Juan Armendáriz-Borunda

Pirfenidone effectively reverses experimental liver fibrosis

BACKGROUND/AIMS Our group has been involved in searching for different strategies to ameliorate hepatic cirrhosis. The aim of this study was to evaluate the effect of Pirfenidone in the reversion or prevention of cirrhosis experimentally induced in rats by chronic administration of CCl(4) and bile-duct ligation (BDL). METHODS Male cirrhotic Wistar rats (8 weeks of intoxication and then hepatotoxin was discontinued) received either oral saline or Pirfenidone at 500 mg/kg per day. RESULTS High levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase decreased significantly (P<0.001) in animals treated with Pirfenidone (n=11) with regard to saline-administrated animals (n=9). Prothrombin activity and bilirubins were also reduced. Computerized fibrosis index demonstrated a 70% decrease (P<0.001) along with less hydroxyproline content, reduction in activated HSC and higher active cell regeneration. A rearrangement of the parenchyma was also noted and gene expression of collagens I, III and IV, transforming growth factor beta-1, Smad-7, TIMP-1 and PAI-1 decreased considerably in treated animals. Cirrhotic rats in which CCl(4) was not discontinued displayed 40% liver fibrosis reduction. In a different cirrhosis model, 4-week BDL rats treated with the drug showed a significant 50% reduction in hepatic fibrosis (P<0.01). CONCLUSIONS This new drug might be useful in healing human disease.

The multifaceted role of pirfenidone and its novel targets.

BackgroundPirfenidone (PFD) is a molecule that exhibits antifibrotic properties in a variety of in vitro and animal models of lung, liver and renal fibrosis. These pathologies share many fibrogenic pathways with an abnormal fibrous wound-healing process; consequently, tissue repair and tissue regeneration-regulating mechanisms are altered.ObjectiveTo investigate the usefulness of PFD as an antifibrotic agent in clinical and experimental models of fibrotic disease.ConclusionsThere is a growing understanding of the molecular effects of PFD on the wound healing mechanism, leading to novel approaches for the management of fibrosis in lung, liver and renal tissues. Although the optimum treatment for fibrosis remains undefined, it is possible that combined therapeutic regimens that include this wide-application molecule, pirfenidone, could offer a useful treatment for fibrotic disease.

TH1/TH2 cytokine profile, metalloprotease-9 activity and hormonal status in pregnant rheumatoid arthritis and systemic lupus erythematosus patients

During the course of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), several immune and neuroendocrine changes associated with pregnancy may exert positive (amelioration) or negative (exacerbation) effects on the clinical outcome. In order to shed light on the mechanisms underlying these responses, we performed a prospective longitudinal study in RA and SLE pregnant women, including healthy pregnant women as a control group. Cytokine messenger RNA (mRNA) expression assessed by quantitative competitive polymerase chain reaction (PCR) in peripheral blood mononuclear cells (PBMC), cytokine levels and lymphocyte proliferation responses (LPR) following phytohaemagglutinin (PHA) stimulation of PBMC, plasma metalloprotease‐9 activity (MMP‐9) and hormonal status during pregnancy were determined. TNFa was the most abundant cytokine mRNA expressed in PBMC in all groups studied (healthy pregnant women, RA and SLE pregnant patients). However, a general TH2 response reflected by high IL‐10 levels was found in RA, as well as SLE, patients. A significant change in IFN‐γ was observed in RA patients but only during the first trimester of pregnancy. This compared with a major TH1 response in healthy pregnant women. Interestingly, our study showed a homogeneous hormonal pattern in RA and SLE patients. Although decreased cortisol levels were observed in all patients studied, this is possibly related to the remission of disease activity status brought about by steroid treatment before and during pregnancy. In summary, we suggest that complex immune and hormonal networks are involved in pregnancy and that rheumatic diseases are very dynamic immune processes that cannot be described with a clear‐cut cytokine profile. Furthermore, the observations in this study may reflect treatment‐related immune effects more than those associated with disease.

Potent antioxidant role of Pirfenidone in experimental cirrhosis

Three important features must be considered when proposing therapeutic strategies in liver cirrhosis: inflammation, oxidative stress and fibrogenesis. Pirfenidone is a synthetic molecule which oxidative action has not been tested in cirrhosis. Cirrhosis was induced in rats by ligation of the common bile duct or carbon tetrachloride (CCl(4)) chronic intoxication and treated with pirfenidone or diphenyleneiodonium (a potent known antioxidant) for the last two weeks for bile duct ligation model or for the last three weeks for CCl(4) chronic intoxication. A 60% reduction in fibrosis index for bile duct ligation model and 42% for CCl(4) along with reduced inflammation was observed. Considerable reduction on hepatic enzymes and total and direct bilirubins were detected with pirfenidone in both models. Pirfenidone antioxidant capacity rendered a 28% and 30% reduction in nitrites and malonyldealdehide concentration in bile duct ligation and 52% and 38% in CCl(4). With respect to gene expression, fibrotic genes like transforming growth factor-beta (TGF-beta) and collagen Ialpha (Col-1alpha) were down-regulated by pirfenidone and increased expression of regenerative genes like hepatocyte growth factor (HGF) and c-met . Superoxide dismutase (SOD), catalase (CAT) and inducible nitric oxide synthase (iNOS) gene expression were importantly down-regulated where nuclear factor kappa B (NF-kappaB) binding activity also decreased with pirfenidone treatment. Also, SOD and CAT functional activity decreased after pirfenidone action. On the other hand, diphenyleneiodonium induced a drop in oxidative stress similar in extent to pirfenidone, but it was not as effective as pirfenidone in reducing fibrosis. In this work, we showed antioxidant properties of pirfenidone beyond its well-known antifibrotic effect. These features make pirfenidone an attractive drug for trying fibrotic diseases accompanied by oxidative stress processes.

Pirfenidone Prevents Capsular Contracture After Mammary Implantation

BackgroundPirfenidone (PFD), a new antifibrotic and antiinflammatory agent, prevents and resolves fibrous tissue. This study evaluated the effect of PFD on adverse events in mammary implants using an animal model. Mammary implantation, the most frequent aesthetic surgery, may present several complications after surgery such as swelling, capsule contracture, hardness, and pain.MethodsWistar rats underwent submammary implantation with either smooth or textured silicone gel implants and were administrated 200 mg/kg of PFD daily. The control group received saline. The animals were killed at 8 weeks. The capsular tissue of both implants was removed for histologic and molecular analyses.ResultsTypical postaugmentation periimplant capsules with opacity on adjacent tissues developed 8 weeks after silicone implantation. No significant differences were observed between the textured and smooth implants in any analyzed parameter. Clearly, PFD reduced capsule thickness around submmamary tissue, fibroblast-like cell proliferation, and recruitment of inflammatory cells. The total cell numbers per field were reduced as well. In contrast, the control group presented abundant mononuclear cell infiltration and fibroblast-like cell proliferation. The total content of collagen in the PFD group was 50% less than in the control group. Fibroblast cells displayed 45% less activated phenotype in the PFD group than in the control group, as determined by immunohistochemistry techniques. In the PFD animals, transforming growth factor-β (TGF-β) decreased 85% and collagen 1 gene expression 60%, compared with the control group.ConclusionThe findings show a positive effect of PFD on mammary contracture in 10 rats. Despite the small number of animals, the differences found in 10 control rats encourage the authors to propose a larger study later and to suggest PFD as a potential preventive strategy in human mammary implantation surgery.

Quercetin improves hepatic fibrosis reducing hepatic stellate cells and regulating pro-fibrogenic/anti-fibrogenic molecules balance

Development of hepatic cirrhosis involves oxidative stress, inflammation, hepatic stellate cells (HSC)s activation and fibrosis. On the other hand, quercetin, a natural flavonoid is a potent antioxidant and activator of superoxide dismutase and catalase. The aim was to determinate the effect of quercetin on HSCs and development of hepatic fibrosis.

Kupffer Cells from CC14-Treated Rat Livers Induce Skin Fibroblast and Liver Fat-Storing Cell Proliferation in Culture

Conditioned media of Kupffer cells from normal rat liver produce in culture two factors that inhibit fibroblast proliferation. The inhibitory factors have molecular masses of approximately 25 and 5 kDa. In contrasts to these results, the conditioned media of Kupffer and mononuclear macrophagic cells, obtained 48 hours after CC1(4) administration to rats, contains a 17 kDa factor that stimulates fibroblast proliferation (FSF). FSF also stimulates [3H]-thymidine incorporation into DNA of cultured rat liver fat-storing cells. Two peaks with FSF activity were demonstrated after isoelectrofocusing; one with a pI of 6.1 and a second with a pI of 7.5. The fraction containing FSF is devoid of interleukin-1 (IL-1) activity and no inhibitory activity is detected in this conditioned media. Production of FSF is inhibitable by colchicine but not by indomethacin, it is thermolabile and trypsin-sensitive. In animals treated chronically with CC1(4) to produce cirrhosis, FSF activity is demonstrable from the first to the 8th week of treatment. However, the activity is lower at 8 weeks post-CC1(4) as compared with 2 weeks. The results suggest that homeostasis of cells in the liver is controlled by factors produced locally, that act by autocrine and paracrine mechanisms. When homeostasis is altered, fibroblast proliferation occurs, and excess collagen deposition leads to fibrosis. We propose that the antifibrogenic activity of colchicine is associated, in part, with its capacity to inhibit the release of FSF by Kupffer cells and liver mononuclear macrophage cells.

Urokinase-type plasminogen activator gene therapy in liver cirrhosis is mediated by collagens gene expression down-regulation and up-regulation of MMPs, HGF and VEGF

Human urokinase‐type plasminogen activator (uPA) gene administration via an adenoviral (Ad)‐vector induced cirrhosis regression and ameliorated hepatic dysfunction in a model of experimental liver cirrhosis. The administration of a single dose of 6 × 1011 viral particles per kilogram of a clinical‐grade Ad‐vector was evaluated after the onset of rat liver cirrhosis via degradation of deposited collagen and a substantial decrease of α‐sma‐positive cells. Also, gene expression for pro‐fibrogenic molecules (Col I, III, IV, TIMP‐1 and PAI‐1) was clearly down‐regulated. In contrast, gene expression for collagen‐degrading enzymes such as MMP‐13 and MMP‐2 was up‐regulated. These events correlated with increased amounts of proteic free‐TIMP‐1, i.e. non‐complexed with metalloproteinases (MMPs), indicating the presence of higher amounts of active MMPs inside the liver of cirrhotic animals treated with Ad‐huPA. The harmonized and concerted expression of HGF and c‐met resulted in exacerbated hepatocyte proliferation, although these events did not induce an abnormal liver growth. Angiogenesis, i.e. formation of new blood vessels, was evaluated by vascular endothelial growth factor (VEGF) expression which was notably detected to be 10 times higher during the first 6 days after Ad‐huPA‐treatment in cirrhotic animals as compared with controls. These events provide a clearer rationale as to how Ad‐huPA‐induced liver regeneration on CCl4‐induced liver fibrosis takes place. Copyright

Treatment with anti-tumor growth factor β antibodies influences an altered pattern of cytokines gene expression in injured rat liver

The role of tumor growth factor L1 (TGF-L1) in mediating hepatic inflammation and regeneration after acute liver injury is beginning to be elucidated, yet its in vivo effect on the gene expression of the major pro-inflammatory and antiinflammatory cytokines produced during that process is unknown. Our previous experiments demonstrated that anti-TGF-Ltreated animals presented profound histological changes as compared with control animals. Therefore, our hypothesis was that by blocking in vivo TGF-L1 action, with polyclonal anti-TGF-L antibodies, we could monitor by RT-PCR significative alterations on the gene expression of IL-1L, IL-6, TGF-L, TNF-K, IL-4 and IL-10 in liver-regenerated rats after administration of a single CCl4 dosing. Accordingly, we here report a completely different pattern of cytokines gene expression amidst those groups of rats. Pro-inflammatory cytokines gene expression in control animals showed a clear-cut pattern peaking at 1^2 days postinjury and declining thereafter. Interestingly, IL-6 was present in the control animals only between 12 and 24 h after CCl4 dosing. In the experimental animals, TGF-L1 was mainly increased at 4 and 6 days, while IL-6 mRNA was completely absent. IL-1L mRNA expression was also altered in the experimental rats, albeit TNF-K was nearly unaffected. IL-4 was fully absent in control rats, but remarkably expressed in experimental animals throughout the study. IL-10 was also more expressed in experimental animals. fl 1998 Elsevier Science B.V. All rights reserved.

A Controlled Clinical Trial With Pirfenidone in the Treatment of Pathological Skin Scarring Caused by Burns in Pediatric Patients

Background:Pathologic skin scarring reversion remains a big challenge for surgeons, as disfiguring scars have a dramatic influence on patients quality of life. Methods:A controlled clinical trial was conducted to evaluate 8% pirfenidone (PFD) gel administered topically 3 times a day during 6 months to 33 pediatric patients with hypertrophic scars caused by burns. A total of 30 patients with hypertrophic scars with identical Vancouver Scar Scale values were treated with pressure therapy and included as controls. Improvements were evaluated by Vancouver Scar Scale and a Visual Analog Scale. Safety parameters were determined by the presence of adverse events and monitoring laboratory and hematology parameters. Results:Patients treated with PFD during 6 months presented a continuous monthly statistically significant scar regression in comparison with the initial Vancouver measurement (P = <0.001). PFD group showed a higher improvement of all scar features as compared with control group treated with pressure therapy (P = <0.001). In the PFD group, 9 of 33 patients (27%) had their scores decreased in Vancouver classification by more than 55%, 22 patients (67%) had a 30% to 45% decrease, whereas 2 patients (6%) had a 30% decrease or less. Control group treated with pressure therapy showed a slight improvement in 16% of cases on an average. Patients did not show serious adverse effects or laboratory alterations throughout the study. Conclusions:Topical administration of 8% PFD gel 3 times a day is more effective and safe in the treatment of hypertrophic scars caused by burns in children, as compared with standard pressure therapy.