PHARMACOKINETIC PROFILES OF ORAL PHENYLBUTAZONE, MELOXICAM, AND FIROCOXIB IN SOUTHERN BLACK RHINOCEROS (DICEROS BICORNIS MINOR).

The pharmacokinetic profile of selected NSAIDs in southern black rhinoceros (Diceros bicornis minor) were studied. Phenylbutazone (PBZ), meloxicam (MEL), and firocoxib (FIR) were administered orally to five captive, black rhinoceros, and blood was collected at predetermined time points for NSAID quantification and noncompartmental pharmacokinetic (PK) analysis. Phenylbutazone 4.0 mg/kg PO q12h for three doses, MEL 0.3 mg/kg PO q24h administered twice, and a single oral dose of FIR 0.1 mg/kg, were tested with a minimum washout time of 2 wk. PBZ reached a median (range) peak concentration (Cmax) of 9.42 (2.74-11.5) g/ml at a mean (range) time (Tmax) of 6.00 (4.00 to >12.00) h, and the median (range) elimination half-life (T1/2) was 6.07 (3.95-6.49) h. Phenylbutazone pharmacokinetic parameters for black rhinoceros in this study were similar to domestic horses. Meloxicam reached a median (range) Cmax of 0.576 (0.357-0.655) µg/ml at a median (range) time (Tmax) of 6.00 (4.00-12.00) h; the median (range) T1/2 of MEL was 14.0 (12.4-17.9) h. These results demonstrate that once-daily administration of MEL at 0.3 mg/kg resulted in a serum concentration of greater than 0.200 µg/ml from 2 to 24 h in four animals, which is within the analgesic range (0.200-0.400 µg/ml) for this drug in other species postulated by other studies. A single dose of firocoxib (0.1 mg/kg) reached a median (range) peak concentration (Cmax) of 15.7 (9.65-17.3) ng/ml at a median (range) Tmax of 4.00 (4.00-6.00) h. The median (range) elimination T1/2 of FIR was 4.96 (4.47-6.51) h, which is faster than in the horse. The data suggest that extrapolation from equine FIR dosage recommendations is inappropriate for black rhinoceros.

Non-antibiotic pharmaceutical phenylbutazone binding to MexR reduces the antibiotic susceptibility of Pseudomonas aeruginosa.

Antimicrobial resistance has been an increasingly serious threat to global public health. The contribution of non-antibiotic pharmaceuticals to the development of antibiotic resistance has been overlooked. Our study found that the anti-inflammatory drug phenylbutazone could protect P. aeruginosa against antibiotic mediated killing by binding to the efflux pump regulator MexR. In this study, antibiotic activity against P. aeruginosa alone or in combination with phenylbutazone was evaluated in vitro and in vivo. Resazurin accumulation assay, transcriptomic sequencing, and PISA assay were conducted to explore the underlying mechanism for the reduced antibiotic susceptibility caused by phenylbutazone. Then EMSA, ITC, molecular dynamic simulations, and amino acid substitutions were used to investigate the interactions between phenylbutazone and MexR. We found that phenylbutazone could reduce the susceptibility of P. aeruginosa to multiple antibiotics, including parts of ß-lactams, fluoroquinolones, tetracyclines, and macrolides. Phenylbutazone could directly bind to MexR, then promote MexR dissociating from the mexA-mexR intergenic region and de-repress the expression of MexAB-OprM efflux pump. The overexpressed MexAB-OprM pump resulted in the reduced antibiotic susceptibility. And the His41 and Arg21 residues of MexR were involved in the phenylbutazone-MexR interaction. We hope this study would imply the potential risk of antibiotic resistance caused by non-antibiotic pharmaceuticals.

Comparative pharmacokinetics of phenylbutazone in healthy young-adult and geriatric horses.

OBJECTIVE: To evaluate the effects of aging on phenylbutazone (PBZ) disposition in older horses (≥ 25 years old) compared to young adults (4 to 10 years old) by characterizing the pharmacokinetic profile of PBZ and its active metabolite, oxyphenbutazone (OPBZ), following a 2.2-mg/kg dose, IV. We hypothesized that the disposition of PBZ will be affected by age. ANIMALS: 16 healthy horses (8 young adults aged 4 to 10 years and 8 geriatric horses ≥ 25 years old). METHODS: Horses were administered a single 2.2-mg/kg PBZ dose, IV. Plasma samples were collected at designated time points and frozen at -80 °C until assayed using liquid chromatography-tandem mass spectrometry. Pharmacokinetic analyses were performed using Phoenix WinNonlin, version 8.0 (Certara). Both clinical and pharmacokinetic data were compared between age groups using independent samples t tests, with P < .05 considered significant. RESULTS: Baseline characteristics did not differ between groups, with the exception of age, weight, and plasma total solids. Plasma concentrations of PBZ were best described by a two-compartment model. The maximum plasma concentration of OPBZ was reached at 5 hours for both age groups, and the metabolite-to-parent-drug area-under-the-curve ratios were approximately 20% for both groups. None of the pharmacokinetic parameters of PBZ or its metabolite, OPBZ, differed significantly between age groups. CLINICAL RELEVANCE: The hypothesis was rejected as there was no significant difference in PBZ disposition in young-adult horses compared to geriatric horses. Our data do not support the need for dose adjustments of PBZ in clinically healthy geriatric horses.

Prolonged administration of oral phenylbutazone and firocoxib in horses has no impact on selected cytokine and growth factor concentrations in platelet-rich plasma and autologous protein solution.

OBJECTIVE: To determine the effects of prolonged administration of the oral NSAIDs phenylbutazone and firocoxib on concentrations of cytokines and growth factors in platelet-rich plasma (PRP) and autologous protein solution (APS). ANIMALS: 6 adult University owned horses. METHODS: Horses were randomized to receive phenylbutazone (1 g, orally, q 12 h) or firocoxib (57 mg, orally, q 24 h) for 6 days. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before the administration of NSAIDs and at 7 days (1 day following cessation of NSAIDs). Horses underwent a two-week washout period, during which blood was obtained at 14 days and 21 days. The protocol was repeated with a crossover design. PRP and APS were analyzed for concentrations of platelets, leukocytes, and several cytokines (IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) and growth factors (PDGF, FGF-2, and TGF-ß1) using immunoassays. Plasma was evaluated for drug concentrations. RESULTS: No significant differences existed in concentrations of growth factors and cytokines before or after prolonged administration of NSAIDs. There were significant differences in concentrations of leukocytes and platelets in PRP compared to APS, with higher concentrations of leukocytes at the day 7 time point (T) in APS (phenylbutazone) and in concentrations of platelets in APS at T0 (firocoxib) and in APS at T7 (phenylbutazone). CLINICAL RELEVANCE: Veterinarians can recommend the administration of these oral NSAIDs prior to obtaining blood for PRP and APS provided a single-day washout period is instituted.

Evaluation of gastrointestinal tract lesions and serum malondialdehyde levels after repeated oral administration of phenylbutazone in horses.

Phenylbutazone (PBZ) is a widely used nonsteroidal anti-inflammatory drug for horses. However, because of its gastrointestinal side effects, its administration requires careful attention in veterinary practice. Malondialdehyde (MDA) is a serum biomarker associated with increased damage to the equine gastrointestinal system. This study investigated the hematological effects and alterations in the gastrointestinal tract and assessed serum MDA concentrations following repeated oral PBZ administration at clinical doses. Fourteen horses were randomly divided into control and treatment groups. All horses in the treatment group were administered 4.4 milligrams per kilogram of body weight of PBZ syrup orally twice a day for 7 days, whereas the control group received syrup as a placebo. The development of gastrointestinal side effects was investigated using gastroscopy, abdominal ultrasound, and fecal pH; serum MDA concentrations were assessed using a commercially available enzyme-linked immunosorbent assay kit. Data were compared between PBZ-treated and control horses before and after the treatment period. The treatment group exhibited decreased albumin and total protein concentrations. Moreover, this group exhibited a higher thickness of the right dorsal colon wall (p = 0.03) and had higher scores for squamous gastric ulcers (p = 0.01). Fecal pH was lower in the treatment group than in the control group after PBZ administration (p < 0.01). Although MDA concentrations were higher in the treatment group after PBZ administration, they did not differ significantly from those of the control group. This study highlighted the changes in hematological and gastrointestinal lesions resulting from PBZ administration in horses at clinical doses, even without clinical signs. However, MDA may not be an optimal biomarker for the early detection of gastrointestinal damage due to PBZ treatment in horses.

Integrated analysis of gut metabolome, microbiome, and exfoliome data in an equine model of intestinal injury.

BACKGROUND: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions. METHODS: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches. RESULTS: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa. CONCLUSIONS: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract.

Treatment of mares with the non-steroidal anti-inflammatory drug (NSAID) phenylbutazone transiently affects in vitro maturation of equine oocytes and blastocyst development after Intracytoplasmic Sperm Injection (ICSI).

Mares enrolled in assisted reproductive technologies (ARTs) programs are often treated with non-steroidal anti-inflammatory drugs (NSAIDs), particularly phenylbutazone (Bute), due to chronic lameness. The current study was performed to determine the effect of Bute administration on the developmental competence of in vitro-matured equine oocytes subjected to Intracytoplasmic Sperm Injection (ICSI). In a Preliminary Study, immature cumulus-oocyte complexes (COCs) recovered by post-mortem ovary harvested from two healthy mares (n = 2) treated for 10 days with Bute (4.4 mg/kg, PO, BID), and four non-treated healthy mares (n = 4), were matured in vitro and subjected to Piezo-driven ICSI. Lower oocyte in vitro maturation [Bute: 25% (3/12) vs. Control: 61% (28/46)] and blastocyst rates [Bute: 0% (0/12) vs. Control: 18% (5/28)] were observed in the Bute-treated when compared to the Control mares (P < 0.05). In the Main Experiment, a group of healthy mares (n = 9) received a daily dose of Bute (4.4 mg/kg, orally, SID) for 10 days. A control group of mares (n = 10) was treated with an equal volume of placebo. Mares in both groups were subjected to ultrasound-guided transvaginal oocyte aspiration (TVA) on days 3, 33, and 77 following the last dose of Bute (PT). Recovered COCs from both mare groups were matured in vitro and subjected to Piezo-driven ICSI. By day-3 PT, oocyte in vitro maturation rate was similar between mare groups [Bute: 65% (36/55) vs. Control: 67% (78/116); P > 0.05], while oocyte recovery [Bute: 53% (55/103) vs. Control: 70% (116/166)], cleavage [Bute: 31% (11/36) vs. Control: 62% (48/78)] and blastocyst rates [Bute: [0%] (0/36) vs. Control: 28% (22/78)] were significantly different (P < 0.05). By day 33 PT and 77 PT, differences on oocyte recovery, in vitro maturation, cleavage, and blastocyst rates were not observed between mare groups. In summary, the administration of Bute for 10 consecutive days (4.4 mg/kg, PO, SID, or BID) is associated with a decrease in the ability of immature equine oocytes to undergo in vitro-maturation (Preliminary Study) and develop to the blastocyst stage following ICSI (Preliminary Study and Main Experiment). This negative effect appeared to be transient, as 30- and 77-days post-treatment, no differences on in vitro maturation, cleavage or blastocyst rates were observed.

Effect of phenylbutazone on insulin secretion in horses with insulin dysregulation.

BACKGROUND: Phenylbutazone is often prescribed to manage pain caused by hyperinsulinemia-associated laminitis, but in diabetic people nonsteroidal anti-inflammatory drugs increase insulin secretion and pancreatic activity. HYPOTHESIS/OBJECTIVES: Investigate the effect of phenylbutazone administration on insulin secretion in horses. It was hypothesized that phenylbutazone will increase insulin secretion in horses with insulin dysregulation (ID). ANIMALS: Sixteen light breed horses, including 7 with ID. METHODS: Randomized cross-over study design. Horses underwent an oral glucose test (OGT) after 9 days of treatment with phenylbutazone (4.4 mg/kg IV q24h) or placebo (5 mL 0.9% saline). After a 10-day washout period, horses received the alternative treatment, and a second OGT was performed. Insulin and glucose responses were compared between groups (ID or controls) and treatments using paired t test and analyses of variance with P < .05 considered significant. RESULTS: In horses with ID, phenylbutazone treatment significantly decreased glucose concentration (P = .02), glucose area under the curve (2429 ± 501.5 vs 2847 ± 486.1 mmol/L × min, P = .02), insulin concentration (P = .03) and insulin area under the curve (17 710 ± 6676 vs 22 930 ± 8788 µIU/mL × min, P = .03) in response to an OGT. No significant effect was detected in control horses. CONCLUSION AND CLINICAL IMPORTANCE: Phenylbutazone administration in horses with ID decreases glucose and insulin concentrations in response to an OGT warranting further investigation of a therapeutic potential of phenylbutazone in the management of hyperinsulinemia-associated laminitis beyond analgesia.

Single-dose nonsteroidal anti-inflammatory drugs in horses have no impact on concentrations of cytokines or growth factors in autologous protein solution and platelet-rich plasma.

OBJECTIVE: To determine the effects of a single dose of the NSAIDs phenylbutazone, firocoxib, flunixin meglumine, and ketoprofen on concentrations of growth factors and cytokines in autologous protein solution (APS) and platelet-rich plasma (PRP). ANIMALS: 6 adult university-owned horses. METHODS: For the first phase, 6 horses were randomized to receive ketoprofen (1,000 mg) or flunixin meglumine (500 mg) IV. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before and 6 hours after administration of NSAIDs. Horses underwent a 2-week washout period, after which the protocol was repeated using a crossover design. For the second phase, following at least a 2-week washout period, the study protocol was repeated with phenylbutazone (1 g) or firocoxib (57 mg) administered orally. Plasma was collected 6 hours after administration for evaluation of drug concentrations, and APS and PRP were analyzed for concentrations of drug, platelets, leukocytes, and several growth factors and cytokines (PDGF, fibroblast growth factor, TGF-ß1, IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) before and 6 hours after administration of NSAIDs using immunoassays. RESULTS: There were no significant differences in concentrations of cytokines or growth factors before or after administration of any NSAID. There were significant differences in concentrations of leukocytes and platelets based on both product and time. NSAID concentrations in plasma were not significantly different from concentrations in APS and PRP. CLINICAL RELEVANCE: These results help guide clinicians on the appropriate use of these NSAIDs in conjunction with the processing of APS and PRP, which is unlikely to significantly alter the final product after single-dose administration.

Clinical effects of a combination of phenylbutazone and omeprazole on chronic lameness in Mongolian horses.

BACKGROUND: Phenylbutazone (PBZ) is the most commonly used drug to treat symptoms of lameness in horses; however, it is associated with adverse effects such as gastric ulcer syndrome (EGUS). Interestingly, many practitioners prescribe omeprazole (OME) concurrently with PBZ to prevent the development of EGUS. However, the efficacy and safety of this practice in Mongolian horses with chronic lameness remain unknown. OBJECTIVES: To evaluate the clinical effects of a combination of PBZ and OME on chronic lameness in Mongolian horses. STUDY DESIGN: Randomised block experimental design. METHODS: Eighteen Mongolian horses with lameness score was ≥3 points, were divided into three treatment groups, with six horses in each group: placebo (CON), PBZ (4.4 mg/kg PO q. 24 h), or PBZ plus OME (4 mg/kg PO q. 24 h; PBZ + OME) in a randomised block design based on the initial lameness score. The horses were treated for 15 days. During this period, weekly gastroscopy, and physiological and biochemical tests were performed. RESULTS: Both PBZ (median 1.0, interquartile range [IQR]: 0.8-1.3; p = 0.01) and PBZ + OME (median 1.0, IQR: 1.0-1.0; p = 0.01) significantly decreased the lameness score compared with before administration. In addition, PBZ significantly increased the equine glandular gastric disease (EGGD) score (3.0 ± 0.6, p < 0.001), GT-17 content (293.4 ± 21.8 pg/mL, p < 0.001), and pepsinogen-1 (PG1) content (295.3 ± 38.3 ng/mL, p < 0.001) compared with CON or PBZ + OME. However, it significantly reduced the total protein (53.6 ± 1.5 g/L, p < 0.05) and albumin (25.5 ± 1.8 g/L, p < 0.05) contents. Nevertheless, compared with PBZ, PBZ + OME significantly decreased the EGGD score (0.3 ± 0.5, p < 0.001) and significantly increased the gastric fluid pH (7.3 ± 0.5, p < 0.001), total protein content (62.5 ± 4.6 g/L, p = 0.009), and albumin content (29.4 ± 1.1 g/L, p = 0.004). Meanwhile, they significantly diminished the gastrin 17 (GT-17) (162.0 ± 21.0 pg/mL, p < 0.001) and PG1 (182.4 ± 22.5 ng/mL, p < 0.001) contents. MAIN LIMITATIONS: Individual differences in horses were larger, but the sample size was small. There was larger interval between observations for each index. CONCLUSIONS: Compared with PBZ alone, PBZ + OME had no therapeutic effect on chronic lameness; however, it reduced the occurrence of EGGD in Mongolian horses. Horses may be protected against chronic lameness and PBZ-induced EGGD by increasing the pH value, decreasing serum PG1 and GT-17 content, and preventing the reduction of myeloperoxidase content.

Effects of phenylbutazone, firocoxib, and dipyrone on the diuretic response to furosemide in horses.

BACKGROUND: Treatment with phenylbutazone (nonselective COX inhibitor) decreases the diuretic and natriuretic effects of furosemide by nearly 30% but the effects of COX-2 specific inhibitors (firocoxib) and atypical NSAIDs (dipyrone) are unknown. HYPOTHESIS: Furosemide-induced diuresis after pretreatment with firocoxib or dipyrone is diminished to a lesser extent than after pretreatment with phenylbutazone. ANIMALS: Eight healthy mares. METHODS: Each mare received 4 treatments in a prospective experimental crossover study using a replicated 4 × 4 Latin Square design: furosemide alone (FU), furosemide and phenylbutazone (PB), furosemide and firocoxib (FX), and furosemide and dipyrone (DP). After 24 hours of NSAID treatment at recommended dosages, ureteral catheters were placed for continual urine collection. After a 30-minute baseline collection period, furosemide (1.0 mg/kg, IV) was administered, and urine and blood samples were collected for 4 hours. Data were assessed by repeated measures ANOVA. RESULTS: Four-hour urine volume was (mean ± SD) ~25% less (P < .001) after pretreatment with all NSAIDs (PB 19.1 ± 2.1 mL/kg, FX 17.7 ± 3.5 mL/kg, DP 19.1 ± 3.9 mL/kg), as compared to FU (23.4 ± 5.1 mL/kg) (P < .001), but there were no differences between PB, FX, or DP. Interindividual variability in furosemide diuresis after pretreatment with different NSAIDs was observed. CONCLUSIONS AND CLINICAL IMPORTANCE: Though COX-2 selective NSAIDs and dipyrone might have less severe or fever gastrointestinal adverse effects in horses, our data suggest minimal differences in effects on furosemide-induced diuresis, and possibly, risk of nephrotoxicosis.

Right dorsal colitis in horses: A multicenter retrospective study of 35 cases.

BACKGROUND: Right dorsal colitis (RDC) is a nonsteroidal anti-inflammatory drug (NSAID) induced, protein losing enteropathy in horses associated with a high case fatality rate. OBJECTIVES: To describe signalment, NSAID usage, clinical presentations, clinical pathology, ultrasonographic findings, treatments, outcomes, and factors associated with survival in horses diagnosed with RDC. ANIMALS: Thirty-five horses from 7 Australian equine hospitals diagnosed with RDC. METHODS: Retrospective case series. Clinical records of cases were accepted if definitively or presumptively diagnosed by an internist with RDC and had ≥3 of: hypoproteinemia or hypoalbuminemia; diarrhea with negative test results for infectious diseases; colic for which other diseases were excluded or right dorsal colon thickening on ultrasound. Descriptive data analysis was performed for categorical and continuous variables. Univariate binominal logistic regressions were used to assess factors associated with survival. RESULTS: An overdose of NSAIDs occurred in 84% (21/25) cases where dose was known. Common clinical presentations included diarrhea (69%; 22/32), colic (61%; 20/33), and tachycardia (53%, 17/32). Common clinicopathological findings included hypoalbuminemia (83%; 26/31), hypocalcaemia (79%, 23/29), and hyperlactatemia (77%, 14/18). The right dorsal colon wall appeared subjectively thickened in 77% (24/31) cases using ultrasonography. Case fatality rate was 43% (15/35). Odds of survival significantly decreased with increasing heart rate (odds 0.84, 95% CI = 0.71-0.92, P = .01), packed cell volume (odds 0.91, 95% CI 0.82-0.98, P = .05) and abnormal appearance of mucous membranes (odds 0.05, 95% CI 0.005-0.28, P = .001) on hospital presentation. CONCLUSIONS AND CLINICAL IMPORTANCE: An overdose of NSAIDs is common in horses diagnosed with RDC. Serum albumin concentrations should be monitored in horses receiving a prolonged course of NSAIDs. Overall prognosis for RDC remains fair.

Preliminary evaluation of hepatitis A virus cell receptor 1/kidney injury molecule 1 in healthy horses treated with phenylbutazone.

OBJECTIVES: To investigate if hepatitis A virus cell receptor 1/kidney injury molecule 1 (HAVCR1/KIM1) in urine is detectable concurrently with increases in serum creatinine concentrations in horses receiving a recommended dose of phenylbutazone (PBZ) for 7 days. DESIGN: Preliminary study. METHODS: Ten clinically healthy horses with normal physical examination and laboratory work were randomly assigned to PBZ or placebo groups (5 each). The PBZ group received PBZ at 4.4 mg/kg mixed with corn syrup orally every 12 hours. The placebo group received corn syrup orally every 12 hours. Both groups were treated for 7 days. Kidney ultrasonography was performed, and venous blood and urine samples were collected prior to commencement and at the end of treatment. Samples from 1 additional healthy horse, 3 horses with acute kidney failure, and 1 horse with chronic kidney failure were also evaluated. RESULTS: None of the 10 horses had detectable HAVCR1/KIM1 in urine at baseline. Serum creatinine concentrations in placebo group did not increase, and HAVCR1/KIM1 was undetectable in urine. At the end of treatment, 3 of 5 horses receiving PBZ developed increases in serum creatinine of >26.5 µmol/L (>0.3 mg/dL), and HAVCR1/KIM1 was detectable in urine, despite normal findings on kidney ultrasonography in all horses. CONCLUSIONS: HAVCR1/KIM1 is detectable in urine and is associated with increases in serum creatinine concentrations of >26.5 µmol/L in horses following treatment with PBZ for 7 consecutive days. Thus, HAVCR1/KIM1 might aid in the early detection of acute kidney injury in horses.

Novel eco-friendly spectrophotometric approaches for resolution of fixed dose formulation of phenylbutazone with minor component of dexamethasone: Greenness assessment by AGREE tool.

Spectrophotometric resolution of severely overlapped binary mixtures with minor component is challenging. Herein, coupling of mathematical manipulation steps with sample enrichment was conducted on the binary mixture spectrum of Phenylbutazone (PBZ) and Dexamethasone sodium phosphate (DEX) to resolve, for the first time each component separately. Simultaneous determination of both components in a mixture ratio of 1:0.002 was achieved in their zero or first order spectra by the recent factorized response method along with ratio subtraction and constant multiplication methods; all coupled with spectrum subtraction. In addition, a novel second derivative concentration value and second derivative constant value methods were developed for PBZ determination. The concentration of the minor component DEX was obtained, without preliminary separation steps by derivative ratio after sample enrichment by either spectrum addition or standard addition. Spectrum addition approach showed superior characteristics compared to standard addition technique. All proposed methods were placed through a comparative study. Linear correlation was found to be 1.5-18.0 µg/mL for PBZ, and 4.0-45.0 µg/mL for DEX. The proposed methods were validated in accordance with ICH guidelines. The greenness assessment of the proposed spectrophotometric methods was evaluated by AGREE software. Results obtained from the statistical data were evaluated by comparing to one another as well as the official USP methods. These methods offer a cost and time effective platform to analyze bulk materials and combined veterinary formulation.

A comparative survey of veterinarians, equine owners, and equine keepers regarding the knowledge and implementation of legal requirements in Germany for the use and documentation of veterinary medicines in equines intended for slaughter.

In Europe, equines destined for human consumption (hereafter called slaughter equines) are subject to the same restrictions of usage of veterinary drugs as other food-producing animals, with amendments regulated in the so-called 'positive list', Regulation (EC) No. 1950/2006. Due to the complex legal requirements for drug administration in slaughter equines, it might be that specific knowledge regarding the legislation of slaughter equines may be insufficient among veterinarians, equine owners, and equine keepers. To study this assumption, three target group-specific surveys were conducted in 2021. Answers from 153 equine treating veterinarians, 170 equine owners, and 70 equine keepers were included in the analysis. In total 68.4% (91/133) of the participating veterinarians, the regulations of the 'positive list', Regulation (EC) No. 1950/2006, were 'rather complicated' to 'complicated'. Among the participating veterinarians, 38.4% (58/151) did not or could not answer correctly how to proceed if a slaughter equine is scheduled to receive phenylbutazone, usage of which is prohibited in all livestock by Regulation (EU) No. 37/2010. Simultaneously, 56.2% (86/153) of the participating veterinarians named phenylbutazone as the, or one of the, most often used non-steroidal anti-inflammatory drugs. Altogether, 41.2% (70/170) of participating equine owners and 42.9% (30/70) of equine keepers did not know under which circumstances an equine can legally be slaughtered for human consumption. In total, 34.3% (24/70) of the equine keepers classified their knowledge of national regulations for animal keepers regarding the documentation of drug usage in equines as 'poor' to 'nonexistent'. This lack of knowledge in all three surveyed groups, combined with the complex legal regulations regarding the usage and documentation of drugs in slaughter equines, could result in missing and false documentation, treatment of slaughter equines with prohibited substances and therefore pose a risk factor for drug residues in equine meat.

A review of horses sent to slaughter for human consumption: impact of horsemeat consumption, residual banned drugs, and public health risks.

Nearly all of the American horses exported to Mexico and Canada are slaughtered for human consumption, and their meat is either exported around the world or consumed locally. Previous work showed that 18 Thoroughbred racehorses purchased by rescues that would have otherwise been sold for export for the sole purpose of slaughter to produce meat for human consumption were administered phenylbutazone. We report the number of American horses exported to Canada and Mexico from 2016 to 2021, the presence of contaminated horsemeat from Canadian slaughterhouses, and the human use and idiosyncratic effects of veterinary phenylbutazone and side effects of clenbuterol, 2 of the drugs that were found in contaminated Canadian horsemeat. The number of live American horses exported to Canada declined precipitously from 2016 to 2017, and a second decline occurred in 2020. All food-producing animals are under strict regulatory control to prevent animals administered banned drugs to enter the food chain. A major principle of this program is zero tolerance for banned drugs and testing for compliance. No regulatory process is in place to remove horses administered banned drugs such as phenylbutazone. The efficacy lasts for more than 24 hours as a result of the irreversible binding to cyclooxygenase, slow elimination, and long elimination half-life of its metabolite oxyphenbutazone. High or frequent doses of phenylbutazone result in disproportionately increased plasma concentrations, which result in the residual presence in tissues. It is this fact that underlies the ban of this drug in food-producing animals. No human clinical surveillance program is in place to monitor individuals on the possible short- and long-term consequences of banned drugs in contaminated horsemeat. If the United States is unable to put in place a regulatory program to remove horses administered banned drugs as exists for all food-producing animals, the exportation of American horses across both borders for the sole purpose of slaughter for human consumption must end.

Pharmacokinetics and clinical efficacy of acetaminophen (paracetamol) in adult horses with mechanically induced lameness.

BACKGROUND: Acetaminophen has been used clinically in horses alone or combined with traditional non-steroidal anti-inflammatory drugs for treatment of musculoskeletal pain in horses. OBJECTIVES: To determine the pharmacokinetics and efficacy of acetaminophen at two doses in horses with mechanically induced lameness compared with phenylbutazone or placebo control. STUDY DESIGN: In vivo experiment. METHODS: Nine healthy mares with mechanical lameness induced via a reversible sole pressure horseshoe model were treated with acetaminophen (20 mg/kg PO; A20), acetaminophen (30 mg/kg PO; A30), phenylbutazone (2.2 mg/kg, PO; PB) and oral placebo (C) in a randomised four-way Latin square model. Plasma concentrations for A20 and A30 were analysed via LC-MS/MS and noncompartmental pharmacokinetic analysis. Heart rate and heart rate variability were measured using a portable telemetry. Lameness was scored by three blinded boarded equine surgeons using the AAEP and 10-point scales. RESULTS: Mean maximum plasma concentration (Cmax ) for A20 was 20.01 µg/ml within 0.66 h (Tmax ) after administration; The mean Cmax for A30 was 30.02 µg/ml with a Tmax of 0.43 h. Post-treatment heart rate for A30 was significantly lower than A20 at 1 and 7 h; lower than PB at 2, 3, 4.5 and 7 h; lower than C at 2, 3.5, 4.5, 6, 7 and 8 h. 10-point Lameness scores were significantly improved for A30 than C at 2 and 4 h post-treatment; PB was significantly improved than C at 8 h post treatment. There were no significant differences in lameness between A20, A30 and PB. MAIN LIMITATIONS: Small sample size, lack of objective lameness measurement. CONCLUSIONS: Acetaminophen at 30 mg/kg produced a more rapid improvement in lameness scores and heart rate compared with other treatments in this model. Further evaluation of the pharmacokinetics and safety of repeated oral dosing of acetaminophen at 30 mg/kg is needed to determine clinical utility.

CONTEXTO: Acetaminofeno tem sido usado rotineiramente em cavalos com dor musculoesquelética, tanto como terapia solo quanto em associação com outros anti-inflamatórios não esteroides tradicionais. OBJETIVOS: Determinar a farmacocinética e eficácia de duas doses de acetaminofeno em cavalos com claudicação mecanicamente induzida, e comparar com fenilbutazona e placebo. DELINEAMENTO DO ESTUDO: Estudo randomizado, cego e controlado utilizando quadrado latino. METODOLOGIA: Nove éguas adultas com claudicação induzida mecanicamente pelo método de aplicação de pressão na sola através de ferradura foram tratadas com acetaminofeno (20 mg/kg VO; A20), acetaminofeno (30 mg/kg VO; A30), fenilbutazona (2.2 mg/kg, VO; PB) e placebo oral (C) em um estudo quadrado latino de forma randômica. Concentração plasmática dos grupos A20 e A30 foram analisadas pelo método LC-MS/MS e análise farmacocinética não compartimentar. Frequência cardíaca e variação da frequência cardíaca foram mensuradas usando telemetria portátil. O grau de claudicação foi avaliado usando a escala de 10 pontos da AAEP por três cirurgiões especialistas (board-certified) que estavam cegos ao tratamento. RESULTADOS: A média máxima da concentração plasmática (Cmax ) do grupo A20 foi 20.01 µg/ml dentro de 0.66 h (Tmax ) da administração. A média Cmax do grupo A30 foi 30.02 µg/ml dentro da Tmax de 0.43 h. A frequência cardíaca do grupo A30 foi significativamente mais baixa do que a do grupo A20 nos momentos 1 e 7 h; mais baixa do que o grupo PB nos momentos 2, 3, 4.5 e 7 h; e mais baixa do que as do grupo C nos momentos 2, 3.5, 4.5, 6, 7 e 8 h. O grau de claudicação diminuiu significativamente no grupo A30 quando comparado com o grupo C nos momentos 2 e 4 h pós tratamento, e no grupo PB quando comparado com o grupo C no momento 8 h pós tratamento. Não houve diferença significativa em grau de claudicação quando os grupos A20, A30 e PB foram comparados. PRINCIPAIS LIMITAÇÕES: Número pequeno de animais, ausência de mensuração de claudicação objetiva. CONCLUSÕES: A dose de 30 mg/kg de acetaminofeno proporcionou uma superior melhora na escala de claudicação e frequência cardíaca quando comparada com os outros tratamentos avaliados neste estudo. Mais informações sobre a farmacocinética e efeitos da repetida dosagem de 30 mg/kg de acetaminofeno precisam ser avaliadas para determinar a sua aplicabilidade clínica.

Prophylactic effects of Glycyrrhiza glabra root extract on phenylbutazone-induced Equine Glandular Gastric Disease (EGGD).

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), like phenylbutazone (PBZ), are prescribed to treat pain and inflammation in the equine. However, experimentally their use has been associated with the development of Equine Glandular Gastric Disease (EGGD). To evaluate the prophylactic effects of Glycyrrhiza glabra root extracts (GGRE) on EGGD induction following oral administration of PBZ, 12 donkeys were used. Animals were divided into three equal groups (A, B and C) in a randomized block design. A: placebo, B: PBZ (4.4 mg/kg PO q 12 h) and C: PBZ plus GGRE (17.6 mg/kg PO q 24h). Gastroscopy and blood sampling for CBC, biochemical tests and antioxidant status were performed days 0 and 7. All but 1 animal had no EGGD lesions on day 0, but on day 7, severe EGGD lesions were seen in group B animals (grade 1/4 (× 1), grade 2/4 (× 2), and grade 3/4 (× 1)). Only mild changes were seen in group C animals, the most severe lesions being grade 1/4 lesions in 2 animals. No change was observed in Group A. Hematology and serum antioxidant status did not change significantly in any group. In group B glucose decreased and ALT and ALP increased, while in group C only ALP increased. In conclusion, GGRE reduced the severity of EGGD caused by PBZ. Further, GGRE prevented PBZ induced hypoglycemia and might reduce the possible hepatic injury of PBZ.

An Evolving Role of Aqueous Piperazine to Improve the Solubility of Non-Steroidal Anti-Inflammatory Drugs.

Piperazine (PIP) is a pharmaceutically acceptable molecule and a good co-conformer in crystallographic engineering. Most of the non-steroidal anti-inflammatory drugs (NSAIDs) have poor aqueous solubility, which hinders their clinical application. The reports show that the solubility of many insoluble drugs can be significantly improved through salt formation with the PIP. In this work, we obtained a series of NSAIDs-PIP salts, such as ibuprofen-piperazine (IBU-0.5PIP) salt, indomethacin-piperazine (IND-0.5PIP) salt, sulindac-piperazine (SUL-0.5PIP) salt, phenylbutazone-piperazine (PBZ-0.5PIP) salt, ketoprofen-piperazine (KPF-0.5PIP) salt and flurbiprofen-piperazine (FLB-0.5PIP) salt. The spatial structure, arrangement, interaction and associations were expatiated by single crystal X-ray diffraction. Powder X-ray diffraction, Fourier transform infrared, differential scanning calorimetry, and thermogravimetric analysis were used to characterize the novel salts. The six new salts had more than 10 folds of solubility and a faster dissolution rate improved corresponding to the bulk drugs in pure water, and the significant improvement of solubility is closely related to the structure of salts.

Effects of cyclooxygenase and soluble epoxide hydrolase inhibitors on apoptosis of cultured primary equine chondrocytes.

BACKGROUND: Apoptosis is an important mechanism underlying chondrocyte loss in osteoarthritis that could be affected by modulation of lipid signaling via inhibition of cyclooxygenases (COX) and soluble epoxide hydrolase (sEH). OBJECTIVE: To determine the impact of inhibiting COX and sEH alone or in combination on apoptosis of equine chondrocytes. METHODS: Cultured primary equine chondrocytes were subjected to serum deprivation or incubation with 1 µg/ml tunicamycin for 24 h to induce apoptosis via caspase activation and endoplasmic reticulum (ER) stress, respectively. Cells were treated with the non-selective COX inhibitor phenylbutazone, the COX-2 selective inhibitor firocoxib and the sEH inhibitor t-TUCB alone or in combination. The inhibitors were used at half-maximal (IC50), 80% of maximal (IC80) and 10-fold the 80% inhibitory concentration (10xIC80) for the equine enzymes. Apoptosis was quantified via ELISA technique. Data were analyzed with unpaired two-tailed t-test or one-way ANOVA followed by Bonferroni's post-hoc while correcting for multiple comparisons via statistical hypothesis testing. P < 0.05 was considered significant. RESULTS: In the caspase model, 10xIC80t-TUCB significantly decreased whereas 10xIC80 phenylbutazone significantly enhanced apoptosis. Apoptosis enhancement by phenylbutazone was significantly attenuated by concurrent 10xIC80t-TUCB. The remaining treatments and concentrations had no effect on apoptosis development. In the ER stress model, IC50 and IC80 phenylbutazone and firocoxib significantly enhanced apoptosis, which was fully prevented by concurrent 10xIC80t-TUCB. MAIN LIMITATIONS: In vitro findings that will need to be verified in vivo. CONCLUSIONS: Chondrocyte apoptosis caused by ER stress can be enhanced by COX inhibition but prevented by concurrent inhibition of sEH.