Doramectin concentration profiles in the gastrointestinal tract of topically-treated calves: Influence of animal licking restriction

Veterinary Parasitology 133 (2005) 61–70 www.elsevier.com/locate/vetpar

Doramectin concentration profiles in the gastrointestinal tract of topically-treated calves: Influence of animal licking restriction J.M. Sallovitz, A. Lifschitz, F. Imperiale, G. Virkel, J. Larghi, C. Lanusse * Laboratorio de Farmacologı´a, Nu´cleo FISFARVET, Departamento de Fisiopatologı´a, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Campus Universitario, 7000 Tandil, Argentina Received 12 April 2005; received in revised form 14 May 2005; accepted 18 May 2005

Abstract Endectocide compounds are extensively used for broad-spectrum parasite control and their topical administration to cattle is widespread in clinical practice. Pour-on formulations of moxidectin, ivermectin, eprinomectin and doramectin (DRM) are marketed internationally for use in cattle. However, variability in antiparasitic efficacy and pharmacokinetic profiles has been observed. Although the tissue distribution pattern for different endectocide molecules given subcutaneously to cattle has been described, only limited information on drug concentration profiles in tissues of parasite location after topical treatment is available. Understanding the plasma and target tissue kinetics for topically-administered endectocide compounds is relevant to optimise their therapeutic potential. The current work was designed to measure the plasma and gastrointestinal (GI) concentration profiles of DRM following its pour-on administration to calves. The influence of natural licking behaviour of cattle on DRM concentration in mucosal tissue and luminal content of different GI sections was evaluated. The trial was conducted in two experimental phases. In Phase I, the DRM plasma kinetics was comparatively characterised in free-licking and in 2-day licking-restricted (non-licking) calves. The pattern of distribution of topical DRM to mucosal and luminal contents from abomasum, duodenum, ileum, caecum and spiral colon was assessed in free-licking and non-licking calves restricted over 10 days post-administration (Phase II). The prevention of licking caused marked changes on the plasma and GI kinetics of DRM administered pour-on. In 2-day licking restricted calves, DRM systemic availability was significantly lower (29%) than in free licking animals during the first 9 days post-treatment. Following a 10day long licking restriction period, DRM concentrations profiles in both mucosal tissue and luminal contents of the GI tract were markedly higher in animals allowed to lick freely. This enhancement in drug concentrations in free-licking compared to non-licking calves, was particularly pronounced in the abomasal (38-fold higher) and duodenal (six-fold higher) luminal content. As shown earlier for ivermectin, licking behaviour may facilitate the oral ingestion of topically-administered DRM in cattle. This would be consistent with the marked lower drug concentration profiles measured in the bloodstream and GI tract of the animals prevented from licking. The work reported here provides relevant information on the pattern of DRM distribution to the GI tract after pour-on treatment, and contributes to understand the variability observed in the antiparasitic persistence of topically-administered endectocides in cattle. The implications of natural licking in topical treatments are

* Corresponding author. E-mail address: [email protected] (C. Lanusse). 0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.05.049

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required to be seriously assessed to achieve optimal parasite control and to design parasitological and pharmacological studies within the drug approval process. # 2005 Elsevier B.V. All rights reserved. Keywords: Doramectin; Licking behaviour; Cattle; Transdermal absorption

1. Introduction The macrocyclic lactone compounds from both the avermectin and milbemycin families, known as endectocides for their activity against both endo-and ectoparasites, are extensively used for broad-spectrum parasite control in livestock. Doramectin (DRM) is an avermectin endectocide compound that exhibits no activity against cestode and trematode parasites, but it is highly effective against the adult, developing and hypobiotic larvae of most gastrointestinal (GI) nematodes, lungworms and many arthropod ectoparasites at extremely low dosages (Vercruysse et al., 1993; Jones et al., 1993). Parasiticides are the most frequently used topical veterinary medications and the use of pour-on endectocide formulations in cattle is widespread (Baynes et al., 1997). DRM is marketed as injectable and topical (pour-on) formulations for use in beef cattle. Additionally, pour-on formulations of ivermectin, eprinomectin and moxidectin are internationally available for use in cattle. They offer considerable practical (less labour-intensive) and pharmacological (avoidance of liver first-pass effect and no drug residue at the injection site) advantages compared to other routes of drug administration (Baggot and Brown, 1998). However, variability in antiparasitic efficacy has been observed. Information on the plasma kinetic behaviour of topical ivermectin, doramectin (Gayrard et al., 1999; Laffont et al., 2001, 2003; Bousquet-Me´lou et al., 2004), eprinomectin (Alvinerie et al., 1999a) and moxidectin (Sallovitz et al., 2002; Bousquet-Me´lou et al., 2004) in cattle is available. Other studies have also investigated the tissue distribution and level of tissue drug residues after the subcutaneous administration of different endectocide molecules (Lifschitz et al., 1999a, 1999b, 2000; Afzal et al., 1994; Zulalian et al., 1994) to ruminant species. The gastrointestinal disposition of DRM has been studied after oral and intravenous administration to sheep (Hennessy et al., 2000). Other studies investigated the effect of different variables on

the plasma kinetics of endectocides in cattle. Among these factors were route of administration and formulation (Herd et al., 1996; Lifschitz et al., 1999b; Alvinerie et al., 1999b), breed (Sallovitz et al., 2002), and natural licking behaviour (Laffont et al., 2001; Bousquet-Me´lou et al., 2004). However, only few studies (Sallovitz et al., 2003) reported on the tissue distribution of endectocides (moxidectin) after its topical (pour-on) administration to cattle. This study was conducted without considering the effect of licking on the pattern of tissue distribution of the drug. It is well established that an optimised full antiparasitic effect can be achieved if pharmacological, parasitological and physiological variables are considered in an integrated manner. The optimisation of the antiparasitic activity of topically administered endectocides can be achieved through a thorough knowledge of their absorption pattern and tissue kinetic behaviour, particularly at those sites where parasites locate, and identification of different factors affecting the performance of the topically administered compound. Hence, the current work was designed to investigate the plasma and GI tract disposition of DRM following pour-on treatment in calves. Also, the influence of animal natural licking behaviour in cattle on DRM concentration profiles in the bloodstream and in the GI mucosas and luminal contents was assessed. The comparative DRM availability in abomasum and in different intestine sections in free-licking and licking-restricted calves topically-treated at recommended dose rates is reported here.

2. Materials and methods The present experiment was conducted in two experimental phases. In Phase I, DRM plasma kinetics was comparatively characterised in free-licking and in 2-day licking restricted (non-licking) calves. In Phase II, the pattern of DRM concentrations in mucosal

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tissue and luminal contents from abomasum, duodenum, ileum, caecum and spiral colon was assessed in free-licking and non-licking calves restricted over 10 days after topical treatment. 2.1. Phase I 2.1.1. Experimental animals, treatments and sample collection Animals were allotted in two groups (n = 8). Sixteen (16) 10-month old, parasite-free Holstein calves (183
30.9 kg) were used in this study. In order to minimise eventual pharmacokinetic variations due to differences in diet intake (Alı´ and Hennessy, 1996), both groups received the same feed. The animals were fed with lucerne hay prior to and during the experimental period. Water was provided ad libitum. The health of the animals was monitored throughout the entire trial period. Natural licking was prevented during 2 days postadministration. Animals in the non-licking group were placed in a separate paddock and each animal was restrained with a stanchion and tied from both sides to restrain from both self- and allo-licking. This system allowed animals to lie down, stand up, eat and drink freely. Animals in the licking group were kept together in a paddock. Both licking and non-licking animals were treated with a commercial formulation of DRM for topical (pour-on) administration (Doramectin1 Pour-On, Pfizer Animal Health, Exton, PA, USA). The formulation containing DRM at 0.5% was administered on the backline from the withers to the tailhead of each animal at a dose rate of 500 mg/kg b.w. in a final volume ranging from 12 to 17 mL. Blood samples were obtained by venipuncture from the jugular vein and collected in heparinised vacutainer tubes. The blood sampling was done prior to and from 2 h and up to 35 days post-administration. After collection, plasma was separated by centrifugation and stored at 20 8C until analysed. 2.2. Phase II 2.2.1. Experimental animals, treatments and sample collection Twelve (12) parasite-free Holstein calves (150
17 kg b.w.) were randomly assigned to two experimental groups. As for Phase I, both groups were

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fed the same feed (lucerne hay) prior to and during the trial. Water was provided ad libitum. In this experimental phase, the licking prevention period was extended over 10 days post-administration. Animals were kept in sheds of two animals per shed. Animals in the non-licking group were restrained with a stanchion and tied from both sides in order to restrain from both self- and allo-licking. Calves in the licking group were kept in a paddock, allowed to lick freely. The sampling times were established prior to trial initiation. Two animals of each experimental group were randomly assigned for sacrifice at one of the following times post-administration: 2, 5 and 10 days. Calves were stunned by a captive bolt and immediately exsanguinated according to the animal euthanasia guidelines of the Animal Welfare Committee, Faculty of Veterinary Science, UNCPBA (Animal Welfare Act, Academic Council Resolution 087/02) and American Veterinary Medical Association (AVMA, 2001). Samples collected included jugular blood and gastrointestinal (GI) tract luminal contents and mucosal tissue from the abomasum, ileum, caecum and spiral colon. Once the abdominal cavity was opened, the different GI sections to be sampled were identified (avoiding excessive manipulation of the bowels) and limited by tying up both extremes of the viscera with a cotton string. After collection of the intestinal and abomasal contents, the mucosal tissues of each GI section were obtained by scraping. Both duodenal content and mucosa were collected before opening the bile duct. All samples were transported on ice from the abattoir to the laboratory and stored at 20 8C and analysed within 2 months of their collection. 2.3. Drug analysis Doramectin concentrations in plasma and in the different GI contents and mucosal tissues were measured by high performance liquid chromatography (HPLC) with fluorescence detection using automated solid phase extraction according to the methods described by Alvinerie et al. (1993) and modified by Lifschitz et al. (1999b). Experimental and DRM spiked luminal content/ tissue samples were homogenised and solid phase extraction was performed, after about 15 min incubation at room temperature. A 1 g (tissue) or 1 mL (luminal content) sample was mixed with 1 mL

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acetonitrile. After mixing, the tube was sonicated (Ultrasound Bath, Lab-Line Instruments, Inc., Melrose Park, IL, USA) and centrifuged at 2000  g. The supernatant was manually transferred into a tube and the procedure repeated. The pooled supernatants of luminal content or tissue samples obtained were then placed on the appropriate rack of an automated solid phase extraction apparatus (Aspec XL, Gilson, Villiers Le Bel, France). The eluent was evaporated to dryness under a gentle stream of nitrogen in a 60 8C water bath, derivatised (De Montigny et al., 1990) and a 100 mL aliquot was injected directly into the chromatographic system (Shimadzu 10 A HPLC System, Shimadzu, Kyoto, Japan). The chromatographic conditions included a mobile phase of water–methanol–acetonitrile (6:40:54, v/v/v) with a flow rate of 1.5 mL/min through a reverse phase C18 column (Phenomenex, 5 mm, 4.6 mm  250 mm) placed in an oven at 30 8C. Fluorescence detection (Spectrofluorometric detector RF 10, Shimadzu, Kyoto, Japan) occurred at an excitation wavelength of 365 nm and readings taken at an emission wavelength of 475 nm. 2.4. Validation procedures The analytical procedures for extraction and quantification of DRM were validated prior to analysing the experimental samples. Two calibration curves for DRM were generated: one in the range of 0.1–5 ng/mL and the other in the range of 5–80 ng/ mL. Method linearity was confirmed by injecting spiked DRM standards in plasma and GI content/ tissues at different concentrations (triplicate determinations). Calibration curves were established using an unweighted least squares linear regression analysis, and the corresponding correlation coefficients (r) and coefficients of variation (CV) were calculated. Linearity was determined by the lack of fitness test (GraphPad InStat, Version 3.00, GraphPad Software, San Diego, CA, USA). Drug recovery was estimated from DRM standards. Percent of DRM recovery was determined for control samples ranging between 0.1 and 50 ng/mL. The inter-assay precision of the extraction and chromatographic procedures was evaluated by processing replicate aliquots (n = 4) of pooled content/tissue samples containing known amounts of DRM (2 and 40 ng/mL) on different days.

The coefficient of variation (CV) was obtained by dividing the standard deviations by the mean recovery values of at least three determinations. The limit of detection for DRM was established by injecting spiked plasma and fluid content/mucosal tissue blanks with the internal standard into the HPLC system and measuring the baseline noise at the DRM retention time. The mean baseline noise at the DRM retention time plus three standard deviations was defined as the theoretical detection limit (LOD), while the addition of six standard deviations was defined as the theoretical limit of quantification (LOQ).

2.5. Pharmacokinetic and statistical analyses of the data The concentration–time curves obtained for plasma and each luminal content/mucosal tissue under study were fitted with the PK Solution 2.0 computer software (Summit Research Services, Ashland, OH, USA). The peak concentrations (Cmax) and times to peak concentrations (Tmax) for each individual animal were read from the plotted concentration–time curves. Both the partial and total (up to the last sampling time) areas under the concentration–time curves (AUC) were calculated by the trapezoidal rule (Gibaldi and Perrier, 1982). Statistical moment theory was applied for calculating DRM mean plasma residence time (MRT) as follows: MRT ¼

AUMC AUC

where AUC is as defined previously and AUMC is the area under the concentration versus time curve of the product of time and drug concentration versus time from zero to the last sampling time (Gibaldi and Perrier, 1982). DRM concentration values are presented as mean
standard deviation. When appropriate, the DRM concentrations and/or AUC values measured were statistically compared across the various tissue and fluid content samples using Student’s t-test. The AUC values for luminal contents and mucosas were calculated using the pooled data of the animals of each sampling time as reported elsewhere (Lifschitz et al., 1999a).

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3. Results The linearity of the analytical method was confirmed by using the lack of fitness test. The linear regression lines for DRM concentrations ranging from 0.1 to 5 ng/mL and from 5 to 80 ng/mL of plasma showed a coefficient of correlation of 0.9933 and 0.9948, respectively. The limit of DRM detection of the analytical method was 0.03 ng/mL and the limit of quantification was 0.05 ng/mL. The inter-assay precision of the analytical technique presented a coefficient of variation of 8.14%. The estimated values of the correlation coefficient for the different analysed mucosal tissues and luminal contents ranged between 0.994 and 0.997. The theoretical quantification limit obtained for the different matrices ranged from 0.05 to 0.14 ng/g or ng/mL. The interassay precision of the analytical method showed a mean coefficient of variation of 4.13% (range 1.99–6%). The extraction recovery in the concentration range between 0.1 and 50 ng/mL, ranged between 60 and 71% (intestinal mucosas) and from 64 to 78% (digestive luminal contents). Doramectin was detected in all harvested plasma samples for up to 35 days post-administration in both experimental groups in Phase I. Fig. 1 depicts the plasma concentrations (mean
S.D.) versus time curves obtained for both experimental groups up to 9

Fig. 1. Doramectin (DRM) plasma concentration profiles (mean
S.D.) (n = 8) obtained during the first 9 days after topical treatment (500 mg/kg) in non-licking (2-days licking prevention) and in free licking calves. The shadowed area represents the posttreatment time period where the natural licking behaviour was restricted.

Fig. 2. Comparison of the mean (
S.D.) maximum plasma concentration (Cmax) and partial area under the plasma concentration vs. time curve up to 9 days post-administration (AUC0–9 days) obtained after topical administration of doramectin (500 mg/kg) to non-licking (2-days prevention period) and free licking calves (n = 8). * and ** stand for statistically significant differences between experimental groups at P < 0.05 and P < 0.01, respectively.

days post-treatment, where differences due to the effect of licking restriction are evidenced. Fig. 2 shows the Cmax achieved in the licking group (mean
S.D.) (20.05
2.57 ng/mL) that was 1.56 times higher than that of the non-licking group (12.85
2.98 ng/mL, P < 0.01). Also, the difference in the partial AUC up to 9 days post-treatment is shown, which was statistically significant (P < 0.05). Fig. 3 shows the plasma concentration–time curves up to 25 days posttreatment where it demonstrates an upward trend in plasma concentrations from the 9th day post-treatment onward in the licking-restricted group. However, by day 20 post-treatment, DRM plasma concentrations of both groups became similar. The partial AUC up to 25 days (Fig. 3, insert) were also similar (P > 0.05) for both groups. There was no statistical difference in the time to peak concentration (Tmax) between groups (3.75
0.5 versus 4.25
1.39 days, P > 0.05). The absorption (1.44
0.43 versus 0.96
0.12 days) and elimination (3.84
0.83 versus 3.60
0.93 days) half-lives showed no statistical differences between the non-licking and licking groups. The 10-day licking prevention period (Phase II) determined marked changes in the pattern of gastrointestinal distribution and availability of topically administered DRM. Plasma availability measured up

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Fig. 3. Doramectin (DRM) plasma concentration profiles (mean
S.D.) (n = 8) measured during 25 days after topical treatment (500 mg/kg) in non-licking (2-days licking prevention) and in free licking calves. The shadowed area represents the post-treatment time period where the natural licking behaviour was restricted. The total AUC value obtained from treatment up to 25 days posttreatment is shown as an insert.

Fig. 4. Comparison of doramectin (DRM) plasma availability, expressed as partial area under the plasma concentration vs. time (AUC0–10 days) measured over 10 days after topical treatment (500 mg/kg) in non-licking (10-day licking restriction period) and free licking calves.

to 10 days post-administration was lower in nonlicking animals (>75%) (Fig. 4). The pattern of distribution differed markedly between experimental groups. Maximum concentrations of DRM along the sampling period in the different intestinal luminal contents were, on average, 4.16-fold higher in the licking compared to the non-licking group, ranging from 2.34- (caecum) to 5.96-fold (duodenum) (Table 1). DRM concentrations in the intestinal mucosal tissue were 1.78-fold higher, with a range between 1.56- (ileum, spiral colon) and 1.99-fold (caecum), in licking calves (Table 2). Similarly, DRM concentrations in abomasal luminal content (38.4-

fold) and abomasal mucosa (1.45-fold) tended to be higher in the licking compared to the non-licking group (Tables 1 and 2). The relationship between DRM concentrations in the mucosa and luminal content (mucosa/luminal content ratio) for each GI section was compared between experimental groups. The mean ratios during the experimental period for each GI section in non-licking and licking animals, respectively, were – abomasum: 64.6 and 14.0; duodenum: 2.74 and 1.34; ileum: 1.25 and 0.82; caecum: 1.69 and 0.94; spiral colon: 0.90 and 0.52. There was a statistically significant difference (P < 0.05) between the duodenal and ileal ratios

Table 1 Doramectin concentrations (ng/mL) measured in different gastrointestinal luminal contents following its topical administration to non-licking (10-day licking restriction) and free licking calves Time post-administration Gastrointestinal luminal content Abomasum

Duodenum

Ileum

Caecum

Spiral colon

Non-licking Licking Non-licking Licking Non-licking Licking Non-licking Licking Non-licking Licking Two days Five days Ten days Peak ratioa a

0.37 3.08 3.99

7.12 16.2 153.6 38.4

7.31 15.7 23.8

12.1 43.5 142.1 5.96

13.6 34.8 36.5

24.7 49.3 148.4 4.07

12.2 22.2 61.3

17.6 73.8 143.4 2.34

20.6 37.5 55.8

37.3 80.4 228.7 4.09

Indicates the ratio between the peak concentrations (Cmax) measured in each gastrointestinal content in non-licking and free licking calves. Concentration values are the mean of two determinations for each assayed gastrointestinal content.

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Table 2 Doramectin concentrations (ng/g) measured in the mucosal tissue of the gastrointestinal tract after its topical administration to non-licking (10day licking restriction) and free licking calves Time post-administration Gastrointestinal mucosal tissue Abomasum

Duodenum

Ileum

Caecum

Spiral colon

Non-licking Licking Non-licking Licking Non-licking Licking Non-licking Licking Non-licking Licking Two days Five days Ten days Ratio a

17.7 55.2 58.7

23.2 16.2 85.2 1.45

18.2 49.3 50.5

21.0 66.9 100.6 1.99

15.7 35.4 58.7

16.8 55.0 91.8 1.56

14.1 40.3 51.1

17.2 82.6 101.9 1.99

15.0 31.2 46.3

16.5 53.9 72.0 1.56

a

Indicates the ratio between the peak concentrations (Cmax) measured in each gastrointestinal content in non-licking and free licking calves. Concentration values are the mean of two determinations for each assayed gastrointestinal content.

throughout the experimental period. Although the greatest differences were observed in the abomasal ratios, these were not statistically significant due to individual variability. The availability of topical DRM in the GI tract followed a similar pattern to that described for plasma, being higher in the licking group than in the nonlicking group. In the GI luminal contents, DRM availability for the licking group was between 132% (caecum) and 1926% (abomasum) higher compared to the non-licking group (Fig. 5). In the GI mucosa, the licking group presented DRM availabilities between 50% (caecum) and 120% (abomasum) higher than the non-licking group (Fig. 6).

4. Discussion

Fig. 5. Doramectin (DRM) availability (as AUC) measured in the gastrointestinal luminal content of abomasum and different intestine sections after its topical administration (500 mg/kg) to non-licking (10-day licking restriction period) and free licking calves. Percentage values indicate differences in DRM availabilities observed between non-licking and free licking claves.

Fig. 6. Doramectin (DRM) availability (as AUC) measured in the mucosal tissue of the abomasum and intestine (different sections) after its topical administration (500 mg/kg) to non-licking (10-day licking restriction period) and free licking calves. Percentage values indicate the differences in DRM availabilities observed between non-licking and free licking claves.

The knowledge of the pattern of tissue distribution for antiparasitic drugs has been shown to be relevant to understanding and optimising their activity. The current work reports detailed information on the distribution of DRM to the GI tract after its pour-on administration to calves. In addition to the evaluation of the effect of licking restriction, this work contributes to a better understanding of the extensive exchange between the bloodstream and digestive tract that occurs once the topically-administered drug is absorbed through the skin, which is consistent with the recognised lipophilicity of this compound and with the

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data available for similar endectocide molecules (Sallovitz et al., 2002). Previous work has reported on the variability in the antiparasitic efficacy/persistence (Williams et al., 1999) and plasma kinetics (Gayrard et al., 1999; Laffont et al., 2001; Bousquet-Me´lou et al., 2004) of endectocides after their topical administration to cattle. This variability is consistent with the previously demonstrated effect of natural licking behaviour on the plasma disposition of ivermectin and doramectin in cattle (Laffont et al., 2001, 2003; Bousquet-Me´lou et al., 2004). Mechanical transfer of topicallyadministered endectocides from treated to untreated animals when the former are licked by the latter has also been demonstrated (Barber and Alvinerie, 2003; Bousquet-Me´lou et al., 2004). It should be kept in mind that efficacy trials did not take into account the licking behaviour when reporting their results. The comparison of DRM plasma profiles measured after pour-on treatment in free-licking and non-licking animals described here agrees with the results previously reported for ivermectin (Laffont et al., 2001; Bousquet-Me´lou et al., 2004). Those results clearly correlate with the licking-induced changes on the GI availability of topically given DRM described in the current work. A 2-day long licking prevention period after the pour-on treatment was sufficient to significantly reduce the DRM concentrations measured in plasma during the first 9 days post-treatment (Fig. 1). DRM systemic availability expressed as a partial AUC value was significantly higher (P < 0.05) in free-licking compared to non-licking calves (Fig. 2). The animals in the free-licking group ingested topically-administered DRM by self- and/or allo-licking, which accounted for the higher peak concentration observed in plasma (P < 0.01) as well as for the enhanced drug availability compared to the licking-restricted animals (Fig. 2). The oral ingestion of DRM by licking the drug from the skin and its subsequent absorption in the GI tract during the first 2 days post-treatment was able to determine significant differences in plasma availability for up to 9 days. However, over the length of the study (up to 25 days post-treatment) the DRM plasma availability (AUC0–25 days) showed no statistically significant differences between experimental groups. This was probably due to the increase in DRM concentrations observed in the non-licking calves after day 9 post-

treatment (Fig. 3), as a consequence of licking occurring after finishing the 2-day restriction period. The licking that took place after the restriction period could compensate the plasma levels by day 9 postadministration and, from there on, outweighed DRM plasma levels measured in the free-licking group (Fig. 3). This increase in drug plasma concentrations was helped by the slow release of DRM from the skin. Even though a greater amount of drug should have been in the skin of non-licker animals, its slow rate of release from the skin and limited cutaneous absorption were unable to counterbalance the intestinal absorption taking place in free licking animals that ingested DRM orally. This is depicted in the plasma profiles measured during the first 2 days post-administration, when DRM plasma levels in non-licking animals were determined only by transdermal absorption (Fig. 1). As earlier discussed for moxidectin (Sallovitz et al., 2003), a slow release of DRM from the skin can be given by formation of a skin depot after the topical treatment, where its high lipophilicity facilitates its retention in the lipid contents of the skin (fat tissue in the dermis and sebaceous secretion). Laffont et al. (2003) demonstrated that after the topical administration of ivermectin to cattle, drug absorption through the GI tract due to licking-induced oral ingestion is more important than the transdermal absorption in determining drug systemic concentrations. This finding was confirmed for DRM in the present work, particularly during the first 2 days postadministration. However, the licking behaviour was unable to remove enough drug from the skin to compensate DRM plasma concentrations from day 2 to 9 post-administration. The prevention of the licking activity during 2 days may have allowed the topical DRM to spread over and deep in the skin, making it difficult to remove the drug by self-licking. The difference in plasma availability in the first 9 days posttreatment and changes in DRM plasma concentrations observed in the present work can be explained by the different physiological role of each tissue involved in the absorption of topically-administered DRM. It is generally accepted that while the GI mucosa, particularly that of the small intestine, is prepared to absorb a great variety of substances, the skin is to function as a barrier that prevent or limit the amount of substances reaching the bloodstream. Hence, in general, the plasma levels of any given substance achieved exclusively by transdermal absorption will be lower than those

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achieved by intestinal absorption. Thus, a more complete absorption of DRM in the GI tract following oral ingestion of the topically-administered formulation, compared to the cutaneous permeation considered as the only absorption process in non-licking animals, would explain the enhanced plasma profiles measured in calves allowed to lick freely in both experimental phases. Licking prevention during 10 days post-treatment permitted the identification of marked changes on the DRM concentration profiles both systemically and, much more importantly, in the GI tract. As it is shown in Tables 1 and 2, DRM concentrations in mucosal tissue and luminal contents from the digestive tract were markedly higher in free-licking animals. DRM concentrations were higher in the fluid contents than in the mucosas in the licking group (Tables 1 and 2), which is in agreement with results previously obtained for moxidectin in free licking animals (Sallovitz et al., 2003). Conversely, in licking-restricted calves, DRM concentrations were higher in the mucosal tissue than in luminal content of the different GI sections (Tables 1 and 2). These observations in non-licking calves are in agreement with the data reported by Lifschitz et al. (2000) after the subcutaneous administration of DRM, where after absorption into the systemic circulation, the drug can reach the GI lumen by a distribution exchange between the bloodstream and the digestive compartment, which must occur through the mucosal layer of the intestine. Altogether, these results show that the large differences on DRM concentrations observed in the digestive contents and mucosal tissues between the experimental groups were due to the licking behaviour. In fact, licking permitted a larger amount of available DRM to be absorbed in the intestine, which determined the observed enhancement of DRM systemic availability in free-licking calves. It is also likely that a higher proportion of the drug bound to solid digestive material would be excreted by faeces, which may have environmental implications as it is well documented for these antiparasitic molecules. The ratio between DRM concentrations measured in the mucosa and luminal content indicates the dynamic relationship between these sites. While licking has a direct and determinant impact on DRM concentrations in the luminal content, the transdermal absorption becomes the sole source of

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drug during licking prevention. The mucosa/luminal content ratio is a useful indicator of the amount of drug reaching that particular GI section when non-licking and licking animals are compared. The differences among mucosa/luminal content ratios of non-licking and licking animals along the experimental period were statistically significant (P < 0.05) for the duodenum and ileum. Even though the highest ratios were observed in the abomasum of non-licking animals, there were no statistically significant differences between experimental groups. This can be explained due to the great individual variation in the concentrations observed in this GI section. Unfortunately, the low number of experimental animals (because of humane and economical reasons) precludes further statistical analyses of the data. However, a well-defined tendency can be observed from the data generated in the current work. In conclusion, as earlier shown for ivermectin (Laffont et al., 2001; Bousquet-Me´lou et al., 2004), the systemic availability of DRM after pour-on administration to calves is markedly influenced by the natural licking behaviour. Licking avoidance during either 2 or 10 days post-treatment was useful to demonstrate that DRM kinetic profiles both in the bloodstream and GI tract, is notably altered by cattle licking/grooming behaviour. Considering the marked licking-induced changes observed in the concentrations of DRM in the mucosal tissue and luminal contents from abomasum and intestine, the persistence claimed for topically administered endectocides in cattle should be fully revised. As recently discussed (Bousquet-Me´lou et al., 2004), there are many therapeutic, practical and regulatory issues to be discussed on the basis of the altered pharmacokinetic behaviour induced by the natural licking in pour-on treated cattle. While the pharmaco-parasitological implications of this animal behaviour need to be further evaluated, the data shown here on the effect of licking prevention on DRM concentrations in the GI tract illustrate on the magnitude of the pharmacokinetic modifications occurring after pour-on treatments in cattle.

Acknowledgments Juan M. Sallovitz is Professional Assistant from Comisio´n de Investigaciones Cientı´ficas de la Pro-

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vincia de Buenos Aires (CICPBA). Research at the Laboratorio de Farmacologı´a, Departamento de Fisiopatologı´a, Facultad de Ciencias Veterinarias (UNCPBA) is supported by CICPBA, Universidad Nacional del Centro de la Provincia de Buenos Aires and Agencia Nacional de Promotio´n Cientı´fica y Tecnolo´gica (PICT 08-07277) (all from Argentina).

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