Concomitant Simultaneous and Consecutive Treatment Of

Concomitant Simultaneous and Consecutive Treatment of Imidacloprid/Moxidectin Spot-on with Emodepside/Praziquantel Tablets in Adult Dogs Eva Maria Krüdewagen1 Annette Schimmel1

1Bayer Animal Health GmbH, Kaiser-Wilhelm-Allee 50, 51373 Leverkusen, Germany

KEY WORDS: : Safety, concomitant and emodepside. Neither compound-related treatment, moxidectin, emodepside, dogs adverse reactions nor treatment-related ABSTRACT changes in the blood hematology or chemistry profile were seen. In this study reported Fourteen healthy adult Beagle dogs were here, it was clearly demonstrated that the included in this clinical study. Dogs ran- concomitant treatment of both products ap- domly allocated to two study groups were plied either simultaneously or consecutively treated on day 0 with a spot-on containing did not lead to any clinical nor laboratory imidacloprid/moxidectin at the maximum abnormalities in adult Beagle dogs. approved dosage of 25 mg imidacloprid/kg INTRODUCTION bodyweight (bw) + 6.25 mg moxidectin/kg bw. Group 1 was treated at the same time A wide range of endo- and ectoparasites can orally with tablets containing emodepside/ concomitantly infect the dog. Although there praziquantel at the maximum approved dos- are already several broad spectrum parasiti- age of 1.9 mg emodepside/kg bw + 9.4 mg cides for dogs on the market, none of them praziquantel/kg bw. Group 2 was treated cover the whole endo- and/or ectoparasitic on day 8 with emodepside/praziquantel spectrum. Concomitant treatment with one tablets at the same dosage. These two dif- or more products is therefore a common ferent treatment scenarios were selected to practice. compare a simultaneous treatment with a In the present study, two broad-spectrum consecutive treatment. In the consecutive antiparasitic products, emodepside/prazi- treatment an 8-day interval was chosen to quantel tablets (Profender®) and imidaclo- ensure overlapping peak serum concentra- prid/moxidectin spot-on (Advocate®), were tions of emodepside and moxidectin. Dogs administered to dogs either simultaneously were fed directly after treatment. Profound or consecutively. safety assessments were performed pre- and Profender® tablets for dogs, authorized post-treatment. Blood samples were drawn in 2008 (Bayer Animal Health GmbH, pre- and post-treatment to evaluate the Leverkusen, Germany), are a combination hematology and clinical chemistry profile product containing emodepside and pra- as well as the serum levels of moxidectin ziquantel. Emodepside is a semi-synthetic 290 Vol. 9, No. 3, 2011 • Intern J Appl Res Vet Med. derivate of PF1022A and belongs to the des felis), treatment of ear mite infestation cyclic depsipeptides. Emodepside binds (Otodectes cynotis), prevention of heart- to a presynaptic latrophilin receptor which worm disease (L3 and L4 larvae of Dirofi- belongs to the group of G-protein coupled laria immitis) and Angiostrongylus vasorum, receptors.33,14. In addition, emodepside acts and treatment of sarcoptic mange (Sarcoptes via a second target, a Ca++-activated K+ scabiei var. canis) and demodicosis (Demo- channel. This channel belongs to the large- dex canis).15,6,20,11,19,12 conductance calcium- and voltage-activated Emodepside/praziquantel tablets and im- potassium channels, termed SLO-1, which idacloprid/moxidectin spot-on were selected generally are important regulators of cell to assess the clinical impact of concomitant excitability. The end effect of emodepside use, since both products may be adminis- is flaccid paralysis and death of the nema- tered simultaneously in a clinical setting. 16 tode. Both emodepside and moxidectin are Praziquantel is an acylated pyrazino- known to be substrates for P-glycoprotein isoquinoline derivative and has been used (P-gp), the product of the multidrug re- in veterinary medicine for many years as an sistance gene 1 (mdr1).7,17 P-gp functions effective active against cestodes and trema- as a drug transport pump in various cell todes.31,5 membranes including the blood brain bar- Emodepside/praziquantel tablets are rier, transporting a variety of drugs from indicated for dogs suffering from, or at risk the brain back into the blood.24Although from, mixed parasitic infections caused by moxidectin is described as a weak P-gp roundworms (immature and mature Toxo- substrate, co-administrations with other P-gp cara canis, Toxascaris leonina, Ancylostoma substrates could cause interactions at the caninum, Uncinaria stenocephala, Trichuris level of the transporter. 13,21 vulpis) and tapeworms (Dipylidium cani- Potential clinical consequences of co-ad- num, Taenia spp, mature and immature ministration of emodepside and moxidectin Echinococcus multilocularis, and E. granu- have not previously been investigated. In the losus).3,26 present study the two parasiticides, emodep- Advocate® spot-on for dogs (Bayer Ani- side/praziquantel tablets and imidacloprid/ mal Health GmbH, Leverkusen, Germany) is moxidectin spot-on, were administered a solution for dermal application containing concomitantly to dogs simulating worst case imidacloprid and moxidectin. Moxidectin scenarios: on the one hand, a simultaneous is a macrocyclic lactone structurally within treatment, and on the other a consecutive the milbemycin family and derived from the treatment with overlapping maximum serum actinomycete Streptomyces cyanogriseus concentrations of emodepside and moxidec- spp Noncyanogenus.28 The primary mode tin. In addition, the maximum therapeutic of action results from moxidectin binding dose of both compounds was applied. To to glutamate-gated chloride channels of the test the worst case scenarios even further, parasites, leading to hyperpolarization of the animals in this study were fed immediately neuronal cells that results in paralysis and after treatment, which is in contrast to the death of the parasite.10 Imidacloprid belongs label instructions to administer emodepside/ to the group of neonicotinoide insecticides praziquantel tablets only to fasted dogs and and acts as an agonist at nicotinic acetylcho- not to feed dogs until 4 hours post-treatment. line receptors (nAChRs) and shows selective MATERIALS AND METHODS 22 toxicity for insects over vertebrates. In Animals addition to efficacy against gastrointesti- Fourteen adult Beagle dogs (1.4 – 7.3 nal nematodes, imidacloprid/moxidection years, 3 male, 11 female) weighing between spot-on is efficacious for treatment and 8.4 and 14.7 kg were enrolled. The dogs prevention of flea infestation Ctenocephali( - were part of a regularly maintained re-

Intern J Appl Res Vet Med • Vol. 9, No. 3, 2011. 291 Table 1: Clinical observation and blood sampling time points Group 1 Simultaneous treatment Physical examination for study inclusion Day -3 weighing, randomisation Clinical assessment Day 0: pre-tr., hourly 1 to 6 h, 8 h Day 1 (24 h p.tr.) Day 0 pre-tr. Blood chemistry/haematology* Day 1 (24 h p.tr.) Day 2 (48 h p.tr.) Day 3 (72 h p.tr.) Day 0: pre-tr., 1, 3, 5, 8 h Serum concentration* Day 1 (24 h p.tr.) (moxidectin, emodepside, praziquantel) Day 3 (72 h p.tr.) Day 8 Group 2 Consecutive treatment Physical examination for study inclusion Day -3 weighing, randomisation Day 0: pre-tr. Clinical assessment Day 8: pre-tr., hourly 1 to 6 h, 8 h Day 9 (24 h p.tr.) Day 0 pre-tr. Day 8: pre-tr. Blood chemistry/haematology* Day 9 (24 h p.tr.) Day 10 (48 h p.tr.) Day 11 (72 h p.tr.) Day 0: pre-tr.

Serum concentration* Day 3 (72 h p.tr.) (moxidectin, emodepside, praziquantel) Day 8 pre-tr., 1, 3, 5, 8 h Day 9 (24 h p.tr.) Day 11 (72 h p.tr.) Pre-tr. = pre-treatment , p.tr. = post-treatment *±12 minutes (mean: 3 minutes/group 1, 4 minutes/group 2) search colony and were returned to the provided ad libitum. All dogs were exposed colony after study completion. All animal to 12 h light and 12 h darkness. Temperature procedures were approved by the respon- range was between 19° and 23°C and rela- sible authorities and the animal welfare tive humidity between 33% and 50%. officer. Husbandry of animals complied with Clinical observations the European Commission guidelines for the On day -3 a physical examination was accommodation of animals used for experi- performed with special emphasis on behav- mental and other scientific purposes (June ioural attitude, nutritional status, respiratory 18, 2007/526/EC). The dogs were identi- system, gastrointestinal system, and car- fied by ear tattoo number and each animal diovascular system. General health obser- was housed in an individual pen during the vations of the dogs were performed daily study. They were fed once daily with a com- during the whole study period at cleaning mercial dry dog food and water supply was and feeding activities. Clinical assessments

292 Vol. 9, No. 3, 2011 • Intern J Appl Res Vet Med. for signs of abnormal behaviour, salivation, peutic dose, ie, the maximum theoretic vomiting, neurological signs, and tremor dose a dog can receive if it is located at the were performed pre- and post-treatment (see lower margin of the body weight range. Table 1). Day -3 body weights were used for both Treatment groups to calculate the dose on day 0, while The dogs were ranked by descending body day 7 body weights were used to calculate weight, placed into blocks, and were al- the dose on day 8 in group 2. Dogs of both located by random number draw within a study groups were treated on day 0 dermally block to two groups of seven dogs each. As with imidacloprid/moxidectin spot-on. The pre-treatment clinical observations and base- dosage applied was 25 mg imidacloprid/kg line blood samples were performed, each bw + 6.25 mg moxidectin/kg bw. This cor- ® dog acted as its own control. Especially for responded to a volume of 0.25 ml Advocate blood parameters with high inter-individual for dogs/kg bw. Treatment was applied in variations, the direct comparison of post- accordance with the label instructions, ie, treatment to pre-treatment parameters was dermally at one spot between the shoulder regarded as an accurate way to reflect the blades. real impact on the individual dog. Group 1 was treated at the same time All dogs received the maximum thera- orally with emodepside/praziquantel tablets at a dosage of 1.9 mg emodepside/kg bw

Table 2: Parameters tested for hematology and clinical chemistry Hematology Clinical Chemistry ADVIA® 120 Hematology System IDEXX VetTest® 8008 Chemistry Analyzer Albumin (ALB) g/l Alkaline phosphatase (ALKP) U/l Alanine-aminotransferase (ALT) U/l Amylase (AMYL) U/l Aspartate-aminotransferase (AST) U/l Blood urea nitrogen (UREA) mmol/l Red blood cell count x106/µl Creatinine (CREA) µmol/l Hemoglobin g/dl Creatinkinase (CK) U/l Hematocrit % Cholesterol (CHOL) mmol/l Mean corpuscular volume (MCV) fl Gamma-glutamyl-transferase (γGT) U/l White blood cell count x103/µl Globulin (GLOB) g/l White blood cell differential count x103/µl / % Glucose (GLU) mmol/l Reticulocyte count x106/µl / % Lactate dehydrogenase (LDH) U/l Platelet count Lipase (LIPA) U/l Ammonium (NH3) µmol/l Total bilirubine (TBIL) µmol/l Total protein (TP) g/l Triglyceride (TRIG) mmol/l Calcium (Ca) mmol/l Magnesium (Mg) mmol/l Phosphorus (PHOS) mmol/l

Intern J Appl Res Vet Med • Vol. 9, No. 3, 2011. 293 Table 3: Between-group comparison: Parameters showing both significant (p < 0.05) differences between group 1 and 2 and values outside the reference ranges Time point Statistical significance Dogs outside Comments to dogs p.tr. # (group comparison) reference range outside reference (high / low) range Clinical chemistry CK 2 days p.tr. Group 2 higher than 1 dog in group 2: Elevation > 3x group 1* high (p = 0.0507) LDH 3 days p.tr. Group 1 higher than 1 dog in group 1: Slight elevation group 2 high (p = 0.0398) # for group 2: after second treatment *not significant, but approached significance (p = 0.0507) p.tr. = post-treatment

+ 9.4 mg praziquantel/kg bw. Group 2 was blood samples were centrifuged, plasma treated with the emodepside/praziquantel was harvested and directly analyzed with an tablets on day 8 at the same dosage. The IDEXX VetTest® 8008 Chemistry Analyzer tablets were weighed and filed off with sand- (IDEXX Laboratories, Inc., Westbrook, ME, paper to get the individual calculated tablet USA) in accordance with the manufacturer´s mass (tolerance + 1 mg). The dogs were instructions. A total of 21 parameters were observed at dosing and shortly after dosing determined (see Table 2). The serum tubes to determine whether any tablet matter was were centrifuged, serum was harvested and regurgitated. All dogs were fed immediately frozen at -18°C until analysis. after treatment on day 0 and 8. Analysis of actives Sampling For analysis, serum was deproteinized by Blood samples were drawn for hematology, mixing with acetonitrile and was subse- clinical chemistry, and for confirmation of quently centrifuged. The quantitative deter- serum levels of moxidectin and emodepside. mination was performed by direct injection For the baseline blood was collected of an aliquot of the supernatant into a High from all dogs on day 0 before treatment, Performance Liquid Chromatograph and enabling the dogs to act as their own control. detection by tandem mass spectrometry. The For sampling time points see Table 1. method was validated in the range from 2 Blood samples were collected from the to 1500 µg/l with mean recovery rates of Vena jugularis. EDTA-K3 and lithium hepa- 96 ± 9.6% for moxidectin and 105 ± 9.1% rin containing blood collection systems were for emodepside. used as anticoagulants for hematological Data were analyzed using non-com- and serum chemistry analyses respectively, partmental pharmacokinetic methods with while plain serum collection systems were a commercial program (WinNonlin version used for measurement of serum concentra- 5.2, Pharsight® Corp., Mountain View, CA, tions. EDTA containing blood samples were USA). directly analyzed with an ADVIA® 120 Statistical analysis Hematology System (Siemens Healthcare Prior to analyses, all post-treatment clini- Diagnostics, Deerfield, IL, USA) in accor- cal chemistry and hematology values were dance with the manufacturer´s instructions. aligned between both treatment groups. A total of 19 parameters were determined Post-treatment values were compared to pre- (see Table 2). Lithium heparin containing treatment values by using the day 0 values

294 Vol. 9, No. 3, 2011 • Intern J Appl Res Vet Med. Table 4: Pharmacokinetic parameters (geometric mean, CV%) of moxidectin and emodepside Parameter Group Moxidectin Emodepside

cmax (µg/l) 1 42.6 CV% = 67.3 140.3 CV% = 28.7 2 51.8 CV% = 42.2 64.5 CV% = 243.1

tmax (h) 1 80.1 ( = 3 days 7.2 h) CV% = 117.8 1.5 CV% = 76.2 2 152.6 ( = 6 days 9.6 h) CV% = 0.7 2.7 CV% = 0.7 cmax = peak serum concentration, tmax = time to peak serum concentration, CV% = coefficient of variation for the baseline in both groups. Comparisons discussed here. between the blood values of the two treat- Statistical analyses were performed on ment groups were performed using group all hematological and clinical chemistry 1 values from days 1, 2, and 3, and group 2 parameters, specifically testing whether the values from days 9, 10, and 11. pre-treatment baseline values were signifi- Comparisons between groups utilized cantly different from any of the three post- day 0 values as a baseline covariate, and a treatment values for each separate treatment repeated measures analysis of covariance or group. There were no significant changes a generalized linear mixed model analy- observed in parameters outside the reference sis using binary data was used, depending ranges. upon the data distributions. In addition, Statistical analyses were also performed separate analyses were performed within comparing the blood values between the two each treatment group, testing whether the treatment groups. Results indicated signifi- pre-treatment baseline values of day 0 were cant changes in two parameters (CK, LDH) significantly different from any of the three that were outside the reference ranges (see post-treatment values. All analyses used Table 3). In one dog in group 2, CK activ- SAS version 9.3 software (SAS Institute ity pre-treatment on day 0 was more than Inc., Cary, NC, USA) with an alpha of 0.05 double the upper reference range (upper or less as statistically significant. limit of reference range 200 U/l), but normal RESULTS on day 8. On day 9 and 10, activity of CK increased again to more than 5-fold (1,059 Clinical observations and 1,907 U/l) with concurrent slight eleva- All dogs tolerated the treatment well. One tions of LDH. On day 11 CK activity had dog in group 2 regurgitated one tablet with decreased to concentrations only slightly a small amount of saliva between 7 to 16 above the reference range and LDH activity minutes after application of the emodepside/ was inside the reference range. As a conse- praziquantel tablets (day 8) before feeding. quence of this high CK activity of this single The tablet that was found was immediately dog on day 10 the CK activity of group 2 reapplied. The observed regurgitation is nearly approached significantly higher levels likely due to stress by restraining and deep on day 10 compared to group 1 (p = 0.0507). tablet application into the mouth and there- On day 9, another two dogs in group 2 had fore, not regarded as product related. CK values that were more than double the Blood hematology and chemistry upper reference range (881 and 453 U/l) Only parameters that were outside the with slightly increased LDH values, but reference ranges given by the respective these were normal on day 10. ® manufacturers of ADVIA 120 Hematology Although on day 3 significantly higher ® System and IDEXX VetTest 8008 Chemis- activity of LDH in group 1 than in group 2 try Analyzer, and that exhibited significant was observed (p = 0.0398), only one dog in differences were considered relevant and are group 1 had LDH activities slightly above

Intern J Appl Res Vet Med • Vol. 9, No. 3, 2011. 295 Figure 1: Mean serum concentration time Figure 2: Mean serum concentration time profile of moxidectin and emodepside for profile of moxidectin and emodepside for group 1 group 2

the reference range on this day. pharmacokinetic studies with imidacloprid/ Serum concentration moxidectin spot-on.30 In our study the time Moxidectin concentrations were first de- to peak serum concentrations of moxidectin tected 3 h after application and reached a was between 3 and 7 days post-treatment, plateau between days 1 and 11 (see Figures but as moxidectin concentration remained 1+2). from day 3 to day 11 on a plateau-like level, the overlapping of peak moxidectin Emodepside concentrations were and emodepside serum concentrations was detected in serum at the first sampling time achieved. point of 1 h post-treatment and reached their peak at 1.5 h in group 1 and 2.7 h in group In previous studies, peak moxidectin 2. Concentrations decreased thereafter with concentrations of 15.3 µg/l and emodepside levels below the limit of quantification on concentrations of 47 µg/l were found after day 8. application of 2.5 mg moxidectin/kg bw and 1.5 mg emodepside/kg bw (G. Beddies, un- The calculated peak serum concentra- published data 2001, G. Ahr and O. Görgen, tions (c ) and time to peak serum concen- max unpublished data 2007). In this study rela- tration (t ) are summarized in Table 4. max tive high mean peak serum concentrations DISCUSSION of moxidectin (day 3-11: 42.6/51.8 µg/l) and The purpose of this study was to evaluate emodepside (64.5/140.3 µg/l) were found, a concomitant treatment of imidacloprid/ but a higher dosage of both actives was moxidectin (Advocate® spot-on) and em- applied. These high serum levels confirmed odepside/praziquantel (Profender® tablets). high resorption and confirmed and met the To simulate a treatment scenario that might demands of the worst case scenarios. have maximum impact, in addition to the Label instructions for Profender® tablets common practice of simultaneous treat- are to treat only fasted dogs and to not feed ment, a consecutive treatment with an 8-day earlier than 4 hours post-treatment, as neu- interval to ensure overlapping peak serum rological signs are more likely to occur in concentrations of emodepside and moxidec- fed dogs.8 Therefore, to simulate additional tin was also investigated. This 8-day interval worst case scenarios that might have an was chosen based on results of previous impact, in this study all dogs were fed im-

296 Vol. 9, No. 3, 2011 • Intern J Appl Res Vet Med. mediately after treatment and the maximum elevations of LDH in these dogs seemed to therapeutic dose of the active ingredients of be based on the same mechanisms. both products was applied. During clinical All the results obtained in this study assessments no compound related adverse prove the safety of a concomitant simultane- reactions were observed. ous or consecutive treatment of imidaclo- Concerning hematology and clinical prid/moxidectin spot-on (Advocate® spot- chemistry, no changes in these profiles could on) and emodepside/praziquantel tablets be seen that were considered to be treatment (Profender® tablets) in adult Beagle dogs. As related. Comparisons to baseline and be- moxidectin and emodepside are known to be tween groups indicated that even when there substrates for P-glycoprotein (P-gp) and in were statistically significant changes, the this study no dogs with the mdr1 gene defect values were nevertheless inside the reference (mdr1-/-) were tested, the safety for this ranges and therefore not clinically relevant. special case has still to be proven in further No relevant significant changes were seen in investigations. comparison to baseline. CONCLUSION In the between-group comparison two None of the 14 dogs dosed with imidaclo- parameters (CK and LDH) were observed prid/moxidectin spot-on and emodepside/ with statistical significant changes and praziquantel tablets showed adverse events values that were outside the reference range. nor changes in their blood haematology or Especially one dog in group 2 had more serum biochemistry profile during the course than 5-fold CK activity on day 9 and 10, of the study that were considered to be treat- while another two dogs in this group also ment related. It was shown that concomitant had slight CK elevations on day 9. The treatment with both products applied either cytosolic enzyme CK is a muscle specific simultaneously or consecutively at the enzyme with its main distribution in skeletal maximum therapeutic dose and with feeding muscle and myocardium, while low concen- directly after treatment was safe in adult 18,1 trations are found in intestine and brain. Beagle dogs. Although CK is considered a good and easily available indicator of muscle damage ACKNOWLEDGEMENTS in dogs, false positives resulting from its The authors express their sincere thanks to high concentration in muscles and its pos- Terry Settje for the statistical analysis, Ralph sible leakage for non-specific reasons may Krebber for the serum analysis and Kristine occur.2 Intramuscular injection itself has Fraatz and Angela Schulten for the kinetic been shown to increase the enzyme levels22 analysis. and even incorrect venipuncture can result in DISCLOSURE STATEMENT serum values elevated by at least 25% due to muscle puncture.9 Hemolysis and hyperbili- The study was sponsored by Bayer Animal rubinaemia can also lead to falsely elevated Health GmbH, all authors were employed CK values but have only slight effects if by Bayer Animal Health GmbH, Germany, moderate. during the conduct of this study. Physical activity was described as the References main factor for intra-individual variation, 1.Aktas M., Auguste D., Lefebvre H., Toutain P. L. & Braun J. P. (1993) Creatin kinase in dog: A review. which occurred in this study during the Veterinary Research Communications 17: 353-369. 1 frequent clinical assessments. Another con- 2.Aktas M., Auguste D., Concordet D., Vinclair P., tributing factor might be accidental muscle Lefebvre H., Toutain P. L. & Braun J. P. (1994) puncture during blood sampling and hemo- Creatin kinase in dog plasma: preanalytic factors of variation, reference values and diagnostic signifi- lysis of the blood samples. As the ubiquitous cance. Research in Veterinary Science 56: 30-36. LDH is found in high concentrations in mus- 3.Altreuther G, Radeloff I, LeSueur C, Schimmel A, cle together with CK,18 the concurrent slight Krieger KJ (2009) Field evaluation of the efficacy and safety of emodepside plus praziquantel tablets

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