Pharmacokinetics in Plants: Carbamazepine and Its Interactions with

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Pharmacokinetics in Plants: Carbamazepine and Its Interactions with Lamotrigine Myah Goldstein, Tomer Malchi, Moshe Shenker, and Benny Chefetz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01682 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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Environmental Science & Technology

ACS Paragon Plus Environment


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Pharmacokinetics in Plants:

1

Carbamazepine and its Interactions with Lamotrigine

2 3

Myah Goldstein,†,‡ Tomer Malchi,†,‡ Moshe Shenker,† and Benny Chefetz†,‡,*

4 5

†

Department of Soil and Water Sciences, The Robert H. Smith Faculty of Agriculture,

6

Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot

7

7610001, Israel

8

‡

9

The Hebrew University Center of Excellence in Agriculture and Environmental

Health

10 11

*

12

Benny Chefetz

13

Tel.: 972-8-9489384; email: [email protected]

14

Corresponding Author:

1 ACS Paragon Plus Environment

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ABSTRACT

15

Carbamazepine and lamotrigine prescribed antiepileptic drugs are highly

16

persistent in the environment, and were detected in crops irrigated with reclaimed

17

wastewater. This study reports pharmacokinetics of the two drugs and their

18

metabolites in cucumber plants under hydroponic culture, testing their uptake,

19

translocation and transformation over 96 h in single and bi-solute systems at varying

20

pH. Ruling out root adsorption and transformations in the nutrient solution, we

21

demonstrate that carbamazepine root uptake is largely affected by the concentration

22

gradient across the membrane. Unlike carbamazepine, lamotrigine is adsorbed to the

23

root and undergoes ion trapping in root cells thus its translocation to the shoots is

24

limited. Based on that, carbamazepine uptake was not affected by the presence of

25

lamotrigine, while lamotrigine uptake was enhanced in the presence of

26

carbamazepine. Transformation of carbamazepine in the roots was slightly reduced in

27

the presence of lamotrigine. Carbamazepine metabolism was far more pronounced in

28

the shoots than in the roots, indicating that most of the metabolism occurs in the

29

leaves, probably due to higher concentration and longer residence time. This study

30

indicates that the uptake of small non-ionic pharmaceuticals is passive and governed

31

by diffusion across the root membrane.

32



INTRODUCTION

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33

Carbamazepine (CBZ) is the most frequently prescribed antiepileptic drug

34

worldwide, used by patients of all ages.1–4 It acts as a voltage-dependent sodium-

35

channel blocker.5–7 Once administered, CBZ is metabolized, mainly in the liver, into

36

various different metabolites8,9 which are subsequently excreted from the body. Of the

37

total administrated dose, 13.8% was reported to be excreted as the parent compound

38

(mostly in the feces); 32% of the total as the metabolite 10,11-dihydro-10,11-

39

dihydroxy-carbamazepine (DiOH-CBZ), 5.1% as 3-hydroxy-carbamazepine (3-OH-

40

CBZ), 4.3% as 2-hydroxy-carbamazepine (2-OH-CBZ), and 1.4% as 10,11-dihydro-

41

10,11-epoxy-carbamazepine (EP-CBZ), all excreted in the urine, with several other

42

metabolites excreted in both feces and urine.10 Low removal efficiency and minimal

43

degradability of CBZ and its metabolites have been reported in municipal wastewater-

44

treatment plants.11–13 Thus CBZ and several of its metabolites are frequently detected

45

in reclaimed-wastewater used for irrigation and in freshwater bodies receiving

46

effluents.10,13–16 Once CBZ is introduced into the soil via irrigation with reclaimed-

47

wastewater or biosolids application, it has been shown to undergo only limited

48

biodegradation17, attributed to binding to soil organic matter and/or clays18, and to be

49

highly resistant to microbial decomposition.17,19 The shortest half-life reported for

50

CBZ in soils is approximately 42 days, whereas in most cases it is more than 200

51

days.17,20 CBZ is a neutral compound within the range of environmental pH.15

52

Lamotrigine (LTG), also a sodium-channel blocker,5–7 is a frequently

53

prescribed antiepileptic drug which may be co-administered with other antiepileptic

54

drug such as CBZ.1,3,4 LTG is excreted in urine mainly in its glucuronide form while

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only 10% is excreted as the parent compound 21,22. LTG is of low removal efficiency

56

in municipal wastewater-treatment plants and is found at concentrations above 1 µg L-

57


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1

in water bodies and in reclaimed-wastewater.13,23 Lamotrigine-N2-glucuronide is the

58

main LTG metabolite found in reclaimed-wastewater.13,23 Following reclaimed-

59

wastewater irrigation LTG was found to accumulate in the top soil exhibiting little to

60

no biodegradation.17,24 LTG is a weak base with a pKa of 5.7 and a Log D of 2.12 (at

61

pH of 7.5).25,26

62

Being a relatively restrictive barrier to organic compounds, the casparian strip

63

in plant roots exhibits traits similar to those of tight junctions which make up the

64

blood–brain barrier in mammals,27,28 thus implying that compounds known to cross

65

the blood–brain barrier through diffusion, such as CBZ29 and LTG,22 may also be

66

taken up and translocated by plant roots. We hypothesize that CBZ and LTG are taken

67

up as non-ionic species by diffusion through root cell membranes and therefore are

68

not expected to directly affect each other. However, based on well documented

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pharmacokinetic interactions reported in humans30, we suggest that co-introduction of

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CBZ and LTG will affect the kinetics of CBZ metabolism within the plant. The main

71

objective of this study was to understand the kinetics governing CBZ and LTG

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uptake, translocation and metabolism in plants and to reveal whether these processes

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are affected by co-introduction with other drugs (i.e., the environmental scenario). We

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also aimed to reveal if the CBZ metabolites found in plant materials are a result of in-

75

plant metabolism or due to direct uptake of the metabolites from the irrigation water

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and/or soil solution.

77 78

EXPERIMENTAL SECTION

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Chemicals. CBZ (>97% purity) was purchased from Sigma-Aldrich Israel Ltd.

80

(Rehovot, Israel), and LTG (>99%) from EnzoBiochem Inc. (New York, NY). The

81

following CBZ metabolites were purchased from Toronto Research Chemicals Inc.

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(Toronto, Canada): EP-CBZ, DiOH-CBZ, and 2-OH-CBZ. Selected properties of the

83

studied compounds are presented in Table 1. The following labeled compounds were

84

purchased from Toronto Research Chemicals: CBZ-13C-D2, LTG-13C3, DiOH-D3,

85

EP-CBZ-D8.

86

Hydroponic Culture Setup. Cucumber seeds (Cucumis sativus, Patriot, Hazera

87

Genetics Ltd., Berurim, Israel) were germinated in vermiculite moistened with

88

CaSO4·H2O-saturated solution. Seven-day-old seedlings were transferred and each

89

single plant was cultivated in 1-L darkened glass jars in a continuously aerated

90

nutrient solution, at an initial volume of 800 ± 10 mL, with the following composition

91

of macronutrients (mM): K2SO4, 0.7; KCl, 0.1; Ca(NO3)2·4H2O, 2.0; MgSO4, 0.5;

92

KH2PO4, 0.1, and micronutrients (µM): Fe-EDTA, 10; MnSO4·H2O, 0.5;

93

ZnSO4·7H2O, 0.5; CuSO4, 0.2; (NH4)6Mo7O24·4H2O, 0.01; H3BO3, 10. The nutrient

94

solution was prepared in deionized water, with a final pH of 5.7. The plants were

95

grown in a temperature-controlled chamber with a 16/8-h day/night cycle at 25 ± 1 °C

96

and 20 ± 1 °C day and night, respectively31 and relative humidity of 60-70% during

97

the day and 80-100% during the night. The nutrient solution was replaced every 3–4

98

days. Following 29 days of cultivation in the nutrient solution, the plants were

99

introduced into a fresh nutrient solution containing: (i) CBZ, 1.82 ± 0.05 µM (430 µg

100

L-1); or (ii) LTG, 1.80 ± 0.03 µM (461 µg L-1); or (iii) EP-CBZ, 1.69 ± 0.00 µM (428

101

µg L-1); or (iv) DiOH-CBZ, 1.78 ± 0.00 µM (480 µg L-1); or (v) a mixture of CBZ and

102

LTG at concentrations of ~2 µM each.

103

Experimental Design. Plants were exposed to either CBZ, LTG, EP-CBZ, or DiOH-

104

CBZ (single-solute experiments), or to CBZ and LTG (bi-solute experiments) for up

105

to 96 h, maintaining a pH of 6-7 in all experiments by adding 4-(2-hydroxyethyl)-1-

106

piperazineethanesulfonic acid (HEPES) to the nutrient solution at a concentration of 5

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mM. During this time, the nutrient solution was not replenished and continuously

108

aerated. The plants exhibited a healthy root system throughout the entire cultivation

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and experimental period. The nutrient solution was sampled at 1, 3, 6, 8, 24, 48, 72

110

and 96 h for the CBZ and LTG single-solute experiments and CBZ and LTG bi-solute

111

experiments, and once after 72 h for the EP-CBZ and DiOH-CBZ single-solute

112

experiments. The plants exposed to CBZ in the presence or absence of LTG were

113

sampled at 8, 24, 48, 72 and 96 h; the plants exposed to the metabolites were sampled

114

after 72 h. The experiments were conducted in 5 replicates for each sampling time. At

115

each sampling time, a whole plant was sacrificed for sap sampling and plant analyses.

116

After plants were detopped, xylem sap was collected over a period of approximately

117

15 min after detopping, collecting 2-3 mL of sap from each plant, while roots were

118

still in the nutrient solution, first drops being discarded. Then roots and shoots were

119

washed, weighed and frozen at -20 °C until extraction.

120

CBZ and LTG uptake and translocation in a bi-solute experiment was also tested

121

comparatively at pH of 4.5 and 7.5. In this experiment, the exposure time was 48 h.

122

To maintain pH 7.5, HEPES was added to the nutrient solution at a concentration of 5

123

mM. To maintain pH 4.5, 2(N-morpholino)ethane sulfonic acid (MES) was added at a

124

concentration of 5 mM. Since the MES buffer alone was not capable of maintaining a

125

steady, low pH, the Ca(NO3)2·4H2O (2 mM) in the nutrient solution was substituted

126

with 0.8 mM NH4NO3, 1.2 mM Ca(NO3)2·4H2O and 0.8 mM CaCl2 in the low pH

127

treatment, so that the N was composed of 20% NH4+ and 80% NO3-.

128

Analysis. Analyte concentrations in all solutions (sap and nutrient solutions) were

129

quantified using a Waters Alliance HPLC system equipped with a LiChrospher 100

130

RP-18 column (25 cm × 4.6 mm, 5 µm particle size; Merck, Darmstadt, Germany)

131

and photodiode array detector. The analytes were eluted from the column at a constant

132



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flow rate of 1 mL min-1 and constant temperature of 45 °C; sample injection volume

133

was 10 µL. The initial mobile phase consisted of a mixture of methanol (14%),

134

acetonitrile (20%) and water (66%) (v/v). A gradient program was applied by raising

135

the level of acetonitrile from 20 to 50% over 12 min. CBZ, EP-CBZ and DiOH-CBZ

136

were quantified based on absorption at 210 nm, and LTG was quantified at 307 nm.

137

Limit of detection (LOD) and limit of quantification (LOQ) values, determined using

138

a signal to noise ratio of 3:1 and 10:1 respectively, were 25 and 50 µg L-1,

139

respectively, for CBZ and its metabolites and 50 and 100 µg L-1, respectively, for

140

LTG.

141

Analyte concentration in the plant material (roots and shoots) was analyzed as

142

detailed in Goldstein et al.15 In brief, plant material was ground to a fine powder and

143

extracted with methanol using accelerated solvent extractor (ASE 350, Dionex,

144

Sunnyvale, CA) in two static 5-min cycles with 100% methanol at 80°C under a

145

constant pressure of 10.34 MPa. Extracts were evaporated to dryness and

146

reconstituted in 1 mL acetonitrile:water:acetic acid (20:80:0.01) spiked with 10 µL of

147

a mixture of isotopically labeled internal standards, centrifuged at 13500 rpm for 20

148

minutes and filtered (0.22 µm PTFE) before LC-MS analysis. Extracts were analyzed

149

by Agilent 1200 Rapid Resolution LC system (Agilent Technologies Inc., Santa

150

Clara, CA) equipped with a Gemini C-18 column (150 × 2 mm, 3-µm particle size;

151

Phenomenex, Torrance, CA) coupled to an Agilent 6410 triple quadruple mass

152

spectrometer with ESI ion source, in multiple reaction monitoring (MRM) mode, with

153

positive or negative ionization. LOD and LOQ, determined using a signal to noise

154

ratio of 3:1 and 10:1 respectively, were 1and 2 µg kg-1, respectively, for LTG; 0.05

155

and 0.1 µg kg-1, respectively, for CBZ; 0.1 and 0.2 µg kg-1, respectively for DiOH-

156


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CBZ; 0.05 and 0.1 µg kg-1, respectively, for EP-CBZ; 0.2 and 0.5 µg kg-1,

157

respectively for 2-OH-CBZ and 0.05 and 0.1 µg kg-1, respectively for 3-OH-CBZ.15

158

Data Analysis. Statistical analysis (non-parametric Wilcoxon/Kruskal–Wallis test and

159

non-parametric multiple comparisons using Dunn method for joint ranking, p < 0.05)

160

was performed using JMP Pro 10 software (JMP®, Version 10. SAS Institute Inc.,

161

Cary, NC, 1989-2007). Mass balance was calculated for CBZ and its metabolites as

162

the ratio between the sum of CBZ and its metabolites at each sampling time (the

163

amount of CBZ in the nutrient solution and CBZ and its metabolites in the plant

164

material) and the initial amount of CBZ in the nutrient solution, and found to be 95.36

165

± 0.76% in the single-solute system and 94.36 ± 0.77% in the bi-solute system. Mass

166

balance calculation for LTG in the bi-solute system revealed a growing deficit over

167

time, starting with 92.08 ± 2.18% at 8 h and declined to 57.33 ± 3.49% at 96 h.

168 169 170



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Table 1. Selected physicochemical properties of the studied compounds Compound

Structure

Carbamazepine (CBZ)

log Kow (ref)

2.77

(24)

171

pKa (ref) pKa1 = -0.5 (24)

pKa2 = 14.4 pKa1 = -0.9 (24)

10,11-dihydro-10,11-epoxy1.97

(24)

carbamazepine (EP-CBZ)

pKa2 = 14.8

pKa1 = -1.5 (24) 10,11-dihydro-10,11dihydroxy-carbamazepine

0.81

(24)

pKa2 = 11.7 pKa3 = 12.3

(DiOH-CBZ)

pKa4 = 14.0 2-hydroxy-carbamazepine 2.66 (32)

pKa = 9.30 (32)

2.66 (32)

pKa = 9.46 (32)

1.93 (24)

pKa = 5. 7 (25)

(2-OH-CBZ) 3-hydroxy-carbamazepine (3-OH-CBZ)

Lamotrigine (LTG)

172


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RESULTS AND DISCUSSION

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CBZ and LTG Uptake. The concentrations of CBZ in the nutrient solution,

174

as well as in the sap, were similar for the single- and bi-solute systems (i.e., plants

175

exposed only to CBZ, and plants exposed to both CBZ and LTG as a mixture,

176

respectively) during the exposure time (Figure 1a). Therefore, the following

177

discussion about CBZ uptake by the roots describes both treatments as a single

178

scenario. CBZ concentration in the nutrient solution was constant (~1.76 µM) for the

179

first 48 h, after which a minor, but statistically significant rise in concentration was

180

observed (1.98 µM at 72 h and 2.3 µM at 96 h; Figure 1a). LTG concentration in the

181

single-solute system (Figure 1b) was constant both in the nutrient solution and in the

182

sap during the exposure time. However, for the bi-solute system LTG concentration in

183

the nutrient solution decreased significantly with time. Unlike the steady increase in

184

CBZ concentration in the nutrient solution, CBZ concentration in the plant sap

185

remained constant from 24 h to the end of the experiment (Figure 1a). It is important

186

to note that the CBZ concentration in the sap was always lower than in the nutrient

187

solution and this difference increased with time from 24 h to the end of the

188

experiment. CBZ, being neutral with an intermediate lipophilicity (Table 1), is

189

relatively easily translocated from root to shoot via the sap.33,34 It is important to note

190

that metabolites of CBZ were not detected in the sap. As for LTG concentration in the

191

sap, it was consistently much lower than in the nutrient solutions in both the single-

192

and bi-solute systems (Figure 1b). The sap LTG in the bi-solute system exhibited a

193

significant decrease in concentration following 48 h of exposure.

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Figure 1. Concentrations of carbamazepine (CBZ; left) and lamotrigine (LTG; right) in the plant sap and in the nutrient solution throughout the exposure period in the absence (single-solute system) and presence (bi-solute system) of the companion compound. Averages and standard errors are shown (n = 5).

196 197 198 199 200

The kinetics analysis of CBZ and LTG influx into the cucumber roots,

201

calculated from the decreased amount of each compound in the nutrient solution, and

202

water uptake are presented in Figure S1. The initial CBZ uptake rate was high (11.98

203

± 2.42 and 13.52 ± 1.48 µmol-1 kg root-1 h-1 in the single- and bi-solute systems

204

respectively; average ± standard error) in the first 8 h, and decreased thereafter to 5.45

205

± 0.33 and 6.30 ± 0.36 µmol-1 kg root-1 h-1, in the single- and bi-solute, respectively.

206

The water-uptake rate largely affected the CBZ-uptake rate, but this does not mean

207

that CBZ was actually taken up with the water-influx stream. Alternatively, we

208

suggest different routes for these two components. Water transport across the roots

209

occurs via three pathways: (i) through the cell walls (apoplastic path), (ii) from cell to

210

cell through plasmodesmata (symplastic path), and (iii) across membranes

211

(transcellular path). In order to pass the casparian band and reach the sap, water and

212

solutes must enter the symplast and pass through root cell membranes in the

213

endodermis.35,36 Water uptake is largely driven by water potential gradients, however

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studies have demonstrated the importance of water channels known as aquaporins in

215

the active regulation of water uptake and for elevated water influx rates.35,37 The non-

216

ionic CBZ and LTG molecules are mainly translocated by a passive diffusion

217

mechanism across root-cell membranes, and is thus largely affected by the

218

concentration gradient across the membrane according to Fick’s law, thus their initial

219

influx rate is rapid and slows down as the concentration gradient decrease (Figure S1).

220

Later on, the influx rate of CBZ depends on a steady concentration gradient that is

221

maintained by the water influx. For LTG, the translocation rate in the sap is much

222

slower, as is evident from its lower concentration in the sap (Figure 1b) and thus its

223

influx rate diminishes with the time of exposure. It is hypothesized that CBZ will be

224

transported with water in both apoplastic and symplastic pathways, and as water

225

influx and transpiration increase, a larger CBZ gradient across the membrane will be

226

maintained and larger rates of CBZ uptake will prevail.

227

The apparent LTG uptake rate, as calculated from LTG disappearance from

228

the nutrient solution, was higher than that of CBZ throughout most of the exposure

229

period (Figure S1). The apparent LTG uptake rate decreased gradually over time. The

230

high initial apparent uptake rate calculated for LTG may be attributed to two separate

231

mechanisms, the first being the high root sorption affinity as demonstrated in the root

232

adsorption experiments where LTG adsorption was shown to be substantial and rapid

233

(Figure S2). The second mechanism is ion trapping within the root vacuole. The root

234

vacuole has a pH of ~5.5 under which ~50% of LTG molecules are positively charged

235

and thus vacuole serve as a sink for LTG,38 and the concentration gradient between

236

the external concentration (nutrient solution) and the internal cytoplasm concentration

237

is preserved, resulting in a greater driving force for diffusion across the root cell

238

membrane.

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To demonstrate the uptake mechanism for both drugs, we calculated the

240

apparent influx concentration (µM) as the ratio between CBZ or LTG uptake rate

241

(µmol kg root-1 h-1) and water-uptake rate (L kg root-1 h-1). This is shown in Figure 2.

242

For CBZ the initial (8 h) apparent influx concentration was about 60% higher than the

243

CBZ concentration in the nutrient solution, similar to it at 24 and 48 h and diminished

244

later to be lower than the nutrient solution concentration (Figure 2a). Since CBZ

245

adsorption to the external root surfaces was shown to be negligible (see SI; Figure

246

S2a) and CBZ transformation products were not detected in the nutrient solution, we

247

conclude that the calculated apparent CBZ-influx concentration truly represents CBZ

248

influx across the root membrane (i.e., uptake).

249

250

Figure 2. Concentrations of carbamazepine (CBZ; left side) and lamotrigine (LTG; right side) in the nutrient solution and the apparent CBZ and LTG influx concentrations in the absence (single-solute system) and presence (bi-solute system) of the companion compound throughout the duration of the exposure period. Average data are shown (n = 5); bars represent standard errors.

251 252 253 254 255 256

Based on the greater initial influx rate of CBZ molecules through the root cell

257

membrane, followed by a slower and steady influx rate we suggest that water and

258

CBZ are taken up through separate pathways, while water uptake occurs mainly

259


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through regulated water channels or aquaporins, and is affected by the plant's demand

260

for water, the non-ionic CBZ molecule is mainly translocated by a diffusion

261

mechanism across root-cell membranes.

The CBZ influx is driven by the

262

concentration gradient between the outer and inner sides of the root cell membrane in

263

accordance with Fick’s law: the initial gradient is steep facilitating the high apparent

264

CBZ-influx concentration; this is followed by a decreasing gradient across the

265

membrane and results in decreased apparent CBZ-influx concentration (Figure 2).

266

This also explains the increase in CBZ concentration in the nutrient solution with time

267

(Figure 1a). The apparent LTG-influx concentration was higher than the LTG

268

concentration in the nutrient solution and significantly higher than the apparent CBZ-

269

influx concentration throughout the entire exposure period, although declining over

270

time (Figure 2b). The large initial difference between the two compounds is probably

271

reflecting not only real uptake but also a rapid adsorption of LTG to the roots, as

272

shown in Figure S2. After this initial step, it reflects a higher concentration gradient

273

that is maintained across the root cell membranes due to the trapping mechanisms

274

described above.

275

The transpiration stream concentration factor (TSCF; Figure S3) is the ratio

276

between the concentration in the xylem sap and the concentration in the nutrient

277

solution. The TSCF indicates the efficiency of the uptake of a chemical from the

278

nutrient solution and efficiency of its translocation from roots to shoot. TSCF values

279

equal to 1 are usually interpreted as indicating that the compound is taken up and

280

translocated with the water transportation stream, values >1 indicate active transport,

281

and values

Pharmacokinetics in Plants: Carbamazepine and Its Interactions with

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