Misoprostol Inhibits Lipopolysaccharide-Induced Pro-inflammatory Cytokine Production by Equine Leukocytes

Pro-inflammatory cytokines including tumor necrosis factor α (TNFα), IL-1β, IL-6, and IL-8 are potent immune mediators that exacerbate multiple equine diseases such as sepsis and laminitis. Unfortunately, safe and effective cytokine-targeting therapies are lacking in horses; therefore, novel mechanisms of inhibiting cytokine production are critically needed. One potential mechanism for inhibiting cytokine synthesis is elevation of intracellular cyclic AMP (cAMP). In human leukocytes, intracellular cAMP production is induced by activation of E-prostanoid (EP) receptors 2 and 4. These receptors can be targeted by the EP2/4 agonist and prostaglandin E1 analog, misoprostol. Misoprostol is currently used as a gastroprotectant in horses but has not been evaluated as a cytokine-targeting therapeutic. Thus, we hypothesized that misoprostol treatment would inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-stimulated equine leukocytes in an in vitro inflammation model. To test this hypothesis, equine leukocyte-rich plasma (LRP) was collected from 12 healthy adult horses and used to model LPS-mediated inflammatory signaling. LRP was treated with varying concentrations of misoprostol either before (pretreated) or following (posttreated) LPS stimulation. LRP supernatants were assayed for 23 cytokines using an equine-specific multiplex bead immunoassay. Leukocytes were isolated from LRP, and leukocyte mRNA levels of four important cytokines were evaluated via RT-PCR. Statistical differences between treatments were determined using one-way RM ANOVA (Holm–Sidak post hoc testing) or Friedman’s RM ANOVA on Ranks (SNK post hoc testing), where appropriate (p < 0.05, n = 3–6 horses). These studies revealed that misoprostol pre- and posttreatment inhibited LPS-induced TNFα and IL-6 protein production in equine leukocytes but had no effect on IL-8 protein. Interestingly, misoprostol pretreatment enhanced IL-1β protein synthesis following 6 h of LPS stimulation, while misoprostol posttreatment inhibited IL-1β protein production after 24 h of LPS stimulation. At the mRNA level, misoprostol pre- and posttreatment inhibited LPS-induced TNFα, IL-1β, and IL-6 mRNA production but did not affect IL-8 mRNA. These results indicate that misoprostol exerts anti-inflammatory effects on equine leukocytes when applied before or after a pro-inflammatory stimulus. However, the effects we observed were cytokine-specific and sometimes differed at the mRNA and protein levels. Further studies are warranted to establish the inhibitory effects of misoprostol on equine cytokine production in vivo

A comparison of serum and plasma cytokine values using a multiplexed assay in cats

Degenerative joint disease (DJD) is highly prevalent in cats, and pain contributes to morbidity. In humans, alterations of cytokine concentrations have been associated with joint deterioration and pain. Similar changes have not been investigated in cats. Cytokine concentrations can be measured using multiplex technology with small samples of serum or plasma, however, serum and plasma are not interchangeable for most bioassays. Correlations for cytokine concentrations between serum and plasma have not been evaluated in cats

Evaluation of serum cytokines in cats with and without degenerative joint disease and associated pain

Degenerative joint disease is common in cats, with signs of pain frequently found on orthopedic examination and radiographs often showing evidence of disease. However, understanding of the pathophysiology of degenerative joint disease and associated pain remains limited. Several cytokines have been identified as having a role in pain in humans, but this has not been investigated in cats. The present study was performed to use a multiplex platform to evaluate the concentration of 19 cytokines and chemokines in serum samples obtained from cats with and without degenerative joint disease and associated pain. Samples from a total of 186 cats were analyzed, with cats representing a range of severity on radiographic and orthopedic evaluations and categorized by degenerative joint disease scores and pain scores. Results showed that cats with higher radiographic degenerative joint disease scores have higher serum concentrations of IL-4 and IL-8, while cats with higher orthopedic exam pain scores have higher concentrations of IL-8, IL-2, and TNF-α increased concentration of IL-8 in degenerative joint disease and pain may be confounded by the association with age. Discriminant analysis was unable to identify one or more cytokines that distinguish between groups of cats classified based on degenerative joint disease score category or pain score category. Finally, cluster analysis driven by analyte concentrations show separation of groups of cats, but features defining the groups remain unknown. Further studies are warranted to investigate any changes in cytokine concentrations in response to analgesic therapies, and further evaluate the elevations in cytokine concentrations found here, particularly focused on studies of local cytokines present in synovial fluid

Efficacy of a single session in‐series hemoperfusion and hemodialysis in the management of carprofen overdose in two dogs

Objective: To describe the efficacy of in-series hemoperfusion and hemodialysis in 2 dogs with carprofen overdose. Case Summary: This report describes the treatment of 2 dogs following accidental carprofen overdoses who underwent a single in-series hemoperfusion and hemodialysis session. Serial serum carprofen concentrations were measured before, during, and after the session. The first patient’s session lasted 5 hours, with the largest decrease in serum carprofen concentrations occurring during the first hour of treatment. The carprofen clearance during the following 4 hours of treatment decreased substantially compared to the first hour and was not different from the patient’s intrinsic clearance of carprofen after the session was completed. Based on the findings from the first case, the second patient was treated with a 1 hour single hemoperfusion and hemodialysis session. Our results support the hypothesis that carprofen is not effectively removed by conventional hemodialysis and the efficacy of hemoperfusion is short lived due to rapid saturation of the charcoal filter. Once filter saturation occurs, the extracorporeal session is no longer efficacious. Using in-series hemoperfusion and hemodialysis is of benefit to correct the side effects seen with hemoperfusion alone, and hourly charcoal filter replacementmay extend the efficacy of treatment in removing carprofen. New or Unique Information Provided: This is the first published report of in-series hemoperfusion and hemodialysis being used to treat carprofen overdose in a dog. In these 2 cases, the intrinsic clearances of the patients were shown to be equivalent to that of standard hemodialysis alone, indicating that hemodialysis does not produce any advantage in carprofen clearance. In this limited report, we suggest that the efficacy of hemoperfusion in removing carprofen is short-lived, and extending the treatment beyond the first hour does not produce any therapeutic benefit. In order to extend the efficacy of hemoperfusion, hourly replacement of the charcoal filter should be considered

Pharmacokinetics and efficacy of orally administered acetaminophen (paracetamol) in adult horses with experimentally induced endotoxemia

Abstract Background Acetaminophen has been evaluated in horses for treatment of musculoskeletal pain but not as an antipyretic. Objectives To determine the pharmacokinetics and efficacy of acetaminophen compared to placebo and flunixin meglumine in adult horses with experimentally induced endotoxemia. Animals Eight university owned research horses with experimentally induced endotoxemia. Methods Randomized placebo controlled crossover study. Horses were treated with acetaminophen (30 mg/kg PO; APAP), flunixin meglumine (1.1 mg/kg, PO; FLU), and placebo (PO; PLAC) 2 hours after administration of LPS. Plasma APAP was analyzed via LC‐MS/MS. Serial CBC, lactate, serum amyloid A, heart rate and rectal temperature were evaluated. Serum IL‐1β, IL‐6, IL‐8, IL‐10, and TNF‐α were evaluated by an equine‐specific multiplex assay. Results Mean maximum plasma APAP concentration was 13.97 ± 2.74 μg/mL within 0.6 ± 0.3 hour after administration. At 4 and 6 hours after treatment, both APAP (P = <.001, P = .03, respectively) and FLU (P = .0045 and P < .001, respectively) had a significantly greater decrease in rectal temperature compared to placebo. FLU caused greater heart rate reduction than APAP at 4 and 6 hours (P = .004 and P = .04), and PLAC at 4 hours (P = .05) after treatment. Conclusions and Clinical Importance The pharmacokinetics of acetaminophen in endotoxemic horses differ from those reported by previous studies in healthy horses. Acetaminophen is an option for antipyresis in clinical cases, particularly when administration of traditional NSAIDs is contraindicated

Pharmacokinetics and toxicity of subcutaneous administration of carboplatin in poloxamer 407 in a rodent model pilot study

<div><p>The objectives of this study were to assess the pharmacokinetics and safety of subcutaneously delivered carboplatin in poloxamer 407 in rats. Carboplatin (5mg/rat) in 0.5ml poloxamer 407 (1.0 ml total volume) was administered subcutaneously in a right subcutaneous perineal incision in all 12 treatment rats. Three control rats received 1.0 ml of poloxamer 407. Total platinum was measured in plasma q24hrs from 0 to 168hrs. Protein-unbound platinum was measured in plasma at 168hrs. After sacrifice on day 7, total platinum was determined in wound bed muscle. Platinum concentrations in all samples were measured by ICP-MS. Wounds were visually assessed daily for 7 days. Perineal tissues (full wound bed including muscle, subcutis, skin) were assessed histologically and scored. Total platinum was detectable in plasma from 24 to 168 hrs. Total plasma platinum AUC and C_max were 9,165.3 ng/mL•h and 129.4 ng/mL. Day 7 total platinum concentration in muscle was approximately 10-fold higher than total plasma platinum concentration. No unbound platinum was detected in plasma samples at 168 hours. No wound healing complications were detected at any time point, nor was tissue necrosis observed histologically. The results of this study suggest that subcutaneous carboplatin in poloxamer 407 can be used in vivo providing direct tissue exposure to carboplatin without significant local effects or systemic absorption and without wound healing complications.</p></div

Non-compartmental pharmacokinetic parameter of Pt.

<p>Non-compartmental pharmacokinetic parameter of Pt.</p

Histology of the subcutaneous perineal implantation site.

<p>A Shows a photomicrograph of the subcutaneous perineal surgical implantation site of rat from the CP cohort. Minimal necrosis of adipose (score = 1) (arrow) is present adjacent to the implantation site (asterisk). B Shows a photomicrograph of the subcutaneous perineal surgical implantation site of a rat from the CP-μD cohort. Minimal fibrosis (score = 1) (arrows) is present adjacent to the implantation site (asterisk) after receiving carboplatin and poloxamer 407 gel and a μD catheter (C) at the site.</p

Histological scores.

<p>Histological scores.</p