Abstract
Objective
To determine the pharmacokinetics of acetaminophen (N-acetyl-para-aminophenol [APAP]) and its metabolites after oral administration of a single dose of APAP, with or without silymarin or N-acetylcysteine (NAC), to orange-winged Amazon parrots (Amazona amazonica).
Methods
Eight parrots received, in 3 separate studies, 1 of the following oral treatments: (1) APAP (100 mg/kg) with silymarin (50 mg/kg, twice, q 12 h); (2) APAP (100 mg/kg) with NAC (400 mg/kg); or (3) APAP (100 mg/kg) alone. For each study, blood samples were collected over 24 hours after drug administration to evaluate plasma concentrations of APAP, APAP-glucuronide, and APAP-sulfate. Pharmacokinetic parameters were calculated. Plasma biochemistry panels were performed before and after each study. In a fourth study, a single oral dose of APAP (100 mg/kg) was administered to 8 additional parrots for adverse effects evaluation alone.
Results
Pharmacokinetic parameters for APAP, APAP-glucuronide, and APAP-sulfate were established. The APAP maximum plasma concentration, time of maximal plasma concentration, and half-life across studies ranged from 2,016.9 to 2,917.2 ng/mL, 1.13 to 2.1 hours, and 1.3 to 1.45 hours, respectively. Acetaminophen had marked metabolism to APAP-glucuronide and negligible to APAP-sulfate. Concurrent administration of APAP with silymarin resulted in a mild but significant elevation in glutamate dehydrogenase.
Conclusions
Acetaminophen plasma concentrations were lower than in other avian species despite a relatively high dose. Acetaminophen has fast absorption, short half-life, and marked glucuronidation. Single oral dose administration of APAP, alone or with NAC, appears safe based on plasma biochemistries. Multidose and pharmacodynamic studies are needed.
Clinical Relevance
This is the first pharmacokinetic study of APAP in psittacines, which has the potential to be an effective and safe component of multimodal analgesia in these species.
Acetaminophen (N-acetyl-para-aminophenol or paracetamol [APAP]) is an antipyretic and analgesic drug commonly used in human and veterinary medicine.1,2 Acetaminophen is used in humans to manage acute and chronic pain and, due to its limited adverse effects at appropriate dosing, can be administered to patients where nonsteroidal anti-inflammatory medications are contraindicated.3 In rat and human pain models, concurrent administration with other analgesics, such as tramadol, can lead to additive or superadditive analgesic and antihyperalgesic effects.4,5 As a result, APAP has the potential to be an effective component of multimodal pain management in psittacines.
Acetaminophen undergoes complex metabolism that depends on species, age, and the dose administered.2,6,7 In humans, APAP is mostly converted to pharmacologically inactive metabolites through conjugation with glucuronic acid (APAP-glucuronide) and sulfate (APAP-sulfate). It is also metabolized to p-aminophenol, which is then metabolized to N-acylphenolamine (AM404) after crossing the blood-brain barrier. A smaller fraction of APAP undergoes N-hydroxylation in the liver to produce the toxic reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). N-acetyl-p-benzoquinone imine is detoxified through a glutathione-mediated pathway and eliminated in the urine.2,3 When glutathione is depleted, such as with an overdose of APAP or in patients with compromised hepatic function, NAPQI can accumulate leading to hepatotoxicity.
Despite its wide and longstanding use, the precise mechanism of action of APAP is still being elucidated. It was previously thought to provide analgesia through the inhibition of cyclooxygenase enzymes.1–3 However, more recent studies3 have shown APAP to have weak cyclooxygenase inhibition that may have little clinical effect. The main analgesic mechanism of APAP is now thought to be through its metabolization to AM404, which then acts on the transient receptor potential vanilloid 1 and cannabinoid 1 receptors in the brain. It was also recently revealed that the APAP metabolite AM404 directly induces analgesia via transient receptor potential vanilloid 1 receptors on terminals of C-fibers in the spinal dorsal horn. It is known that, similar to the brain, the spinal dorsal horn is critical to pain pathways and modulates nociceptive transmission. Therefore, APAP induces analgesia by acting not only on the brain but also on the spinal cord.3
Pharmacokinetic studies have been performed in several species, including dogs,7,8 rats,7,9 horses,7,10 pigs,7 chickens (Gallus gallus domesticus),7 turkeys (Meleagris gallopavo domesticus),7 and domestic geese (Anser anser domesticus).11 Studies7,11 in avian species have shown lower oral bioavailability of APAP compared to humans. No clinical adverse effects were noted with administration of a single dose of 10 mg/kg APAP orally to domestic geese; however, minor changes to the liver consistent with fatty degeneration of hepatocytes were noted on histopathology 24 hours after drug administration.11 A dosage of 10 mg/kg has been administered once daily for 7 days to 5-week-old chickens without reported adverse effects.12 To the investigators’ knowledge, there are no reports on the pharmacokinetics, pharmacodynamics, or safety of APAP administration in any psittacine species.
Silymarin is an antioxidant complex of flavonolignans and flavonoids that is extracted from the seeds of the milk thistle plant Silybum marianum.13 Administration of silymarin is thought to decrease glutathione depletion in the liver both through direct oxidative radical scavenging and by increasing glutathione synthesis.14 Silymarin has been shown to have hepatoprotective effects in mice,14–16 rats,17 cats,18 and pigeons19 in instances of experimentally induced liver injury due to APAP overdose. In pigeons, administration of 35 mg/kg silymarin orally every 12 hours for 3 days starting 12 hours after a single oral dose of 3,000 mg/kg APAP prevented mortality and significantly remediated bloodwork changes associated with APAP toxicity.19 While silymarin has anecdotally been used in psittacines to manage hepatopathies, pharmacokinetic and pharmacodynamic studies in these species are lacking.
N-acetylcysteine (NAC) is an acetylated precursor of the amino acid l-cysteine. N-acetylcysteine is the primary treatment for APAP toxicity/overdose in humans20 and has been shown to reduce oxidative damage from toxic compounds in cats18 and rats.21,22 N-acetylcysteine increases hepatic levels of glutathione, resulting in increased scavenging of toxic APAP metabolites and reactive oxygen species, thereby ameliorating the hepatotoxic effects. N-acetylcysteine also has potential analgesic properties, which have been demonstrated in a rat hot plate model23 and radiant heat models in mice and humans.24 Its analgesic effects are thought to be mediated through its antioxidant effects and reduction of nitrous oxide metabolites in the spinal cord.25
N-acetylcysteine has been administered to poultry at varying doses to counteract the negative effects of toxic compounds and oxidative stress.26–30 Administration of 800 mg/kg/day NAC orally to broiler chickens significantly reduced the nephrotoxic and hepatotoxic effects of aflatoxin B1 administration, with no adverse effects reported at dosages of 400 to 800 mg/kg/day.26 N-acetylcysteine administered as a feed additive at 1 g/kg feed to broiler chickens27,29 and 2.5 to 5 g/kg of feed to Japanese quail (Coturnix japonica)28 was protective against the effects of temperature stress in those species. The pharmacokinetics and pharmacodynamics of NAC in psittacines are unknown.
The objectives of this study were to (1) determine the single-dose pharmacokinetics of APAP and its metabolites APAP-glucuronide and APAP-sulfate after oral administration of APAP, with and without concurrent administration of silymarin or NAC; and (2) to evaluate possible adverse effects of APAP administration in orange-winged Amazon parrots (Amazona amazonica). It was hypothesized that APAP administered orally at a single dose would result in plasma concentrations considered to be therapeutic in mammalian species. We also hypothesized that potential adverse effects of APAP administration would be ameliorated by concurrent administration of silymarin or NAC without significant effects on pharmacokinetic parameters.
Methods
Animals
Sixteen (8 male and 8 female) adult orange-winged Amazon parrots (age, 8 to 23 years) maintained in a research flock at the University of California-Davis were used. Body weights ranged from 319 to 453 g (mean ± SD of 398 ± 40 g). For the studies, birds were individually housed in a single room in stainless steel cages (dimensions of 91 X 71 X 165 cm) that contained at least 2 perches and hanging toys and allowed visual and auditory socialization with each other. The birds were provided with a 12-hour day-night cycle, a commercial pelleted diet (Maintenance diet; Roudybush Inc), and water ad libitum. They were considered healthy based on physical examination, CBC count, and plasma biochemical analysis performed before the start of the study. The parrots did not receive any other medications or undergo any anesthetic procedures for at least 1 month before the start of the study. The study protocol was approved by the IACUC at the University of California-Davis (protocol No. 23478).
Drug formulations
A commercially available oral formulation of APAP (32 mg/mL; Infants’ Tylenol Dye-Free; Johnson & Johnson) was used in this study. A compounded oral formulation of silymarin (80 mg/mL; Wedgewood Pharmacy; Unispend Anhydrous sweetened oral suspension vehicle, sorbitan monooleate, and almond oil in proprietary ratios) and a commercially available solution of NAC (10% solution; Fresenius Kabi) were also used.
Pilot study
A pilot study was first performed to investigate the appropriate and safe dose of APAP to be administered in the single-dose pharmacokinetic studies. Two birds (1 male and 1 female) were allocated to receive doses of oral APAP at 5, 10, and 20 mg/kg. In addition, 2 birds (1 male and 1 female) received 10 mg/kg APAP orally with concurrent oral silymarin 35 mg/kg every 12 hours for 2 doses. The drugs were administrated directly into the crop via a metal gavage tube. The parrots were not fasted before drug administration. Blood samples were collected under manual restraint from each of the 8 birds at a total of 8 time points over 24 hours (0.5, 1, 2, 3, 4, 8, 12, and 24 hours). For blood collection, a volume of no more than 1% of a bird’s body weight was collected during each study portion. For each time point, 0.25 to 0.3 mL of whole blood was collected using a 0.3-mL tuberculin syringe and 29-gauge needle and sampled from the jugular, brachial, or metatarsal veins. The collected blood samples were placed in lithium-heparin pediatric collection tubes, kept in refrigeration at 4 °C, and centrifuged for 6 minutes at 3,500 X g within 4 hours of collection. Plasma was then collected into labeled 0.5-mL cryovials and stored at −80 °C until analysis.
Single-dose pharmacokinetic and safety studies
Following a 1-month washout period, the same 8 orange-winged Amazon parrots used previously in the pilot study were used in a nonrandomized cross-over single-dose study. The birds were each manually restrained to allow for direct drug administration into the crop via a metal gavage tube. The parrots were not fasted before drug administration. Each parrot received a single oral dose of 100 mg/kg APAP based on individual body weight. During the first single-dose study, silymarin was also administered concurrently at 50 mg/kg orally every 12 hours for 2 doses (APAP-silymarin group). During the second single-dose study, NAC was also administered concurrently at 400 mg/kg orally once (APAP-NAC group). During the third single-dose study, APAP was administered alone without other concurrent drug administration (APAP-alone group). There was a 1- to 3-month washout period between studies.
Blood samples were collected under manual restraint from each of the 8 birds at a total of 8 time points over 24 hours (0.5, 1, 2, 3, 4, 8, 12, and 24 hours). Blood collection and processing were performed as described for the pilot study.
Before the start of each study and 24 hours after APAP administration, 0.4 to 0.5 mL blood was collected using a 1-mL syringe and 25-gauge needle from the jugular, brachial, or metatarsal veins to be used for serial plasma biochemical analysis. The collected blood samples were placed in lithium-heparin pediatric collection tubes and centrifuged for 6 minutes at 3,500 X g within 1 hour of collection, and plasma samples were kept in refrigeration at 4 °C until plasma biochemistry analysis. The total volume of blood collected for each study phase was no more than 1% of a bird’s body weight. The birds were monitored throughout the studies for evidence of adverse effects including changes in mentation, weight, appetite, eliminations, mucous membrane color, and evidence of vomiting, regurgitation, or peripheral edema.
Single-dose safety study
Eight parrots (4 male and 4 female) that had not been used in the pilot or single-dose studies were used in a subsequent safety study to further evaluate for potential clinical adverse effects of APAP administration alone. A separate group of birds was used to avoid possible carryover effects from prior drug administration. A single oral dose of 100 mg/kg APAP was administered as previously described. Before and 24 hours after APAP administration, 0.5 mL whole blood was collected using a 1-mL syringe and 25-gauge needle from the jugular vein. The collected blood samples were placed in lithium-heparin pediatric collection tubes and centrifuged for 6 minutes at 3,500 X g within 1 hour of collection, and plasma samples were kept in refrigeration at 4 °C until plasma biochemistry analysis. The birds were also monitored for evidence of adverse effects as previously described.
Plasma concentration determinations of APAP and its metabolites APAP-glucuronide and APAP-sulfate
Plasma calibrators were prepared by dilution of the APAP (Cerilliant), APAP-sulfate, and APAP-glucuronide (Toronto Research Chemicals) working standard solutions with drug-free parrot plasma to concentrations ranging from 0.001 to 80 mg/mL and up to 250 mg/mL for APAP-glucuronide. Calibration curves and negative control samples were prepared fresh for each quantitative assay. As a check of accuracy, quality control samples (drug-free parrot plasma fortified with analyte at 3 concentrations within the standard curve) were included.
Before analysis, 50 µL of plasma was diluted with 250 µL of acetonitrile (ACN):1 M acetic acid (9:1 [vol:vol]) containing 50 ng/mL of the internal standards D4APAP, D3APAP-sulfate, and D3APAP-glucuronide to precipitate proteins. The samples were vortexed for 2 minutes to mix, refrigerated for 20 minutes, vortexed for an additional 1 minute, and centrifuged in a Sorvall ST 40R centrifuge (Thermo Scientific) at 4,300 rpm/3,830 X g for 10 minutes at 4 °C. The supernatant (200 µL) was dried under nitrogen at 55 °C and redissolved in 120 µL water, and 20 µL was injected into the liquid chromatography–tandem mass spectrometry (LC-MS/MS) system.
The concentrations were measured in plasma by LC-MS/MS using positive and negative heated electrospray ionization. Quantitative analysis was performed on a TSQ Altis triple quadrupole mass spectrometer coupled with a Vanquish liquid chromatography system (Thermo Scientific). Chromatography employed an ACE 3 C18 10-cm X 2.1-mm 3-μm column (Mac-Mod Analytical) and a linear gradient of ACN in water with a constant 0.2% formic acid at a flow rate of 0.35 mL/min. The initial ACN concentration was held at 1% for 0.5 minutes, ramped to 30% over 4 minutes, and flushed at 90% ACN for 0.2 minutes before reequilibration for 2.3 minutes at initial conditions.
Detection and quantification were conducted using selective reaction monitoring of initial precursor ion for APAP (m/z, 152.1), APAP-sulfate (m/z, 229.9), APAP-glucuronide (m/z, 328.2), and the internal standards D4APAP (m/z, 156.1), D3APAP-sulfate (m/z, 232.9), and D3APAP-glucuronide (m/z, 331.2). The responses for the product ions for APAP (m/z, 65.1, 93.1, and 110.1), APAP-sulfate (m/z, 107.1 and 150.1), APAP-glucuronide (m/z 110.0 and 152.0), D4APAP (m/z, 114.1), D3APAP-sulfate (m/z, 107.1 and 153.1), and D3APAP-glucuronide (m/z, 155.1) were plotted, and peaks at the proper retention time were integrated, using Quanbrowser software (Thermo Scientific). Quanbrowser software was used to generate calibration curves and quantitate analytes in all samples by linear regression analysis. A weighting factor of 1/X was used for all calibration curves. Accuracy and precision values for LC-MS/MS analysis of APAP and its metabolites in parrot plasma are shown in Supplementary Table S1.
Plasma biochemistry analysis
Plasma biochemistry panels were performed through the Veterinary Medical Teaching Hospital Clinical Diagnostic Laboratory Service at the University of California-Davis on a Roche Cobas c501 Analyzer. Measurands evaluated were anion gap, sodium, potassium, chloride, bicarbonate, phosphorus, calcium, BUN, glucose, total protein, albumin, globulin, AST, creatinine kinase (CK), ALP, cholesterol, glutamate dehydrogenase (GLDH), uric acid, triglycerides, and bile acids. Plasma biochemistry panels were performed within 4 hours of sample collection.
Pharmacokinetic analysis
Noncompartmental analysis (NCA) using a commercially available computer software program (Phoenix WinNonlin v8.3; Certara) was used to generate initial estimates of pharmacokinetic parameters for subsequent model fitting. After NCA, a nonlinear mixed effects modeling (NLME) approach with the Phoenix NLME software program was used to fit a population compartmental model to the data. For the model-building process, the first-order conditional estimation method with interaction was used and 1- and 2-compartment models were evaluated. Several error models including additive, multiplicative, Poisson, and mixed additive/multiplicative residual error models were assessed. Random effects were included for all structural parameters and were modeled with log-linear functions. Both a full and diagonal variance-covariance matrix were assessed for the random effects. Visual analysis of the observed versus predicted concentration graphs, residual plots, Akaike information criterion, percent coefficient of variation, and minus twice the log likelihood were all considered in assessing which model provided the best fit. Pharmacokinetic parameters for the glucuronide and sulfate metabolites were determined using NCA and the Phoenix WinNonlin software.
Statistical analysis
The effects of treatment on pharmacokinetic parameters were analyzed using the NCA pharmacokinetic parameters of APAP, APAP-glucuronide, and APAP-sulfate and comparing treatment groups using linear mixed models with individual birds as the random effect. The effects of treatment on biochemistry measurands were also analyzed using linear mixed models with measurands as the outcome variables, treatment as the fixed variable, and individual birds as the random variable. A compound symmetry covariance structure was used. Assumptions of linearity, normality, homoscedasticity of residuals, and lack of outliers were assessed graphically on standardized residual plots. An ANOVA was performed on the fixed effect. Post hoc analysis was performed using a Tukey adjustment. R (version 4.2.0, 2022; R Foundation for Statistical Computing) was used for statistical analysis. An alpha of 0.05 was used for statistical significance.
Results
Pilot study
The single oral dose of APAP at 5, 10, and 20 mg/kg resulted in maximum APAP plasma concentrations of 125.32 ± 72, 254.51 ± 157.37, and 748.68 ± 294.29 ng/mL, respectively. Administration of a single oral dose of 10 mg/kg APAP with concurrent administration of silymarin resulted in a maximum APAP plasma concentration of 313.52 ± 201.59 ng/mL. Maximum plasma concentrations achieved in the pilot were markedly below plasma concentrations considered therapeutic in humans, which range from 5,000 to 20,000 ng/mL.31 Due to the 5 to 20 times lower maximum plasma concentration than target concentrations obtained when administered at 20 mg/kg, a higher APAP dose of 100 mg/kg was chosen for subsequent phases of the study to balance potential adverse effects. No adverse clinical signs were noted in the pilot study.
Single-dose pharmacokinetic and safety studies
Plasma concentrations of APAP and its metabolites APAP-glucuronide and APAP-sulfate after a single oral administration of 100 mg/kg APAP with and without concurrent administration of 50 mg/kg silymarin or 400 mg/kg NAC are depicted in Figure 1. Select pharmacokinetic parameters from noncompartmental analysis are described in Tables 1–3. A single-compartment population pharmacokinetic analysis was also performed, with select parameters listed in Supplementary Table S2. Diagnostic plots and the visual predictive check plots are shown in Supplementary Figures S1–S6.
Mean ± SD plasma concentrations of acetaminophen (N-acetyl-para-aminophenol [APAP]; A), APAP-glucuronide (B), and APAP-sulfate (C) after a single oral dose of 100 mg/kg of APAP, 100 mg/kg of APAP and 400 mg/kg of N-acetylcysteine, and 100 mg/kg of APAP and 50 mg/kg of silymarin every 12 hours twice to orange-winged Amazon parrots (n = 8).
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.24.12.0402
Noncompartmental pharmacokinetic parameters of acetaminophen (N-acetyl-para-aminophenol [APAP]) after single oral administration of 100 mg/kg of APAP with and without concurrent oral administration of 50 mg/kg of silymarin or 400 mg/kg of N-acetylcysteine (NAC) to orange-winged Amazon parrots (n = 8).
| Pharmacokinetic parameter | APAP + silymarin | APAP + NAC | APAP alone |
|---|---|---|---|
| Cmax (ng/mL) | |||
| Mean ± SD | 2,308.1 ± 1,304.7 | 2,016.9 ± 889.9 | 2,917.2 ± 2,890.7 |
| Range | 1,025.8–4,568.9 | 955.4–3,211.1 | 592.9–9572.7 |
| 95% CI | 1,404.0–3,212.2 | 1,400.3–2,633.6 | 914.0–4,920.3 |
| tmax (h) | |||
| Mean ± SD | 2.10 ± 1.40 | 1.19 ± 0.88 | 1.13 ± 0.79 |
| Range | 1.00–4.00 | 0.50–3.00 | 0.50–3.00 |
| 95% CI | 1.19–3.06 | 0.58–1.80 | 0.58–1.67 |
| t½ (h) | |||
| Mean ± SD | 1.30 ± 0.60 | 1.45 ± 0.27 | 1.33 ± 0.87 |
| Range | 0.70–2.40 | 1.19–2.01 | 0.49–1.81 |
| 95% CI | 0.92–1.69 | 1.26–1.64 | 0.72–1.93 |
| Lambda Z (1/h) | |||
| Mean ± SD | 0.62 ± 0.25 | 0.49 ± 0.08 | 0.70 ± 0.37 |
| Range | 0.29–1.00 | 0.34–0.58 | 0.21–1.43 |
| 95% CI | 0.45–0.79 | 0.43–0.55 | 0.44–0.95 |
| AUC0-inf (h·ng/mL) | |||
| Mean ± SD | 7,329.1 ± 3,009.4 | 5,441.3 ± 1,869.8 | 6,063.7 ± 2,790.9 |
| Range | 4,449.1–13,372.0 | 3,432.8–9,028.2 | 2,899.4–12,083.0 |
| 95% CI | 5,243.6–9,414.5 | 4,145.6–6,737.0 | 4,129.8–7,997.7 |
| CL/F (mL/h/kg) | |||
| Mean ± SD | 15,604.4 ± 5,816.0 | 20,222.2 ± 6,413.7 | 19,344.2 ± 8,047.1 |
| Range | 7,478.3–22,311.1 | 11,076.4–29,130.6 | 8,276.1–34,490.1 |
| 95% CI | 11,574.1–19,634.7 | 15,777.7–24,666.7 | 13,767.9–24,920.6 |
AUC0-inf = AUC-versus-time curve from time 0 to infinity. CL/F = Drug clearance rate. Cmax = Maximum plasma concentration. t1/2 = Elimination half-life. tmax = Time of maximal plasma concentration. Lambda Z = Terminal rate constant.
Noncompartmental pharmacokinetic parameters of APAP-glucuronide after single oral administration of 100 mg/kg of APAP with and without concurrent oral administration of 50 mg/kg of silymarin or 400 mg/kg of NAC to orange-winged Amazon parrots (n = 8).
| Pharmacokinetic parameter | APAP + silymarin | APAP + NAC | APAP alone |
|---|---|---|---|
| Cmax (ng/mL) | |||
| Mean ± SD | 67,814.0 ± 16,842.3* | 88,299.9 ± 26,007.5 | 105,813.7 ± 54,319.5 |
| Range | 39,883.7–93,204.4 | 55,804.8–131,509.0 | 54,182.4–215,892.6 |
| 95% CI | 56,142.9–79,485.1 | 70,277.6–106,322.1 | 68,172.2–143,455.1 |
| tmax (h) | |||
| Mean ± SD | 3.00 ± 1.07* | 2.00 ± 0.93 | 2.13 ± 0.99 |
| Range | 1.00–4.00 | 1.00–3.00 | 1.00–4.00 |
| 95% CI | 2.26–3.74 | 1.36–2.64 | 1.44–2.81 |
| t½ (h) | |||
| Mean ± SD | 2.24 ± 0.57 | 2.83 ± 0.68 | 2.18 ± 0.43 |
| Range | 1.61–3.03 | 1.96–3.76 | 1.53–2.85 |
| 95% CI | 1.85–2.63 | 2.35–3.30 | 1.88–2.48 |
| Lambda Z (1/h) | |||
| Mean ± SD | 0.33 ± 0.08 | 0.26 ± 0.06 | 0.33 ± 0.07 |
| Range | 0.23–0.43 | 0.19–0.35 | 0.24–0.45 |
| 95% CI | 0.27–0.38 | 0.21–0.30 | 0.28–0.38 |
| AUC0-inf (h·ng/mL) | |||
| Mean ± SD | 347,506.7 ± 63,679.7 | 384,876.8 ± 97,732.8 | 366,340.4 ± 56,877.4 |
| Range | 279,518.1–474,610.4 | 268,190.0–548,665.0 | 300,005.0–458,487.4 |
| 95% CI | 303,378.9–391,634.5 | 317,151.4–452,602.2 | 326,926.3–405,754.4 |
*Significantly different from other treatment groups (P < .05).
Noncompartmental pharmacokinetic parameters of APAP-sulfate after single oral administration of 100 mg/kg of APAP with and without concurrent oral administration of 50 mg/kg of silymarin or 400 mg/kg of NAC to orange-winged Amazon parrots (n = 8).
| Pharmacokinetic parameter | APAP + silymarin | APAP + NAC | APAP alone |
|---|---|---|---|
| Cmax (ng/mL) | |||
| Mean ± SD | 135.8 ± 51.1 | 77.1 ± 28.6 | 96.8 ± 89.5 |
| Range | 71.8–204.0 | 49.9–123.8 | 26.0–304.2 |
| 95% CI | 100.4–171.2 | 57.3–97.0 | 34.8–158.8 |
| tmax (h) | |||
| Mean ± SD | 3.00 ± 1.07 | 2.38 ± 1.19 | 2.38 ± 0.92 |
| Range | 1.00–4.00 | 1.00–4.00 | 1.00–4.00 |
| 95% CI | 2.26–3.74 | 1.55–3.20 | 1.74–3.01 |
| t½ (h) | |||
| Mean ± SD | 1.59 ± 1.02 | 1.39 ± 0.35 | 1.49 ± 0.80 |
| Range | 0.80–4.05 | 0.96–2.10 | 0.87–3.08 |
| 95% CI | 0.89–2.30 | 1.14–1.63 | 0.94–2.04 |
| Lambda Z (1/h) | |||
| Mean ± SD | 0.53 ± 0.19 | 0.53 ± 0.12 | 0.56 ± 0.22 |
| Range | 0.17–0.87 | 0.33–0.72 | 0.23–0.80 |
| 95% CI | 0.40–0.66 | 0.44–0.61 | 0.41–0.71 |
| AUC0-inf (h·ng/mL) | |||
| Mean ± SD | 565.7 ± 208.6* | 315.0 ± 129.4 | 329.3 ± 179.3 |
| Range | 305.1–885.8 | 180.8–545.0 | 140.5–585.8 |
| 95% CI | 421.1–710.2 | 225.4–404.7 | 205.01–453.52 |
*Significantly different from other treatment groups (P < .05).
There were no differences in the NCA pharmacokinetic parameters of APAP between treatment groups (all P > .05). For APAP-glucuronide, the maximum plasma concentration (Cmax) and half-lives significantly differed between treatment groups (P = .018 and P = .033, respectively). On post hoc analysis, the Cmax was significantly higher in the APAP-alone group than in the APAP + silymarin group (P = .015). The half-life was significantly longer in the APAP + NAC group than in the APAP-alone group (P = .045). For APAP-sulfate, the AUC-versus-time curve from time 0 to infinity was significantly different between treatment groups (P < .001). Specifically, the APAP + silymarin group had significantly higher AUC than the APAP-alone (P < .001) and APAP + NAC groups (P < .001).
The results from the plasma biochemistry panels are summarized in Table 4. Sufficient blood volume for the 24-hour sample was not able to be obtained from 1 of the parrots in the APAP-silymarin group, and as a result, a limited postdrug administration plasma biochemistry panel was performed for that parrot (GLDH, uric acid, and bile acids). Aspartate aminotransferase (P < .0001) and CK (APAP-silymarin, P < .001; APAP-NAC, P < .0003; APAP alone, P < .0015) were significantly elevated after treatment for all single-dose studies. Potassium (P < .0001) and phosphorous (APAP-silymarin, P < .001; APAP-NAC, P < .0003; APAP alone, P < .0002) were also elevated after treatment in all study phases. Glutamate dehydrogenase was significantly higher only in the APAP-silymarin group (P < .007). Three out of 8 birds that received APAP with silymarin had mild elevation in GLDH, and 1 out of 8 birds had mild elevation in bile acids outside of the reported reference range for this species.32 Liver marker values remained within normal limits in all birds that received APAP alone or with NAC. One out of 8 birds exhibited regurgitation in the APAP-silymarin phase, with no other clinical adverse effects noted in any birds in any other single-dose studies.
Mean ± SD plasma biochemistry panels pre- and postadministration and blood sample collection for the pharmacokinetic analysis of a single oral dose of 100 mg/kg of APAP with or without oral administration of 50 mg/kg of silymarin or 400 mg/kg of NAC to orange-winged Amazon parrots (n = 8).
| APAP + silymarin | APAP + NAC | APAP alone | |||||
|---|---|---|---|---|---|---|---|
| Measurand | Reference interval | Pre | Post** | Pre | Post | Pre | Post |
| Anion gap (mmol/L) | 17.9–39.4 | 31.4 ± 5.1 | 31.0 ± 3.1 | 29.5 ± 2.3 | 32.5 ± 4.0 | 28.8 ± 3.2 | 30.0 ± 2.5 |
| Sodium (mmol/L) | 146.2–154.2 | 155.9 ± 1.1 | 150.9 ± 3.1 | 153.5 ± 2.1 | 158.5 ± 6.3 | 155.6 ± 3.3 | 152.5 ± 2.9 |
| Potassium (mmol/L) | 1.29–5.04 | 2.5 ± 0.5 | 4.5 ± 0.6* | 3.1 ± 0.5 | 4.9 ± 0.6* | 2.5 ± 0.4 | 4.9 ± 0.9* |
| Chloride (mmol/L) | 105.4–114.2 | 113.9 ± 1.6 | 109.7 ± 2.8 | 112.1 ± 3.3 | 116.3 ± 5.5 | 111.8 ± 2.1 | 112.1 ± 2.0 |
| Bicarbonate (mmol/L) | 6.6–21.8 | 13.3 ± 3.0 | 14.6 ± 2.1 | 15.0 ± 1.6 | 14.6 ± 0.9 | 17.4 ± 1.5 | 15.1 ± 2.5 |
| Phosphorous (mg/dL) | 1.16–5.0 | 2.7 ± 0.7 | 5.1 ± 1.2* | 3.8 ± 0.6 | 5.7 ± 1.1* | 2.7 ± 0.9 | 4.7 ± 0.9* |
| Calcium (mg/dL) | 7.74–10.36 | 9.9 ± 0.3 | 9.6 ± 0.5 | 9.8 ± 0.8 | 11.0 ± 2.9 | 10.6 ± 0.6 | 10.2 ± 0.6 |
| BUN (mg/dL) | 0–2 | 0.8 ± 0.9 | 1.3 ± 1.1 | 0.3 ± 0.7 | 5.1 ± 1.2* | 0.5 ± 0.9 | 0.8 ± 1.0 |
| Glucose (mg/dL) | 213–371 | 283.8 ± 15.9 | 298.4 ± 35.5 | 291.4 ± 25.4 | 288.3 ± 20.9 | 275.8 ± 27.7 | 295.0 ± 26.9 |
| Total protein (g/dL) | 3.44–4.91 | 4.2 ± 0.4 | 3.6 ± 0.3* | 4.1 ± 0.3 | 4.1 ± 0.5 | 4.1 ± 0.4 | 3.6 ± 0.3* |
| AST (U/L) | 125–375 | 174.3 ± 37.4 | 1,085.9 ± 494.1* | 176.1 ± 29.4 | 866.4 ± 247.7* | 181.9 ± 29.9 | 872.8 ± 345.5* |
| ALP (U/L) | 17.5–119.6 | 51.4 ± 25.8 | 38.0 ± 16.0 | 58.9 ± 29.4 | 68.8 ± 66.8 | 48 ± 18.2 | 45.6 ± 17.0 |
| Cholesterol (mg/dL) | 110.4–363 | 266.8 ± 55.5 | 186.4 ± 40.9 | 279.0 ± 91.0 | 246.5 ± 69.4 | 236.6 ± 65.8 | 199.9 ± 39.5 |
| GLDH (U/L) | 0–3.6 | 0.9 ± 0.4 | 4.0 ± 3.5* | 1.1 ± 0.6 | 2.3 ± 1.5 | 1.5 ± 0.8 | 1.9 ± 0.8 |
| Uric acid (mg/dL) | 1.86–12.66 | 4.0 ± 1.2 | 4.7 ± 1.9 | 8.9 ± 1.4 | 4.5 ± 1.9* | 6.6 ± 2.0 | 6.1 ± 1.3 |
| Creatinine kinase (U/L) | 182–1,459 | 649.6 ± 643.1 | 20,344.3 ± 144,470.4* | 501.5 ± 318.1 | 15,614.6 ± 8,670.9* | 374.1 ± 212.2 | 13,608.6 ± 7,980.4* |
| Triglycerides (mg/dL) | 69–234 | 167.4 ± 44.7 | 219.4 ± 159.3 | 189.0 ± 52.0 | 296.3 ± 362.7 | 152.8 ± 32.3 | 450.1 ± 677.6 |
| Bile acids (μmol/L) | 8.1–88 | 34.1 ± 15.8 | 48.6 ± 42.7 | 35.3 ± 17.5 | 40.3 ± 20.8 | 36.5 ± 14.1 | 32.6 ± 14.2 |
*Significant difference between pre- and posttreatment.
**Incomplete chemistry profile obtained for 1 bird. For glutamate dehydrogenase (GLDH), uric acid, and bile acids, n = 8; for all other parameters, n = 7.
Single-dose safety study
The 8 additional birds that received a single oral dose of 100 mg/kg APAP in the safety study showed no adverse clinical effects. The results from the plasma biochemistry panels are summarized in Table 5. Uric acid was significantly elevated (P = .0003) after treatment; however, individual values remained within reference intervals for this species. Creatinine kinase was also significantly higher (P = .0334), and chloride was significantly lower (P = .0003) after treatment. There were no significant changes in liver marker values after APAP administration.
Mean ± SD plasma biochemistry panels pre- and postadministration of a single oral dose of 100 mg/kg of APAP to orange-winged Amazon parrots (n = 8) in the safety study.
| APAP-alone safety study | |||
|---|---|---|---|
| Measurand | Reference interval | Pre | Post |
| Anion gap (mmol/L) | 17.9–39.4 | 32.1 ± 2.7 | 33.4 ± 2.7 |
| Sodium (mmol/L) | 146.2–154.2 | 156.8 ± 2.8 | 155.8 ± 2.3 |
| Potassium (mmol/L) | 1.29–5.04 | 3.5 ± 0.6 | 3.7 ± 0.4 |
| Chloride (mmol/L) | 105.4–114.2 | 114.6 ± 2.7 | 109.9 ± 3.1* |
| Bicarbonate (mmol/L) | 6.6–21.8 | 13.3 ± 2.1 | 16.4 ± 2.9 |
| Phosphorous (mg/dL) | 1.16–5.0 | 3.4 ± 0.8 | 3.5 ± 0.5 |
| Calcium (mg/dL) | 7.74–10.36 | 10.6 ± 1.4 | 10.9 ± 0.7 |
| BUN (mg/dL) | 0–2 | 0.5 ± 0.9 | 0.4 ± 0.7 |
| Glucose (mg/dL) | 213–371 | 272.5 ± 18.8 | 271.4 ± 32.5 |
| Total protein (g/dL) | 3.44–4.91 | 4.3 ± 0.3 | 4.2 ± 0.4 |
| AST (U/L) | 125–375 | 181.5 ± 60.6 | 306.8 ± 199.5 |
| ALP (U/L) | 17.5–119.6 | 101.3 ± 83.8 | 80.3 ± 58.0 |
| Cholesterol (mg/dL) | 110.4–363 | 278.0 ± 52.1 | 265.3 ± 51.8 |
| GLDH (U/L) | 0–3.6 | 0.8 ± 0.7 | 0.3 ± 3.5 |
| Uric acid (mg/dL) | 1.86–12.66 | 3.7 ± 0.8 | 8.2 ± 2.3* |
| Creatinine kinase (U/L) | 182–1,459 | 475.0 ± 304.8 | 1,314.9 ± 1,154.9* |
| Triglycerides (mg/dL) | 69–234 | 179.8 ± 47.8 | 150 ± 44.4 |
| Bile acids (μmol/L) | 8.1–88 | 25.6 ± 21.9 | 28.9 ± 21.7 |
*Significant difference between pre- and posttreatment.
Discussion
This is the first study to evaluate the pharmacokinetics and safety of oral APAP in a psittacine species. Noncompartmental pharmacokinetic parameters were established for APAP and its metabolites APAP-glucuronide and APAP-sulfate after a single oral administration of 100 mg/kg APAP with and without concurrent oral administration of silymarin or NAC in orange-winged Amazon parrots. Single compartment population pharmacokinetic models were also established for APAP, which revealed moderate to high variability between individuals as demonstrated by the coefficient of variation for many of the parameters assessed.33 This variability is consistent with what has been reported in some human population pharmacokinetic studies.34,35
The metabolism of APAP in orange-winged Amazon parrots is markedly different than other species. Despite using a 5- to 10-fold higher dose of APAP in this study, the maximum APAP plasma concentrations were notably lower than those reported in other species. The Cmax ± SD in parrots across treatment groups was 2,308.1 ± 1,304.7 (APAP-silymarin), 2,016.9 ± 889.9 (APAP-NAC), and 2,917.2 ± 2,890.7 ng/mL (APAP alone). In comparison, domestic geese,11 chickens,7 and turkeys7 receiving 10 mg/kg APAP orally reached mean maximum plasma concentrations of 5,310, 8,610, and 1,070 ng/mL, respectively. In mammals, oral administration of 20 mg/kg APAP similarly resulted in higher Cmax values of 11,110 ng/mL in dogs and 20,020 ng/mL in horses.8,36 The half-life of APAP in parrots was also short, ranging from 1.30 to 1.45 hours across treatment groups. This is similar to the APAP half-life in dogs (1.25 hours)8 and turkeys (1.14 hours)7 but shorter than that reported in geese (5.01 hours)11 and horses (3.5 hours)36 and longer than that reported in chickens (0.45 hours).7 The AUC-versus-time curve from time 0 to infinity in parrots across treatment groups was 7,329.1 ± 3,009.4 (APAP-silymarin), 5,441.3 ± 1,869.8 (APAP-NAC), and 6,063.7 ± 2,790.9 ng/mL (APAP alone). This is similar to the AUC achieved in chickens (6,700 ng/mL)7 but higher than that achieved in turkeys (2,200 ng/mL)7 and lower than that achieved in geese (11,550 ng/mL),11 dogs (34,610 ng/mL),8 and horses (115,700 ng/mL).36
Acetaminophen was relatively quickly absorbed in orange-winged Amazon parrots, reaching maximum plasma concentrations at 1 to 2 hours across treatment groups, and cleared, with no detectable levels of APAP after 12 hours in the APAP-NAC and APAP-alone groups. Minimal APAP plasma concentrations were quantifiable in the APAP-silymarin group at 24 hours. In comparison, geese,11 chickens,7 and turkeys7 have even faster absorption and elimination of APAP, reaching maximum plasma concentrations at 0.17 to 0.77 hours and having no detectable APAP plasma concentrations after 8 to 10 hours depending on species. These findings highlight significant interspecies differences in APAP pharmacokinetics, emphasizing the need for species-specific dosing considerations.
This study demonstrated that orange-winged Amazon parrots have a marked metabolism of APAP to APAP-glucuronide, with minimal conversion to APAP-sulfate. The Cmax of APAP-glucuronide after administration of APAP alone, with silymarin, and with NAC was 105,813.7 ± 54,319.5, 67,814.0 ± 16,842.3, and 88,299.9 ± 26,007.5 ng/mL, respectively. These maximum plasma concentrations are 29 to 44 times higher than the corresponding APAP Cmax in each respective group. Comparatively, domestic geese receiving 10 mg/kg APAP had an APAP-glucuronide Cmax of 16,550 ng/mL, only 3 times higher than that of APAP. The high level of glucuronidation in orange-winged Amazon parrots may be contributing to the relatively low plasma concentrations of APAP achieved in this study compared to other species. Individual variation, differences in oral bioavailability, and conversion to other metabolites not measured in this study may also be affecting APAP plasma concentrations.
Pharmacodynamic studies of APAP have not been performed in any bird species, and consequently, therapeutic plasma concentrations in orange-winged Amazon parrots are unknown. However, the maximum plasma concentrations of APAP achieved in this study are below the reported therapeutic plasma concentrations in other species. In horses, concentrations of 8,000 to 12,000 ng/mL are associated with analgesia in lameness pharmacodynamic models.36,37 In humans, target plasma concentrations are reported to range from 5,000 to 20,000 ng/mL, with a possible ceiling effect for analgesia occurring at 14,000 ng/mL.31 However, in humans the analgesic effect of APAP is directly related to an effect compartment, which is approximately equal to CSF concentrations, rather than to blood plasma concentrations.2 Concurrent administration with NAC may also potentiate APAP’s analgesic effects, which has been demonstrated in rats.23 As a result, plasma APAP concentrations and the metabolites measured in this study cannot be used to predict the analgesic effect in birds, and further studies are needed to evaluate whether the dose of APAP used in this study, with or without concurrent NAC administration, produces an analgesic effect in orange-winged Amazon parrots.
Administration of APAP alone and with NAC was well tolerated, with no adverse clinical signs or significant changes to plasma ALP, GLDH, or bile acids that may be associated with hepatotoxicity. However, GLDH was mildly but significantly elevated in birds that received APAP with silymarin, indicating mild hepatotoxic effects in this group. This is in contrast to other species, where silymarin acts as a hepatoprotectant for APAP toxicity. In mice, pretreatment with multiple doses of silymarin before a toxic dose of APAP prevented hepatotoxicity but did not change plasma concentrations of APAP, APAP-glucuronide, or APAP-sulfate.14 Posttreatment with silymarin after a toxic dose of APAP in mice,15 cats,18 and pigeons,19 as well as concurrent administration of silymarin and APAP in rats,17 has also been shown to prevent hepatotoxicity. The occurrence of adverse clinical signs in the APAP-silymarin group, but not the APAP-alone or APAP-NAC groups, was a surprising finding and the reason for it remains unknown. While silymarin is used anecdotally for the management of hepatopathies in psittacines, no pharmacokinetic or pharmacodynamic studies have been performed in these species, and current dosing is empirical. The formulation of silymarin used in this study was compounded due to a lack of suitable commercially available formulations. While the ingredients used in the formulation are commonly used as pharmaceutical and feed additives,38 there are no studies specifically evaluating their safety in psittacines. As a result, we cannot rule out that another compound in the formulation, rather than silymarin itself, contributed to the adverse clinical effects seen in this treatment group.
There are several limitations of this study. While there were few statistically significant differences in pharmacokinetic parameters between treatment groups, the small sample size in this study may have been insufficient to detect significant differences between treatment groups, and the presence of multiple comparisons may have further limited the ability to achieve statistical significance. Acetaminophen hepatotoxicity is mediated through the effects of the toxic metabolite NAPQI.2 As a result, direct measurement of this metabolite would be beneficial to more thoroughly understand the metabolism and safety of APAP administration in orange-winged Amazon parrots. However, NAPQI is highly reactive and unstable in its native form in the blood and therefore was unable to be measured in this study.39 Given the unknown safety profile of APAP in psittacines and the relatively high dose being used in the single-dose studies compared to other species, APAP was first concurrently administered with silymarin, then with NAC, and then alone to the same 8 birds to try to decrease risks for morbidity and mortality. Even though there was at least a 1-month washout period between study phases, we cannot rule out that there were carryover effects from prior drug administration in subsequent treatment trials given the lack of crossover design. A separate group of birds was used for the safety evaluation of APAP alone to avoid a possible carryover effect when evaluating safety using biochemistry panels. A lack of a control group that did not receive any drugs during the different studies additionally limits the interpretation of blood work changes. Repeated venipuncture can result in changes to biochemistry parameters, as demonstrated by the significant elevation in AST, CK, phosphorous, and potassium after treatment for all single-dose study phases. These changes are suspected to be due to repeated handling of the birds for venipuncture,40 rather than a consequence of APAP administration itself. In contrast, the birds in the safety study, which were handled for venipuncture only twice within 24 hours, had significant elevation in CK, but not AST, potassium, or phosphorous, after treatment, consistent with reduced handling.
In conclusion, a single oral dose of 100 mg/kg APAP with and without concurrent administration of NAC was well tolerated in healthy orange-winged Amazon parrots; however, it did not reach target therapeutic plasma concentrations established in mammalian species. Concurrent administration of APAP with silymarin resulted in a mild but significant elevation in GLDH. Further studies are needed to establish the pharmacodynamics of APAP with and without concurrent administration of NAC, as well as the pharmacokinetics and safety of multidose administration.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors thank Mariana Sosa-Higareda, Lexi Durant, Rachel Hirota, Sebastian Elsenbroek, and Jaimie Brown for their help in parrot handling and sample processing. The authors also thank Kevin Bellido for his direct support with husbandry and handling of the orange-winged Amazon parrots throughout this project.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
This study was funded by the Center for Companion Animal Health, School of Veterinary Medicine, University of California-Davis, and by the Richard M. Schubot Parrot Wellness and Welfare Program.
ORCID
H. Beaufrere https://orcid.org/0000-0002-3612-5548
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