J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12178
Disposition of ampicillin trihydrate in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle B. C. CREDILLE* , 1 S. GIGU ERE* T. W. VICKROY †
Credille, B. C., Giguere, S., Vickroy, T. W., Fishman, H. J., Jones, A. L., Mason, M. E., DiPietro, R. O., Ensley, D. T. Disposition of ampicillin trihydrate in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12178.
H. J. FISHMAN* A. L. JONES ‡ M. E. MASON* R. O. DIPIETRO* & D. T. ENSLEY § *Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA; †Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA; ‡Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA; §Boehringer Ingelheim Vetmedica, Inc, St. Joseph, MO, USA
The objective of this study was to determine the disposition of ampicillin in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. Ampicillin trihydrate was administered by intramuscular (i.m.) injection at a dose of 11 mg/kg of body weight every 24 h (n = 6, total of 3 doses) or every 12 h (n = 6, total of 5 doses) for 3 days. Concentrations of ampicillin were measured in plasma, uterine tissue, lochial fluid, and milk using HPLC with ultraviolet absorption. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (every 12 vs. every 24 h) and no significant interactions between dosing interval and sampling site on the pharmacokinetic variable measured or calculated. Median peak ampicillin concentration at steady-state was significantly higher in lochial fluid (5.27 lg/mL after q 24 h dosing) than other body fluids or tissues and significantly higher in plasma (3.11 lg/mL) compared to milk (0.49 lg/mL) or endometrial tissue (1.55 lg/mL). Ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle. (Paper received 22 April 2014; accepted for publication 18 September 2014) Brent C. Credille, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. E-mail: [email protected] 1 Present address: Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA Supported by Boehringer Ingelheim Vetmedica, Inc
INTRODUCTION Dairy cattle are susceptible to numerous infectious and metabolic disorders in the immediate postparturient period. Acute puerperal metritis (APM) is one of the most common clinical conditions seen in modern dairy cattle, affecting up to onethird of all cattle that calve (Sheldon et al., 2009). Consequences of APM include reduced milk production, impaired reproductive performance, increased culling risk and, in severe cases, death (Overton, 2008). The economic impact of APM has been studied extensively and current estimates suggest that the disease can cost individual producers $358 per case and the United States dairy industry $650 000 000 over the course of a single year (Overton & Fetrow, 2008). After calving, more than 90% of cattle experience some degree of contamination of the uterine lumen with bacteria. © 2014 John Wiley & Sons Ltd
Through the processes of uterine involution and normal immune function, most cattle clear this contamination and experience no complications. However, cattle with retained placenta, hypocalcemia, and significant negative energy balance fail to clear uterine contamination and develop APM (Overton, 2008). While a variety of micro-organisms may be isolated from the reproductive tract of both healthy postparturient cattle and cattle with APM, E. coli and Trueperella pyogenes represent the bacteria most commonly associated with clinical disease (Sheldon et al., 2009). Antimicrobials are a mainstay of therapy for cattle with APM. Currently, three antimicrobials are labeled for systemic use in cattle with APM: oxytetracycline dihydrate (Liquamycin LA200), ceftiofur hydrochloride (Excenel RTU), and ceftiofur crystalline free acid (Excede). The United States Food and Drug Administration (FDA) recently passed an order prohibiting 1
2 B. C. Credille et al.
extra-label use of all cephalosporins except cephapirin in major food producing species because of concerns over the development of resistance to similar 3rd generation cephalosporin compounds that are used commonly in critically ill human patients. While the use of ceftiofur-based products is allowed for cattle with APM, it is prudent to investigate the efficacy of other antimicrobials for treating cattle with this important clinical disease to reserve cephalosporin antibiotics for critically ill veterinary and human patients. Ampicillin trihydrate is an aminobenzyl penicillin labeled for the therapy of infections in cattle and calves caused by susceptible strains of Aerobacter spp., Klebsiella spp., Staphylococcus spp., Streptococcus spp., E. coli, and Pasteurella spp. In addition, the activity of ampicillin against anaerobes and some organisms encountered in cattle with APM is, in some cases, broader than that of ceftiofur (Samitz et al., 1996; Prescott, 2013). Recently, two clinical trials comparing ampicillin sodium to ceftiofur hydrochloride in cattle with APM demonstrated comparable clinical efficacy between the two compounds, (Moore et al., 2012; Lima et al., 2014). In addition, economic modeling has shown that, when used at current label dose and dosing frequency, ampicillin trihydrate costs less than ceftiofur hydrochloride when treating APM (Overton & Fetrow, 2008). Nevertheless, using ampicillin trihydrate to treat cattle with APM is currently considered extra label drug use and, as such, its use in these cases must be justified by the prescribing veterinarian. While emerging evidence would suggest that ampicillin trihydrate would have value as a therapeutic agent in dairy cattle with APM, the disposition of the drug in plasma, uterine tissue and lochial fluid has not been evaluated in postparturient dairy cattle, precluding the development of rational dosage regimens for this compound (Lima et al., 2014). Thus, the objectives of the study reported here were to determine the disposition of ampicillin trihydrate in plasma, uterine tissue, lochial fluid, and milk following once and twice daily intramuscular administration to healthy postpartum dairy cattle. MATERIALS AND METHODS Animals Twelve Holstein and Holstein cross-cattle between 2 and 6 years of age and weighing between 364 and 654 kg were selected for this study. All animals were enrolled in the study within 24 h of calving. The cattle were considered healthy on the basis of physical examination, complete blood count, and plasma biochemical profile. The cattle were milked 3 times daily and housed in a free-stall barn for the duration of the study. All procedures were approved by the Clinical Research Committee of the University of Georgia. Study design and sample collection Ampicillin trihydrate, in its market formulation (Polyflex Injectable Suspension, Boehringer Ingelheim Vetmedica, St
Joseph, MO, USA), was administered at a dose of 11 mg/kg of body weight every 24 h for 3 days via the intramuscular route in the cervical musculature of six cattle and at a dose of 11 mg/kg of body weight every 12 h for 3 days via the intramuscular route in the cervical musculature of a second group of six cattle. No more than 10 mL of suspension was deposited in any one site, and each injection site was separated by three inches. Blood samples for plasma separation were collected from the jugular vein via direct venipuncture at 30 min and at 1, 2, 4, 6, 8, 12, 24, 48, 48.5, 49, 50, 52, 56, 58, 60, and 72 h after initial drug administration in the 24 h dosing interval group and at 30 min, 1, 2, 4, 6, 8, 12, 48, 48.5, 49, 50, 52, 56, 58, and 60 h after initial drug administration in the 12 h dosing interval group. Lochial fluid and milk were collected at 2, 4, 8, 12, 24, 50, 52, 56, 60, and 72 h after initial drug administration in the 24 h dosing interval and at 2, 4, 8, 12, 50, 52, 56, and 60 h after initial drug administration in the 12 h dosing interval. Uterine biopsy was performed in all cattle in both dosing intervals at 50, 52, 56, 60, and 72 h after initial drug administration. All samples were stored at 80 °C until analysis. Collection of milk, lochial fluid and endometrial tissue Each teat end was cleaned by wiping with 70% isopropyl alcohol prior to sampling. The foremilk was removed from each quarter of the mammary gland and 1 mL of milk from each quarter was collected into 4 mL plastic tubes. The animal’s tail was held to the side and the external genitalia cleaned with alternating applications of 4% chlorhexidine and 70% isopropyl alcohol. A gloved hand was inserted into the uterine lumen and approximately 4 mL of lochial fluid was collected. Endometrial tissue (approximately 4 g) was collected using the Hauptner equine endometrial biopsy instrument. Measurement of ampicillin concentrations The concentration of ampicillin in plasma, milk, uterine tissue, and lochial fluid was measured using a validated analytical procedure based on modifications of a method reported previously by Nelis and coworkers (Nix et al., 1991). Samples of lochial fluid and endometrial biopsies were extracted and prepared in a manner different from samples of plasma and milk to eliminate interferences from sample matrices. In brief, frozen samples of plasma and milk were thawed, and aliquots (1 mL) were transferred into clean glass tubes containing 0.05 mL of cephalexin (20 lg/mL) as internal standard. In addition, milk samples were mixed with 2 mL of 0.067 M K2HPO4 (pH 4.0), vortex mixed and centrifuged for 20 min at 2200 g (room temperature). The supernatant fraction was removed and the extraction process repeated and concluded with combination of both supernatant fractions. Milk extracts and plasma samples were transferred onto C-18 SPE columns that were preconditioned sequentially with methanol, water and 0.067 M phosphate buffer (2 mL each). Columns were rinsed with 0.067 M phosphate buffer (2 mL) prior to elution © 2014 John Wiley & Sons Ltd
Disposition of ampicillin trihydrate in dairy cattle 3 Table 1. Plasma, milk, lochial fluid, and endometrial tissue noncompartmental pharmacokinetic variables (median and range) after i.m. administration of ampicillin trihydrate to cows at a dose of 11 mg/kg every 24 h (n = 6) or every 12 h (n = 6) for 3 days Variable First dose Cmax (lg/mL) Tmax (h) AUC0–t (lgh/mL) Clast (lg/mL) Last dose Cmax (lg/mL) Tmax (h) AUC0–t (lgh/mL) Clast (lg/mL) t1/2 (h)
Dosing interval
Plasma
Milk
Lochial fluid
q q q q q q q q
12 24 12 24 12 24 12 24
h h h h h h h h
1.66 2.61 6 4 33 32.1 0.23 0
(0.91–2.14)a (0.44–4.5)a (0.5–8) (2–12) (19–88.7)a (3.8–62.4)a (0–0.64)a (0–0.34)a
1.63 2.12 5 6 12.4 31.7 1 1.08
(0.91–3.98)a (0.93–3.27)a (2–12) (2–24) (7.9–34.4)a (3.1–46.5)a (0.18–3.98)a (0–2.57)a
75.7 55.7 3 6 423 343 13.40 6.97
(3.63–382)b (6.40–313)b (2–24) (2–12) (29.6–2710)b (117.3–1510)b (0–200.6)b (1–99.03)b
q q q q q q q q q q
12 24 12 24 12 24 12 24 12 24
h h h h h h h h h h
3.07 3.11 1 3 19.5 55.6 0.64 0.60 2.60 6.36
(1.67–4.7)a (2.2–5.4)a (1–4)a (1–4)a (7.6–65.1)a (24.1–106.3)a (0.22–1.34)a,b (0.13–1.50)a,b (1.21–14.9) (4.41–11.7)
0.78 0.49 3 6 21.2 5.35 0.96 0.24
(.68–1.7)c (0–1.48)c (2–8)a,b (2.0–24)a,b (0.4–56.5)a (0–53.9)a (0.04–1.36)a,b (0–1.09)a,b N/A
76.7 5.27 4 8 557 152 16.6 2.95
(29.9–153) b (2.4–129)b (2–12)a,b (2–24)a,b (436–4250)b (24.9–5031)b (0–41.7)a (1.8–29.6)a N/A
Endometrial tissue
N/A N/A N/A N/A
1.25 1.55 10 5 9.8 35.6 0 0
(0–1.9)c (0–13.8)c (2–24)b (2–12)b (0.5–63.5)a (0.5–183)a (0–5.1)b (0.40–1.1)b N/A
t1/2, terminal half-life; AUC0-t, Area under the plasma concentration vs. time curve from time 0 to the last quantifiable time point; Tmax, Time to maximum concentration; Cmax, Maximum concentration; Clast, Last quantifiable concentration. a,b Different letters within a row indicate statistically significant differences between sample types (P < 0.05).
Variable A (lg/mL) ka (1/h) kel (1/h) t1/2ka (h) t1/2kel (h) V/F (mg)/(lg/mL) Cl/F (mg)/(lg/mL)/h Tmax (h) Cmax (lg/mL) AUC0-∞ (lgh/mL)
Value 3.70 0.859 0.107 0.807 6.48 1447.5 165.6 2.77 2.41 30.26
A, intercept; Ka, absorption rate constant; Kel, elimination rate constant; t1/2ka, absorption half-life; t1/2kel, elimination half-life; F, bioavailability; V/F, apparent volume of distribution per fraction absorbed; CL/F, Systemic clearance per fraction absorbed; AUC0-∞, area under the plasma concentration vs. time curve extrapolated to infinity; Cmax, maximum plasma concentration; Tmax, time to maximum plasma concentration.
with methanol (2 mL for plasma and 2.5 mL of milk extract). Methanolic extracts were dried under a gentle stream of nitrogen at 40 °C, reconstituted in 1 mL of phosphate buffer and passed through 0.45 micron syringe filter prior to analysis by HPLC. Lochial fluid and endometrial tissue samples were prepared in a different manner to remove interfering matrix peaks. In brief, samples were weighed and approximately 1 g was placed in © 2014 John Wiley & Sons Ltd
10
Plasma ampicillin concentration (µg/mL)
Table 2. Compartmental pharmacokinetic variables derived from mean plasma concentration vs. time data after the first i.m. administration of ampicillin trihydrate to cows at a dose of 11 in the group treated every 24 h
q 24 h
q 12 h
1
0.1
0.01
0
12
24
36
48
60
72
Time (h)
Fig. 1. Mean (+ SD) plasma ampicillin concentration (lg/mL) after i.m. administration of ampicillin trihydrate at a dose of 11 mg/kg every 24 h (n = 6) or every 12 h (n = 6) for 3 days. Plasma was collected after administration of the first (time 0) and last (48 h) dose.
10 mL of acetonitrile:water (90:10) containing cephalexin internal standard then homogenized with a Polytron tissue digester (30 sec at setting 20). Homogenates were centrifuged (30 min at 2500 g), and aliquots of supernatant were transferred to clean tubes and mixed with hexane (10 mL). Samples were shaken vigorously (1 min), centrifuged (30 min at 2500 g), and the lower (aqueous) layer was transferred to a clean glass tube and dried under nitrogen at 50 °C. Dried extracts were reconstituted and filtered as described above prior to analysis. The concentration of ampicillin in all sample extracts was determined by high-performance liquid chromatography with ultraviolet
4 B. C. Credille et al.
model with 1/y2 weighting was most appropriate based upon computer assisted examination of residual plots, goodness of fit, and the Akaike information criterion: C¼
Fig. 2. Mean (+ SD) lochial fluid ampicillin concentration (lg/g) after i.m. administration of ampicillin trihydrate at a dose of 11 mg/kg every 24 h for 3 days. Lochial fluid was collected after administration of the first (time 0) and last (48 h) dose. The dotted horizontal line represents the MIC90 of E. coli isolates cultured from various sites in cattle.
absorption (210 nm). Ampicillin and internal standard were separated using an ODS Hypersil C-18 (5 lm) analytical column (4.6 9 250 mm, Thermofisher) and a two-step gradient with mobile phase containing a 94:6 mixture of 0.067 M phosphate buffer (pH 4): acetonitrile (0–35 min and 45–75 min) and a 50:50 mixture from 35 to 45 min at a flow rate of 1.0 mL/min. Peak areas were compared to a standard curve for ampicillin (0.025–10.0 lg/mL, 8 nonzero concentrations) that exhibited good linearity (R2 values above 0.98) and a RSD < 3.0% at 1 lg/mL. Average recovery of ampicillin (0.25, 1 and 2.5 lg/ mL) and cephalexin (1 lg/mL) was >90 percent from all tissue matrices. The limit of detection (LOD) for ampicillin was 0.05 lg/mL in plasma and milk and 0.10 lg/mL in lochial and endometrial samples. The lower limit of quantification (LLOQ) was 0.25 lg/mL in all matrices as determined by guidelines set forth in Guidance for Industry #145 (Bioanalytical Method Validation) from the FDA Center for Drug Evaluation and Research. Pharmacokinetic analysis For each cow, plasma, milk, lochial fluid, and endometrial tissue median ampicillin trihydrate concentration vs. time data were analyzed based on noncompartmental pharmacokinetics using commercial software (PK Solutions 2.0; Summit Research Services, Montrose, CO, USA). For plasma samples, the rate constant of the terminal phase (kz) was determined by linear regression of the terminal phase of the logarithmic plasma concentration vs. time curve using a minimum of three data points. Half-life of the terminal phase (t½kz) was calculated as ln 2 divided by kz. The area under the concentration–time curve (AUC) was calculated using the trapezoidal rule. For each animal, maximum concentration (Cmax), time to maximum concentration (Tmax), and concentration at the last sampling time (Clast) were calculated for each type of sample. Compartmental analysis was also used to model the mean plasma concentration vs. time data of the first dose in the q 24 h treatment group(Zhang et al., 2010). A one compartment
ka F D ½ekelt ekat V ðka kel Þ
where C is the plasma concentration, t is time, Ka is the nonIV absorption rate, assuming first-order absorption, Kel is the elimination rate constant, V is the apparent volume of distribution, F is the bioavailability, and D is the dose. Secondary parameters from the model included Cmax, Tmax, clearance per fraction absorbed (CL/F), AUC, and the respective absorption and terminal half-lives (t½). Statistical analysis Normality of the data and equality of variances were assessed using the Shapiro–Wilk and Levene’s tests, respectively. Variables that did not meet the assumptions for parametric testing were rank-transformed prior to analysis. A two-way ANOVA with one factor repetition was used to assess the effect of sample type (plasma, milk, lochial fluid, endometrial tissue), dosing interval (12 h vs. 24 h) and interactions between sample type and dosing interval each on measured and calculated pharmacokinetic variable. When applicable, multiple pairwise comparisons were made using the Holm–Sidak method. Differences were considered significant at P < 0.05.
RESULTS No adverse effects were noted in any cow during the course of the study. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (q 12 vs. q 24 h) and no significant interactions between dosing interval and sampling site on Cmax, Tmax, AUC0-t, t1/2, or Clast after administration of the first or last dose of ampicillin trihydrate (Table 1). Pharmacokinetic variables derived from the compartmental analysis are presented in Table 2. The plasma concentration vs. time profile for both dosing intervals is presented in Fig. 1. After administration of the first dose, there was a significant effect of sample type on Cmax (P = 0.004), AUC0-t (P = 0.007), and Clast (P = 0.017). The lochial fluid concentration vs. time profile for the once daily dosing interval is presented in Fig. 2. Median Cmax, AUC0-t, and Clast were significantly higher in lochial fluid than in plasma or milk (Table 1). After administration of the last dose, there was a significant effect of sample type on Cmax (P ≤ 0.001), Tmax (P = 0.031), AUC0-t (P ≤ 0.001), and Clast (P = 0.016). Median Cmax was significantly higher in lochial fluid than in other sample types and significantly higher in plasma than in milk or endometrial tissue (Table 1). Median AUC0-t was significantly higher in lochial fluid than in other sample types (Table 1). Median Tmax © 2014 John Wiley & Sons Ltd
Disposition of ampicillin trihydrate in dairy cattle 5
was significantly higher for endometrial tissue than for plasma, and median Clast was significantly higher for lochial fluid than for endometrial tissue (Table 1).
DISCUSSION Because ampicillin is active against many bacterial pathogens associated with APM and in light of recent evidence that ampicillin trihydrate is effective for the treatment of APM, we investigated the disposition of ampicillin trihydrate in plasma, uterine tissue, and lochial fluid of postpartum dairy cattle(Lima et al., 2014). Median peak plasma concentrations obtained in the present study (1.66–2.61 lg/mL) were lower than those previously reported after i.m. administration of a lower dose (7.7 mg/kg) of ampicillin trihydrate to calves (3.7 lg/mL) (Nouws et al., 1982). Nevertheless, the elimination half-life was similar (3.7 h) in this study was similar to what has been previously reported. However, the variability observed in the elimination half-life in the two groups in this study, particularly the q12 h dosing group, precludes further comparison. The inclusion of additional sampling times after the 12 h time point in this group would have allowed for more accurate determination of half-life and is a limitation of this study. Median peak drug concentrations in milk were higher in the present study (1.63–2.12 lg/mL) compared to levels following i.m. administration of an oil based suspension of ampicillin trihydrate (12.5 mg/kg) to milk from cows without (0.03 lg/mL) or with (0.2 lg/mL) mastitis (Gehring et al., 2005). While ampicillin bioavailability was not evaluated in the present study, the bioavailability of ampicillin trihydrate in calves after i.m. administration is nearly 100% (Nouws et al., 1982). Development of an optimal dosage regimen for a specific antimicrobial is dependent on both the pharmacokinetic and the pharmacodynamic parameters of the drug product being used (Giguere & Tessman, 2011). The most important factor determining the efficacy of b-lactam antimicrobials such as ampicillin trihydrate is the amount of time that concentrations of the drug exceed the MIC (minimum inhibitory concentration) against a given pathogen (Prescott, 2013). The goal of therapy with time dependent antimicrobials is to maintain plasma drug concentrations above the MIC of the gram-positive organisms for 40–50% of the dosing interval and above the MIC of gram-negative pathogens for 80–100% of the dosing interval (Toutain et al., 2002; McKellar et al., 2004; Giguere & Tessman, 2011; Martinez et al., 2012; Papich, 2014). Increasing the dose of antimicrobial administered or shortening the duration of time between doses are methods that are used to prolong the duration of time that concentrations of drug remains above the MIC of infecting pathogens (Giguere & Tessman, 2011). Therefore, it is conceivable that shortening the dosing interval of ampicillin trihydrate to every 12 h could potentially increase the duration of time that ampicillin concentrations remain above the MIC of potentially pathogenic bacteria. However, in the present study, administration of ampicillin trihydrate every 12 h, as compared to every 24 h, did not have any significant effect on the pharmacokinetic variables assessed. © 2014 John Wiley & Sons Ltd
Traditionally, most pharmacokinetic/pharmacodynamic studies of antimicrobials have held that drug concentration in plasma is an indicator of potential efficacy. While plasma drug concentration is an important driving force for penetration to the site of infection, the drug-concentration–time relationship at the site of infection may be quite different from that of plasma (Liu et al., 2002). The rate and extent of drug penetration into most sites outside the vascular space are also determined by the drug’s molecular charge and size, lipid solubility, extent of plasma protein binding, and by blood flow at the site of infection (Nix et al., 1991). The difference between total plasma concentration and free tissue concentrations can be substantial, particularly for drugs that are highly bound to plasma proteins. However, only the free fraction of the antimicrobial in the fluids at the target site is ultimately responsible for therapeutic success (Liu et al., 2002). In the uterus, a barrier consisting of endothelial cells, endometrial epithelial cells and the tight junctions between cells exists between the vascular compartment and lochial fluid. In the present study, median Cmax of ampicillin was significantly greater in lochial fluid than plasma for animals treated at either 12 h intervals (75.7 vs. 1.66 lg/mL) or 24 h intervals (55.7 vs. 2.61 lg/mL). In addition, concentrations of ampicillin achieved in lochial fluid in the present study were considerably above the MIC that inhibits 90% of the isolates of T. pyogenes (0.25 lg/mL) and E. coli (8.0 lg/mL) cultured from cattle (Yoshimura et al., 2000; Thomson et al., 2009; Wasyl et al., 2013). In the present study, ampicillin trihydrate was found to concentrate in lochial fluid as compared to plasma with a mean ratio of maximum lochial fluid to plasma ampicillin trihydrate concentration of 21.3 after the first dose and 1.7 following the last dose in the every 24 h dosing group and may reflect the effects of the attainment of a steady state. Currently, both ceftiofur hydrochloride and ceftiofur crystalline free acid (CCFA) are labeled for parenteral treatment of cattle with APM. Previous work evaluating ceftiofur hydrochloride has found that, although ceftiofur and its derivatives achieve therapeutic concentrations in both plasma and lochial fluid, the mean ratio of maximum lochial fluid to plasma ceftiofur concentrations is lower (0.34), suggesting that ceftiofur hydrochloride does not preferentially accumulate in lochial fluid (Okker et al., 2002). Studies evaluating CCFA have yielded similar results and found that the maximum lochial fluid to plasma concentration ratio of CCFA was 0.79, a finding that also suggests lack of preferential accumulation in lochial fluid (Witte et al., 2011). The preferential accumulation of ampicillin trihydrate in lochial fluid as compared to milk is important as antimicrobial accumulation at the site of infection is a primary determinant of efficacy and further supports the potential for ampicillin trihydrate as a therapeutic agent in cattle with APM. The reasons for this phenomenon are not clear. One potential explanation for the preferential accumulation of ampicillin in lochial fluid as compared to plasma is that lochial fluid is not continuously recirculated as is the case for other fluids (i.e., synovial fluid) in the body and this may allow for the accumulation of drug within the uterine lumen. Another potential explanation for the preferential accumulation of ampi-
6 B. C. Credille et al.
cillin in lochial fluid as compared to plasma is that, due to the relatively low degree of protein binding by ampicillin, the drug is better able to penetrate into sites with large amounts if inflammatory and cellular exudate (Barza & Weinstein, 1974). Indeed, the process of uterine involution is intimately associated with inflammatory processes and the accumulation of acute phase proteins may account for the penetration of ampicillin into lochial fluid in this study (Sheldon et al., 2014). Based on the results of the present study, ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle. In addition, administration of ampicillin trihydrate at the label dose once daily is equivalent to twice daily dosing and allows veterinarians to avoid using an extra-label dosing interval that might result in residues in meat and milk of treated cattle. Further work is needed to characterize the activity of ampicillin against common uterine pathogens of cattle and establish susceptibility breakpoints to better guide therapy.
REFERENCES Barza, M. & Weinstein, L. (1974) Penetration of antibiotics into fibrin loci in vivo. I. Comparison of penetration of ampicillin into fibrin clots, abscesses, and ‘interstitial fluid’. Journal of Infectious Diseases, 129, 59–65. Gehring, R., van der Merwe, D., Pierce, A.N., Baynes, R.E., Craigmill, A.L. & Riviere, J.E. (2005) Multivariate meta-analysis of pharmacokinetic studies of ampicillin trihydrate in cattle. American Journal of Veterinary Research, 66, 108–112. Giguere, S. & Tessman, R.K. (2011) Rational dosing of antimicrobial agents for bovine respiratory disease: the use of plasma vs. tissue concentrations in predicting efficacy. International Journal of Applied Research in Veterinary Medicine, 9, 342–355. Lima, F.S., Vieira-Neto, A., Vasconcellos, G.S., Mingoti, R.D., Karakaya, E., Sole, E., Bisinotto, R.S., Martinez, N., Risco, C.A., Galvao, K.N. & Santos, J.E. (2014) Efficacy of ampicillin trihydrate or ceftiofur hydrochloride for treatment of metritis and subsequent fertility in dairy cows. Journal of Dairy Science, 97, 5401–5414. Liu, P., Muller, M. & Derendorf, H. (2002) Rational dosing of antibiotics: the use of plasma concentrations vs. tissue concentrations. International Journal of Antimicrobial Agents, 19, 285–290. Martinez, M.N., Papich, M.G. & Drusano, G.L. (2012) Dosing regimen matters: the importance of early intervention and rapid attainment of the pharmacokinetic/pharmacodynamic target. Antimicrobial Agents and Chemotherapy, 56, 2795–2805. McKellar, Q.A., Sanchez Bruni, S.F. & Jones, D.G. (2004) Pharmacokinetic/pharmacodynamic relationships of antimicrobial drugs used in veterinary medicine. Journal of Veterinary Pharmacology and Therapeutics, 27, 503–514. Moore, D.A., Overton, M.W. & Sischo, W.M. (2012). Randomized clinical trial to compare the efficacy of two different antimicrobials on cure, milk production, and reproductive performance of dairy cows diagnosed with metritis. In Randomized clinical trial to compare the efficacy of two different antimicrobials on cure, milk production, and reproductive performance of dairy cows diagnosed with metritis p 236, Montreal, Quebec, Canada.
Nix, D.E., Goodwin, S.D., Peloquin, C.A., Rotella, D.L. & Schentag, J.J. (1991) Antibiotic tissue penetration and its relevance: impact of tissue penetration on infection response. Antimicrobial Agents and Chemotherapy, 35, 1953–1959. Nouws, J.F., van Ginneken, C.A., Hekman, P. & Ziv, G. (1982) Comparative plasma ampicillin levels and bioavailability of five parenteral ampicillin formulations in ruminant calves. Veterinary Quarterly, 4, 62–71. Okker, H., Schmitt, E.J., Vos, P.L., Scherpenisse, P., Bergwerff, A.A. & Jonker, F.H. (2002) Pharmacokinetics of ceftiofur in plasma and uterine secretions and tissues after subcutaneous postpartum administration in lactating dairy cows. Journal of Veterinary Pharmacology and Therapeutics, 25, 33–38. Overton, M. (2008) Periparturient management for post-parturient success. Proceedings of the North American Veterinary Conference, pp. 30– 38. Orlando, Florida. Overton, M.W. & Fetrow, J. (2008) Economics of post-partum uterine health. Proceedings of the Dairy Cattle Reproductive Council Convention, pp. 39–44. Omaha, Nebraska. Papich, M.G. (2014) Pharmacokinetic-pharmacodynamic (PK-PD) modeling and the rational selection of dosage regimes for the prudent use of antimicrobial drugs. Veterinary Microbiology, 171, 480–486. Prescott, J.F. (2013). Beta-lactam antibiotics: penam penicillins. In Antimicrobial Therapy in Veterinary Medicine, 5th edn. Eds Giguere, S., Prescott, J.F. & Dowling, P.M.. Wiley-Blackwell, Ames, IA. Samitz, E.M., Jang, S.S. & Hirsh, D.C. (1996) In vitro susceptibilities of selected obligate anaerobic bacteria obtained from bovine and equine sources to ceftiofur. Journal of Veterinary Diagnostic Investigation, 8, 121–123. Sheldon, I.M., Cronin, J., Goetze, L., Donofrio, G. & Schuberth, H.J. (2009) Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle. Biology of Reproduction, 81, 1025–1032. Sheldon, I.M., Cronin, J.G., Healey, G.D., Gabler, C., Heuwieser, W., Streyl, D., Bromfield, J., Miyamoto, A., Fergani, C. & Dobson, H. (2014) Innate immunity and inflammation of the bovine female reproductive tract in health and disease. Reproduction, 148, R41–51. Thomson, D.U., Taylor, W., Noffsinger, T., Christoper, J.A., Wileman, B.W. & Ragsdale, J. (2009) Case Report – Tail tip necrosis in a confined cattle feeding operation. Bovine Practitioner, 43, 18–22. Toutain, P.L., del Castillo, J.R. & Bousquet-Melou, A. (2002) The pharmacokinetic-pharmacodynamic approach to a rational dosage regimen for antibiotics. Research in Veterinary Science, 73, 105–114. Wasyl, D., Hoszowski, A., Zajac, M. & Szulowski, K. (2013) Antimicrobial resistance in commensal Escherichia coli isolated from animals at slaughter. Frontiers in Microbiology, 4, 221. Witte, T.S., Iwersen, M., Kaufmann, T., Scherpenisse, P., Bergwerff, A.A. & Heuwieser, W. (2011) Determination of ceftiofur derivatives in serum, endometrial tissue, and lochia in puerperal dairy cows after subcutaneous administration of ceftiofur crystalline free acid. Journal of Dairy Science, 94, 284–290. Yoshimura, H., Kojima, A. & Ishimaru, M. (2000) Antimicrobial susceptibility of Arcanobacterium pyogenes isolated from cattle and pigs. Journal of Veterinary Medicine B, Infectious Diseases and Veterinary Public Health, 47, 139–143. Zhang, Y., Huo, M., Zhou, J. & Xie, S. (2010) PKSolver: an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Computer Methods and Programs in Biomedicine, 99, 306–314.
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Disposition of ampicillin trihydrate in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle B. C. CREDILLE* , 1 S. GIGU ERE* T. W. VICKROY †
Credille, B. C., Giguere, S., Vickroy, T. W., Fishman, H. J., Jones, A. L., Mason, M. E., DiPietro, R. O., Ensley, D. T. Disposition of ampicillin trihydrate in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12178.
H. J. FISHMAN* A. L. JONES ‡ M. E. MASON* R. O. DIPIETRO* & D. T. ENSLEY § *Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA; †Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA; ‡Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA; §Boehringer Ingelheim Vetmedica, Inc, St. Joseph, MO, USA
The objective of this study was to determine the disposition of ampicillin in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. Ampicillin trihydrate was administered by intramuscular (i.m.) injection at a dose of 11 mg/kg of body weight every 24 h (n = 6, total of 3 doses) or every 12 h (n = 6, total of 5 doses) for 3 days. Concentrations of ampicillin were measured in plasma, uterine tissue, lochial fluid, and milk using HPLC with ultraviolet absorption. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (every 12 vs. every 24 h) and no significant interactions between dosing interval and sampling site on the pharmacokinetic variable measured or calculated. Median peak ampicillin concentration at steady-state was significantly higher in lochial fluid (5.27 lg/mL after q 24 h dosing) than other body fluids or tissues and significantly higher in plasma (3.11 lg/mL) compared to milk (0.49 lg/mL) or endometrial tissue (1.55 lg/mL). Ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle. (Paper received 22 April 2014; accepted for publication 18 September 2014) Brent C. Credille, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. E-mail: [email protected] 1 Present address: Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA Supported by Boehringer Ingelheim Vetmedica, Inc
INTRODUCTION Dairy cattle are susceptible to numerous infectious and metabolic disorders in the immediate postparturient period. Acute puerperal metritis (APM) is one of the most common clinical conditions seen in modern dairy cattle, affecting up to onethird of all cattle that calve (Sheldon et al., 2009). Consequences of APM include reduced milk production, impaired reproductive performance, increased culling risk and, in severe cases, death (Overton, 2008). The economic impact of APM has been studied extensively and current estimates suggest that the disease can cost individual producers $358 per case and the United States dairy industry $650 000 000 over the course of a single year (Overton & Fetrow, 2008). After calving, more than 90% of cattle experience some degree of contamination of the uterine lumen with bacteria. © 2014 John Wiley & Sons Ltd
Through the processes of uterine involution and normal immune function, most cattle clear this contamination and experience no complications. However, cattle with retained placenta, hypocalcemia, and significant negative energy balance fail to clear uterine contamination and develop APM (Overton, 2008). While a variety of micro-organisms may be isolated from the reproductive tract of both healthy postparturient cattle and cattle with APM, E. coli and Trueperella pyogenes represent the bacteria most commonly associated with clinical disease (Sheldon et al., 2009). Antimicrobials are a mainstay of therapy for cattle with APM. Currently, three antimicrobials are labeled for systemic use in cattle with APM: oxytetracycline dihydrate (Liquamycin LA200), ceftiofur hydrochloride (Excenel RTU), and ceftiofur crystalline free acid (Excede). The United States Food and Drug Administration (FDA) recently passed an order prohibiting 1
2 B. C. Credille et al.
extra-label use of all cephalosporins except cephapirin in major food producing species because of concerns over the development of resistance to similar 3rd generation cephalosporin compounds that are used commonly in critically ill human patients. While the use of ceftiofur-based products is allowed for cattle with APM, it is prudent to investigate the efficacy of other antimicrobials for treating cattle with this important clinical disease to reserve cephalosporin antibiotics for critically ill veterinary and human patients. Ampicillin trihydrate is an aminobenzyl penicillin labeled for the therapy of infections in cattle and calves caused by susceptible strains of Aerobacter spp., Klebsiella spp., Staphylococcus spp., Streptococcus spp., E. coli, and Pasteurella spp. In addition, the activity of ampicillin against anaerobes and some organisms encountered in cattle with APM is, in some cases, broader than that of ceftiofur (Samitz et al., 1996; Prescott, 2013). Recently, two clinical trials comparing ampicillin sodium to ceftiofur hydrochloride in cattle with APM demonstrated comparable clinical efficacy between the two compounds, (Moore et al., 2012; Lima et al., 2014). In addition, economic modeling has shown that, when used at current label dose and dosing frequency, ampicillin trihydrate costs less than ceftiofur hydrochloride when treating APM (Overton & Fetrow, 2008). Nevertheless, using ampicillin trihydrate to treat cattle with APM is currently considered extra label drug use and, as such, its use in these cases must be justified by the prescribing veterinarian. While emerging evidence would suggest that ampicillin trihydrate would have value as a therapeutic agent in dairy cattle with APM, the disposition of the drug in plasma, uterine tissue and lochial fluid has not been evaluated in postparturient dairy cattle, precluding the development of rational dosage regimens for this compound (Lima et al., 2014). Thus, the objectives of the study reported here were to determine the disposition of ampicillin trihydrate in plasma, uterine tissue, lochial fluid, and milk following once and twice daily intramuscular administration to healthy postpartum dairy cattle. MATERIALS AND METHODS Animals Twelve Holstein and Holstein cross-cattle between 2 and 6 years of age and weighing between 364 and 654 kg were selected for this study. All animals were enrolled in the study within 24 h of calving. The cattle were considered healthy on the basis of physical examination, complete blood count, and plasma biochemical profile. The cattle were milked 3 times daily and housed in a free-stall barn for the duration of the study. All procedures were approved by the Clinical Research Committee of the University of Georgia. Study design and sample collection Ampicillin trihydrate, in its market formulation (Polyflex Injectable Suspension, Boehringer Ingelheim Vetmedica, St
Joseph, MO, USA), was administered at a dose of 11 mg/kg of body weight every 24 h for 3 days via the intramuscular route in the cervical musculature of six cattle and at a dose of 11 mg/kg of body weight every 12 h for 3 days via the intramuscular route in the cervical musculature of a second group of six cattle. No more than 10 mL of suspension was deposited in any one site, and each injection site was separated by three inches. Blood samples for plasma separation were collected from the jugular vein via direct venipuncture at 30 min and at 1, 2, 4, 6, 8, 12, 24, 48, 48.5, 49, 50, 52, 56, 58, 60, and 72 h after initial drug administration in the 24 h dosing interval group and at 30 min, 1, 2, 4, 6, 8, 12, 48, 48.5, 49, 50, 52, 56, 58, and 60 h after initial drug administration in the 12 h dosing interval group. Lochial fluid and milk were collected at 2, 4, 8, 12, 24, 50, 52, 56, 60, and 72 h after initial drug administration in the 24 h dosing interval and at 2, 4, 8, 12, 50, 52, 56, and 60 h after initial drug administration in the 12 h dosing interval. Uterine biopsy was performed in all cattle in both dosing intervals at 50, 52, 56, 60, and 72 h after initial drug administration. All samples were stored at 80 °C until analysis. Collection of milk, lochial fluid and endometrial tissue Each teat end was cleaned by wiping with 70% isopropyl alcohol prior to sampling. The foremilk was removed from each quarter of the mammary gland and 1 mL of milk from each quarter was collected into 4 mL plastic tubes. The animal’s tail was held to the side and the external genitalia cleaned with alternating applications of 4% chlorhexidine and 70% isopropyl alcohol. A gloved hand was inserted into the uterine lumen and approximately 4 mL of lochial fluid was collected. Endometrial tissue (approximately 4 g) was collected using the Hauptner equine endometrial biopsy instrument. Measurement of ampicillin concentrations The concentration of ampicillin in plasma, milk, uterine tissue, and lochial fluid was measured using a validated analytical procedure based on modifications of a method reported previously by Nelis and coworkers (Nix et al., 1991). Samples of lochial fluid and endometrial biopsies were extracted and prepared in a manner different from samples of plasma and milk to eliminate interferences from sample matrices. In brief, frozen samples of plasma and milk were thawed, and aliquots (1 mL) were transferred into clean glass tubes containing 0.05 mL of cephalexin (20 lg/mL) as internal standard. In addition, milk samples were mixed with 2 mL of 0.067 M K2HPO4 (pH 4.0), vortex mixed and centrifuged for 20 min at 2200 g (room temperature). The supernatant fraction was removed and the extraction process repeated and concluded with combination of both supernatant fractions. Milk extracts and plasma samples were transferred onto C-18 SPE columns that were preconditioned sequentially with methanol, water and 0.067 M phosphate buffer (2 mL each). Columns were rinsed with 0.067 M phosphate buffer (2 mL) prior to elution © 2014 John Wiley & Sons Ltd
Disposition of ampicillin trihydrate in dairy cattle 3 Table 1. Plasma, milk, lochial fluid, and endometrial tissue noncompartmental pharmacokinetic variables (median and range) after i.m. administration of ampicillin trihydrate to cows at a dose of 11 mg/kg every 24 h (n = 6) or every 12 h (n = 6) for 3 days Variable First dose Cmax (lg/mL) Tmax (h) AUC0–t (lgh/mL) Clast (lg/mL) Last dose Cmax (lg/mL) Tmax (h) AUC0–t (lgh/mL) Clast (lg/mL) t1/2 (h)
Dosing interval
Plasma
Milk
Lochial fluid
q q q q q q q q
12 24 12 24 12 24 12 24
h h h h h h h h
1.66 2.61 6 4 33 32.1 0.23 0
(0.91–2.14)a (0.44–4.5)a (0.5–8) (2–12) (19–88.7)a (3.8–62.4)a (0–0.64)a (0–0.34)a
1.63 2.12 5 6 12.4 31.7 1 1.08
(0.91–3.98)a (0.93–3.27)a (2–12) (2–24) (7.9–34.4)a (3.1–46.5)a (0.18–3.98)a (0–2.57)a
75.7 55.7 3 6 423 343 13.40 6.97
(3.63–382)b (6.40–313)b (2–24) (2–12) (29.6–2710)b (117.3–1510)b (0–200.6)b (1–99.03)b
q q q q q q q q q q
12 24 12 24 12 24 12 24 12 24
h h h h h h h h h h
3.07 3.11 1 3 19.5 55.6 0.64 0.60 2.60 6.36
(1.67–4.7)a (2.2–5.4)a (1–4)a (1–4)a (7.6–65.1)a (24.1–106.3)a (0.22–1.34)a,b (0.13–1.50)a,b (1.21–14.9) (4.41–11.7)
0.78 0.49 3 6 21.2 5.35 0.96 0.24
(.68–1.7)c (0–1.48)c (2–8)a,b (2.0–24)a,b (0.4–56.5)a (0–53.9)a (0.04–1.36)a,b (0–1.09)a,b N/A
76.7 5.27 4 8 557 152 16.6 2.95
(29.9–153) b (2.4–129)b (2–12)a,b (2–24)a,b (436–4250)b (24.9–5031)b (0–41.7)a (1.8–29.6)a N/A
Endometrial tissue
N/A N/A N/A N/A
1.25 1.55 10 5 9.8 35.6 0 0
(0–1.9)c (0–13.8)c (2–24)b (2–12)b (0.5–63.5)a (0.5–183)a (0–5.1)b (0.40–1.1)b N/A
t1/2, terminal half-life; AUC0-t, Area under the plasma concentration vs. time curve from time 0 to the last quantifiable time point; Tmax, Time to maximum concentration; Cmax, Maximum concentration; Clast, Last quantifiable concentration. a,b Different letters within a row indicate statistically significant differences between sample types (P < 0.05).
Variable A (lg/mL) ka (1/h) kel (1/h) t1/2ka (h) t1/2kel (h) V/F (mg)/(lg/mL) Cl/F (mg)/(lg/mL)/h Tmax (h) Cmax (lg/mL) AUC0-∞ (lgh/mL)
Value 3.70 0.859 0.107 0.807 6.48 1447.5 165.6 2.77 2.41 30.26
A, intercept; Ka, absorption rate constant; Kel, elimination rate constant; t1/2ka, absorption half-life; t1/2kel, elimination half-life; F, bioavailability; V/F, apparent volume of distribution per fraction absorbed; CL/F, Systemic clearance per fraction absorbed; AUC0-∞, area under the plasma concentration vs. time curve extrapolated to infinity; Cmax, maximum plasma concentration; Tmax, time to maximum plasma concentration.
with methanol (2 mL for plasma and 2.5 mL of milk extract). Methanolic extracts were dried under a gentle stream of nitrogen at 40 °C, reconstituted in 1 mL of phosphate buffer and passed through 0.45 micron syringe filter prior to analysis by HPLC. Lochial fluid and endometrial tissue samples were prepared in a different manner to remove interfering matrix peaks. In brief, samples were weighed and approximately 1 g was placed in © 2014 John Wiley & Sons Ltd
10
Plasma ampicillin concentration (µg/mL)
Table 2. Compartmental pharmacokinetic variables derived from mean plasma concentration vs. time data after the first i.m. administration of ampicillin trihydrate to cows at a dose of 11 in the group treated every 24 h
q 24 h
q 12 h
1
0.1
0.01
0
12
24
36
48
60
72
Time (h)
Fig. 1. Mean (+ SD) plasma ampicillin concentration (lg/mL) after i.m. administration of ampicillin trihydrate at a dose of 11 mg/kg every 24 h (n = 6) or every 12 h (n = 6) for 3 days. Plasma was collected after administration of the first (time 0) and last (48 h) dose.
10 mL of acetonitrile:water (90:10) containing cephalexin internal standard then homogenized with a Polytron tissue digester (30 sec at setting 20). Homogenates were centrifuged (30 min at 2500 g), and aliquots of supernatant were transferred to clean tubes and mixed with hexane (10 mL). Samples were shaken vigorously (1 min), centrifuged (30 min at 2500 g), and the lower (aqueous) layer was transferred to a clean glass tube and dried under nitrogen at 50 °C. Dried extracts were reconstituted and filtered as described above prior to analysis. The concentration of ampicillin in all sample extracts was determined by high-performance liquid chromatography with ultraviolet
4 B. C. Credille et al.
model with 1/y2 weighting was most appropriate based upon computer assisted examination of residual plots, goodness of fit, and the Akaike information criterion: C¼
Fig. 2. Mean (+ SD) lochial fluid ampicillin concentration (lg/g) after i.m. administration of ampicillin trihydrate at a dose of 11 mg/kg every 24 h for 3 days. Lochial fluid was collected after administration of the first (time 0) and last (48 h) dose. The dotted horizontal line represents the MIC90 of E. coli isolates cultured from various sites in cattle.
absorption (210 nm). Ampicillin and internal standard were separated using an ODS Hypersil C-18 (5 lm) analytical column (4.6 9 250 mm, Thermofisher) and a two-step gradient with mobile phase containing a 94:6 mixture of 0.067 M phosphate buffer (pH 4): acetonitrile (0–35 min and 45–75 min) and a 50:50 mixture from 35 to 45 min at a flow rate of 1.0 mL/min. Peak areas were compared to a standard curve for ampicillin (0.025–10.0 lg/mL, 8 nonzero concentrations) that exhibited good linearity (R2 values above 0.98) and a RSD < 3.0% at 1 lg/mL. Average recovery of ampicillin (0.25, 1 and 2.5 lg/ mL) and cephalexin (1 lg/mL) was >90 percent from all tissue matrices. The limit of detection (LOD) for ampicillin was 0.05 lg/mL in plasma and milk and 0.10 lg/mL in lochial and endometrial samples. The lower limit of quantification (LLOQ) was 0.25 lg/mL in all matrices as determined by guidelines set forth in Guidance for Industry #145 (Bioanalytical Method Validation) from the FDA Center for Drug Evaluation and Research. Pharmacokinetic analysis For each cow, plasma, milk, lochial fluid, and endometrial tissue median ampicillin trihydrate concentration vs. time data were analyzed based on noncompartmental pharmacokinetics using commercial software (PK Solutions 2.0; Summit Research Services, Montrose, CO, USA). For plasma samples, the rate constant of the terminal phase (kz) was determined by linear regression of the terminal phase of the logarithmic plasma concentration vs. time curve using a minimum of three data points. Half-life of the terminal phase (t½kz) was calculated as ln 2 divided by kz. The area under the concentration–time curve (AUC) was calculated using the trapezoidal rule. For each animal, maximum concentration (Cmax), time to maximum concentration (Tmax), and concentration at the last sampling time (Clast) were calculated for each type of sample. Compartmental analysis was also used to model the mean plasma concentration vs. time data of the first dose in the q 24 h treatment group(Zhang et al., 2010). A one compartment
ka F D ½ekelt ekat V ðka kel Þ
where C is the plasma concentration, t is time, Ka is the nonIV absorption rate, assuming first-order absorption, Kel is the elimination rate constant, V is the apparent volume of distribution, F is the bioavailability, and D is the dose. Secondary parameters from the model included Cmax, Tmax, clearance per fraction absorbed (CL/F), AUC, and the respective absorption and terminal half-lives (t½). Statistical analysis Normality of the data and equality of variances were assessed using the Shapiro–Wilk and Levene’s tests, respectively. Variables that did not meet the assumptions for parametric testing were rank-transformed prior to analysis. A two-way ANOVA with one factor repetition was used to assess the effect of sample type (plasma, milk, lochial fluid, endometrial tissue), dosing interval (12 h vs. 24 h) and interactions between sample type and dosing interval each on measured and calculated pharmacokinetic variable. When applicable, multiple pairwise comparisons were made using the Holm–Sidak method. Differences were considered significant at P < 0.05.
RESULTS No adverse effects were noted in any cow during the course of the study. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (q 12 vs. q 24 h) and no significant interactions between dosing interval and sampling site on Cmax, Tmax, AUC0-t, t1/2, or Clast after administration of the first or last dose of ampicillin trihydrate (Table 1). Pharmacokinetic variables derived from the compartmental analysis are presented in Table 2. The plasma concentration vs. time profile for both dosing intervals is presented in Fig. 1. After administration of the first dose, there was a significant effect of sample type on Cmax (P = 0.004), AUC0-t (P = 0.007), and Clast (P = 0.017). The lochial fluid concentration vs. time profile for the once daily dosing interval is presented in Fig. 2. Median Cmax, AUC0-t, and Clast were significantly higher in lochial fluid than in plasma or milk (Table 1). After administration of the last dose, there was a significant effect of sample type on Cmax (P ≤ 0.001), Tmax (P = 0.031), AUC0-t (P ≤ 0.001), and Clast (P = 0.016). Median Cmax was significantly higher in lochial fluid than in other sample types and significantly higher in plasma than in milk or endometrial tissue (Table 1). Median AUC0-t was significantly higher in lochial fluid than in other sample types (Table 1). Median Tmax © 2014 John Wiley & Sons Ltd
Disposition of ampicillin trihydrate in dairy cattle 5
was significantly higher for endometrial tissue than for plasma, and median Clast was significantly higher for lochial fluid than for endometrial tissue (Table 1).
DISCUSSION Because ampicillin is active against many bacterial pathogens associated with APM and in light of recent evidence that ampicillin trihydrate is effective for the treatment of APM, we investigated the disposition of ampicillin trihydrate in plasma, uterine tissue, and lochial fluid of postpartum dairy cattle(Lima et al., 2014). Median peak plasma concentrations obtained in the present study (1.66–2.61 lg/mL) were lower than those previously reported after i.m. administration of a lower dose (7.7 mg/kg) of ampicillin trihydrate to calves (3.7 lg/mL) (Nouws et al., 1982). Nevertheless, the elimination half-life was similar (3.7 h) in this study was similar to what has been previously reported. However, the variability observed in the elimination half-life in the two groups in this study, particularly the q12 h dosing group, precludes further comparison. The inclusion of additional sampling times after the 12 h time point in this group would have allowed for more accurate determination of half-life and is a limitation of this study. Median peak drug concentrations in milk were higher in the present study (1.63–2.12 lg/mL) compared to levels following i.m. administration of an oil based suspension of ampicillin trihydrate (12.5 mg/kg) to milk from cows without (0.03 lg/mL) or with (0.2 lg/mL) mastitis (Gehring et al., 2005). While ampicillin bioavailability was not evaluated in the present study, the bioavailability of ampicillin trihydrate in calves after i.m. administration is nearly 100% (Nouws et al., 1982). Development of an optimal dosage regimen for a specific antimicrobial is dependent on both the pharmacokinetic and the pharmacodynamic parameters of the drug product being used (Giguere & Tessman, 2011). The most important factor determining the efficacy of b-lactam antimicrobials such as ampicillin trihydrate is the amount of time that concentrations of the drug exceed the MIC (minimum inhibitory concentration) against a given pathogen (Prescott, 2013). The goal of therapy with time dependent antimicrobials is to maintain plasma drug concentrations above the MIC of the gram-positive organisms for 40–50% of the dosing interval and above the MIC of gram-negative pathogens for 80–100% of the dosing interval (Toutain et al., 2002; McKellar et al., 2004; Giguere & Tessman, 2011; Martinez et al., 2012; Papich, 2014). Increasing the dose of antimicrobial administered or shortening the duration of time between doses are methods that are used to prolong the duration of time that concentrations of drug remains above the MIC of infecting pathogens (Giguere & Tessman, 2011). Therefore, it is conceivable that shortening the dosing interval of ampicillin trihydrate to every 12 h could potentially increase the duration of time that ampicillin concentrations remain above the MIC of potentially pathogenic bacteria. However, in the present study, administration of ampicillin trihydrate every 12 h, as compared to every 24 h, did not have any significant effect on the pharmacokinetic variables assessed. © 2014 John Wiley & Sons Ltd
Traditionally, most pharmacokinetic/pharmacodynamic studies of antimicrobials have held that drug concentration in plasma is an indicator of potential efficacy. While plasma drug concentration is an important driving force for penetration to the site of infection, the drug-concentration–time relationship at the site of infection may be quite different from that of plasma (Liu et al., 2002). The rate and extent of drug penetration into most sites outside the vascular space are also determined by the drug’s molecular charge and size, lipid solubility, extent of plasma protein binding, and by blood flow at the site of infection (Nix et al., 1991). The difference between total plasma concentration and free tissue concentrations can be substantial, particularly for drugs that are highly bound to plasma proteins. However, only the free fraction of the antimicrobial in the fluids at the target site is ultimately responsible for therapeutic success (Liu et al., 2002). In the uterus, a barrier consisting of endothelial cells, endometrial epithelial cells and the tight junctions between cells exists between the vascular compartment and lochial fluid. In the present study, median Cmax of ampicillin was significantly greater in lochial fluid than plasma for animals treated at either 12 h intervals (75.7 vs. 1.66 lg/mL) or 24 h intervals (55.7 vs. 2.61 lg/mL). In addition, concentrations of ampicillin achieved in lochial fluid in the present study were considerably above the MIC that inhibits 90% of the isolates of T. pyogenes (0.25 lg/mL) and E. coli (8.0 lg/mL) cultured from cattle (Yoshimura et al., 2000; Thomson et al., 2009; Wasyl et al., 2013). In the present study, ampicillin trihydrate was found to concentrate in lochial fluid as compared to plasma with a mean ratio of maximum lochial fluid to plasma ampicillin trihydrate concentration of 21.3 after the first dose and 1.7 following the last dose in the every 24 h dosing group and may reflect the effects of the attainment of a steady state. Currently, both ceftiofur hydrochloride and ceftiofur crystalline free acid (CCFA) are labeled for parenteral treatment of cattle with APM. Previous work evaluating ceftiofur hydrochloride has found that, although ceftiofur and its derivatives achieve therapeutic concentrations in both plasma and lochial fluid, the mean ratio of maximum lochial fluid to plasma ceftiofur concentrations is lower (0.34), suggesting that ceftiofur hydrochloride does not preferentially accumulate in lochial fluid (Okker et al., 2002). Studies evaluating CCFA have yielded similar results and found that the maximum lochial fluid to plasma concentration ratio of CCFA was 0.79, a finding that also suggests lack of preferential accumulation in lochial fluid (Witte et al., 2011). The preferential accumulation of ampicillin trihydrate in lochial fluid as compared to milk is important as antimicrobial accumulation at the site of infection is a primary determinant of efficacy and further supports the potential for ampicillin trihydrate as a therapeutic agent in cattle with APM. The reasons for this phenomenon are not clear. One potential explanation for the preferential accumulation of ampicillin in lochial fluid as compared to plasma is that lochial fluid is not continuously recirculated as is the case for other fluids (i.e., synovial fluid) in the body and this may allow for the accumulation of drug within the uterine lumen. Another potential explanation for the preferential accumulation of ampi-
6 B. C. Credille et al.
cillin in lochial fluid as compared to plasma is that, due to the relatively low degree of protein binding by ampicillin, the drug is better able to penetrate into sites with large amounts if inflammatory and cellular exudate (Barza & Weinstein, 1974). Indeed, the process of uterine involution is intimately associated with inflammatory processes and the accumulation of acute phase proteins may account for the penetration of ampicillin into lochial fluid in this study (Sheldon et al., 2014). Based on the results of the present study, ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle. In addition, administration of ampicillin trihydrate at the label dose once daily is equivalent to twice daily dosing and allows veterinarians to avoid using an extra-label dosing interval that might result in residues in meat and milk of treated cattle. Further work is needed to characterize the activity of ampicillin against common uterine pathogens of cattle and establish susceptibility breakpoints to better guide therapy.
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