Gamithromycin in swine: Pharmacokinetics and clinical evaluation against swine respiratory disease

Abstract The pharmacokinetics of gamithromycin were evaluated in 26 male castrated and female crossbred swine administered gamithromycin 15% w/v (Zactran®, Boehringer Ingelheim) intravenously at 6 mg/kg bodyweight or intramuscularly at 3, 6 or 12 mg/kg bodyweight. Blood samples were collected up to Day 10 to establish the plasma profile of gamithromycin, bioavailability and dose proportionality. When administered by intramuscular injection at 6 mg/kg BWT, pharmacokinetic parameters were as follows: area under the curve until last quantifiable plasma concentration, 5.13 ± 0.957 µg*hours/ml; maximum plasma concentration, 960 ± 153 ng/ml at 5 to 15 min; terminal half‐life of 94.1 ± 20.4 hr. Absolute bioavailability was 92.2%. Increase in systemic exposure was proportional to the gamithromycin dose level over the range 3–12 mg/kg BWT. No gender‐related statistically significant difference in exposure was observed. For clinical evaluation of Zactran® against swine respiratory disease, 305 pigs from six commercial farms in three countries in Europe with signs associated with Actinobacillus pleuropneumoniae and/or Haemophilus parasuis and/or Pasteurella multocida and/or Bordetella bronchiseptica were used. At each site, animals were treated once in a 1:1 ratio with a single intramuscular dose of Zactran® (6 mg gamithromycin/kg bodyweight) or Zuprevo® (4% w/v tildipirosin at 4 mg/kg bodyweight; MSD Animal Health) at the recommended dose respectively. Animals were observed and scored daily for 10 consecutive days for signs of swine respiratory disease (depression, respiration and rectal temperature), and animals presenting signs of clinical swine respiratory disease (Depression Score 3 and/or Respiratory Score 3 associated with Rectal Temperature > 40.0°C) were removed from the study and considered as treatment failure. Animals which remained in the study were individually assessed for ʽtreatment successʼ or ʽtreatment failureʼ (Depression Score ≥ 1 and Rectal Temperature > 40.0°C or Respiratory Score ≥ 1 and Rectal Temperature > 40.0°C). Using a non‐inferiority hypothesis test (non‐inferiority margin = 0.10), the proportion of treatment successes in the Zactran® group (97%) was equivalent to or better than that in the Zuprevo® group (93%).


| INTRODUC TI ON
belongs to the 15-membered semisynthetic macrolide antibiotics and, as an azalide, is characterized by a uniquely positioned alkylated nitrogen at 7a-position of the lactone ring.
Similar to its efficacy in the treatment and control of bacterial bovine respiratory disease following a single subcutaneous injection with GAM 15% w/v (Baggott et al., 2011;Linhardt & Brumbaugh, 2019;Torres et al., 2017), GAM should display favourable characteristics in the treatment of clinical swine respiratory disease. This multifactorial condition results from mixed infection of viral and/or bacterial agents and is characterized by anorexia, fever (>40°C), cough and dyspnoea, ultimately leading to decreased feed conversion and growth rate (Opriessnig et al., 2011).

Gamithromycin 15% w/v injection (Zactran®, Boehringer
Ingelheim) is currently licensed in Europe, the Americas and other regions for the treatment and control of bovine respiratory disease in cattle caused by Mannheimia haemolytica, Pasteurella multocida and Histophilus somni, and/or swine respiratory disease caused by Actinobacillus pleuropneumoniae, P. multocida, Haemophilus parasuis and Bordetella bronchiseptica. In addition, the product was licensed in Europe for the treatment of footrot in sheep caused by Dichelobacter nodosus and Fusobacterium nodosus (EMA, 2018a).
This study describes the results of studies on the pharmacokinetic profile of GAM in swine following intravenous and intramuscular (IM) injection and reports results of an European multicentre field study on the clinical evaluation of a single GAM 15% w/v injection at a dose of 6 mg/kg bodyweight (BWT) IM against swine respiratory disease.

| General study design
The pharmacokinetic study was conducted in accordance to GLP and to "Guidelines for the Conduct of Pharmacokinetic Studies in Target Animal Species, EMEA/CVMP/133/99-FINAL". The European multicentre field study was in accordance with the Committee for Veterinary Medicinal Products "Guideline on Good Clinical Practice -VICH Topic GL9 GCP, CVMP/VICH/595/98-Final" and the "Guideline for the demonstration of efficacy for veterinary medicinal products containing antimicrobial substances, EMEA/ CVMP/627/01-FINAL". All study procedures complied with the appropriate local animal welfare regulations, and were approved by applicable legal bodies and by the company´s animal welfare committees. The on-farm procedures in the field study were performed with the informed consent of the animal owners.
None of the swine had received any macrolide antibiotic treatment within 1 month prior to Day 0 (=day of treatment). All animals were fitted with two surgically implanted jugular vein catheters 1 week prior to treatment. The animals were allocated to treatment groups based on Day −1 BWT; the heaviest pig within each sex was selected to form the non-treated control group (Group 1), the remaining animals were ranked by decreasing BWT within sex and formed in three blocks of five, four and four animals, and were randomly allocated to Groups 2, 3, 4 and 5 consisting of total eight, six, six and six animals, respectively. Gamithromycin 15% w/v was administered once on Day 0 either by intravenous (IV) (Group 2) or by IM injection (Groups 3, 4 and 5) based on Day −1 BWT. Animal and dosing details are given in Table 1.
Gamithromycin 15% w/v was given to Group 2 pigs via one IV catheter; following administration, the catheter was flushed with ~20 ml of physiological saline solution to ensure delivery of the targeted dose. In animals of Groups 3, 4 and 5, GAM 15% w/v was administered IM in the dorsal part of the left neck side, in front of the shoulder.
Gamithromycin 15% w/v was administered using 0.1 ml graduated syringes in Groups 2, 3 and 4 or using 0.2 ml graduated syringes in Group 5. Intramuscular doses were administered with sterile disposable hypodermic 19 G x 1'' needles.
Following GAM 15% w/v administration, the animals were observed hourly for the first four hours for reactions to treatment.
Animals were housed individually in pens with straw bedding and they were fed a complete fattening ration offered for ad libitum consumption and had free access to water.
Prior to blood sampling, catheters were flushed with physiological saline solution. The first ~5 ml of blood was discarded, and thereafter, blood for plasma processing was collected. Following blood collection, catheters were flushed with ~10 ml of physiological saline solution and a ~5 ml bolus of anticoagulant in saline was placed in the catheter. Plasma was separated by centrifugation and stored at ≤-20°C until assayed for GAM concentrations.

| Analytical method
Plasma samples were analysed for GAM using a validated LC-MS/ MS method (Huang et al., 2010). The lower limit of quantitation in plasma was established as 2.0 ng/ml and the lower limit of detection as 1.0 ng/ml. The method performed well during the analysis of the samples. Quality control samples had GAM recoveries from 85%-114% (mean 102 ± 5%). The maximum concentration and observed time to maximum concentration, and time to last quantifiable concentration were determined directly from plasma concentration data. The first-order rate constant λ z , associated with the terminal log-linear portion of the curve was estimated via linear regression of the log drug plasma concentration versus time curve, and the terminal plasma half-life (T 1/2 ) concentration was calculated using T 1/2 = ln(2)/ λ z . The area under the curve (AUC) was determined using the linear trapezoidal rule for increasing and the logarithmic trapezoidal model for decreasing plasma concentrations from Day 0 to the last time plasma drug concentrations were above the lower limit of quantitation (AUC last ). AUCs were extrapolated to infinity using the formula AUC inf = AUC last + C last /λ z .

| Pharmacokinetic analysis
Dose proportionality of GAM following IM administration over the range 3-12 mg/kg BWT was assessed by analysing the dose-normalized average AUC inf at each dose level from Groups 3, 4 and 5 and also using a power model: Linear regression analysis was performed using Proc Reg in SAS 9.0 (SAS Corporation, Cary, NC, USA) with weighting of (Dose)-1.
The residuals were normally distributed, independent and randomly distributed around zero. The 95% confidence limits on the parameters were determined at α = 0.05.
A t test was utilized to determine statistical differences between the pharmacokinetics of male castrate and female swine.

| European multicentre field study
Various commercial cross-breed pigs were used in this study conducted in six commercial fattening farms located in France, Germany and Spain (Table 2). Only swine respiratory disease-positive animals from available stock were eligible for enrolment. At each site, animals were housed in group pens, study animals together with nonstudy animals, within one airspace. They were managed according to the normal husbandry practices at each site and fed according to local practice ensuring adequate nutrients for their age and condition. Routine disease control measures were similar for all animals at a study site and were limited to the administration of (but not necessarily any or all of) viral vaccines and endectocides. None of the animals had received bacterial vaccines against swine respiratory disease pathogens (A. pleuropneumoniae, P. multocida, B. bronchiseptica and H. parasuis). Animals at three of the six sites were vaccinated  Day 0 (= day of treatment) varied by site and by animal. Upon enrolment, animals were weighed and allocated randomly by means of site-specific randomization lists to treatments in a 1:1 ratio: Treatment Group 1, GAM 15% w/v (Zactran®); Treatment Group 2, tildipirosin (TIL) 4% w/v (Zuprevo®) in blocks of two animals.
Treatments (commercial doses) were administered as described in Table 2. The doses were rounded up to the next 0.1 ml increment for doses up to 2 ml and were rounded up to the next 0.2 ml increment for doses >2 ml. Treatments were administered IM on the dorsal left part of the neck. For Zuprevo®, total doses of >5 ml were divided into 5 ml (primary injection) and the remainder (secondary injection on the dorsal right part of the neck).
Animals in a block were treated the same day. Enrolment at each site was performed for up to 3 consecutive days after enrolment of the first block. Animals of both treatment groups were housed comingled at all sites.

| Enrolment
Animals were scored for clinical signs of swine respiratory disease based on depression and respiratory signs as given in Table 3  removed from the study, euthanized and necropsied for confirmation of swine respiratory disease. Animals developing a non-swine respiratory disease concurrent pathological condition were also removed from the study.
Personnel involved with post-treatment evaluations were masked; they were not present during treatments and did not have access to the allocation/treatment assignments.

| General and local tolerance
The general reactions to treatments were assessed daily from Day 0 until Day 10 inclusive. The injection sites were observed approximately 1 hr post-treatment, on the day of removal or at final assessment on Day 10, as applicable. In the event that injection site reactions were present on Day 0, the injection site was observed daily until resolution.

| Clinical evaluation and data analysis
All animals removed from the study according to swine respiratory were dichotomized as "Acceptable" (0 or 1) and "Not acceptable" (>1). Rectal temperatures were also dichotomized as "Acceptable" (≤40.0°C) and "Not acceptable" (>40.0°C). Data from all six sites were combined for analysis. The percentage of treatment success and each clinical parameter were compared between groups using a non-inferiority hypothesis test (non-inferiority margin = 0.10) (SAS® 9.1.3, Cary, NC, USA). The hypothesis of non-inferiority between the two Treatment Groups was tested for the proportion of treatment success, and the proportions of acceptable Depression Score, Respiratory Score and Rectal Temperature.

| Pharmacokinetic study
A summary of the basic pharmacokinetic parameters is given in Table 4.
Following a single IV injection of GAM at 6 mg/kg BWT, the AUC inf was 5.89 ± 0.658 µg*hr/ml and the T 1/2 was 76.1 ± 23.7 hr.
The full AUCs were captured in this study with less than 10% ex-  (Figure 1) was also assessed based on regression of AUC inf versus dose at 0.5, 1 and 2 times of the target dose rate with the following resulting equation: The coefficient of determination was 0.8277, and the slope and intercept were highly statistically significant (p < .0001) and, therefore, not equal to zero. The mean value for the slope was 0.8102.
There was no statistical difference between T 1/2 and the AUCs of male castrate and female swine (Student's T test, 95% confidence), indicating that there was no gender-related difference in the pharmacokinetics.
No abnormal health observation or adverse experience occurred during the study except in one animal in Group 2. The animal exhibited 40.6°C rectal temperature, increased respiration and tachycardia in the afternoon of Day 2 and received anti-inflammatory treatment (meloxicam) for 3 days. The condition had improved on Day 3, and the animal was normal at Day 6.  Results of the data analysis are summarized in Table 5. The proportion of treatment successes was 97% in the GAM group and 93% in the TIL group. As the lower limit of the two-sided 95% confidence interval on the difference was greater than the non-inferiority threshold, data supported that GAM treatment was comparable with or better than TIL treatment.

| Pharmacokinetic study
Based on the results of this study, GAM administered once to swine as 15% w/v injectable solution by IM route at three different doses demonstrated high bioavailability, fast absorption, rapid and extensive distribution to tissues, high clearance and approximate dose proportionality of AUC inf .
The pharmacokinetic profile of GAM following IM administration at 6 mg/kg BWT in the present study was largely similar to that established by Wyns et al. (2014), indicating that the pharmacokinetic Ln AUC inf = 7.1420 + 0.8102Ln (Dose) F I G U R E 1 Gamithromycin Log AUC inf (hr*µg/ml) versus Log Dose (mg/kg) following intramuscular administration of gamithomycin 15% w/v (Zactran ® ) at 3, 6 and 12 mg per kg body weight to swine GAM has overall a comparable pharmacokinetic profile across several species and irrespective of the route of administration as shown in domestic animals other than swine including cattle (DeDonder et al., 2016;Giguère et al., 2011;Huang et al., 2010), sheep (Kellermann et al., 2014), young foals (Berghaus et al., 2012;Berlin et al., 2014) and poultry (Watteyn et al., 2013;2015) ( Table 6).
Comparable with cattle (Huang et al., 2010), average C max values in swine did not increase proportionally over the dose range 3-12 mg/kg BWT, indicating that the absorption of GAM did not follow linear processes. The rate of absorption from the administration site is limited by blood flow to the injection site and, thus, the maximum plasma concentration did not increase proportionally with a higher dose. Similarly, a slow but continuous release following injection was observed in swine for tylosin and TIL, related 16-membered semisynthetic macrolide antibiotics (Prats et al., 2002;Rose et al., 2012), which may be attributed to concentration-dependent plasma protein binding .
However, as found in cattle with GAM doses of 3, 6 and 9 mg (Huang et al., 2010), approximate dose proportionality was evident in swine for the AUC inf values for doses of 3, 6 and 12 mg/ kg BWT. This indicates that the increase in the administered dose was accompanied by a proportional increase in the overall exposure to GAM over the dose range tested. Apart from potential species-specific physiological differences, absorption in cattle of the 9 mg/kg BWT dose may not be impacted in the same manner as the higher dose of 12 mg/kg BWT in swine. Nonetheless, all doses administered in the present pharmacokinetic study were highly bioavailable (average 93.3%) and absorbed within hours.

| Field study
The results of the European multicentre field study show that a single IM injection of GAM 15% w/v at the dose of 6 mg/kg The percentage of animals that had an acceptable outcome for the listed endpoint ("success" for Overall Evaluation and "acceptable" for Depression Score, Respiration Score and Rectal Temperature).
The numbers in parentheses are the number of animals with acceptable outcome over total number of animals in the group which were included in the analysis.
b Two-sided 95% confidence interval was computed using the formula, (P control in accordance with the recommendations for the design of non-inferiority studies (Freise et al., 2013).
No adverse reactions related to treatments were observed during this field study confirming that GAM 15% w/v is a well-tolerated and safe product for use in swine.

E TH I C S S TATEM ENT
All study procedures complied with the appropriate local animal welfare regulations, and were approved by applicable legal bodies and by the company´s animal welfare committees. The on-farm procedures in the field study were performed with the informed consent of the animal owners.

ACK N OWLED G EM ENTS
We gratefully acknowledge Ted Chester, PhD, former employee of Merial, for the statistical analysis of data generated in the field study.

D I SCL A I M ER
ZACTRAN® is a registered trademark of Merial. All other marks are the property of their respective owners. This document is provided for scientific purposes only. Any reference to a brand or trademark herein is for informational purposes only and is not intended for a commercial purpose or to dilute the rights of the respective owner(s) of the brand(s) or trademark(s). Merial is now a part of Boehringer Ingelheim.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.375.