ABSTRACT

PROCEDURES

Each calf was implanted with an intravenous jugular catheter and a subcutaneous ultrafiltration probe on the lateral side of the cervical neck 24 hours prior to dosing with tulathromycin. Tulathromycin was injected SC and blood samples from the jugular vein and ISF samples from the ultrafiltration probe were collected immediately before and at predetermined times for 312 hours after dosing. PELF concentrations were calculated by a urea dilution assay of the bronchoalveolar lavage fluids collected a predetermined time points. Plasma protein binding was measured using a microcentrifugation system. PELF, Plasma and ISF tulathromycin concentrations were determined by ultra high-performance liquid chromatography with mass spectrometry.

RESULTS

The maximum total (free plus protein bound) concentrations in the plasma (Cmax) were greater in 3-week old calves (1.34 ± 0.45 µg/mL) as compared to that of the 6-month old calves (0.82±0.45 µg/mL). Clearance values (CL/F) were significantly lower in 3-week old calves but the volume of distribution (Vd/F) significantly higher in 6-month old calves. A downward trend in the plasma protein binding of tulathromycin was observed in plasma derived from 6-month old calves (63% to 39% for tulathromycin concentrations ranging from 0.1 to 1.0 µg/mL) but a lower and relatively constant fraction bound was observed in 3-week old calves (22% – 24% bound at tulathromycin concentrations ranging from 0.1 to 1.0 µg/mL).  The older calves maintained higher ISF concentrations throughout the study period compared to those seen in younger calves. PELF concentrations of tulathromycin tended to be higher in 3-week old calves and reached higher maximum concentrations than seen in plasma in both groups.

CONCLUSIONS AND CLINICAL RELEVANCE

An age-associated difference in plasma and ISF concentration time curves is consistent with maturational changes in calf physiology, resulting in altered tulathromycin exposure characteristics in the plasma, lungs and ISF of healthy calves.

ABBREVIATIONS

BRD – Bovine Respiratory Disease

PK – Pharmacokinetic

SC – subcutaneous

IM – Intramuscular

PELF – Pulmonary epithelial lining fluid

ISF – Interstitial Fluid

BAL – Bronchoalveolar lavage

UPLC-MS/MS – Tandem mass spectrometry

CL/F – Clearance

Vd/F – Volume of distribution

NLME – Non-linear mixed effects

Tmax

Cmax – Maximum plasma concentration

AGP – α1-acid glycoprotein

P-gp – P-glycoprotein

T½ – elimination half life

PK-PD – pharmacokinetic-pharmacodynamic

Introduction

BRD continues to be a significant cause of morbidity and mortality in young dairy and veal calves.1, 2 Pneumonia in dairy calves is multifactorial, and its occurrence and severity is impacted by herd management, animal age, calf immunity, and the environment.3

Tulathromycin, a semi-synthetic macrolide antibiotic of the subclass triamilide, has been shown to be safe and effective for the treatment of BRD associated with Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis, and for the control of respiratory disease in cattle at high risk of developing BRD associated with Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Tulathromycin has excellent bacteriostatic and some bactericidal activity against many of these pathogens.4 Currently, it is approved for use in multiple ages of calves, including those to be processed for veal, with an extended meat withdrawal time in veal calves.

Similar to other macrolide antibiotics, PK studies of tulathromycin in cattle, swine, deer, bison and foals demonstrate rapid absorption following SC and IM injection, extensive accumulation in lung tissue, and prolonged T1/2 in lung homogenate and PELF.5-10 In previous PK studies, tulathromycin was measured in plasma, PELF, and the ISF of mature, clinically healthy calves. 10 Although a plasma PK characterization in pre-ruminating calves has been previously reported,11 similar assessments comparing blood, ISF and PELF have not as yet been reported for pre-ruminating calves. Maturation can affect drug metabolism,12,13 transporter function14 body composition [including body fat, muscle and water content15 blood flow16 and blood composition in terms of plasma proteins and cellular constituents.17

With regard to the latter, bovine neutrophils exhibit a high-affinity for tulathromycin.18 The number of circulating neutrophils decrease with age from birth to 30 days of age in Holstein heifer calves, with maximum values occurring between birth and 8 hours of life.19 In cattle, endotoxins induced a higher rate of neutrophil migration in neonatal calves as compared to that of adults.20 In rats, previous studies have correlated an immature neutrophil function with a decrease in lung injury,21 indicating that age impacts not only the neutrophil function but also the pathogenesis and progression of disease.

Ultimately, a determination of effective dosing strategies should relate the concentrations at the site of action to the in vitro susceptibility of the target bacteria. For some drugs, free drug concentrations in the blood can be used to approximate concentrations available to treat extracellular infections. This is not the case for macrolides where drug concentrations in the lung, ISF and blood can be markedly different.  Accordingly, appreciating the distribution patterns of antimicrobials into the ISF and PELF in different subpopulations may improve the PK-PD correlations for many bacterial pathogens. However, the extent to which age influences drug exposure (clearance), and plasma versus tissue (lung and ISF) tulathromycin concentrations have yet to be evaluated relative to that seen in ruminating (mature) calves.

With these points in mind, the objective of this study was to characterize tulathromycin drug penetration into PELF and ISF of different age calves after administration of a single SC dose of 2.5 mg/kg body weight.  The results of this study will provide insights into the ways that drug PK in cattle may differ as a function of age.

Materials and methods

Animals

This study was approved by the North Carolina State University Institutional Animal Care and Use Committee. Holstein steer calves were bought from the North Carolina State University Dairy herd. Eight unweaned Holstein calves, two to three weeks of age, weighing between 41-53 kg were housed at the University Dairy and fed a commercial non-medicated milk replacer twice a day, and had free access to water and calf starter ration ad libitum throughout the study. Eight weaned calves, 6 months of age and weighing between 151-214 kg at time of study, were also enrolled. These older calves were group housed indoors at the University lab animal facility. The 6-month old calves provided ad libitum access to grass hay and water and their diets were supplemented with grain. All calves were confirmed healthy via physical exams conducted prior to the start of the study and none of the calves had a previous history of disease or antibiotic administration.

Drug Administration and Blood Collection 

All calves were weighed on a digital scale on the morning of the study commencement for determination of the administered dose. Approximately 24 h prior to start of the study, calves were restrained for intravenous catheter placement. The area where the catheter was to be placed was clipped and cleaned with alternating scrubs of chlorhexidine and isopropyl alcohol. A 14 G x 3.25 mm catheter was inserted into the right jugular vein with an extension set. The catheter was sutured to the skin using a 2-0 monofilament suture and were flushed four times a day using 6 mLs of 10 units/mL of heparin saline.  A single SC injection of tulathromycina (2.5 mg/kg) was administered to each calf in the neck per label instructions. Blood samples were taken from the jugular vein 0 (pretreatment), 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 post administration of tulathromycin and the samples were transferred to lithium heparinized tubes. These samples were stored on ice until centrifugation at approximately 3500 g for 10 min to collect plasma. The plasma samples were stored -80°C until analysis.

ISF Collection 

All calves were implanted with SC ultrafiltration probes  on the side of the neck opposite site of the SC tulathromycin injection.b  Each probe contained three semi-permeable loops connected to a non-permeable tube that extended outside the animal and attached to a 3 mL plastic vacutainer tube without clotting agents. This tube provided negative pressure for fluid collection through small pores in the probe membranes. These pores allowed for the movement of water, electrolytes and low molecular weight molecules (<30,000 Da) to pass into collection tube while excluding large molecules such as proteins, protein bound drugs, and cells. Probes were placed while the calves were sedated with xylazinec at a dose of 0.05-0.1 mg/kg in the cervical neck muscles. Probes were placed twenty-four hours before the start of the trial to provide the time necessary to allow for equilibration with the surrounding ISF. One probe was inserted into each calf SC in the area cranial to the scapula. The ISF was collected at 0 (pretreatment), 2, 3, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, and 312 hours following SC tulathromycin administration. Since each ISF sample represents fluid collection over a certain amount of time (i.e. not an instantaneous sample), a lag time was calculated based on the length of the tube and the fluid volume collected over time for each sample. The fluid collected was frozen at -80°C until analysis.

Lung Fluid Collection 

To determine drug concentrations in the PELF, a BAL was performed using a method previously described.23 Briefly, BALs were performed in all calves using either a sterilized, flexible 10 French X 36 inch catheter with a 3-cc balloon cuff (3-week old calves) or a 24 French X 59 inch catheter (6-month old calves).d  At each time point, the calf was restrained and the head and neck of the calf were extended to facilitate passage of the sterile catheter.  The BAL catheter was introduced into the ventral meatus of the nose and advanced down the trachea until it 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 guide-wire was removed. At each time point, 100 mL of sterile saline were infused into the lungs and the fluid was immediately aspirated, limiting the dwell time in the lungs. The volume of fluid retrieved ranged from 0 to 42.5 mLs of clear to mildly turbid foamy fluid. The sample was placed into a sterile collection tube, the total volume recorded, and sample subsequently placed on ice until centrifugation. The BAL samples were centrifuged at 300 g for 10 min, and the supernatant fluid separated from cell pellet and the supernatant stored frozen at -80°C until analysis. Prior to the start of the study, all 8 calves were randomly allocated to one of two groups. Group 1 calves were sampled at 3, 12, 24 and 72 hours postdose and Group 2 was sampled at 3, 12, 48 and 96 hours postdose.

Drug Analysis

Quantitative analysis of tulathromycin concentrations in plasma, ISF and PELF was accomplished using UPLC-MS/MS.e An analytical method for determination of tulathromycin concentrations in various matrices was developed for this study. Plasma samples for UPLCMS/MS analysis were prepared using a solid phase extraction cleanup. For each assay matrix, calibration curves were constructed spanning over the calibration range 5–1000 ng/mL. The R2 values for the calibration curves were 0.99.  Intraday and interday percent coefficient of variation were less than 20%, and the accuracy ranged from 102 to 106%. The lower limit of quantification was 5 ng/mL with a precision of 8% and an accuracy of 105%.

Plasma: 500-µL of plasma was mixed with 500-µL of 4% phosphoric acid, vortexed for 10 seconds to pre-treat samples. The 1 mL pretreated sample was then loaded onto cartridges with and pulled through with a vacuum at ~3 psi.f Each sample was then washed with 1 mL of 5:95 (v:v) Methanol:water. Samples were then eluted from cartridge using 400-µL of 60:40 (v:v) Acetonitrile:Water with 0.1% formic acid. The collected liquid was then transferred to a vial and 5-µL aliquot was analyzed by the UPLC-MS/MS.

Interstitial Fluid: 100-µL of ISF from each sample was loaded into UPLC vials and a 5-µL aliquot was injected onto the UPLC-MS/MS.  

PELF/BAL Supernatant: 100-µL of BAL supernatant from each sample was filtered through a MS/MS.

serum (Ureaserum to manufacturer guidelines . The volume of PELF (V ) in BAL fluid was derived from the following equation:  

In which VBAL is the volume of recovered BAL fluid. The concentration of tulathromycin in PELF (TULPELF) was derived from the following equation:  

TULPELF= TULBAL×

In which TULBAL is the measured concentrations of tulathromycin in BAL fluid.

Plasma Protein Binding 

Plasma from five healthy calves of both ages (3-weeks and 6-months) were pooled to determine in vitro plasma protein binding. Plasma was spiked at three different concentrations (0.1, 0.5 and 1.0 µg/mL in triplicate. All samples were allowed to stand at room temperature for 30 minutes in the dark to equilibrate. A 1 mL sample of each standard was loaded onto an ultrafiltration device and was centrifuged for 2,000 g for 10 minutes. h The ultrafiltrate was analyzed using UPLC-MS-MS to determine unbound concentration and plasma protein binding.

Non-specific binding of tulathromycin was determined to be <1% in the device and filter.

PK Analysis

Unless otherwise indicated, all PK parameter values are expressed relative to the unbound tulathromycin concentrations determined from in-vitro protein binding assays performed in pooled 3-week old and 6-month old calf plasma.

The influence of age and weight on primary and secondary PK parameters were determined using a NLME model.i The population base model was fitted as a multiplicative twocompartment model parameterized by clearance. Model selection was based upon precision of parameter estimates, and goodness-of-fit plots (e.g., residual plots) and statistical significance between models using lowest log-likelihood ratio values obtained in the software.  A preliminary non-compartmental and compartmental analysis was conducted in order to obtain the initial estimates for the parameters of the basic model (i.e. no covariates).

Assessment for age and weight was conducted by the individual addition of each term to the base model and the change in the objective function was noted. A box plot of effect of the covariate (age and weight) on each parameter showed 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 (where 3-week old calves = 3 and 6 month old calves = 24), and its effect on the PK parameters, the apparent Vd/F expressed as a function of bioavailability and CL/F was evaluated using a likelihood ratio test. A P-value < 0.05 was considered to be significant. Age was found to significantly improve model predictions for CL/F, but not Vd/F (p < 0.001).   After age was determined to improve the model, this covariate remained in the final model. All PK parameters, except for T½, were reported as a geometric mean. The T½ was

/

= ! ×”

Hi is the harmonic value (λz) and HM is the harmonic mean being reported (T1/2).

ISF/plasma concentration ratios at each time point were determined for each calf using the concentration of ISF (unbound drug) to the total (bound and unbound) and unbound tulathromycin plasma concentration. Statistical analyses were performed on the ISF concentrations of both groups of calves.j Levels of significance were set at P-values of ≤0.05 using a two-tailed test. The normality assumption was tested for each variable using the Shapiro– Wilk W-test, which is the preferred method for testing normality of data when the sample size is small.25 Efforts to employ NLME modeling of the PELF was unsuccessful due to the apparent random fluctuations of values within an individual. Therefore, the evaluation of the data generated from the PELF was limited to a simple numerical comparison of the average concentrations at each time for each group of calves.

Results

No adverse reactions were observed following placement of the ISF probes, jugular catheters, or tulathromycin injection. However, due to malfunctions in the ISF probes in several calves, fluid was not collected over some sampling intervals. Therefore, only incomplete ISF concentration versus time profiles were available in some calves from the two ages groups.

During BAL collection, sample volumes ranged from 0 to 42.5 mLs. At the 72 h sample for 6-month old calves, the lavage yielded no lung fluid for two of the four of the calves. The variability of fluid retrieved with the BAL procedure in the 3-week old calves could be attributed to difficulties in achieving proper placement of the catheter in their terminal bronchus.

Plasma

Based upon free plus protein bound (total) plasma tulathromycin concentrations, age associated differences were observed in several of the primary and derived PK parameters estimated via the NLME model (Table 2). The individual plasma-concentration time curves showed high variability and oscillations in the concentrations determined at the later time points in both groups of calves (Fig. 1). The Tmax values tended to occur earlier in 3-week versus 6month old calves.  CL/F and Vd/F determined from total plasma concentrations were significantly higher in 6-month old calves. The harmonic mean T1/2, was estimated as 67.6 and 44.4 h for 3-week and 6-month old calves respectively.  Based upon the fitted estimates of the rate of drug distribution from the peripheral back to central compartments in plasma (K21), values tended to be smaller (slower partitioning back into the plasma) in the 6-month vs. 3-week old calves, which is consistent with the larger Vd/F values estimated in the 6-month old calves.  Compartmental parameter rate constant estimates in the individual calves are depicted in Appendix (Table. S1).

After SC injection, the tulathromycin plasma Cmax values, estimated directly from individual calf data, achieved 1.34 µg/mL in 3-week old calves and 0.82 µg/mL in 6-month old calves. The unbound fraction of drug in plasma varied from 0.36 to 0.61 in 6-month old calves and 0.75-0.82 in 3-week old calves as tulathromycin concentrations varied from 0.1 to 1.0 µg/mL (Table 3).  Although a shift from proportionality in protein binding was seen in the older calves, the majority of the profile was constrained within a range where the protein binding was relatively stable. A significant difference was found in the unbound fraction at all tested concentrations in plasma pooled from healthy 3-week and 6-month old calves.

ISF

The collection of ISF using ultrafiltration probes allowed for the monitoring of unbound drug concentration in repeated samples. These devices were well tolerated and were able to collect between 0.05 mL to >2 mL of fluid in most samples. 6-month old calves showed detectable concentrations in ISF at 12 h post dosing.  In contrast, tulathromycin was not detected in the ISF of 3-week old calves until 51 h. In both age groups, maximum ISF tulathromycin concentrations were obtained later than that of the plasma. Regardless of whether expressed relative to total or unbound plasma drug concentrations, the ISF/Plasma concentration ratios were significantly higher in the 6-month versus 3-month old calves (Fig. 2).

PELF

Average PELF concentrations determined from BAL samples are reported in Table 4 and Figure 3. The average maximum concentrations in PELF occurred at 96 hours. Large variability in drug concentrations in PELF was seen across both groups at all time points. Concentrations in PELF exceeded blood and ISF concentrations for both ages of calves.

Discussion

Developmental changes in body composition, organ functions, ontogeny of drug biotransformation pathways and elimination pathways can impact drug PK in calves (Table 1). This is the first study to evaluate the effect of age on tulathromycin concentrations in plasma, PELF and ISF and plasma protein binding in calves. The use of ultrafiltration probes and the collection of PELF allows for a continuous assessment of active drug in sites of action.  Accordingly, the results of this investigation provide an important step toward our understanding of how drug PK, and therefore the targeted dose needed to achieve some desired level of tissue exposure, can be influenced by a calf’s age.

To determine active drug concentrations at infection sites, the accurate measurement of antimicrobial concentrations is crucial for the prediction of drug antimicrobial efficacy. Typical methods for obtaining this information includes the quantification of drug concentrations in plasma, tissue cages, and homogenized lung tissue. The latter is a poor predictor of drug concentrations in the PELF, which is where many lung bacterial infections are localized.26-28 Alternatively, the ISF has been considered a potential surrogate for drug lung concentrations since it reflects the unbound drug concentration and the partitioning of drug into a tissue compartment.  However, such extrapolations are inappropriate for macrolides.  For example, tulathromycin, PELF exposure has been shown to reach over 9x higher than plasma and ISF concentrations in ruminating calves.10 Lung concentrations of tulathromycin have been shown to be different in bovine pneumonic vs healthy lung homogenates but whole tissue homogenate do not allow for the evaluation of free versus bound drug concentrations in the pulmonary tissues.29

In contrast to our study results, it has been previously reported that there were no statistically significant differences in tulathromycin plasma PK parameters between preruminant calves (4-7 weeks of age) and adult cattle.11 What is not clear is whether or not this discrepancy may be due to PK differences between 3-week old versus 4-7 week old calves (even though both age groups are classified as pre-ruminants). We note that the PK parameter values for tulathromycin in 6-month old calves in our investigation are within the same range as reported in previous studies.10,30

Irrespective of age, plasma tulathromycin concentrations were relatively low. SC administration was associated with rapid absorption and a subsequent slow decline (Fig. 1). The younger calves had statistically significantly lower CL/F and Vd/F values as compared to those of the 6-month old calves. Across age groups, the T1/2 was similar, although was an observed trend for a longer T1/2 in the 3-week old (67.6 hours) versus 6-month old (44.4 hours) calves. Since disposition of drugs refers to the simultaneous effects of elimination and distribution, the observed age-related changes in these two processes could account for similarity in T1/2 values.

Assuming similar bioavailability following SC injection, the CL/F values in 6-month old calves (0.33 L/hr/kg) were were higher than those reported by.27 Although calves enrolled in that study were beef calves with body weights similar to those in current study (181-246kg), the age of the calves used were not reported. We also cannot discount the possibility that the observed differences CL/F values observed in the Nowakowski versus our study was a function of breed.

The lower CL/F values observed in the 3-week old calves in the current investigation could potentially be attributed to age-associated differences in kidney and liver function. Regarding liver function, hepatic clearance depends on several factors including blood flow, hepatic enzyme activities, transport systems and plasma protein binding.31 Hepatic blood flow has been shown to be lower in 3-month old calves compared to adults and can impact the metabolism of high extraction ratio drugs.32,33 Since tulathromycin is a low extraction ratio drug, it is unlikely that hepatic blood flow will be a contributing factor to the lower CL/F in young calves.34 Moreover, most the administered drug is eliminated unchanged and only about 10% is metabolized in cattle. Less than 10% each of the metabolites in excreta and tissues were formed by N-demethylation or N-oxidation.35 Therefore, it is highly unlikely that age-associated differences in CL/F were due to differences in drug metabolism.

Alternatively, tulathromycin is predominantly excreted unchanged in the feces.  Therefore, maturation differences in elimination pathways in the biliary excretion (i.e., via transporter mechanisms) may be responsible for differences in drug elimination. To date, little is known with regard to the maturation of liver efflux transporters in humans or veterinary species.  However, ontological changes in glomerular filtration, renal tubular secretion and tubular reabsorption have been well characterized in pediatric patients and could potentially have a profound impact on drug PK profiles.36 Therefore, it would not be surprising to find similar maturation-associated changes in the hepatic efflux transporters.

The Vd/F is affected by plasma protein binding, tissue binding and the lipid solubility of drugs. Lipid soluble drugs like tulathromycin have very high apparent volumes of distribution, as seen in this study with 3.5 L/kg and 10.5 L/kg in 3-week old and 6-month old calves respectively (Table 2). As animals mature, the body fat:water ratio increases, leading to a greater sequestration of lipid soluble drugs in the adipose tissue. Volume of distribution also takes into account drug distribution into immune cells. Macrolides (which are weak bases) tend to ionize in an acidic environment and therefore can accumulate in cells and tissues, particularly polymorphonuclear cells due to lysosomal trapping. This lysosomal trapping has been suggested to serve as a vehicle that transports drug to an infection site.37 While some argue that this may be a mechanism for high drug concentrations at the site of infection38, 39, others have refuted this assumption.40 In vitro, macrolides have demonstrated high accumulation in neutrophils and macrophages.  Irrespective of whether or not these do in fact as a drug delivery mechanism, differences in development of the immune system may also have contribute to the larger volume of distribution in older calves. Neutrophil counts were demonstrated to be higher than adult reference values at 1 day of age but returned to adult levels by 28 days of age.41

The changes noted in the plasma kinetics of tulathromycin could be attributed to differences in plasma protein binding. Most previous studies determined plasma PK parameters using total (unbound + bound) concentrations.  Such measures may miss significant changes as a function of age in intrinsic clearance or unbound systemic drug concentrations. As only unbound drug is free to move across biological barriers and to fight infections, unbound concentrations are the more clinically relevant measurement. Reported bovine plasma tulathromycin protein binding is approximately 40% [i.e., fraction unbound (fu)= 0.53-0.68 in 6-month old calves.27,10 The in vitro plasma protein binding estimated in the current study was similar for 6-month old calves (Table 3). In contrast, we estimated the protein binding in 3-week old calves to be approximately 17-24%. Similar to our study in calves, age has been found to influence the plasma protein binding of a variety of drugs in humans.42

In calves, concentrations of major drug binding proteins such as albumin, and AGP showed significant changes during the first three months of life.43,44 The higher unbound fraction of basic drugs, like tulathromycin, in 3-week old calves may be due to the drug binding properties of AGP.45 Since the plasma concentration of AGP is relatively low in neonates and there is only one drug-binding site in each AGP molecule, drug binding to AGP is typically saturable and is readily displaceable.46

For macrolides, the use of blood levels for the assessment of dose when treating BRD does not directly mirror drug concentrations at the site of action. Although not the targeted infection site, measuring drug concentrations within the ISF was considered to provide an additional layer of information regarding the way tulathromycin moves through the various tissue compartments and how the observed plasma PK differences in 3-week old and 6-month old calves may have influenced the distribution in ISF vs plasma. Drug concentrations in the ISF have been evaluated for several drug classes, including cephalosporins, fluoroquinolones, and macrolides.10,23 Overall, many macrolides have limited penetration into ISF compartments, as shown by consistently lower drug concentrations in the ISF compared to free drug concentrations in the plasma. These results are consistent with the observed difficulty in achieving therapeutic drug concentrations in human ISF for a wide range of macrolides.47 Therefore, it may be difficult to achieve the macrolide concentrations necessary to successfully treat soft tissue infections.

Tulathromycin concentrations in the ISF were significantly higher in 6-month old calves as compared to 3-week old calves.  This could reflect age-associated differences in body composition and immune system constituents (Fig. 2A). Tulathromycin was below the limit of detection (0.005 µg/mL) in ISF until 48 h after dosing for the young calves, but was detectable in 6-month old calves by 8 h postdose. In addition, ISF/plasma ratios were examined before and after correcting the plasma concentrations for protein binding (Fig. 2B, 2C). Differences were noted in ISF concentrations and ISF/Plasmaunbound ratios as a function of time in both groups. For the 3-week old calves, the ISF/plasma (total and unbound) ratios peaked at 168 hours after dosing while that of the 6-month old calves reached peak concentrations much earlier at around 48 hours (Fig. 2C).  These differences in both concentration and kinetics of ISF drug accumulation may reflect the change in adipose and muscle growth that occurs as animals mature.  The older calves tend to have a smaller proportion of total extracellular water as compared to that of veal calves.  In younger animals, larger proportion of total body water (especially in adipose tissue, which is where the SC probes were placed) could lead to a dilution effect.  The latter would result in the lower drug concentrations found in the ISF. Understanding the partitioning of unbound antimicrobial drug into the ISF and plasma may not be the best modality in determining appropriate therapy in different ages of calves.

Regarding lung concentrations, previous studies have estimated these by such methods as tissue homogenates, lung biopsies, bronchial microsampling and bronchoalveolar lavage.23,28,48 Comparing tilmicosin concentrations (another macrolide) estimated using direct sampling (bronchial swabs) vs bronchoalveolar lavage, it was concluded that the concentrations estimated using either of these two techniques were not significantly different.49 In contrast, the use of lung/tissue homogenates does not allow for a differentiation between intracellular vs. extracellular drug concentrations or for the binding of drug to the tissues. Therefore, lung homogenates typically overestimate the active concentration of macrolides at the site of infection.50

Alternatively, PELF is secreted extracellularly in the respiratory tract and is a potential site of respiratory bacterial infections in cattle. Previous studies demonstrated that some antimicrobials exhibit higher penetration into the PELF than others and therefore are more effective in the control of BRD.10 Accordingly, we sought to measure tulathromycin PELF concentrations as a function of calf age.  Determining drug concentration in the PELF allows for the evaluation of plasma/ISF/PELF PK-PD exposure relationships, thereby facilitating an assessment of potential therapeutic dosing strategies as a function of the calf age. PELF concentrations of tulathromycin tended to be higher in 3-week old calves (Fig. 3). Interestingly, this contrasts with the relationship observed in the ISF. The higher PELF concentrations in 3week old calves cannot reflect immature physiological barrier function in the blood-alveolar interface in the lungs.   Since P-gp is believed to efflux substances into the alveolar sac serving a protective function51, the presence of an immature P-gp activity cannot explain the higher concentration of tulathromycin in the PELF in 3-day old calves. Efflux across the pulmonary membrane would increase tulathromycin concentrations in the PELF.  Therefore, the involvement of an immature P-gp is not likely.  However, if the pH of the PELF were lower in young versus mature calves, ion trapping may have contributed to these observed differences.  The pH of the PELF of young versus mature calves have yet to be measured.

When evaluating the mean concentrations in PELF at each time point, interpretation of age related differences is hindered by the high variability seen across both groups (Table 4). Although concentrations in the PELF was higher than that in the ISF and plasma irrespective of calf age, the variability in PELF concentrations may have been biased by the use of urea to determine volume of PELF. When urea is used as a dilution marker, the dwell time of fluid infused in the airways during the BAL procedure may represent an important source of experimental variability52. Since tulathromycin concentrates at high levels in neutrophils, contamination from ruptured cells during BAL fluid centrifugation process, as well as irritation from repeated BAL sampling, may over-estimate drug concentration in the PELF. Although contamination from lysed cells is possible, the ruptured neutrophils alone cannot explain the high concentrations in the extracellular matrix40 Given these limitations and uncertainties, PELF values should be considered as a rough approximation of pulmonary drug concentrations.

Determining the right dose for drugs used to treat diseases in neonates is critically important.  Physiological differences affecting drug absorption, distribution, metabolism, and elimination render the extrapolating of doses from mature to pre-ruminating calves unreliable.  Movement of tulathromycin from plasma to the ISF and PELF has been shown to be significantly different in young vs older calves, which in turn may indicate functional changes in body composition. The therapeutic success of tulathromycin for the treatment of BRD relies not only on the drug reaching efficacious concentrations at the site of infection, but also on the functionality of a calf’s immune system. Although there are greater numbers of phagocytic cells in the neonatal calf, the function of these cells is decreased until around 4 months of age.53 Each of these components need to be considered when assessing he magnitude of dose adjustment necessary when treating disease in young calves.  

Conclusions

In veterinary species, data are limited with respect to the physiological changes associated with age and its corresponding influence on drug PK. The results of the present study lead us to conclude that age will influence the PK and distribution of tulathromycin when administered as a 2.5 mg/kg SC injection to 3-week old and 6-month old calves.  Rapid absorption and extensive distribution to PELF is advantageous when treating bovine respiratory disease, but differences in age clearly impact the disposition of tulathromycin.  Lower Vd/F and CL/F estimates resulted in higher plasma levels but lower ISF concentration levels in 3-week vs 6-month old calves. Although concentrations in the PELF were highly variable, they were consistently higher than plasma values at all time points.

Based upon our findings, there is a need for additional studies to compare distribution of tulathromycin in diseased vs healthy calves, as well as the relationship between drug exposure vs. response in different ages of calves.

Footnotes

1. Draxxin, Zoetis, Parsippany, NJ
2. Reinforced Ultrafiltration probe, BASi Inc., West Lafayette, IN
3. Rompun Injectable (20 mg/mL), Bayer Animal Health Division, Morrisville, NC
4. Foley Urinary Catheters with Wire Stylet, MILA International, Inc., Florence, KY
5. Tandem Mass Spectrometry Triple Quad, Waters, Milford, MS
6. Oasis 3 cc PRiME HLB cartridges, Waters, Milford, MS
7. Urea test kit, Sigma Chemical, St. Louis, MO
8. Centrifree Ultrafiltration Device, Millipore Sigma, St. Louis, MO
9. Phoenix WinNonlin/NLME, Version 1.3 Certara, Cary, NC
10. SigmaPlot, Systat Software, Inc., San Jose, CA

References

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Figure Legends

Fig. 1. Individual Plasma (bound and unbound) concentration time curves after single SC. injection of 2.5 mg/kg tulathromycin in eight 3-week old (○) and seven 6-month old (▲) calves.

Fig. 2. Mean ISF concentrations ± SD (A), ISF:Plasma (total) ratios (B) and ISF:Plasma (unbound) ratios (C) of tulathromycin in 3-week old and 6-month old calves. Each point is a calves.

American Journal of Veterinary Research

671  Table 1

Developmental Factors Affecting Drug Pharmacokinetics in Neonatal Calves

672

673

674

675

Table 2

Population pharmacokinetic parameters from NLME analysis (n = 7 six-month old calves and n = 8 three-week old calves) after SC administration of 2.5 mg/kg tulathromycin in Holstein calves.

Population Values – Final Model

θV	L/kg	3.7	0.0045	28.3
θV2	L/kg	16.4	4.8	20.2
Cl	L/kg/h	0.10	0.14	21.5
Cl2	L/kg/h	0.92	0.02	20.2
T½*	h	54.3	24.1	17.2

 

Parameter	Units	Mean	Range	Mean	Range  681
TMAX   CMAX θV/F θV2/F Cl/F Cl2/F T½*	h   µg/mL L/kg L/kg L/kg/h L/kg/h h	0.68   0.82 10.5 24.03 0.33 1.07 44.4	0.25 – 1.0   0.32 – 1.63 5.2-20.4 16.5-37.5 0.16-0.49 0.37-1.42 30.4-123.8	0.38   1.34 3.5 16.47 0.14 0.91 67.6	0.25 – 0.50 682   0.61 – 1.84 683 2.0 – 7.03 684 7.5 – 32.7 685 0.07 – 0.25 686 0.53 – 1.63 687 43.7 – 163.5 688

Concentration   (µg/mL)	6-Month Old   % Bound	Unbound   fraction	3-Week Old   % Bound	Unbound   fraction
0.1	63	0.36	22	0.77
0.5	49	0.54	17	0.82
1.0	39	0.61	24	0.75

Table 3

Average %

Plasma protein binding and calculated %

CL, clearance per fraction absorbed; C 689  bound of

half-life; TMAX 690  tulathromycin

691  using

value for the population.   692  microcentrifug

693  ation.

Percent plasma protein binding of tulathromycin spiked plasma from 3-week-old and

6-month-old calves and calculated % bound using the equation from (Toutain &

 

Bousquet-Melou, 2002). Samples were spiked in triplicate at 3 concentration levels 694

(0.1, 0.5, and 1 µg/mL) and averaged. 695

696

Table 4

Mean Tulathromycin Concentrations in PELF

 

Time 6-Month Old  3-Week Old

(hours)  µg/mL ± SD  µg/mL ± SD

3  2.9 ± 2.2  4.9 ± 3.1 715

12  2.1 ± 1.9  5.3 ± 4.6 716

717  24 0.5 ± 0.3 4.7 ± 5.1

718

48  1.9 ± 0.2  3.6 ± 5.0

719

Figure 1: Total tulathromycin plasma concentration vs time curve in all calves

Figure 2: Interstitial Fluid Concentrations and ISF/Plasma ratios (total and unbound)

A) Mean Interstitial Fluid Concentrations ± SD

Figure 3.  Tulathromycin Concentrations in PELF