1 Fibrosis leads to chronic impairment of cardiac and renal function and thus reversal of existing ®brosis may improve function and survival. This project has determined whether pirfenidone, a new anti®brotic compound, and spironolactone, an aldosterone antagonist, reverse both deposition of the major extracellular matrix proteins, collagen and ®bronectin, and functional changes in the streptozotocin(STZ)-diabetic rat. 2 Streptozotocin (65 mg kg 71 i.v.)-treated rats given pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone; approximately 200 mg kg 71 day 71 as 0.2 ± 2g 1 71 drinking water) or spironolactone (50 mg kg 71 day 71 s.c.) for 4 weeks starting 4 weeks after STZ showed no attenuation of the increased blood glucose concentrations and increased food and water intakes which characterize diabetes in this model. 3 STZ-treatment increased perivascular and interstitial collagen deposition in the left ventricle and kidney, and surrounding the aorta. Cardiac, renal and plasma ®bronectin concentrations increased in STZ-diabetic rats. Passive diastolic sti ness increased in isolated hearts from STZ-diabetic rats. Both pirfenidone and spironolactone treatment attenuated these increases without normalizing the decreased +dP/dt max of STZ-diabetic hearts. 4 Left ventricular papillary muscles from STZ-treated rats showed decreased maximal positive inotropic responses to noradrenaline, EMD 57033 (calcium sensitizer) and calcium chloride; this was not reversed by pirfenidone or spironolactone treatment. STZ-treatment transiently decreased GFR and urine¯ow rates in isolated perfused kidneys; pirfenidone but not spironolactone prevented the return to control values. 5 Thus, short-term pirfenidone and spironolactone treatment reversed cardiac and renal ®brosis and attenuated the increased diastolic sti ness without normalizing cardiac contractility or renal function in STZ-diabetic rats.

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1 This study has administered pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) or amiloride to attenuate the remodelling and associated functional changes, especially an increased cardiac stiness, in DOCA-salt hypertensive rats. body wt) without lowering systolic blood pressure. 4 Collagen deposition was signi®cantly increased in the interstitium after 2 weeks and further increased with scarring of the left ventricle after 4 weeks; pirfenidone and amiloride reversed the increases and prevented further increases. This accumulation of collagen was accompanied by an increase in diastolic stiness constant; both amiloride and pirfenidone reversed this increase. 5 Noradrenaline potency (positive chronotropy) was decreased in right atria (neg log EC50: control 6.92+0.06; DOCA-salt 6.64+0.08); pirfenidone but not amiloride reversed this change. Noradrenaline was a more potent vasoconstrictor in thoracic aortic rings (neg log EC50: control 6.91+0.10; DOCA-salt 7.90+0.07); pirfenidone treatment did not change noradrenaline potency. 6 Thus, pirfenidone and amiloride reverse and prevent cardiac remodelling and the increased cardiac stiness without reversing the increased vascular responses to noradrenaline. British Journal of Pharmacology (2002) 135, 961 ± 968

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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.

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.

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.

SUMMARYPirfenidone 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-1a in the presence or absence of pirfenidone (range 0-1000 mM), 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 Eselectin was not affected. Furthermore, we examined whether pirfenidone affects the cellular binding between cultured lymphocytes and IL-1a-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.