Should licking behavior be factored into topical product bioequivalence trials?

1. Should licking behavior be factored into topical product bioequivalence trials? AAVPT/ECVCP Workshop on Bioequivalence Issues in Veterinary Medicine Potomac, June 2010 A. Bousquet-Mélou & P.L. Toutain National Veterinary School, Toulouse, France

2. The beginning of a strange story • Comparison of PK profiles of doramectin and ivermectin pour-on formulation in cattle • 24 young beef cattle • 2 groups (parallel design)

3. AUC range (ng.day.mL-1) DORAMECTINE IVERMECTINE Doramectin vs. ivermectin:pour-on administration (500 µg/kg) 104-258 51-182 Highly variable drugs

4. Pour-on formulations of endectocides are HVD products • It is generally accepted that a drug or a drug product exhibiting within-subject variability of 30% or greater in the measure of the AUC or Cmax can be classified as a highly variable drug or drug product. • For this highly variable drug product, it is very difficult to demonstrate BE using standard design/sample size

5. Crossover or parallel design for PO formulations of endectocides? • Difficult for very long half-life (long washout period) Crossover design Parallel design • Within-subject variability • Between- plus within-subject variability • Attractive for very long half-life Parallel design / Within subject variability ? Cloned twin cattle

6. Parallel design with cloned cattle • Hypothesis : cloned twin cattle can be considered as replicates of the same animal • Question : Is a parallel design with pairs of cloned cattle statistically equivalent to a conventional crossover design ? Measure of the repeatability and reproducibility of ivermectin kinetics in 6 pairs of monozygotic twins

7. Pharmacokinetics homogeneity of twin cattle: IV ivermectin study • very high intra-pair reproducibility of ivermectin disposition (CV=5%) • Cloned cattle could be considered as replicates of the same animal for a BE trial PO are highly variable drug product

8. Inter-occasion variability for the PO formulation in a given subject it was concluded that the erratic absorption of IVM was not of genetic origin and that the intra-animal variability for PO formulations would unlikely be much lower than the inter-animal variability.

9. What is the origin of the large intra-individual variability of the drug exposure after a pour-on administration ? • There was a secondary question in this experiment: Is it possible to compute, using only faecal clearance (easily obtainable after any route of administration), the total amount of drug eliminated by the faeces (environmental issue) ? Total amount eliminated by faeces = Faecal clearance  AUC(plasma)

10. The faecal clearance of IVM after a PO administration Faecal clearance Plasma clearance Rate of drug eliminated in feces Plasma concentration Overall rate of drug elimination Plasma concentration >> Conceptually impossible if the plasma is the only driving force for ivermectin excretion into feces !

11. ! s.c. s.c. pour-on pour-on s.c. pour-on Faecal elimination of IVM: PO vs SQ Similar inconsistent results from observational study on higher fecal concentrations of ivermectin reported following pour-on application than SQ injection (Herd et al. 1996, Int. J. Parasitol. 26: 1087) Faeces Plasma IVM Metabolism

12. Origin of the high “faecal exposure” Licking behaviour Skin Faeces Plasma IVM Metabolism 12

13. Licking cattle Origin of the high “faecal exposure” • Hypothesis: licking behavior plays a role Licking is a form of grooming behavior directed to the skin. Cattle can lick themselves (self-licking) or another conspecific animal (mutual/social or allo-grooming). Cattle can lick most areas of their body and mutual licking (social grooming) has a social meaning, helping to establish and maintain social bonds within the herd. According to (Sato et al. 1991) heifers and steers had 15.0 and 15.2 social licking interactions per hour which lasted for 37.8 and 41.0 s.

14. LICKING (n=6) NON-LICKING (n=6) Licking vs. non-licking cattle: Pour-on Administration 6 pairs of twin cattle Starsky Hutch 14

15. Licking Non-licking Results : plasma disposition after pour-on administration • Half-life • lickers: 144 ± 3h (no flip-flop) • non lickers: 363 ± 16h (flip-flop) • Bioavailability • lickers: 33 ± 18% • non lickers: 19 ± 4.9% 60 100 50 40 10 Ivermectin (ng/mL) 30 Concentrations (ng/mL) 1 20 10 0.1 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (day) Time (days) Laffont et al., Int J Parasitol, 2001

16. 55 % IVM A model of ivermectin disposition after a pour-on formulation administration Assumption: the 2 twin cattle are analyzed as the “same individual” observed twice 500 µg/kg Pour-on Laffont et al., Vet Res, 2003 ingestion 58 - 87% 7 - 14 % of the dose absorbed by skin Oral absorption 22 % Plasma overall bioavailability 13 % of the dose Ftotal = 18 - 68 % FECES

17. PO formulation are actually oral route of administration • By the means of pharmacokinetic modeling, we estimated that from 58 to 87% of the PO dose was actually ingested, while only 7-14% was absorbed percutaneously (Laffont et al. Vet Res, 2003). • By modeling plasma vs time curves, it was estimated that 78 to 82% of the total drug amount recovered in the bloodstream was due to oral ingestion after licking (Sallovitz et al., unpublished data).

19. Material and methods • 4 pairs of twin cattle Control DORA MOXI IVM MOXI IVM DORA Dose : 500 µg/kg Blood and fecal samples

20. Non-treated 10 10 8 8 6 6 4 4 2 2 0 0 0 10 20 30 40 50 0 10 20 30 40 50 Results: plasma exposure Treated Doramectin Ivermectin Moxidectin 4 3 2 Plasma conc. (ng/mL) 1 0 0 10 20 30 40 50 Time (day)

21. Lanusse group results Direct measurements of different MLs in GIT (fluids, mucosa) confirmed that PO formulation of MLs are extensively orally ingested by cattle allowed to lick.

24. >138% DRM oral ingestion LICKING IN TOPICALLY TREATED GRAZING CATTLE -Assessment of DRM concentrations in intestinal content- 600 500 400 DRM availability (AUC) 300 <40% 200 100 0 SC Pour-on non-lickers Pour-on lickers Sallovitz J, Lifschitz A, Lanusse C. Vet Parasitol. 2005 ;133:61-70

25. Non-lickers >300% Free lickers >1900% 10-day LICKING RESTRICTION PERIOD: DOR in gastrointestinal contents in GRAZING ANIMALS 700 600 500 400 DRM availability (AUC) 300 200 100 0 Abomasal fluid content Duodenal fluid content

26. General conclusion • At the herd level, and considering the 2 sources of drug ingestion (self- and allo-licking), the oral (licking) route is the main pathway for an endectocide poured on the back of cattle, to enter the plasma and to have a systemic effect

27. What is actually a PO drug product • A Pour-on administration is not a truly individual topical treatment but also a collective oral treatment raising for BE testing not only a question of high variability but also a question of population dimension as for all collective oral treatments

28. Concerns raised by this behavior-driven drug disposition • Efficacy • Drug resistance • Safety • Environment • Regulation (GLP study) • Bioequivalence

29. Questions addressed • Requirement or not of an in vivo BE • Average BE: should licking behavior be factored into topical product bioequivalence trials? • “Licking effect” vs. “licking X formulation interaction effect” • PO products are HVD products: scaling average BE (SABE) • PO products are collective treatments: is really average BE appropriate ? • GLP (lab) or GCP (field) conditions to perform a PO BE trial?

30. Question 1: Is there or not a requirement for an in vivo BE for Pour-on formulations or can BE be considered as self-evident ?

31. Criteria for Waiver of In Vivo Bioequivalence Study : FDA • Categories of products which may be eligible for waivers include, but are not limited to, the following: • 1. Parenteral solutions (IV, SQ, IM). • 2. Oral solutions….. • 3. Topically applied solutions intended for localtherapeuticeffects. Other topically applied dosage forms intended for local therapeutic effects for non-food animals only. • PO formulation are mainly for systemic effect • 4.Inhalant volatile anesthetic solutions.

32. NOROMECTIN (ivermectin) Pour-On for Cattle FREEDOM OF INFORMATION SUMMARY AN ORIGINAL ABBREVIATED NEW ANIMAL DRUG APPLICATION (ANADA) ANADA 200-272 Based on the formulation characteristics of the generic product, Norbrook Laboratories Ltd. was granted a waiver from the requirement for an in vivo bioequivalence study for NOROMECTIN(ivermectin) Pour-On for Cattle. The generic product is administered as pour-on (topical) contains the same active ingredient in the same concentration and dosage form as the pioneer product, and contains no inactive ingredients that may significantly affect the absorption of the active ingredient. The pioneer product IVOMEC (ivermectin) Pour-on for Cattle, the subject of Merial Ltd’s NADA 140-841, was approved on December 4, 1990.

33. Criteria for Waiver of In Vivo Bioequivalence Study : EMEA • No waiving for PO formulations • Several generic PO dossiers were granted a marketing authorization in EU after a successful demonstration of BE • Crossover design with 10 to 24 animals under GLP conditions

34. Question 2: Should licking behavior be factored into topical product bioequivalence trials?

35. A B A B A licking effect The 2 formulations (A and B) are BE in both free-licking and non-licking groups AUC Licker Non Licker Licking effect is not an issue for average BE testing

36. BE in non lickers B A B A not BE in lickers e.g. due to the poor taste of test formulation Interaction licking X formulation The 2 formulations (A and B) are BE in non-licking condition but not in free-licking condition AUC Interaction is an issue

37. Question 3: How to manage BE for Highly Variable Drug Products ?

38. Types of Bioequivalence • Average Bioequivalence (ABE) • Current regulatory requirement • Population Bioequivalence (PBE) • Prescribability • Individual Bioequivalence (IBE) • Switchability

39. Average BE: concerns raised by licking • Due to the high inter-individual variations, how is it possible to demonstrate a bioequivalence with the currently recommended interval (0.8-1.25) ? • If CV% = 45%  84 to 140 subjects are necessary

40. Available OptionsBioequivalence of Highly Variable PO products • Prevent licking: no due to a possible bias • Increase number of subjects: possible but costly and inefficient • Multiple dose (steady-state) studies: no • Replicate design to determine intra-individual variability: possible but long washout • Widen the a priori BE interval 80-125 for both AUC and Cmax (scaled average BE) • Need for point estimate constraint

41. How to establish average BE for a HVD product: scaling average BE (SABE) * sw0 is the SD at which the BE limits are permitted to be widened (set by FDA to 0.2) (20%) * swr is either the residual SD (ABE) or the SD of the reference product (replicate design) e.g 0.4 (40%) A priori BE interval for PO formulation: 0.64-1.56

42. Limitations of ABE • Focuses only on population average • Ignores distribution of the metric • Does not address the right question for a collective treatment as it is the case for a PO formulation.

43. Question 4: Is really average BE appropriate for PO formulations of endectocides ?

44. Types of Bioequivalence • Average Bioequivalence (ABE) • Current regulatory requirement • Population Bioequivalence (PBE) • Prescribability • Individual Bioequivalence (IBE) • Switchability ! Question of the experimental unit : Are observations on one animal independent of the presence of other animals ?

45. Situations where the dose depends on the animal behavior, in interaction with others in the group Oral collective treatments • Pour-on of endectocides

46. Situations where the dose depends on the animal behavior, in interaction with others in the group “ Unlike traditional dosage forms where the administration (intake) is under the control of the investigator …intake is dependant upon animal behavior The inconsistent intake … influenced by animal … behavior, is a determinant of rate and extent of drug exposure … This source of variability complicates the assessment of formulation effects … the inherent nature of … is that intake is free choice. … the times of intake and the duration of intake … are dictated by the animal.”

47. Population BE (PBE) • Population BE is a method for widening the BE limits using total variance (sum of within-and between animal variance) for test and reference products (σ2TT and σ2TR ) (US FDA , 2001)

48. Question 5: Value of laboratory (GLP) vs field (GCP) condition to demonstrate a BE

49. Regulatory concerns raised by licking • BE trial is a “clinical trial” performed in lab conditions!!! • Is there a possible interaction between lab conditions and field conditions vs. formulations? • i.e.2 formulation can be BE in lab conditions (thanks to preventing licking) but not in field conditions)

50. OECD principles of GLP • According to the OECD the principles of GLP have been developed to promote not only the quality but also the validityof test data. • Validity of results requires the absence of a major bias • e.g. . the presence of factors in the experimental design that cause the results to deviate systematically from those that would have been observed in regular husbandry conditions. • Validity of the results requires that inter-animal and intra-animal variability should be considered as a biological fact, not as a noise that should be reduced or even deliberately suppressed in an experimental setting while these behaviors are actually major factors of drug disposition in the target population