Effects of three anti-TNF-alpha drugs: etanercept, infliximab and pirfenidone on release of TNF-alpha in medium and TNF-alpha associated with the cell in vitro.

Tumor necrosis factor-alpha (TNF-alpha) is a vital component of the inflammatory process and its aberrant over-expression has been linked to numerous disease states. New treatment strategies have sought to reduce circulating TNF-alpha, either with neutralizing anti-TNF-alpha binding proteins such as etanercept or via drugs that inhibit de novo TNF-alpha synthesis like pirfenidone. In the present study, we examined the effects of both classes of drugs on secreted and cell-associated TNF-alpha produced by THP-1 cells. All of the tested drugs significantly reduced secreted levels of bioactive TNF-alpha following stimulation with LPS as measured by bioassay. However, etanercept-treated cells had approximately six-fold higher levels of cell-associated TNF-alpha compared with that of the LPS-alone treatment group. Surprisingly, LPS+infliximab treated cells did not increase cell-associated TNF-alpha relative to the LPS-alone treatment. Pirfenidone significantly reduced both secreted and cell-associated TNF-alpha levels. These drug-related differences in cell-associated TNF-alpha may have broad implications in the future for the therapeutic uses of anti-TNF-alpha drugs in the management of TNF-alpha diseases.

Pharmacokinetics of orally administered pirfenidone in male and female beagles.

Pirfenidone, a promising antifibrotic agent, was administered orally to dogs at 0, 40, 140, and 400 mg/kg/day. Serum was collected for pirfenidone assay at 0, 26 and 39 weeks of treatment. From the pirfenidone concentrations, pharmacokinetic parameters were determined for each dog at each treatment interval. The only significant differences because of gender were for concentration maxima. Unsurprisingly, there were many significant differences because of dose in concentration maximum and area under curve (AUC), and significant, positive linear correlations of both parameters with dose. There were few significant differences in time of maximal concentration and no correlation with dose. The mean +/- SE clearances were 1.99 +/- 0.13, 1.64 +/- 0.13 and 1.78 +/- 0.14 L/h/kg for doses of 40, 140, and 400 mg/kg, respectively, with no significant differences attributable to dose. There was an unexplained pattern in maximal concentration and AUC with regard to duration of treatment, with the parameters being highest at week 0, lowest at week 26, and intermediate at week 39. Clearance had the reverse pattern; time of maximal concentration had no pattern.

Antirheumatic effect of pirfenidone in a double blind clinical pilot trial in humans.

The antirheumatic effect of pirfenidone was compared with a positive control drug, oxyphenbutazone which is used in patients suffering from rheumatoid arthritis, in a double blind clinical trial in humans. The data collected in this pilot project revealed that pirfenidone was more effective (p < 0.025) than oxyphenbutazone in providing relief from arthritic pain. In addition, a greater number (p < 0.025) of patients reported favorable response to oral pirfenidone than oral oxyphenbutazone. However, there were no significant differences in the number of patients who dropped out from the trial and the number of patients who tolerated the drugs for 21 days of the trial between the pirfenidone and oxyphenbutazone groups. It was concluded from this pilot study that pirfenidone potentially offers a novel therapeutic modality for the management of rheumatoid arthritis with little or no adverse effects unlike steroidal and non-steroidal anti-inflammatory drugs which are frequently used for this chronic debilitating disease.

Pirfenidone suppressed the development of glomerulosclerosis in the FGS/Kist mouse.

Pirfenidone (PFD) is a newly developed anti-fibrotic agent. We evaluated the effect of PFD for the prevention of renal fibrosis using a spontaneous progressive glomerulosclerosis animal model, FGS/Kist mice. Male and female FGS/Kist mice were fed a diet containing 0.5% PFD or the same control diet (CD) without PFD, for 1, 2, or 3-month periods. Body weight was monitored for the general effect of PFD on the mice. Proteinuria and glomerular filtration rate (GFR) were evaluated for renal function. The sclerosis index was examined for the morphological changes. There were no significant changes in body weight between the PFD and control groups in both sexes. Proteinuria levels were low in all the PFD groups compared to the corresponding CD groups. The sclerosis scores were also reduced in both sexes of the 3-month PFD groups (p<0.05), and glomerular filtration rates were increased in both sexes of the 3-month PFD groups compared to the CD groups. The treatment of PFD for 1 or 2-month periods did not have statistic significances but the treatment for 3 months had statistic significances in sclerosis and GFR compared to CD groups. These results suggested that long-term administration of PFD suppressed the progression of glomerulosclerosis and improved renal function of the FGS/Kist mice.

Pirfenidone inhibits early myointimal proliferation but has no effect on late lesion size in rats.

AIMS: intimal hyperplasia is mediated by smooth muscle cell proliferation, migration and deposition of extracellular matrix. The anti-fibrotic agent pirfenidone has been shown to inhibit pro-fibrotic growth factors in non-vascular inflammatory models. This study investigated the effect of the novel anti-fibrotic agent pirfenidone on the development of neointima. METHODS: male Sprague-Dawley rats received either standard diet or diet supplemented with pirfenidone (250, 500, 1000 mg/kg/day). Animals underwent left common carotid balloon angioplasty and were explanted at 4, 8, 14 and 28 days and analysed for intimal thickening, pro-fibrotic gene expression, extracellular matrix deposition and metalloproteinase activity. RESULTS: neointimal thickness was significantly reduced in a dose-dependent manner at 14 days; pirfenidone 250 mg/kg (p<0.005), pirfenidone 500 mg/kg (p<0.001), pirfenidone 1 g/kg ( p<0.001). There were no significant differences in intimal thickening at 28 days. Expression of MMP-2, MMP-9, TIMP-1, collagen III and TGF-beta were all significantly inhibited at 14 days. Both collagen III expression and ECM deposition were reduced at 28 days ( p<0.05 and <0.002 respectively). CONCLUSION: pirfenidone reduces expression of MMPs governing smooth muscle cell proliferation and migration (MMP-2 and 9), and genes favouring ECM accumulation (TIMP-1 and collagen III). This study shows that inhibition of MMP activity is not sufficient to inhibit late lesion size.

Pirfenidone for chronic progressive multiple sclerosis.

Current treatment of secondary progressive multiple sclerosis is unsatisfactory in stabilizing or reversing the disabilities associated with the disease. Pirfenidone is a new non-peptide drug which has been shown in vitro and in vivo to decrease synthesis of Tumor Necrosis Factor-alpha (TNF-alpha) and block receptors for TNF-alpha. Since TNF-alpha seems to be a key cytokine in demyelination, a pilot study of oral pirfenidone was undertaken in an open-label baseline vs treatment protocol over a 2-year period in 20 patients. Fourteen (14120) patients (70%) remained in the study for 2 years. Three (3/20) patients dropped out early because of gastrointestinal adverse reactions, and another three patients dropped out for personal reasons after 1 year (not because of adverse reactions). The remaining patients did not manifest any other drug-related adverse reactions and complications. Improvement or stabilization occurred in most patients at about 3 months, and it was sustained at 6, 12 and 24 months as evaluated by both primary and secondary outcome measures. Magnetic resonance imaging foiled to reveal any new lesions. Thus, pirfenidone appears to offer protection against the usual slow progression of the disease. Most patients experienced a distinct decrease in their neurological disability. These findings indicate that an extensive multi-center double blind and placebo controlled trial is warranted.

Pirfenidone reduces in vitro rat renal fibroblast activation and mitogenesis.

BACKGROUND: Fibroblasts have been universally recognised in tubulointerstitial injury, where their presence has been shown to be a marker of disease progression. Recently, pirfenidone (PF) has been shown to both ameliorate progressive fibrosis and reduce established scarring after ureteric obstruction (UUO) in the rat, suggesting that it is a novel anti-fibrotic agent. The objective of this study was therefore to determine if these effects include down-regulation of fibroblast function. METHODS: Cortical fibroblasts were obtained from outgrowth cultures of renal tissue isolated from kidneys 3 days after UUO and constituted 100% of cells studied. Functional studies examined the effects of 20 and 200 microg/ml PF on basal serum stimulated activity. Activation was examined by western blotting for alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Cell proliferation, collagenase activity and collagen production were determined from kinetic studies, zymography for MMP2 and [3H] proline incorporation in collagenous proteins respectively. RESULTS: Proliferation, as measured by [3H] thymidine incorporation, was reduced in dose dependent manner by 20 and 200 microg/ml PF (p<0.05; 200 vs 0 microg/ml). Likewise, 200 microg/ml PF reduced cell population growth over 5 days of culture (p<0.05 vs 0 microg/ml). PF (200 microg/ml) decreased alphaSMA and CTGF protein expression to 66+/-13 and 37+/-26% of basal levels respectively (both p<0.05 vs 0 microg/ml). Synthesis of collagen was unaffected by PF. Maximal dose of PF produced a modest reduction in MMP2 lytic activity (p=0.05). Effects of PF were independent of cell toxicity. CONCLUSIONS: Down-regulation of renal fibroblast activation and proliferation are specific actions of PF.

Effects of pirfenidone on the generation of reactive oxygen species in vitro.

Pirfenidone is a newly developed antifibrotic drug that has been reported to retard the progression of pulmonary fibrosis induced by bleomycin and cyclophosphamide in animal models of lung fibrosis. The present in vitro studies using noncellular and cellular systems evaluated the antioxidant and cytotoxic properties of this drug. The Fenton reaction [Fe(II) + H2O2 --> Fe(III) + *OH + OH-] and the xanthine/xanthine oxidase system were used as sources of hydroxyl (*OH) and superoxide anion (O2*-) radicals, respectively. Electron spin resonance spin trapping was used for free radical detection and measurement. The reaction rate of pirfenidone with *OH was found to be 1.63 x 10(10) M(-1) s(-1), which is comparable to several well-established antioxidants, such as ascorbate, glutathione, cysteine, azide, and lipoic acid. Compared to *OH radicals, the O2*- scavenging was less efficient 42.36 M(-1) s(-1) with pirfenidone. Pirfenidone was also effective in inhibiting zymosan-stimulated chemiluminescence. In a noncellular model of lipid peroxidation, pirfenidone inhibited crystalline silica-induced lipid peroxidation. The inhibition of crystalline silica-induced cytotoxic reactions and lipid peroxidation combined with the efficient antioxidant properties of pirfenidone indicate that this agent may express its antifibrotic effects partly through its ability to scavenge reactive oxygen species.

Involution of keloid implants in athymic mice treated with pirfenidone or with triamcinolone.

The experimental drug pirfenidone (PFD) has been evaluated as an inhibitor of keloid proliferation and compared with triamcinolone (TAC) injections by studying the involution of active human keloid implants in athymic nude mice (nu-nu). PFD was fed to mice with keloid implants at a level of 2.75 mg/g of feed. At this level PFD had no adverse effect on the body weights of the mice. Implant weights in both PFD-fed and control mice decreased with time. The weights of the implants from the PFD group were significantly lower than those of the control implants at 60 and 90 days after implantation. Consequently PFD may cause an increased degradation and absorption of keloid tissue. The implants from the PFD mice were not significantly different histologically from the implants of the mice with corresponding implants. The chondroitin-4-sulfate (C4S) levels of the implants from PFD-fed mice were not significantly different from those of the implants from control mice. Therefore the mechanism of action of PFD apparently is not mediated by an effect on C4S metabolism. In contrast, the injections of TAC at a level that caused temporary body weight loss in the mice resulted in significant decreases in both hyaluronic acid (HA) and C4S in the keloid implants. Histologically, fibroblasts disappeared from the implants treated with TAC by 20 days after injection. At 30 days after TAC injection, HA and C4S were not detected by electrophoresis in keloid implants; only dermatan sulfate appeared to be present.

Pirfenidone induces intercellular adhesion molecule-1 (ICAM-1) down-regulation on cultured human synovial fibroblasts.

Pirfenidone has been shown to modify some cytokine regulatory actions and inhibit fibroblast biochemical reactions resulting in inhibition of proliferation and collagen matrix synthesis by fibroblast. We have investigated the effect of pirfenidone on the expression of cell adhesion molecules. The synovial fibroblasts were treated with IL-1alpha in the presence or absence of pirfenidone (range 0-1000 microM), and assayed for the expression of adhesion molecules such as ICAM-1 and endothelial-leucocyte adhesion molecule-1 (E-selectin) by cell ELISA. Pirfenidone significantly down-regulated the expression of ICAM-1 on cultured synovial fibroblasts in a dose-dependent manner. In contrast, expression of E-selectin was not affected. Furthermore, we examined whether pirfenidone affects the cellular binding between cultured lymphocytes and IL-1alpha-stimulated synovial fibroblasts by in vitro binding assay and found their mutual binding was significantly suppressed in a dose-dependent manner by pirfenidone. It is speculated that down-regulation of ICAM-1 might be one of the novel mechanisms of action of pirfenidone. These data indicate a novel mechanism of action for pirfenidone to reduce the activation of synovial fibroblasts.

Pirfenidone improves renal function and fibrosis in the post-obstructed kidney.

BACKGROUND: Pirfenidone (PFD) is a novel anti-fibrotic agent that can prevent and even reverse extracellular matrix accumulation in several organs, as shown by experimental and clinical studies. Unilateral ureteral obstruction (UUO) is a well-characterized model of experimental renal disease culminating in tubulointerstitial fibrosis. METHODS: UUO or sham-operated rats were administered PFD (500 mg/kg/day) in their food for 21 days to examine the effect on collagen production. The renal function was measured in the kidney after release of obstruction which had been maintained for one week to examine the effects of PFD on restoration after renal dysfunction. RESULTS: The collagen content detected by hydroxyproline progressively increased in kidney with UUO for 21 days. These increases were significantly suppressed by administration of PFD. PFD had no effect on collagen production in sham-operated rats. Expression of mRNA for type IV and I collagen and matrix metalloproteinase-2 in the cortex increased with UUO, but was inhibited by PFD treatment. The levels of cortical transforming growth factor-beta (TGF-beta) mRNA progressively rose with UUO for 21 days, but this increase also could be suppressed by PFD. Inulin clearance of the obstructed kidney was markedly depressed and remained low at five weeks after release. A progressive increase in hydroxyproline content was also observed in the post-obstructed kidney despite the release of obstruction. Administration of PFD following the release not only attenuated collagen accumulation, but also induced recovery of the impaired renal function. CONCLUSIONS: These results demonstrate that PFD can attenuate both renal fibrosis and renal damage in this model, and suggest that PFD can be clinically useful for preventing progressive, irreversible renal failure.

Lung fibrosis is ameliorated by pirfenidone fed in diet after the second dose in a three-dose bleomycin-hamster model.

Interstitial lung fibrosis (ILF) is a life-threatening disease which has no known drug for prevention and cure. In the present study, we evaluated the antifibrotic potential of pirfenidone (PD) (5-methyl-1-phenyl-2-(1H)-pyridone) in a three-dose bleomycin (BL)-hamster model of lung fibrosis. Hamsters were intratracheally (IT) instilled with three consecutive doses of bleomycin sulfate (2.5 U/kg/5mL, 2.0 U/kg/5mL, 1.5 U/kg/3.75 mL) or an equivalent volume of saline at weekly intervals. Hamsters were fed a diet after the second dose of BL containing 0.5% PD and hamsters in the control groups were fed the same diet without the drug. The four groups were saline-instilled fed control diet (SCD); saline-instilled fed the same diet containing PD (SPD); BL-instilled fed control diet (BCD); and BL-instilled fed the diet containing PD (BPD). Hamsters were sacrificed at 28 days after IT instillation of last dose of saline or BL and their lungs processed for various assays. Lung hydroxyproline, an index of fibrosis, in SCD, SPD, BCD and BPD were 830, 804, 1609, 1235 micrograms/lung, respectively. Lung prolyl hydroxylase activities in the SPD, BCD and BPD groups were 103%, 313%, 157% of the control SCD group (5.99 x 10(4) dpm/lung/30 min) respectively. Malondialdehyde equivalent levels and superoxide dismutase activity in the corresponding groups were 99, 79, 240 and 145 nmoles/lung and 412, 433, 538 and 410 units/lung respectively. Lung myeloperoxidase activities in the corresponding groups were 56%, 179%, and 116% of the control group (0.44 units/lung). It is concluded that PD is a novel antifibrotic drug that has therapeutic potential in arresting the progression of an ongoing fibrotic process in the lung.

Pirfenidone: a novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production.

There are currently no effective, long-term drug therapies for the treatment of leiomyomas. Pirfenidone (Marnac, Inc.) is an antifibrotic agent that is being tested for use in patients with pulmonary fibrosis. Because leiomyomas are characterized also by increased cell proliferation and tissue fibrosis, we examined the effects of pirfenidone on cell proliferation and collagen expression in cultured myometrial and leiomyoma smooth muscle cells. Effects of pirfenidone on proliferation of myometrial and leiomyoma cells were measured using tritiated thymidine incorporation assays and changes in actual cell numbers. Possible cytotoxic effects were examined using lactate dehydrogenase assays and trypan blue exclusion. Effects on collagen type I and type III production were assessed by Northern blotting. Doses of pirfenidone tested were: 0, 0.01, 0.1, 0.3, and 1.0 mg/mL. Serum-stimulated increases in DNA synthesis and cell proliferation by myometrial and leiomyoma cells were significantly inhibited in a dose-dependent manner by pirfenidone. Densitometric analysis of Northern blots showed significantly decreased expression of collagen type I and type III messenger RNAs in both leiomyoma and myometrial cells. Lactate dehydrogenase assays and trypan blue exclusion measurements showed no cytotoxic effect of pirfenidone at concentrations that inhibited cell proliferation and collagen production. Pirfenidone is an effective inhibitor of myometrial and leiomyoma cell proliferation in vitro and reduces the messenger RNA levels of collagen types I and III in a dose-dependent manner. This compound may prove to be an effective nonsteroidal therapy for treatment of uterine leiomyomas.

Pirfenidone diminishes cyclophosphamide-induced lung fibrosis in mice.

The deposition of excess or abnormal collagen characteristic of pulmonary fibrosis can disrupt gas exchange resulting in severe respiratory impairment. There currently are no effective pharmacologic agents available that inhibit the fibrotic process. Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is an investigational drug that, when administered at 0.5% (w/w) of the diet, decreases both histologic and biochemical evidence of lung fibrosis in hamsters treated intratracheally with bleomycin. The effectiveness of pirfenidone against lung fibrosis initiated by a systemically administered agent was investigated in mice treated intraperitoneally with 200 mg/kg cyclophosphamide (CP). Control and treated animals were fed a diet containing 0.277% (w/w) pirfenidone beginning 1 day after CP. Despite anorexia in the CP-treated mice the first day after treatment, they ingested a greater average pirfenidone dose over 20 days than saline-treated control mice (717 +/- 44 versus 564 +/- 30 mg/kg per day, respectively). Total lung hydroxyproline content, an index of fibrosis, was significantly lower 21 days after treatment with CP plus pirfenidone as compared to mice treated with CP alone. Although microscopic lung fibrosis scores were not significantly decreased by pirfenidone in CP-treated mice, the overall incidence of fibrosis was significantly decreased. Histologically, mice treated with CP showed fibrosis while mice treated with CP plus pirfenidone exhibited fewer abnormalities. The rate of hydroxyproline synthesis by lung tissue 9 days after treatment with CP was significantly elevated. This rate was not affected by pirfenidone treatment. Overall, these data support an antifibrotic effect of pirfenidone against CP-induced lung fibrosis in mice. The mechanism of its effect is not known, but appears to be unrelated to an inhibition of collagen synthesis.

Pirfenidone prevents collagen accumulation in the remnant kidney in rats with partial nephrectomy.

Pirfenidone (PFD) is a new compound that prevents and even reverses the extracellular matrix accumulation in several organs as shown by experimental and clinical studies. In the present study, we examined the effect of PFD (500 mg/kg daily in the food) on the progression of chronic renal failure (CRF) in the 5/6 nephrectomy rat model. Proteinuria progressively increased in rats with renal ablation (C) at 12 weeks. Urinary protein excretion in PFD-treated rats (P) was numerically lower than C, but the difference did not reach statistical significance. In contrast, in the chronic phase, PFD improved renal function and reduced collagen accumulation detected by hydroxyproline content (OH-Pro) in the cortex of the remnant kidney. Although creatinine clearance decreased with time in C, the values in P were significantly better at 10 and 12 weeks. The OH-Pro in C at 12 weeks was significantly higher than that of no-ablation, sham-operated rats, whereas OH-Pro in CRF was lower in (P). Expression of mRNA for type IV and I collagen in the cortex also increased in C, but it was inhibited in (P). To study the role that TGF-beta plays in the regulatory process following CRF, we examined the expression of TGF-beta mRNA in this model. Levels of cortical TGF-beta mRNA in C were significantly elevated at 12 weeks. The increase was suppressed by PFD. These results demonstrate that PFD attenuates the development of CRF by preventing collagen accumulation in this model, and suggest that PFD can be clinically useful for treating CRF.

Dietary intake of pirfenidone ameliorates bleomycin-induced lung fibrosis in hamsters.

There are no clinically efficacious drugs available for preventing the development of pulmonary fibrosis (PF). In the present study, we tested the antifibrotic potential of pirfenidone (PD) in the bleomycin (BL) hamster model of PF. Hamsters were intratracheally instilled with isotonic saline solution or BL (7.5 U/kg/5 ml). The animals were fed control diet containing 0.5% PD or the same diet without the drug 2 days before and throughout the study. The four groups were as follows: saline-instilled and fed the control diet (SCD); saline-instilled and fed the same diet containing PD (SPD); BL-instilled and fed the control diet (BCD); and BL-instilled and fed the same diet containing PD (BPD). The animals were killed at 21 days after intratracheal instillation and their lungs processed for various assays. The lung hydroxyproline levels, an index of PF, in SCD, SPD, BCD, and BPD groups were 949, 970, 1759, and 990 micrograms/lung, respectively. The SOD activity and malondialdehyde equivalent levels in the corresponding groups were 443, 524, 612, and 499 units/lung and 56, 49, 108, and 63 nmol/lung, respectively. The lung prolyl hydroxylase activities in the SPD, BCD, and BPD groups were 87%, 147%, and 93% of the control (SCD) group (4.2 x 10(4) dpm/lung/30 minutes), respectively. The lung myeloperoxidase activities were 97%, 236%, and 159% of the control group (0.73 units/lung), respectively. BL alone caused significant increases in all the biochemical markers of lung toxicity, and dietary intake of PD minimized the BL toxicity as reflected by significant decreases in all the above markers.(ABSTRACT TRUNCATED AT 250 WORDS)