Determining the Topical and Oral Pharmacokinetics of Flunixin Meglumine in Pre-wean Piglets and Developing Tools for Drug Study in Pre-Wean Piglets

Nearly all pigs experience tail-docking and/or castration in the United States and yet alternatives to castration and tail docking have not been adopted by US pork producers due to inherent challenges with alternatives. Public expectations to mitigate pain continues to build and influence producer decisions. Even in the absence of routine castration and tail-docking, disease and injury create conditions that are painful for piglets. Given that flunixin meglumine (FM) is already approved for IM use in pigs and extra-label use of drugs for pain is allowed, the main hurdle to potential application is confirmation that it reaches useful serum levels and to develop a dose regimen that is safe for the pre-wean piglet. An understanding of drug pharmacokinetics (PK) is a prerequisite to efficacy studies. Additionally, investigation of two additional routes of administration could lead to use of flunixin that does not require injection of every pig treated.
The primary objectives were as follows: Objective 1: Find the spot on a piglet where topical application of drug will most likely lead to transdermal absorption of drug and and quantify parameters of that area. The deliverable from this objective was to determine an anatomical target area on the piglet for testing and administration of topical drugs. Topical drugs are dosed differently than oral or injectable drugs and absorption can be affected by skin composition, thickness, and subcutaneous fat. Ten healthy pigs, 4-5 days of age were purchased and humanely euthanized. Full thickness skin biopsies were collected from 32 anatomical locations on each pig. The biopsies were fixed with formalin and examined microscopically for thickness of the skin and subcutaneous fat and density of hair follicles. The values were reported as an average, range and standard deviation (Table 1). Objective 2: Adapt a procedure to place indwelling, dual lumen intravenous (IV) jugular catheters in pre-wean piglets. The deliverables from this objective were to describe the new procedure in a peer-reviewed, published report and to develop a video demonstrating the procedure. The dual-lumen IV jugular catheter in pre-wean piglets allows for IV dosing without contamination of blood samples and eliminates the need for two catheters. The catheter also provides an alternative to jugular venipuncture during serial serum sampling for pharmacokinetics studies in relatively fragile pre-wean piglets. Ten healthy piglets, 9 days of age, were randomly assigned to one of two groups: anesthesia or non-anesthesia. Isoflurane gas anesthesia was administered to the anesthesia group with the use of a pediatric gas mask. Anesthetic sedation was maintained throughout the duration of the catheterization procedure. Piglets were allowed to recover at the end of the catheterization procedure. The non-anesthetic group did not receive anesthesia. For jugular vein catheterization, each piglet was restrained in a V-tray. The jugular vein was catheterized with the use of a guide wire technique and a dual-lumen catheter. The catheter was sutured in place and bandaged (Fig. 1 and 2) to prevent self-mutilation by the piglets. Blood was collected from the catheter and the catheter flushed with heparinized saline twice daily, for three consecutive days. Following completion of the three days, the piglets were euthanized via anesthetic overdose with the administration of sodium pentobarbital through the jugular catheter. A necropsy was performed at the site of the jugular catheter, to the level of the cranial vena cava for verification of catheterization in the jugular vein (Figure 3). Objective 3: Measure and compare the pharmacokinetics of intramuscular (IM), oral (O) and topical (T) administration of flunixin meglumine in pre-wean piglets. The deliverables from this phase were to create an understanding of the fate of the drug in the pre-wean piglet including what concentrations are achieved in the serum and how long those are maintained using the oral, topical and intramuscular routes. Determining the fate of flunixin for pre-wean piglets by alternate routes, would provide the foundations necessary to develop effective treatment regimens. Twenty-four piglets, 9 days of age, weighing 5-10 pounds, were randomly allotted to 3 treatments (8 pigs / treatment): IM, O, or T administration of FM at 2.2mg/kg of body weight for oral and intramuscular administration and 3.3mg/kg of body weight for topical administration. Serum samples were collected at 0, 15, 30, 45, 60, 90, 120 minutes and 3, 6, 12, 24, 36, 48, 60, 72 hours post treatment, via jugular venipuncture. Samples were transferred to a sodium heparin blood collection tube and stored on ice for no longer than 130 minutes. Samples were centrifuged for 10 minutes at 1,500 g and collected plasma was placed in cryovials and frozen at -70℃ until analysis. Plasma FM concentration were determined using ultrahigh performance liquid chromatography Q Exactive Focus Orbitrap mass spectrometry. Analysis produced plasma FM concentrations over time for each pig (Fig. 4). Objective 4: Precisely determine bioavailability of the topical route of administration. The deliverables from this objective, combined with parameters measured in objective 3, were to allow adjustment of the drug administration required to achieve target serum levels and develop dose regimens for topical application in pre-wean piglets. Sixteen piglets, 9 days of age, 5-10 pounds, were randomly allocated to two treatments in a cross-over study design with two rounds. In round one, eight pigs received FM via the T route and eight pigs received FM via the IV route. Serum samples (1.0 mL volume) were collected at 0, 5, 15, 30, 45, 60, 90, 120 minutes and 3, 6, 12, 24, 36, 48, 60 hours post treatment for the IV route and 0, 15, 30, 45, 60, 90, 120 minutes and 3, 6, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, and 216 hours for the topical route. Following the sampling, a washout period of nine days was used to ensure that the previous drug would not interfere with the next round of drug administration and sample collection. The groups then switched routes of administration and the sampling was repeated. The samples were prepared and submitted for analysis as previously outlined in Objective 3.

Objective 1: Statistical analysis of the data discovered significant differences (p <0.0092) between the highest and lowest average sample site measurement for each skin characteristic evaluated (Table 1). These results reflect the importance of site selection for topical pharmaceutical application. Variations in skin thickness and hair follicle density across the body of a processing aged piglet reflect key factors that may influence transdermal absorption and pharmacokinetic principles of a drug.

Objective 2: Jugular catheterization was successful in 3 of 10 piglets (all from the non-anesthesia group). Patency was maintained in the three catheters for three consecutive days (Table 2). If successful catheterization can be achieved in pre-wean piglets, the technique is useful and beneficial during pharmacology studies, however the ability to achieve and maintain catheterization was poor in this study. Due to the inconsistency of catheterization success, the blood collections for objective 3 and 4 were completed via serial jugular venipuncture.

Objective 3: All three routes of administration produced measurable concentrations of FM (Fig. 4). Intramuscular and oral administration produced concentrations in a range that is consistent with pain mitigation in species where this has been investigated. These routes had short half-lives and clearance rates consistent with FM in older animals. The topical route produced much lower concentrations that persisted beyond 72 hours post administration. Objective 4: The bioavailability (F) of the topical route of administration was calculated using the area under the plasma concentration-time curve (AUC) for IV and topical (Table 3, Fig. 5) dosing and the bioavailability formula: F=(〖AUC〗_█(Topical @)* 〖Dose〗_IV)/(〖AUC〗_█(IV @)* 〖Dose〗_█(Topical @)). The bioavailability for the topical route of administration of FM was calculated to be approximately 6.3%.

Industry Takeaways
Variation in skin characteristics show that topical application of pharmaceuticals should be researched further to determine if other site locations may lend better absorption of the NSAID. Much more variation was observed than expected and parameters did not trend in similar directions where a single spot optimized the factors expected to impact transdermal absorption.
Percutaneous, jugular catheterization is possible but piglet anatomical variation at this age makes it an inconsistent technique for research that would require further refinement.
Maximum serum concentration of the topical product was >100X lower than oral and intramuscular administration and is not consistent with levels demonstrated to mitigate pain. The high serum concentration of the oral route suggests that topical administration in a litter environment (versus in isolation as was done in this study) is warranted to include potential litter effects of licking and peer grooming on serum levels.
In order to maintain consistency, only one location was used for topical application of FM. Future studies are warranted to determine if a more ideal location may lend better absorption of FM.