1. Anthelmintic discovery and registration Nick Sangster Faculty of Veterinary Science University of Sydney

2. Worms

3. Objectives Be aware of research into new modes of action Explain screening for the discovery of AH Explain the development process for AH Detail the important steps in registration Understand the needs for trials Describe Good Clinical Practice Understand the components in the calculation of withholding periods

4. Requirements of anthelmintics Effective, safe, non toxic to consumer. Narrow spectrum, broad spectrum Stock Medicines Act 1989 and vets –Anyone can give stock medicines to non-food producing animals (we eat horses!) –Only vets can vary label directions in food producers but MUST supply (in writing) species, withholding, dose rate, frequency of treatment, manner of administration. –No one can use an unregistered stock medicine except under permit or order

5. Aspects of commercial anthelmintics Establishing a need/market Research Screening Refinement Development Marketing Technical support

6. Research - pharyngeal pumping L1 pumping video

7. 5-HT stimulates pharyngeal pumping in T.colubriformis 5HT 10- 5 M

8. ICC of H. contortus pharynx

9. Screening for anthelmintic activity Primary in vitro: –Screens are muscle contraction, larval death in vitro, failure to hatch, loss of motility. –False positives: pineapple juice kills worms in vitro but is not an anthelmintic –False negatives: some drugs are activated by host metabolism so are not active in vitro

10. Screening for anthelmintic activity Secondary: –Controlled tests in infected lab animals like mice Syphacia Hymenolepis Heligosomoides Tertiary: –Controlled tests in target animal eg. sheep with Haemonchus Field tests: –Trials with natural infections in target animal in real environments

11. Screening for anthelmintic activity High throughput screens and combinatorial chemistry –Decide on a target eg. 5-HT action –Proof of concept eg. physiological effects of 5-HT, different receptor compared with host –Clone, express receptor and develop assay adapted for HTS (multiwell plate, robotics, automated readout) –Screen 1 million compounds –Take ’hits’ and synthesise better analogues –Move to primary or secondary screens

12. Development and Registration Identified compound Pharmaceutical form Analytical techniques Teratoge icity Toxicity Stability, dose rate, spectrum, safety (rats, target), Presentation, field studies Efficacy studies Safety studies Stability studies

13. Why we need Registration Ensure quality of product Ensure safety of product Employ Good Clinical Practice Define efficacy requirements Consider toxicity to the environment Check for exposure of humans to drugs

14. Drug registration and VICH International harmonisation of registration Standard requirements for all countries Overseen by VICH (US, EU, Japan) Under the OIE Align technical requirements Can use trial & testing data from other countries to reduce costs Aim for consistency and safety

15. Registration of anthelmintics Stability testing Analytical procedures Bioequivalence Good Clinical Practice Specific guidelines available for: –General requirements –Swine –Equines –Canines –Bovines –Ovines –Caprines

16. Guidelines for Bovines Controlled tests –Egg counts –Worm counts Natural or induced infections –Minimum infection rates Claims for different species: adults, larvae Trials –Two dose confirmation studies –At least 6 animals per group –Animals >3 mo. old and groups randomised –Administration as for product –Significant difference between treated and control –Effectiveness >90%

17. Other guidelines, for example: Can register as “aids in control” Need 100% efficacy for some parasites like Echinococcus Claims for persistent action Claims for control or protection Equivalence requires blood level measurements in blood Local trails for food producing animals under different climatic conditions Monitor for adverse reactions.

18. Some aspects of “Good Clinical Practice” Accuracy, integrity and correctness of data Standard Operating Procedures for ALL activities Qualified persons to perform them Informed consent of owner Quality control of substances Independent audit Record adverse findings Record keeping and reporting

19. Withholding Withdrawal of animal products from human consumption after treatment Important for registration and food export Calculated on the persistence of chemicals in animal organs after treatment

20. Profile of a drug Log [A] Time absorption Partitioning (depot) metabolism elimination Efficacy driven by C max and time above threshold Dose rate driven by potency, bioavailability

21. Calculation of withholding period Only applies to food producing animals. Period before which it is unsafe (illegal) to slaughter for meat or use milk or eggs for human consumption because of chemical residues. For some compounds, there is NO withholding period, meaning the levels in the product never reach threshold levels. The withholding period is calculated by considering the drug dose rate, the expected toxic level for humans and the drug elimination kinetics from edible organs.

22. MRL Maximum Residue Level is the highest allowable level in food products. Chemicals are eliminated from the body of the animal over time, so it is a matter of waiting long enough. There is general harmonisation of levels ‘agreed’ to by Governments, EPA, Food Standards, WHO

23. Calculation of MRL for drug A for humans eating sheep meat Say the dose rate of A in sheep is 0.2 mg/kg. Let’s say the human ‘no observable effect level’ (NOEL) based on animal studies for A has been determined as 1.25 mg/kg body weight. The safety factor applied is generally 200. So, a the adult daily intake (ADI) is 6  g/kg, 200 fold less than the NOEL. A 60 kg adult can eat 360  g of A per day.

24. MRL A number of sheep are killed at intervals after treatment with A and the concentration of A is measured in various edible tissues. By looking at this data you can identify a time (12 +2 days) where eating the following every day you will, on average, just get your dose (=397  g) Organ[A]kg eaten Muscle 10  g/kg0.3 Fat10  g/kg0.05 Liver100  g/kg0.1 Kidney10  g/kg0.05

25. MRL and elimination profile of A to determine witholding period Log [A] Time MRL Witholding period 14 days