() (+)

LD50a,b (mg/kg)

95% C.I.c (mg/kg)

EC50d (mM)

95% C.I. (mM)

7.0 7.7 12.1

(5.7-7.4) (6.9-8.8) (11.5-14.0)

0.1 0.3 0.9

(0.01-0.3) (0.1-0.9) (0.7-1.3)

a I.V. route of administration. b Median lethal dose. c Ninety-five percent confidence interval. d Median effective concentration.

cies of the coniine enantiomers at 1 mM concentration relative to the maximal epibatidine response were 76 ( 13%, 62 ( 13%, and 52 ( 10% for the (-)-, (()-, and (+)-forms of coniine, respectively. Therefore, the relative order of potency for the enantiomers of coniine on TE-671 cells was (-)-coniine > (()coniine > (+)-coniine. The toxicities of the coniine (1) enantiomers were compared using a mouse bioassay. Onset of clinical signs was almost immediate after injection, beginning with piloerection, tail flicking, and rapidly progressing to intention tremors, clonic convulsions, muscular weakness, lateral recumbency, and death. Typically, death occurred within five minutes of injection, or recovery was imminent and complete within 1 h. The acute lethality of the enantiomers, as depicted by their LD50 value in mice, were 7.0, 7.7, and 12.1 mg/kg for (-)-coniine, (()coniine, (+)-coniine, respectively (Table 1). Although the 95% confidence intervals of the LD50 values for (-)-coniine and (()coniine overlapped (Table 1), the relative order of potency for the enantiomers of coniine in the mouse LD50 bioassay was also (-)-coniine > (()-coniine > (+)-coniine. In addition to acute toxicity, poison hemlock is also reported to cause chronic toxicity and teratogenicity with coniine (1),

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N-methyl coniine, and δ-coniceine as the principle toxic compounds (14, 15). Chronic toxicity is manifested as birth defects in offspring from pregnant animals exposed to poison hemlock during critical periods of gestation, for example, days 40-100 in cattle (16, 17) and days 30-60 in swine (18, 19) and sheep (20). Even though no teratogenic experiments were performed in this study, it is quite likely that there are also differences in the teratogenic potential of the two coniine (1) enantiomers. The relative teratogenic activity of the coniine enantiomers is planned using a goat model characterized for livestock and biomedical research (21-23). In a previous study using (+)-and (-)-anabasine (2), toxic piperidine alkaloids from Nicotiana glauca, (+)-anabasine, was more potent than (-)-anabasine in TE-671 cells and in the mouse LD50 bioassay (10). Similar results were obtained using (+)- and (-)-ammodendrine (3), toxic piperidine alkaloids found in Lupinus spp., where the (+)-ammodendrine was more toxic in the mouse LD50 bioassay (9). The results from this study demonstrating that (-)-coniine is more toxic than (+)-coniine in both the TE-671 cell assay and in the mouse LD50 bioassay is opposite with respect to optical activity of what was previously observed for the anabasine (2) and ammodendrine (3) enantiomers but consistent with mouse i.v. LD50 information where (-)-nicotine is more potent than (+)-nicotine (24). The opposite nature of the potencies and toxicities of the enantiomers of coniine (1) and nicotine (4) compared to the enantiomers of anabasine (2) and ammodendrine (3) is likely due to undetermined stereochemical interactions between the toxins and the nAChR. Further experiments are needed to determine the exact mechanism behind these differences. In conclusion, the results from this study demonstrate that complete separation of enantiomers of coniine (1) was achieved using preferential crystallization with (+)- and (-)-mandelic acid. The in Vitro results suggest that the (-)-enantiomer of coniine is a more potent agonist than the (+)-enantiomer for the nAChR. Additionally, the in Vitro data was corroborated by the in ViVo acute lethality data, where the (-)-enantiomer was more toxic to mice than the (+)-enantiomer. These results indicate that the binding of coniine (1) to the nAChR and the toxicity of coniine is a stereospecific process. Acknowledgment. We thank I. McCollum and A. Dolbear for technical assistance.

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