Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
Veterinary Diagnostic and Production Animal Medicine
Non Technical Summary
Currently in the US, there are no antimicrobials labeled for usage in goats. This leaves goat producers using dosage forms produced for cattle in a species that has a much smaller udder mass. In addition, there is virtually no research looking at the pharmacokinetics of drugs in goats and goat dairymen are very concerned about creating violative drug residues in their saleable milk. In this study, we will determine the pharmacokinetics of cephaparin in milk and determine the residue depletion time necessary. Following completion of the animal portion of the study and determination of drug residues in the collected milk samples, we will use computer modeling to predict drug concentrations in milk at other time points that were not collected and to predict drug concentrations when utilizing other doses of the drug.
Animal Health Component
80%
Research Effort Categories
Basic
(N/A)
Applied
80%
Developmental
20%
Goals / Objectives
The objective of year one of this proposal is to determine the pharmacokinetics and withdrawal period for cephapirin in lactating does using the labeled dose for cattle and then predict the pharmacokinetics and withdrawal period at various dosage regimens. Our specific aims for this proposal are as follows:Specific Aim 1: Following FDA guidelines for drug withholding periods, determine the pharmacokinetics in milk following intramammary administration of cephapirin at the on-label dosage regimen for cattle to lactating does. Specific Aim 2: Utilizing FDA published guidelines and software, determine appropriate milk withholding periods following intramammary administration of cephapirin. Specific Aim 3: Assimilate data collected from specific aims 1 and 2 into computer generated models that will be used to predict the pharmacokinetics and withdrawal periods at different dosage regimens.
Project Methods
Live animals will be selected from and the trial work will be completed on commercial goat dairy farms as ISU has no research facilities that house goats. ISU's Institutional Animal Use and Care Committee will approve the trial protocol and owner consent will be obtained before the trial work begins. Animals will be managed per the normal husbandry practices for the herd, with the exception that milk from treated does will be discarded until milk drug residue concentration is sufficiently depleted to a point where a violative residue will not occur.To complete Specific Aim 1, a cooperator herd will be identified that will allow us to treat twenty lactating animals simultaneously. Twenty healthy lactating animals in various stages of lactation will be treated according to the FDA document Guidance for Industry #207 - Studies to evaluate the metabolism and residue kinetics of veterinary drugs in food-producing animals: marker residue depletion studies to establish product withdrawal periods.8 Briefly, twenty does will be infused with one tube (200 mg) of cephapirin sodium (ToDay®, Boehringer Ingelheim Vetmedica, Duluth, GA) in each udder half at 24-hour intervals, for two treatments. Before the first treatment (T0), milk will be collected in floor pails, it will be well-stirred, and duplicate 50 mL composite milk samples will be taken and frozen for future analysis. Following treatment, triplicate, well-stirred composite 50 mL milk samples will be collected from floor pails one time per day for a period of seven days. Additionally, on the day of the second cephapirin administration, a second sample will be taken at twelve hours after drug administration. At each milking, does will be prepped using the farm's written protocol, except does will not be fore-stripped. Clean milking equipment fitted with a floor pail will be used to collect all harvested milk. Following milking unit attachment, milk flow will be observed until milk flow completely ceases from all udder halves for a minimum of 15 seconds and then the unit will be removed. Following sampling, the milking unit will be thoroughly flushed with water and dried to prevent drug carryover from animal to animal.One milk sample will be utilized for determination of milk withdrawal using commercially available tanker truck milk residue detection tests and the other two milk samples will be stored at -20 C until analyzed for determination of drug concentration using LC/MS.Tanker truck milk residue test kit analysisStarting prior to drug infusion (T0) and then with the milk samples collected twenty-four hours after the final drug infusion, milk from each animal will be tested for cephapirin residues above the tolerance using commercially available test kits that are approved for goat milk (Charm SL ß-Lactam Test, Charm Science, Lawrence, MA). To mimic the herd design described in the FDA's approach for determining milk withdrawal periods,9 milk will be diluted 1:9 with blank milk prior to testing. This will continue on a daily basis until the test kit determines the milk to be non-violative for each animal. As these test kits would be the only kits available for testing milk from goats as prescribed by the Pasteurized Milk Ordinance,10 it is expected that the tolerance limit applied to cow's milk for cephapirin would also be applied to goats. These tolerance limits are established by the FDA upon drug approval and then monitored and regulated in Grade A milk through guidelines set forth in Appendix N of the Pasteurized Milk Ordinance. The PI for this proposal (Gorden) is a member of the Appendix N committee and is currently communicating with the FDA and the Appendix N committee regarding the approach that would be taken by regulatory officials in a commercial setting for use of cephapirin sodium in goats. Once this procedure is clarified, we will analyze the data accordingly.In addition, we will run each milk sample without dilution to understand how long it will take the individual sample to clear the drug residue. While this is not the way the test kit will be used in the regulatory setting, it is the way many producers utilize the test in an attempt to prevent residues in their saleable milk.Cephapirin concentration determination in milkMilk concentrations of cephapirin and its metabolite desacetylcephapirin will be determined using liquid chromatography coupled with mass spectrometry (LC-MS) by the ISU VDL Analytical Chemistry Lab using a validated method adopted from cow milk. This assay is commonly done on cow's milk within the Analytical Chemistry Lab. A partial validation will be performed prior to the beginning of animal trials to assure sample stability, analyte extraction technique, and analytical performance of the method in goat milk. The limit of detection for both analytes using this method with cow milk is 0.5 ng/mL and the limit of quantitation is 1 ng/mL.Non-compartmental pharmacokinetic analysis of milk dataPharmacokinetic parameters will be determined in milk using appropriate methods in a commercially available software program (Pkanalix v.2019, Lixoft, France).Determination of withdrawal periodUtilizing the regulatory method established by the FDA9 and a freely available software package (R Core Team, 2013), drug withdrawal times for milk will be approximated using the tolerance levels established for cephapirin in cow's milk or the tolerance value suggested following conversations with Appendix N and the FDA. The use of tolerance limits accounts for the degree of variation in the population and the sample size used to make the measurements. When using the approach9, a 99th percentile tolerance limit with 95% confidence is used to determine the time necessary for milk cephapirin concentration to be at or below the US tolerance limit of 20 ng/mL (or value determined following conversations with Appendix N and the FDA). If the time determined is a fraction of the period between milkings, when does are milked 2x/day, the milk withdrawal period will be increased to the time of the next milking. Using this procedure, 99% or more of the milk residue samples at or beyond the withdrawal time are expected to fall below the tolerance limit for the drug in question.9Pharmacokinetic modeling and simulation of time-course dataUsing data accumulated from this work, time-varying changes of drug concentrations in milk will be modeled using nonlinear mixed-effects in commercially available software program (Monolix 2019 R1, Lixoft), as previously described for other disease modalities by Mochel et al.11-13 This method will be complementary to the non-compartmental analysis as it will allow us to simulate milk concentrations of cephapirin for dosing schedules that were not tested in this study for further optimization of ToDay® dosing practices in goats.