Xenobiotics and Food-Producing Animals - American Chemical Society

Chapter 4 Design and Conduct of Studies To Meet Residue Chemistry Requirements

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Residue Depletion and Metabolism of Flunixin in Cattle R. P. Clement, R. D. Simmons, R. J. Christopher, S. F. Charles, C. N. Casciano, C. B. McCullough, J. F. Lamendola, and M . N. Cayen Schering-Plough Research, Lafayette, NJ 07848

The safe concentration of drug-related residue must be known in order to determine the withdrawal period for a veterinary product. Often the toxicity data is incomplete and an estimate must be made to progress with requisite residue studies. One approach is to conduct a total residue study with sufficiently widely-spaced sacrifice intervals to assess the rate of depletion of total residue over the projected range of probable safe concentrations. A zero-withdrawal sacrifice interval should be included. The target tissue and marker residue are identified and surveillance/confirmatory assays developed. If a major portion of residue is non-extractable (bound) and the marker is undetectable at times when total residue is still significant, a residue bioavailability study may be necessary. To complete the data package, final residue and comparative metabolism studies are conducted. Studies on the metabolism of flunixin in cattle will illustrate this approach.

To r e g i s t e r a new animal drug f o r use i n food-producing animals, the sponsor of the compound must demonstrate that drug-related residues i n the edible tissues of treated animals c o n s t i t u t e no p o t e n t i a l hazard when consumed by humans Q ) . To t h i s end, the sponsor must develop information on the amount, persistence and chemical nature of the drug-derived residue i n the edible tissues of treated animals. This information must then be correlated with that on the metabolism of the compound i n the laboratory animal species used f o r t o x i c i t y t e s t i n g . Because of the time required to complete long-term t o x i c i t y studies, residue depletion studies should be designed to develop residue data over the projected range of probable safe concentrations. Close communication between the t o x i c o l o g i s t and residue chemist i s r e q u i s i t e to adequate study design. I n the following discussion, 0097-6156/92/0503-0037$06.00/0 © 1992 American Chemical Society In Xenobiotics and Food-Producing Animals; Hutson, D. H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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XENOBIOTICS AND FOOD-PRODUCING ANIMALS

t h i s approach v i l l be i l l u s t r a t e d for residue depletion studies conducted with f l u n i x i n meglumine i n c a t t l e . F l u n i x i n meglumine (Banamine; 2[[2-methyl-3-(trifluoromethyl)phenyl]amino]-3pyridinecarboxylic a c i d , N-methylglucamine s a l t ) i s a nons t e r o i d a l anti-inflammatory drug with analgesic and a n t i p y r e t i c properties which i s registered or i s under development i n many countries as a veterinary pharmaceutical f o r use i n c a t t l e , swine, dogs and horses. The current c a t t l e i n j e c t a b l e product being developed i n the United States i s intended for use as adjunctive therapy i n the treatment of bovine r e s p i r a t o r y disease. The proposed treatment regimen for c a t t l e i s 2.2 mg f l u n i x i n a c t i v e per kilogram body weight per day administered intravenously as the N-methylglucamine (NMG) s a l t once a day for up to three consecutive days. T o t a l Residue Depletion Study The goal of the t o t a l residue depletion study i s to define the time-dependent depletion of t o t a l drug-related residue i n the edible tissues (muscle, l i v e r , kidney, f a t and, where appropriate, milk for c a t t l e ) of target animals (species for which the drug i s being developed) following administration of the drug. Radiolabeled (preferably C) compound of high radiochemical p u r i t y (>98X) i s u t i l i z e d to study the t i s s u e d i s t r i b u t i o n and depletion of r a d i o a c t i v i t y (representing drug-derived residue). At least 12 animals of the correct species, gender and maturity are treated with the drug by the intended route of administration and highest intended dosage regimen. Groups of three animals are s a c r i f i c e d at s p e c i f i e d i n t e r v a l s a f t e r the l a s t treatment and tissues c o l l e c t e d . Urine and feces are also c o l l e c t e d from at least three animals/sex for a s u f f i c i e n t l y long period to characterize the major routes of e l i m i n a t i o n of r a d i o a c t i v i t y . Following c o l l e c t i o n , tissue and excreta samples are analyzed for t o t a l r a d i o a c t i v e content and the pooled samples (by sex and/or s a c r i f i c e i n t e r v a l ) are processed and analyzed to determine the metabolic p r o f i l e of tissue and excreta r a d i o a c t i v i t y . Following p r o f i l i n g , major tissue and excreta metabolites are i s o l a t e d and i d e n t i f i e d . Proper conduct of the t o t a l residue depletion study i s important not only because the r e s u l t s define the depletion of t o t a l drug-related residue from the e d i b l e tissues of treated animals, but also because t h i s data w i l l be u t i l i z e d to i d e n t i f y the target t i s s u e (the edible tissue selected to monitor t o t a l residue - u s u a l l y the l a s t t i s s u e i n which residues deplete to the safe concentration) and marker residue (residue, i . e . drug and/or metabolite(s), selected to monitor the concentration of the t o t a l residue i n the target t i s s u e ) . These r e s u l t s w i l l be u t i l i z e d to e s t a b l i s h the r e l a t i o n s h i p between depletion of t o t a l residue and the marker residue i n the target t i s s u e . I d e n t i f i c a t i o n of major metabolites i n the urine and feces i s important because these products must undergo environmental impact assessment. Tissue and excreta p r o f i l e s w i l l also be used

In Xenobiotics and Food-Producing Animals; Hutson, D. H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


4.

CLEMENT ET AL.

Residue Depletion and Metabolism of Flunixin in Cattle

to e s t a b l i s h comparative metabolism i n the target and laboratory animal species. I f l i t t l e or no previous data on the metabolism of the compound i n the target species i s a v a i l a b l e , i t i s prudent to consider conducting a probe residue depletion study i n three animals s a c r i f i c e d at three widely spaced i n t e r v a l s following the l a s t dose. For example, one animal might be s a c r i f i c e d at 12 hours (zero withdrawal) and another each at 72 and 120 hours post f i n a l dose (the exact times selected w i l l depend on the tissue clearance of the drug under development). The information obtained w i l l allow one to select more accurately s a c r i f i c e i n t e r v a l s for the d e f i n i t i v e study which encompass proposed safe concentrations of t o t a l drug-related residue. A d d i t i o n a l l y , the probe study residue data w i l l allow for adjustments i n the s p e c i f i c a c t i v i t y of the radiolabeled dose to ensure tissue concentrations of t o t a l r a d i o a c t i v i t y at l a t e r s a c r i f i c e i n t e r v a l s which are s u f f i c i e n t for metabolite p r o f i l i n g and isolation. I n i t i a l T o t a l Residue Depletion Study WITH FLUNIXIN IN C a t t l e . In the i n i t i a l t o t a l residue depletion study, C - f l u n i x i n NMG was administered once d a i l y for two consecutive days by intravenous i n j e c t i o n to three l a c t a t i n g cows and three steers at a dose of 2.2 mg/kg/day (based on f l u n i x i n free a c i d ) . One cow and one steer per time point were s a c r i f i c e d at 24, 72 and 120 hours a f t e r the f i n a l dose, and selected t i s s u e s , including l i v e r , kidney, muscle and f a t , were c o l l e c t e d and analyzed for radioactive content. Highest l e v e l s of t o t a l r a d i o a c t i v i t y were noted i n the l i v e r and kidneys. Average values of radiolabeled residues i n the l i v e r at 24, 72 and 120 hours were 530 ±226, 145 ±49 and 85 ±35 ng equivalents /g, respectively. In kidneys, the average values of t o t a l radiolabeled residues at 24, 72 and 120 hours were 520 ±212, 115 ±21, and 55 ±7 ng equivalents/g, respectively. Levels of t o t a l r a d i o a c t i v i t y i n fat and muscle were below the l i m i t of r e l i a b l e detection (defined as 30 dpm above the background count rate) at each s a c r i f i c e i n t e r v a l . These r e s u l t s indicate that l i v e r i s the target tissue. Metabolites were i s o l a t e d and i d e n t i f i e d i n the urine, feces, l i v e r and kidneys of animals.administered two consecutive d a i l y intravenous doses of 2.2 mg C - f l u n i x i n active/kg body weight. Approximately 90X of the t o t a l administered dose was recovered i n the urine and feces w i t h i n 24 hours a f t e r the second dose. R a d i o a c t i v i t y was excreted i n approximately equal percentages i n the urine and feces. Urine and feces were extracted with methanol and the extracts analyzed by HPLC and TLC. Urinary and f e c a l metabolites were i d e n t i f i e d by co-chromatography with known synthetic metabolite standards. The major r a d i o a c t i v e component i n both cow and steer urine had s i m i l a r chromatographic retention c h a r a c t e r i s t i c s to unchanged f l u n i x i n . Two minor metabolites, with retention times corresponding to the 2'-methylhydroxyf l u n i x i n and 5-hydroxyflunixin metabolite standards, were also detected. The only r a d i o a c t i v e component present i n both cow and steer feces had s i m i l a r e l u t i o n c h a r a c t e r i s t i c s to 5-hydroxyflunixin.


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To examine the nature of radiolabeled residues i n l i v e r and kidney, the tissues from treated animals were homogenized i n methanol, extracted, further cleaned up by s o l i d phase e x t r a c t i o n , and analyzed by HPLC. Only the 24-hour post f i n a l dose kidney and l i v e r samples were analyzed because of the low l e v e l s of t o t a l r a d i o a c t i v i t y present i n the other tissues. Three compounds, including f l u n i x i n and i t s 5-hydroxy- and A'-hydroxy- metabolites were i d e n t i f i e d by c o - e l u t i o n with authentic standards i n the l i v e r and kidney e x t r a c t s from male and female c a t t l e . The structures of f l u n i x i n and i t s metabolites are presented i n Figure 1. Downloaded by UNIV OF PITTSBURGH on February 17, 2016 | http://pubs.acs.org Publication Date: August 24, 1992 | doi: 10.1021/bk-1992-0503.ch004

SECOND TOTAL RESIDUE DEPLETION STUDY VITH FLUNIXIN IN CATTLE.

Because f l u n i x i n vas only administered for two consecutive days i n the i n i t i a l study, A second t o t a l residue depletion study vas conducted i n vhich the compound vas administered f o r the maximum proposed treatment duration (3 consecutive days). Tvelve Hereford crossbred feeder c a t t l e (6 steers and 6 h e i f e r s ) received three consecutive d a i l y doses of 2.2 mg/kg f l u n i x i n meglumine. Three animals (2 males and 1 female or v i c e versa) vere s a c r i f i c e d at each of four time points (12, 24, 72 and 120 hours post f i n a l dose). ^£nimals i n the 12- and 24-hour s a c r i f i c e groups vere given C-flunixin vith a specific activity of 0.7 mC^g. Animals i n the 72- and 120-hour s a c r i f i c e groups received C - f l u n i x i n v i t h increasing a c t i v i t i e s of 1.4 mCi/g and 2.7 mCi/g, respectively. Increasing the s p e c i f i c a c t i v i t y of the dose at the l a t e r slaughter groups (72 and 120 hours post f i n a l dose) assured that there vould be adequate r a d i o l a b e l present i n the tissue samples to provide meaningful r e s u l t s . Samples of l i v e r , kidney, muscle and fat vere removed at s a c r i f i c e for a n a l y s i s of r a d i o a c t i v i t y . Of the tissues examined, highest l e v e l s of t o t a l r a d i o a c t i v i t y vere found i n the l i v e r and kidneys. No r a d i o a c t i v i t y vas detected i n any of the fat or muscle samples c o l l e c t e d at 12, 24, 72, or 120 hours. For each animal, the l e v e l of t o t a l r a d i o a c t i v i t y i n the l i v e r vas higher than that i n the kidney. Total radiolabeled residues i n l i v e r and kidneys vere 1,656 ±1,143 ng equivalents/g and 1,123 ±590 ng equivalents/g, respectively at 12 hours a f t e r the f i n a l dose of f l u n i x i n . There vas a marked decrease i n t o t a l r a d i o a c t i v i t y i n both the l i v e r and kidney by 24 hours. Liver and kidney samples at 24 hours post f i n a l f l u n i x i n dose had l e v e l s of t o t a l r a d i o a c t i v i t y of 327 ±121 ng equivalents/g and 308 ±68 ng equivalents/g, respectively. At 72 and 120 hours, t o t a l radiolabeled residues i n l i v e r vere 143 ±36 ng equivalents/g and 121 ±25 ng equivalents/g, respectively. In kidney, the l e v e l s of t o t a l r a d i o a c t i v i t y at 72 and 120 hours vere 107 ±9 ng equivalents/g and 79 ±15 ng equivalents/g, r e s p e c t i v e l y . This data confirms that l i v e r i s the target tissue as i t i s the e d i b l e tissue v i t h the greatest amount of t o t a l r a d i o a c t i v i t y p e r s i s t i n g for the longest period of time. P r o f i l i n g and i d e n t i f i c a t i o n of f l u n i x i n and i t s metabolites i n the l i v e r and kidneys of male and female feeder c a t t l e vas also c a r r i e d out during the second t o t a l residue depletion study. I n i t i a l l y , l i v e r tissue from c a t t l e treated intravenously v i t h



F i g u r e 1. S t r u c t u r e s o f f l u n i x i n and i t s major ( a s t e r i s k d e n o t e s p o s i t i o n o f carbon-14 l a b e l ) .

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2.2 rag f l u n i x i n NMG/kg body weight/day for three consecutive days vas homogenized i n 60% p e r c h l o r i c a c i d , incubated at 25°C overnight to digest the t i s s u e , and then extracted with chloroform or methylene c h l o r i d e under a c i d i c , n e u t r a l and basic conditions. The combined organic extracts were evaporated to dryness under reduced pressure, redissolved i n methanol and analyzed by HPLC. Recovery of sample r a d i o a c t i v i t y i n the combined organic extracts ranged from 69Z to 83X f o r the 12-hour l i v e r samples and SX to 22% f o r the 24-hour samples. F l u n i x i n was the major residue i n the organic extract from l i v e r of c a t t l e at 12 and 24 hours post f i n a l dose, representing greater than 952 of the extractable r a d i o a c t i v i t y . In an attempt to enhance recovery, two a l t e r n a t e e x t r a c t i o n methods, including aqueous hydrochloric acid e x t r a c t i o n and methanol-hydrochloric acid:potassium hydroxide e x t r a c t i o n were employed. In each case, recoveries were comparable to the i n i t i a l r e s u l t s , and f l u n i x i n s t i l l represented the major residue detected i n the 12- and 24-hour l i v e r samples. Following i n i t i a l p r o f i l i n g of the 12- and 24-hour post f i n a l dose l i v e r t i s s u e , the l i v e r s from two of three animals at each s a c r i f i c e i n t e r v a l (12, 24, 72 and 120 hours post f i n a l dose) as w e l l as kidney tissue from each animal at each s a c r i f i c e i n t e r v a l were extracted and the metabolite p r o f i l e s determined. Each l i v e r and kidney sample was homogenized i n hexane to remove l i p i d , centrifuged, the tissue p e l l e t hydrolyzed i n hydrochloric a c i d , the hydrolyzed homogenate extracted with e t h y l acetate at pH 4-4.5, and the e t h y l acetate extract concentrated and analyzed by HPLC. With few exceptions, f l u n i x i n accounted f o r at least 50X of extractable tissue r a d i o a c t i v i t y and was the major residue in the l i v e r s and kidneys of male and female feeder c a t t l e . The 4'-hydroxy metabolite of f l u n i x i n was also present and represented a major residue i n female l i v e r samples at 12 and 24 hours post f i n a l dose and i n selected male and female kidney samples at 72 and 120 hours. Minor amounts of the other hydroxylated metabolites, including 5-hydroxyflunixin and 2'-methylhydroxyflunixin, were also detected. These r e s u l t s are in agreement with the p r o f i l i n g data from the i n i t i a l t o t a l residue study and indicate that the primary routes of metabolism of f l u n i x i n are v i a oxidation of the pyridine and phenyl r i n g systems as w e l l as the methyl substituent on the phenyl moiety. The data also indicate that f l u n i x i n i s the most appropriate marker residue i n the target tissue ( l i v e r ) . Despite use of a l t e r n a t e methods of tissue e x t r a c t i o n , the percent of t o t a l radiolabeled residue extractable from the l i v e r or kidney decreased with increasing length of time a f t e r dosing. In l i v e r , the percent recovery of t o t a l r a d i o a c t i v i t y i n the organic extract ranged from 70-942 at 12 hours, 61-72X at 24 hours, and had decreased to between 21 and 48% at 72 and 120 hours post f i n a l dose. These r e s u l t s suggest that bound residues may be formed during the metabolism of f l u n i x i n i n cattle.


4.

CLEMENT ET AL.

Residue Depletion and Metabolism of Flunixin in Cattle


Surveillance and Confirmatory Assay Development To e s t a b l i s h a q u a n t i t a t i v e r e l a t i o n s h i p between the depletion of t o t a l residue of t o x i c o l o g i c a l concern and the marker residue i n the target t i s s u e , a r e l i a b l e a n a l y t i c a l method for the marker residue must be developed. This method w i l l undergo interlaboratory v a l i d a t i o n t r i a l s and must, therefore, be rugged and of s u f f i c i e n t s e n s i t i v i t y to detect the marker residue at concentrations at which the t o t a l residue i s at or below the safe concentration. The method u s u a l l y consists of two components: a s u r v e i l l a n c e (determinative) assay to quantify the marker residue, and a confirmatory assay to v e r i f y the i d e n t i t y of the compound. Each assay must be of acceptable s p e c i f i c i t y ( i n c l u d i n g freedom from interference by p o t e n t i a l l y coadministered medications), s e n s i t i v i t y (accurate detection of the marker compound at a concentration equal to one-half the concentration at a time when t o t a l residue i s at the safe concentration), accuracy and p r e c i s i o n . Each method should u t i l i z e commercially a v a i l a b l e reagents and standard a n a l y t i c a l laboratory instrumentation, and be capable of being performed by experienced analysts w i t h i n a 48-hour period. For a detailed discussion of assay requirements, the reader i s referred to guideline V of "General P r i n c i p l e s for Evaluating the Safety of Compounds Used i n Food-Producing Animals' (1). 1

Assay Development f o r F l u n i x i n (Marker Residue) i n L i v e r (Target Tissue). A s u r v e i l l a n c e assay was developed which detects and quantitates f l u n i x i n i n bovine l i v e r by ion-pair reverse phase high performance l i q u i d chromatography. In the assay, l i v e r samples are homogenized i n T r i s Buffer (0.1 M, pH 10.7), extracted with dichloromethane/isopropyl a l c o h o l (9:1 v/v) to extract l i p i d s and p r e c i p i t a t e proteins, and then, following c e n t r i f u g a t i o n , the aqueous layer i s a c i d i f i e d to pH

Xenobiotics and Food-Producing Animals - American Chemical Society

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