Egg Residue

Subscriber access provided by UNIV OF LETHBRIDGE

Article

IVERMECTIN PHARMACOKINETICS, METABOLISM, AND TISSUE/EGG RESIDUE PROFILES IN LAYING HENS Laura Moreno, Paula Domínguez, Cristina Farias, Lucila Cantón, Guillermo Virkel, Laura Maté, Laura Ceballos, Carlos E. Lanusse, and Luis Alvarez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04632 • Publication Date (Web): 10 Nov 2015 Downloaded from http://pubs.acs.org on November 14, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

Journal of Agricultural and Food Chemistry

IVERMECTIN PHARMACOKINETICS, METABOLISM, AND TISSUE/EGG RESIDUE PROFILES IN LAYING HENS

LAURA MORENO*, PAULA DOMINGUEZ, CRISTINA FARIAS, LUCILA CANTON, GUILLERMO VIRKEL, LAURA MATÉ, LAURA CEBALLOS, CARLOS LANUSSE, LUIS ALVAREZ

Laboratorio de Farmacología, Centro de Investigación Veterinaria de Tandil (CIVETAN), CONICET, Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Argentina.

*Corresponding author: Dra. Laura Moreno (Phone: +54-249-4385850¸E-mail address: [email protected])

1 ACS Paragon Plus Environment


Page 2 of 26

1 2

ABSTRACT

3

The goals were to determine the ivermectin (IVM) plasma pharmacokinetics, tissue

4

and egg residue profiles, and the in vitro metabolism in laying hens. Experiments

5

conducted were: 1) Eight hens were intravenously treated with IVM and blood

6

samples taken; 2) Eighty-eight hens were treated with IVM administered daily in

7

water (5 days): forty were kept and their daily eggs collected; forty-eight were

8

sacrificed in groups (n=8) at different times and tissue samples taken and

9

analysed;

3)

IVM

biotransformation

was

studied

in

liver

microsomes.

10

Pharmacokinetic parameters were AUC 85.1 ngday/mL; Vdss 4.43 L/kg and T½el

11

1.73 days. Low IVM tissue residues were quantified with the highest measured in

12

liver and skin+fat. IVM residues were not found in egg white, but significant

13

amounts were quantified in yolk. Residues measured in eggs were greater than

14

some MRLs values, suggesting that a withdrawal period would be necessary for

15

eggs after IVM use in laying hens.

16 17 18 19 20 21

KEYWORDS: ivermectin, plasma pharmacokinetics, egg residue, tissue residue

22

profiles, laying hens


Page 3 of 26


23

INTRODUCTION

24

Ivermectin (IVM) is an important broad-spectrum antiparasitic drug belonging to the

25

macrocyclic lactone family. Since it was introduced in the veterinary market in

26

1981, it has been the most commonly used antiparasitic agent in cattle, horses,

27

sheep and pigs in many countries. Its spectrum of activity covers a wide variety of

28

nematodes, microfilaria, and external parasites of domestic species1. The

29

pharmacokinetic properties of IVM have been widely studied in domestic animals1,

30

for which authorized veterinary medicinal products containing IVM are available. A

31

huge number of IVM formulations such as slow delivery systems, oral, topic or

32

injectable intended for domestic animals are available. The recommended doses

33

range from 6 µg/kg for dogs (as prophylaxis for filariasis), up to 200 µg/kg for

34

ruminants (500 µg/kg for pour on administration) and 300 µg/kg for pigs. In

35

addition, the efficacy of this compound has also been investigated in other animal

36

species like non-traditional domesticated wild ruminants (reindeer, deer, buffaloes,

37

camels, yaks, etc.), for which IVM extra-label use has been reported2. The

38

remarkable utility of the macrocyclic lactones was also demonstrated in laboratory

39

mammals, birds, fish, and reptiles many years ago3. IVM is reported to be effective

40

against nematode infections in poultry but it is not approved for use in avian

41

species. IVM administered in water was effective in removing A. galli, H.

42

gallinarum, and Capillaria in poultry4-6. The available data describing the

43

pharmacokinetic behaviour of IVM in avian species is scarce. In some countries,

44

until recently there were some oral formulations to control internal and external

45

parasites in pet or game birds and fighting cock, but most of them are no longer in

46

the market. In addition, no IVM formulations for avian production were available. 3 ACS Paragon Plus Environment


Page 4 of 26

47

However, extralabel use of this drug has been reported7, as evidenced in web-

48

based online discussion forums for avian producers. To our knowledge, IVM is

49

used mainly to control internal and external parasites in chicks and hens,

50

respectively. It is administered by applying available formulations in food, water or

51

topically in the cloaca in an empirical way. At present, there are some IVM

52

formulations intended for avian production in some Latin American countries.

53

The European Commission adopted a Regulation establishing maximum residue

54

limits for IVM in all mammalian food producing species. Extrapolation of MRLs

55

(Maximum Residue Limits) to the relevant minor species has been considered. In

56

addition, it is possible to further extrapolate the existing MRLs to other species and

57

foodstuffs with a view to ensuring availability of veterinary medicinal products for

58

conditions affecting food producing animals while ensuring a high level of

59

protection of human health. However, taking into account the current scientific

60

knowledge, the extrapolation of MRLs to poultry (including eggs) was not

61

recommended. The justification was based on metabolism patterns, which can be

62

significantly different in poultry as compared with mammals. Consequently, species

63

specific metabolism and residue data are considered necessary to allow adequate

64

evaluation of the risk to consumer safety regarding to residues in poultry-derived

65

food commodities8.

66

In order to contribute to the correct use of IVM in laying hens, the goals of the

67

present work were 1) investigate the pharmacokinetic behaviour of IVM in laying

68

hens following a single IV administration; 2) to describe the IVM tissue and egg

69

residue profiles in laying hens after oral administration in water; 3) to evaluate the

70

IVM liver microsomal metabolism in hens in vitro. 4 ACS Paragon Plus Environment

Page 5 of 26


71 72

MATERIALS AND METHODS

73

Reagents and chemicals. Pure reference standards (97–99% purity) of IVM and

74

the internal standard (IS) abamectin (ABM) were obtained from Sigma Chemical

75

Company (Saint Louis, MO, USA). Acetonitrile solvent used during the extraction

76

and drug analysis was HPLC grade and purchased from J.T. Baker (Phillipsburg,

77

USA). The N-methylimidazole and trifluoroacetic anhydride used for derivatization

78

reaction were from Aldrich (Milwaukee, WI, US). Nicotinamide adenine dinucleotide

79

phosphate (NADP+) tris (base and acetate) were purchased from Sigma-Aldrich

80

Chemical Company (St. Louis, USA). Glucose-6-phosphate and glucose-6-

81

phosphate dehydrogenase were purchased from Roche Applied Science (Buenos

82

Aires, Argentina). Buffer salts (KCl, Na2HPO4, NaH2PO4, K2HPO4, KH2PO4 and

83

MgCl2) were purchased from J.T. Baker (Phillipsburg, USA). Water was double-

84

distilled and deionized using a water purification system (Simplicity; Millipore, Sao

85

Paulo, Brazil).

86

Animals. One hundred healthy laying hens (Plymouth Rock Barrada), 6 months

87

old, and 2.2 ± 0.3 kg body weight (b.w.), were used in the experiment. The hens

88

were monitored daily during a 2-week acclimatization period. They were housed

89

with water and balanced commercial food ad libitum. Before the experiments, the

90

hens were not medicated with any anthelmintic drug.

91

IVM dosing. IVM treatments to laying hens were dosed using Ivomec® 1% (Merial)

92

diluted depending on the trial as follows: For the Intravenous (IV) treatment, a 400

93

µg/kg dose was administered. Ivomec 1% was diluted to 0.2% (1:5) with 40%

94

propylene glycol:ethanol (1:2) and 60% sterile physiological saline solution. For the 5 ACS Paragon Plus Environment


Page 6 of 26

95

Oral treatment, a dose (400 µg/kg/day) was administered in water for five days.

96

The daily volume of Ivomec® 1% to be administered (1 mL per 25 kg b.w.) was

97

diluted to 0.1% (1:10) with propylene glycol. Then, it was diluted in 25% of the total

98

volume of daily water intake. To ensure uptake of medicated water, the regular

99

drinking water was removed 2 h before administration.

100

Experimental design. All the experiments were carried out following ethical

101

guidelines of the Animal Welfare Committee of the Faculty of Veterinary Medicine,

102

Universidad Nacional del Centro de la Provincia de Buenos Aires (Internal

103

Protocol: 01/2014; Approval date: 28,04,2014).

104

Intravenous administration. Eight animals were administered a single dose (400

105

µg/kg) of IVM by IV route using a catheter (MCM 24 G, China) previously placed

106

into the right wing vein. After treatment, blood samples (1 mL) were taken by an IV

107

catheter previously placed into the left wing vein as follows: 0 (blank) and 0.25, 0.5,

108

1, 3, 6, 9, 12, 15, 24, 30, 36, and 48 h; and 3, 5, 8, and 10 days post-treatment.

109

The volume of blood taken in each sample was replaced by sterile physiological

110

saline solution (1 mL) by 10-15 second IV infusion. Blood samples taken in

111

heparinized tubes were centrifuged at 3000 rpm for 10 min, and the plasma

112

collected placed into plastic tubes and frozen at -20 °C to be analysed later.

113

Oral therapeutic treatment. Eighty-eight laying hens were treated with IVM

114

administered daily in water at 400 µg/kg dose for a five-day period. Animals were

115

divided into two groups: Group 1, Forty hens were kept and their daily eggs were

116

collected from the beginning of the treatment up to 20 days post-treatment. After

117

collection, the eggs were opened, yolk and white separated and samples placed

118

into plastic tubes, then frozen at -20 ºC to be analysed by HPLC later. Group 2, 6 ACS Paragon Plus Environment

Page 7 of 26


119

Forty-eight hens were sacrificed (following ethical guidelines) in six groups of eight

120

at the following times: 1, 3, 5, 7, 10, and 15 days post-treatment. Samples of blood,

121

muscle, liver, kidney, and skin and fat in natural proportions (skin+fat) were taken

122

after sacrifice. Blood samples were centrifuged (3000 rpm for 10 min) and the

123

plasma collected. Tissue samples were wrapped with aluminium foil and labelled.

124

All samples were frozen at -20 ºC until analysis by HPLC.

125

In vitro metabolism experiments. Samples of liver parenchyma were obtained

126

from four untreated laying hens, slaughtered following the above mentioned ethical

127

guidelines. Liver samples were rinsed with ice-cold KCl 1.15 %, covered with

128

aluminium and chilled in ice. Tissue samples were brought to the laboratory for

129

subsequent procedures, which started within 1 hour from sample collection and

130

were carried out between 0 and 4 °C. The liver microsomes were prepared as

131

previously described9. Microsomal suspensions were stored at -70 °C until used in

132

the different biotransformation assays. Protein contents of each microsomal

133

preparation were measured according to Lowry et al. (1951)10.

134

The enzymatic biotransformation of IVM was studied in liver microsomes by

135

assessing the percentages of both unchanged parent drug and its formed

136

metabolites. A typical reaction mixture contained (in a final volume of 0.5 mL):

137

sodium phosphate buffer 0.1 M (pH 7.4) containing 5 mM MgCl2 and 0.8 mM EDTA,

138

1.25 mg of microsomal protein, NADPH-generating system in phosphate buffer

139

(0.32 mM NADP+, 6.4 mM glucose-6-phosphate and 1.25 U of glucose-6-phosphate

140

dehydrogenase) and 250 ng (0.3 nmol) of IVM (dissolved in 5 µL methanol). All

141

incubation mixtures were prepared and allowed to equilibrate (1 min at 37 °C).



Page 8 of 26

142

Then, the reaction was started by the addition of 0.2 mL of the NADPH-generating

143

system. Incubations (30 min at 37 °C) were carried out in glass vials in an

144

oscillating water bath under aerobic conditions. Blank samples, containing all

145

components of the reaction mixture except the NADPH-generating system, were

146

also incubated under the same conditions. These incubations were used as

147

controls for possible non enzymatic drug conversion. All reactions were stopped by

148

the addition of 0.2 mL of acetonitrile and stored at -20 °C until analysis.

149

Samples analysis. All samples were analysed by HPLC with fluorescent detection

150

following an adapted version of the methodology previously described11.

151

Plasma extraction. Samples (0.5 mL) were placed into a 5 mL plastic tube and

152

spiked with 50 µL of the internal standard (IS) abamectin (2 ng/10 µL). Acetonitrile

153

(1 mL) was added to the sample and then mixed for ten minutes with a high speed

154

vortexing shaker (Multi-tube Vortexer, VWR Scientific Products, West Chester, PA,

155

US). After mixing, the sample was sonicated (Ultrasound Bath, Lab-Line

156

Instrument, Inc., Melrose Park, OL, US) and centrifuged (BR 4i Centrifuge, Jouan,

157

Saint Herblain, France) at 3000 rpm for 10 min at 5 ºC. The clear supernatant was

158

transferred to a tube, and the procedure repeated. The total supernatant was

159

transferred to C18 cartridges (100 mg/mL, Strata C18-T, Phenomenex) using a

160

manifold vacuum (Baker spe-24G, Phillipsburg, US). The cartridges were

161

previously conditioned with 2 mL of methanol (HPLC grade), followed by 2 mL of

162

water (HPLC grade). All samples were applied and then sequentially washed with

163

1 mL of water, 1 mL methanol:water (1:4), dried with air for 5 min and eluted with

164

1.5 mL of methanol (HPLC grade). The eluted volume was evaporated at 60 ºC to


Page 9 of 26


165

dryness in a vacuum concentrator (Speed-Vac, Savant, Los Angeles, CA, US). The

166

dry residue was dissolved in 100 µL of a N-methylimidazole:acetonitrile solution

167

(1:1 v/v). To initiate the derivatization, 150 µL trifluoroacetic anhydride:acetonitrile

168

solution (1:2 v/v) were added12 and an aliquot of 100 µL was injected into the

169

chromatographic system.

170

Tissue and egg extraction. Tissue samples (muscle, liver, kidney and skin+fat)

171

were thinly sliced, 1 g was placed into a 5-mL plastic tube and spiked with 10 µL of

172

the IS abamectin (4 ng per 10 µL). A volume (500 µL) of water was added to egg

173

white or yolk samples (500 mg) before extraction. After fortification with the IS, 1

174

mL of acetonitrile was added to each sample and mixed for 10 min on a high-

175

speed vortexing shaker. After mixing, the sample was sonicated and centrifuged at

176

2000 g for 10 min at 5 ºC. The clear supernatant was transferred to a tube and the

177

procedure repeated. The total supernatant was transferred to C18 cartridges for

178

solid phase extraction following the procedure described for plasma. An aliquot

179

(100 µL) of the derivatised sample was injected in the HPLC system.

180

Microsomal sample extraction. Microsomal sample extraction. Ten ng of ABM

181

(internal standard), dissolved in 25 µL methanol, were added to each microsomal

182

incubation. Then, samples were mixed with 0.5 mL acetonitrile, shaken for 5 min

183

and centrifuged (10000 g, 10 min). An aliquot (100 µL) of the clear supernatant

184

was evaporated to dryness and, after derivatization, injected into the HPLC

185

system.

186

HPLC

187

Corporation, Kyoto, Japan) was used to quantify IVM by HPLC. A mobile phase of

188

water/methanol/acetonitrile (6:40:54, v/v) was pumped into the system through a

quantification.

A

Shimadzu

chromatography

system

(Shimadzu



Page 10 of 26

189

C18 column (Kromasil 100-5C18, 5 µm, 4.6x250 mm) placed in an oven at 30 ºC.

190

Fluorescence detection (Spectrofluorometric detector RF 10; Shimadzu, Kyoto,

191

Japan) was performed at 365 nm excitation and 475 nm emission wavelength.

192

Validation procedure. A complete validation of the analytical procedures for the

193

extraction and quantification of IVM in the different matrixes (plasma, muscle, liver,

194

kidney, skin+fat, egg white, egg yolk of hens) was performed. The linearity of the

195

method was tested after elaboration of analytical calibration curves. Blank tissue

196

samples were fortified with IVM in different ranges of calibration: plasma (0.2-200

197

and 200-800 ng/mL); muscle (0.2-10 ng/g); liver, kidney, and skin+fat (0.5-10

198

ng/g); egg white (0.2-10 ng/g); egg yolk (0.2-100 ng/g). The extraction efficiency of

199

the analytes was determined by comparison of the peak areas from fortified blank

200

samples with the peak areas from direct injections of equivalent quantities of

201

standards. Precision and accuracy (intra- and inter-assay) of the method were

202

determined by evaluation of replicates of drug-free samples (n=5) fortified with

203

each compound at different concentrations (0.2, 5, 50 ng/g) depending on the

204

matrix. Precision was expressed as coefficient of variation (% CV). The limit of

205

quantification (LOQ) was calculated as the lowest drug concentration (n=5) on the

206

standard curve that could be quantitated with precision not exceeding 20% and

207

accuracy within 20% of nominal. The linear regression lines for IVM showed

208

correlation coefficients ≥ to 0.995. Mean absolute recovery percentages ranged

209

between 74.8-97.1%. The inter assay precision of the analytical procedures

210

obtained after HPLC analysis of IVM on different working days showed CV

211

between 0.90% and 14.1%. The LOQ values were established at 0.2 ng/mL for


Page 11 of 26


212

plasma; 0.2 ng/g for muscle, egg yolk and egg white; and 0.5 ng/g for kidney, liver

213

and skin+fat.

214

Data analysis. Tissue and egg concentrations were expressed as ng/g. The

215

pharmacokinetic parameters and concentration data are reported as mean ± SEM.

216

The pharmacokinetic analysis of the plasma concentration (ng/mL) vs. time curves

217

for IVM for each animal after IV administration was carried out using the PK

218

Solution 2.0 software (Summit 10 Research Services, CO, USA). Pharmacokinetic

219

analysis of the experimental data was performed using non-compartmental (area)

220

and compartmental (exponential terms) methods without presuming any specific

221

compartmental model. The concentration–time profile for IVM in plasma after its IV

222

administration was best fitted to a bi-exponential equation: Cp = A-αt + B

223

A and B are primary and secondary disposition intercepts; α and β were the

224

primary and secondary disposition rate constants (h-1); and Cp was the plasma

225

concentration of IVM at time t. Regression parameters from this equation were

226

then used to calculate the presented pharmacokinetic parameters. The elimination

227

half-live (T1/2el) was calculated as ln 2 divided by β rate constant. The estimated

228

plasma concentration of IVM at time zero (C0) was the sum of the extrapolated

229

time zero concentrations of the coefficients A and B. The area under the

230

concentration–time curve (AUC), clearance (Clarea) and apparent volume of

231

distribution at steady-state (Vdss) were calculated according to the equations of

232

Gibaldi and Perrier (1982)13. Statistical moment theory was applied to calculate the

233

mean residence time (MRT) in plasma13.

-βt

, where

234 235 11 ACS Paragon Plus Environment


Page 12 of 26

236

RESULTS

237

Intravenous administration. The mean (± SEM) plasma concentrations (ng/mL)

238

vs. time profile after IVM administration (400 µg/kg) by IV route to laying hens is

239

plotted in Figure 1. The highest IVM concentration (739.6 ± 50.2 ng/mL) was

240

quantified at 30 min (first sampling time), decreasing until the lowest concentration

241

(0.38 ± 0.06 ng/mL) attained at day 10, showing a typical IV profile.

242

Pharmacokinetic parameters obtained for IVM after its administration, are shown in

243

Figure 1. The plasma availability represented by AUC was 85.1 ± 4.9 ngday/mL.

244

The clearance was 4.8 ± 0.1 L/day/kg, with a T½el and MRT of 1.73 ± 0.08 and 0.95

245

± 0.11 days, respectively.

246 247

Oral therapeutic treatment. The tissue residue concentrations (mean ± SEM)

248

quantified after IVM administration in water to hens for five days are shown in

249

Table 1. Residues were quantified for a longer time in skin+fat until 15 days post-

250

treatment. Plasma, muscle and liver residues were quantified until 7 days after the

251

end of treatment, while IVM kidney residues were quantified only until 3 days post-

252

treatment. The residue levels were low, with the highest concentration measured

253

the first day post-treatment in all tissues. The tissue with the highest concentration

254

was liver, followed by skin+fat, kidney, plasma and muscle.

255

After IVM oral administration in water to laying hens for five days residues were

256

found in eggs. Yolk and egg white were analysed separately. Although IVM

257

residues were not found in egg white, significant residues were quantified in yolk

258

(Figure 2). The residue levels were higher than those found in tissues. IVM

259

residues were quantified in yolk from the second day of treatment to 17 days post12 ACS Paragon Plus Environment

Page 13 of 26


260

treatment. The maximum concentration of residues nearly 50 ng/g was quantified

261

three days post-treatment.

262

In vitro hepatic biotransformation of IVM. After 30 min incubation, mean IVM

263

concentrations decreased (27.8±4.4%, p

Egg Residue

Recommend Documents