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Comparative plasma dispositions of ivermectin and doramectin following subcutaneous and oral administration in dogs
Veterinary Parasitology 135 (2006) 347–354 www.elsevier.com/locate/vetpar
Comparative plasma dispositions of ivermectin and doramectin following subcutaneous and oral administration in dogs Cengiz Gokbulut a,b,*, Umit Karademir a, Murat Boyacioglu a, Quintin A. McKellar c a
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Adnan Menderes, Isikli Koyu, Aydin, Turkey b Research and Development Laboratory, University of Adnan Menderes, Aydin, Turkey c The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK Received 11 July 2005; received in revised form 30 September 2005; accepted 6 October 2005
Abstract This study evaluates the comparative plasma dispositions of ivermectin (IVM) and doramectin (DRM) following oral and subcutaneous administration (200 mg/kg) over a 40-day period in dogs. Twenty bitches were allocated by weight in to four groups (Groups I–IV) of five animals each. Animals in the first two groups (Groups I and II) received orally the injectable solutions of IVM and DRM, respectively, at the dose of 200 mg/kg bodyweight. The other two groups (Groups III and IV) received subcutaneously injectable solutions at the same dose rate. Blood samples were collected between 1 h and 40 days after treatment and the plasma samples were analysed by high performance liquid chromatography (HPLC) using fluorescence detection. The results indicated that IVM produced a significantly higher maximum plasma concentration (Cmax: 116.80 10.79 ng/ml) with slower absorption (tmax: 0.23 0.09 day) and larger area under the concentration versus time curve (AUC: 236.79 41.45 ng day/ml) as compared with DRM (Cmax: 86.47 19.80 ng/ml, tmax: 0.12 0.05 day, AUC: 183.48 13.17 ng day/ml) following oral administration of both drugs; whereas no significant differences were observed on the pharmacokinetic parameters between IVM and DRM after subcutaneous administrations. In addition, subcutaneously given IVM and DRM presented a significantly lower maximum plasma concentration (Cmax: 66.80 9.67 ng/ml and 54.78 11.99 ng/ml, respectively) with slower absorption (tmax: 1.40 1.00 day and 1.70 0.76 day, respectively) and larger area under the concentration versus time curve (AUC: 349.18 47.79 ng day/ml and 292.10 78.76 ng day/ml, respectively) as compared with the oral administration of IVM and DRM, respectively. No difference was observed for the terminal half-lives (t1=2lz ) and mean residence times (MRT) of both molecules. Considering the pharmacokinetic parameters, IVM and DRM could be used by the oral or subcutaneous route for the control of parasitic infection in dogs. # 2005 Elsevier B.V. All rights reserved. Keywords: Anthelmintics; Endectocides; Ivermectin; Doramectin; Pharmacokinetics; Dogs
* Corresponding author. Tel.: +90 256 247 03 40; fax: +90 256 247 07 00. E-mail address: [email protected] (C. Gokbulut). 0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.10.002
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1. Introduction Ivermectin (IVM) and doramectin (DRM) are members of avermectins and have a similar antiparasitic activity at extremely low dosage rates based on a common mode of action. Both chemicals are naturally derived 16-membered macrocyclic lactones (Takiguchi et al., 1980) produced by the soil dwelling actinomycetes, Streptomyces spp. Doramectin differs from IVM through substitution of a cyclohexyl group at the C-25 position (Fig. 1). They are highly effective against nematode and ectoparasitic arthropods in different host species. However, neither molecule is effective against trematodes or cestodes (Shoop et al., 1995).
The pharmacokinetic behaviour of avermectins and milbemycins are significantly affected by route of administration, formulation of the drug, and interspecies and interindividual variation (McKellar and Benchaoui, 1996). These anthelmintics are highly lipophilic substances, and are extensively distributed throughout the body and slowly eliminated from tissues such as liver and fat (Zulalian et al., 1994). Consequently, these compounds have large volumes of distribution (Lanusse et al., 1997). However, bioavailability of endectocides following oral administration may result in lower efficacy and it has been shown that subcutaneous administration of endectocides is a much more efficient route for
Fig. 1. Chemical structures of ivermectin (IVM) and doramectin (DRM).
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administration in sheep, cattle and goats as compared to the oral route (Alvinerie, 1997; Gayrard et al., 1999; Laffont et al., 2001; Lespine et al., 2003). Although, a significant first-pass metabolism of IVM is not present in ruminant species (Prichard et al., 1985), oral administration confers relatively lower bioavailability of endectocides because of binding with the organic gut content in sheep (Hennessy et al., 2000) and cattle (Alvinerie et al., 1999). Plasma dispositions of IVM and DRM have been studied in different animal species. In these studies, it has been demonstrated that DRM has a greater bioavailability and a longer persistence than IVM when administered by the same routes in sheep (Atta and Abo-Shihada, 2000; Barber et al., 2003), cattle (Toutain et al., 1997; Lanusse et al., 1997; Gayrard et al., 1999), horse (Gokbulut et al., 2001; Perez et al., 2002) and donkey (Gokbulut et al., 2005), suggesting that DRM elimination is slower than IVM. Doramectin is relatively a newer avermectin, licensed for use cattle, sheep and swine, its extralabel use in dogs as an endectocide has been increasing in recent years. The pharmacokinetic behaviour of IVM has been investigated more extensively than that of DRM as IVM is so far the most widely used endectocide across animal species. However, there is a lack of information in the literature on the plasma pharmacokinetics of DRM in dogs. The present study describes the comparative plasma kinetic profile of IVM and DRM in dogs for a 40-day period following oral and subcutaneous administration at a dose of 200 mg/kg bodyweight.
2. Material and methods 2.1. Experimental animals A total of 20 cross-bred bitches, 2–5 years old and weighing 15–30 kg were used in the study. For the duration of the study, the animals in each group were housed in appropriate pens and each dog was identified by natural markings. Water was supplied ad libitum and animals were fed once daily with an appropriate quantity of feed during the experiment period. This study was approved by Animal Ethic Committee of University of Adnan Menderes.
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2.2. Treatments and sampling The animals were allocated into four groups of five such that the mean weight of animals in each group was similar. Group I (IVM-OR) and group II (DRMOR) received orally the commercially available injectable solution of IVM (Ivomec1, 1%, w/v, formulated in propylene glycol and glycerol formal, 60:40, v/v) and DRM (Dectomax1, 1%, w/v, formulated in sesame oil and ethyl oleate, 90:10, v/ v), respectively, as a single dose on the back of the tongue each at the dose of 200 mg/kg bodyweight. Group III (IVM-SC) and group IV (DRM-CS) received the injectable solutions subcutaneously at the same dose rate. The drugs were administered by subcutaneous injection in the lateral midline of the back as a single dose. After the treatment the animals were observed continuously for any adverse reactions within the first day. Heparinized blood samples (5 ml) were collected by cephalic venepuncture 1 day prior to drug administration and 1, 2, 4, 8, 12, 16, 24, 32, 48, 72, 96 h and 6, 9, 12, 15, 20 25, 30, 35 and 40 days posttreatment. Blood samples were centrifuged at 2000 g for 20 min and plasma was transferred to plastic tubes. All the plasma samples were stored at 20 8C until estimation of drug concentration. 2.3. Analytical procedures The parent compounds of IVM and DRM in plasma were analysed using validated high performance liquid chromatography (HPLC) with a liquid–liquid phase extraction procedures. Plasma concentrations were measured by modification of the methods of Scott and McKellar (1992) and Gokbulut et al. (2001). Stock solutions (100 mg/ml) of pure standard of IVM (Merck, Rahway, NJ, USA) and DRM (Pfizer Inc., Groton, USA) were prepared using acetonitrile as the solvent. These were diluted to give 5, 10, 100, 250, 500 and 1000 ng/ml standard solutions for calibration as standard curves and to add to drug-free plasma samples to determine the recovery. Drug-free plasma samples (1 ml) were spiked with either IVM or DRM standards to reach the following final concentrations: 0.5, 1, 10, 25, 50 and 100 ng/ml. Doramectin (50 ng) and IVM (50 ng) were used as an internal standard for IVM and DRM studies,
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respectively. Acetonitrile (1 ml) was added, and after vortexing for 15 s, 5 ml chloroform was also added. The tubes were shaken on a slow rotary mixer for 15 min. After centrifugation at 2000 g for 15 min, the supernatant was removed with a Pasteur pipette. The organic phase (3 ml) was transferred to a thinwalled 10-ml conical glass tube and evaporated to dryness at 45 8C in a sample concentrator (Maxi-dry plus, Heto Lab. Equipment, Denmark). The dry residue was dissolved in 100 ml of N-methylimidazole (Aldrich Chemical Company Inc., WI, USA) solution in acetonitrile (1:1). To initiate the derivatization, 150 ml trifluoroacetic anhydride (Aldrich Chemical Company Inc.) solution in acetonitrile (1:2) was added. Finally, 35 ml of this solution was injected into the chromatographic system. The mobile phase consisted of acetonitrile and methanol (66:34, v/v) for IVM and DRM and was delivered (Agilent 1100 Series QuatPump, Waldron, Germany) at a flow rate of 1.5 ml min1. A nukleosil C18 analytical column (4 mm, 250 mm 4.6 mm Macherey-Nagel) with nucleosil C18 guard column (Phenomenex, Cheshire, UK) was used for analysis of both molecules. Fluorescence detection (Agilent 1100 Series, FLD Waldron, Germany) was at an excitation wavelength of 365 nm and an emission wavelength of 475 nm.
response relationship. The detection limit of the IVM or DRM was established with HPLC analysis of blank plasma fortified with the standard, measuring the baseline noise at the retention time of the peak. The mean baseline noise at the peak retention time plus three standard deviations was defined as the detection limit (LOD). The mean baseline noise plus six standard deviations was defined as the limit of quantification (LOQ). 2.5. Pharmacokinetic and statistical analysis of data The plasma concentration versus time curves obtained after each treatment in individual animals, were fitted with the WinNonlin software program (Scientific Consulting Inc.). Pharmacokinetic parameters for each animal were analysed using noncompartmental model analysis with extravascular input. The maximum plasma concentration (Cmax) and time to reach maximum concentration (tmax) were obtained from the plotted concentration–time curve of each drug in each animal. The area under the plasma concentration time curve (AUC) and mean residence time (MRT) from time zero to last time with a measurable concentration was calculated by trapezoidal rule. Terminal half-life (t1=2lz ) was calculated as:
2.4. Validation procedures The analytical method used for IVM and DRM in dog plasma was validated prior to the start of the studies. The analytes were identified with the retention times of pure reference standards. Recoveries of the two molecules under study were measured by comparison of the peak areas from spiked plasma samples with the areas resulting from direct injections of standards prepared in acetonitrile. The inter-assay precision of the extraction and chromatography procedures was evaluated by processing replicate aliquots of drug-free dog plasma samples containing known amounts of the drugs on different days. Calibration graphs for IVM and DRM were prepared (linear range 0.25–100 ng/ml). The slope of the lines between peak areas and drug concentration was determined by least squares linear regression and correlation coefficient (r) and coefficient of variations (CV) calculated. Linearity was established to determine the IVM and DRM concentration/detector
t1=2lz ¼
lnð2Þ lz
where lz represents the first order rate constant associated with the terminal (log linear) portion of the curve. The pharmacokinetic parameters are reported as mean S.D. Mean pharmacokinetic parameters were statistically compared by an analysis of variance (ANOVA). Mean values were considered significantly different at P < 0.05.
3. Results The analytical procedures for the determination of IVM and DRM plasma concentrations were validated. The linear regression lines for IVM and DRM under the study in the range between 0.25 and 100 ng/ml showed correlation coefficients as 0.996 (IVM) and
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Table 1 Validation of analytical method used to determination of ivermectin (IVM) and doramectin (DRM) concentration in dog plasma
Limit of quantification (ng/ml) Recovery (%) Linearity (r) Coefficient of variation (%)
IVM
DRM
0.25 96.4 (6.51) 0.996 4.75
0.25 94.29 (6.39) 0.999 3.53
Values in the brackets represent the coefficient of variations for the recovery assays; r: correlation coefficient.
0.999 (DRM). The mean extraction recoveries were 96.4% for IVM and 94.29% for DRM. The inter-assay precision of the extraction and chromatographic procedures for IVM and DRM showed coefficients of variations 4.75 and 5.53%, respectively. The validation parameters for both molecules are summarised in Table 1. No adverse response was observed for any of the treatments during the study. The parent molecule was detected in plasma between 1 h and either 25 days after oral administration of both molecules and subcutaneous administration of DRM, whereas the last detectable plasma concentration extended to 30 days following subcutaneous administration of IVM. Mean pharmacokinetic parameters of IVM and DRM after oral and subcutaneous administration of 0.2 mg/kg bodyweight are shown in Table 2 with the linear and semi log plot of mean plasma concentration versus time curves (Fig. 2). In addition, the mean plasma concentrations after oral and subcutaneous
Fig. 2. Mean plasma concentration of ivermectin (IVM) and doramectin (DRM) over the first 15 days following oral and subcutaneous administration to dogs at a dose rate of 200 mg/kg. (Smaller graph: semi log plot of mean plasma concentration vs. time curves.)
administrations of IVM and DRM are compared in Figs. 3 and 4, respectively. The results indicated that IVM produced a significantly higher maximum plasma concentration (Cmax: 116.80 10.79 ng/ml) with slower absorption (tmax: 0.23 0.09 day) and larger area under the concentration versus time curve (AUC: 236.79 41.45 ng day/ml) as compared with DRM (Cmax: 86.47 19.80 ng/ml, tmax: 0.12 0.05 day, AUC: 183.48 13.17 ng day/ml) following oral administration of both drugs. Although no significant differences were found for the pharmacokinetic parameters, Cmax (66.80 9.67 ng/ml) and AUC (349.18 47.79 ng day/ml) values for the IVM group were higher than those obtained for the DRM (Cmax:
Table 2 Mean (S.D.) pharmacokinetic parameters of ivermectin (IVM) and doramectin (DRM) following oral and subcutaneous administration to dogs at a dose rate of 200 mg/kg Kinetic parameters tmax (day) Cmax (ng/ml) AUClast (ng day/ml) t1=2lz (days)d AUMClast (ng day2/ml) MRTlast (day)
IVM-OR
DRM-OR a
0.23 0.09 116.80 10.79a,c 236.79 41.45a,c 3.32 1.56 1074.16 569.94 4.35 1.78
b
0.12 0.05 86.47 19.80b 183.48 13.17b 3.75 0.89 864.10 256.95 4.66 1.11
IVM-SC
DRM-SC
1.40 1.00 66.80 9.67 349.18 47.79 3.19 0.95 1904.22 695.50 5.32 1.26
1.70 0.76 54.78 11.99 292.10 78.76 3.09 0.99 1548.68 688.07 5.07 1.18
tmax: time to reach peak plasma concentration, Cmax: peak plasma concentration, AUClast: area under the (zero moment) curve from time 0 to the last detectable concentration, t1=2lz : terminal half-life, AUMClast: area under the moment curve from time 0 to t last detectable concentration, MRTlast: mean residence time. a IVM-OR significantly different (P < 0.05) from IVM-SC. b DRM-OR significantly different (P < 0.05) from DRM-SC. c IVM-OR significantly different (P < 0.05) from DRM-OR. d Values represent the harmonic means for t1=2lz (days).
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4. Discussion
Fig. 3. Semi log plot of mean (S.D.) plasma concentration of ivermectin following oral (IVM-OR) and subcutaneous (IVM-SC) administration to dogs at a dose rate of 200 mg/kg.
54.78 11.99 ng/ml, AUC: 292.10 78.76 ng day/ ml) group after subcutaneous administrations. In addition, subcutaneously given IVM and DRM presented a significantly lower maximum plasma concentration (Cmax: 66.80 9.67 ng/ml and 54.78 11.99 ng/ml, respectively) with slower absorption (tmax: 1.40 1.00 day and 1.70 0.76 day, respectively) and larger area under the concentration versus time curve (AUC: 349.18 47.79 ng day/ml and 292.10 78.76 ng day/ml, respectively) as compared with the oral administration of IVM and DRM, respectively. The terminal half-life (t1=2lz ) and MRT of the IVM and DRM observed in the present study were not significantly different from each other after both oral and subcutaneous administrations.
Fig. 4. Semi log plot of mean (S.D.) plasma concentration of doramectin following oral (DRM-OR) and subcutaneous (DRM-SC) administration to dogs at a dose rate of 200 mg/kg.
The plasma pharmacokinetic profiles obtained following oral and subcutaneous administration of IVM and DRM were different in dogs when compared with the other animals. In ruminants and equine species, DRM produced greater bioavailability and longer persistence compared with IVM when administered by same route of administration (Toutain et al., 1997; Atta and Abo-Shihada, 2000; Barber et al., 2003; Lanusse et al., 1997; Gayrard et al., 1999; Gokbulut et al., 2005; Perez et al., 2002). The findings of the present study do not support the previous studies. In contrast to ruminants and equine species, DRM generated a relatively lower plasma concentration and bioavailability than those of IVM following oral administration, whereas no significant difference was absorbed between both molecules following subcutaneous administration in dogs. These differences may be attributable to using different injectable formulation of IVM and DRM solutions for oral administration and the physiological or body composition differences between dogs and the other animals. It has been suggested that the major factors contributing to the differences could be a consequence of differences in the physico-chemical properties of the drug and in the formulations of both drugs (Wicks et al., 1993; Toutain et al., 1997; Lanusse et al., 1997). IVM is chemically different from DRM which has cyclohexyl group at a position 25 and this group may confer reduced polarity on DRM (Toutain et al., 1997). Moreover, IVM is formulated in propylene glycol and glycerol formal whereas DRM is in sesame oil and ethyl oleate. Previous studies have shown that the plasma disposition of avermectins was considerably affected by the route of administration. A significant difference in the bioavailability and elimination half-life of IVM was observed between oral and subcutaneous/intramuscular administration in sheep and horses (Marriner et al., 1987; Perez et al., 2003) and cattle (Chiu et al., 1990). Similarly, in swine higher bioavailability and slower absorption were observed following subcutaneous than oral administration (Fink and Porras, 1989). In the present study, the slow absorption of IVM and DRM resulted in larger AUC when the drugs were given subcutaneously as compared with their oral administration in dogs. Such slow adsorption was
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attributed to precipitation of the drug at the injection site (Lo et al., 1985), which was later supported by the findings of Scott and McKellar (1992), who reported the presence of large amount of IVM at the injection site 24 h after subcutaneous administration. It has been suggested that the peak concentration (Cmax) increased directly with the oral doses, suggesting a linear relationship between the amount of drug absorbed and the administered dose in dogs (Fink and Porras, 1989). Following oral tablet administration formulation of IVM at three dose levels (6, 27 and 100 mg/kg body weight) produced linear increased peak plasma concentrations (2.97, 19.1 and 44.3 ng/ml, respectively) (Daurio et al., 1992, Kojima et al., 1987) in dogs. The ratios of dose-Cmax were 2.0, 1.4 and 2.2, respectively. This was also supported by the findings of the present study, since Cmax was 116.8 ng/ml after oral administration of IVM at 200 mg/kg and the corresponding ratio was 1.7. The antiparasitic spectrum and efficacy pattern of the different avermectin molecules are similar; however, differences in physico-chemical properties among them may account for differences in formulation flexibility, kinetic behaviour, and in the potency and persistence of their antiparasitic activity. It has been demonstrated that plasma availability of IVM (Lo et al., 1985) and doramectin (Wicks et al., 1993) in cattle is profoundly affected by the solvent vehicle in which the drug is formulated. Although IVM and DRM are widely used in veterinary medicine as an anthelmintic with a wide margin of safety, some dogs of collie breed and colliecross have extreme IVM sensitivity (Paul et al., 1987; Pulliam et al., 1985; Hopper et al., 2002) and they display clinical signs of neurointoxication such as salivation, dilated pupils, vomiting, tremors, ataxia and depression at much lower doses of ivermectin than the Beagles did. The hypersensitivity and neurointoxication was associated with increased ivermectin accumulation in the brain of sensitive Collies (Paul et al., 1987; Pulliam and Preston, 1989). It was suggested that these dogs had a genetic deficiency in a MDR1 (multi drug resistance, MDR)-type P-glycoprotein which is an important component of the blood-brain barrier (Neff et al., 2004; Roulet et al., 2003; Mealey et al., 2001, 2005). More recently, a similar toxicity was reported for DRM in a Collie following subcutaneous administra-
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tion of 200 mg/kg bodyweight (Yas-Natan et al., 2003). Ivermectin sensitive collies tolerate 50 mg/kg, and therefore the therapeutic administration at the recommended dose of 6 mg/kg confers a large safety margin on the commercialize product (Pulliam et al., 1985; Paul et al., 1987). Although no adverse response was observed for any of the dogs during present study, a high dose (200 mg/kg) of IVM and DRM could be neurotoxic to some hypersensitive dogs.
5. Conclusion In conclusion, the present study indicated that oral and subcutaneous treatment of IVM and DRM at doses as high as 200 mg/kg did not cause any clinically detectable adverse affects in dogs. In contrast with ruminants and equine species, oral administration of IVM displayed a significantly higher plasma concentration than that of DRM, whereas no significant differences were observed on the pharmacokinetic parameters between both molecules after subcutaneous administrations. Considering the pharmacokinetic disposition, IVM and DRM could be used by oral or subcutaneous route for the control and treatment of parasitic diseases in dogs. However, because of avermectin hypersensitivity, much lower therapeutic dose of both drugs must be used in some dog breeds. Moreover, development and optimization of appropriate formulations of DRM for oral and subcutaneous administration may be required to use in dogs.
Acknowledgements This study was supported by Scientific Research Committee of Adnan Menderes University (VTF05005), Aydin, Turkey.
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Comparative plasma dispositions of ivermectin and doramectin following subcutaneous and oral administration in dogs Cengiz Gokbulut a,b,*, Umit Karademir a, Murat Boyacioglu a, Quintin A. McKellar c a
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Adnan Menderes, Isikli Koyu, Aydin, Turkey b Research and Development Laboratory, University of Adnan Menderes, Aydin, Turkey c The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK Received 11 July 2005; received in revised form 30 September 2005; accepted 6 October 2005
Abstract This study evaluates the comparative plasma dispositions of ivermectin (IVM) and doramectin (DRM) following oral and subcutaneous administration (200 mg/kg) over a 40-day period in dogs. Twenty bitches were allocated by weight in to four groups (Groups I–IV) of five animals each. Animals in the first two groups (Groups I and II) received orally the injectable solutions of IVM and DRM, respectively, at the dose of 200 mg/kg bodyweight. The other two groups (Groups III and IV) received subcutaneously injectable solutions at the same dose rate. Blood samples were collected between 1 h and 40 days after treatment and the plasma samples were analysed by high performance liquid chromatography (HPLC) using fluorescence detection. The results indicated that IVM produced a significantly higher maximum plasma concentration (Cmax: 116.80 10.79 ng/ml) with slower absorption (tmax: 0.23 0.09 day) and larger area under the concentration versus time curve (AUC: 236.79 41.45 ng day/ml) as compared with DRM (Cmax: 86.47 19.80 ng/ml, tmax: 0.12 0.05 day, AUC: 183.48 13.17 ng day/ml) following oral administration of both drugs; whereas no significant differences were observed on the pharmacokinetic parameters between IVM and DRM after subcutaneous administrations. In addition, subcutaneously given IVM and DRM presented a significantly lower maximum plasma concentration (Cmax: 66.80 9.67 ng/ml and 54.78 11.99 ng/ml, respectively) with slower absorption (tmax: 1.40 1.00 day and 1.70 0.76 day, respectively) and larger area under the concentration versus time curve (AUC: 349.18 47.79 ng day/ml and 292.10 78.76 ng day/ml, respectively) as compared with the oral administration of IVM and DRM, respectively. No difference was observed for the terminal half-lives (t1=2lz ) and mean residence times (MRT) of both molecules. Considering the pharmacokinetic parameters, IVM and DRM could be used by the oral or subcutaneous route for the control of parasitic infection in dogs. # 2005 Elsevier B.V. All rights reserved. Keywords: Anthelmintics; Endectocides; Ivermectin; Doramectin; Pharmacokinetics; Dogs
* Corresponding author. Tel.: +90 256 247 03 40; fax: +90 256 247 07 00. E-mail address: [email protected] (C. Gokbulut). 0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.10.002
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1. Introduction Ivermectin (IVM) and doramectin (DRM) are members of avermectins and have a similar antiparasitic activity at extremely low dosage rates based on a common mode of action. Both chemicals are naturally derived 16-membered macrocyclic lactones (Takiguchi et al., 1980) produced by the soil dwelling actinomycetes, Streptomyces spp. Doramectin differs from IVM through substitution of a cyclohexyl group at the C-25 position (Fig. 1). They are highly effective against nematode and ectoparasitic arthropods in different host species. However, neither molecule is effective against trematodes or cestodes (Shoop et al., 1995).
The pharmacokinetic behaviour of avermectins and milbemycins are significantly affected by route of administration, formulation of the drug, and interspecies and interindividual variation (McKellar and Benchaoui, 1996). These anthelmintics are highly lipophilic substances, and are extensively distributed throughout the body and slowly eliminated from tissues such as liver and fat (Zulalian et al., 1994). Consequently, these compounds have large volumes of distribution (Lanusse et al., 1997). However, bioavailability of endectocides following oral administration may result in lower efficacy and it has been shown that subcutaneous administration of endectocides is a much more efficient route for
Fig. 1. Chemical structures of ivermectin (IVM) and doramectin (DRM).
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administration in sheep, cattle and goats as compared to the oral route (Alvinerie, 1997; Gayrard et al., 1999; Laffont et al., 2001; Lespine et al., 2003). Although, a significant first-pass metabolism of IVM is not present in ruminant species (Prichard et al., 1985), oral administration confers relatively lower bioavailability of endectocides because of binding with the organic gut content in sheep (Hennessy et al., 2000) and cattle (Alvinerie et al., 1999). Plasma dispositions of IVM and DRM have been studied in different animal species. In these studies, it has been demonstrated that DRM has a greater bioavailability and a longer persistence than IVM when administered by the same routes in sheep (Atta and Abo-Shihada, 2000; Barber et al., 2003), cattle (Toutain et al., 1997; Lanusse et al., 1997; Gayrard et al., 1999), horse (Gokbulut et al., 2001; Perez et al., 2002) and donkey (Gokbulut et al., 2005), suggesting that DRM elimination is slower than IVM. Doramectin is relatively a newer avermectin, licensed for use cattle, sheep and swine, its extralabel use in dogs as an endectocide has been increasing in recent years. The pharmacokinetic behaviour of IVM has been investigated more extensively than that of DRM as IVM is so far the most widely used endectocide across animal species. However, there is a lack of information in the literature on the plasma pharmacokinetics of DRM in dogs. The present study describes the comparative plasma kinetic profile of IVM and DRM in dogs for a 40-day period following oral and subcutaneous administration at a dose of 200 mg/kg bodyweight.
2. Material and methods 2.1. Experimental animals A total of 20 cross-bred bitches, 2–5 years old and weighing 15–30 kg were used in the study. For the duration of the study, the animals in each group were housed in appropriate pens and each dog was identified by natural markings. Water was supplied ad libitum and animals were fed once daily with an appropriate quantity of feed during the experiment period. This study was approved by Animal Ethic Committee of University of Adnan Menderes.
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2.2. Treatments and sampling The animals were allocated into four groups of five such that the mean weight of animals in each group was similar. Group I (IVM-OR) and group II (DRMOR) received orally the commercially available injectable solution of IVM (Ivomec1, 1%, w/v, formulated in propylene glycol and glycerol formal, 60:40, v/v) and DRM (Dectomax1, 1%, w/v, formulated in sesame oil and ethyl oleate, 90:10, v/ v), respectively, as a single dose on the back of the tongue each at the dose of 200 mg/kg bodyweight. Group III (IVM-SC) and group IV (DRM-CS) received the injectable solutions subcutaneously at the same dose rate. The drugs were administered by subcutaneous injection in the lateral midline of the back as a single dose. After the treatment the animals were observed continuously for any adverse reactions within the first day. Heparinized blood samples (5 ml) were collected by cephalic venepuncture 1 day prior to drug administration and 1, 2, 4, 8, 12, 16, 24, 32, 48, 72, 96 h and 6, 9, 12, 15, 20 25, 30, 35 and 40 days posttreatment. Blood samples were centrifuged at 2000 g for 20 min and plasma was transferred to plastic tubes. All the plasma samples were stored at 20 8C until estimation of drug concentration. 2.3. Analytical procedures The parent compounds of IVM and DRM in plasma were analysed using validated high performance liquid chromatography (HPLC) with a liquid–liquid phase extraction procedures. Plasma concentrations were measured by modification of the methods of Scott and McKellar (1992) and Gokbulut et al. (2001). Stock solutions (100 mg/ml) of pure standard of IVM (Merck, Rahway, NJ, USA) and DRM (Pfizer Inc., Groton, USA) were prepared using acetonitrile as the solvent. These were diluted to give 5, 10, 100, 250, 500 and 1000 ng/ml standard solutions for calibration as standard curves and to add to drug-free plasma samples to determine the recovery. Drug-free plasma samples (1 ml) were spiked with either IVM or DRM standards to reach the following final concentrations: 0.5, 1, 10, 25, 50 and 100 ng/ml. Doramectin (50 ng) and IVM (50 ng) were used as an internal standard for IVM and DRM studies,
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respectively. Acetonitrile (1 ml) was added, and after vortexing for 15 s, 5 ml chloroform was also added. The tubes were shaken on a slow rotary mixer for 15 min. After centrifugation at 2000 g for 15 min, the supernatant was removed with a Pasteur pipette. The organic phase (3 ml) was transferred to a thinwalled 10-ml conical glass tube and evaporated to dryness at 45 8C in a sample concentrator (Maxi-dry plus, Heto Lab. Equipment, Denmark). The dry residue was dissolved in 100 ml of N-methylimidazole (Aldrich Chemical Company Inc., WI, USA) solution in acetonitrile (1:1). To initiate the derivatization, 150 ml trifluoroacetic anhydride (Aldrich Chemical Company Inc.) solution in acetonitrile (1:2) was added. Finally, 35 ml of this solution was injected into the chromatographic system. The mobile phase consisted of acetonitrile and methanol (66:34, v/v) for IVM and DRM and was delivered (Agilent 1100 Series QuatPump, Waldron, Germany) at a flow rate of 1.5 ml min1. A nukleosil C18 analytical column (4 mm, 250 mm 4.6 mm Macherey-Nagel) with nucleosil C18 guard column (Phenomenex, Cheshire, UK) was used for analysis of both molecules. Fluorescence detection (Agilent 1100 Series, FLD Waldron, Germany) was at an excitation wavelength of 365 nm and an emission wavelength of 475 nm.
response relationship. The detection limit of the IVM or DRM was established with HPLC analysis of blank plasma fortified with the standard, measuring the baseline noise at the retention time of the peak. The mean baseline noise at the peak retention time plus three standard deviations was defined as the detection limit (LOD). The mean baseline noise plus six standard deviations was defined as the limit of quantification (LOQ). 2.5. Pharmacokinetic and statistical analysis of data The plasma concentration versus time curves obtained after each treatment in individual animals, were fitted with the WinNonlin software program (Scientific Consulting Inc.). Pharmacokinetic parameters for each animal were analysed using noncompartmental model analysis with extravascular input. The maximum plasma concentration (Cmax) and time to reach maximum concentration (tmax) were obtained from the plotted concentration–time curve of each drug in each animal. The area under the plasma concentration time curve (AUC) and mean residence time (MRT) from time zero to last time with a measurable concentration was calculated by trapezoidal rule. Terminal half-life (t1=2lz ) was calculated as:
2.4. Validation procedures The analytical method used for IVM and DRM in dog plasma was validated prior to the start of the studies. The analytes were identified with the retention times of pure reference standards. Recoveries of the two molecules under study were measured by comparison of the peak areas from spiked plasma samples with the areas resulting from direct injections of standards prepared in acetonitrile. The inter-assay precision of the extraction and chromatography procedures was evaluated by processing replicate aliquots of drug-free dog plasma samples containing known amounts of the drugs on different days. Calibration graphs for IVM and DRM were prepared (linear range 0.25–100 ng/ml). The slope of the lines between peak areas and drug concentration was determined by least squares linear regression and correlation coefficient (r) and coefficient of variations (CV) calculated. Linearity was established to determine the IVM and DRM concentration/detector
t1=2lz ¼
lnð2Þ lz
where lz represents the first order rate constant associated with the terminal (log linear) portion of the curve. The pharmacokinetic parameters are reported as mean S.D. Mean pharmacokinetic parameters were statistically compared by an analysis of variance (ANOVA). Mean values were considered significantly different at P < 0.05.
3. Results The analytical procedures for the determination of IVM and DRM plasma concentrations were validated. The linear regression lines for IVM and DRM under the study in the range between 0.25 and 100 ng/ml showed correlation coefficients as 0.996 (IVM) and
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Table 1 Validation of analytical method used to determination of ivermectin (IVM) and doramectin (DRM) concentration in dog plasma
Limit of quantification (ng/ml) Recovery (%) Linearity (r) Coefficient of variation (%)
IVM
DRM
0.25 96.4 (6.51) 0.996 4.75
0.25 94.29 (6.39) 0.999 3.53
Values in the brackets represent the coefficient of variations for the recovery assays; r: correlation coefficient.
0.999 (DRM). The mean extraction recoveries were 96.4% for IVM and 94.29% for DRM. The inter-assay precision of the extraction and chromatographic procedures for IVM and DRM showed coefficients of variations 4.75 and 5.53%, respectively. The validation parameters for both molecules are summarised in Table 1. No adverse response was observed for any of the treatments during the study. The parent molecule was detected in plasma between 1 h and either 25 days after oral administration of both molecules and subcutaneous administration of DRM, whereas the last detectable plasma concentration extended to 30 days following subcutaneous administration of IVM. Mean pharmacokinetic parameters of IVM and DRM after oral and subcutaneous administration of 0.2 mg/kg bodyweight are shown in Table 2 with the linear and semi log plot of mean plasma concentration versus time curves (Fig. 2). In addition, the mean plasma concentrations after oral and subcutaneous
Fig. 2. Mean plasma concentration of ivermectin (IVM) and doramectin (DRM) over the first 15 days following oral and subcutaneous administration to dogs at a dose rate of 200 mg/kg. (Smaller graph: semi log plot of mean plasma concentration vs. time curves.)
administrations of IVM and DRM are compared in Figs. 3 and 4, respectively. The results indicated that IVM produced a significantly higher maximum plasma concentration (Cmax: 116.80 10.79 ng/ml) with slower absorption (tmax: 0.23 0.09 day) and larger area under the concentration versus time curve (AUC: 236.79 41.45 ng day/ml) as compared with DRM (Cmax: 86.47 19.80 ng/ml, tmax: 0.12 0.05 day, AUC: 183.48 13.17 ng day/ml) following oral administration of both drugs. Although no significant differences were found for the pharmacokinetic parameters, Cmax (66.80 9.67 ng/ml) and AUC (349.18 47.79 ng day/ml) values for the IVM group were higher than those obtained for the DRM (Cmax:
Table 2 Mean (S.D.) pharmacokinetic parameters of ivermectin (IVM) and doramectin (DRM) following oral and subcutaneous administration to dogs at a dose rate of 200 mg/kg Kinetic parameters tmax (day) Cmax (ng/ml) AUClast (ng day/ml) t1=2lz (days)d AUMClast (ng day2/ml) MRTlast (day)
IVM-OR
DRM-OR a
0.23 0.09 116.80 10.79a,c 236.79 41.45a,c 3.32 1.56 1074.16 569.94 4.35 1.78
b
0.12 0.05 86.47 19.80b 183.48 13.17b 3.75 0.89 864.10 256.95 4.66 1.11
IVM-SC
DRM-SC
1.40 1.00 66.80 9.67 349.18 47.79 3.19 0.95 1904.22 695.50 5.32 1.26
1.70 0.76 54.78 11.99 292.10 78.76 3.09 0.99 1548.68 688.07 5.07 1.18
tmax: time to reach peak plasma concentration, Cmax: peak plasma concentration, AUClast: area under the (zero moment) curve from time 0 to the last detectable concentration, t1=2lz : terminal half-life, AUMClast: area under the moment curve from time 0 to t last detectable concentration, MRTlast: mean residence time. a IVM-OR significantly different (P < 0.05) from IVM-SC. b DRM-OR significantly different (P < 0.05) from DRM-SC. c IVM-OR significantly different (P < 0.05) from DRM-OR. d Values represent the harmonic means for t1=2lz (days).
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4. Discussion
Fig. 3. Semi log plot of mean (S.D.) plasma concentration of ivermectin following oral (IVM-OR) and subcutaneous (IVM-SC) administration to dogs at a dose rate of 200 mg/kg.
54.78 11.99 ng/ml, AUC: 292.10 78.76 ng day/ ml) group after subcutaneous administrations. In addition, subcutaneously given IVM and DRM presented a significantly lower maximum plasma concentration (Cmax: 66.80 9.67 ng/ml and 54.78 11.99 ng/ml, respectively) with slower absorption (tmax: 1.40 1.00 day and 1.70 0.76 day, respectively) and larger area under the concentration versus time curve (AUC: 349.18 47.79 ng day/ml and 292.10 78.76 ng day/ml, respectively) as compared with the oral administration of IVM and DRM, respectively. The terminal half-life (t1=2lz ) and MRT of the IVM and DRM observed in the present study were not significantly different from each other after both oral and subcutaneous administrations.
Fig. 4. Semi log plot of mean (S.D.) plasma concentration of doramectin following oral (DRM-OR) and subcutaneous (DRM-SC) administration to dogs at a dose rate of 200 mg/kg.
The plasma pharmacokinetic profiles obtained following oral and subcutaneous administration of IVM and DRM were different in dogs when compared with the other animals. In ruminants and equine species, DRM produced greater bioavailability and longer persistence compared with IVM when administered by same route of administration (Toutain et al., 1997; Atta and Abo-Shihada, 2000; Barber et al., 2003; Lanusse et al., 1997; Gayrard et al., 1999; Gokbulut et al., 2005; Perez et al., 2002). The findings of the present study do not support the previous studies. In contrast to ruminants and equine species, DRM generated a relatively lower plasma concentration and bioavailability than those of IVM following oral administration, whereas no significant difference was absorbed between both molecules following subcutaneous administration in dogs. These differences may be attributable to using different injectable formulation of IVM and DRM solutions for oral administration and the physiological or body composition differences between dogs and the other animals. It has been suggested that the major factors contributing to the differences could be a consequence of differences in the physico-chemical properties of the drug and in the formulations of both drugs (Wicks et al., 1993; Toutain et al., 1997; Lanusse et al., 1997). IVM is chemically different from DRM which has cyclohexyl group at a position 25 and this group may confer reduced polarity on DRM (Toutain et al., 1997). Moreover, IVM is formulated in propylene glycol and glycerol formal whereas DRM is in sesame oil and ethyl oleate. Previous studies have shown that the plasma disposition of avermectins was considerably affected by the route of administration. A significant difference in the bioavailability and elimination half-life of IVM was observed between oral and subcutaneous/intramuscular administration in sheep and horses (Marriner et al., 1987; Perez et al., 2003) and cattle (Chiu et al., 1990). Similarly, in swine higher bioavailability and slower absorption were observed following subcutaneous than oral administration (Fink and Porras, 1989). In the present study, the slow absorption of IVM and DRM resulted in larger AUC when the drugs were given subcutaneously as compared with their oral administration in dogs. Such slow adsorption was
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attributed to precipitation of the drug at the injection site (Lo et al., 1985), which was later supported by the findings of Scott and McKellar (1992), who reported the presence of large amount of IVM at the injection site 24 h after subcutaneous administration. It has been suggested that the peak concentration (Cmax) increased directly with the oral doses, suggesting a linear relationship between the amount of drug absorbed and the administered dose in dogs (Fink and Porras, 1989). Following oral tablet administration formulation of IVM at three dose levels (6, 27 and 100 mg/kg body weight) produced linear increased peak plasma concentrations (2.97, 19.1 and 44.3 ng/ml, respectively) (Daurio et al., 1992, Kojima et al., 1987) in dogs. The ratios of dose-Cmax were 2.0, 1.4 and 2.2, respectively. This was also supported by the findings of the present study, since Cmax was 116.8 ng/ml after oral administration of IVM at 200 mg/kg and the corresponding ratio was 1.7. The antiparasitic spectrum and efficacy pattern of the different avermectin molecules are similar; however, differences in physico-chemical properties among them may account for differences in formulation flexibility, kinetic behaviour, and in the potency and persistence of their antiparasitic activity. It has been demonstrated that plasma availability of IVM (Lo et al., 1985) and doramectin (Wicks et al., 1993) in cattle is profoundly affected by the solvent vehicle in which the drug is formulated. Although IVM and DRM are widely used in veterinary medicine as an anthelmintic with a wide margin of safety, some dogs of collie breed and colliecross have extreme IVM sensitivity (Paul et al., 1987; Pulliam et al., 1985; Hopper et al., 2002) and they display clinical signs of neurointoxication such as salivation, dilated pupils, vomiting, tremors, ataxia and depression at much lower doses of ivermectin than the Beagles did. The hypersensitivity and neurointoxication was associated with increased ivermectin accumulation in the brain of sensitive Collies (Paul et al., 1987; Pulliam and Preston, 1989). It was suggested that these dogs had a genetic deficiency in a MDR1 (multi drug resistance, MDR)-type P-glycoprotein which is an important component of the blood-brain barrier (Neff et al., 2004; Roulet et al., 2003; Mealey et al., 2001, 2005). More recently, a similar toxicity was reported for DRM in a Collie following subcutaneous administra-
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tion of 200 mg/kg bodyweight (Yas-Natan et al., 2003). Ivermectin sensitive collies tolerate 50 mg/kg, and therefore the therapeutic administration at the recommended dose of 6 mg/kg confers a large safety margin on the commercialize product (Pulliam et al., 1985; Paul et al., 1987). Although no adverse response was observed for any of the dogs during present study, a high dose (200 mg/kg) of IVM and DRM could be neurotoxic to some hypersensitive dogs.
5. Conclusion In conclusion, the present study indicated that oral and subcutaneous treatment of IVM and DRM at doses as high as 200 mg/kg did not cause any clinically detectable adverse affects in dogs. In contrast with ruminants and equine species, oral administration of IVM displayed a significantly higher plasma concentration than that of DRM, whereas no significant differences were observed on the pharmacokinetic parameters between both molecules after subcutaneous administrations. Considering the pharmacokinetic disposition, IVM and DRM could be used by oral or subcutaneous route for the control and treatment of parasitic diseases in dogs. However, because of avermectin hypersensitivity, much lower therapeutic dose of both drugs must be used in some dog breeds. Moreover, development and optimization of appropriate formulations of DRM for oral and subcutaneous administration may be required to use in dogs.
Acknowledgements This study was supported by Scientific Research Committee of Adnan Menderes University (VTF05005), Aydin, Turkey.
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