Biopesticides: State of the Art and Future Opportunities : Activity of

Chapter 10

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Activity of Plant-Based Compounds on Anthelmintic-Resistant Caenorhabditis elegans B. W. Bissinger,*,1 C. T. Knox,1 S. M. Mitchell,1 and R. M. Kaplan2 1TyraTech,

Inc., Morrisville, North Carolina 27560, U.S.A. of Infectious Diseases, University of Georgia, Athens, Georgia 30602, U.S.A. *E-mail: [email protected].

2Department

Anthelmintic resistance is a major global problem for livestock production and its incidence and severity have increased in recent years. With a paucity of new anthelmintics on the market, there is a need for efficacious treatments with novel modes of action. Four monoterpenoids, α-pinene, linalool, p-cymene, and thymol were examined for antinematodal activity in the free-living nematode, Caenorhabditis elegans. The order of toxicity was thymol > p-cymene > linalool > α-pinene, although no significant difference in mortality was observed between thymol, p-cymene, and linalool. Further tests were conducted using TT-7001, a blend of these monoterpenoids that exhibits 7.5-fold greater activity than expected, on three anthelmintic-resistant strains of C. elegans. Mortality did not differ between wild-type, benzimidazole-, levamisole-, and ivermectin-resistant C. elegans strains. Additionally, TT-1013, a variant of TT-7001 formulated for enteric release and to mask the flavor and fragrance of the oils, caused dose-dependent mortality after exposure to simulated small intestine conditions, while mortality did not differ from negative controls for formulated TT-1013 exposed only to simulated stomach conditions.

© 2014 American Chemical Society In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Introduction Gastrointestinal nematodes are a major threat to the health of humans and domestic animals worldwide. Three major drug classes exist for the treatment of these internal parasites. These are the benzimidazoles that target β-tubulin in nematodes leading to inhibition of microtubule formation, macrocyclic lactones which cause paralysis of pharyngeal pumping by increasing the opening of glutamate-gated chloride channels, and levamisole/pyrantel derivatives that act on nicotinic acetylcholine receptors in nematode muscles leading to spastic paralysis (reviewed by (1)). Extensive use of these anthelmintics has led to widespread resistance in parasites of livestock, including multi-drug resistance to all three classes (2, 3). Nematodes of veterinary importance exhibit a high probability of developing anthelmintic resistance due to rapid nucleotide sequence evolution, sizable populations, and host movement leading to increased gene flow (reviewed by (2)). Apart from the recent launches of the amino-acetonitrile derivative monepantel (4) and the spiroindole derquantel (5), no new drug class has entered the market since the introduction of ivermectin in 1981. Veterinary drug development has declined over the past two decades because of consolidation of animal health companies and the high cost associated with development of new pharmaceutical therapeutics (6). Plant-based compounds have demonstrated nematicidal activity against multiple helminth species (7–10) and may serve as an alternative to synthetic chemical anthelmintics or complement them in an integrated pest management program (IPM). Many plant compounds are commonly used in the flavor and fragrance industries and are classified as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Terpenoids and related aromatic compounds are the primary constituents of many plant essential oils. In plants, terpenoids function as secondary metabolites and play important roles in plant defense against herbivory (11). These compounds appear to exert their activity on the nervous system of nematodes and other invertebrates by acting on various targets including the invertebrate-specific G protein-coupled receptors for octopamine and tyramine (10, 12–15), acetylcholine esterase (16–18), and ionotropic gamma amino butyric acid (GABA) receptors (19, 20). Multiple target sites of action may slow the development of resistance in nematodes. Additionally, targeting alternative modes of action to traditional anthelmintics may play a role in resistance management. TT-7001, a synergistic blend of four monoterpenoid plant essential oil constituents: α-pinene, linalool, p-cymene, and thymol was developed for use as a functional food additive to control parasitic helminths in humans. One of these constituents, thymol has shown activity on the tyramine receptor, Ser2 in the free-living soil-dwelling nematode, Caenorhabditis elegans (11). Caenorhabditis elegans has served as a model for anthelmintic screening for decades (21). Here we describe the activity of four monoterpenoids in C. elegans, and show synergy of a blend of these compounds, TT-7001 against wild-type and anthelmintic drug-resistant C. elegans. We also demonstrate the activity against C. elegans of TT-1013, a microencapsulated blend of α-pinene, linalyl acetate, 134 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

p-cymene, and thymol octanoate designed for enteric release and taste-masking of the aromatic essential oil compounds.

Materials and Methods


Caenorhabditis elegans Maintenance Wild-type (N2 strain) and three anthelmintic-resistant C. elegans strains, genotypes unc-29 (e1072) I resistant to levamisole, ben-1 (e1880) III resistant to benzimidazole, and avr-14 (ad1302) I resistant to ivermectin were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis, MN) on nematode growth media (NGM). All nematode strains were maintained under monoxenic conditions using the OP50 strain of Escherichia coli received with each culture on NGM (22). Caenorhabditis elegans Mortality Bioassay Ivermectin and levamisole hydrochloride were purchased from SigmaAldrich (St. Louis, MO). Essential oil constituents (a-pinene, linalool, p-cymene, and thymol) were purchased from The Lebermuth Company (Mishawaka, IN). A stock solution of the essential oil blend was prepared using soybean oil (Harris Teeter, Matthews, NC) as a carrier. When examining synergy of the four constituents, TT-7001 was formulated without soybean oil. Working solutions of essential oils were made by diluting stock solutions in distilled water containing 1.2% Ryoto sugar ester S-1590 (RSE) (Mitsubishi Food Ingredients, Inc., Dublin, OH) as a surfactant. Serial dilutions of TT-7001 were tested against wild-type and anthelmintic-resistant strains of C. elegans to determine the LC50 for each strain. Levamisole hydrochloride was dissolved in distilled water. Ivermectin was first dissolved in dimethyl sulfoxide (DMSO) with further dilutions made with M9 buffer. Benzimidazole was not tested on wild-type or benzimidazole-resistant C. elegans because its insolubility in water made it incompatible with our testing methods. Doses are presented as ppm (µg/g) unless otherwise noted. Caenorhabditis elegans were collected for mortality bioassays by rinsing plates with 1.5 mL M9 buffer (3.8 g Na2PO4, 1.5 g KH2PO4, 2.5 g NaCl, 0.5 mL 1M MgSO4, Q.S. to 500 mL with dH2O). L4 and adult nematodes were selected by straining the M9 solution containing the worms through a 40 µm nylon cell strainer (BD Falcon, Franklin Lakes, NJ) allowing L1-L3 nematodes to pass through. L4 and adult nematodes were then rinsed from the strainer into a 50 mL conical tube (BD Falcon, Franklin Lakes, NJ) using M9 buffer and allowed to settle in the bottom of the tube before the supernatant was removed leaving 0.5-1.0 mL of worms in the tube. The nematode solution was then diluted to obtain a density of approx. 100 nematodes/750 µL. To test compounds for mortality against C. elegans, 750 µL of diluted worm suspension was added to a 20 mL screw top scintillation vial (VWR, Radnor, PA). Two hundred fifty microliters of each dilution of the test compounds was then added separately to vials containing worms. Vials were swirled gently and the top was placed loosely on the vial. Vials were then placed in the dark and mortality 135 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


was assessed 16 h after treatment. Mortality was considered a lack of body and pharyngeal pump movement. Each treatment concentration and no-drug controls were replicated in duplicate or triplicate. Average mortality was calculated as, (mean no. of dead worms / total number of worms)*100. Schneider-Orelli’s formula (23) was used to account for mortality of C. elegans treated with the control vehicle (distilled water containing RSE for essential oils, DMSO + M9 buffer for ivermectin, and water for levamisole). Regression analysis was used to approximate the LC50 concentration for each monoterpenoid constituent and for the blend of these constituents, TT-7001, by plotting probit mortality versus log dose (24) using the method of least squares and inverse predictions of 95% fiducial ranges (25). The methods of (26) and (27) were used to estimate the variance-covariance matrix and 95% confidence intervals, respectively. Calculations were carried out in Excel spreadsheets as described previously (28). Average mortality caused by treatment with ivermectin or levamisole for wild-type and the respective anthelmintic-resistant strains were compared using the Student’s t-test (P < 0.05). To examine synergy of the essential oil components of TT-7001, α-pinene, linalool, p-cymene, and thymol were combined without soybean oil in their appropriate ratios on a weight:weight basis. Soybean oil aids in solubilization of the essential oil constituents, but does not contribute to the mortality of C. elegans (data not shown). Actual and expected mortalities for each constituent and the blend were compared to determine whether a synergistic effect was present. Expected mortality was calculated as,

Where, A1 is the percentage of compound 1 in the mixture and Z1 is the LC50 for compound 1 (29). The percentages of each essential oil are not provided because of the proprietary nature of the blend technology. Simulated Digestion Protocol The simulated digestion protocol was kindly provided by Kraft Foods (unpublished). Due to the light sensitivity of enzymes used in this protocol, all experiments were conducted under yellow light (CFL bug light, Sylvania, Winchester, KY). Stomach simulation solution (500 mL of 3 mg/mL pepsin, 2 mg/mL NaCl, pH 2.1) was prepared by adding 1.0 g of NaCl to 600 mL of deionized water in a beaker containing a magnetic stir bar on a stir plate set to medium speed. Upon total dissolution, stirring was reduced to minimum, and concentrated HCl was added until the pH reached 1.95-2.10 at 22.3° C. One and a half grams of pepsin from porcine gastric mucosa (Sigma-Aldrich, St. Louis, MO) was added and allowed to dissolve for 10 min. The contents of the beaker were then transferred to a plastic Nalgene bottle and refrigerated. Two concentrations (0.27 and 1.35%) of TT-1013 and empty encapsulate (negative control) (Kraft Foods, Glenview, IL) were tested for mortality to C. elegans. Samples were weighed into 20-mL glass screw cap headspace vials (Sigma-Aldrich, St. Louis, MO) and 7.3 g of stomach solution were added to each vial. Vials were capped 136 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


and rotated 360° on a rotator base (Glas-Col tissue culture rotator, VWR, Radnor, PA) at an angle of 60°, speed of 4 rpm at 37° C for 30 minutes. Small intestine simulation solution (200 mL of 3.24X pancreatin, 8X concentrate NaOH, and bile salts, pH 6.8) was prepared as follows. Porcine bile extract (Sigma-Aldrich, St. Louis, MO) was preconditioned according to USP specifications by heat treating 1.98 g at 105° C for 4 h followed by 20 minutes in a desiccator at room temperature. Forty milliliters of deionized water (25 ± 1° C) were placed into a beaker containing a magnetic stir bar on a stir plate set to low. To this, 6.71 g pancreatin from porcine pancreas (Sigma-Aldrich, St. Louis, MO) was added and stirred for 15 min. In a separate beaker with a magnetic stir bar, 50 mL of deionized water (25 ± 1° C) and the heat-treated bile salts were added and mixed on low until dissolved. The pH was adjusted to 7.1 by the addition of 0.5 N NaOH. The pancreatin solution was then added to the bile salts solution by gently pouring down the side of the beaker wall. The beaker that previously contained the pancreatin was rinsed twice with 2 mL of DI H2O. The pH was adjusted to 7.05 by the addition of 0.5 N NaOH. One hundred and fifty milliliters of DI H2O was added to the beaker. The mixture was then transferred to 50-mL conical tubes and centrifuged at 10,000 x g for 20 min at 22°C. The supernatant was transferred to a plastic Nalgene container and immediately refrigerated. A premix solution was prepared in a glass beaker by combining 87.50 g of small intestine solution with 182.50 g pepsin and 6.85 g 0.5 N NaOH each per sample on an ice bath. Final pH of the premix solution was 7.74 at 7° C. For small intestine simulation bioassays, 11.0 g of premix solution was added to each labeled vial. Vials were capped and stored on an ice bath at 7° C. Samples (0.02 and 0.10 g TT-1013 or empty encapsulate) were weighed into headspace vials containing premix solution, capped and stored at room temperature after sample addition. Samples were then rotated for 6 h on a rotator base as previously described. After incubation, samples were removed from the rotator and cooled to ambient temperature in a water bath before being tested in mortality bioassays as described above. Each concentration of treatment and control was run through the stomach and small intestine digestion simulations in duplicate (biological replicates) with three technical replicates conducted from each biological replicate. Mean percentage mortality was calculated as previously described with simulated stomach or small intestine solution serving as the negative controls for control mortality adjustment using the Schneider-Orelli formula.

Results Of the four monoterpenoids tested for C. elegans mortality, thymol exhibited the lowest LC50, followed by p-cymene and linalool. The efficacy among these three compounds, however, did not differ significantly (log probit analysis, P < 0.05) (Table I). No difference in mortality was found between C. elegans treated with TT-7001 containing soybean oil (Table I) and TT-7001 without soybean oil (Table II) based on 95% fiducial limits. α-Pinene, linalool, p-cymene, and thymol were 7.5-fold more active than expected when combined in the ratio in TT-7001, demonstrating synergy. 137 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

138


Table I. Median Lethal Concentration of Monoterpenoids and TT-7001 (without the Carrier, Soybean Oil) against Wild-Type (N2 strain) C. elegans Measured 16 h after Treatments Were Applied to Worms in Scintillation Vials (3 Replicates Per Treatment Dose) Compound

na

LC50 (ppm)b

95% FLc (ppm)

χ2d

Slope ± SE

r2

α-pinene

1943

11797 Ce

6382 - 21644

4.38

2.90 ± 0.26

0.98

linalool

2557

529 B

310 – 827

17.05

10.55 ± 1.63

0.96

p-cymene

2359

486 B

244 - 931

21.08

2.94 ± 0.38

0.97

thymol

1802

121 AB

58 – 388

170.88

6.82 ± 1.88

0.94

TT-7001 without soybean oil

1891

120 A

76 – 165

7.98

10.91 ± 1.65

0.98

Number of C. elegans tested b Median lethal concentration from probit analysis. Mortality was considered the lack of motility (straightened body and lack of pharyngeal pumping). c Fiducial limits from probit analysis d Chi-square value from probit analysis e Values followed by a different letter indicate a statistically significant difference based on fiducial limits (log probit analysis, P < 0.05). a

In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

na

LC50 (ppm)b

95% FLc (ppm)

χ2d

Slope ± SE

r2

Wild-type (N2)

961

162 Ae

66 - 347

19.93

6.73 ± 1.22

0.94

Benzamidazole-resistant (ben-1)

1179

172 A

30 - 690

73.93

5.43 ± 1.28

0.90

Ivermectin-resistant (avr-14)

813

202 A

123 - 326

4.23

5.74 ± 0.56

0.98

Levamisole-resistant (unc-29)

724

78 A

31 - 168

160.86

2.64 ± 0.44

0.96

Worm strain

Number of C. elegans tested b Median lethal concentration from probit analysis. Mortality was considered the lack of motility (straightened body and lack of pharyngeal pumping). c Fiducial limits from probit analysis d Chi-square value from probit analysis e Values followed by a different letter indicate a statistically significant difference based on fiducial limits (log probit analysis, P < 0.05). a

139


Table II. Median Lethal Concentration of TT-7001 against Wild-Type and Three Anthelmintic-Resistant Strains of C. elegans Measured 16 H after Treatments Were Applied to Worms in Scintillation Vials (2-3 Replicates Per Treatment Dose)

In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Activity of TT-7001 did not differ between wild-type, benzimidazole-, levamisole-, or ivermectin-resistant C. elegans strains (Table II). Ivermectin and levamisole were tested against their respective anthelmintic strains to verify phenotypic resistance. Ivermectin was 20.0-fold more active against the N2 strain compared to the avr-14 strain when tested at 1 ppm (Table III). Similarly, levamisole at 10 ppm was 27.1-fold more active against the N2 strain compared to the unc-29 strain. In both cases, mortality was significantly greater (t-test, P < 0.0001) for wild-type vs. anthelmintic-resistant nematodes when treated with their respective drug, demonstrating active resistance.

Table III. Mean Percentage Mortality (± 1 SEM) of Wild-Type and Anthelmintic-Resistant C. elegans 16 h after Treatments (Ivermectin or Levamisole) Were Applied to Worms in Scintillation Vials (2-3 Replicates Per Treatment Dose) Ivermectin (1 ppm)a

Levamisole (10 ppm)b

Wild-type (N2)

79.8 ± 2.2*

100 ± 0.0*

Ivermectin-resistant (avr-14)

4.0 ± 1.1

ntc

Levamisole-resistant (unc-29)

nt

3.7 ± 0.3

Control mortality = 0.00% b Control mortality = 2.27% c nt = not tested * Indicates a significant difference (t-test, P < 0.0001) between the wild-type and anthelmintic-resistant C. elegans strain for a given treatment (ivermectin or levamisole). a

Table IV. Mean Percentage Mortality (± 1 SEM) of C. elegans Exposed to Encapsulated TT-1013 and Empty Encapsulate under Simulated Stomach or Small Intestine Conditions (2 Biological with 3 Technical Replicates Per Treatment) Sample

Concentration (%)

Stomach simulation (30 min exposure)

Small intestine simulation (6 h exposure)

Empty encapsulate

2.74

2.4 ± 0.7

0.0 ± 0.5*

13.70

2.3 ± 0.6

1.3 ± 0.7*

2.74

5.8 ± 1.1

68.6 ± 1.7

13.70

7.7 ± 1.4

100 ± 0.0

TT-1013

*

Indicates a significant (t-test, P < 0.0001) difference between the empty encapsulate and TT-1013 at the same dose.

Microencapsulated TT-1013 and the empty encapsulate were tested against C. elegans after exposure to simulated stomach or small intestine conditions. Mortality of C. elegans did not differ between TT-1013 and the control at the low or high dose (P = 0.45 and 0.16, respectively) after a 30-minute exposure to the pH 140 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


and enzymatic conditions of the stomach, suggesting that the essential oils were not released under these conditions. However, under simulated small intestine conditions, C. elegans treated with TT-1013 demonstrated dose-dependent mortality, with the top dose achieving 100% mortality. Conversely, under the same conditions, no mortality was observed when C. elegans were treated with the low dose of empty encapsulate, and mortality was only 1.3% for the high dose (Table IV). These large differences in mortality of C. elegans under simulated small intestine conditions were significant between both doses of TT-7001 and the empty encapsulate controls (t-test, P < 0.0001; Table IV).

Discussion Monoterpenoids from plant essential oils have demonstrated nematicidal activity (8, 11, 30) and are documented to act upon multiple target sites in nematodes (11) and other invertebrates (16–20). These sites are all different from the known modes of action of the three major anthelmintic drug classes. In this study, four monoterpenoids were tested, alone and in combination, for activity against wild-type C. elegans. Similar to the results of other studies (8, 11, 30), thymol was a potent nematicide. Evidence suggests that thymol, a major constituent of English thyme (Thymus vulgaris L.) essential oil (31), exerts its activity by binding to the tyramine receptor, Ser2 in C. elegans. This compound is also toxic to the parasitic pig roundworm, Ascaris suum Goeze (11), presumably by the same mechanism of action. Like thymol, linalool appears to exert its activity on GPCRs, as intracellular calcium release is observed in calcium mobilization assays in HEK293 cells stably transfected with the C. elegans Ser2 receptor (data not shown). Neither p-cymene (11) nor α-pinene (data not shown) elicit calcium mobilization in the transfected cell system. To our knowledge, the molecular mode of action for these compounds in nematodes has yet to be elucidated, although p-cymene demonstrated agonistic behavior for the American cockroach (Periplaneta americana (L.)) octopamine receptor in ligand binding assays (12), and linalool is an inhibitor of acetylcholinesterase (32). Activity of TT-7001 against drug-resistant C. elegans strains from each of the three major anthelmintic classes was equivalent to that of the N2 wild-type strain. This verifies that the activity of monoterpenoid blend is unaffected by the genes that confer resistance in these C. elegans. TT-7001 with its apparent alternative mode of action versus these drugs, combined with the different mechanism of action of α-pinene and p-cymene compared to linalool and thymol, could serve as an alternative control measure to the current treatments, particularly if implemented in a multi-approach IPM program. Essential oils are comprised of highly volatile, aromatic compounds. One difficulty with formulating these compounds for consumption by humans or domestic animals is that their flavor and odor profiles can be unpleasant. Microencapsulation is a technique used in the pharmaceutical industry to mask the flavors of unpalatable compounds (33). The encapsulation technique used in the formulation of TT-1013 was developed for delivery of TT-7001 as a functional food additive to reduce parasitic helminth infections of humans. When 141 In Biopesticides: State of the Art and Future Opportunities; Coats, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


fed daily to pigs at a dose of 1 mg/kg body weight, TT-1013 reduced the number of A. suum by 76.8% and fecal egg counts by 68.6% compared to placebo-treated pigs (Kaplan et al., unpublished). Although not tested, it is plausible that infection by A. suum could be eliminated by treatment with TT-1013 at a higher dose. Anthelmintic-resistant gastrointestinal nematodes threaten the health and well-being of all classes of livestock animals and widespread multi-drug resistance is common (3). TT-1013 was formulated for enteric release in the monogastric human gut. Results of the present study along with those conducted in pigs (Kaplan et al., unpublished) suggest that the active compounds survive the pig stomach and simulated conditions of the human stomach but are released under those of small intestine. Further formulation would be necessary to overcome the multiple stomach compartments and different enzymatic environment of the ruminant gut. Additionally, testing would need to be carried out against ruminant parasites of the abomasum (true stomach) and small intestine such as Haemonchus contortus (Rudolphi) and Trichostrongylus colubriformis (Giles), respectively. Widespread and multi-drug resistance in gastrointestinal nematodes of animals necessitates the development of alternative control strategies and therapeutics. Furthermore, the development of resistance to currently used anthelmintics in human mass drug treatment programs for nematode control is a serious concern (34). Drug development and registration of a new active ingredient is expensive and time consuming. Alternative treatments based on plant compounds, particularly those that are GRAS-listed leading to reduced registrarion time, may play an important role in managing parasite resistance. The current study demonstrates that four individual essential oil constituents and a blend of these compounds provide nematicidal activity. Additionally, these compounds have successfully been formulated so that their flavors and fragrances are masked, and release occurs under the conditions of the small intestine of monogastrics. TT-7001 and TT-1013, with their activity upon resistant nematode strains, may provide a means to improve parasite control and help to manage the problems posed by anthelmintic-resistant nematode parasites.

Acknowledgments The authors are grateful to Dr. George Haas and Ms. Dana Sebesta (Kraft Foods) for direction and advice on the simulated digestion protocol and to Mr. Ahmad Akashe (Kraft Foods) for his formulation expertise. This project was funded by TyraTech, Inc. BWB, CTK, and SMM were employees of TyraTech, Inc. at the time these studies were conducted.

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Biopesticides: State of the Art and Future Opportunities : Activity of

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