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Pharmacokinetics of harpagoside in horses after intragastric administration of a Devil's claw (Harpagophytum procumbens) extract

Abstract Devil's claw is used for the treatment of inflammatory symptoms and degenerative disorders in horses since many years, but without the substantive pharmacokinetic data. The pharmacokinetic parameters of harpagoside, the main active constituent of Harpagophytum procumbens DC ex Meisn., were evaluated in equine plasma after administration of Harpagophytum extract FB 8858 in an open, single‐dose, two‐treatment, two‐period, randomized cross‐over design. Six horses received a single dose of Harpagophytum extract, corresponding to 5 mg/kg BM harpagoside, and after 7 days washout period, 10 mg/kg BM harpagoside via nasogastric tube. Plasma samples at certain time points (before and 0–24 hr after administration) were collected, cleaned up by solid‐phase extraction, and harpagoside concentrations were determined by LC‐MS/MS using apigenin‐7‐glucoside as internal standard. Plasma concentration‐time data and relevant parameters were described by noncompartmental model through PKSolver software. Harpagoside could be detected up to 9 hr after administration. C max was found at 25.59 and 55.46 ng/ml, t 1/2 at 2.53 and 2.32 hr, respectively, and t max at 1 hr in both trials. AUC 0–inf was 70.46 and 117.85 ng hr ml−1, respectively. A proportional relationship between dose, C max and AUC was observed. Distribution (V z/F) was 259.04 and 283.83 L/kg and clearance (CL/F) 70.96 and 84.86 L hr−1 kg−1, respectively. Treatment of horses with Harpagophytum extract did not cause any clinically detectable side effects.

In cases of chronic pain wherein the equine patient requires ongoing, daily pharmaceutical support to maintain functionality and quality of life, the usefulness of NSAIDs is offset by the potential of their toxicological implications like the development of gastric and intestinal ulcer (Reed, Messer, Tessman, & Keegan, 2006). In addition, long-term treatment with NSAIDs has been associated with the suppression of normal proteoglycan synthesis in horses (Beluche, Bertone, Anderson, & Rohde, 2001). The well-known adverse effects in long-term use of NSAIDs are likely the contributors to the growing use of "alternative" treatments for chronic inflammatory disorders in horses (Trumble, 2005).
As a result of a number of recent studies, Harpagophytum is proposed as a complementary treatment for chronic rheumatism, tendonitis, osteoarthritis, and arthritis (Colas, Garcia, & Popot, 2006).
Pharmacokinetic data of H. procumbens have previously been documented in human plasma (Loew et al., 2001). Transferring the pharmacokinetic profile of a drug from one species to another might cause problems in clinical application concerning the design of dosage schedules (Lees, 2003). Although a liquid chromatography/electrospray ionization mass spectrometric method was published to detect iridoids in equine urine and plasma by the Association of Official Racing Chemists (Colas et al., 2006), there is no report to our knowledge on the pharmacokinetic data of harpagoside in equine plasma after administration of a therapeutic dose of Harpagophytum extract.
The objective of the present study was to evaluate the main pharmacokinetic parameters of harpagoside after single intragastric administration of two different doses of the H. procumbens extract FB 8858 in horses. As biopharmaceutical data are primarily important to link results from in vitro assays and clinical studies, the study of the pharmacokinetic parameters of harpagoside (the main active constituent of H. procumbens) will also provide basic data in order to design a rational dosage regimen for extracts of devil's claw and obtain useful information for clinical treatment.

| Experimental trials: Horses and equine samples
Six adult clinically healthy warm-blooded horses (1 gelding, 5 mares), with no history of joint disease, weighing 495-560 kg, were included in the study. They received three meals of a commercial feed per day.
During the experimental period, each horse was kept in an individual straw-bedded stable and given water ad libitum. The horses were kept on paddocks between the experiments.
The horses were randomly assigned to two treatment groups, using an open, single-dose, two-treatment, two-period, and randomized cross-over design. A 7 days washout period was arranged between administrations. The horses were weighed the day before each drug administration to exactly calculate the required dose of the Harpagophytum extract. The dosage was calculated based on the recommended daily dosage in humans (Chrubasik, 2004;ESCOP Monographs, 2009) considering metabolic body weight and equine body mass, respectively, and is reflecting the empirical applied dosages in horses based on the results of a clinical trial with the same H. procumbens extract conducted at the University of Veterinary Medicine Vienna, data not published. Each horse received a single harpagoside-dose of 5 mg/kg BM (trial 1) and 10 mg/kg BM (trial 2) via nasogastric tube. A catheter was placed into the left jugular vein to facilitate blood sampling. Blood samples (8 ml) were collected into heparinized tubes before and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 hr after intragastric administration. The blood samples were immediately centrifuged at 3900 g for 10 min. The plasma collected was stored at −20°C until analysis.
The study was discussed and approved by the institutional ethics committee (BMWF-68.205/0109-II/10b/2009) according to international guidelines.

| Sample preparation and plasma analysis
Plasma samples were cleaned up by solid-phase extraction (SPE), according to the method described by Colas et al. (2006)

| Validation of the LC-MS/MS method
To determine the selectivity of the method, the chromatograms of blank, spiked and drug administered equine plasma were compared to determine the level of interference by endogenous coeluent components.
Quantification of harpagoside was performed using apigenin-7-glucoside as internal standard. Stock standard solutions of harpagoside (100 ng/ml) and apigenin-7-glucoside (IS, 1 μg/ml) were prepared separately in methanol. These solutions were stored at 4°C away from light and were found to be stable for at least 6 months.
For the calibration curves, a series of 1 ml blank equine plasma was spiked with harpagoside and IS from the stock standard solutions to give harpagoside concentrations of 2.5, 5, 10, 15, 20 and 30 ng/ml.
Sample preparation and LC-MS/MS analysis of the calibration samples were performed as described above. Quantification was based on the ratio of the harpagoside peak area to internal standard peak area.
The limit of detection (LOD) was defined as the lowest concentration of spiked plasma samples resulting in a signal-to-noise ratio (S/N) of 3:1, and for the limit of quantification (LOQ) signal-to-noise ratio of 10:1. Both were determined using plasma samples spiked with harpagoside standards in concentrations of 2.5-30 ng/ml (n = 6 each). For each spiked standard the peak intensity of harpagoside was compared to the noise in the near neighborhood of the harpagoside peak. The resulting linear regression of S/N versus concentrations was used for determination of LOD and LOQ.
The accuracy of the method was assessed by adding known amounts (10 ng/ml) of the active pharmaceutical ingredient (harpagoside) to the equine blank plasma before SPE enrichment. Recovery (%) was calculated analysing four replicate QC samples. These were prepared in the same way as the calibration samples.
In order to evaluate precision, the repeatability (intra-day and inter-day) was analysed using the same QC samples spiked with 10 ng/ml harpagoside. They were injected into the LC-MS/ MS system six times on two different days. This allowed the percentage relative standard deviations (RSD, %) of the data to be calculated.

| Pharmacokinetic analysis
For determination of pharmacokinetic parameters, the software PKSolver (Zhang, Huo, Zhou, & Xie, 2010), an add-in programme for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel, was used. The utilized model was a noncompartmental analysis of plasma data after extravascular input. Linear trapezoidal calculation method was used for AUC.

| Safety assessment
Participant horses were monitored for adverse reactions, if any, with special attention to gastrointestinal disturbances as reported in humans. Safety monitoring included clinical investigation and evaluation of clinical signs as appetite and feed intake.

| Optimization of sample preparation and LC-MS/MS conditions
Selectivity of the extraction process was verified for plasma samples by comparison of a blank with the same sample spiked at a known concentration of harpagoside. No interfering peaks were observed at the expected retention time.
Linearity was obtained in the harpagoside concentration range 2.5-30 ng/ml. Samples were analysed in two batches. For each batch, six sets of standard calibration curves were measured.
The determination of precision using plasma samples spiked with 10 ng harpagoside per ml showed 6.3 % RSD intra-day and 12.8 % inter-day reproducibility. Accuracy of the sample preparation method including SPE showed a mean recovery of 96.8 %.
Harpagoside was stable under these conditions, storage did not affect concentrations.

| Pharmacokinetics
Plasma samples of six horses following single intragastric administration of H. procumbens extract FB 8858 were analysed by the above described method. The active pharmaceutical ingredient harpagoside was detected in equine plasma at 30 min after administration via nasogastric tube and reached the maximum plasma con- Note. AUC 0-inf , Area under the plasma concentration-time curve from time zero to infinity; AUC 0-t , Area under the plasma concentration-time curve from time zero to time t; AUMC 0-inf , Area under the first moment of the plasma concentration-time curve from time zero to infinity; CL/F, apparent total clearance of the drug from plasma after extravascular administration; C max , maximum plasma drug concentration; MRT, mean residence time from time zero to infinity; t max , time to maximum plasma concentration; V z /F, apparent volume of distribution during terminal phase after extravascular administration; λ z , terminal rate constant; t 1/2 , half-life.
TA B L E 1 Mean pharmacokinetic parameters calculated from equine drug plasma concentrations of harpagoside in six horses after single intragastric administration of Harpagophytum procumbens extract FB 8858 corresponding to 5 mg/kg BM versus 10 mg/kg BM harpagoside by PKSolver using a noncompartmental analysis of plasma data after extravascular administration. Linear trapezoidal calculation method was used for AUC respectively, in six horses are presented in Table 1. There was a proportional relationship between dose and C max or dose and AUC.
The mean harpagoside plasma concentration-time profiles are depicted in Figure 1.
No clinical signs of systemic drug intolerance or adverse reactions, such as gastrointestinal disturbances, were seen during the period of observation after the administration in any of the treated horses.

| D ISCUSS I ON
Extracts of devil's claw show analgetic and anti-inflammatory effects and improve significantly the symptomatology of chronicinflammatory diseases like e.g., arthrosis of the hips and knees in human (Chrubasik, Pollak, & Black, 2002;Mahomed & Ojewole, 2004;Wegener & Lüpke, 2003). In addition, an increasing amount of in vitro studies confirm the anti-inflammatory, anti-oxidative and analgetic properties of H. procumbens making it a promising medicinal plant (Mncwangi et al., 2012). In traditional medicine, H. procumbens preparations are mainly used against chronic inflammation of the joints (Schopohl et al., 2016). In Veterinary Medicine, they are used for the prevention of inflammatory symptoms in competition horses and in the treatment of animals with lameness (Colas, Garcia, & Popot, 2008).

The Food and Drug Administration of the United States of
America claimed that botanical drug products should be investigated with regard to the blood levels of known active constituents, representative markers and major chemical compounds.
Some pharmacokinetic data of devil's claw have been published in human (Loew et al., 2001) and analytical methods for analysis of harpagoside in equine urine and plasma have been established by Colas et al. (2006Colas et al. ( , 2008, but to the best of our knowledge this is the first study presenting substantive pharmacokinetic data of harpa-  Loew et al. (2001) and FB 8858 in our study differ, these extracts are identical coming both from Andernach with the same content of 25% harpagoside. Therefore, human and equine pharmacokinetic data of these extracts can be directly compared. Assuming a body weight of 70 kg for a man, in the study of Loew et al. (2001) a dose corresponding to 2.14 mg/kg BM harpagoside was administered. This led to a C max of 32.2 ng/ml at a t max of 1.3 hr. These values are very similar to our findings, although the dosage was more than twice as high. There was a proportional relationship between the intragastric administered dose and C max F I G U R E 1 Mean plasma concentration-time profile of harpagoside after single intragastric dosing of Harpagophytum procumbens extract FB 8858 corresponding to 5 mg/kg BM harpagoside (trial 1) and 10 mg/kg BM harpagoside (trial 2) to six horses [Colour figure can be viewed at wileyonlinelibrary.com] of harpagoside concentrations in postadministration equine plasma samples, which has also been reported for humans (Loew et al., 2001).
In contrast to humans, for whom in time course of plasma levels a second increase in harpagoside 8 hours after oral administration of the extract WS 1531 was found (Loew et al., 2001), potentially indicating the enterohepatic circulation of harpagoside, there was no second harpagoside peak in equine plasma in the present study.
Conjugates ( (Colas et al., 2006). In the present study, it was possible to determine the concentration of harpagoside in equine plasma up to 9 hr after single intragastric administration of a Harpagophytum extract.
The fact that harpagoside could be detected up to 24 hr in urine in the study of Colas et al. (2006) might be important for analysis of postcompetition samples for controlling drug exposure in the horse. However, controlling the drug effect, rather than the drug exposure, makes urine a less than ideal matrix (Toutain & Lassourd, 2002).
The present data showed low inter-individual variability in harpagoside plasma concentrations in the horse. In consideration that this is a characteristic for drugs which have limited biotransformation, harpagoside seems to be metabolized to a relatively low extent, minimizing the risk of metabolic drug-drug interactions. There are no reports on Harpagophytum regarding other drug-interactions in various species (Harman, 2002); moreover, no evidence of interactions has been reported in horses (Williams & Lamprecht, 2008).
The half-life of harpagoside 2.53 and 2.32 hr, respectively, in the present study, is also indicative that multiple doses of Harpagophytum extract are required throughout the day. This also brings up the importance of the galenic formulation. By the way, detection time is a variable influenced by many factors unrelated to the drug properties, such as dose, route of administration, pharmaceutical formulation or LOD (Toutain & Lassourd, 2002).
There are no published data about the distribution of harpagoside in human plasma to be compared. A low volume of distribution of a substance indicates that the drug does not have the attempt to go specifically to "deep" tissues in the peripheral compartments or is stored or pooled somewhere in the peripheral compartment, such as fat or is bound to specific biological material (Ritschel, Banarer, Diaz, Margary, & Otero, 1980). It is suggested that harpagoside could not easily be accumulated in equine plasma.
As arthrosis and related orthopedic disorders are chronic, com-  (Montavon, 1994). Even in the present study, no clinical signs of gastrointestinal adverse reactions could be seen in any of the treated horses. However, further investigations should be done to confirm long-term safety of Harpagophytum preparations in horses.

| CON CLUS ION
The main pharmacokinetic data of harpagoside in equine plasma are essential to understand the time course of this efficacious substance after administration of an extract of Harpagophytum procumbens (Devil's Claw) in horses. Devil's claw is a safe drug and well-tolerated on oral administration route. Treatment of horses with Harpagophytum extract did not cause any clinically detectable side effects such as gastrointestinal irritation in our study. The knowledge of basic pharmacokinetics of devil's claw in the horse, based on the results of this study, will help to link results from in vitro assays and clinical studies and optimize therapeutic efficacy.

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest.