The Pharmacokinetics And Metabolism Of A Novel Antifibrotics Drug Pirfenidone In Rats And Beagle Dogs

Idiopathic pulmonary fibrosis (IPF) is the most serious form of idiopathic interstitial pneumonia due to excessive accumulation of collagen in the lung interstitium(Weissler, 1989; Nicod, 1998). Once symptoms appear, there is a relentless deterioration of pulmonary function and median survival of 2.8 years after diagnosis. Conventional treatment with corticosteroids and immunosuppressive therapy has been disappointing , and the severe systemic side effects are well-established (Hunninghake et al., 1995; PLoS Med, 2005). The disease affects more than 5 million people worldwide, of whom 40, 000 die every year, and approximately 75, 000 patients suffer from this disease in the United States alone (Izumi et al., 1993; Andrew, 2003 ). There are currently no drugs approved by the U.S. and China for the treatment of IPF.Pirfenidone (PF , RUIXTNG Genomics , Inc , Shanghai, China), 5-methyl-l-phenyl-2-(1 H) pyridone (Fig 1), is an investigational new drug that shows a wide range of biologic activities. In vitro evidence, it has shown that pirfenidone inhibits collagen synthesis, down-regulates profibrotic cytokines, and decreases fibroblast proliferation (Giri et al., 1999; Lurton et al., 1996 ). In vivo studies have demonstrated the ability of this compound to minimize lung fibrosis in several models of drug-induced fibrosis including the bleomycin-hamster model (Iyer et al., 1995; Iyer et al., 1998) Pirfenidone has been granted orphan drug designation by the U.S. Food and Drug Administration (FDA) for the treatment of IPF. Data presented from phase II clinical trials in America suggest that pirfenidone may impact disease progression in patients with IPF (Medical Lette, 2004; Selman et al., 2003). In a double-blind, randomized, placebo-controlled trial in Japan, 107 patients with IPF were prospectively evaluated for efficacy of a this novel compound. It canobviously improve vital capacity, and prevented acute exacerbation of IPF during the nine months of follow-up(Azuma et al, 2005). However, there are only limited studies on the Pharmacokinetics of PF. One study dealt with the 0-4 h AUC values oral administration of PF for three human patients who were undergoing hemodialysis (Taniyama et al, 1997). Another study utilized ¹⁴C-PF given i.v. to mice in which its metabolites were identified and pharmacokinetic parameters were calculated (Giri et al., 2002). And on the recent research paper, only limited parameters were available on the pharmacokinetic profiles after oral PF in beagle dogs (Bruss et al., 2004). But the pharmacokinetic behavior of pirfenidone in rats is not yet known. Therefore, the objective of this study was to determine the pharmacokinetic, metabolic disposition and tissue distribution of PF following oral and intravenous administration in rats and beagle dogs.Part I Pharmacokinetics of pirfenidone in rats and beagle dogs1. Pharmacokinetics of PF in Sprague-Dawley rats and Beagle dogsThe HPLC method of PF plasma concentrations plasma after oral or i.v administration in rats and dogs had been estabolished. The parent compound ( t_R=7.5 min) and 2 major metabolites ( M1 t_R1=2.4 min, M2 t_R2=3.2 min, ) were successfully separated by using the mobile phase. A one-compartment pharmacokinetic model was best fitted to plasma PF concentration-time curves after oral administration, and two-compartment model was best fitted to that of PF after i.v .both in rats and dogs.After oral administration at 25, 50, 100, 450 mg·kg^-1(n=5) and intravenous administration of 50 mg·kg^-1 of PF in rats, the plasma PF concentration reached a peak rapidly at about 17-27 min and also fell rapidly with a terminal half-life of 59-74 min after oral over the range of 25-100 mg·kg^-1. The major pharmacokinetic parameters were as follows: T_max 21, 17, 27 and 48 min; C_max 6.2, 8.6, 23.1 and 85.7 mg·kg^-1; T_1/2 60.6, 58.9, 73.7 and 543 min; AUC_{0→300min} 535, 880 , 2842 and 16200 mg·min·L^-1, respectively. After intravenous injection of 50 mg·kg^-1, thepharmacokinetic parameters were C_max45.1 mg·L^-1, T_1/(2α) 6.8 min, T_1/(2β) 109 min and AUC_{0→300min} 1706 mg·min·L^-1 . After oral administration at 50 mg·kg^-1, PF was rapidly absorbed in the rats, and possessed absolute bioavailability of 51.59% with the comparison between AUC of oral and intravenous administration. The proportionality between dosage and C_max or AUC was calculated , A good correlation was found in oral administration between the dosages (X) and resulting value (Y): Y = 0.1877X + 1.5581 (r=0.9978 ) for C_max and Y = 42.04X - 932.68 (r=0.9980 ) for AUC in the range of 25-100 mg·kg^-1. But when oral dosage at 450 mg·kg^-1 in rats, PF exhibited dose-disproportional Pharmacokinetics with nonlinear kinetic character.Each dog was given a single dose at random by oral or intravenous administration.Plasma samples were obtained up 480 min after single oral administration of 18, 36 or 100 mg·kg^-1 of PF , and after single intravenous administration of 18 mg·kg^-1 . The HPLC method with DAD detection was applied for the measurement of PF in plasma samples , and HPLC -MS/ESI analysis was used for identified parent compound and its metabolites in plasma samples after single oral doses at 36 mg·kg^-1. After oral administration, plasma concentration-time (C-T) curves of PF in beagle dogs are best fitted to one-compartment model. The major pharmacokinetic parameters were as follows: T_max: 15, 32, and 33 min; C_max: 11.7, 20.2 and 47.1 mg·L^-1 ; T_1/2. 41 , 49 and 47 min; AUC_{0→480min} : 711, 1457 and 4039 mg·min·L^-1, respectively. After intravenous injection of 18 mg·kg^-1, the C-T curve could be described by two-compartment model. The pharmacokinetic parameters were C_max 22.6 mg·L^-1, T_1/(2α)6.2 min, T_1/(2β) 50 min and AUC_{0→480min} 883 mg·min·L^-1.Comparison of AUC_{0→480min} following intravenous and oral administrations at dose of 18 mg·kg^-1, it indicated the absolute bioavailability of 80.59 % in dogs. After a single oral dose of 36 mg·kg^-1, the parent drug and its metabolites were detected in plasma rapidly. Two major metabolites were identified as the alcohol (M1) and the carboxylic acid ( M2) derivatives of PF . PF was rapidly absorbed, metabolized and cleaned from plasma after administration. A good linearity was display between the dosages and C_max or AUC in the range of 18-100 mg·kg^-1 after oral and show a good absolute bioavailability. Totally, PF was rapidly absorbed, metabolized and cleaned fromplasma after administration.2. Tissue distribution of PF in Sprague-Dawley ratsPirfenidone was widely distributed to tissues of most organs in rats after oral administration of 100 mg·kg^-1. 5 min afteroral administration of PF, most of tissues , such as liver, kidneys, lung, heart, and fat pad, had the high parent drug concentration and the major metabolites M1 , M2 also appeared. The levels, in descending order, rank as: stomach, duodenum, spleen, kidneys , liver, ventricle, brain and lung, etc. PF and its major metabolites could also across blood-brain barrier . For all tissues, PF concentrations had significantly decreased at the time point of 150 min after oral administration.3. Disposition studies of PF in Sprague-Dawley ratsPirfenidone was rapidly eliminated from systematic circulation after oral administration at 100 mg·kg^-1. The parent PF is rapidly and completely metabolized, little parent compound was detected in 24 h urine after administration and metabolism is the primary mechanism of drug clearance.4. Plasma Protein BindingThe plasma protein binding of pirfenidone in rats and human was assayed by equilibrium dialysis . The concentration of PF in two sides of dialyzator was determined by HPLC. One side is blank plasma, the other is the NaH₂PO₄ buffer solution which consisted of 2 mg·L^-1, 20mg·L^-1, 100 mg·L^-1 of PF. The equilibrium time is 24 h. The ratio of plasma protein binding of the PF from rats and human were found to be 64.09%-84.92% and 66.19 %-77.78 % in the range of 2-100 mg·L^-1, and shown that there were no species dependence.Part II Study the effect of pirfenidone on CYP in rats1. The effect of pirfenidone on P450 total enzyme in ratsThe absorbance of P450 in 450nm and 490nm was assayed by difference spectrum to calculate the contents of P450 in liver microsome .48 SD rats were divided into 6 groups in random, in which they received CMC, Dexamethasone 100 mg·kg^-1, Ketoconazole 40 mg·kg^-1, PF 25 mg·kg^-1, PF 50 mg·kg^-1, PF 100 mg·kg^-1 respectively. After administration for 6 days, getting their livers to prepare livermicrosome, we determined the concentration of proteinum in microsome and shade selection to plasma samples by spectrophotometer. The result indicate PF has effect of induce to drug-metabolizing enzyme in high dose. 2. The effect of pirfenidone on CYP3A and CYP2E1 in ratsSD rats were divided into 5 groups(n=6) in random and received an oral dosage of Dexamethasone 100 mg·kg^-1, 50% Alcohol 5ml·kg^-1, PF 50 mg·kg^-1, PF 100 mg·kg^-1 and control (1% CMC-Na) on 6 or 12 consecutive days respectively. After 24h following the last administration, Liver microsome were freshly extracted from liver tissue. The activities of ERD(erythrornycin N-demethylase) and NDMA (N- nitrosodimethyl -amine) were measured as described by Nash et al (Nash ,1953).The results showed that PF could significantly increase activities of erythromycin N-demethylase (ERD ) in rats after oral administration for 6 days (P<0.05). After administration for 12 days, the ERD activities in each drug treatment group were increased further, especially in 100 mg·kg^-1 PF groups (P<0.01). Both 50 mg·kg^-1 PF groups and 100 mg·kg^-1 PF groups had no significant effect on N- nitrosodimethylamine (NDMA). The results indicate that PF has effect of induce CYP3A and no influence on CYP2E1. The significant induction of pirfenidone on ERD has possesses the character of time dependence.Part III Metabolism study of pirfenidone in rats1. LC- MS/ MS identification of major metabolites in urine and bile of rats.Metabolism studies were performed to identify the major metabolic pathways and metabolites of PF. PF eluted at 16.3min under the mobile phase systems used. A totall of 7 phase I and 3 phase II metabolites of PF were found in the urine and bile of Sprague-Dawley rats that received 100 mg·kg^-1 PF via the oral route, comparing with the blank urine and bile samples collected from the same rat. Identification of the metabolites of PF was based on the understanding of the fragmentation pathway of the parent compound. The major metabolites were identified as the alcohol ( M1) and the carboxylic acid ( M2) derivatives of PF. The three phase II metabolites were also found , two of them(M9, M10) were glucuronide conjugated with M2 and the other (M8) was conjugated with M1. The metabolites M3 and M4 were dihydric derivatives of parent compound and followed by futher metabolism to the carboxylic acid M5, M6and M7, the constructions of which were unidentified .The mass spectrum of the peak eluting from the HPLC column at 12.9 min had an intense signal at m/z 216 mass. This signal represents the (M+H)⁺ of a metabolite with a mass of 215 Da (M2). The product spectrum of the molecular ion of metabolite m/z 216 shows a strong fragment to m/z 170 and m/z 140 ions, which occur through the loss of-COOH and loss of phenyl from the pyridone ring .The other intense product is the m/z 122 ion. It is highly probable that this process occurs through the loss of —H₂O and loss of phenyl from the pyridone ring . The peak eluting at 6.9 min had an intense signal at m/z 202 that represents the (M+H)⁺ ion of a metabolite with mass of 201 Da (M1). The product spectrum of the molecular ion of this metabolite (m/z 202 ) shows a strong fragment to m/z 186, 172 , 154 and108 ions. The peak eluting at 4.6 min had an intense signal at m/z 3 78 that represents the (M+H)⁺ ion of a metabolite with mass of 377 Da (M 8). The product spectrum of the molecular ion of this metabolite shows a strong fragment to m/z 202 and m/z 184 ions, which occur through the loss of glucuronide and loss of -OH from the teminal -CH₂OH group . 2. Identification the chemical structure of major metabolites (M2) of rats.Pool plasm samples from 30 rats after oral administration at the dosage of 100 mg·kg^-1. Metabolites and parent compound were separated by semipreparative HPLC using a Eclipse XDB-C18 column(9.4mm ×25cm ) Solvent M2 were collected and extracted by ethyl acetate and evaporated to dryness. The purity of M2 was determined as 97.2% by HPLC. The chemical structure of M2 was identified by IR, NMR and MS . It was carboxylic acid derivatives of PF parent compound.