Pharmacokinetics and ex vivo anti-inflammatory effects of oral misoprostol in horses.

BACKGROUND: Misoprostol is an E prostanoid (EP) 2, 3 and 4 receptor agonist that is anecdotally used to treat and prevent NSAID-induced GI injury in horses. Misoprostol elicits anti-inflammatory effects in vivo in men and rodents, and inhibits TNFα production in equine leucocytes in vitro. OBJECTIVE: Define the pharmacokinetic parameters of oral misoprostol in horses, and determine the inhibitory effect of oral misoprostol administration on equine leucocyte TNFα production in an ex vivo inflammation model. STUDY DESIGN: Pharmacokinetic study, ex vivo experimental study. METHODS: Six healthy adult horses of mixed breeds were used. In phase one, horses were given 5 µg/kg misoprostol orally, and blood was collected at predetermined times for determination of misoprostol free acid (MFA) by UHPLC-MS/MS. Pharmacokinetic parameters were calculated. In phase two, horses were dosed as in phase one, and blood was collected at T0, 0.5, 1 and 4 h following misoprostol administration for leucocyte isolation. Leucocytes were stimulated with 100 ng/mL LPS, and TNFα mRNA concentrations were determined via quantitative real-time PCR. RESULTS: About 5 µg/kg oral misoprostol produced a rapid time to maximum concentration (Tmax ) of 23.4 ± 2.4 min, with a maximum concentration (Cmax ) of 0.29 ± 0.07 ng/mL and area under the curve (AUC0-∞ ) of 0.4 ± 0.12 h ng/mL. LPS stimulation of equine leucocytes ex vivo significantly increased TNFα mRNA concentrations, and there was no significant effect of misoprostol even at the Tmax . MAIN LIMITATIONS: Only a single dose was used, and sample size was small. CONCLUSIONS: Misoprostol is rapidly absorbed following oral administration in horses, and a single 5 µg/kg dose had no significant inhibitory effect on ex vivo LPS-stimulated TNFα mRNA production in leucocytes. Further studies analysing different dosing strategies, including repeat administration or combination with other anti-inflammatory drugs, are warranted.

Oral reserpine administration in horses results in low plasma concentrations that alter platelet biology.

BACKGROUND: Reserpine is a popular drug in the equine industry for long-term tranquilisation. Clinical observations revealed that blood from horses receiving oral reserpine was hypercoagulable. No studies have documented the pharmacokinetics of orally administered reserpine nor the effects of reserpine on platelets in horses. OBJECTIVES: To evaluate the pharmacokinetics of oral reserpine in horses and the effects of clinically relevant concentrations of reserpine on platelet functionality in vitro. STUDY DESIGN: Experimental controlled study. METHODS: The pharmacokinetics of oral reserpine (2.5 mg/horse, once) were determined in six healthy adult horses. Plasma samples were collected and concentrations of reserpine were determined by UPLC-MS/MS. Using this data, the in vitro effects of reserpine on platelets were examined. Aggregation, adhesion and releasate assays for serotonin and thromboxane B2 were performed on platelets exposed to varying concentrations of reserpine (0.01-10 ng/mL), aspirin (negative control) and saline (unexposed control). RESULTS: Oral reserpine administration demonstrated low plasma concentrations with a Cmax of 0.2 ± 0.06 ng/mL and a prolonged half-life of 23.6 ± 6.24 h. Simulations over a dose range of 2-8 µg/kg predicted Cmax at steady state between 0.06-0.9 ng/mL. Platelets exposed to these reserpine concentrations in vitro displayed increased aggregation and adhesion compared to unexposed or aspirin-exposed platelets as well as compared to higher concentrations of reserpine. These functional changes correlated with lower concentrations of serotonin and higher concentrations of thromboxane B2 in the platelet suspension supernatant. MAIN LIMITATIONS: This study used a small number of horses and only in vitro platelet experiments. CONCLUSIONS: Oral reserpine demonstrates low plasma concentrations and a prolonged half-life in horses. At these concentrations, reserpine causes significant changes in platelet function, most likely due to serotonin release and re-uptake which primes platelets for activation and thromboxane B2 release. These findings suggest that clinicians should harvest blood for biological processing prior to the onset of reserpine administration.

Effects of quinapril on angiotensin converting enzyme and plasma renin activity as well as pharmacokinetic parameters of quinapril and its active metabolite, quinaprilat, after intravenous and oral administration to mature horses.

REASONS FOR PERFORMING STUDY: Angiotensin converting enzyme (ACE) inhibitors improve survival and quality of life in human patients and small animals with cardiovascular and renal disease. There is limited information regarding their effects in horses. OBJECTIVES: The purpose of this study was to determine the pharmacokinetics of quinapril and its effects on ACE and renin in horses. STUDY DESIGN: Experimental study using healthy mature horses. METHODS: Six healthy horses were administered quinapril at 120 mg i.v., 120 mg per os and 240 mg per os in a 3-way crossover design. Blood was collected for measurement of quinapril and quinaprilat concentrations using ultra-high pressure liquid chromatography with mass spectrometry. Angiotensin converting enzyme activity and renin activity were measured using a radioenzymatic assay. Noncompartmental pharmacokinetic modelling and statistical analyses were performed. RESULTS: No adverse effects were observed during the study period. Intravenous and oral administration significantly inhibited ACE activity. Renin concentrations increased in all groups, but this increase was not statistically significant. Following i.v. administration of quinapril, mean terminal half-life was 0.694 h and 1.734 h for quinapril and quinaprilat, respectively. The mean volume of distribution and clearance for quinapril were 0.242 l/kg bwt and 11.93 ml/kg bwt/min, respectively. Maximum concentration for quinaprilat was 145 ng/ml at 0.167 h. Bioavailability of quinapril following oral administration was <5%. Quinaprilat was detected in all horses following oral administration of quinapril; however, it was below the limit of quantification of the assay (2.5 ng/ml) for most horses in the 120 mg dosing group. CONCLUSIONS: These results suggest that, despite low plasma concentrations, quinapril has sufficient oral absorption to produce inhibition of ACE in healthy horses. Controlled studies in clinically affected horses are indicated. Quinapril provides a potential treatment alternative for horses with cardiovascular and renal disease.

Complement-mediated hemodynamic depression in the early postburn period.

This study examines the influence of complement on systemic hemodynamics following severe thermal injury in rats. Animals were injected intraperitoneally at t = -36 and t = -24 hours with either cobra venom factor (20 units/kg/dose; n = 56) to delete circulating complement or with saline alone (n = 64). Rats within each subset were then subjected to either a 50% TBSA full-thickness scald burn or sham burn. Cardiac output (CO), mean arterial pressure (MAP), heart rate, systemic vascular resistance (SVR), stroke volume, and cardiac power as well as hematocrit and the change in per cent complement activity were determined at various time periods between 15 minutes and 25 hours after the burn. In normocomplementemic animals the burn produced a marked early (t = 3-6 hours) depression in CO and MAP with a rise in SVR. Over time the hemodynamics returned to normal (t = 12 hours) and eventually approached a hyperdynamic response (t = 24 hours). Serum hemolytic complement activity fell immediately and progressively after the burn, indicating significant complement activation. Complement depletion attenuated the early decline in CO and sharply lowered the rise in SVR in the early postburn period. In addition, complement depletion improved heart rate and stroke volume and appeared to preserve/enhance late (t = 24 hours) cardiac function. This study suggests that complement activation contributes to the depression in cardiac output in the early postburn period.

Recombinant human tumor necrosis factor produces hemodynamic changes characteristic of sepsis and endotoxemia.

Tumor necrosis factor (TNF) is a macrophage-derived peptide mediator released during endotoxemia and sepsis. We examined the systemic and visceral hemodynamic response to low doses of human recombinant TNF in rats. Each animal received a 30-minute intravenous infusion of either saline solution (n = 8) or TNF (n = 8) in a dose of 0.25 mg/kg or 1.0 mg/kg. Thermodilution cardiac output, blood pressure, pulse, vascular resistance, effective hepatic blood flow (galactose clearance), and effective renal plasma flow (p-aminohippurate clearance) were determined at time = 2 hours. The 0.25-mg/kg dose had no apparent effect on systemic hemodynamics. The 1.0-mg/kg dose produced a hyperdynamic systemic circulatory response with an elevated cardiac output, tachycardia, and a diminished systemic vascular resistance. Effective hepatic blood flow was exquisitely sensitive to even the lowest dose of TNF, with a 29% reduction despite the normal cardiac output. Renal flow was unaffected by either dose. Tumor necrosis factor-induced systemic and visceral hemodynamic changes are remarkably similar to those seen in gram-negative sepsis, suggesting that TNF may occupy a proximal position in the pathogenesis of overwhelming infection.

Visceral perfusion abnormalities following complement activation. Clues to the mediators of organ ischemia in trauma and sepsis. First place winner: Conrad Jobst Award.

Complement, activated during infection and injury, has been implicated as a mediator of microvascular injury and obstruction. This study examines how two potent activators of complement, zymosan, and cobra venom factor (CVF), affect systemic and visceral perfusion. Rats were injected with either saline (1 ml/kg), zymosan (5 mg/kg) or CVF (5 units/kg) at t = 0 and 30 minutes. Thermodilution cardiac output, mean arterial pressure, heart rate, systemic vascular resistance, and hematocrit were determined at t = 2 hours. Effective hepatic and renal blood flows, by clearance of galactose and p-aminohippurate respectively, were determined over the next hour. The per cent change in total hemolytic complement from t = 0 to t = 3 hours was determined by immune hemolysis of sheep erythrocytes. There was no difference in systemic hemodynamic parameters between the three groups. Hepatic blood flow was depressed in both the zymosan (3.83 +/- 0.23 ml/min/100 g) and CVF (3.72 +/- 0.20 ml/min/100 g) groups compared with controls (4.62 +/- 0.19 ml/min/100 g, P less than 0.05). Renal blood flow in the zymosan-treated group (6.40 +/- 0.24 ml/min/100 g) increased over control (4.80 +/- 0.40 ml/min/100 g, P less than 0.05) but was unchanged in the CVF group (5.06 +/- 0.23 ml/min/100 g). The amount of complement activated correlated with the change in hepatic (r = -0.419, P less than 0.05) but not renal (r = -0.008, P = 0.917) flow. Complement activation may occupy a proximal position in the pathogenesis of hepatic ischemia associated with trauma and sepsis.

Contribution of toxic oxygen intermediates to complement-induced reductions in effective hepatic blood flow.

This study examines the effects of complement activation and of complement-induced oxygen radical production on the principal determinant of hepatic function, i.e., effective hepatic blood flow (EHBF). Female Sprague-Dawley rats received cobra venom factor, 40 units/kg, in two divided doses at 30-minute intervals. At t = 2 hours, thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, and EHBF by galactose clearance were determined. Complement activation produced a significant depression in EHBF independent of changes in systemic perfusion. To determine whether oxygen radicals participated in the insult, additional animals were pretreated with superoxide dismutase, 6 mg/kg, plus catalase, 15 mg/kg, immediately before complement activation. Concomitant treatment with the oxygen radical scavengers attenuated the degree of complement-induced hepatic ischemia, again independent of effects on systemic perfusion. This study suggests that the reduction in hepatic blood flow that accompanies animal models of trauma and sepsis may result, in part, from the sequelae of complement activation with oxygen radicals as secondary mediators.

Allopurinol and lodoxamide in complement-induced hepatic ischemia.

Intravascular complement activation with either zymosan or cobra venom factor (CVF) impairs hepatic blood flow. Oxygen radical scavengers given at the time of complement activation attenuate the resulting hepatic ischemia. It is not clear whether complement-stimulated phagocytes or transiently ischemic then reperfused endothelial and parenchymal cells generated the toxic oxygen radicals. In this study, a group of rats were given allopurinol (50 mg/kg/day postoperatively X 3 days plus 100 mg/kg iv at t = 0), a specific inhibitor of xanthine oxidase, prior to complement activation with CVF (20 units/kg iv at t = 30 and 60 min) to determine whether xanthine oxidase-derived oxygen radicals contributed significantly to the hepatic perfusion abnormalities. Additional rats received lodoxamide tromethamine (10 mg/kg iv bolus at t = 0 followed by 20 mg/kg/hr iv infusion), a novel and potent inhibitor of mast cell release and inhibitor of xanthine oxidase, prior to the same CVF challenge to determine whether mast cell mediators were involved in the flow disturbance. Thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, and effective hepatic blood flow (EHBF) by galactose clearance were determined at t = 2 hr. The percentage change in total hemolytic complement activity (% delta CH50) was determined between serum obtained prior to sacrifice and at t = 0. Systemic hemodynamics and HCT were for the most part unaffected regardless of pretreatment group or challenge with CVF or saline. CVF challenge produced a 25% reduction (P less than 0.05) in EHBF in vehicle-pretreated rats compared to saline challenge. Neither allopurinol nor lodoxamide tromethamine significantly improved EHBF when given prior to CVF challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Femur fracture with associated soft-tissue injury produces hepatic ischemia. Possible cause of hepatic dysfunction.

Clinical studies demonstrate that early débridement and operative fixation of femur fractures in multiply injured patients lowers both the incidence and severity of hepatic failure. Perhaps the single most important determinant of hepatic function is nutrient hepatic perfusion. This study compares systemic and hepatic blood flow in rats that have sustained femur fractures with or without associated soft-tissue injury. Femur fracture without soft-tissue trauma resulted in a hyperdynamic state with normal blood flow distribution at 24 hours after injury and normal hemodynamics at 48 hours. When femur fracture was associated with soft-tissue trauma, the elevated cardiac output at 24 hours was not matched by a proportionately elevated hepatic blood flow. In this latter group, the cardiac output was normal at 48 hours, but the hepatic perfusion defect remained. Retained fracture fragments, hematoma, and injured and necrotic soft tissue may serve as a stimulus leading to a pathologic reduction in hepatic perfusion.

Systemic complement activation produces hemodynamic changes characteristic of sepsis.

Zymosan was administered intravenously in graded doses to Sprague-Dawley rats to investigate the hemodynamic effects of systemic complement activation. At two hours, thermodilution cardiac output, mean arterial pressure, heart rate, systemic vascular resistance, hematocrit, effective hepatic and renal blood flows, and percent change in total hemolytic complement activity were measured on all animals. Progressively increasing doses of zymosan produced characteristic hemodynamic changes of progressively deteriorating stages of hyperdynamic sepsis. In addition, complement activation resulted in a redistribution of systemic blood flow with hepatic hypoperfusion similar to that seen in sepsis. Renal blood flow was unaffected early after complement activation. Additional rats were studied from the control and a representative zymosan-treated group at 24 and 48 hours to determine if the hemodynamic changes observed at two hours persisted or resolved. All systemic hemodynamic measures returned to normal at both 24 and 48 hours. Liver blood flow, however, remained depressed and actually deteriorated over time. Renal perfusion, which was stable at both two and 24 hours, fell below control values in the zymosan-treated group at 48 hours. We conclude that complement may be a mediator of both systemic and visceral flow abnormalities that precede, and perhaps precipitate, organ failure in trauma and sepsis.

Complement activation in peritonitis. Association with hepatic and renal perfusion abnormalities. First place winner: Conrad Jobst award.

The authors have shown that systemic activation of the complement system with either zymosan or cobra venom factor produces some of the hemodynamic changes characteristic of sepsis, specifically, a reduction in hepatic perfusion despite a normal or hyperdynamic systemic circulation. This study was undertaken to determine whether complement activation accompanied reductions in effective hepatic and renal blood flow (EHBF and ERBF, respectively) in a septic murine model previously demonstrated to be associated with flow redistribution. Rats underwent either cecal ligation and puncture (CLP) or sham laparotomy after a baseline blood sample was collected for complement assay. Eighteen hours later, thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, EHBF by galactose clearance, and ERBF by p-aminohippurate (PAH) clearance were determined. A second blood sample was collected for measurement of total hemolytic complement (CH50) by immune hemolysis of sheep erythrocytes and was compared to the t = 0 sample for calculation of per cent change in CH50. The cardiac output and hematocrit were normal in the CLP group relative to sham. The septic animals were tachycardic and slightly hypotensive, suggesting a diminished systemic vascular resistance. EHBF and ERBF fell dramatically in the septic group despite the normal cardiac output. Residual hemolytic complement activity was reduced to less than 40% of preseptic levels in the CLP group while sham values were no different than baseline, indicating massive complement activation in the septic animals. This study demonstrates an association between complement activation and hepatic and renal perfusion abnormalities in murine peritonitis. Work is underway to establish the temporal relationship between complement activation and visceral flow changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow redistribution in a hyperdynamic small animal burn: comparison to patterns in sepsis.

Visceral hypoperfusion with local accumulation of lactate in the ischemic tissues has been reported in a septic rat model despite a hyperdynamic systemic circulation. This visceral ischemia is felt to contribute to the multiple system organ failure (MSOF) syndrome associated with sepsis. The purpose of this study was to determine whether a similar redistribution of blood flow existed in rats after a severe thermal injury as it too is associated with MSOF. Twenty-four hours after animals were subjected to either a resuscitated 50% scald burn (BURN) or sham treatment (SHAM), thermodilution cardiac output (CO), effective hepatic blood flow (EHBF) by galactose clearance at low concentrations, effective renal plasma flow (ERPF) by para-aminohippurate clearance, and blood, liver, and skeletal muscle pyruvate (P), and lactate (L) concentrations were determined. CO increased 52% in BURN (46.5 +/- 2.8 ml/min/100 g, n = 21) versus SHAM (30.7 +/- 1.0 ml/min/100 g, n = 22; P less than 0.001) while EHBF increased only 18% (BURN: 6.81 +/- 0.36 ml/min/100 g, n = 8 vs SHAM: 5.77 +/- 0.29 ml/min/100 g, n = 8; P less than 0.025) and ERPF showed an insignificant 24% increase (BURN: 2.98 +/- 0.32 ml/min/100 g, n = 6 vs SHAM: 2.40 +/- 0.40 ml/min/100 g, n = 6; P less than 0.10), demonstrating a redistribution of flow. There was no local accumulation of lactate in blood, liver, or skeletal muscle and no derangement in P/L ratios. This study when compared to previous observations in sepsis suggests that (1) the flow redistribution of sepsis has features differentiating it from solely a "stress response".(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in renal perfusion and renal energy charge in murine peritonitis.

Whether acute renal failure following overwhelming bacterial septicemia is a initially a consequence primarily of a cytotoxic insult or a perfusion insufficiency remains unclear. To assess the effects of intra-abdominal sepsis on the distribution of renal blood flow and renal cell bioenergy status, the glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and energy-charge ratios were measured in rats following cecal ligation/puncture (CLP) or sham laparotomies. The CLP animals demonstrated a decrease in ERPF of 42% and 58% from sham groups at ten and 20 hours, respectively. The GFR showed similar but more severe impairments of 53% and 71% at ten and 20 hours, respectively, following insult despite moderate increases in cardiac output. The disproportionate decrease in GFR over ERPF supports the hypothesis of a corticomedullary redistribution of renal blood flow in sepsis. Renal energy charge, unchanged at ten hours, decreased significantly at 20 hours. Diminished renal perfusion and the redistribution of renal blood flow precedes and may contribute to the renal cell bioenergy derangements in septic acute renal failure.

Galactose elimination kinetics in sepsis. Correlations of hepatic blood blow with function.

To study hepatic blood flow with clearance techniques during sepsis, it is essential to work within the limitations of the test being applied. Based on galactose elimination kinetics, this study validates galactose clearance at low concentrations as an estimate of effective hepatic blood flow in a rat peritonitis model of cecal ligation and puncture. Hepatic function as determined by galactose elimination capacity fell 25% at ten hours after induction of peritonitis, which correlated closely with the 20% reduction in effective hepatic blood flow at the same time point despite a normal cardiac output. The pattern of reduced flow and reduced function is consistent with intrahepatic flow redistribution. Inadequate flow at the microvascular level with secondary cellular injury may explain the liver dysfunction observed during sepsis.

Low-dose dopamine improves effective hepatic blood flow in murine peritonitis.

Diminished effective hepatic blood flow (EHBF) has been postulated as contributory to hepatocellular dysfunction in sepsis. In addition, dopamine has been demonstrated to increase perfusion to selective viscera. In order to examine the effects of peritonitis upon hepatic perfusion and its response to dopamine infusion, peritonitis was induced in rats via cecal ligation and perforation (CLP). Sixteen to 20 hours following insult, cardiac outputs were determined by thermodilution, and effective hepatic blood flow was determined by low-dose galactose clearance. Studies were performed in peritonitis-induced rats (CLP) and sham-operated controls with and without dopamine infusion for 30 minutes (0.5 microgram/100 g/min). Peritonitis resulted in a significant reduction in effective hepatic blood flow (p less than 0.01) despite a maintained cardiac output. Low-dose dopamine infusion resulted in a significant restoration of effective hepatic blood flow in CLP rats without altering cardiac output or hemodynamic status significantly. Dopamine may be beneficial in the maintenance of effective hepatic perfusion in peritonitis.

Imidazole and indomethacin improve hepatic perfusion in sepsis.

Visceral microcirculatory insufficiency has been demonstrated in sepsis despite a hyperdynamic systemic circulation. This study examines the effect of the cyclo-oxygenase inhibitor indomethacin and the thromboxane synthetase inhibitor imidazole on septic hemodynamics and visceral perfusion in a septic rat model of cecal ligation and puncture. Animals received either indomethacin (INDO), imidazole (IMID), or saline intramuscularly at t = 0, 6, and 12 hr of peritonitis. Thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, effective hepatic blood flow, effective renal plasma flow, and arterial and mixed venous blood gases were determined at 15 hr. Both INDO and IMID improved effective hepatic blood flow in the septic animals to virtually sham, nonseptic levels without significantly altering systemic hemodynamics. This study suggests that the reduction in hepatic perfusion in sepsis may be mediated by thromboxane, the synthesis of which is suppressed by both INDO and IMID.