Chapter 9 Pirlimycin in the Dairy Cow
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Metabolism and Residue Studies R. E. Hornish, T. S. Arnold, L. Baczynskyj, S. T. Chester, T. D. Cox, T. F. Flook, R. L. Janose, D. A. Kloosterman, J. M. Nappier, D. R. Reeves, F. S. Yein, and M. J. Zaya Animal Health Drug Metabolism, Upjohn Laboratories, The Upjohn Company, Kalamazoo, MI 49001 Pirlimycin hydrochloride (I), a lincosaminide antibiotic, is a new therapeutic agent under development for the treatment of mastitis in the dairy cow. Absorption, distribution, metabolism, excretion and residue decline studies of I have been conducted in the dairy cow following intramammary infusion of an aqueous gel formulation of I into all four quarters of the udder via the teat canals. Total milk residues accounted for only 50% of the dose and the milk residue concentration- time course was bi-phasic. Nearly half of the dose was thus absorbed for systemic circulation. Drug residue concentrations in blood were best fit to a two -compartment pharmacokinetic model. Pirlimycin accounted for ≥95% of the drug residue in milk and was excreted predominantly as parent compound in the urine and feces. Pirlimycin sulfoxide was the major residue found in the liver, the target tissue for residue analysis. GI tract microflora converted part of the fecal drug residue to 3-(5'-ribonucleotide) adducts of pirlimycin and pirlimycin sulfoxide. The comparative metabolism of I in the rat following oral administration was nearly identical to that in the cow following intramammary infusion.
Pirlimycin (I), Figure 1, is a semi-synthetic member of the lincosaminide antibiotics derived from lincomycin (II) and clindamycin (III). Its activity against most grampositive organisms is comparable to clindamycin and several times as active against Staph.aureus (1,2). The proposed use of pirlimycin as a therapeutic agent for the treatment of bovine mastitis was initially investigated by Yancey in an in vitro lactating mouse model developed for estimating mastitis activity (3). Pirlimycin hydrochloride is now under development as a dairy cow mastitis therapeutic agent. The development of any drug or chemical entity targeted for food-producing animals must undergo a six-step safety evaluation process encompassing the study of the adsorption, distribution, metabolism and excretion ( A D M E studies) to address drug residue concerns in the consumable products as outlined in the landmark papers by
0097-6156/92/0503-0132$06.00/0 © 1992 American Chemical Society
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Perez (4) and Weber (5). This report describes part of the A D M E studies carried-out for the safety evaluation of pirlimycin in the dairy cow for the treatment of mastitis. All four udder quarters of twelve dairy cows in mid-lactation were treated at 4 times the therapeutic dose of 50 mg/ quarter by intramammary infusion of an aqueous gel containing 200 mg of pirlimycin free base equivalents, including the labeled C-pirlimycin hydrochloride. The pharmacokinetic parameters for total pirlimycin residue in blood and milk were determined. Three cows were sacrificed at each of four post-treatment intervals (4, 6, 14, and 28 days) to establish tissue residue depletion kinetics. Metabolite profiles of the residues in milk, liver, urine and feces were obtained and the unknown radiolabeled components identified. A comparative metabolism study in the rat was also conducted to address its relevancy as a toxicological test species. The residue concentration decline kinetics were also determined for pirlimycin in milk from cows treated at a dose rate of 50 mg per quarter in all 4 quarters to establish a milk discard interval for the proposed use. 14
Materials and Methods 14
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C-pirIimycin. C-Pirlimycin hydrochloride was synthesized by the sequence shown in Figure 2. Final purification was accomplished by recrystallization to a radiochemical purity >98% as measured by H P L C radioactivity monitoring techniques (HPLC/RAM). The specific activity of the purified material was 11.7 mCi/mmole. Cow animal husbandry for radiolabeled studies. Twelve Holstein cows in mid-2nd or mid-3rd lactation were housed individually in stainless-steel metabolism stalls and maintained therein through 4 or 6 days post-last-treatment then allowed freedom of movement in an enclosed corral until sacrificed. The cage floor was fitted with a plastic-coated rubber mat to reduce the stress of standing for long periods of time. The cages were equipped with manual-fill feed bins and automatic-fill water troughs as well as a drainage system to collect urine and a rear access door to approach the animal for milking and feces collection. 14
Dose preparation and administration. A mixture of C-pirlimycin hydrochloride and non-labeled pirlimycin hydrochloride (99% chemical purity) was prepared to adjust the specific activity to ^ 10,000 dpm^g. The dose formulation was prepared to contain total pirlimycin free base equivalents at 20 mg per mL in an aqueous gel containing 2% by weight carboxymethylcellulose. Plastets, polyethylene dosing devices used for udder infusions, were filled with 10.1 mL of formulation. The contents of one Plastet was administered into each quarter the udder through the teat canal immediately after milk-out. All four quarters were treated to simulate the rare maximal use situation. A second dose was administered similarly 24 hours following the first dose. Collection of samples and total residue analysis. Blood (10-15 mL) was collected by jugular venipuncture into heparinized syringes at 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 24, 30, 36, 48, 60, 72 and 96 hours after the 1st dose. Sub-samples of 200-300 mg were immediately weighed in triplicate for radiolabel quantitation by combustion analysis. The remainder of the sample was centrifuged for the separation of plasma, which was
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Pirlimycin HCI 14
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iso-Pirlimycin HCI
Figure 2. Synthetic sequence for C-pirlimycin hydrochloride.
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Pirlimycin in the Dairy Cow
then sub-sampled and stored at -20°C for subsequent analysis. The cows were milked by commercial single cow milking machines at regular 11-13 hour intervals to provide a composite milk sample (4 quarters combined) per cow per time interval. Milk was collected for analysis through 6 days post-last-treatment or until the animal was sacrificed (4 day animals). Urine was collected at 24- hour intervals through the drainage system of the metabolism cage into 5 gallon plastic containers. Each collection was weighed, homogenized and sub-sampled for analysis. Total feces was collected at 24-hour intervals into 5 gallon plastic containers. The net fecal weight was measured and an equal weight of water added to prepare a homogenate slurry for sub-sampling and analysis. Each animal was sacrificed by captive bolt after the appropriate withdrawal interval (4, 6, 14 and 28 days after the 2nd dose) and processed as in an abattoir. The entire liver, kidneys and udder were excised and 1-2 kg samples of muscle from both the flank and the udder diaphragm and 1-2 kg samples of fat from the abdominal area were collected. Each organ and tissue was minced and processed three times through a commercial meat grinder to prepare respective homogenate samples. Sub-samples (200-300 mg) were prepared in triplicate for total residue analysis. Total radioactivity concentrations, expressed as pirlimycin free base equivalents, were determined by direct liquid scintillation counting (liquids) or combustion analysis (solids) following standard techniques. Metabolite/residue analysis. Milk, urine and plasma samples were first analyzed by a microbiological cylinder/plate procedure against M.luteus which has a limit of detection of 0.02 ppm. A sub-sample of the milk was prepared for this assay by a centrifugation step followed by a pH adjustment to 8.5. In addition, an HPLC/RAM analysis was conducted after treating another sub-sample with FTSH (10% formic acid, 30% trifluoroacetic acid, 2% sodium chloride, 2N hydrochloric acid) followed by centrifugation to precipitate the proteins. The supernatant was basified and concentrated by C-18 solid phase extraction (SPE) techniques. The HPLC conditions were: Column - 20 cm χ 4.8 mm C-8; Mobile-phase -linear gradient at 5%/minute from 90:10 0.1M pH 7 phosphate buffenmethanol to 20:80; Detectors - UV operated at 214 nm and a radioactivity flow detector operated in the C DPM mode. 14
Tissues and feces were processed as follows: The extraction of >90% of the total C residue was accomplished for all samples by homogenizing 1 part sample with 2 parts FTSH, centrifugation, followed by a second extraction of the solids with 20% FTSH. The acid extracts were combined, basified to pH 8.5 ± 0.5 with cone, ammonium hydroxide and processed through C-18 solid phase extraction columns. Pirlimycin and the metabolites were eluted from the columns with methanol and 1% HC1 in methanol, respectively. These samples were evaporated to dryness and takenup in buffer for microbiological or HPLC/RAM analysis. 14
FAB/MS and NMR. FAB/MS spectra were obtained on a VG magnetic sector instrument. The samples were placed on the FAB target probe containing 2-hydroxyethyldisulfide as the matrix solvent. The target was bombarded with xenon at 8-9 KV and the data recorded with a UPACS II data system. Matrix ions were subtracted from the samples ions. All NMR experiments were performed at 25° on a Bruker
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AM-500 spectrometer operating at 500.13 MHz for proton magnetic resonance. Samples were prepared in d -DMSO and 1-D and 2-D spectra obtained. Several experiments were run for the 2-D spectra: COSY, Relay COSY, HOHAHA, and NOESY which were zero-filled in the F l dimension only. D 0 exchange spectra were also obtained. 6
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Reference standards. Pirlimycin was obtained as an Upjohn Control Reference Standard of purity >99% as the hydrochloride hydrate. Pirlimycin sulfoxide was prepared from the treatment of pirlimycin with hydrogen peroxide followed by recrystallization. Samples of pirlimycin adenylate and pirlimycin sulfoxide adenylate were prepared by the procedures described by Argoudelis et al (6). Comparative metabolism in the rat. Adult male (6) and female (6) Sprague-Dawley rats were housed individually in polycarbonate metabolism cages and were orally administered by gavage an aqueous solution of C-pirlimycin HCI. Five daily doses of 29 mg/kg/day were administered at 24-hour intervals to each rat. Urine and feces were collected at 24-hour intervals just before dose administration. The animals were sacrificed at 2 to 3 hours post-last-dose and liver, kidneys, and samples of flank muscle and abdominal fat carefully excised and placed into tared bottles. Homogenates of 2:1 water.tissue were prepared for combustion/LSC analysis. Metabolite profiles were obtained for liver, urine and feces as described above. 14
Milk decline study at 50 mg/quarter (IX). Twenty-six lactating cows (Holstein) identified to be mastitic in one or more quarters were treated with two intramammary infusions of 50 mg/quarter of pirlimycin hydrochloride into all four quarters at a 24hour interval. All cows were milked at 11-13 hour intervals following standard dairy farm practices. Samples from the individual cows (composite of all 4 quarters) were taken for analysis out to 96 hours post-last-dose. The samples were analyzed by the M.luteus cylinder/plate microbiological assay. Results And Discussion i4
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Radiolabeled C-pirlimycin was readily synthesized from C sodium cyanide and 4ethyl-pyridine N-oxide as shown in Figure 2. The final reduction step produced a 2:1 mixture of desired product and a biologically inactive stereoisomer. Recrystallization preferentially produced pirlimycin HCL in >98% radiochemical purity and >99% chemical purity with an overall radiochemical yield of 25%. 14
ADME studies. Twelve cows were administered two doses of C-pirlimycin at a dose rate of 200 mg/quarter into all 4 quarters at a 24-hour interval. This dose rate was selected as the highest potential dose rate before the final efficacious dose of 50 mg/quarter had been firmly established. This treatment rate thus resulted in a 4-fold overdose. Blood, milk, urine and feces were collected at various times following the first dose. Combustion analysis of whole blood produced the time course of total residue, as illustrated in Figure 3 for three of the cows. There was a slow absorption of pirlimycin across the udder membrane/blood barrier with maximum concentrations occurring in the 6- to 12-hour posttreatment period. The terminal depletion of the
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total residue following the second dose appeared to correspond to a two-compartment pharmacokinetic model and suggests a very slow overall elimination. Various pharmacokinetic parameters were derived following noncompartmental analysis (7) and are summarized in Table I. Subsequent analysis of plasma, which contained total residue at a concentration approximately equal to whole blood, showed that the plasma residue consisted almost exclusively of unchanged pirlimycin. Thus, the parameters in Table I are useful indicators of the overall pharmacokinetic behavior of pirlimycin in the dairy cow following intramammary administration.
Table I. Whole Blood Pharmacokinetics of Pirlimycin in the Dairy Cow by the Intramammary Route Parameter
Mean Value, η = 12
AUCo_ t abs
2.27 to 7.11 μg/hr/mL 2.89 ± .46 hours 12 hours 6-12 hours 0.083 ± .03 μ g / m L 0.131 ± .047 μ g / m L 0.0224 ± .009 hour 37.6 ± 17.4 hours
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Typical depletion of total residue in milk, expressed as a concentration-time course, is illustrated in Figure 4 for three of the cows. Milk can be treated as an elimination pathway and estimates of pharmacokinetic parameters can be made by the Sigma minus technique (7). However, the true focus of milk residue concentration determinations as a function of time is the decline of these residues to levels below the "safe concentration." This will be addressed later when the 50 mg/quarter dose rate study is discussed. The important observation made clear by Figure 4 is the biphasic shape of the concentration-time course following the second dose. We interpret this to reflect an initial rapid udder emptying of unabsorbed pirlimycin during the first 2 to 3 milkings post-treatment since each milking is not 100% efficient in milk removal. The slow terminal depletion phase represents systemic elimination of absorbed drug as it is transported back across the udder membrane/blood barrier.
Tissue residues.
The concentrations of total residues resulting from the 200 mg/quarter/ dose study in the various tissues at various withdrawal times are presented in Table II. Muscle and fat contained little or no detectable residue beyond day 6. Liver is clearly the target tissue for residue analysis and showed a first order depletion (r = .995) with a t of 5.7 days. Kidney depleted at a faster rate, with a t of 3.3 days. Pirlimycin was not sequestered in the udder as demonstrated by the relatively low concentration of total residue detected in udder. 1/2
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Figure 3. Time-course of C-pirlimycin total residue in whole blood in 3 cows treated twice at a 24-hour interval by the intramammary infusion of Cpirlimycin hydrochloride into all 4 quarters at 200 mg/quarter. 14
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Figure 4. Time-course of C-pirlimycin total residue in milk in 3 cows treated twice at a 24-hour interval by the intramammary infusion of C-pirlimycin hydrochloride into all 4 quarters at 200 mg/quarter. 14
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Table Π. Total
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in the Dairy Cow
C Pirlimycin Residues in Tissues - 4X I M M Dose Mean Concentration (n=3) in Parts Per Million 28 Day 14 Day 4 Day 6 Day
Tissue
Liver Kidney Muscle Fat Udder
9.18 1.96 0.10 0.22 0.97
± 1.37 ± 0.71 ± 0.04 ± 0.22 ± 0.62
7.13 0.78 0.05 0.03 0.13
± 1.28 ±0.17 ± 0.01 ± 0.01 (n=l)
0.50 ± .37 0.01 ± .01 (0) (0) (0)
3.57 ± .39 0.26 ± .05 0.02 ±
