Effects of intravenous administration of pirfenidone on horses with experimentally induced endotoxemia.

OBJECTIVE: To characterize effects of IV administration of pirfenidone on clinical, biochemical, and hematologic variables and circulating tumor necrosis factor (TNF)-alpha concentrations in horses after infusion of a low dose of endotoxin. ANIMALS: 18 healthy adult horses. PROCEDURES: Horses were randomly assigned to 3 groups (n = 6 horses/group) and administered an IV infusion of 30 ng of endotoxin/kg or saline (0.9% NaCl) solution during a 30-minute period. Lipopolysaccharide-pirfenidone horses received endotoxin followed by pirfenidone (loading dose of 11.6 mg/kg and then constant rate infusion [CRI] at 9.9 mg/kg/h for 3 hours). Lipopolysaccharide-saline horses received endotoxin followed by infusion (loading dose and CRI for 3 hours) of saline solution. Saline-pirfenidone horses received saline solution followed by pirfenidone (loading dose and then CRI for 3 hours). Physical examination variables were recorded and blood samples collected at predetermined intervals throughout the 24-hour study period. Blood samples were used for CBCs, biochemical analyses, and determinations of TNF-alpha concentrations. RESULTS: IV infusion of pirfenidone after administration of a low dose of endotoxin failed to attenuate the clinical, clinicopathologic, or cytokine alterations that developed secondary to endotoxin exposure. Intravenous infusion of pirfenidone after administration of saline solution induced mild transient clinical signs, but associated clinicopathologic changes were not detected. CONCLUSIONS AND CLINICAL RELEVANCE: IV administration of pirfenidone was tolerated with only mild transient clinical adverse effects during infusion. However, administration of pirfenidone did not protect horses from the systemic effects of experimentally induced endotoxemia. Further studies of related, but more potent, drugs may be warranted.

Effects of 5-ethyl-1-phenyl-2-(1H) pyridone on serum biomarkers of multiorgan dysfunction and mortality in lipopolysaccharide/galactosamine and cecal ligation and puncture models of septic shock in mice.

Septic shock results from a systemic host response to infection, in particular, and is associated with multiorgan dysfunction (MOD). Effective preventive measures against organ failure are essential as it is the cumulative burden of MOD that invariably leads to death. The aim of this study was to determine if a novel compound, 5-ethyl-1-phenyl-2-(1H) pyridone (5-EPP), could decrease the increased serum levels of various biomarkers of MOD in LPS/D-Galactosamine (LPS/D-GalN) and cecal ligation and puncture (CLP) models of septic shock in mice. Treatment with 5-EPP minimized the liver dysfunction as assessed by its ability to decrease the increased serum levels of aminotransferases. It also reduced proinflammatory cytokines such as TNF-alpha, IL-6 and IL-12, and offered complete protection against mortality in LPS/D-GalN model. 5-EPP treatment also offered a significant protection against LPS alone- induced mortality. Pretreatment with 5-EPP minimized the kidney, heart and muscle damage as assessed by its ability to decrease the CLP-induced increases in the serum levels of blood urea nitrogen, creatine kinase, glucose and mortality. Several possible mechanisms for the beneficial effects of 5-EPP in the LPS/D-GalN, LPS alone, and CLP models of septic shock have been discussed. It was concluded from the findings of this study that 5-EPP, a novel pyridone, is a promising candidate for the management of septic shock by offering protection against MOD and mortality clinically seen in septic patients.

Effects of 5-ethyl-1-phenyl-2-(1H) pyridone on serum biomarkers of multiorgan dysfunction and mortality in lipopolysaccharide/glactosamine and cecal ligation and puncture models of septic shock in mice.

Septic shock results from a systemic host response to infection, in particular, and is associated with multiorgan dysfunction (MOD). Effective preventive measures against organ failure are essential as it is the cumulative burden of MOD that invariably leads to death. The aim of this study was to determine if a novel compound, 5-ethyl-1-phenyl-2-(1H) pyridone (5-EPP), could decrease the increased serum levels of various biomarkers of MOD in LPS/D-Galactosamine (LPS/D-GalN) and cecal ligation and puncture (CLP) models of septic shock in mice. Treatment with 5-EPP minimized the liver dysfunction as assessed by its ability to decrease the increased serum levels of aminotransferases. It also reduced proinflammatory cytokines such as TNF-alpha, IL-6 and IL-12, and offered complete protection against mortality in LPS/D-GalN model. 5-EPP treatment also offered a significant protection against LPS alone- induced mortality. Pretreatment with 5-EPP minimized the kidney, heart and muscle damage as assessed by its ability to decrease the CLP-induced increases in the serum levels of blood urea nitrogen, creatine kinase, glucose and mortality. Several possible mechanisms for the beneficial effects of 5-EPP in the LPS/D-GalN, LPS alone, and CLP models of septic shock have been discussed. It was concluded from the findings of this study that 5-EPP, a novel pyridone, is a promising candidate for the management of septic shock by offering protection against MOD and mortality clinically seen in septic patients.

Pharmacokinetics and clinical effects of pirfenidone administered intravenously in horses.

OBJECTIVE: To characterize the plasma pharmacokinetics and clinical effects of pirfenidone administered IV in healthy horses. ANIMALS: 6 adult horses. PROCEDURES: A 15 mg/kg dose of pirfenidone was administered IV over 5 minutes. Physical variables were recorded and blood samples collected prior to infusion; 2.5 minutes after beginning infusion; at the end of infusion; and at 3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 60, 75, and 90 minutes and 2, 2.5, 3, 4, 6, 8, 12, and 24 hours after completion of infusion. Plasma concentrations of pirfenidone and its metabolites were determined. RESULTS: Mild clinical effects, including tachycardia and muscle fasciculations, were observed during drug administration but stopped at the end of the infusion. Pirfenidone and 2 metabolites, hydroxypirfenidone and carboxypirfenidone, were detected by the end of the 5-minute infusion. Mean peak plasma concentration of pirfenidone was 182.5 micromol/L, detected at the end of the infusion. Mean peak plasma concentrations of hydroxypirfenidone and carboxypirfenidone were 1.07 and 3.4 micromol/L, respectively, at 40 minutes after infusion. No parent drug or metabolites were detected at 24 hours. Distribution of pirfenidone best fit a 2-compartment model, and the drug had mean +/- SEM elimination half-life of 86.0 +/- 4.7 minutes, mean body clearance of 6.54 +/- 0.45 mL/kg/min, and apparent volume of distribution at steady state of 0.791 +/- 0.056 L/kg. CONCLUSIONS AND CLINICAL RELEVANCE: Intravenous administration of pirfenidone was tolerated with transient adverse affects during infusion, and drug clearance was rapid.

Pharmacokinetics and metabolism of intravenous pirfenidone in sheep.

Pirfenidone, a promising antifibrotic agent, was administered intravenously to six female sheep at 30 mg/kg. Four sheep received 14C-pirfenidone simultaneously. Plasma and urine were obtained for assay of pirfenidone and its metabolites over two days, and tissues were obtained via necropsy. Samples were analysed for pirfenidone and metabolites using HPLC-MS and flow scintillation spectrometry. Plasma pirfenidone disappeared with first order kinetics with a clearance of 1.2 l/kg/h, half-life of 24 min, and distribution volume of 0.71 l/kg. After 48 h, the organs containing the largest quantity of 14C were lungs, liver and intestinal wall. Tissues with the highest concentration of 14C were lung, kidney, brain, liver, lymph node and adipose. Metabolites found in plasma and urine were hydroxypirfenidone (half-life of 44 min) and carboxypirfenidone. Additional metabolites found in urine were hydroxypirfenidone glucuronide and acetoxypirfenidone. Approximately, 80% of the tracer eventually appeared in the urine, and approximately 50% of it was in the form of identifiable metabolites. Less than 1% of the dose appeared in the urine in the form of the parent drug. Quantitatively, most of the metabolites appeared in the urine within 2 h. Thus, the drug is rapidly and completely metabolized.

Influence of pirfenidone on airway hyperresponsiveness and inflammation in a Brown-Norway rat model of asthma.

Pirfenidone was administered to sensitized Brown Norway rats prior to a series of ovalbumin challenges. Airway hyperresponsiveness, inflammatory cell infiltration, mucin and collagen content, and the degree of epithelium and smooth muscle staining for TGF-beta were examined in control, sensitized, and sensitized/challenged rats fed a normal diet or pirfenidone diet. Pirfenidone had no effect on airway hyperresponsiveness, but reduced distal bronchiolar cell infiltration and proximal and distal mucin content. Statistical analysis showed that the control group and sensitized/challenged pirfenidone diet group TGF-beta staining intensity scores were not significantly different from isotype controls, but that the staining intensity scores for the sensitized/challenged normal diet group was significantly different from isotype controls. These results suggest that pirfenidone treatment is effective in reducing some of the components of acute inflammation induced by allergen challenge.

A double-blind, randomized, controlled study of oral pirfenidone for treatment of secondary progressive multiple sclerosis.

Currently, there are no approved treatments for secondary progressive multiple sclerosis (MS) that stabilize or reverse the neurological disabilities associated with this disease. Oral pirfenidone was found to stabilize and overcome the disabilities in two published independent open-label studies in secondary progressive MS. This led us to study pirfenidone in a phase II double-blind, randomized and controlled, clinical trial in patients with advanced secondary progressive MS for 12 months. Forty-three patients met the eligibility criteria approved by the IRB and accepted by the FDA. Of these patients, 18 were randomly assigned to placebo and 25 patients to oral pirfenidone groups. All eligible patients were included in the statistical analysis of the data according to intention-to-treat principles. Some patients on oral pirfenidone manifested mild drug-related adverse effects, but it was well tolerated overall. By one month, pirfenidone significantly (P < 0.05) improved the Scripps Neurological Rating Scale (SNRS) scores, and scores remained significantly improved for 3, 6 and 12 months when compared to the baseline SNRS scores. In contrast, the SNRS scores of patients on oral placebo were not significantly improved at 1, 3, 6 or 12 months of the study, when compared with baseline scores. Oral pirfenidone significantly (P <0.04) reduced the incidence of relapses (27.8% on placebo versus 8.0% on pirfenidone). Furthermore, oral pirfenidone treatment was associated with a marked improvement in bladder dysfunction (40.0% on pirfenidone versus 16.7% on placebo). Expanded Disability Status Scale scores and MRI lesion count were not significantly different in the placebo and pirfenidone groups. These findings indicate a significant effect of pirfenidone on clinical disability and bladder function for secondary progressive MS patients. A major multicentre, double-blind, randomized, controlled trial is justified.

Effects of topical application of pirfenidone ointment on thermoplasty-induced acute lameness in a double-blind and acute and chronic lameness of musculoskeletal origin in an open multi-centered field trial in horses.

The effectiveness of pirfenidone ointment against thermoplasty-induced acute foreleg lameness in a double-blind study, and against acute and chronic lameness of musculoskeletal origin in an open multi-centered field trial was evaluated in this study. Thermoplasty was performed on both inner forelegs at designated locations of each horse under anesthetics. A 10% pirfenidone or placebo ointment was topically applied starting 24 hours after the thermoplasty three times daily for 7 days. For acute and chronic lameness of musculoskeletal origin, pirfenidone ointment was also applied one to three times daily for 7 to 10 days and continued for an additional 20 to 30 days. A marked swelling around the locations occurred in 24 hours post-thermoplasty. The topical application of pirfenidone ointment not only caused a significant reduction in the circumference measurements at 5, 6 and 7 days, but it also decreased the changes in the circumferences from pre-thermoplasty as an index of edema, at 3, 4, 5, 6, and 7 days when compared to the placebo ointment at the corresponding times. Although treatment for 7 days of acute leg lameness of musculoskeletal origin with topical pirfenidone ointment caused significant decreases in swelling, heat, and pain, and improved the degree of flexion when compared with the pretreatment, it had little effect on chronic lameness except that it improved the flexion at the second-exam interval. It was concluded that topical application of pirfenidone is effective for treatment of acute lameness resulting from thermoplasty or from various types of musculoskeletal disorders, suggesting that pirfenidone offers a promising therapeutic potential to manage acute inflammation, an important component of lameness.

Amelioration of doxorubicin-induced cardiac and renal toxicity by pirfenidone in rats.

PURPOSE: Doxorubicin (DXR) is an anthracycline glycoside with a broad spectrum of therapeutic activity against various tumors. However, the clinical use of DXR has been limited by its undesirable systemic toxicity, especially in the heart and kidney. This study was designed to test the effectiveness of dietary intake of pirfenidone (PD) against DXR-induced cardiac and renal toxicity. METHODS: Male Sprague Dawley rats were placed into four treatment groups: saline injected intraperitoneally (i.p.) plus regular diet (SA+RD); DXR i.p. plus regular diet (DXR+RD); saline i.p. plus the same diet mixed with 0.6% PD (SA+PD); and DXR i.p. plus the same diet mixed with 0.6% PD (DXR+PD). The animals were fed regular or regular plus PD diets 3 days prior to i.p. injections of either saline or DXR and continuing throughout the study. A total dose of DXR (16.25 mg/kg) or an equivalent volume of saline was administered in seven injections (2.32 mg/kg per injection) three times per week with an additional dose on the 12th day. At 25 days following the last DXR or saline injection, some animals were anesthetized for the measurement of cardiac and pulmonary function, and others were killed by an overdose of pentobarbital. At the time the animals were killed, abdominal fluid was collected. Kidney and heart were removed, weighed, fixed with 10% formalin or frozen in liquid nitrogen. The fixed tissues were used for histological examination and the frozen tissues were used for biochemical studies. RESULTS: The average volumes of abdominal fluid in the DXR+RD and DXR+PD groups were 9.42 ml and 3.42 ml and the protein contents of abdominal fluid in the DXR+RD and DXR+PD groups were 218 mg and 70 mg, respectively. A 12.5% mortality occurred in the DXR+RD group as compared to 0% in DXR+PD group. There were no changes in any of the cardiac or pulmonary physiological parameters in any of the four groups. The changes in the heart and kidney of the DXR+RD group included reduction in organ weight, increase in hydroxyproline content of heart, increase in hydroxyproline, and lipid peroxidation in the kidney and plasma, and increase in protein concentration in urine as compared to rats in the control, SA+RD and SA+PD groups. Treatment with PD abrogated the DXR-induced increases in hydroxyproline content in the heart and kidney, lipid peroxidation of the kidney and plasma, and protein content of the urine in the DXR+PD group. DXR treatment alone caused disorganization of cardiac myofibrils, vacuolization of the myofibers, and renal tubular dilation with protein casts in both the cortical and medullary regions. Treatment with PD minimized the DXR-induced histopathological changes of heart and kidney in the DXR+PD group. CONCLUSIONS: Treatment with PD reduced the severity of DXR-induced toxicity as assessed by reduced mortality, diminished volume of recovered fluid in the abdominal cavity, and severity of cardiac and renal lesions at both the biochemical and morphological levels. These results indicate that PD has the potential to prevent DXR-induced cardiac and renal damage in humans on DXR therapy.

Novel pharmacological approaches to manage interstitial lung fibrosis in the twenty-first century.

Pharmacological agents currently in use to treat interstitial lung fibrosis are either ineffective or too toxic in humans. This review addresses mechanistically based novel approaches that have the potential to minimize the accumulation of collagen in the lung, a hallmark of lung fibrosis. These approaches include maintaining the intracellular levels of NAD(+) and ATP, blocking the biological activities of TGF-beta and integrins, evaluating the effectiveness of PAF-receptor antagonists and NOS inhibitors, and developing a new generation of cysteine pro-drugs with an adequate degree of bioavailability. A critical analysis of each approach as it relates to management of IPF in humans is presented.

Modulation of articular chondrocyte activity by pirfenidone.

Pirfenidone is under investigation as an anti-inflammatory and anti-fibrotic agent in several organs including lung. Since important features of arthritic conditions include inflammation and long-term damage to articular cartilage, we have investigated whether PD can suppress chondrocyte responses to bacterial lipopolysaccharide (LPS) and interleukin 1 (IL-1); modulators that induce a cascade of inflammatory responses that lead to articular joint tissue damage. PD (0 - 5microM) showed no effect on cell number or viability when incubated with high density primary equine chondrocyte cultures for a 24 hr period. PD did not stimulate nitric oxide (NO) release by chondrocytes when added alone but LPS and IL-1-induced NO release was inhibited by PD, in a dose-dependent manner. PD did not significantly influence GAG release from cartilage matrix nor did it stimulate or suppress the GAG releasing actions of LPS or IL-1. We conclude that PD is capable of attenuating the cytokine-induced production of the inflammatory mediator, NO by chondrocytes, without stimulating matrix glycosaminoglycan loss from cartilage. PD may have potential as an anti-inflammatory agent in the joint.

Effects of delayed treatment with transforming growth factor-beta soluble receptor in a three-dose bleomycin model of lung fibrosis in hamsters.

Transforming growth factor-beta (TGF-beta) plays a pivotal role in an exaggerated synthesis and accumulation of collagen in fibrotic disorders of many organs. We have previously demonstrated that repeated intratracheal (IT) instillation of TGF-beta soluble receptor (TR) in hamsters markedly decreased the bleomycin (BL)-induced lung fibrosis in response to a single dose. The present study was carried out in a 3-dose BL-hamster model of lung fibrosis to better evaluate the therapeutic potential of TR. Three doses of BL (2.5, 2.0, and 1.5 U/4 mL/kg) or an equivalent volume of isotonic saline was administered IT consecutively at weekly intervals, and phosphate-buffered saline (PBS) or TR (4 nmol/0.3 mL/hamster) by the same route twice a week, starting after the 2nd BL or 3rd BL dose. Twenty-one days after the 3rd dose of BL instillation, the hamsters were killed for bronchoalveolar lavage (BAL) and biochemical and histopathological analyses. The results showed that treatment with TR starting after either the 2nd or 3rd dose of BL caused significant reduction in BL-induced lung fibrosis, as demonstrated by marked decreases in the hydroxyproline level and prolyl hydroxylase activity of the lungs. Histopathological evaluation of the lungs also revealed that the hamsters in both BL+TR groups had markedly fewer fibrotic lesions than hamsters in BL+PBS group. These results demonstrate the beneficial effects of delayed treatment with TR in attenuating the progression of ongoing fibrotic process and suggest its potential therapeutic uses in the management of lung fibrosis in humans.

Pharmacokinetics and metabolism of a novel antifibrotic drug pirfenidone, in mice following intravenous administration.

The present study describes the pharmacokinetics and metabolism of pirfenidone (PD), a compound which has been shown to have significant antifibrotic effects in rodent models of pulmonary and cardiac fibrosis. Despite the fact that this compound is currently in phase II clinical trials, little data are available on the metabolism and disposition of this agent in rodents or humans. Radioactive PD [benzene ring (14)C(U)] was administered i.v. to mice at 40 mg PD/kg body weight, and animals were killed at varying times for determination of parent compound and metabolites in various tissues. The disappearance of parent compound from the plasma followed apparent 2-compartment elimination kinetics with a terminal elimination half-life of 8.6 min. Cl (0.10 ml/min/g) and V(d(ss)) (0.67 ml/g) indicated that PD was rapidly distributed in body water. This is consistent with the finding that peak tissue radioactivity occurred within 5 min following the i.v. administration of [(14)C]-PD and that well-perfused tissues, kidney>liver>lung have much higher levels of parent compound and metabolites than did fat. Two peaks isolated from plasma samples by HPLC yielded mass spectra that were consistent with initial oxidation to the alcohol followed by further metabolism to the carboxylic acid. The radioactivity recovered in the 24 h urine samples averaged 97% of the administered dose and none of that was associated with the parent compound. The short plasma half-life of parent compound in mice supports the need for additional studies in humans where the compound has been shown to have clinical benefits.

Effect of pirfenidone against vanadate-induced kidney fibrosis in rats.

Renal fibrosis is a complication of kidney injury and can contribute to organ failure. Currently, there are no drugs for the treatment of renal fibrosis. Pirfenidone (PD) has been proven to have antifibrotic effects in animal models of fibrosis. We tested the ability of PD against vanadate-induced kidney fibrosis in rats. The rats were injected subcutaneously with vehicle or vanadate solution (1mg vanadate/kg/day) for 12 or 16 days to produce varying degrees of kidney fibrosis. The vanadate- and vehicle-treated rats were fed a laboratory diet or the same diet mixed with 0.6% PD ad lib. One vanadate-injected group was initially fed the same diet without PD and later switched to the diet containing PD 2 days after the last injection. The rats were killed at 12 and 25 days following the last dose. The changes found in the kidney of vanadate-treated rats included increases in RNA and DNA content and increases in kidney weight. Treatment with PD diminished the vanadate-induced increases in kidney weight and RNA content. The hydroxyproline content of the kidney in vanadate-treated animals was increased significantly (P< or =0.05) from the control level of 1452 microg/kidney to 1765 microg/kidney. Treatment with PD for 37 days caused significant reductions in the vanadate-induced increases in the hydroxyproline level. Similarly, treatment for 41 days also caused significant reductions (1744 microg/kidney) in vanadate-induced increases in the hydroxyproline level (1996 microg/kidney). The histological evaluation revealed that the severity of the lesions in the vanadate-treated group was moderate to severe, and treatment with PD for 41 days decreased the severity to a mild level. In addition, the delayed treatment with PD also minimized the vanadate-induced increases in the collagen content of the kidney. Although it is speculative, PD may potentially be therapeutic in the management of renal fibrosis.