Evaluation of pharmacokinetics and efficacy of ivermectin following oral administration in dogs against experimental infection of Ctenocephalides felis felis and Rhipicephalus sanguineus

Veterinary Parasitology 228 (2016) 167–171

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Research paper

Evaluation of pharmacokinetics and efficacy of ivermectin following oral administration in dogs against experimental infection of Ctenocephalides felis felis and Rhipicephalus sanguineus Viviane S. Magalhães a,∗ , Yara P. Cid b , Thais P. Ferreira a , Deborah M.V. Medeiros c , Lilian C. de S. O. Batista a , Thais R. Correia a , André L.M. Albert d , Fabio B. Scott a a

Animal Parasitology Department, Federal Rural University of Rio de Janeiro (UFRRJ), BR 465, Km. 7, 23897-000 Seropédica, RJ, Brazil Pharmaceutical Science Department, Federal Rural University of Rio de Janeiro, BR 465, Km. 7, 23897-000 Seropédica, RJ, Brazil c Chemical Department, Federal Rural University of Rio de Janeiro, BR 465, Km. 7, 23897-000 Seropédica, RJ, Brazil d National Institute for Quality Control in Health (INCQS), Oswaldo Cruz Foundation (Fiocruz), Av. Brazil, 4365, 21040-900 Manguinhos, RJ, Brazil b

a r t i c l e

i n f o

Article history: Received 17 June 2016 Received in revised form 5 September 2016 Accepted 7 September 2016 Keywords: Macrocyclic lactones Pharmacokinetics Ticks Fleas

a b s t r a c t With the increasing number of pets in home the human-animal relationship is increasingly close and care about control disease growing. Ivermectin (IVM) is frequently used because its proven safety. IVM is recommended for the treatment of demodectic scabies and prevention of heartworm in dogs, but informally is extremely used to control of Ctenocephalides felis felis and Rhipicephalus sanguineus. The aim of this study is evaluate the use of IVM in dogs, by the oral route at 0.6 ␮g/kg dose, against experimental infection of these parasites using the construction of the plasma concentration curve and efficacy study. A IVM quantification method in canine plasma using HPLC-FL was developed and validated based on RE n◦ 899/03 ANVISA. The samples collected during the efficacy test was analyzed by this validated method and prove Cmax of 350 ng/mL at 4 h (tmax ) and AUC of 8411 ng/h/mL. Spite of formulation have shown good absorption, the highest efficiency values found for Rhipcephalus sanguineus and Ctenocephalides felis felis were very low, 35% and 67% respectively, demonstrating this not be the most appropriate treatment for the control of these parasites. © 2016 Elsevier B.V. All rights reserved.

1. Introduction In the last decades, the pets have become increasingly important in the people’s lives, being considered friends and/or family. Dogs are the most common pet at home and as a result, the increasing of parasitic transmission, from animals to human, is not uncommon. Ectoparasites of domestic animals are the veterinary interest by fleecing action and transmission of pathogens to their hosts and the human population (Ribeiro et al., 1997). Among the most important ectoparasites that infest the dogs we can mention the fleas, as Ctenocephalides felis felis, ticks as Rhipicephalus sanguineous (Taylor, 2001). Thus, the ectoparasites control are the great importance to public health professionals, pet owner and veterinary medical (Dantas-Torres, 2008).

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (V.S. Magalhães). http://dx.doi.org/10.1016/j.vetpar.2016.09.004 0304-4017/© 2016 Elsevier B.V. All rights reserved.

To this end, there are several strategies for controlling infestations and the use of chemical agents are the most common. According to Scott et al. (2002) this control can be performed with the use of various chemical groups and associations such as organophosphates, carbamates, the formamidine, pyrethrins, pyrethroids, the phenylpyrazoles and macrocyclic lactones in different formulations and application methods. Ivermectin is a drug of macrocyclic lactones family with exceptional potency against endo- and ectoparasites at extremely low doses, highly active against a wide spectrum of nematode species, including most larvae and adult forms, and many arthropod parasites of domestic animals (González Canga et al., 2009). The excellent spectrum of activity of IVM resulted in the all-embracing name ‘endectocide’ (Lifschitz et al., 1999). The mode of action of IVM involve the act on GABA neurotransmission and glutamate gate Cl− , leading to a flaccid paralysis, death and elimination of parasites (Taylor, 2001). The selectivity of pharmacological action is associated with lack of target glutamate receptors in mammalian species and relative restriction of GABA receptors to the central nervous system in mammals. However adverse effects are attributed to

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neuro-intoxication with GABA receptor stimulation in some sensitive animals. In these sensitive breeds of dogs, the concentration of IVM in the brain were 2–31 times higher than liver and plasma. This dogs have a gene mutation that reduce the P-glycoprotein carries increasing IVM brain levels (McKellar and Gokbulut, 2012). Informally, at Brazil, clinics and specialist shops indicate IVM for the treatment of infestations by R. sanguineus using an injectable formulation for cattle at doses equal to or greater than 0.5 mg/kg. (Frederico, 2016). However their effectiveness to control ticks is not well established, because most of available studies the dogs are naturally infested (Dias et al., 2005; Morsy and Haridy, 2000; Roy and Roy, 2010). Since 1981, when IVM was has marketed, several pharmacokinetic studies were published. This drug can be administered orally (PO), intramuscularly (IM), subcutaneously (SC) or topically (TR) depending on the pet species. Route of administration will directly affect its pharmacokinetic profile (González Canga et al., 2009) and higher bioavailability can be observed with IVM subcutaneously, followed by PO and finally TR (Danaher et al., 2006; Õmura, 2008). The rational use of medicine requires basic knowledge of pharmacokinetics in the target species. It helps optimize clinical efficacy (González Canga et al., 2009). The aim of this study is establish the relationship between the control of ticks and fleas and IVM plasma concentration in Beagle dogs treated with oral ivermectin. 2. Material and methods 2.1. Experimental animals A total of 17 experimental dogs, male Beagle, 1, 5–6 years old and weighing 9–17 kg were used in this study. The animals were individually allocated in cages seven days before the treatment (day −7) and infested with 50 male fleas and 50 female fleas. The fleas were adult, not fed, aged 14 days and Ctenocephalides felis felis species. On the same day, the dogs were infested with 25 couple of ticks, Rhipicephalus sanguineus. After 48 h (day −5), the dogs were combed and all the ticks and fleas were removed and counted. Free dogs were separated into two groups. The treated was 10 dogs and control group with 7 animals. The number of ticks and fleas of animals in each group was similar. Two days before the treatment (day −2) the dogs were infested with 50 adult ticks not fed and 100 adult fleas not fed. After 48 h (day 0) the dogs were treated. During the study water was supplied ad libitum and animals were fed a standard commercial diet twice daily with an appropriate quantity of feed during the experimental period. This study was approved by Animal Ethic Committee of FAPUR. 2.2. Treatment and sampling The tablets formulation of IVM for dogs (Mectimax® , Agener União, Brazil) was administered orally, in fasting, as a single dose in the per os group at 0.6 mg/kg bodyweight (BW). After the treatment the animals were fed. This medicine was chosen because, at date, was the only product containing IVM orally registered in Brazil indicated for dogs. Heparinized blood samples were collected by venipuncture prior to drug administration then at 1, 4, 6, 8, 10, 24 and 32 h and 2, 3, 4, 7, 9, 11, 15 and 18 days. Blood samples were centrifuged at 756 × g for 15 min and plasma was transferred to plastic tubes. All the samples were stored at −20 ◦ C until the estimation of drug concentration. 2.3. Analytical procedure A stock solution (1 mg/mL) of IVM and abamectin, using as internal standard (IS), was prepared using acetonitrile (Vetec, RJ, Brazil)

as the solvent. Drug-free plasma samples (0.5 ml) were spiked with IVM and IS to reach the following final concentrations: 0,2; 20; 50; 100; 200 and 300 ng/mL for IVM and 200 ng/mL for IS. All the samples were stored at −20 ◦ C. The plasma samples (spiked and experimental) were mixed with 0.5 ml acetonitrile. After mixing for 5 min, the solvent sample mixture was centrifuged at 756 × g for 10 min. The supernatant was transferred to plastic tubes. This procedure was repeated once more and the supernatants were filtered together with HV membrane (0,45 ␮m – MILLIPORE). The filtered sample was concentrated to dryness at 70 ◦ C in a sample concentrator (TE-0197, Tecnal, RJ, BR). Derivatization was initiated by adding 100 ␮l N-N dimethylformamide (Merck, Alemanha), 40 ␮l 1 methylimidazole (Sigma) and 60 ␮l acetic anhydride (Spectrum) and subsequent incubation at 70 ◦ C for 1 h (TE-019, Tecnal). After completion of the reaction, an aliquot was injected directly into the HPLC system (Chen et al., 1996; Fink et al., 1997; Kojima et al., 1987). The plasma concentrations of IVM were analyzed by high performance liquid chromatography (HPLC—Dionex Ultimate 3000) with fluorescence detection (RF2000 DIONEX). The mobile phase consisted of methanol and water (97:3, v/v), a flow rate of 1.2 ml/min and 30 ␮l injection volume. A nucleosil C18 analytical column (MACHEREY-NAGUEL, 100-5, 125 × 4,6 mm) with C18 guard column (KROMASIL, 100-5, 10 × 4,6 mm) was used for the analysis of IVM. Fluorescence detection was at an excitation wavelength of 480 nm and an emission wavelength of 360 nm, gain of 4, medium sensibility and 10 min of run time. The analytical methods used for IVM in plasma were validated according to RE 899/03 ANVISA, to selectivity, linearity, accuracy, precision, limit of quantification, stability and recovery. The recovery was performed according AOAC/2002 since ANVISA does not have accept values for this test.

2.4. Pharmacokinectis analysis The plasma concentration vs. time curves obtained after each treatment in individual animals were fitted with the PK Solutions software program (Version 2.0 Summit Research Services). Pharmacokinetic parameters for each animal were analyzed using non-compartmental model analysis according Lifschitz et al. (2009). The maximum plasma concentration (Cmax) and time to reach maximum concentration (tmax) were obtained from the plotted concentration–time curve in each animal. The trapezoidal rule was used to calculate the area under the plasma concentration–time curve (AUC). Terminal half-life (T1/2␤ ) was calculated as ln 2/K␤ where K␤ represent the first order rate constant associated with the terminal (log linear) portion of the curve and distribution half-life (T1/2␣ ) was calculated as ln 2/K␣ where K␣ represent the constant associated with the distribution portion of the curve. The pharmacokinetic parameters are reported as mean ± SD.

2.5. Parasitological analysis To determine the efficacy of oral IVM to control C. f. felise R. sanguineus, infestations were performed and then, 48 h after, the dogs were combed and counted the parasites. The cycles of infestations and counted were repeated successively until a lower 50% efficacy was found. At the end of the study and start of the cycle the animals were dewormed mechanically until no parasite was found. To evaluate the parasites load, the number of ticks were counted by manual inspection and visual with subsequent counting of fleas. For flea counts the animals were combed with the aid of a proper comb with approximately 13 teeth per linear centimeter (“comb

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test”) as described Marchiondo et al. (2007). The efficacy of IVM was calculated according to the formula: Efficacy(%) = [(MPC–MPT) × 100]/MPC Where: MPC = mean live parasites of control group; MPT = mean live parasites of treated group. Data were analyzed by use Shapiro-Wilk normality test. The normal data was analyzed by Student t-test and non-normal by Kruskal-Wallis test. All tests were performed with a significance level set at 5%. All statistical analyses were performed by using Bioestat 5.0 (Ayres et al., 2007). Mean values were considered significantly different at p < 0.05.

3. Results 3.1. Pharmacokinectis analysis

Plasma log Concentration (ng/mL)

The analytical procedures and HPLC analysis of IVM were validated. The linear regression lines for IVM in the range between 0.2 and 300 ng/mL showed correlation coefficients upper 0.99. The mean recovery of IVM from plasma was 89,9%. The quantification limit of the analytical technique was 0.2 ng/mL. The mean interand intra-assay precisions of the analytical procedure obtained after HPLC analysis of spiked standards of IVM (0.2–50–100 ng/mL) showed a CV of 6.3% and 6.6%, respectively (Table 1). For accuracy test the mean inter and intra-assay was 93% and 99%, respectively. Stability tests assess sample stability after undergoing the extraction process (post-processing) and in case of freezing and thawing (freeze-thaw cycle). No significant interference or matrix effect was observed at the retention times of IVM and IS in spiked plasma samples. To correlate the data of plasma concentration over time of collection, each animal was examined individually. To determine the IVM distribution model has been observed the correlation coefficient obtained from the linear regression of the plasma profile of the exponential curve of concentration versus time. Indicative values of two compartments were found, with the view of two distinct lines. During this study the IVM was quantified in plasma at 1 h to 18 days, the AUC0-␣ observed was 8490,7 ngh/mL and AUC0-t was de 8411,2, corresponding to 99% of the AUC0-t . Peak plasma concentration was about 4 h and AUC was 350,7 ng/mL (Table 2).

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Table 1 Results of Validation for Ivermectin Quantification in Dogs Plasma. Validation

Ivermectin

Linearity (k = 3; m = 6; n = 2)

Serie 1

Serie 2

Serie 3

0.0069 0.0107 0.9990

0.0071 0.0105 0.9980

0.0072 0.0118 0.9990

Recovery (K = 1,m = 3, n = 5) 0.2 ng/mL 50 ng/mL 200 ng/mL

76.7 96.1 98.8

76.9 94.9 97.3

71.0 97.4 99.6

Precision (k = 3, m = 3, n = 5) Intra-day/Inter-day (RSD) 0.2 ng/mL 50 ng/mL 200 ng/mL

13.8/13.9 2.8/2.6 2.4/3.5

Accuracy (k = 3, m = 3, n = 5) Intra-day/Inter-day (%) 0.2 ng/mL 50 ng/mL 200 ng/mL

93/88 100/100 101/101

LOQ (K = 1, m = 1, n = 5)

0.2 ng/mL

Range (ng/mL) a b r2

K = number of series; m = number of concentrations levels; n = number of replicates; a = slope; b = intercept; r2 = correlation coefficient; RSD = relative standard deviation.

The plasma concentration vs. time curves fits can be observed at Fig. 1. 3.2. Efficacy studies The results of ticks and fleas score can have observed in Table 3. For C. felis felis, there was no statistical difference between the averages of the treated and control groups on days +2 and +7. At days +2 and +7 efficacies were 35% and 18% respectively, less than 50%, so the study was ended with seven days after the treatment. Pearson teste was performed to correlated the flea counts in the group treated with the concentrations found in plasma at days +2 and +7. The correlated values were found (r = −0.19; −0.64 respectively) indicates complete lack of correlation between counts of fleas and IVM plasma concentration on days +2 to +7. For R. sanguineus, there was no statistical difference between the averages of the treated and control groups on day +7. At days Semi-Log Plot Concentration - Time Curve

1000.0

100.0

10.0

1.0 0

50

100

150

200

250

300

350

400

450

500

Time (h) Fig. 1. Plot of the mean plasma concentration vs. time curves of ivermectin following orally (0.6 mg/kg), administration to dogs (n = 10).

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Table 2 Median (±SD) pharmacokinetic parameters of ivermectin administration to dogs (n = 10). Parameters

Animals

Mean± SD

1

2

3

4

5

6

7

8

9

10

Cmax (ng/mL)

340.5

390.0

404.8

304.4

506.7

177.2

318.0

333.8

384.0

347.2

350.7 ± 84.1

Tmax (h)

4

4

4

4

4

4

4

4

4

4

4.0 ± 0

AUC0-␣ (ng h/mL)

13201

10095

9315

6654

9186

4742

5580

6780

10957

8392

8490.7 ± 2599.9

AUC0-t (ng h/mL)

13107

10086

8717

6643

9183

4726

5580

6741

10942

8382

8411.2 ± 2570.4

T1/2␣ (h)

2.3

1.4

3.3

4.7

4.2

2.5

2.9

8.1

9.3

6.2

4.5 ± 2.6

T1/2 ␤ (h)

56.5

31.0

104.1

46.9

44.8

28.0

24.9

50.2

51.2

33.5

47.1 ± 22.8

Vd (L/kg)

4.4

3.6

12.1

7.5

4.3

6.2

5.3

8.7

5.5

4.8

6.3 ± 2.6

Bodyweight (kg)

12.7

11.0

16.0

12.1

9.7

12.5

11.0

11.0

11.0

10.8

Cmax : peak plasma concentration; Tmax : time to reach Cmax ; AUC0-t : area under the curve from time 0 to the last detectable concentration; AUC0-␣ : area under the curve from time 0 to infinite; T1/2␣ : distribution half-life; T1/2␤ : terminal half-life; Vd : volume of distribution.

Table 3 Median ± SD of fleas and ticks score, efficacy in controlling parasites and Person’s test for correlated score of parasites and plasma concentrated ivermectin in dogs treated orally with ivermectin tablets (0.6 mg/kg bw). Parasites

Fleas

Days

+2

+7

+2

+7

Control Group—Median ± SD Treated group—Median ± SD Eficacy r2 P

31a ± 13 20.3a ±18 35.1 −0,19 0,61

60 a ± 20 49.6a ± 16 17.9 −0.64 0,05

19a ± 7 6.3b ± 4 67.1 0,12 0,73

25a ± 8 18.5a ± 16 24.7 0,05 0,89

Ticks

SD = standard deviation; r2 = correlation coefficient; p = p-value of Person’s test; ab = Columns with same letters have no statistical difference.

+2 and +7 efficacies were 67% and 24% respectively, less than 50%, so the study was ended with seven days after the treatment. Pearson teste was performed to correlated the flea counts in the group treated with the concentrations found in plasma at days +2 and +7. The correlated values were found (r = 0.12; 0.05 respectively) indicates complete lack of correlation between counts of fleas and IVM plasma concentration on days +2 to +7 4. Discussion Several studies have been published reporting the plasma profile of IVM in dogs to 0.2 mg/kg dose (Eraslan et al., 2010; Gokbulut et al., 2006). These studies also demonstrate two-compartment models in pharmacokinetics profiles. IVM has lipophilic characteristic that tends to deposit in the subcutaneous tissues and have constant distribution of different values of the tissues and the blood compartment (Borges et al., 2008; Chittrakarn et al., 2009; Eraslan et al., 2010; González Canga et al., 2009). The median Cmax to SC was 40 ng/mL while PO presents very disparate data ranging from 117 ng/mL in Gokbulut et al. (2006) and 19 ng/mL by Kojima et al. (1987). The Cmax value found for this study was much higher than reported in previous studies. This difference can be attributed to the increased dose of 0.6 mg/kg but correcting the dose to 0.2 mg/kg assuming proportionality the Cmax was 116.9 ng/mL. This values corroborate those found by Gokbulut et al. (2006). The terminal half-life presented by previous studies for SC formulations were much higher than found for PO, about 112 and 59 h respectively. This can be explained by the lipophilicity of IVM which tends to be deposited at the application site resulting in an extended release formulations for SC route (Danaher et al., 2006; Löffler and Ternes, 2003; Taylor, 2001). The same does not occur in oral formulations decreasing its half-life. The present study shown a terminal

half-life value slightly lower than the previous studies, however it can be observed that the animals in the study with lower weights showed values of terminal half-life lower when compared to animals with higher weights. Heavier animals, in general, have greater adipose tissue mass. Ivermectin has a lipophilic character and tends to be released more slowly at adipose tissues increasing your elimination half-life. Correlations of pharmacokinetic parameters with body-weights in different species were showed by McKellar and Gokbulut (2012) with negative correlations between bodyweight and volume of distribution (Vd ) or clearance (Cl) and positive correlations for area under the concentration time curve (AUC) and elimination half-life (T1/2 ). Eraslan et al. (2010) found a median AUC 4054 ngh/mL for SC formulation at a dose of 0.2 mg/kg. The plasma profile presented in this study found a AUC 8491 ngh/mL, value little more than twice the findings by Eraslan et al. (2010). The findings corroborate with the average AUC value observed in the study. Higher doses of IVM increase bioavailability and consequently the AUC because the bioavailability of IVM is dose linear as described above. Zakson-Aiken et al. (2001) demonstrated in studies in vitro and in vivo that the avermectin, in general, not show high effectiveness to controling infestations of C. f. felis and IVM is one of avermectins less effective in vitro. Calves infested with C.f.felis were not wormed with ivermectin SC to (Araújo et al., 1998). Phipps et al. (2005) showed increased permeability of selamectin in the brain of ivermectin in comparison fleas and suggested that permeability differences can be responsible for their low effectiveness in combating the C.f. felis. These results corroborate with the findings in this study because independent of plasma concentrations of IVM was no significant effectiveness. Few studies to evaluate the effectiveness against R. sanguineus using protocols based on WAAVP guide (Worl Association for de advancement of Veterinary Parasitology) (Marchiondo et al., 2007) are found. Morsy and Haridy (2000) demonstrated that IVM subcutaneously at a dose of 0.3 mg/kg was sufficient to eliminate 100% of the ticks in four days in vivo assay. Although the demonstrated efficacy, Morsy uses a very small number of animals, only two per group, and it is not clear the parasite load of individual animals because they are infested naturally. The results of this study do not corroborate with demonstrated by Morsy probably due to lack of standardization of the parasite load of study and the low number of animals used by Morsy. Another trial (Roy and Roy, 2010) demonstrates the efficacy of 100% after 14 days to a formulation pour on the dose of 0.5 mg/kg bw applied to seven-day intervals on tick combat. This efficacy may be related to the fact that the product is topical, the direct contact acaricide effect with the IVM and the weekly re-

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treatment. In this study the treatment was orally. This explains the difference of effectiveness found between studies. In a study in Ibiúna region, São Paulo, naturally infected dogs were treated with two doses of IVM subcutaneously 0.3 mg/kg, with an interval between doses of 15 days and had total absence of ticks after a week of treatment. This study was educational character only to guide dog owners about veterinary primary care. The parasite control was performed only visually during the visit of the veterinary team home owners. No control was carried out to monitor the effectiveness of the insecticide, not even the period when the survey was conducted was documented. Thus it is not possible to guarantee that no other insecticide method was used and that the IVM was solely responsible for the disinfestation of animals (Nogari et al., 2004). Treatment of dogs with IVM subcutaneously at a dose of 20 mg/kg was shown to be effective in combating tick at study carried out Dias et al. (2005). This study aimed to evaluate the possibility of R. sanguineus be vector of Trypanossoma cruzi. Therefore, was investigated tick control with high doses of IVM and subsequent analysis of the same to verify the presence or absence of T. cruzi. It is important to say that the dose used in this study is 100 times higher than recommended for the use of IVM and even at high doses was not possible to prevent reinfestation new tick in treated animals. Efficacy values found in this study are lower than reported and does not suggest a residual effect of the product, however none of the above studies determined appropriate protocols for assessing the effectiveness and for this reason discrepant values reported previously may have been found. According to the WAAVP guide (Marchiondo et al., 2007) product with acaricide and pulgicida efficacy should promote reduction of 90% of the population, in the other words, have 90% effectiveness. These results were not found to oral formulation of IVM single dose of 0.6 mg/kg for dogs. 5. Conclusion In conclusion, the present study indicated that ivermectin orally at a dose of 0.6 mg/kg administered as a single dose for Beogle dogs showed a higher AUC and Cmax when compared with IVM orally at a dose of 0.2 mg/kg (Gokbulut et al., 2006), however it was not effective for controlling of C. f. felis and R. sanguineus. Acknowledgments This study was supported by Fundac¸ão de Apoio à Pesquisa Tecnológica da Universidade Federal Rural do Rio de Janeiro (FAPUR), Coordenac¸ão de Aperfeic¸oamento Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References Õmura, S., 2008. Ivermectin: 25 years and still going strong. Int. J. Antimicrob. Agents 31, 91–98. Araújo, F., Silva, M., Lopes, A., Ribeiro, O., Pires, P., Carvalho, C.M., Balbuena, C., Villas, A., Ramos, J.K., 1998. Severe cat flea infestation of dairy calves in Brazil. Vet. Parasitol. 80, 83–86. Ayres, M., Ayres Junior, M., Ayres, D.L., Santos, A.A., 2007. Bioestat, Aplicac¸ ões estatísticas nas áreas das ciências bio-médicas, 5th ed. Ong Mamiraua, Belém, PA.

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