Bisphosphonates (BPs) have been used for over 40 years in human medicine and their use in veterinary medicine has gained interest in the last 10–20 years due to their effects on osteoclast inhibition.1 In 2014, 2 BPs, tiludronate disodium (C7H9ClO6P2S) and clodronate disodium (CH2Cl2Na2O6P2), were approved by the Food and Drug Administration under the New Animal Drug Application (FDA NADA) 141-420 (tiludronate disodium) and 141-427 (clodronate disodium [CLO]) for the treatment of navicular syndrome in horses 4 years of age and older.2,3 BPs’ ability to impair osteoclast function4 with possible concomitant pain-relieving effects5 makes them potentially useful for a myriad of musculoskeletal conditions, including fetlock and distal tarsal osteoarthritis6,7 and thoracolumbar vertebral disease.8 After administering BPs, the drug that is not absorbed by the skeleton is excreted unchanged by the kidneys.9 In veterinary medicine, clinicians have raised concern over adverse effects from extra-label use in young and exercising horses including impediment of growth, masking of pain, and fracture predisposition, in addition to known potentially adverse effects such as renal toxicity and gastrointestinal discomfort.1,10,11
Sheep have been used to assess the efficacy of BPs for osteoporosis and to investigate adverse effects experienced by humans such as necrosis of the jaw and atypical femoral fractures.12–16 In addition, sheep have been used in exercise studies as a model for horses.17 Therefore, juvenile sheep may be suitable for investigating the effects of BPs under exercise, providing a novel animal model to assess the effects of BPs in juvenile, exercising horses.
To use sheep as a model for juvenile, exercising horses, an appropriate dose of CLO must be determined. The ideal dose would result in pharmacokinetic and plasma protein binding profiles similar to those observed in horses. Pharmacokinetics (PK) focus on how drugs enter the body, travel to their site of action, and are removed from the organism.18 Drug accumulation and elimination rates from an organism are determined by the absorption, distribution, metabolism, and excretion of a specific drug over time.19 The quantification of these parameters helps establish a safe and effective dosage of a drug.19 Plasma protein binding (PPB) determines the fractions of drug bound (fb) and unbound (fu) to plasma proteins in the blood, which influence the volumes of distribution (Vd)20 and clearance (CL) of drugs.21–23 Not evaluating PPB leads to misinterpretations of the total drug plasma concentration, as fu is directly associated with the CL of the total drug plasma concentration.24 Hence, differences in PPB values between species may affect the safety margin of drugs, leading to potential incompatibility between species.25 The PK parameters of a commercially available, intramuscular (IM) formulation of CLO have been published for horses2,26,27 but not sheep and the authors are unaware of studies that describe the PPB of CLO in sheep or horses.
The current study compared the PK of a single dose of CLO (0.6 mg/kg IM) in juvenile sheep and determined the PPB of CLO in both sheep and horses. The CLO dose of 0.6 mg/kg IM was based on a preliminary study.28 We hypothesized that a single 0.6 mg/kg dose of CLO administered IM in sheep would lead to similar PK parameters as a single dose of CLO administered at 1.8 mg/kg IM in adult horses. Furthermore, we hypothesized that there would be less than a 5-fold difference between the fu of CLO in sheep and horses.
Methods
Sheep
The animal use experimental protocols was approved by the Michigan State University (MSU) Institutional Animal Care and Use Committee (202000264). All sheep were obtained from an established flock at the MSU Sheep Teaching and Research Center (Lansing, MI).
Eleven juvenile crossbred (Dorset X Polypay, 76 ± 8 kg, 252 ± 6 days of age, 5 castrated male and 6 female) sheep were used in the PK study. A juvenile sheep was defined as an animal that had reached approximately 80% of its adult weight.29,30 All sheep had a physical examination, including heart rate (HR), respiratory rate (RR), and rectal temperature (RT), before sample collection.
Three additional sheep (Dorset X Polypay, 68 ± 11 kg, 281 ± 4 days of age, 1 castrated male and 2 females) were used as PPB study subjects; these sheep had never received CLO and had a physical examination before the sample collection.
All sheep were housed in a 21.6 m2 pen in an indoor facility at the MSU Bennett Road Farm. The sheep underwent a 2-week acclimation period before sampling. All sheep had free access to a 90% dry matter (DM) total mixed ration that contained chopped hay (82%), a corn/soybean blend (16.5%), and 1.5% of mineral blend (Caledonia Farmers Elevator). The sheep ingested approximately 2.0% to 2.3% of their body weight (BW) in DM per day and had ad libitum access to water.
Horses
The animal use experimental protocols was approved by the Texas A&M Institutional Animal Care and Use Committee (2019-0325). All horses were obtained from Texas A&M Dick Freeman Arena (College Station, TX).
Horses were group housed in dry lots at the time of sample collection for PPB. Three juvenile horses (Quarter Horses, 411 ± 18 kg, 599 ± 25 days of age, 1 castrated male and 2 females) were used for the PPB. A juvenile horse was defined as an animal that had reached approximately 80% of its adult weight.31 Horses were fed coastal Bermuda grass hay ad libitum and supplemented twice daily with 1.4 kg of a 12% protein, 8% fat commercially available concentrate. All animals were used as part of an equine behavior and training course and health status was monitored daily.
Pharmacokinetic study dose selection
The CLO dose was determined by a preliminary study in which 12 adult sheep were administered 3 different single doses of CLO (n = 4/treatment group: 0.6, 1.8, 3.0 mg/kg IM, respectively).28 Preliminary study findings suggested a single dose of 0.6 mg/kg IM resulted in similar plasma concentrations of CLO as reported in mature horses for 48 hours after administration.2,28
Pharmacokinetic study design
Sheep were moved through a chute system in a randomized order and restrained in a crate for sampling. Ten milliliters of blood were collected from the right jugular vein using an 18-gauge needle and vacutainer before single administration (hour 0) of CLO (OSPHOS®, Dechra Veterinary Products) at 0.6 mg/kg IM in the right side of the neck. An additional 10 mL of blood was collected from the right jugular vein at 0.5, 1, 3, 6, 12, 24, 48, and 72 hours in the same collection order established at hour 0. Blood was collected in a K2 EDTA blood collection plastic tube and immediately placed on ice for transport followed by sample centrifugation at 2,000 X g for 10 min.
Plasma was aliquoted into 2.0 mL microcentrifuge tubes and frozen at −80 °C until analysis. All samples were analyzed within 4 months of collection. Animals were monitored hourly during the first 6 hours and then daily after drug administration for acute adverse effects (eg, syncope and sudden death), side effects described in other species (eg, gastrointestinal discomfort and/or swelling at the injection site), and/or changes in normal behavior (eg, agitation and depression).2,32,33
Bioanalytical methods
The liquid chromatography-mass spectrometry (LC-MS/MS) analysis was based on the methods of Hasan and colleagues.34 Samples were removed from −80 °C and thawed to room temperature. An internal standard of 20 µL (10 ng/µL of etidronate solution, Sigma-Aldrich) was added to 1.0 mL of plasma. Then, 200 µL of perchloric acid (10%) was added to precipitate proteins. The solution was thoroughly mixed using a vortex mixer and centrifuged at 17,000 X g for 10 min. The supernatant was evaporated until visibly dry under a stream of nitrogen at ∼65 °C, then dissolved in 150 µL glacial acetic acid and mixed with 500 µL trimethyl orthoacetate. The solution was incubated at 100 °C for 30 min for derivatization. Samples were allowed to cool to room temperature and then 300 µL formic acid and 500 µL DI water were added. The solution was then transferred to a screw-top tube, followed by liquid-liquid extraction with methyl tert-butyl ether. The tube was capped, placed in a rotorack for 10 min, and centrifuged at 12,000 X g for 5 min. The bottom layer was removed by aspiration. The supernatant was transferred to a new glass tube and evaporated until visibly dry under a stream of nitrogen at ∼65 °C. The residue was reconstituted in 80 µL of a 1:1 methanol-water mixture and transferred to an autosampler vial (Zorian, Inc) for the LC-MS/MS analysis. Plasma clodronate concentrations were quantified by LC-MS/MS analysis conducted in a Thermo TSQ Altis™ (ThermoFisher Scientific) triple quadrupole mass spectrometer with an electrospray ionization source, with a lower limit of detection (LOD) of 10 ng/mL for CLO.
Pharmacokinetics data analysis
Noncompartmental PK parameters were determined using commercially available software (Phoenix WinNonLin 8.3). The PK parameters included the time of maximum concentration (Tmax), maximum concentration (Cmax), the slope of the second phase (slow distribution phase) of the drug’s concentration curve (λz), terminal half-life (t1/2λ), area under the curve estimated to the last observation (AUCall), area under the curve extrapolated to infinity (AUC0-inf), and mean residence time (MRT).
Plasma protein binding assay
Twenty milliliters of blood were collected from the jugular vein of sheep and horses using an 18-gauge with a hub. Blood was immediately placed in a K2 EDTA blood collection plastic tube and placed on ice for transport. Plasma was harvested and stored as previously described for no longer than 3 months. Plasma samples were thawed at room temperature and CLO (pharmaceutical grade, Millipore Sigma) was added to 2 mL of plasma to obtain the desired concentrations for sheep (0, 100, 1,000, 10,000, 20,000, and 40,000 ng/mL) and horses (0, 100, 1,000, 7,500, 10,000, and 15,000 ng/mL). These concentrations were selected based on our preliminary sheep study (Cmax 11,605 ng/mL)28 and a previously disclosed equine study (Cmax 7,460 ng/mL).2 A single-use rapid equilibrium dialysis (RED®, ThermoFisher Scientific) system was used according to the manufacturer’s recommendations.
In brief, 500 µL of the spiked samples were added to the plasma chamber and 750 µL of 1X PBS was added to the buffer chamber. The kit was covered with a sealing plate and incubated at 37 °C for 6 hours on an orbital shaker at 250 rpm. The content of the plasma chamber and buffer chamber were then pipetted into separate microcentrifuge tubes and stored at −80 °C. Samples were diluted with distilled water to increase the volume for laboratory analysis. Each biological replicate was measured in duplicate as a technical replicate. The CLO concentrations within both chambers were calculated using LC-MS/MS as described. The technical replicates for each biological replicate were averaged to create an individual data point for the analysis. The percentage of the unbound drug was calculated as follows: %free drug = (drug concentration buffer chamber)/(drug concentration plasma chamber) X 100.
Statistical analysis
Normality of the PK data was assessed by a Shapiro-Wilk test using Phoenix WinNonLin 8.3. Normally distributed data including physical examination, PPB, and PK parameters, except for Tmax, were reported as means and ranges. Nonnormally distributed data (Tmax) were reported as median and range.
Results
Pharmacokinetics of clodronate in sheep
Sheep were determined to be bright and alert before PK sampling. Physical examination parameters reflected mild stress associated with temporary restraint: HR (138 [102–160] beats per min), RR (97 [60–132] breaths per min), and RT (39.5 [39.1–39.8] °C]). No animals or data were excluded from the analyses. No adverse effects were detected after the administration of CLO to juvenile sheep (n = 11).
Mean values of CLO plasma concentration in sheep over time are presented (Figure 1). After IM administration of CLO, the Tmax was reached at 0.5 (0.5–1.0) hours postadministration, with a Cmax of 5,596 (2,396–8,613) ng/mL. The λz was 0.034 (0.023–0.042) 1/h, t1/2λ reached 21.2 (16.4–30.3) hours with an AUCall of 12,831 (7,590–17,593) X ng/mL, and AUC0-inf of 13,334 (7,947–17,973) X ng/mL (Table 1).
Plasma clodronate disodium concentration (ng/mL) time curves over time (72 h) after a single IM administration of 0.6 mg/kg (OSPHOS®) in 11 juvenile sheep.
Citation: American Journal of Veterinary Research 84, 8; 10.2460/ajvr.23.03.0051
Plasmatic pharmacokinetics (PK) parameters (mean or median [Tmax] and range) for clodronate disodium (OSPHOS®) after single-IM administration in juvenile sheep (0.6 mg/kg) determined through liquid chromatography-mass spectrometry and noncompartmental analysis compared with a previously published single dose (1.8 mg/kg) PK study in mature horses.27
| Mean | Range | ||||
|---|---|---|---|---|---|
| Parameter | Unit | Sheep (n = 11) | Horse (n = 7)27 | Sheep (n = 11) | Horse (n = 7)27 |
| Tmax | h | 0.5 | 0.5 | 0.5–1.0 | 0.25–0.75 |
| Cmax | ng/mL | 5,596 | 4,155 | 2,396–8,613 | 1,969–5,541 |
| λz | 1/h | 0.034 | 0.002 | 0.023–0.042 | 0.0001–0.016 |
| t1/2λa | h | 21.2 | 141.7 | 16.4–30.3 | 43.1–6,814.8 |
| AUCall | h X ng/mL | 12,831 | 11,564 | 7,590–17,593 | 6,393–17,026 |
| AUC(0-inf) | h X ng/mL | 13,334 | 11,912 | 7,947–17,973 | 6,507–17,606 |
| MRT(0-inf) | h | 12.1 | — | 8.1–22.3 | — |
AUC0-∞ = Area under the curve extrapolated to infinity. AUCall = Area under the curve to the last observation. Cmax = Maximal concentration. λz = Slope of the second phase. MRT(0-inf) = Mean resident time from 0 to infinity. t1/2λ = Terminal half-life. Tmax = Time of maximal concentration.
Harmonic mean.
Plasma protein binding
The mean percentage of unbound CLO in sheep plasma was 26.1 ± 5.1% (n = 3 for each of 5 concentrations) from 100 to 40,000 ng/mL of CLO (Table 2). The mean percentage of unbound CLO in horse plasma was 18.7 ± 7.5% (n = 3 for each of 5 concentrations) from 100 to 15,000 ng/mL of CLO.
In vitro unbound fraction (fu, mean ± SD) of clodronate disodium (ng/mL) in juvenile sheep and horses.
| Clodronate disodium concentrations (ng/mL) | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| 0 | 100 | 1,000 | 7,500 | 10,000 | 15,000 | 20,000 | 40,000 | Average fu (%) | |
| Ovine (%) | <LOD | 26.1 ± 0.1 | 34.5 ± 0.1 | ND | 25.5 ± 0.1 | ND | 22.7 ± 0.1 | 21.7 ± 0.1 | 26.1 ± 5.1 |
| Equine (%) | <LOD | 26.4 ± 0.1 | 21.9 ± 0.1 | 23.7 ± 0.1 | 11.8 ± 0.0 | 9.8 ± 0.0 | ND | ND | 18.7 ± 7.5 |
The table indicates the unbound clodronate disodium fractions in sheep plasma (0, 100, 1,000, 10,000, 20,000, and 40,000 ng/mL, n = 3) and horse plasma (0, 100, 1,000, 7,500, and 15,000 ng/mL, n = 3), determined using single-use rapid equilibrium dialysis and liquid chromatography-mass spectrometry analysis. ND = not determined. <LOD = below lower level of detection of 10 ng/mL).
Discussion
Sheep have been used as a model to evaluate potential adverse effects of BPs in humans12–16 and exercise on horses,17 suggesting that sheep may be a useful model species to evaluate the effects of BPs in juvenile, exercising horses. The PK and PPB of CLO must be similar between sheep and horses for sheep to be a reasonable model for exercising horses. Therefore, this study sought to determine the PK of CLO in sheep and determine the PPB of CLO in horses and sheep. Sheep PK parameters, including Cmax, Tmax, AUCall, and AUC0-inf, were similar to the horse’s PK parameters, and fu in sheep was less than a 5-fold difference from the fu of horses. These results support sheep as an appropriate model for CLO administration in horses.25
This study measured the PK response in juvenile sheep over 72 hours to compare with recently available data in horses.27 The PK parameters such as Cmax, Tmax, AUCall, and AUC0-inf were similar between sheep and recent findings by Knych and colleagues for horses,27 providing evidence that a 0.6 mg/kg IM dose in sheep may be appropriate to model a 1.8 mg/kg IM dose in horses. Despite the total duration of the PK study being longer (182 days) and the assay sensitivity being greater in the study by Knych and colleagues,27 our study reports sheep blood concentrations of CLO that appear to mirror what is reported in the horse for the initial 72 hours after administration. Although there are no clear criteria to define equivalent PK parameters between species, previous studies have suggested similar PK parameter values with up to a ∼40% difference in AUC and Cmax.35–37 The current study indicates that sheep had 10% and 26% higher values for AUCall and Cmax, respectively, in comparison to published equine data,27 further supporting the validity of CLO administration in sheep as a model for the horse. A statistical analysis was not performed comparing sheep PK parameters with historical PK parameters in horses due to the multiple limitations of utilizing historical data. However, an a priori sample size analysis indicated 6 animals would be needed in each group to achieve a power of 0.8 with a mean difference between groups of 4,600 h X ng/mL and SD of 2,500 h X ng/mL. A post hoc power analysis comparing the AUCall reported previously from 7 horses27 to the AUCall from the current study of 11 sheep, results in a power of 0.14, and a post hoc sample size analysis indicates that 81 animals would be required to obtain a power of 0.8 using the mean difference of 1,267 h X ng/mL between the AUCall for sheep and that reported in horses.27 The decreased post hoc power and increased post hoc sample size are reflective of the similarity found between the AUCall for sheep and horses.
The majority of PK parameters between sheep and horses were similar, however, t1/2λ and λz were the exception. Differences in t1/2λ and λz between our study and the study by Knych and colleagues27 may be explained by differences in sample analysis and study durations. The study by Knych and colleagues had a more sensitive limit of quantification (0.1 ng/mL) than our study, allowing them to report the terminal phase of elimination of CLO.27 Our analytical technique was not able to detect the terminal phase of elimination as reported by Knych et al.27 Instead, our study determined the slope of the drug slow distribution phase or second phase. Future studies should include additional sample times and more sensitive methods of BP detection to determine the terminal phase of elimination in sheep. This analysis would help to elucidate the reattachment and recirculation of CLO over time associated with bone turnover.38 This would be particularly interesting to determine in exercising and growing animals that may experience frequent bone turnover and recirculation.26
Physiologic differences between sheep and horses may provide an alternative explanation for the differences in λz and t1/2λ between studies.39 These PK parameters have inverse proportionality (t1/2λ = 0.693/λz), where λz is dependent upon the drug elimination from the body, CL, and the ability of the drug to distribute to extravascular tissues, Vd (λz = CL/Vd).20,39,40 Vd can be considered a proportionality constant between the amount of drug in the body and plasma concentrations at a given time.20 Different versions of Vd may be used as the proportionality ratio may have different values depending on the state of drug disposition.20 BPs largely bind to bone,41 and horses have significantly more bone mass than sheep (horse: 12–15% and sheep: 5.5% of BW).42–44 This increased bone mass may increase the terminal phase volume of distribution (Vdarea) in horses (compared with sheep) resulting in a lower λz and increased t1/2λ.20 However, Vdarea of CLO cannot be measured in this study as this drug was not administered intravenously20 in the previously published horse data 27 or the present sheep data. Therefore, the discussion of Vd must be theoretical in nature. This study illustrates that horses have a higher, although similar, PPB for CLO when compared with sheep. Increased PPB should have the opposite effect on Vd, leading to decreased Vd and resulting in an increase in λz and lower t1/2λ. Therefore, PPB is unable to explain the differences in λz and t1/2λ between sheep and horses. Although t1/2λ and λz are important PK parameters, they have the greatest influence on the dosing regimen of drugs.45 As CLO is administered at large dosing intervals,2 these PK parameters may be less important for determining the viability of sheep as a model for horses. Similarities in the maximum concentration (Cmax) and exposure to the drug (AUCall) are arguably more relevant.
Two additional studies have analyzed the PK parameters of IM clodronate administration in horses,2,26 but neither provides the ideal comparison for the current study. The FDA NADA for OSPHOS® describes both a single-dose and multi-dose study.2 Unfortunately, in the single-dose administration study, the maximal proposed dose (900 mg) was administered to each animal without consideration of the animal’s weight. Further, the study reported only mean values for limited PK parameters.2 A PK study was then conducted during the multi-dose safety assessment where CLO was administered every 28 days at 1.8 mg/kg IM; the PK study was conducted on day 84 after the fourth dose.2 The PK parameters reported in this multidose study in horses were similar to our ovine study, including AUCall (12,150 ± 1,830 h X ng/mL), Cmax (5,360 ± 980 ng/mL), and Tmax (0.33 ± 0 h), which could further support the use of sheep as a model for horses.2 However, the multidose study FDA NADA for OSPHOS® had a shorter sample time (48 hours), different methodology of CLO measurement (gas chromatography-mass spectrometry), and shorter t1/2λ in horses (1.7 ± 0.5 h),2 making it difficult to propose a meaningful comparison to our study. The authors are aware of one additional PK study of CLO in horses.26 However, that study returned markedly lower plasma CLO concentrations (eg, AUCall: 703 ± 158 h X ng/mL) in comparison to other published reports,26 possibly due to the frequent administration of xylazine to facilitate arthrocentesis.26 Xylazine increases osmotic diuresis, increasing CLO excretion.46 Therefore, considering the multidose study used in the FDA NADA for OSPHOS®2 and the administration of xylazine by Kreuger et al,26 the authors recognize that the historical data obtained by Knych and colleagues27 is most appropriate as a comparison to our ovine PK study.
The current study also presents novel information regarding the PPB of CLO in horses and sheep. The concentrations tested for the sheep (100–40,000 ng/mL) and horses (100–15,000 ng/mL) encompass the CLO concentrations observed after 0.6 mg/kg IM single administration in sheep and 1.8 mg/kg IM single administration in horses.27 The PPB has been defined as high if less than 20% of the drug is unbound to plasmatic proteins, as moderate PPB if between 20% to 60% is unbound, and as low PPB if more than 40% is unbound.47 Our results indicated a moderate to low fu of CLO in both species. Although differences between sheep and horses were found, these differences were modest (not greater than 1.4-fold), suggesting that the plasmatic binding of CLO was similar between the 2 species. These findings further highlight the usefulness of sheep as an animal model for studying the effects of CLO and its potential implications for horses.
The main limitation of the present study is the lack of a parallel PK study in juvenile horses with an identical duration and assay sensitivity. In addition, measuring CLO excreted in urine would have helped to more accurately determine renal CL and Vdarea for CLO in sheep,20,48 as BPs are mainly excreted in urine.9 Future studies could also include biochemical profiles, urinalysis, and kidney histological assessment to identify adverse renal effects of CLO administration in sheep.41 Bone biopsy samples would be valuable for determining the concentration of CLO absorbed in the cortical and trabecular bone at different locations within the skeleton and could be performed under different physiologic conditions, such as routine exercise or growth.
Despite these limitations, this study provides valuable insights into the PK properties of CLO in juvenile sheep and its potential as an animal model for assessing the effects of BPs in horses. Our findings demonstrated that a 0.6 mg/kg IM administration of CLO (OSPHOS®) in sheep resulted in similar PK parameters (Cmax, Tmax, AUCall, and AUC0-∞) and PPB values when compared with 1.8 mg/kg IM single administration of CLO (OSPHOS®) in horses, without any measurable adverse effects. Therefore, an ovine model of CLO administration presents a promising and previously unexplored approach for assessing and translating the effects of CLO on juvenile exercising horses.
Acknowledgments
Funding for this study was provided by the National Institute of Food and Agriculture – United States Department of Agriculture [2021-67015-34079].
The authors declare that there are no conflicts of interest concerning the research, authorship, and/or publication of this article.
The authors would like to thank Rachel Postello, Julia Baker, Taylor Collier, and Rebekah Agnew for their assistance in the data collection. The authors additionally acknowledge the support of Fernando Vergara-Hernandez through the Fulbright Foreign Student Program and the National Agency for Research and Development [56150020].
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