SCIENTIFIC DISCUSSION Cylap Pfizer Limited

I. INTRODUCTION

Cylap is a vaccine for the prevention of rabbit haemorrhagic disease (RHD) in rabbits. This disease, which was first identified in China in 1984, is characterised by haemorrhagic lesions, particularly in the liver and lung. It affects mostly rabbits over three months of age and starts with depression and fever. It is fatal in 90% of cases.

RHD is a comparatively new disease and at the time of its first appearance, there was no means of preventing or treating it. Because it takes a considerable length of time to develop and comprehensively test a new vaccine, the VMD has the authority to issue a provisional marketing authorisation for a new product which has not undergone the full range of tests, provided that the quality of the product is adequate, that the major safety questions have been answered and that there is some evidence that the product will work. Cylap was first approved in this way, but the full range of tests have subsequently been completed and some changes made to the original composition of the product. Thus, the information submitted to the VMD in support of the application for a full marketing authorisation includes both the original data (generated on the earlier formulation) and new data demonstrating the quality, safety and efficacy of the new formulation.

Cylap contains inactivated RHD virus and is administered by subcutaneous injection. The initial vaccination programme consists of a single injection of 1 ml, and an annual booster is recommended.

II. QUALITY ASPECTS

Product Development and Composition Cylap has been developed for the active immunisation* of susceptible rabbits against rabbit haemorrhagic disease. It is intended to prevent clinical signs of disease, and mortality.

As well as the inactivated RHD virus, the product also contains an adjuvant** comprising Marcol 82, Eumulgin M-8 and Montanide 80. Thiomersal is included in the formulation, as this substance has been shown effective in controlling microbial contamination which might be introduced during removal of doses. The product is presented in saline solution in clear Type 1 glass vials with red Type 1 bromobutyl rubber stoppers and aluminium overseals. Two sizes of vial are used – 5 ml vials containing enough vaccine for one rabbit, and 20 ml vials containing enough vaccine for 10 rabbits.

Active Substance There is no pharmacopoeial monograph for the active substance, rabbit haemorrhagic disease virus (RHDV). It was first obtained from a rabbit which had died from RHD in 1989 and has since been replicated and stored frozen. The company has provided a detailed description of the production process. Despite extensive investigations it has not been possible to identify a means of producing the virus other than by collecting relevant organs from rabbits infected with the virus. The virus is separated from the organs by homogenisation and centrifugation, prior to

* This means that it will cause the vaccinated rabbits to produce antibodies to the viral antigens. ** An adjuvant is a substance (or mixture of substances) that is administered with the objective of enhancing the immune response to the organism(s).

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the addition of suitable antibiotics to ensure no growth of bacteria. After mixing, the virus is inactivated, filtered and the preservative thiomersal is added.

The tests which are carried out to ensure that the correct virus has been produced and that it is not contaminated with other organisms or agents have been described and justified. The potency of the virus produced is assayed so that the appropriate amount can be added to the finished product. The active substance may be stored for up to one week prior to incorporation into the product.

Other Ingredients Other ingredients comply with the relevant pharmacopoeial monographs where these exist. In other cases, the company has identified the source of each ingredient, explained how its quality is controlled and provided relevant certificates of analysis.

Packaging materials The Type 1 glass vials and rubber stoppers comply with the requirements of the European Pharmacopoeia, and are sterilised and depyrogenated*. There is no pharmacopoeial monograph for the aluminium overseals as these do not come into contact with the product.

Manufacture of the Finished Product The product is manufactured in accordance with Good Manaufacturing Practice and, where applicable, conditions, equipment and materials are sterile. The components of the adjuvant are mixed together at an appropriate temperature, with stirring, and these are added to the virus preparation, again with stirring. The resulting emulsion is held for up to three weeks prior to filling into vials to enable relevant quality checks to be made.

In-process control tests have been described in detail, and include tests for content of RHDV, adequacy of the inactivation process, sterility, and absence of contamination with other viruses, particularly those likely to cause disease in rabbits.

All components of the product have been demonstrated to comply with relevant guidance on minimising the risk of transmitting animal spongiform encephalopathy agents via veterinary medicines.

Finished Product Quality Control Standard tests (visual examination, pH, sterility, extraneous agents, etc.) are routinely applied to the finished product. The content of RHDV is determined using a haemagglutination inhibition (HI) assay, which is a well-established way of quantifying viruses which have the property of causing red blood cells to agglutinate (clump together). The content of preservative and adjuvant is also determined by means of validated assays.

Results of the analysis of three production batches have demonstrated that a product of consistent quality can be produced.

* This means they are treated to get rid of bacterial endotoxins which, if present in the vials, could have adverse effects on rabbits following vaccination.

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Stability of the product

Active substance The production criteria require that the bulk virus is prepared not more than one week prior to production of the product, and data have been provided to show that it is stable for this period.

Finished Product The company provided data on four batches packed in different sizes of container stored refrigerated, and these data showed that the virus potency, the quantity of adjuvant and preservative, and the efficacy of the preservative remain satisfactory over a period of twenty four months.

In-Use No data are available on the stability of the product once the first dose has been removed from the vial. Therefore the shortest practicable in-use shelf life has been agreed – three hours.

CONCLUSIONS ON QUALITY The data provided by the company included satisfactory descriptions of the production and quality control procedures, including appropriate diagrams. The in-process and finished product tests ensure an efficacious, safe and consistent product. The stability data provided show that a shelf-life of 24 months for the product when stored at 2 – 8°C, protected from light is justified.

III. SAFETY ASPECTS

Introduction The vaccine is intended for use in rabbits from ten weeks of age. A primary course consists of one subcutaneous injection of 1 ml, and an annual booster vaccination is recommended. Because an earlier version of the product had been given a provisional marketing authorisation, the safety data available include data on this earlier version as well as new data relating to the current product.

Laboratory Tests A series of studies using the original formulation, investigated the safety of the product in three- month old New Zealand rabbits given the recommended dose of vaccine, with the dose being repeated after 30 days, 2½ months or 3 months. No local reactions were observed in any of these rabbits and their temperatures remained normal throughout the studies. These studies demonstrated that a single dose of the old formulation was safe for three-month old New Zealand rabbits; a second dose given after an interval of between one and three months was also safe for these animals. It should be noted that the old formulation contained a higher content of virus, and therefore the amount of virus received by the rabbits in these studies was higher than that currently recommended. On the other hand, there was a slight difference in the proportion of the adjuvant components, so these data are only partially relevant to the new formulation.

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A pivotal new study, conducted in accordance with Good Laboratory Practice, investigated the safety of the new formulation of Cylap in three different breeds of rabbit: dwarf lop, crossbred dwarf brown and New Zealand. The product was administered to ten-week old animals by subcutaneous injection at double the recommended dose, and a second injection was given 28 days later. After the first vaccination, all breeds showed some incidence of raised temperature and reactions (redness and swelling) at the site of injection, but the New Zealand rabbits appeared to be more susceptible to these reactions than the other breeds. The incidence of reactions tended to be greater after the second injection in all breeds. After the second injection there were sometimes additional effects, such as lameness and hair loss. All the reactions resolved spontaneously.

A further new study was also conducted in which a group of rabbits without antibodies to RHDV received a double dose of maximum potency vaccine by the recommended route of administration. Rabbits were examined daily from the day before vaccination until 14 days after. Half of the rabbits had slightly raised temperature, which lasted from one to three days. Redness at the injection site was noted in some animals, again lasting up to three days. Two per cent of rabbits developed palpable swellings of less than 2 cm diameter which lasted for one day. This study demonstrated the safety of Cylap at maximum potency, even when administered at twice the recommended dose.

In August 2009, the variation was submitted to add a safety warning advising of the potential side effect of lameness following administration of the product. The following statement was included on the SPC and package leaflet:

‘’Following vaccination animals may exhibit transient forelimb lameness which normally resolves within 48 hours’’

Another study which investigated the effects of a five times overdose of the original formulation in three-month old New Zealand rabbits identified no adverse effects. Similarly, no adverse effects were noted in adults or young, when pregnant Californian does were vaccinated with a two-fold dose of the original formulation.

Field Studies Three field trials were conducted in which a total of 119 rabbits of various ages were vaccinated, 26 of which received a second injection 30 days after the first. Adverse reactions to the vaccine were limited to a short period of anorexia and some slight swellings which disappeared spontaneously. The vaccine was tolerated well even in pregnant does, although there appeared to be a slight reduction in the number of live young, with a similar increase in the number of stillborn pups. There was also a slight reduction in the number of pups weaned.

Pharmacovigilance Data The product has been used in various provinces in Spain, and has been shown to have a low incidence of adverse effects and an 89% reduction in mortality from RHD. Pharmacovigilance data from use of the product under its provisional marketing authorisation in the UK also indicated an acceptable incidence of adverse reactions.

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Environmental Safety The company considered the environmental impact of Cylap in accordance with the relevant European guideline on environmental risk assessment. The conclusion of this was that the route of injection, and individual handling of small animals means that environmental contamination is unlikely. Furthermore, the product is an inactivated vaccine which would not cause a threat to the environment if it were excreted by vaccinated rabbits.

CONCLUSIONS ON SAFETY AND RESIDUES

The safety part of the company’s dossier fulfilled the legislative requirements, and provided adequate information to assess the safety of the product.

The laboratory tests and field trials demonstrated the overall safety of the vaccine for rabbits of 2½ months of age, but it was noted that local reactions may occur in rabbits; these are explained in the summary of product characteristics. The data also indicated that, with care, the product may be used in pregnant does.

A user risk assessment indicates that the main risk to people administering the product to rabbits is the possibility of acidentally injecting themselves. The possible outcome of injecting a product containing a mineral oil is well-documented and a detailed warning on the appropriate action to take is included in the summary of product characteristics and on the information provided with the product.

None of the ingredients of Cylap are such as would cause unacceptable residues in meat from treated rabbits. There is no need for a withdrawal period* and no consumer safety concerns.

An environmental risk assessment indicates that the risk to the environment from the use of Cylap in rabbits in minimal. Appropriate disposal advice has been agreed.

IV. EFFICACY ASPECTS

Introduction The vaccine is intended for use in rabbits from ten weeks of age. A primary course consists of one subcutaneous injection of 1 ml, and an annual booster vaccination is recommended.

Laboratory Trials In a study designed to identify the concentration of virus that is needed to induce the development of protective levels of antibody, three groups of New Zealand rabbits aged between 2½ and 3 months and with no antibodies to RHDV at the start of the trial were vaccinated with different amounts of vaccine. A further group of rabbits was left unvaccinated for comparative purposes. Twenty one days after vaccination all the rabbits were exposed to a virulent form of RHDV and were then observed for fourteen days. All the unvaccinated rabbits died whereas 70% of the rabbits given the lowest dose of vaccine survived, and all the rabbits

* A withdrawal period is the interval which must elapse between the time when an animal is treated with a veterinary medicine and the time when meat from that animal may be used for human consumption.

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given the middle or high dose survived. This demonstrated the minimum amount of vaccine virus that is needed for efficacy, and this is specified in the production procedures and stated in the summary of product characteristics.

Another study was specifically designed to demonstrate how long it took for immunity to develop following vaccination with the minimum effective amount of vaccine virus, and how long this immunity lasted. As before, some rabbits were left unvaccinated for comparison. All rabbits were exposed to virulent RHDV 366 days after vaccination. The unvaccinated rabbits all died, whereas almost 90% of the vacinated ones survived. Of the rabbits which died, it was noted that the level of antibodies to RHDV found in their bloodstream was significantly lower than in those which survived. The level of antibodies required for protection from RHDV was identified in this study. The study confirmed that such levels had developed by 21 days after vaccination, and that antibody levels remain elevated for up to one year.

Field Trials As already indicated in the section on safety aspects, three field trials have been conducted, in which a total of 119 rabbits of various ages were vaccinated, 26 of which received a second injection 30 days after the first. Some rabbits remained unvaccinated for comparison. Eight months after vaccination, some rabbits from each group (i.e. unvaccinated, vaccinated once or vaccinated twice) were exposed to virulent RHDV.

Vaccinated rabbits showed an antibody response that was similar to that observed in the laboratory trials except in the case of one animal which failed to develop antibodies. This was also the only animal to die when exposed to virulent RHDV. All unvaccinated rabbits died when exposed to virulent RHDV. This demonstrated the efficacy of the vaccine in typical field conditions.

There was little difference between the results obtained following one and two injections, and this indicated that a second dose was unnecessary for efficacy. This finding was consistent with that found in the early safety studies (discussed in the section on safety aspects), in which a second dose was not found necessary for the development of immunity, but did seem to increase the incidence of adverse reactions. The studies demonstrated that immunity lasts for at least eight months under field conditions. Vaccinated does passed on antibodies to their young during pregnancy, and these maternal antibodies persisted for 15 days after birth.

CONCLUSIONS ON EFFICACY ASPECTS

The company provided the reports of two new laboratory trials on the efficacy of Cylap in rabbits of 2½ months or more. The findings of these studies were supported by the results of the rather older field trials. It was considered unnecessary to conduct further field trials because of the reports of the product’s successful use in preventing disease caused by RHDV during the time it has been used under its provisional marketing authorisation.

A single dose of vaccine has been shown to prevent mortality caused by RHDV by three weeks after vaccination, and this protection lasts for one year.

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PART V. OVERALL CONCLUSION ON THE PRODUCT

The data submitted in the dossier demonstrate that when the product is used in accordance with the Summary of Product Characteristics, the risk benefit profile for the target species is favourable and the quality and safety of the product for man and the environment is acceptable.

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POST-AUTHORISATION ASSESSMENTS The SPC and package leaflet may be updated to include new information on the quality, safety and efficacy of the veterinary medicinal product. The current SPC is available on the Product Information Database of the Veterinary Medicines Directorate website. (WWW.GOV.UK/CHECK-ANIMAL-MEDICINE-LICENSED)

The post-authorisation assessment (PAA) contains information on significant changes which have been made after the original procedure which are important for the quality, safety or efficacy of the product.

The PAA for this product is available on the Product Information Database of the Veterinary Medicines Directorate website. (WWW.GOV.UK/CHECK-ANIMAL-MEDICINE-LICENSED)

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