Bovine respiratory tract disease continues to cause substantial morbidity and death in young dairy and veal calves.1,2 In dairy calves, pneumonia is a multifactorial disease, and its incidence and severity are affected by the environment, herd management factors, and calf age and immunity.3 Tulathromycin, a semisynthetic macrolide of the subclass triamilide, is safe and effective for the treatment of BRD associated with Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis and also for the control of disease in cattle at high risk of developing BRD associated with M haemolytica, P multocida, H somni, and M bovis. Tulathromycin has excellent bacteriostatic and some bactericidal activity against many of those pathogens, although results of a recent study4 indicate several bovine pathogens have developed substantial resistance to the drug. Currently, tulathromycin is approved for use in cattle of all ages, including calves intended to be processed for veal, as long as an appropriately extended meat withdrawal time is observed.
Similar to other macrolides, results of pharmacokinetic studies of tulathromycin in cattle,5 swine,6,7 deer,8 bison,9 and foals10 indicate that the drug is rapidly absorbed following SC and IM injection, readily accumulates in lung tissue, and has a prolonged t1/2 in lung homogenate and PELF. In one5 of those studies, tulathromycin was measured in plasma, PELF, and ISF of healthy 6-month-old calves (ie, calves with fully functional rumens). Although the pharmacokinetics of tulathromycin in plasma has been described for preruminant calves,11 to our knowledge, the pharmacokinetics of the drug has not been compared among blood, ISF, and PELF of preruminant calves. Maturation from preruminant to ruminant can affect drug metabolism,12,13 transporter function,14 body composition (including body fat, muscle, and water content),15 blood flow,16 and blood composition in terms of plasma proteins and cellular constituents.17
Bovine neutrophils have a high affinity for tulathromycin.18 In a study19 of neonatal Holstein calves, the number of circulating neutrophils peaked between birth and 8 hours old and then gradually decreased between peak and 30 days old (ie, end of the observation period). The rate of endotoxin-induced neutrophil migration is greater in neonatal calves than in adult cattle.20 In rats, immature neutrophil function was associated with a decrease in zymosan-induced lung damage,21 which indicated that age affects neutrophil function as well as the pathogenesis and progression of disease.
Ultimately, effective dosing strategies should be determined on the basis of the drug concentration at the site of action relative to the in vitro susceptibility (ie, minimal inhibitory concentration) of the target bacteria to that drug. Additionally, for any drug, randomized controlled clinical trials should be conducted to determine the accuracy of pharmacokinetic-pharmacodynamic predictions. For some drugs, the blood free-drug (unbound) concentration can be used to approximate the drug concentration available to treat extracellular infections. Unfortunately, that principle does not apply to macrolides, for which drug concentrations can differ markedly among lung, ISF, and blood. Thus, elucidation of the distribution patterns of antimicrobials in the ISF and PELF of various subpopulations of cattle may improve the pharmacokinetic-pharmacodynamic correlations for many bacterial pathogens. However, the extent to which age affects drug exposure (clearance) and plasma versus tissue (lung and ISF) tulathromycin concentrations has yet to be evaluated in cattle. The purpose of the study reported here was to compare the pharmacokinetics of tulathromycin between healthy 3-week-old (preweaned) and 6-month-old (weaned) calves and to characterize the distribution of the drug into PELF and ISF of preweaned and weaned calves following administration of a single dose of the drug (2.5 mg/kg, SC).
Materials and Methods
Animals
All study procedures were reviewed and approved by the North Carolina State University Institutional Animal Care and Use Committee. Holstein steer calves were purchased from the North Carolina State University dairy herd for the study. The study population consisted of 8 preweaned calves and 8 weaned calves. The preweaned calves were 2 to 3 weeks old and weighed between 41 and 53 kg. Those calves were housed at the university dairy farm where they had ad libitum access to water and a calf starter ration and were fed a commercial nonmedicated milk replacer twice daily. The weaned calves were 6 months old and weighed between 151 and 214 kg. Those calves were housed indoors at a university laboratory animal facility and had ad libitum access to water and grass hay and received a grain supplement on a daily basis. None of the calves had a history of disease or antimicrobial treatment, and all were considered healthy on the basis of results of a physical examination performed immediately prior to study initiation.
Tulathromycin administration
All calves were individually weighed on a digital scale the morning of tulathromycin administration so that the volume of the drug to be administered could be calculated. Each calf received 1 dose of tulathromycina (2.5 mg/kg) SC in the neck region on the side opposite the ultrafiltration probe in accordance with the label instructions.
Blood sample collection
Approximately 24 hours prior to tulathromycin administration, a 14-gauge, 3.25-inch IV catheter was aseptically placed in the right jugular vein of each calf to facilitate collection of serial blood samples. The catheter was sutured to the skin with 2–0 monofilament suture and was flushed 4 times daily (at approx 6-hour intervals) with 6 mL of heparinized saline (0.9% NaCl) solution (10 U of heparin/mL of saline solution). A blood sample (6 mL) was obtained from the catheter immediately before (0 hours) and 0.25, 0.5, 1, 2, 3, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, and 312 hours after tulathromycin administration. At each sample acquisition time, approximately 6 mL of blood was aspirated from the catheter to ensure that the analyzed blood sample was not diluted with any residual heparinized saline solution in the catheter from the previous flush. Then, the blood sample to be analyzed was collected, the blood sample aspirated from the catheter before the analyzed blood sample was reinfused, and the catheter was flushed with 6 mL of heparinized saline solution. The blood samples for analysis were immediately transferred to blood collection tubes containing lithium heparin as an anticoagulant and stored on ice. All blood samples were centrifuged at approximately 3,500 × g for 10 minutes within 1 hour after collection. The plasma was harvested from each sample, placed in a cryovial, and stored frozen at −80°C until analysis.
ISF collection
Approximately 24 hours prior to tulathromycin administration, an ultrafiltration probeb was aseptically placed SC in the left side of the neck of all calves (ie, side opposite that used for the tulathromycin injection) just cranial to the scapula. Each calf was sedated with xylazinec (0.05 to 0.1 mg/kg, IM in the cervical muscles) for probe placement.
Each probe contained 3 semipermeable loops connected to a nonpermeable tube that extended outside the calf and was attached to a 3-mL evacuated plastic blood collection tube that did not contain any anticoagulants. The tube provided negative pressure for collection of fluid through small pores in the probe membranes. Those pores allowed for the movement of water, electrolytes, and low-molecular-weight molecules (< 30,000 Da) but not large molecules, such as proteins, protein-bound drugs, and cells, to pass into collection tube. Calves were instrumented with the probes 24 hours before tulathromycin administration to allow for equilibration of ISF around the probe prior to initiation of sample collection.
The tube used to collect the ISF was replaced immediately before (0 hours) and at 2, 3, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, and 312 hours after tulathromycin administration. Because each ISF sample represented fluid collected over a certain amount of time (ie, was not collected instantaneously as for a blood sample), a lag time was calculated for each sample on the basis of the duration that the tube was attached to the probe and the volume of ISF collected.
BALF collection
Bronchoalveolar lavage fluid samples were collected as described.22 Briefly, a sterile 36-inch flexible 10F catheterd with a 3-mL balloon cuff was used to collect BALF samples from preweaned calves, and a sterile 59-inch flexible 24F catheterd with a 30-mL balloon cuff was used to collect BALF samples from weaned calves. For each sample acquisition, the calf was manually restrained and its head and neck were extended to facilitate passage of the catheter over a sterile guidewire. The guidewire and catheter were introduced into the ventral meatus of a nostril and advanced into the trachea until the catheter was wedged in a terminal bronchus. The balloon cuff was inflated to create a seal, and the catheter was held firmly in place while the guidewire was removed. Sterile saline solution (100 mL) was infused into the catheter, and the fluid was immediately aspirated, which limited the time that the fluid was allowed to dwell in the bronchus. At each sample acquisition, the volume of fluid retrieved ranged from 0 to 42.5 mL, and the fluid was generally clear to mildly turbid and foamy. The total volume of the recovered fluid was recorded. Then, the fluid was placed into a sterile collection tube and placed on ice. The fluid samples were centrifuged at 300 × g for 10 minutes within 1 hour after collection. The supernatant (BALF) was decanted, placed in a cryovial, and stored frozen at −80°C until analysis.
Because BAL is a fairly invasive procedure, it was not performed on all calves at each sample acquisition time. The 8 calves in each age group (preweaned and weaned) were randomly allocated to 2 groups (1 and 2) of 4 calves prior to tulathromycin administration. Bronchoalveolar lavage was performed at 3, 12, 24, and 72 hours after tulathromycin administration for calves assigned to group 1 and at 3, 12, 48, and 96 hours after tulathromycin administration for calves assigned to group 2.
Tulathromycin concentration in plasma, ISF, and BALF
The concentration of tulathromycin in plasma, ISF, and BALF samples was quantified by use of UPLC-MS-MS.e An analytic method for quantification of tulathromycin concentration in various matrices was developed for this study. For each matrix (plasma, ISF, and BALF), calibration curves were constructed for tulathromycin concentrations ranging from 5 to 1,000 ng/mL. The R2 value was 0.99 for each calibration curve. For each matrix assay, intraday and interday coefficients of variation were < 20%, and accuracy ranged from 102% to 106%. The lower limit of quantification for tulathromycin was 5 ng/mL, with a precision of 8% and accuracy of 105%.
Solid-phase extraction cleanup was used to prepare plasma samples for UPLC-MS-MS. During pretreatment, 500 μL of each plasma sample was mixed with 500 μL of 4% phosphoric acid and vortexed for 10 seconds. The resulting 1-mL pretreated sample was loaded onto and pulled through a hydrophilic-lipophilic balanced sorbent cartridgef with a vacuum at a pressure of approximately 3 psi. The sample was washed with 1 mL of 5:95 (vol/vol) methanol:water, then eluted from the cartridge by use of 400 μL of 60:40 (vol/vol) acetonitrile:water with 0.1% formic acid. The collected liquid was transferred to a UPLC vial, and a 5-μL aliquot was injected for UPLC-MS-MS.
For each ISF sample, 100 μL of the sample was loaded into a UPLC vial, and a 5-μL aliquot was injected for UPLC-MS-MS. For each PELF sample, 100 μL of the sample was filtered through a 0.2-μm filter and loaded into a UPLC vial, and a 5-μL aliquot was injected for UPLC-MS-MS.
Estimation of PELF tulathromycin concentration
The PELF tulathromycin concentration was estimated from the BALF tulathromycin concentration. Briefly, the volume of PELF within each BALF sample was estimated by means of a urea dilution method previously described22 in cattle. Briefly, the SUN and BALF urea concentrations (ureaBALF) were measured by spectrophotometryg in accordance with the manufacturer's guidelines. The volume of PELF was calculated as the volume of recovered BALF × (ureaBALF/SUN). Then, the estimated PELF tulathromycin concentration was calculated as the measured BALF tulathromycin concentration × (volume of BALF/volume of PELF).
In vitro plasma protein binding by tulathromycin
For each age group, plasma samples obtained from 5 calves prior to tulathromycin administration were pooled for determination of in vitro plasma protein binding by tulathromycin. The pooled plasma was divided into 9 replicates, and each of 3 replicates was spiked with 1 of 3 concentrations of tulathromycin (0.1, 0.5, or 1.0 μg/mL). The spiked samples were allowed to equilibrate in the dark at room temperature (approx 22°C) for 30 minutes. A 1-mL sample of each standard was loaded onto an ultrafiltration deviceh and was centrifuged at 2,000 × g for 10 minutes. The ultrafiltrate was analyzed with UPLC-MS-MS as previously described to determine the unbound tulathromycin concentration. Nonspecific binding of tulathromycin was < 1% in the ultrafiltration device and filter. The plasma unbound tulathromycin concentration was used to calculate the percentage of tulathromycin bound to plasma proteins by use of the equation described by Toutain and Bousquet-Melou.23
Pharmacokinetic and statistical analyses
Unless otherwise indicated, all pharmacokinetic parameters were expressed in terms of unbound tulathromycin concentrations determined from in vitro protein binding assays for calves of both age groups. Preliminary noncompartmental and compartmental analyses were conducted to obtain initial estimates for the parameters of the base model (ie, model with no covariates). The effects of age and weight on primary and secondary pharmacokinetic parameters were determined with an NLME model.i Briefly, a population-based model was fitted as a multiplicative 2-compartment model parameterized by clearance. Model fit was assessed on the basis of the precision of parameter estimates and goodness-of-fit plots (eg, residual plots). Log likelihood ratios were compared among competing NLME models, and the model with the lowest log likelihood ratio was selected as the preferred model.
Age and weight were individually added to the base NLME model, and the change in the objective function was noted. Evaluation of a box plot of the effect of each covariate (age and weight) on each parameter suggested that Cl/F expressed as a function of bioavailability was the parameter most likely affected by age. Age was added to the base model as a categorical covariate (3 or 24 weeks old), and its effect on pharmacokinetic parameters, the apparent Vd/F expressed as a function of bioavailability, and Cl/F was evaluated with a likelihood ratio test for which a value of P ≤ 0.05 was considered significant. Results indicated that age significantly (P < 0.001) improved the model-predicted Cl/F but not Vd/F; therefore, age was retained in the final model. All pharmacokinetic parameters were reported as the geometric mean ± SD, except for t1/2, which was reported as the harmonic mean ± pseudo-SD. The harmonic mean and pseudo-SD were estimated by the delta method as described.24
The tulathromycin concentration in plasma, ISF, and PELF was plotted over time for each calf. The ratios of ISF tulathromycin concentration to plasma total (bound and unbound) tulathromycin concentration and ISF tulathromycin concentration to plasma unbound tulathromycin concentration were likewise calculated for each calf, and the mean for each group was plotted over time. The distribution of tulathromycin concentration data within each matrix (plasma, ISF, and PELF) was assessed for normality by means of the Shapiro-Wilk W test, which is the preferred method for testing the normality of data when the sample size is small.25 Nonlinear mixed-effects models were used to compare plasma and ISF tulathromycin concentrations between preweaned and weaned calves over time. Efforts to use an NLME model to compare the PELF tulathromycin concentration between the 2 age groups over time were unsuccessful because of substantial intraindividual variation. Therefore, a t test was used to compare the mean PELF concentration between preweaned and weaned calves at each sample acquisition time. Two-tailed tests were used for all analyses, and values of P ≤ 0.05 were considered significant. All statistical analyses were performed with a commercially available software program.j
Results
No adverse reactions were observed in any of the calves following placement of the ISF probes, jugular catheters, or tulathromycin injection. However, the ISF probes malfunctioned in several calves. Consequently, ISF samples were not collected from all calves during some sampling intervals, and only incomplete ISF tulathromycin concentration versus time profiles were available for some calves in both age groups. The volume of BALF obtained during each BAL procedure ranged from 0 to 42.5 mL. Variation in the amount of BALF retrieved from preweaned calves was attributed to difficulty in achieving proper placement of the catheter in a terminal bronchus. No BALF was retrieved from 2 of the 4 weaned calves that underwent BAL 72 hours after tulathromycin injection.
Plasma
The plasma total (bound and unbound) tulathromycin concentration for individual calves at each sample acquisition time was plotted (Figure 1). There was substantial variability and oscillation among the individual plasma concentration-time curves within each age group, especially at the later sample acquisition times. In general, the tmax occurred sooner in preweaned calves than in weaned calves. A 2-compartment model provided the best fit for the plasma total tulathromycin concentration data, and results of the NLME model indicated that several of the primary and derived pharmacokinetic parameters differed significantly between the 2 age groups (Table 1). Specifically, the Cl/F and Vd/F in both the central (circulation; first) and peripheral (noncirculation; second) compartments were significantly greater for weaned calves than preweaned calves. The fitted estimates of the rate of drug distribution from the peripheral compartment to the central compartment were lower for weaned calves than preweaned calves, which was consistent with the greater Vd/F values estimated for the weaned calves.
Plasma total (bound and unbound) tulathromycin concentration for healthy 3-week-old (preweaned; n = 8; circles) and 6-month-old (weaned; 8; triangles) Holstein steers at various times following SC administration of a single dose (2.5 mg/kg) of the drug. Each symbol represents the result for 1 calf.
Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1193
Plasma total (bound and unbound) tulathromycin concentration for healthy 3-week-old (preweaned; n = 8; circles) and 6-month-old (weaned; 8; triangles) Holstein steers at various times following SC administration of a single dose (2.5 mg/kg) of the drug. Each symbol represents the result for 1 calf.
Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1193
Plasma total (bound and unbound) tulathromycin concentration for healthy 3-week-old (preweaned; n = 8; circles) and 6-month-old (weaned; 8; triangles) Holstein steers at various times following SC administration of a single dose (2.5 mg/kg) of the drug. Each symbol represents the result for 1 calf.
Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1193
Pharmacokinetic parameters derived by NLME analysis of plasma total tulathromycin concentrations for preweaned (3-week-old; n = 8) and weaned (6-month-old; 7) Holstein steers as well as the overall study population following SC administration of a single dose of tulathromycin (2.5 mg/kg).
| Population | ||||
|---|---|---|---|---|
| Parameter | Preweaned calves | Weaned calves | Mean (SD) | Coefficient of variation (%) |
| tmax (h) | 0.38 (0.25–0.50) | 0.68 (0.25–1.0) | 0.02 (0.81) | 21.8 |
| Cmax (μg/mL) | 1.34 (0.61–1.84) | 0.82 (0.32–1.63) | 0.67 (3.1) | 18.8 |
| Vd1/F (L/kg) | 3.5 (2.0–7.03) | 10.5 (5.2–20.4)* | 3.7 (0.005) | 28.3 |
| Vd2/F (L/kg) | 16.47 (7.5–32.7) | 24.03 (16.5–37.5)* | 16.4 (4.8) | 20.2 |
| Cl1/F (L/kg/h) | 0.14 (0.07–0.25) | 0.33 (0.16–0.49)* | 0.10 (0.14) | 21.5 |
| Cl2/F (L/kg/h) | 0.91 (0.53–1.63) | 1.07 (0.37–1.42)* | 0.92 (0.02) | 20.2 |
| t1/2 (h)† | 67.6 (43.7–163.5) | 44.4 (30.4–123.8) | 54.3 (24.1) | 17.2 |
A 2-compartment model provided the best fit to the data. Values represent the geometric mean (range) unless otherwise indicated.
Cl1/F = Clearance from central (first) compartment per fraction absorbed. Cl2/F = Clearance from peripheral (second) compartment per fraction absorbed. Vd1/F = Volume of distribution within the central compartment per fraction absorbed. Vd2/F = Volume of distribution within the peripheral compartment per fraction absorbed.
Value differs significantly (P ≤ 0.05) from the corresponding value for the preweaned calves.
Harmonic mean.
Results of the in vitro protein-binding assays indicated that the unbound fraction of tulathromycin in plasma ranged from 0.75 to 0.82 for preweaned calves and 0.36 to 0.61 for weaned calves (Table 2). Although the fraction of tulathromycin that remained unbound to plasma protein for weaned calves was significantly lower than that for preweaned calves, it was constrained within a range where protein binding was fairly stable.
Mean proportion of tulathromycin bound and unbound to plasma proteins as estimated by in vitro assays.
| Weaned calves | Preweaned calves | |||
|---|---|---|---|---|
| Tulathromycin concentration (μg/mL) | Bound (%) | Unbound (%) | Bound (%) | Unbound (%) |
| 0.1 | 63 | 36 | 22 | 77* |
| 0.5 | 49 | 50 | 17 | 82* |
| 1.0 | 39 | 61 | 24 | 75* |
For each calf group described in Table 1, plasma samples obtained from 5 calves prior to tulathromycin administration were pooled. The pooled samples were divided into 9 replicates, and each of 3 replicates was spiked with 1 of 3 concentrations of tulathromycin (0.1, 0.5, or 1.0 μg/mL). The tulathromycin-spiked plasma samples were allowed to equilibrate in the dark at room temperature (approx 22°C) for 30 minutes. The samples then underwent UPLC-MS-MS to determine the unbound tulathromycin concentration, which was then used to calculate the percentage of tulathromycin bound to plasma proteins as described.23
Value differs significantly (P ≤ 0.05) from the corresponding value for the weaned calves.
ISF
Only tulathromycin that remained unbound to plasma protein (unbound tulathromycin) could pass through the ultrafiltration probes used to collect ISF. The probes were well tolerated by the calves, and the volume of ISF collected at each sample acquisition time generally ranged from 0.05 to just > 2 mL. Tulathromycin was detectable in ISF samples of weaned calves beginning 12 hours after injection but was not detectable in the ISF samples of preweaned calves until 48 hours after injection (Figure 2). The time to maximum ISF tulathromycin concentration was greater than the time to Cmax for both age groups. The mean ratios for ISF tulathromycin concentration to plasma total (bound and unbound) tulathromycin concentration and ISF tulathromycin concentration to plasma unbound tulathromycin concentration were significantly greater for weaned calves than preweaned calves at all sample acquisition times.
Geometric mean ISF tulathromycin concentration (A), ratio of ISF tulathromycin concentration to plasma total tulathromycin concentration (B), and ratio of ISF tulathromycin concentration to plasma unbound (active) tulathromycin concentration (C) over time for the preweaned and weaned calves of Figure 1. Brackets represent the SDs for the means. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1193
Geometric mean ISF tulathromycin concentration (A), ratio of ISF tulathromycin concentration to plasma total tulathromycin concentration (B), and ratio of ISF tulathromycin concentration to plasma unbound (active) tulathromycin concentration (C) over time for the preweaned and weaned calves of Figure 1. Brackets represent the SDs for the means. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1193
Geometric mean ISF tulathromycin concentration (A), ratio of ISF tulathromycin concentration to plasma total tulathromycin concentration (B), and ratio of ISF tulathromycin concentration to plasma unbound (active) tulathromycin concentration (C) over time for the preweaned and weaned calves of Figure 1. Brackets represent the SDs for the means. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 11; 10.2460/ajvr.79.11.1193
PELF
The mean estimated PELF tulathromycin concentrations over time for both age groups were summarized (Table 3). It varied substantially among calves of both groups, as evidenced by the fairly large SDs relative to the corresponding means. For both age groups, the mean maximum PELF tulathromycin concentration was greater than the mean Cmax and mean maximum ISF tulathromycin concentration.
Mean ± SD PELF tulathromycin concentration (μg/mL) for the calves described in Table 1.
| Time after tulathromycin administration (h) | Preweaned calves | Weaned calves |
|---|---|---|
| 3 | 4.9 ± 3.1 | 2.9 ± 2.2 |
| 12 | 5.3 ± 4.6 | 2.1 ± 1.9 |
| 24 | 4.7 ± 5.1 | 0.5 ± 0.3 |
| 48 | 3.6 ± 5.0 | 1.9 ± 0.2 |
| 72 | 2.0 ± 1.1 | 1.1 ± 0.5 |
| 96 | 3.5 ± 2.0 | 4.3 ± 2.0 |
The PELF tulathromycin concentration was estimated from the BALF tulathromycin concentration. Because BAL is a fairly invasive procedure, it was not performed on all calves at each acquisition time. The 8 calves in each group were randomly assigned to 2 groups (1 and 2) of 4 calves. Bronchoalveolar lavage was performed at 3, 12, 24, and 72 hours after tulathromycin administration for calves assigned to group 1 and at 3, 12, 48, and 96 hours after tulathromycin administration for calves assigned to group 2. Therefore, within each group, the values for 3 and 12 hours after tulathromycin administration represent the mean ± SD for 8 calves, whereas those for 24, 48, 72, and 96 hours after tulathromycin administration represent the mean ± SD for 4 calves.
Discussion
Multiple physiologic factors change as calves mature from preruminants to ruminants, and those factors can affect drug biotransformation and elimination pathways, which in turn can alter drug pharmacokinetics (Appendix). To our knowledge, the present study was the first to evaluate the effect of age on the tulathromycin concentration in plasma, ISF, and PELF following SC administration of a single dose (2.5 mg/kg; ie, label dose) of the drug to 3-month-old (preweaned) and 6-month-old (weaned) calves. Collection of ISF by means of indwelling ultrafiltration probes and PELF by BAL allowed us to assess active drug concentrations over time at the site of action. Information regarding how the pharmacokinetics of tulathromycin change as calves mature is essential for the development of targeted dosing schedules to maximize the likelihood that therapeutic drug concentrations will be achieved in infected tissues.
Accurate measurement of active antimicrobial concentrations at the site of infection is crucial for prediction of treatment efficacy. Typical methods for obtaining that information include quantification of drug concentrations in plasma, tissue cages, and homogenized lung tissue. Results of other studies32–34 indicate that drug concentration in homogenized lung tissue is a poor predictor of drug concentration in PELF. The antimicrobial concentration in PELF may be the most clinically relevant predictor of treatment efficacy depending on the specific site within the lung that becomes infected with a bacterial pathogen.
Drug concentrations in ISF are considered a potential surrogate for drug concentrations in lung tissue because only unbound (active) drug is present in ISF, which provides an accurate reflection of drug partitioning into a tissue compartment. However, such a surrogate is inappropriate for a macrolide such as tulathromycin. In 6-month-old calves (ie, calves with mature rumens) that received the same dose of tulathromycin (2.5 mg/kg, SC) as the calves of this study, the tulathromycin concentration in PELF was 9 times that in plasma and ISF.5 Tulathromycin concentrations vary between bovine pneumonic and healthy lung tissue homogenates; moreover, lung tissue homogenates do not allow for evaluation of free (unbound) versus bound drug concentrations in those tissues.35
Several plasma pharmacokinetic parameters for tulathromycin differed significantly between the preweaned and weaned calves of the present study. Results of another study11 indicate that the plasma pharmacokinetic parameters for tulathromycin did not differ significantly between preruminant calves (age, 4 to 7 weeks old) and adult cattle. It is unclear whether the apparently contradictory findings between the present study and that study5 was the result of actual pharmacokinetic differences between 3-week-old calves and 4- to 7-week-old calves (although all calves were considered preruminants) or variation between the 2 studies. The plasma pharmacokinetic parameters for tulathromycin for the weaned calves of this study were similar to those reported in similarly aged calves of other studies.5,36
Fairly low plasma tulathromycin concentrations were achieved for both the preweaned and weaned calves of the present study. Subcutaneous administration of tulathromycin was characterized by rapid absorption followed by slow clearance. The Cl/F and Vd/F for preweaned calves were lower than those for weaned calves. The mean t1/2 of tulathromycin for preweaned calves (67.6 hours) was 1.5 times that for weaned calves (44.4 hours), but that difference did not quite reach significance (P = 0.10). Drug disposition (and thus t1/2) is dependent on the simultaneous effects of drug elimination and distribution, and those effects might have contributed to the lack of significance in t1/2 observed between the preweaned and weaned calves.
In the present study, the population tmax estimated by the NLME model was 0.02 hours, which translated to approximately 1 minute after tulathromycin administration. For all calves, the first postbaseline blood sample was collected 15 minutes after tulathromycin injection. Therefore, it is likely that the true tmax was missed because blood samples were not collected prior to 15 minutes after drug administration.
The Cl/F (0.33 L/h/kg) for the weaned dairy calves of the present study was greater than the Cl/F (0.18 L/h/kg) reported for weaned beef calves of another study.33 We found that interesting because we assumed that the bioavailability of tulathromycin following SC injection would be similar among similarly aged cattle regardless of breed, but that assumption might be incorrect. Although the body weight range (181 to 246 kg) for the beef calves of that study33 was similar to the body weight range (151 to 214 kg) for the dairy calves of this study, the age of the beef calves was not reported. Also, dairy calves are typically weaned at a younger age than beef calves. Therefore, it is possible that the weaned calves of the 2 studies were at different stages of rumen development, which could have affected tulathromycin bioavailability and thus Cl/F.
In the present study, the Cl/F for the preweaned calves was significantly slower than that for the weaned calves. That finding might have been the result of age-associated differences in kidney and liver function. Hepatic drug clearance is dependent on several factors including blood flow, hepatic enzyme activities and transport systems, and binding of the drug to plasma proteins.37 Hepatic blood flow in 3-month-old calves is less than that in adult cattle and can affect the metabolism of drugs with high extraction ratios.38,39 Tulathromycin has a low extraction ratio40; therefore, it was unlikely that hepatic blood flow was a contributing factor to the discrepancy in the Cl/F between preweaned and weaned calves. Moreover, in cattle, approximately 90% of the tulathromycin dose administered is eliminated unchanged in the bile and only about 10% is metabolized, and < 10% of the tulathromycin metabolites in excreta and tissues are formed by N-demethylation or N-oxidation.41 Thus, it was unlikely that the discrepancy in Cl/F observed between the preweaned and weaned calves of this study was caused by differences in the metabolism of tulathromycin.
In cattle, tulathromycin is predominantly excreted unchanged in the feces. Therefore, differences in the maturation of elimination pathways (eg, via biliary excretion or transporter mechanisms) can affect the elimination rate of drugs. Currently, little is known regarding the maturation of liver efflux transporters in humans and veterinary species. The ontogeny of renal glomerular filtration and tubular secretion and reabsorption has been well characterized for human pediatric patients and can have a profound effect on the pharmacokinetic profiles of drugs.37 It seems likely that hepatic efflux transporters also undergo ontogenesis as animals mature, and those changes could also affect the pharmacokinetic profiles of drugs.
The Vd/F of a drug is affected by its propensity to bind to plasma proteins and tissues and its lipid solubility. Lipid-soluble drugs such as tulathromycin have high apparent volumes of distribution, as evidenced by the high Vd/F of tulathromycin observed for the preweaned (3.5 L/kg) and weaned (10.5 L/kg) calves of the present study. As calves mature, the body fat-to-water ratio increases, which results in an increase in the sequestration of lipid-soluble drugs in adipose tissue. The distribution of a drug into immune cells also contributes to its Vd/F. Macrolides are weak bases and tend to become ionized in acidic environments; thus, they can accumulate in cells and tissues, particularly polymorphonuclear cells, owing to lysosomal trapping. It has been suggested that lysosomal trapping may serve as a vehicle by which drugs are transported to the site of infection.42 Some investigators43,44 believe that mechanism may account for the high concentrations of lipophilic drugs at infection sites, whereas others45 refute that assumption. Macrolides readily accumulate in neutrophils and macrophages in vitro.10 Regardless of whether immune cells serve as vehicles for drug delivery, differences in the development of the immune system may have contributed to the greater Vd/F in weaned calves relative to that in preweaned calves. For calves of another study,46 the neutrophil count was greater than the upper limit of the reference range for adult cattle at 1 day of age but was within the reference range for adult cattle at 28 days of age.
Some of the differences in the plasma pharmacokinetics of tulathromycin observed between the preweaned and weaned calves of the present study might be attributable to age-related differences in the binding of tulathromycin to plasma proteins. Results of in vivo studies5,33 estimate that approximately 40% of the tulathromycin administered to 6-month-old calves becomes bound to plasma proteins (ie, unbound fraction of tulathromycin, 0.53 to 0.68). In the present study, the in vitro plasma protein binding of tulathromycin for weaned calves (39% to 63%) was similar to that reported for 6-month-old calves of the other studies5,33; however, in vitro plasma protein binding of tulathromycin for preweaned calves (17% to 24%) was approximately half that for weaned calves. Thus, age appeared to affect the extent of plasma protein binding of tulathromycin for the calves of this study. Age also affects the extent of plasma protein binding of multiple drugs in human patients.31
In calves, concentrations of major drug-binding proteins, such as albumin and AGP, change substantially during the first 3 months of life.47,48 In the present study, the unbound fraction of tulathromycin (a weak base) was greater in the preweaned calves than weaned calves, which might have been the result of age-related differences in the drug-binding properties of AGP.49 In neonates, the plasma concentration of AGP is fairly low and each AGP molecule has only 1 drug binding site; therefore, drug binding to AGP is typically saturable and readily displaceable.50
For cattle with BRD, the blood or plasma concentration of a macrolide does not accurately reflect the macrolide concentration at the infection site (eg, lung tissue).45 Although ISF is not the targeted infection site for tulathromycin, we decided to determine the ISF tulathromycin concentration for the calves of the present study because we believed it would provide valuable information regarding the way tulathromycin moves through various tissue compartments and how age-related effects on the plasma pharmacokinetics of the drug might affect its distribution in ISF versus plasma. Interstitial fluid concentrations of various drugs including cephalosporins, fluoroquinolones, and macrolides have been evaluated in cattle.5,22 In general, macrolides have only limited penetration into the ISF compartment. That characteristic was evident in the present study, as the mean ISF tulathromycin concentration was consistently lower than the mean plasma unbound (free) tulathromycin concentration in both preweaned and weaned calves. Those findings were also consistent with the observation in human medicine that achieving therapeutic drug concentrations in ISF is difficult for many macrolides.51
The mean ISF tulathromycin concentration for weaned calves was consistently greater than that for preweaned calves at all sample acquisition times. That finding may be the result of age-associated differences in body composition and immune system constituents. Tulathromycin was below the limit of detection (0.005 μg/mL) in the ISF of preweaned calves until 48 hours after injection but was detectable in the ISF of weaned calves by 12 hours after injection. Additionally, the ratio of tulathromycin concentration in ISF relative to tulathromycin concentration in plasma before and after correction for plasma protein binding was significantly associated with time in both preweaned and weaned calves. The ratio of ISF tulathromycin concentration to plasma total tulathromycin concentration and ratio of ISF tulathromycin concentration to plasma unbound tulathromycin concentration both peaked at 168 and 48 hours after drug administration for preweaned and weaned calves, respectively. The differences in both tulathromycin concentration and kinetics in ISF are likely attributable to changes in adipose and muscle content as calves mature. Total body water percentage decreases as calves mature. For preweaned calves, the large proportion of total body water, especially in adipose tissue, which is where the SC ultrafiltration probes were placed, could result in a dilution effect that may decrease the ISF tulathromycin concentration. Therefore, assessment of the partitioning of unbound antimicrobials in the ISF and plasma may not be the best modality for determining appropriate drug doses for calves of varying ages.
Lung tissue homogenates, biopsy specimens, and collection of samples by means of bronchial microsampling and BAL have been used to estimate pulmonary drug concentrations.22,34,52 In another study,53 the tilmicosin (another macrolide) concentration estimated by use of bronchial swab specimens did not differ significantly from that estimated by use of BALF samples. The use of lung or tissue homogenates for estimation of drug concentrations does not allow for differentiation between intracellular and extracellular drug concentrations or account for binding of the drug to tissues. Therefore, macrolide concentrations determined from lung tissue homogenates typically overestimate the active macrolide concentration at the infection site.54
In cattle, PELF is secreted extracellularly in the respiratory tract and is a potential matrix for bacterial infections. Results of another study5 indicate that some antimicrobials are able to penetrate the PELF better than others. In the present study, we sought to assess PELF tulathromycin concentration as a function of calf age. Determination of PELF drug concentrations allows for the evaluation of plasma-ISF-PELF pharmacokinetic-pharmacodynamic exposure relationships, thereby facilitating assessment of potential therapeutic dosing strategies as a function of age. Interestingly, the PELF tulathromycin concentrations for preweaned calves were consistently higher than those in weaned calves, which was in contrast to ISF tulathromycin concentrations that were consistently highest in weaned calves. We do not believe that the reason the PELF tulathromycin concentration was greater in preweaned calves than weaned calves was the result of immature physiologic barrier function at the blood-alveolar interface in the lungs or immature p-glycoprotein activity. P-glycoprotein is believed to transport substances into alveolar sacs and thereby has a protective function.55 Efflux of tulathromycin across the pulmonary membrane via p-glycoprotein would increase PELF concentrations of the drug. We believe a more likely cause for the discrepancy in the PELF tulathromycin concentrations between preweaned and weaned calves was PELF pH differences between the 2 groups. If the PELF pH in preweaned calves was lower than that in weaned calves, it could have led to ion trapping, which could increase the PELF tulathromycin concentration. To our knowledge, changes in the pH of PELF as calves mature from preruminants to ruminants have not been evaluated.
In the present study, the mean PELF tulathromycin concentration was substantially greater than corresponding mean plasma and ISF tulathromycin concentrations at all sample acquisition times. The PELF tulathromycin concentration also varied greatly among the calves of both age groups, which might have affected interpretation of the effect of age on that variable. The variation in PELF tulathromycin concentrations might have been biased when urea was used to determine the volume of PELF. When urea is used as a dilution marker, the duration of time that the fluid infused during the BAL procedure is exposed to the airways can be an important source of experimental variability.56 Tulathromycin accumulates in neutrophils; therefore, airway irritation caused by serial collection of BALF samples and sample contamination from ruptured neutrophils during centrifugation of those samples could cause over-estimation of the PELF tulathromycin concentration. However, although tulathromycin contamination from lysed leukocytes was possible, the high PELF drug concentrations observed cannot be explained by ruptured neutrophils alone.45 Despite those limitations and uncertainties, we believe that the PELF tulathromycin concentration should be considered a rough approximation of the lung tulathromycin concentration.
Accurate determination of the appropriate dose of a drug for treatment of disease is critically important for patients of all ages, but especially so for neonates. Ruminant species in particular undergo major physiologic changes as they mature from preruminants to ruminants that affect drug absorption, distribution, metabolism, and elimination. Thus, extrapolation of drug doses for adult cattle to preruminant calves can be unreliable and inadvisable. Results of the present study indicated that movement of tulathromycin from plasma to the ISF and PELF differed significantly between preweaned and weaned calves, which was suggestive of functional differences in the body composition between the 2 age groups. The success of tulathromycin for the treatment of BRD is dependent on achieving efficacious drug concentrations at the site of infection in conjunction with an adequately functioning immune system. Although neonatal calves have a greater number of phagocytic cells than older calves, those cells do not function at full capacity until the calves reach approximately 4 months of age.57 Consequently, each of these factors needs to be considered during assessment of the magnitude of dose adjustment necessary for the treatment of disease in young calves.
Veterinary drug approval and residue studies generally involve the use of healthy animals. Results of the present study indicated that the pharmacokinetics of tulathromycin differed significantly between healthy preweaned and weaned calves. Disease will also likely affect the pharmacokinetics of the drug, and the data generated in this study can be used as a baseline for subsequent studies involving diseased calves. The data generated by this study also suggested that age should be considered a potential covariate that could affect drug distribution during the development of clinical drug studies in young cattle.
In veterinary medicine, data regarding the effects of age-related physiologic changes on drug pharmacokinetics are limited. Results of the present study provided insight into how the pharmacokinetics of tulathromycin changes as cattle mature from preruminants to ruminants. All calves of the present study were administered a single dose of tulathromycin (2.5 mg/kg, SC) in accordance with the product label. The data generated suggested that the extent of in vitro tulathromycin plasma protein binding was significantly greater in weaned calves than preweaned calves, which could affect the amount of unbound (active) drug available for distribution to the site of infection as well as its elimination from the body. Rapid absorption and extensive distribution of an antimicrobial into PELF are desirable when treating cattle for BRD. Although the PELF tulathromycin concentration was highly variable among calves regardless of age, the mean PELF tulathromycin concentration for preweaned calves was consistently greater than that for weaned calves. The mean PELF tulathromycin concentration was also consistently greater than the mean plasma and ISF tulathromycin concentrations at all sample acquisition times for both preweaned and weaned calves. Additional studies are necessary to compare the pharmacokinetics of tulathromycin in diseased versus healthy calves and assess the relationship between tulathromycin exposure and clinical response in diseased calves of various ages.
Acknowledgments
This manuscript represents a portion of a thesis submitted by Dr. Mzyk to the North Carolina State University College of Veterinary Medicine as partial fulfillment of the requirements for a Doctor of Philosophy degree.
Supported by the USDA for the Food Animal Residue Avoidance and Depletion Program (USDA 2013-41480-21001). Zoetis Inc supplied the standard tulathromycin used for analysis in this study but had no role in the study design; collection, analysis, and interpretation of data; or decision to submit the manuscript for publication.
The authors declare that there were no conflicts of interest.
The authors thank Jim Yeatts and Jenna Schirmer for assistance with assay development and Dr. Jennifer Davis for technical assistance and assistance with manuscript preparation.
ABBREVIATIONS
| AGP | α1 Acid glycoprotein |
| BAL | Bronchoalveolar lavage |
| BALF | Bronchoalveolar lavage fluid |
| BRD | Bovine respiratory disease |
| Cl/F | Clearance per fraction absorbed |
| Cmax | Maximum plasma concentration |
| ISF | Interstitial fluid |
| NLME | Nonlinear mixed-effects |
| PELF | Pulmonary epithelial lining fluid |
| t1/2 | Half-life |
| tmax | Time to maximum plasma concentration |
| UPLC | Ultrahigh-performance liquid chromatography |
| UPLC-MS-MS | Ultrahigh-performance liquid chromatography-tandem mass spectrometry |
| Vd/F | Volume of distribution per fraction absorbed |
Footnotes
Draxxin, Zoetis Inc, Parsippany, NJ.
Reinforced ultrafiltration probe, BASi Inc, West Lafayette, Ind.
Rompun injectable (20 mg/mL), Bayer Animal Health, Morrisville, NC.
Foley urinary catheters with wire stylet, MILA International Inc, Florence, Ky.
Tandem Mass Spectrometry Triple Quad, Waters Corp, Milford, Mass.
Oasis 3-cc PRiME HLB cartridges, Waters Corp, Milford, Mass.
Urea test kit, Sigma Chemical Co, St Louis, Mo.
Centrifree ultrafiltration device, Millipore Sigma, St Louis, Mo.
Phoenix WinNonlin/NLME, version 1.3, Certara, Cary, NC.
SigmaPlot, Systat Software Inc, San Jose, Calif.
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Appendix
Summary of physiologic factors that change as calves mature from preruminants to ruminants and the effects those factors can have on the pharmacokinetics of drugs as reported in the scientific literature.
| Physiologic factor | Nature of change as the animal matures | Drug factor affected | Pharmacokinetic implications | Reference No. |
|---|---|---|---|---|
| Abomasal pH | Increases | Absorption | Decreases bioavailability of weak acids and increases bioavailability of weak bases | 26 |
| Abomasal emptying time | Increases | Absorption | Increases drug absorption time | 27 |
| Intestinal drug transporters | Variable | Absorption | Unknown in cattle | - |
| Skeletal muscle blood flow | Variable | Absorption | Unknown in cattle | - |
| Body water-to-fat ratio | Increases | Distribution | Increases volume of distribution for hydrophilic drugs and decreases volume of distribution for lipophilic drugs | 28, 29 |
| Protein binding | Decreases | Distribution | Increases the free (unbound) fraction of drugs | 30 |
| Phase I enzyme activity | Decreases | Hepatic metabolism | Decreases hepatic drug clearance | 12 |
| Phase II enzyme activity | Decreases | Hepatic metabolism | Decreases hepatic drug clearance | 12 |
| Glomerular filtration rate | Decreases | Renal excretion | Decreases renal drug clearance | 31 |
| Renal tubular absorption and secretion | Decreases | Renal excretion | Decreases renal drug clearance | 31 |
