Hello to Aivlosin, the brand new macrolide antibiotic

Technical Monograph 01/04/2005 11:59 Page 1 Technical Monograph Hello to Aivlosin, the brand new macrolide antibiotic - premix against Enzootic Pneumonia 1

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Technical Monograph 01/04/2005 12:00 Page 3 Contents Page 1. Introduction 4 Chemistry................................... 7 Dosage Information........................... 8 Mode of Action............................... 8 2. Pharmacokinetics 9 Absorption................................... 9 Distribution................................. 10 Metabolism................................. 10 Excretion.................................. 10 3. Antimicrobial spectrum of activity 11 Mycoplasma hyopneumoniae................. 11 4. Toxicology 12 Tolerance studies in pigs..................... 12 Withdrawal period........................... 12 5. Environmental considerations 13 6. Efficacy 14 Dose Titration and Dose Confirmation.......... 14 European Field Trials......................... 16 7. References 17 - premix against Enzootic Pneumonia

Technical Monograph 01/04/2005 12:00 Page 4 Introduction Enzootic pneumonia is caused by Mycoplasma hyopneumoniae (M.hyo). The organism progressively colonises the respiratory tract, attaches to the endothelial cells and damages the ciliated cells, impairing the muco-ciliary escalator system, finally leading to the formation of the classic purple consolidated lesions in the ventral part of the pig s lungs. Primarily the apical, cardiac, intermediate and anteroventral parts of the diaphragmatic lobes are affected. The organism is spread from pig to pig by aerosol droplet, hence the condition is worse where there is poor ventilation, overcrowding and mixed ages of growing pigs. The lesions can take 3-4 weeks to form and remain for several weeks. Histologically there are typical accumulations of lymphoid tissue causing peribronchiolar and perivascular cuffing. In immunologically naive herds, it can affect all ages of pigs, including breeding stock, when first Cilia with M. hyo on the mucosal surface in the respiratory tract. challenged but usually almost all herds are chronically infected and the condition is seen mainly in the growing & finishing pig from 8-26 weeks of age. In slaughterhouse surveys on average 40-50% of pig lungs have lesions and in severely affected herds over 90% of the pigs would be affected. The damage to the cilia and also other immunodefence mechanisms in the respiratory tract and lung leads to poor local and systemic antibody production and impairment of macrophage activity. This facilitates secondary bacteria in particular Pasteurella multocida, but also Haemophilus parasuis, Streptococcus suis and Actinobacillus pleuropneumoniae to penetrate the respiratory tract and cause a complicated severe bronchopneumonia. Mycoplasma on its own causes a relatively chronic but mild respiratory infection, characterized by a dry cough starting from about 10 days after infection, uneven growth and poor feed conversion efficiency. It is mainly when the secondary bacteria invade that the disease becomes acute, the pigs may have high temperatures, show dyspnoea, inappetence and may even die. Over the last decade the on-farm disease has changed significantly with the introduction of both porcine reproductive and respiratory syndrome virus (PRRSv), which further damages the immune system and destroys lung macrophages and also the advent of post-weaning multi-systemic wasting syndrome (P.M.W.S.) associated with porcine circovirus type 2, which further damages the pig s immune system. Both viruses permit other viruses such as swine influenza virus (SIV) and porcine respiratory coronavirus (PRCV) to become endemic in herds as well as increase the susceptibility of pigs to further bacteria including salmonella. This has led to the term the porcine respiratory disease complex (PRDC) being used to refer to this mix of respiratory diseases which has had a significant impact on pig production and mortality rates across Europe. 4

Technical Monograph 01/04/2005 12:00 Page 5 M. hyopneumoniae is recognised as playing a major underlying role in this complex and in spite of the widespread use of vaccines, there is still a need for additional medication to either treat or prevent the development of the severer forms of this condition associated with M.hyopneumoniae and the resulting secondary bacterial infections. A variety of antimicrobials have been used for the treatment and prevention of M. hyopneumoniae such as tetracyclines, macrolides, pleuromutilins and lincosamides. Aivlosin is an in-feed premix containing the macrolide antibiotic, acetylisovaleryltylosin, in a 4.85% formulation, for the prevention and treatment of Enzootic Pneumonia in pigs caused by M. hyopneumoniae. Acetylisovaleryltylosin administered to pigs in feed at a dose rate of 2.125 mg/kg per day as a sole ration for seven consecutive days prevents weight loss and lung lesions. Key characteristics Administered in feed Well tolerated in pigs High concentration in target organs (i.e lungs, liver, small intestine) No resistance by M. hyo has been reported Mycoplasmacidal at concentrations similar to MIC Macrophage stimulation Minimal environmental effect Acetylisovaleryltylosin is a member of the chemical class, macrolide antibiotics which are produced by fermentation of soil organisms. Chemical modification of these fermentation product has yielded a lot of analogues including erythromycin, spiramycin, tylosin and tilmicosin which are widely used for control of animal and human disease complexes (Enzootic pneumonia, Ileitis or PPE, Swine Dysentery etc.). In the past, macrolide antibiotics have also been used as growth promoters in growing pigs, but this use has been banned in the EU. Acetylisovaleryltylosin tartrate is rapidly absorbed and widely distributed in the tissues after oral administration of Aivlosin. It has been proven that Aivlosin has Mycoplasmacidal activity at concentrations similar to Minimum Inhibitory Concentration (MIC), and to date, no resistance by M. hyopneumoniae has been reported in the field. Macrophages in the respiratory tract have an important role in clearance of M. hyopneumoniae by Phagocytosis. Aivlosin is distributed intracellular in macrophages and prevents microorganisms from hiding and protecting themselves inside the cells. A special feature of some macrolides and especially Aivlosin, is the ability to stimulate macrophage growth. Aivlosin was developed specifically to provide producers with a product that combines highly effective control of Enzootic Pneumonia with safety in pigs, ease of administration and which minimise any risk for the consumers. 5

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Technical Monograph 01/04/2005 12:01 Page 7 Chemistry Chemical structure of acetylisovaleryltylosin The following data describes the active ingredient: Acetylisovaleryltylosin tartrate is the active ingredient in Aivlosin. Acetylisovaleryltylosin has a 16-membered lactone ring and is derived from tylosin. Aivlosin is an acetylated derivative with an acetyl group attached at the hydroxyl group at the 3-carbon position and an isovaleryl group at the 4 carbon position, which enhance absorption and antimicrobial activity respectively. Generic name: Acetylisovaleryltylosin tartrate Molecular Formula: C 53 H 87 NO 19 C 4 H 6 O 6 Due to the dimethylamino group Aivlosin is basic. 7

Technical Monograph 01/04/2005 12:01 Page 8 Dosage Information Premix Formula for Aivlosin (Acetylisovaleryltylosin tartrate 4.25% Premix) The product is a medicated feed premix for inclusion into pig feed. The active is made into a granule with hydroxypropylcellulose and non-fat soybean powder and blended with mineral (clay) and grain carriers and anti-dusting agent light liquid paraffin. Supplied in 20 kg polyethylene lined paper sacks Aivlosin Premix 4.25mg/g has a shelf life of 24 months. After incorporation into meal or pelleted feed the shelf life is 1 month. Medicated Feed Production (see label for explanation) Mixing instructions: A horizontal ribbon mixer should be used to incorporate the product into the feeding stuff. It is recommended that Aivlosin is first mixed into 10 kg of the feeding stuff, followed by the rest of the feeding stuff and mixed well. Medicated feed may then be pelleted. Pelleting Medicated feed may then be pelleted. Pelleting conditions involve pre-conditioning ingredients with steam for 5 minutes and pelleting at 70 C under normal conditions. Rate of incorporation into non-medicated feeds (50 ppm): 1 kg of Aivlosin premix + 999 kg feed = 1000kg medicated feed. 20 kg (1 sack) of Aivlosin premix + 19,980 kg feed = 20,000 kg medicated feed. Dose rate Recommended dose rate is 2.125 mg acetylisovaleryltylosin per kg bodyweight per day in-feed for 7 consecutive days. This is normally achieved by incorporating Aivlosin at a level of 1 kg per tonne of feed to provide 42.5 mg acetylisovaleryltylosin per kg of feed, assuming that a pig s appetite is 5% of its bodyweight per day. In older pigs, or in pigs with reduced appetite, or on restricted feed intake, inclusion levels may need to be increased to achieve target dosage. Mode of action Aivlosin acts by inhibiting bacteria from synthesising protein. Like other macrolides Aivlosin interferes with protein synthesis by reversibly binding to the 50S ribosome subunit of the 70S ribosome. They bind to the donor site and prevent the translocation of amino acids, thus preventing the peptide chain from growing. Aivlosin has a higher level of antibacterial activity than tylosin, which may be due to a stronger binding potential or due to an extra binding site, thanks to its additional side chains. 8

Technical Monograph 01/04/2005 12:01 Page 9 Pharmacokinetics Absorption, distribution, metabolism and excretion studies were conducted in pigs. Acetylisovaleryltylosin is rapidly absorbed following a single oral bolus administration. Peak plasma concentrations are achieved two hours after oral administration in the pig. In a seperate trial, pigs received 2.125mg acetylisovaleryltylosin per Kg bodyweight for 7 days. Samples taken 2 and 12 hours after withdrawal of the medication showed levels in serum were below detection, but levels in the lung were maintained. Mean concentrations of acetylisovaleryltylosin tartrate in lung tissue were 0.066 and 0.060 µg/g at 2 and 12 hours postwithdrawal respectively. These levels are above themic90for all EU isolates studied. Excretion is primarily via the liver, bile and faeces and to a minor extent via the kidney and urine. Graph 2: Comparison of Aivlosin and tylosin plasma levels in pigs. Area under the curve (AUC) was 60.1µg/hour/ml for Aivlosin and 20.3µg/hour/ml for tylosin. The bioavailability of Aivlosin was nearly 3 times higher than tylosin. Aivlosin is rapidly metabolized in the body to 3-O-acetyltylosin (3AT). This compound, the major metabolite of Aivlosin also has antimicrobial activity. Absorption Atsuko and Kido (1988) recorded absorption, distribution and excretion of Aivlosin dosed at 50 mg/kg in pigs. Graph 1: Absorption of Aivlosin T max was 2 hours and the biological half-life was 2.21 hours. Graph 3: Comparison of Aivlosin and 3-AT plasma levels The kinetics of the two substances differ, the C max was 3.06 g/ml for Aivlosin and 5.4 g/ml for 3-AT. The T max for Aivlosin was 2 hours but 4 hours for 3-AT. Combined, the figures match quite well the microbiological assay that was achieved previously. This demonstrates the potential significance of the microbiological effect of the metabolite and the speed of its formation. In a further study the blood levels of Aivlosin and tylosin were compared following a 50 mg/kg bodyweight dose given orally. 9

Technical Monograph 01/04/2005 12:01 Page 10 Distribution Distribution of Aivlosin has been reported by Atsuko and Kido (1988). Aivlosin was given orally at a dose of 50 mg/kg. Tissue Conc. µg/ml/g Ratio to serum Serum 4.8 1 Lung 8.0 1.7 Liver 23.8 5.0 Bile 140.1 29.2 Kidney 17.0 3.5 Small intestine 15.4 3.2 Table 1: Distribution of Aivlosin in tissues 2 hours after dosing Metabolism In vitro metabolism of Aivlosin in liver homogenate has been reported by Iguchi et al (1988). A known amount of Aivlosin was added to the liver homogenate and incubated for 0.25, 0.5, 1, 2 and 4 hours at 37 C. The recovery of Aivlosin was 90%. Aivlosin was rapidly converted to 3-AT in 4 hours. Hours Aivlosin g/g 3-AT g/g Total g/g 0 10.9 0 10.9 0.25 8.1 2.7 10.8 0.5 5.4 5.1 10.5 1 2.2 8.0 10.2 2 0.4 10.0 10.4 4 <0.1 10.1 10.1 Another study by Iguchi et al (1988) compares the distribution of Aivlosin and the major metabolite 3-AT after oral dosing at 50 mg/kg bodyweight Tissue Aivlosin µ/g/ml 3-AT µ/g/ml Serum 1.77 3.53 Lung 4.07 3.63 Liver 2.27 19.9 Bile 12.6 117.8 Kidney 2.63 12.8 Small intestine 2.03 12.4 Table 2: Distribution of Aivlosin and 3-AT in tissues 2 hours after dosing Table 3: Metabolism of Aivlosin in swine liver homogenate. Aivlosin is rapidly metabolized in the liver and excreted in the bile. 3-AT is a major metabolite which is also microbiological active. Eight other metabolites have been found in urine and faeces, but would appear to have a relatively minor microbiological activity. Excretion The major route of elimination was via the faeces (averaged 71.6%), with excretion via the urine averaging 3.35% Interestingly the Aivlosin lung concentration was 2.3 times the Aivlosin serum concentration, yet the 3-AT concentrations were similar. 10

Technical Monograph 01/04/2005 12:01 Page 11 Aivlosin like other macrolides has shown to be active against mycoplasma, Gram-positive microorganisms such as Staphylococci, Streptococci, Gram-negative anaerobes such as Brachyspira, Campylobacter and Lawsonia and also against Chlamydia (Prescott, 2000) but is naturally not active against the enterobacteriaceae such as E.coli, Salmonella and Pseudomonas. Mycoplasma hyopneumoniae susceptibility testing Aivlosin is very active against Mycoplasma hyopneumoniae. Results from many studies show that the overall MIC 90 is as low as 0.06 µg/ml. Furthermore the MMC s (minimum mycoplasmacidal concentration) were found to be very close to the MIC which is unusual for Mycoplasma. This means that Aivlosin at concentrations similar to MIC is not only a bacteriostatic antibiotic but also has bactericidal activity. Antimicrobial spectrum of activity The killing activity is both concentration and time dependent and because Mycoplasmas are slow growing organisms, it is suggested that a 24-hour period with concentrations in the lungs above or similar to MIC is needed. At the recommended dose rate of 2.125 mg/kg bodyweight the Aivlosin concentration in the lungs is 0.06-0.066 µg/g. If the lung concentrations are assumed to be steady over a 24-hour period and the MIC 90 for European strains is 0.06 µg/ml the activity period for Aivlosin is 24 hours. But if the MIC is only 0.01 µg/ml as shown in many studies the concentration in the lungs will be above MIC for 144 hours. This is remarkably high, and highlights the sensitivity of Mycoplasma hyopneumoniae to Aivlosin. Antimicrobial Matsubara Yamamoto Inamoto Ayling (UK) Windsor range µg/ml MIC 90 µg/ml MIC 90 µg/ml MIC 90 µg/ml MIC 90 µg/ml Aivlosin 0.006 0.05 <0.013 <0.0125 0.11 0.01 tylosin 0.05 0.2 0.1 0.1 0.5 - lincomycin - 0.2 0.1 - - chlortetracycline - - >100 - - tiamulin - - 0.025 - - Table 4: Sensitivity testing of Mycoplasma hyopneumoniae. (MIC: minimum inhibitory concentration). 11

Technical Monograph 01/04/2005 12:01 Page 12 Toxicology A complete toxicological evaluation has been conducted with acetylisovaleryltylosin tartrate. From the acute and chronic toxicity tests in mice and rats, Aivlosin appears to have a wide margin of safety between the proposed usage level (2.125 mg/kg) and toxic levels in laboratory species. LD 50 in mice is 790 mg/kg bodyweight and toxicological effects are not noted until a doses of 1000 mg/kg is reached. In rats LD 50 is >2900 mg/kg. Aivlosin s metabolites and breakdown products all have much higher LD 50 s than the parent compound. There were no major effects on fertility, gestation, litter or individual birth-weights at doses of 21-113 mg/kg bodyweight in rats and on gestation below 200 mg/kg in mice and no teratogenic effects noted in rats even up to 2000 mg/kg, although maternal toxicity was noted. Conclusion: Aivlosin appears to be relatively safe in breeding animals and does not appear to exert a teratogenic effect. Tolerance studies in pigs This study measured the tolerance of pigs to an orally administered Aivlosin-medicated feed for 7 consecutive days. Each treatment group contained 8 pigs 10 weeks of age. The treatment groups were fed an Aivlosin-medicated feed of nominal dose rates of 0, 50 (normal dose), 150 (3X), and 250 ppm (5X). Daily Rates Multiple of target dose Observed results (ppm) (based on 50 ppm) 0 0X No adverse reaction 50 1X No adverse reaction 150 3X No adverse reaction 250 5X No adverse reaction Table 5: Results of Pig Tolerance Study Feeding Even at 5 times the normal dosage Aivlosin is safe. Additionally no appetite or palatability problems were noted. In addition to this, a recent report by King (2003) looked at levels of 500 ppm (10X normal dosing) Aivlosin in feed in comparison with an untreated control. 6 pigs weighing 15-20 kg were given the medicated feed for 14 days (twice the normal duration of treatment) and then the diets were replaced with a basal diet for a further 7 days. No adverse reactions were present during the 3 week period (clinical observation, body weight recordings, haematology and biochemistry in blood samples, necropsy on day 21). It was concluded that there was no effect on health associated with feeding Aivlosin at 500 ppm for 14 consecutive days and there was no intolerance to the test diet. In fact the overall intake was marginally higher in the treated group. Withdrawal period The withdrawal period is based on Acceptable Daily Intake (ADI) and Maximum Residue Level (MRL). ADI is 1.02 µg/kg bodyweight. MRL is 50 µg/kg muscle, skin and fat, liver and kidney respectively. After one day Aivlosin is no longer detectable in any tissues. Withdrawal period for meat and offal: 2 days. 12

Technical Monograph 01/04/2005 12:02 Page 13 Environmental Considerations All antimicrobials currently available for treatment and prevention of Enzootic pneumonia have the potential of causing environmental damage, depending on the exposure level. Environmental exposure is dependent upon a variety of factors including: the amount of active ingredient(s) used for treatment, the frequency of use, the biological activity of the active ingredient, the biological activity of any metabolites or degradation products, the degree of deposition and the sensitivity of the surrounding biota. A complete set of trials has been made, including: Soil microflora, Carbon transformation, Koc, Kow, Degradation, Earthworm toxicity, Phytotoxicity, Daphnia acute toxicity, Dissociation constant, UV-visible absorption, Melting point, Solubility, Hydrolysis. Conclusion: No concerns were raised in any of the studies and it was concluded that Aivlosin, if used as recommended, was expected to be of low risk to the environment. 13

Technical Monograph 01/04/2005 12:02 Page 14 Efficacy Dose Titration and Dose Confirmation in pigs A dose titration trial for the prevention of Enzootic Pneumonia was carried out by Takamura (1987) testing the efficacy of 0, 10, 20, 50 and 100 ppm of Aivlosin against 110 ppm of tylosin. 10 days old piglets from a Specific Pathogen Free (SPF) herd were infected with an isolate of M. hyopneumoniae intranasally. They were given medicated feed for seven days (13-19 days after infection) and sacrificed 4 weeks after infection. Body weights were measured weekly, blood was taken to observe increases in antibody to M. hyopneumoniae and they were checked clinically on a daily basis. At sacrifice, the lung lesions were scored and tested for recovery of M. hyopneumoniae. Treatment No of pigs Lung lesion (ppm) score(%) Uninfected control 4 0 Infected control 5 16.1 Tylosin 110 5 3.5 Aivlosin 10 5 8.5 Aivlosin 20 5 2.2 Aivlosin 50 6 2.3 Aivlosin 100 4 1.2 Table 6: Results of dose titration study against tylosin Significant reductions in lung lesion scores were made with Aivlosin 20, 50 and 100 ppm and tylosin 110 ppm all given in feed for seven days in comparison with infected untreated controls (p< 0.01). There is a good dose titration effect with levels of Aivlosin at 20, 50 and 100 ppm showing significant improvements over the negative control and numerical improvements over tylosin 110 ppm the positive control. Graph 4: Reduction in lung lesion scores dose titration effect In another experimental study, (Burrows et al. 2002) examined Aivlosin 50 ppm for prevention and Aivlosin 50 ppm and 100 ppm for treatment of Enzootic Pneumoniae (E.P.) against an untreated control and valnemulin 200 ppm (Econor R, Novartis) as a positive control. Sixty 4-5 week old pigs were allocated to 5 groups of 12 pigs, which were infected intranasally with a lung homogenate containing M. hyopneumoniae and a further 9 pigs were uninfected and acted as controls. Treatment in the prevention part of the study started the day before infection. They were infected on two consecutive days and the medication was withdrawn after seven days. For the treatment part medication was started 4 days after the last infection and continued for seven days. All pigs were sacrificed 19-20 days post infection. The pigs were inspected clinically daily for signs of respiratory disease, feed intake pre-treatment, during treatment and post-treatment was recorded, body weights were recorded at day -14, -6, -1, 0, 7 and 13. At the end of the trial the pigs were sacrificed and the lungs removed, weighed, scored for lesions and the challenge controls were cultured for M. hyopneumoniae. The results were compared statistically using an analysis of variance, with the lung lesion percentage as the primary criterion. 14

Technical Monograph 01/04/2005 12:02 Page 15 Coughing was observed as early as 4 days after infection in the infected untreated controls and subsequently all of the challenged groups but not in the uninfected controls. Growth rate in the Aivlosin 50 ppm group was statistically better days -14 to 14, the Food Conversion Efficiency (FCE) was numerically better during the medication period but similar over the whole trial period. The drug intake was estimated at 2.54 mg/kg bodyweight. similar over the whole trial period but with the treated groups showing minor numerical improvements. The Aivlosin 50 ppm group showed a highly significant (**p<0.01) reduction, (see table: 8) in lung lesion score (-51%) and the other treatments (Aivlosin 100 ppm and valnemulin 200 ppm) were just short of the 5% level of significance. In lung weight as a percentage of bodyweight the Aivlosin 100 ppm showed a statistically Treatment No. Weight gain (kg) Weight gain (kg) FCE Lung lesion Lung weight (ppm) of pigs day 6to0 day 14to14 day 6to1 score(%) as % of bodyweight Uninfected control 9 3.0 12.4-1.3 1.10 Infected control 1 1.4 9.4 1.71 26.3 1.77 Aivlosin 50 12 2.2 11.4 2.52 14.4 1.57 Table 7: Prevention trial results with Aivlosin 50 ppm dose confirmation The lung lesion score was significantly (p<0.05) reduced (-45%) as well as the lung weightasa percentage of bodyweight confirming Aivlosin 50 ppm s efficacy in preventing the development of lung lesions associated with EnzooticPneumonia. In the treatment part, significant (*p<0.01) weight gains were shown by all of the treatment groups during the trial period over the infected untreated controls (see table 8). FCE was generally significant improvement, (see table: 8) and the Aivlosin 50 ppm and valnemulin 200 ppm were just short of significance at the 5% level. In this study there were no major improvements seen with Aivlosin at 100 ppm over 50 ppm especially with regard to lesion score reduction, therefore the dosage of Aivlosin at 50 ppm administered in the feed for 7 days was deemed the appropriate effective dose. Treatment No. Weight gain (kg) Weight gain (kg) FCE Lung lesion Lung weight (ppm) of pigs day 0-7 day -14-14 day 14-14 score(%) as % of bodyweight Uninfected control 9 2.6 12.4 1.78 1.3 1.1 Infected control 12 2.7 9.4 1.76 26.3 1.77 Aivlosin 50 12 2.6 11.2* 1.60 12.9** 1.56 Aivlosin 100 12 3.3 11.9* 1.63 17.9 1.53*** Valnemulin 200 12 2.8 11.5* 1.59 16.2 1.55 Table 8: Result of treatment with Aivlosin 50 & 100ppm and valnemulin 200ppm 15

Technical Monograph 01/04/2005 12:02 Page 16 European field trials Clinical trials were carried out in Ireland looking at Aivlosin 50 ppm given in feed for 7 days for the prevention and treatment of Enzootic Pneumonia in comparison with a positive control tylosin 100 ppm given for 21 days. Kavanagh (2001) carried out a trial using 203 growing pigs, weight range 25-60 kg to GCPV standards. Lung examinations and serological tests on a previous group of slaughter pigs had 79% incidence of Enzootic Pneumonia Lesions, an average lung lesion score of 12.1% and over 87% were serologically positive for M. hyopneumoniae using an ELISA test, demonstrating a high level of infection. The pigs were arranged in paired pens (8 pens altogether) with matching numbers of pigs, sexes and total pen weight in 3 separate rooms. Numbers of pigs per pen ranged from 14-30 depending on pen size. Medicated feed was given to the Aivlosin group for 1 week and then un-medicated feed for 2 weeks and the tylosin group was treated for 3 weeks. The feed intake was recorded for this period only. Both groups were then given unmedicated feed for a further 3 weeks. The pigs were weighed at the start of the trial, on day 21 and day 42. The pigs were checked clinically during the trial and scored, according to the severity of the condition, the temperatures of sick pigs were recorded and if elevated they were treated by injection (benzyl penicillin) and any dead pigs were necropsied during days 0-21. The pigs were sent for slaughter over 2 weeks and approximately half of the lungs were examined and scored. A further study was carried out in the UK on 100 finishing pigs using Valnemulin as a positive control, against Aivlosin (Report: ECO/UK/003/A - Appendix 38, Part IV). Because of the size of the animals (weight range 44-72kgs) and their low feed intake (approx. 3% bodyweight per day), the level of Aivlosin and Valnemulin were increased by 50% in the feed to achieve the target dosage rate for Aivlosin (2.125mg acetylisovaleryltylosin per kg bodyweight) and that for Valnemulin (10mg/kg bodyweight). The herd had their lungs examined at slaughter prior to the trial and the average score was 5.5% lung consolidation. (M. hyopneumoniae) was both cultured and detected by PCR. One hundred pigs were fed Aivlosin 64ppm or Valnemulin 300ppm for 7 days. They were then given unmedicated feed for a further 14 days whilst clincal observations/examinations were made. The trial continued with plain feed until day 43. The pigs were weighed on day 0, 7, 21 and 43. Significant differences were seen for Food Conversion Efficiency (FCE) over trial days 0-43, where Aivlosin had an FCE of 2.15 and Valnemulin of 2.39. The individual pen results were almost identical making the SE very small. This represents a marked numercial improvement of 10%. Average lung lesion scores in the Aivlosin 64ppm group were 43% lower than in the valnemulin group (3.45% vs. 6.04% lung consolidation.overall the Aivlosin group showed numercial improvements over valnemulin in weight gain during medication period days 0-7 (31%), again during the post-medication phase, days 21-43 (10.6%) and overall days 0-43 (9.4%). Similarly there were numerical improvements in FCE for these periods with a satistically significant improvement for days 0-43 (10%). The MICs of isolates pre-trial and post treatment were similar, showing no development of resistance to Aivlosin. In summary, Aivlosin gave numerical improvements in liveweight gain and lung scores and also, a statistically highly significant improvement in FCE over the positive control Valnemulin during the 43 day trial period. Aivlosin 50 ppm tylosin 100 ppm Start weight (kg) day 0 42.74 43.21 Weight (kg) day 21 57.22 56.55 Weight (kg) day 42 73.11 72.22 Weight gain (kg)day 0-21 14.48 13.34 Weight gain (kg)day 21-42 15.89 15.67 Weight gain (kg)day 0-42 30.37 29.01 Lung scores 8.67 9.47 Respiratory disease score grade 2 7 4 Respiratory disease score grade 3 2 4 Table 9: Field trial results of Aivlosin 50 ppm for 7 days versus tylosin 100 ppm for 21 days Aivlosin Valnemulin Start weight (kg) day 0 57.53 60.58 Weight gain (kg) day 0-7 6.42 4.90 Weight gain (kg) day 21-43 24.03 21.71 Weight gain (kg)day 0-43 44.22 40.41 Lung scores 3.45 6.04 FCE day 0-7 2.01 2.28 FCE day 7-21 2.23 2.31 FCE day 21-43 2.15 2.48 FCE day 0-43 2.15** 2.39 Feed intake day 0-7 (kg) 12.75* 11.00 Table 10: Field trail results of Aivlosin 64ppm for 7 days versus Valnemulin 300ppm for 7 days in the prevention and treatment of Enzootic Pneumonia*p<0.05;**p<0.01 16

Technical Monograph 01/04/2005 12:02 Page 17 References Aitken, I.A., Morgan,J.H., Dalziel, R., Burch, D.G.S., Ripley, P.H. (1999) Comparative in-vitro activity of valnemulin against bacterial pathogens. Vet. Rec., 144,128. Ayling (2000) MIC and MMC for Aivlosin against M. hyo field isolates from UK and The Netherlands. Burrows, M.R., Morgan, J.H., Tasker, J.B., Martin, M. (2002) In-feed Aivlosin for control of experimental enzootic pneumonia in pigs. Proc. 17th IPVS 2002. Diarra, M.S., Malouin, F., Jacques, M. (1999) Post-antibiotic and physiological effects of tilmicosin, tylosin and apramycin at subminimal and supra-inhibitory concentrations on some swine and bovine respiratory tract pathogens. Int. Journ. of Antimicrobial Agents, 12, 3, 229-237. Friis, N.F., Szancer,J. (1994) Sensitivity of Danish field isolates of Mycoplasma hyosynoviae and Mycoplasma hyopneumoniae to antimicrobial agents. Proc. 13th IPVS 1994. Ibayashi, T., Okada, M., Ando, N. (1994) Pulmonal and Tracheal concentrations of lincomycin, tylosin, acetylisovaleryltylosin and tiamulin, given as feed additives in swine. Proc. 13th IPVS 1994. Inamoto, T., Takahashi, H., Yamamoto, K., Nakai, Y, Ogimoto, K. (1994) Antibiotic susceptibility of Mycoplasma hyopneumoniae isolated from swine. Journ. of Veterinary Medical Science, 56, 2, 393-394. Kavanagh, 2001 (Report: ECO/MHYO/1/NK/015 - Appendix 36, Part IV Matsubara, Nishi (1985) Mycoplasma hyopneumoniae sensitivity for Aivlosin, tylosin, erythromycin and leucomycin. Undated report 1985. Morgan, J.H., Aitken, I.A., Collins, P., Burch, D.G.S. (1996) Prevention of experimentally-induced enzootic pneumonia in pigs using the novel compound SDZ PMD 296 (Econor-Sandoz Ltd.). Proc. 14th IPVS 1996. Nielsen, P. (1997) The influence of feed on the oral bioavailability of antibiotics/chemotherapeutics in pigs. Journ. of Veterinary Pharmacology and Therapeutics, 20 (Supplement 1), 30-31. Okamoto, R., Tsuchiya, M., Nomura, H., Iguchi, H., Kiyoshima, K., Hori, S., Inui, T. (1980) Biological properties of new acyl derivatives of tylosin. Journ. of Antibiotics, 33, 11, 1309-1315. Prescott, J.F. (2000) Section on macrolides erythromycin and tylosin. Antimicrobial Therapy, 3th edition. Editors: prescot, J.F., Baggot, J.D., Walker, R.D. Iowa State University Press, Ames, Iowa, USA, pp 237-247. Thongkamkoon, P., Worarach, A., Kortheerakul, K., Chamsong, N. (2002) In-vitro susceptibility of Mycoplasma hyopneumoniae to antimicrobial agents. Proc. 17th IPVS 2002. Yamamoto, K. (1988) Aivlosin, tylosin, josamycin and lincomycin sensitivity of 30 field strains of M. hyopneumoniae. Windsor (2002) Aivlosin MIC-determination in M. hyo field strains from UK and Germany. 17

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Technical Monograph 01/04/2005 12:03 Page 20 AIVLOSIN 42.5 mg/g Premix Presentation Aivlosin 42.5 mg/g premix for medicated feeding stuff for pigs. Contains Acetylisovaleryltylosin 42.5 mg/g (as Acetylisovaleryltylosin tartrate) in a magnesium trisilicate, wheat feed flour carrier. Uses Treatment and prevention of Swine Enzootic Pneumonia caused by susceptible strains of Mycoplasma hyopneumoniae. At the recommended dose, lung lesions and weight loss are reduced but infection with Mycoplasma hyopneumoniae is not eliminated. Dosage and administration In-feed use. The dosage is 2.125 mg acetylisovaleryltylosin per kg bodyweight per day. This is normally achieved by incorporating Aivlosin at a level of 1 kg/tonne feed to provide 42.5 mg acetylisovaleryltylosin per kg feed, assuming that the pig eats 5 % of its bodyweight in feed. The medicated feed should be fed as the sole ration for 7 days. In older pigs, or in pigs with reduced appetite, or on restricted feed intake, inclusion levels may need to be increased to achieve target dosage. Acute cases and severely diseased pigs with reduced food or water intake should be treated with a suitable injectable product. Secondary infection by organisms such as Pasteurella multocida and Actinobacillus pleuropneumoniae may complicate Enzootic Pneumonia and require specific medication. Mixing Instructions: A horizontal ribbon mixer should be used to incorporate the product into feeding stuff. It is recommended that Aivlosin is first mixed with 10 kg of the feeding stuff, followed by the rest of the feeding stuff and mixed well. Medicated feed may then be pelleted. Pelleting conditions involve preconditioning ingredients with steam for 5 minutes and pelleting at 70 C under normal conditions.interaction with other medicinal products and other forms of interaction: None Known Contra-indications, warnings etc Contra-indications: None. Undesirable Effects: None known. If you notice any side effects, please inform your veterinary surgeon. Withdrawal Period: Meat and offal: Two days. For animal treatment only. Special Warning(S): When mixing the veterinary medicinal product and handling the medicated feed, direct contact with eyes, skin and mucous membranes should be avoided. Personal protective equipment should be worn when mixing the veterinary medicinal product or handling the medicated feed: overalls, impervious gloves and either a disposable half-mask respirator conforming to European Standard EN 149 or a non-disposable respirator conforming to European Standard EN 140, with a filter to European Standard EN 143. Wash contaminated skin. In case of accidental ingestion seek medical advice immediately and show the label to the physician. Because Aivlosin has been shown to cause hypersensitivity reactions in laboratory animals people with known hypersensitivity to acetylisovaleryltylosin tartrate should avoid any contact with the product. The safety of Aivlosin during pregnancy and lactation has not been established in pigs. Use only in accordance with risk/benefit assessment by the responsible veterinarian. Studies in laboratory animals have not produced any evidence of teratogenicity. Maternal toxicity in rodents has been observed at doses of 400 mg acetylisovaleryltylosin per kg bodyweight and above. In mice, a slight reduction in the foetal bodyweight was seen at doses causing maternal toxicity. Special Precautions For The Disposal Of Unused Veterinary Medicinal Products Or Waste Materials Derived From Such Veterinary Medicinal Products, If Appropriate: Any unused veterinary medicinal product or waste materials derived from such veterinary medicinal products should be disposed of in accordance with local requirements. Conditions Or Restrictions Regarding Supply And Use: Veterinary medicinal product subject to prescription. Consideration should be given to official guidance on the incorporation of medicated premixes in final feed. Pharmaceutical precautions Keep out of the reach and sight of children. Do not store above 25 C. Keep the container tightly closed. Store in the original container. Do not use after the expiry date stated on the label. Shelf life after incorporation into meal or pelleted feed: 1 month. Legal category Packaging quantities MFS 20kg Number in the Community Register of Medicinal Products: EU/2/04/044/001 Distributor (UK) Schering-Plough Animal Health, Breakspear Road South, Harefield, Uxbridge, Middlesex, UB9 6LS, United Kingdom. Date of Preparation: November 2004 SP001019 Schering-Plough Animal Health Breakspear Road South Harefield Uxbridge Middlesex UB9 6LS Tel: +44 (0) 1895 626000 Fax: +44 (0) 1895 672429