Milk thistle derivatives are frequently recommended to treat humans and other animals with hepatic conditions. Most beneficial effects of milk thistle extracts are attributed to the active flavonolignan component, silibinin, which accounts for 50% to 70% of standardized silymarin milk thistle extracts.1 Multiple human clinical trials have been performed to assess the efficacy of silibinin treatment for various acute and chronic hepatic diseases.2 Human clinical trials support the use of silibinin as a treatment for viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease and cirrhosis, and following ingestion of hepatotoxins.3 Moreover, silibinin toxicosis and adverse events are reported to be uncommon in humans and other animals.3
Basic pharmacokinetics of silibinin administered to humans and other animals have been documented.4 Milk thistle extracts have low oral bioavailability across species and following absorption are primarily conjugated via glucuronidation and excreted into bile and urine with minimal phase I metabolism.5 Oral bioavailability of silibinin is known in humans,6 dogs,7 cats,8 and rats.9 However, despite its clinical use, there is a general lack of information about silibinin toxicosis and pharmacokinetics in horses. Thus it follows that if silibinin is to be prescribed to treat equine liver disease, the basic pharmacokinetics should be evaluated first. To address this deficiency, the purpose of the study reported here was to determine the oral bioavailability and single and multidose pharmacokinetics of silibinin in healthy horses. A secondary objective was to determine whether increasing doses of silibinin consumed by healthy horses resulted in adverse events, as assessed by clinical and clinical laboratory measurements. It was hypothesized that oral silibinin administration would not result in alterations in clinical examination variables and plasma biochemical profiles in healthy horses, regardless of dose administered.
Materials and Methods
Experiments were performed in 2 phases during which pharmacokinetics of single-dose and multidose treatments of silibinin were studied. All procedures involving the use of animals in research were approved by the Colorado State University Institutional Animal Care and Use Committee as described in protocols #08-144A-01 and #09-011A-01. Horses owned by the Colorado State University Veterinary Teaching Hospital and acclimatized to their housing were used with permission, and environmental conditions were not changed. Horses were group housed in a paddock without access to grass. Horses received water ad libitum and timothy grass hay once daily, providing for consumption of approximately 12 kg of hay/horse/d. Horses were screened prior to inclusion in the study for evidence of gastrointestinal tract or liver dysfunction by physical examination and serum biochemical analyses and were selected only if they readily consumed the carrier diet. Body weight was measured with a commercial scale. Signalment was recorded.
Phase I drug administration—Nine healthy horses with no abnormal results of physical examination were studied in phase I. All horses were geldings with a mean ± SD age of 15 ± 6 years (median, 15 years [range, 4 to 29 years]). Breed of horses included Quarter Horse (n = 7), Arabian (1), and Andalusian (1). Mean ± SD weight was 589 ± 48 kg (median, 602 kg [range, 500 to 640 kg]).
In phase I, the 9 horses were administered silibinin once, via 3 routes (PO, via nasogastric tube, and IV, with a 30-day washout period between routes) preceding single-dose pharmacokinetic analyses. Silibinin (in a 32.7% silibinin phospholipida formulation) was administered via the 2 enteral routes, orally in feed and via nasogastric tube, at a dose of 6.5 mg of silibinin/kg of body weight.
For IV administration, a solution of silibininb was prepared with 99% dimethyl sulfoxide as a carrier, and the drug concentration of the resulting solution (166 mg of silibinin/mL) was confirmed via liquid chromatography–mass spectrometry. The silibinin solution was filtered with a 0.22-μm filterc and administered IV by direct injection at a dose of 6.5 mg of silibinin/kg. Effects of the dimethyl sulfoxide carrier were not specifically evaluated. Blood samples collected in plain evacuated tubes were obtained 0.5, 1, 2, 4, and 8 hours following enteral administration and 0.083, 0.25, 0.5, 2, 4, and 8 hours following IV administration. The blood samples were allowed to clot for 20 minutes in a 37°C water bath and were then centrifuged for 5 minutes at 4,234 × g. The separated serum was collected and stored at −80°C until analysis.
Phase II drug administration—Five healthy horses with no abnormal results of physical examination were studied in phase II. All horses were geldings with a mean ± SD age of 13 ± 5 years (median, 14 years [range, 5 to 17 years]). Breeds included Quarter Horse (n = 3), Arabian (1), and Andalusian (1). Mean ± SD weight was 582 ± 65 kg (median, 615 kg [range, 472 to 625 kg]). The 5 horses studied in phase II were also studied in phase I, with a 6-month interval between phases. In phase II, the 5 horses received identical nonrandomized treatments, resulting in consumption of increasing orally administered doses of silibinin phospholipid twice daily during 4 nonconsecutive weeks. Horses consumed a particular dose twice daily each day for 7 days during 4 administration periods, with progressively higher doses of silibinin phospholipid administered during each period, and with each period separated by a washout period (minimum of 2 weeks). During week 1, twice daily, horses consumed a diet without silibinin phospholipid that consisted of a combination of 400 g of pelleted feed,d 50 g of wheat bran, and 150 mL of water per meal. During week 2, horses were fed the diet plus the same silibinin phospholipid formulation as in phase I, such that they were administered 6.5 mg of silibinin/kg of body weight. During week 3, horses were fed the diet plus silibinin phospholipid formulation such that they received 13 mg of silibinin/kg of body weight. During week 4, horses were fed the diet plus silibinin phospholipid formulation such that they received 26 mg of silibinin/kg of body weight. Blood sampling for single-dose pharmacokinetics was performed on day 1 of weeks 3 and 4. Blood samples were taken 0.5, 1, 2, 4, and 8 hours following the first dose administered and prepared for analysis as described for phase I of the study. Multiple-dose silibinin trough concentrations were evaluated on day 7 immediately prior to consumption of the final meal of each the 4 study weeks. Silibinin concentration was measured in samples taken 1 hour after final meal consumption on day 7 and compared with concentrations in samples taken 1 hour after final meal consumption on day 1 by use of a paired t test, to assess drug accumulation secondary to repetitive administration.
Drug analysis—Silibinin concentration was measured in serum samples by use of liquid chromatography coupled to tandem mass spectrometry with a published method6 modified and validated for silibinin analysis in horse serum. Standard dilutions of silibininb (0, 2.5, 5, 10, 50, 100, 250, 500, and 1,000 ng/mL) were prepared in acetonitrile and control serum collected from healthy horses that were not exposed to silibinin or milk thistle derivatives. Inclusion of quality control samples of silibininb (5 ng/mL, 50 ng/mL, and 250 ng/mL) interspersed among unknown samples was used to assess batch assay performance; control samples comprised at least 10% of all samples analyzed. The criterion for batch acceptance was that in each batch, > 75% of the quality control samples had ≥ 85% accuracy. Quality control samples were prepared in the same manner as the standard silibinin dilution curve samples. Each standard and sample contained 200 ng of naringenine as the internal standard. Naringenin stock solution was prepared in methanol at 10 μg/mL. Serum samples (500 μL) were extracted with 2.5 mL of acidified ethyl acetate (0.1% formic acid) with vortexing for 10 minutes. Organic and aqueous layers were separated by centrifugation for 10 minutes at 3,000 × g, and the organic layer was removed, evaporated, and reconstituted in 250 μL of beginning mobile phase. Samples were centrifuged for 10 minutes at 10,600 × g, and 200 μL of supernatant was transferred to high-performance liquid chromatography vials for liquid chromatography–mass spectrometry analysis.
Negative-ion electrospray ionization mass spectra were obtained with a triple quadrupole mass spectrometer with a turbo ion spray sourcef interfaced with a high-performance liquid chromatography system.g The column used was a YMC ODS-AQ type (5 μm, 12 nm, 50 × 2 mm) with a carbon 18 pre-column.h The liquid chromatography elution gradient began with 20% acetonitrile-80% ammonium acetate plus 0.1% acetic acid and progressed to 90% acetonitrile-10% ammonium acetate plus 0.1% acetic acid over 3.5 minutes with a flow rate of 500 μL/min, sample injection volume of 10 μL, and a total run time of 5.5 minutes. The mass spectrometer settings were turbo ion spray temperature, 500°C; spray needle, −4,500 V; declustering potential, −55 V; entrance potential, −6 V; collision energy, −37; collision gas, N2, low; curtain gas, 10; ion source gas 1, 60; ion source gas 2, 80; collision exit potential, 0; and needle position, 6. Samples were quantified by the internal standard reference method in the MRM mode by monitoring the transition m/z 481 to m/z 125 for silibinin and m/z 271 to m/z 119 for naringenin (internal standard). Lower limit of quantitation of the assay was 2.5 ng/mL
Quantitation of silibinin was performed on the basis of standard curves derived from prepared matrix by use of the ratio of silibinin peak area to the naringenin peak area. Assay performance was linear to 1,000 ng/mL. Further dilution (1:10 or 1:100) was required to evaluate unknown samples obtained within 30 minutes of IV sampling. Batch analysis results were verified by inclusion of quality control samples in prepared matrix. Accuracy of batches analyzed was 94 ± 7.3%, 91.9 ± 4.9%, and 94.2 ± 5.2%.
Safety—Evaluation of safety was performed concurrently with the phase II pharmacokinetic study. As described, 5 horses consumed increasing orally administered doses of silibinin phospholipid during 4 nonconsecutive weeks, resulting in consumption of silibinin concentrations of 0 mg/kg, 6.5 mg/kg, 13 mg/kg, and 26 mg/kg twice daily. Blood samples were obtained from horses on day 1, prior to administration of unsupplemented diet or diet supplemented with silibinin phospholipid and on day 7, 1 hour following the final meal of the weekly study diet. Blood was collected directly into evacuated tubes containing lithium heparin anticoagulant. Plasma biochemical analysis that included hepatic enzyme activity evaluation was performed by the Colorado State University Veterinary Diagnostic Laboratory. The analysis included all relevant core biochemical tests recommended for nonclinical toxicity and safety studies, including those specifically recommended to evaluate hepatocellular and hepatobiliary health.10 Reference intervals of the Colorado State University Veterinary Diagnostic Laboratory were used to interpret clinical laboratory results. Horses were monitored daily for abnormal clinical signs by a board-certified veterinarian and weekly for plasma biochemical abnormalities. Dose-limiting toxicosis was defined as an increase in either GGT or sorbitol dehydrogenase activity > 3 times the upper reference limit, with a concurrent increase in plasma total bilirubin concentration greater than the upper reference limit, or any health abnormality evident by means of clinical examination by authors (who were not unaware of group assignments). Interpretation of clinical laboratory results was consistent with current (human) National Cancer Institute Common Terminology Criteria for Adverse Events guidelines11 (corresponding to grade 2 hepatic enzyme activity increase) and FDA recommendations.12 Specific criteria for clinical adverse events included gastrointestinal tract signs (eg, diarrhea, colic, or anorexia), neurologic signs (eg, ataxia, seizures, abnormal mentation), respiratory tract signs (eg, stridor or tachypnea), and vascular signs (eg, venous thrombosis, hemorrhage, or petechiation). In phase II, the dose of silibinin was increased only if no signs of toxicosis were observed at the previous dose. As a precondition, if signs of toxicosis occurred, individual horses were to be withdrawn from the study.
Statistical analysis—Pharmacokinetics were determined by use of noncompartmental analysis and expressed as mean ± SD values. Peak serum concentration was reported in nanograms per milliliter. Pharmacokinetics for oral, nasogastric, and IV administration were calculated with commercial software.i Bioavailability (%) was determined by dividing the enteral AUC0–∞ by the IV AUC0–∞ and multiplying by 100%. Relative bioavailability (%) between differing routes of enteral administration was calculated by dividing the oral AUC0–∞ by the nasogastric AUC0–∞ and multiplying by 100%. Repeated-measures ANOVA, with Bonferroni multiple comparisons for post hoc pairwise comparisons, was used to analyze for differences in peak serum concentrations, peak serum concentration divided by dose, and AUC divided by dose, with values of P < 0.05 considered significant.
Results
Phase I—Total dose of silibinin phospholipid administered by nasogastric tube and oral feeding was a mean ± SD of 11.8 ± 0.9 g (median, 12.0 g [range, 10.0 to 12.8 g]). Horses were administered IV a mean ± SD volume of silibinin solution of 23.1 ± 1.9 mL (median, 23.6 mL [range, 19.6 to 25.1 mL]). Pharmacokinetics for different routes of administration of identical doses of silibinin evaluated in phase I were determined (Table 1). Maximum serum concentration and AUC were greater with the nasogastric route of administration, compared with the PO route (Figure 1). Bioavailability for the orally administered silibinin was 0.6% and for nasogastrically administered silibinin was 2.9%. Relative bioavailability of orally administered silibinin, compared with nasogastric administration, was 21%.
Mean ± SD serum concentrations (log scale) of silibinin versus time in 9 healthy horses administered a single dose of silibinin (6.5 mg/kg, IV) or silibinin phospholipid (6.5 mg/kg, PO [in feed] or via nasogastric tube). Serum concentrations 8 hours after PO administration in feed were less than the lower limit of quantitation of the assay.
Citation: American Journal of Veterinary Research 74, 10; 10.2460/ajvr.74.10.1327
Pharmacokinetics (mean ± SD) of a single dose (6.5 mg/kg) of silibinin administered to 9 horses PO in feed, via nasogastric tube, or IV.
| Route of administration of silibinin | |||
|---|---|---|---|
| Parameter | P0 in feed | Nasogastric tube | IV |
| T1/2 elimination (h) | 1.1 ± 0.5 | 2.9 ± 1.2 | 1.69 ± 0.21 |
| Cmax (ng/mL) | 44.8 ± 52.8 | 165.6 ± 119.9 | NA |
| Tmax (h) | 0.5 | 0.5 | NA |
| AUC0–∞ (h•ng/mL) | 45.3 ± 38.6 | 214.7 ± 154.1 | 7,462 ± 1,266 |
| Vdss (mL/kg) | NA | NA | 657 ± 145 |
| Vdarea (mL/kg) | NA | NA | 2,243 ± 313 |
| Cl (mL/h/kg) | NA | NA | 900 ± 201 |
| F (%) | 0.6 | 2.9 | NA |
Cl = Clearance. F = Bioavailability. NA = Not applicable. Tmax = Time of maximum serum concentration. T1/2 = Half life. Vdarea = Volume of distribution area. Vdss = Volume of distribution at steady state.
Phase II—Mean ± SD total dose of silibinin phospholipid administered in feed in week 2 was 11.6 ± 1.3 g (median, 12.3 g [range, 9.4 to 12.5 g]), in week 3 was 23.3 ± 2.6 g (median, 24.6 g [range, 18.9 to 25.0 g]), and in week 4 was 46.5 ± 5.2 g (median, 49.2 g [range, 37.8 to 50.0 g]). On day 7 of week 4 (highest-dose week), the meal was incompletely consumed by 1 horse and consumed slowly by 1 horse.
Pharmacokinetics for the 3 doses of silibinin phospholipid given with feed were determined (Table 2). A more than proportionate increase in maximum serum concentration as well as AUC was detected at the highest silibinin phospholipid dose administered (Figure 2), and this was supported by significant differences between maximum serum concentration divided by dose (P = 0.020) and AUC divided by dose (P < 0.001) indicating nonlinearity.
Mean ± SD serum concentrations (log scale) of silibinin versus time in 5 healthy horses. Each horse was administered a single dose of silibinin phospholipid at each concentration (separated by a washout period) mixed with feed. For the 6.5 mg/kg dose, serum concentrations measured 8 hours after administration were less than the lower limit of quantitation of the assay.
Citation: American Journal of Veterinary Research 74, 10; 10.2460/ajvr.74.10.1327
Pharmacokinetics (mean ± SD) of a single dose (6.5 mg/kg, 13 mg/kg, or 26 mg/kg, each separated by a washout period) of silibinin administered in feed to 5 healthy horses.
| Dose of silibinin | |||
|---|---|---|---|
| Parameter | 6.5 mg/kg | 13 mg/kg | 26 mg/kg |
| T1/2 elimination (h) | 1.1 ± 0.5a | 2.1 ± 0.4a | 2.8 ± 0.7b |
| Cmax (ng/mL) | 44.8 ± 52.8 | 81.7 ± 51.6 | 407 ± 247 |
| Tmax (h) | 0.5 | 0.5 | 0.5 |
| AUC0–∞ (h•ng/mL) | 45.3 ± 38.6 | 113 ± 60.1 | 839 ± 216 |
| MRT (h) | 1.49 ± 0.77 | 2.53 ± 0.55 | 4.19 ± 1.86 |
| Cmax/dose (kg/L) | 0.001 ± 0.008a | 0.006 ± 0.004a | 0.016 ± 0.010b |
| AUC0–∞/dose (h•kg−3mL) | 6.97 ± 5.94a | 8.69 ± 4.62a | 32.27 ± 8.30b |
MRT = Mean residence time.
Within a row, values with different superscript letters are significantly (P < 0.05) different.
See Table 1 for remainder of key
On day 7 of each week of the trial, trough concentrations of silibinin were less than the lower limit of quantitation of the assay. Concentration of silibinin measured 1 hour after oral administration did not differ significantly between day 1 and day 7 for horses that received 6.5 mg/kg (P = 0.356), 13 mg/kg (P = 0.414), or 26 mg/kg (P = 0.053). If the horses that either incompletely consumed the dose (1 horse) or consumed the dose slowly (1 horse) were censored, the 1-hour serum silibinin concentrations did not differ significantly (P = 0.134) between day 1 and day 7.
No dose-limiting toxicosis was observed in any group. Plasma GGT activity was a mean ± SD of 20 ± 17 U/L (median, 14 U/L [range, 9 to 50 U/L]) in horses prior to week 1 and a mean of 20 ± 14 U/L (median, 17 U/L [range, 9 to 43 U/L]) at the end of week 1; 20 ± 9 U/L (median, 18 U/L [range, 11 to 35 U/L]) prior to week 2 and a mean of 16 ± 12 U/L (median, 17 U/L [range, 0 to 33 U/L]) at the end of week 2; 22 ± 7 U/L (median, 25 U/L [range, 12 to 29 U/L]) prior to week 3 and a mean of 23 ± 7 U/L (median, 26 U/L [range, 14 to 30 U/L]) at the end of week 3; and 22 ± 9 U/L (median, 20 U/L [range, 11 to 31 U/L]) prior to week 4 and a mean of 23 ± 8 U/L (median, 21 U/L [range, 14 to 33 U/L]) at the end of week 4. Plasma GGT activity increased to values greater than the upper limit of the reference range (20 U/L) in 3 of 5 horses after 7 days of twice daily treatment with silibinin at 13 mg/kg or 26 mg/kg. Maximal increase was observed at the 13 mg/kg dose, at which the median concentration was 1.4 times the upper reference limit. In horses consuming 6.5 mg/kg, median plasma bicarbonate concentration was 1.02 times the upper reference limit. Median values of other clinical laboratory variables were within the laboratory reference ranges in all 3 groups. Therefore, all clinical laboratory measurements were within reference ranges or met the current definition of a grade 1 adverse event in humans.11
Discussion
To the authors’ knowledge, this is the first reported study of the bioavailability and pharmacokinetic disposition of milk thistle extracts in horses. Bioavailability of orally administered silibinin phospholipid was < 1%. Silibinin did not accumulate when given twice daily for 7 days. An unexpected finding was the observation of nonlinear pharmacokinetics of silibinin in horses, because doses as high as approximately 80 mg of silibinin/kg evaluated in humans with normal hepatic function have linear pharmacokinetics.6 This apparent anomaly in the pharmacokinetics of silibinin phospholipids could have ramifications in the study of its effects when used to treat clinical hepatic disease. Silibinin administration to horses did not result in alterations in health, which was consistent with reports in other species. Despite minor alterations in clinical laboratory measurements, no dose-limiting toxicosis was observed.
Oral silibinin bioavailability was tested by administering silibinin complexed with phospholipids both by nasogastric tube administration and by administration in typical feedstuffs. As reported in the literature, when silibinin alone is administered, absorption from the gastrointestinal tract is so low that concentrations are undetectable in serum.13 Therefore, most investigators, as in the present study, use commercially available silibinin complexed with phosphatidylcholine, which forms a phytosome and enhances bioavailability. The present study confirmed that the bioavailability of the silibinin compound was low in horses, as it is in other species. For comparison, oral bioavailability of silibinin is estimated to be 0.73% in rats9 and oral bioavailability of silibinin phospholipid is estimated to be 6% to 7% in cats.8 In the present study, silibinin phospholipid was readily consumed by the horses at low doses when mixed with feed. Even though the data indicated that relative bioavailability was improved with nasogastric administration, compared with feeding the compound, this method of administration is impractical for horse owners, especially for long-term administration regimens. Although Cmax and AUC are greater when feed is supplemented with 80 mg of silibinin phospholipid/kg, feed refusal may be a problem when feeding higher doses for an extended period.
The nonlinear pharmacokinetics of silibinin observed in this study may have been associated with saturation of conjugation pathways, as seen at higher doses in humans with hepatitis.14 Silibinin is primarily conjugated by glucuronidation and undergoes minimal phase I metabolism.1 High plasma concentrations could result if high dosages overwhelm hepatic metabolic capacity. Plasma concentrations of drugs that have nonlinear pharmacokinetics are inherently unpredictable and, although silibinin has a wide margin of safety, toxicosis could result.1 Therapeutic drug monitoring could be useful to avoid this problem if appropriate laboratory resources are available.14,15
Because of hepatobiliary excretion, maximum silibinin concentration in bile is greater than twice that in plasma after IV administration in rats9 and approximately 100 times that in serum after oral administration in humans.16 High biliary concentrations and hepatobiliary recycling of silibinin and its related compounds may be responsible for the hepatoprotective effects seen following ingestion, despite poor bioavailability. In addition, the pharmacokinetics of silibinin are altered in hepatic disease.17 For example, human patients with hepatic cirrhosis have nearly 5 times the serum concentrations of silymarin flavolignans (active milk thistle derivatives) following ingestion, compared with healthy volunteers.17 This suggests that further study is needed to determine whether silibinin phospholipid pharmacokinetics are altered in horses with hepatic disease.15
In the present study, the increase in serum GGT activity was unexpected. Median GGT activity was highest in horses that consumed the intermediate dose (13 mg/kg) of silibinin, suggesting that the magnitude of increase was not strictly dose related. γ-Glutamyl transferase is associated with microsomal membranes in biliary epithelium, and production and release of GGT is increased by cholestasis.18 The magnitude of GGT activity increase was considered small and clinically unimportant. Other liver-related values were not abnormal, and no horses had evidence of dose-limiting toxicosis. Increases in plasma GGT activity have not been reported in other species administered silibinin, but further evaluation may be warranted in horses.
Results suggested that silibinin was safe and nontoxic in the horses evaluated. Silibinin had low bioavailability and did not accumulate when administered twice daily for 7 days. The finding of nonlinear pharmacokinetics was unexpected and may have been caused by saturation of conjugation pathways, but further study is necessary to confirm the mechanism. Pharmacokinetic and safety evaluation in horses with liver disease is necessary to confirm the most appropriate dose and administration interval.
ABBREVIATIONS
| AUC | Area under the serum concentration-time curve |
| AUC0–∞ | Area under the plasma concentration-time curve from time 0 to infinity |
| Cmax | Peak serum concentration |
| GGT | γ-Glutamyl transferase |
Siliphos, Indena Pharmaceuticals Inc, Milan, Italy.
SO417, Sigma-Aldrich, St Louis, Mo.
Sterivex GP 0.22-μm filter unit, Millipore Corp, Billerica, Mass.
Equine Senior Horse Feed, Purina Mills, St Louis, Mo.
N5893, Sigma-Aldrich, St Louis, Mo.
Applied Biosystems 3200 Q Trap, Applied Biosystems, Foster City, Calif.
Shimadzu LC 20A, Columbia, Md.
Phenomenex, Torrance, Calif.
WinNonlin, version 4.1, Pharsight Corp, St Louis, Mo.
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