Fibrogenesis in cirrhosis. Potential for therapeutic intervention

Pharraac. Ther.Vol. 53, pp. 81-104, 1992 Printed in Great Britain. All rightsreserved

0163-7258/92$15.00. C) 1992PergamonPress Ltd

Associate Editor: J. REICrmN

FIBROGENESIS IN CIRRHOSIS. POTENTIAL FOR THERAPEUTIC INTERVENTION MARCOS ROJKIND Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, U.S.A. Abstract--Liver cirrhosis is an end stage of several diseases that affect the liver chronically. It is characterized, among other things, by excess collagen deposition, distortion of liver architecture, tissue malfunction and hemodynamic alterations. Many of the complications of cirrhosis may result from excess matrix deposition. Therefore, prevention of collagen accumulation or removal of collagen deposits could ameliorate the disease. In this article we discuss the pathophysiology of liver fibrosis and we describe various compounds with antiinflammatory and antifibrogenic activity. We discuss their possible mechanism of action and we describe animal and clinical studies in which these compounds have been utilized.

CONTENTS 1. Introduction 2. Prophylactic Treatment 3. Treatment to Eradicate the Etiologic Agent 3.1. From arabinosides and praziquantal to gene targeting 3.2. Interferons 4. Hepatoprotection 4.1. Prostaglandins 4.2. Ursodeoxycholic acid (UDCA) 5. Antiinflammatory and Immunosuppressor Therapy 5.1. Steroids 5.2. Colchicine 5.3. Progesterone 5.4. Methotrexate 5.5. Cyclosporin 6. Antifibrogenic Therapy 6.1. The proline analogs 6.2. Inhibitors of prolyhydroxylase 6.3. Peptides containing oxaproline 6.4. 2-Oxoglutarate inhibitors 6.5. Lysyloxidase inhibitors 6.6. Penicillamine and/~-APN 7. Collagenase Inducers 7.1. Polyunsaturated lecithins 8. Miscellaneous Acknowledgements References

81 83 84 84 84 85 85 85 87 87 88 91 91 91 91 92 93 93 93 94 94 94 94 95 95 95

1. I N T R O D U C T I O N

Liver cirrhosis is a terminal pathologic state induced by several biological and chemical agents capable of producing chronic generalized liver damage. Although individual agents may act by diverse mechanisms, they all trigger a c o m m o n final pathway that leads to excess collagen deposition (Rojkind and Mourelle, 1988; Tsukamoto et aI., 1990). Irrespective of the mechanism by which the etiology agent induces liverinjury, the net resultis an alteration in the homeostatic mechanisms that maintain the structure and function of the liverecosystem (Rojkind and Gr~nwel, 81

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1988). Liver structure and function are maintained by the multiple interactions established among the various liver cell types with themselves and with the different components of the extracellular matrix. The types, quantity and distribution of cells and extracellular matrix components are important in maintaining homeostasis. Accordingly, specific cell--cell and cell-matrix interactions, as well as the cytokines and metabolites produced by the various cells in response to these interactions, are responsible for sustaining the group of biological activities that generically receive the name of liver function. Thus, when a chemical or biological agent alters one of the various interactions, either by producing overt cell damage (alcohol and hepatitis viruses), by modifying cell function (hemochromatosis and Wilson's disease), or by altering cell-matrix balance (schistosomiasis), homeostasis is disturbed. New cell-cell and cell-matrix interactions are established and in response to these alterations a new set of cytokines and metabolites is produced. These events, that lead to cell damage, fibrosis and cirrhosis are listed below: (1) Activation of Kupffer cells with increased production of cytokines (Rojkind and Valadez, 1985; Shiratori et al., 1986; Armendariz-Borunda et al., 1989, 1991) and proteolytic enzymes (Fujiwara et al., 1973). (2) Chemotactic attraction of inflammatory cells and fibroblasts to the injured sites by products derived from extracellular matrix components and/or chemoattractants produced by the ceils (Postlethwaite et al., 1978, 1981; Meyer et al., 1988; Heidel et al., 1989; Lohr et al., 1990; Fukai et al., 1991; Roll et al., 1991). (3) Increased production of cytokines (Sherry and Cerami, 1988; Dinarello and Savage, 1989; Heinrich et al., 1990) and chemoattractants by recruited monocytes and macrophages (Issekutz et al., 1987; Nathan, 1987). (4) Induction of fat-storing cell proliferation (Armendariz-Borunda et al., 1989; Bachem et al., 1989; Greenwel et al., 1991a; Shiratori et al., 1986), transformation of these cells into myofibroblasts (Geerts et al., 1989; M a k e t al., 1984; Mak and Lieber, 1988) and increased production of extracellular matrix components in response to cytokines and growth factors produced by various cell types (Czaja et al., 1989; Greenwel et al., 1990; Matsuoka et al., 1989; Milani et al., 1989, 1990; Nakatsukasa et al., 1990; Weiner et al., 1990a). (5) Decreased collagenolytic activity which results from decreased enzyme production (Maruyama et al., 1982) or from increased secretion of protease inhibitors such as the tissue inhibitor of metalloproteinases (TIMP) (Wright et al., 1991). The aforementioned concepts, that represent the ground substance from which antifibrogenic therapy is being developed, evolved over a period of approximately 20 years. For many years liver cirrhosis was considered an irreversible, untreatable and incurable disease. Therefore, medication provided for the patient was symptomatic and its only purpose was to relieve specific complications of the disease. The belief in the 1960s was that collagen was an insoluble protein with a long halflife, even perhaps inert and that once deposited in the extracellular matrix, it was there to stay for as long as the subject lived (Hartroft, 1954). In addition, increased collagen content of the cirrhotic liver was thought to result from collapse of the stroma adjacent to injured cells. Accordingly, if collagen was inert and fibrosis was a passive process, it was sinful to think of treating liver cirrhosis and it was sacrilegious to consider the possibility of developing a specific antifibrogenic therapy. It was in the late 1960s when Hirayama et al. (1968) and Huberman et al. (1969) demonstrated that fibrogenesis was an active process and that excess collagen deposition in man and rat livers, respectively, resulted from increased biosynthesis of collagen. In addition, Gross and Lapiere (1962) described the existence of mammalian collagenases and Rojkind and Perez-Tamayo (1962) demonstrated active collagenolytic activity in rats with a carrageenin-induced granuloma. These new concepts implied that, if collagen was actively produced by cells within the liver, attempts could be made to prevent its accumulation. Furthermore, if there was an active mechanism to degrade collagen, it could be possible to remove fibrous tissue from cirrhotic liver, thus making liver fibrosis a reversible process (See Perez Tamayo, 1979; Rojkind and Dunn, 1979). Indeed, in a thesis submitted in 1960 as partial fulfillment to obtain a medical degree from the National University of Mexico, Rojkind (1960) wrote: "Knowing that there are some substances capable of activating existing mechanisms (collagenase), we have to find the ideal substance that will work selectively, that will have low toxicity and that will stimulate the resorption of the connective tissue". Thus, what in the 1960s was only a dream, may turn out to be a reality in the 1990s. A better

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understanding of the collagen biosynthetic pathway in general and the knowledge of the mechanisms that regulate liver collagen metabolism, in particular, could result in development of specific agents with potential antifibrogenic activity. Although in the 70s people were reluctant to accept the concept of antifibrogenic therapy, in the 80s many trials with various degrees of success were performed and in the 90s it has attracted the interest of various pharmaceutical companies. As a result of that, several trials are underway and many new agents are being produced and investigated and some will be available for phase 1 and 2 studies in the near future. Therapy of liver cirrhosis could be directed at various targets. These include (a) prevention of the disease, (b) elimination of the etiologic agent once the disease has been established, (c) prevention of inflammation and/or fibrosis and (d) elimination of the residual scar. Ideally therapy should be aimed at more than one of the above mentioned targets, or should attempt to modify regulatory mechanisms that modulate fibrogenesis. The first and most important site of intervention ought to be prevention of the disease itself. This strategy could be applied to infectious diseases such as schistosomiasis and hepatitis. In the former, education of the people to interrupt the life cycle of the parasite. In the latter, the development of vaccines against the various hepatitis viruses. Unfortunately, prevention of alcohol-induced liver disease is more difficult, since there are important socio-economic issues, including tax revenues collected by the governments, that prevent strong actions. Furthermore, the moralistic attitude that considers alcohol-liver disease a sinful, self-inflicted illness, also impedes development of proper preventive programs. Cirrhosis o f the liver is a silent disease. In general, it develops slowly and only produces symptoms when liver function has been compromised and complications of cirrhosis occur, or when an acute injurious episode further compromises cell function. Therefore, many cases seen by physicians in the clinic represent terminal stages of the disease with little hope for success with any type of medical therapeutic intervention. Some of these patients will benefit only from a liver transplant. This procedure, although greatly successful since the advent of cyclosporin, is not the solution for the treatment of a disease that affects hundred of thousands of individuals worldwide. Since possibilities of therapeutic success are greatest when the disease is caught early on, attempts should be made to develop diagnostic procedures easily applicable to large masses of individuals. Thus far, such procedures are missing (Risteli and Risteli, 1990). In this review, we shall briefly describe possible sites of therapeutic intervention in preventing the disease or eliminating the causative agent. We shall describe in greater detail steps involved in collagen synthesis, compounds with potential or proven antiinflammatory and antifibrogenic activity and we shall review some of the trials in which these agents have been tested. I do not have to apologize to the readers if I am biased towards the proline analog azetidine carboxylic acid and to colchicine. After all, this work was carried out in my laboratory and were the first two compounds with proven antifibrogenic activity in vivo (Rojkind, 1973; Rojkind et al., 1973). For a recent review on the treatment of hepatic fibrosis, the readers are referred to an article by Brenner and Alcorn (1990).

2. PROPHYLACTIC TREATMENT As indicated previously, the ideal treatment is not to treat but rather to prevent. Accordingly, vaccination against the various hepatitis viruses in populations at risk should greatly decrease the incidence of post-hepatitic cirrhosis. However, we should not be overconfident, since mutation of the viral genome is known to occur (Carman et al., 1990). New epitopes not recognized by the previously developed vaccines could help the virus skip immunological surveillance and produce disease. The incidence of liver cirrhosis induced by the parasite Schistosoma mansoni could be drastically reduced and perhaps eradicated by proper education of people living in endemic areas. Elimination of the snail needed for the completion of the life cycle of the parasite is quite difficult. However, if farmers of endemic areas were taught not to defecate on the ground and were provided with portable latrines to avoid contamination of the soil, the cycle of the parasite could be interrupted. Proper genetic counseling could help in decreasing inherited diseases such as Wilson's disease and hemochromatosis. As the loci of the defective genes are being localized, specific and sensitive

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probes to detect carriers will be developed. Accordingly, proper counseling of affected individuals may decrease the number of new cases.

3. TREATMENT TO ERADICATE THE ETIOLOGIC AGENT 3.1. FROMARABINOSIDESAND PRAZIQUANTEL TO GENE TARGETING If the above described preventive measures fail, methods should be developed to treat the patient and eliminate the etiologic agent responsible of producing liver damage. Unfortunately, all the arabinosides developed for treatment of hepatitis virus have failed to produce encouraging results (Hoofnagle et al., 1984; Lok et al., 1985; Marcellin et al., 1989, 1990). Hepatologists are still awaiting for the magic antiviral bullet that will change the meaning of viral infections. In the treatment of schistosomiasis, more promising results have been obtained. It has been shown that praziquantel effectively kills the adult worms (Emonard and Grimaud, 1989; Mohamend-All et al., 1991). It has also been shown in the experimental models of schistosomiasis, that early elimination of the parasite may facilitate removal of the granulomas with complete resorption of the connective tissue (Emonard and Grimaud, 1989). With regard to the inherited diseases, it is foreseeable that in the future, their treatment could be accomplished by genetical engineering. Transfection of diseased hepatocytes with plasmids containing the normal gene could permit the expression of the otherwise defective proteins (Wu et al., 1989; Wu, G. Y. et al., 1991). It is also possible that methods currently being developed for the transplantation of isolated hepatocytes (Gupta et al., 1990; Ponder et al., 1991; Wilson et al., 1990) may be of help in correcting inherited diseases. 3.2. INTERFERONS

In this section the interferons were included, since these cytokines are normally produced by various cells and play a role in controlling viral infections (Bonnem and Oldham, 1987). However, interferons are also antifibrogenic, since they affect collagen metabolism. It has been shown that interferon gamma inhibits the expression of type I collagen and TGF-# mRNAs in cultured fat-storing cells and in livers of mice infected with Schistosoma mansoni (Czaja et al., 1987a,b). Interferon-), also induces the expression of colony stimulating factor 1 (CSFI) (Mori et al., 1990), a known chemoattractant for monocytes/macrophages. In a recent study, Castilla et al. (1991) analyzed the effect of interferon-or therapy on a group of patients with hepatitis C. Four of these patients had chronic hepatitis and 4 had cirrhosis. They analyzed steady-state levels of • 1 (I) procollagen and TGF-# mRNAs in liver biopsies taken at the end of one year of treatment. They showed that while patients with a similar disease, not included in this trial, had elevated levels of ct 1 (I) procollagen and TGF-# mRNAs, all the treated patients that responded to interferon had 1 (I) procollagen and TGF-fl mRNA levels similar to normal controls without liver disease. Unfortunately, similar determinations were not available in biopsy material obtained at the beginning of the treatment in patients included in the trial. Histological score was greatly improved in treated patients. The authors found a direct correlation between TGF-fl and ctl (I) procollagen levels. Furthermore, they found direct correlations between serum type III procollagen peptide (P-Ill-P) and between TGF-fl and • 1 (I) procollagen mRNA, as well as between alanine amino transferase and P-III-P. This latter finding indicates that for practical purposes, ALT measures the same that P-III-P and both are indicative of activity of the process. Both suggest the presence of necrosis, inflammation and repair (Risteli and Risteli, 1990; Rojkind, 1984). The question that remains to be answered is whether interferon-or has antifibrogenic activity or whether its effect is mediated through the inhibition of viral replication and indirectly, prevents fibrosis. Special attention should be given to determine possible differences between responders and non-responders. It has been shown in other studies that interferon-0t therapy is useful for 50% of patients with hepatitis C studied (Davis et al., 1989; Hoofnagle et al., 1986). However, of this 50%, one half has long-term remission but the other half relapsed upon discontinuation of treatment. Thus, it would be important to determine whether the markers of fibrogenesis also deteriorate during relapse.

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4. HEPATOPROTECTION The concept of hepatoprotection although a noble one has been of little use in the treatment of liver cirrhosis. Firstly, because the vast majority of patients sccn in the clinic are already cirrhotic and, secondly, because each of the various etiologic agents capable of producing liver damage may act by completely different mechanisms. Accordingly, specific hepatoprotector drugs will be required to inhibit cell damage induced by the various chemical and biological agents that produce cirrhosis. A third problem with regard to hepatoprotective drugs, which is also applicable to antifibrogenic therapy, is the lack of early methods of diagnosis to identify patients with early disease or subjects with high risk to develop cirrhosis. In spite of the above mentioned limitations, there are specific situations in which hepatoprotector therapy can be used. These include: (a) the use of prostaglandins in the treatment of acute liver failure and some experimental models of liver cirrhosis and (b) treatment of primary biliary cirrhosis (PBC) and sclerosing cholangitis (PSC) with ursodeoxycholic acid (UDCA). Other hepatoprotector agents, such as silimarin and malotilate nccd to be evaluated further (see Brenner and Alcorn, 1990). 4.1. PROSTAGLANDINS Prostaglandins are products of the lipooxygenase pathway that are being used in the treatment of peptic ulcer disease because of their protective effect on epithelial cells (Robert et al., 1976). Prostaglandins also protect hepatocytes from various acute or chronic insults (Ruwart et al., 1986, 1988; Stachura et al., 1981). Prostaglandins that have been used are analogs of the E series. In a murine hepatitis model, prostaglandins prevented liver injury (Abecassis et al., 1987). In murine schistosomiasis and CC14-induced liver injury in rats, prostaglandins decreased fibrosis and cell necrosis (Degli Esposti et al., 1991). The mechanism by which prostaglandins exert their action is unknown. They have many biological activities that could result in improvement of liver cell function, in decreasing the extent of inflammation that leads to fibrosis and in reducing the amount of extraceUular matrix that accumulates in scar tissue. Recently, it has been shown that prostaglandins inhibit the expression of ~2 (I) collagen, fibronectin, colony stimulating factor and TGF-fl mRNAs in mice infected with cercarie of Schistosoma mansoni and in rats treated with CCI4 (Degli Esposti et al., 1991). However, when the authors explored the effect of prostaglandins on TGF-fl, a mediator of hepatic fibrogenesis, contradictory results were obtained. While prostaglandins inhibited the expression of TGF-fl mRNA in vivo, they had no effect on the expression of the cytokine in cultured fat-storing cells. Another possible mechanism of prostaglandin action is related to their effect on cAMP (Knudsen et al., 1986). Prostaglandins increase cellular levels of cAMP, a second messenger that is involved in modulation of many biological activities of various cells (Knudsen et al., 1986; Wahl et al., 1977). Through this mechanism, prostaglandins could prevent cell damage, decrease the production CSF1 and diminish the number of inflammatory cells (Mori et al., 1990) and decrease deposition of collagen. However, although cAMP levels modulate intracellular degradation of collagen (Baum et al., 1978; Bienkowski and Ripley, 1990), they also inhibit the production of coUagenase by various cells types (Koob and Jeffrey, 1980; McCarthy et al., 1980). However, the actual role of cAMP in development of cirrhosis must be properly evaluated. As described below, cAMP levels are increased in rats with cirrhosis. As a consequence of this increase, glycogen levels of the hepatocytes are depleted (Mourelle et al., 1981). This lack of carbohydrate-derived fuel could play an important role in the progression of the disease and in body wasting observed in terminal stages of the disease. This latter effect could also be mediated by tumor necrosis factor ct (TNF-ct, cachectin) (Sherry and Cerami, 1988), since TNF-~ values are elevated in patients with cirrhosis (Sweeting, 1989; Yoshioka et al., 1989). 4.2. UR$ODEOXYCHOLICACID (UDCA) As indicated previously, in the specific cases of PBC or PSC, hepatoprotectors are of special interest. It has been suggested that the accumulation of the more hydrophobic bile acids could play a role in destruction of bile ducts (Armstrong and Carey, 1982; Attili et al., 1986; Hofmann and Popper, 1987; Miyai et al., 1982; Miyazaki et al., 1984; Scholmerich et al., 1984). Accordingly, compounds that prevent the accumulation of noxious bile acids, or that protect the membranes

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M. ROJKIND TABLE 1. Clinical Trials o f Ursodeoxycholic Acid in the Treatment o f Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis Author

Year

Etiology

Poupon et al. Podda et al. Chretien et al. Mitzoguchi et al. Podda et al. Batta et al. Leuschner et al. Osuga et al. Roda et al. Matsuzaki et al. Lotterer et al. Poupon et al. Steihl et al. Oka et al. Podda et al. Chazouilleres et al. Poupon et al. Crosignani et al.

1987 1988 1989 1989 1989 1989 1989 1989 1989 1990 1990 1990 1990 1990 1990 1990 1991 1991a

PBC PBC and CH PBC IC PBC, Sc and CH PBC PBC CH and PBC PBC PBC PBC PBC PBC PBC CH SC PBC CH

No. Patients 18 and 22 9 16 4 20 45 and 8 7 10 9 70 29 23 24 15 73 18

Results + + + + + + + + + + + + ? +_ + + + +

PBC = Primary biliary cirrhosis. CH = Chronic hepatitis. SC = Primary sclerosing cholangitis. IC = Intrahepatic cholestasis.

from the toxic effects of the more hydrophobic bile acids, may have a beneficial effect in the outcome of the disease. In this regard, it was shown several years ago that UDCA, a normal bile acid in bears bile, reduced serum transaminases in a small group of patients with chronic active hepatitis and improved the clinical and biochemical parameters in patients with PBC (Poupon et al., 1987). A summary of publications in which UDCA has been used for the treatment of PBC or PSC is presented in Table 1. Although the exact mechanism of action of UDCA is not known, four possible mechanisms have been suggested. (1) Direct protection of plasma membrane from toxic effects of accumulated bile acids (Galle et al., 1990; Koga, 1987; Miyai et al., 1982; Miyazaki et al., 1984; Scholmerich et al., 1984). (2) Decreasing the pool of noxious bile acids by increasing their intestinal excretion and decreasing their enterohepatic circulation (Dumont et al., 1980; Erlinger and Dumont, 1990; Guranz et al., 1985). (3) A decrease in the expression of HLA Class I antigens on the surface of hepatocytes (Calmus et al., 1990). (4) Increased degradation of H D L by hepatocytes by increasing receptor binding and uptake (Malavolti et al., 1977). Serum bile acids have been determined in patients with PBC or PSC that received UDCA treatment. While in the group of PBC patients treated with UDCA primary bile acids in serum decreased significantly (O'Brien et al., 1991), no significant changes were reported after one year of treatment of PSC patients with UDCA (Poupon et al., 1991). Recently, Crosignani et al. (1991b) determined blood bile acids in PBC patients that received UDCA therapy. They found no significant change in hydrophilicity pattern of circulating bile acids. In addition, the results obtained did not support the notion that choleresis plays a role in the beneficial effects of UDCA (Crosignani et al., 1991b). A general concern regarding to the use of UDCA or any other agent that modifies secondary events leading to cirrhosis and not the main pathophysiologic factors, is related to the outcome of the disease upon discontinuation of treatment. Will discontinuation of therapy exacerbate the disease? Do we have to treat patients for life? What are the side effects of long-term therapy with UDCA? In another study, Poupon et al. (1991) reported the results of a multicenter trial of UDCA which included 146 patients with PBC, half of which received UDCA and half an identical placebo. Patients were studied for a 2 year period. The results demonstrated a beneficial effect in clinical and biochemical parameters, as well as a significant reduction in the mean histologic score, except for fibrosis, in the patients with UDCA as compared with those on placebo. However, similar to other studies, not all the patients responded to therapy. No significant side reactions have been reported in this or other studies (Crosignani et al., 1991a; O'Brien et al., 1991; Poupon et al., 1991).

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In a recent publication, O'Brien et al. (1991) reported the effect of UDCA in the treatment of PSC. In their study, they first observed the patients for 3 months, after which time, they were treated for 6 months with UDCA. Therapy was discontinued for 3 months and then reinstalled and continued for an 18 month period. Although a significant improvement in clinical and biochemical parameters was observed during both UDCA treatment periods, deterioration was observed during drug withdrawal. It is noteworthy to mention, that transaminases were highest during the withdrawal period (O'Brien et al., 1991).

5. ANTIINFLAMMATORY AND IMMUNOSUPPRESSOR THERAPY Inflammation is a common feature of chronic liver disease. However, it is not an obligatory component. In many patients with hemochromatosis or Wilson's disease liver inflammation could be minimal or non existing (Anderson and Popper, 1960; Kent and Popper, 1968). Similarly, alcoholic baboons (Popper and Lieber, 1980) and some chronic alcoholics with liver cirrhosis (Nakano et al., 1983) never develop overt alcoholic hepatitis. Nevertheless, inflammation is an important component that precedes and accompanies fibrogenesis and plays a role in signal amplification and in recruiting and/or activating collagen producing cells. Therefore, antiinflammatory drugs have potential as therapeutic agents for cirrhosis. It is important to note that although we have artificially separated antiinflammatory from antifibrogenic drugs, both processes occur simultaneously and that growth factors, hormones and cytokines produced during inflammation are also mediators of fibrosis. Therefore, in this section we included drugs that have primarily antiinflammatory activity, irrespective of whether they modify collagen metabolism. In this category we included steroids, colchicine and progesterone. In the section dealing with antifibrogenic therapy we included exclusively the drugs that have a primary effect on one or more steps of collagen synthesis. 5.1. STEROIDS Glucocorticoids are potent antiinflammatory agents with strong antifibrogenic activity that have been used in the treatment of hepatitis (Ballardini et al., 1984; Brissot et al., 1991; Kirk et al., 1980; Lee et al., 1991), PBC (Matloff et al., 1982) and autoimmune chronic hepatitis (Kirk et al., 1980) and some forms of cirrhosis (Oikarinen et al., 1986). It inhibits collagen synthesis and the expression of collagen mRNA in various cell culture systems (Hamalainen et al., 1985; Oikarinen et al., 1988; Raghow et aL, 1986) and in vivo (Weiner et al., 1987a). Although a glucocorticoid responsive element has been found in the promoter region of ct2 (I) collagen gene (Weiner et al., 1987b) the exact mechanism of action of glucocorticoids is unknown. It has been suggested that it inhibits transcription of the 0t 1 (I) collagen gene in hepatocytes and in livers of mice infected with cercarie of Schistosoma mansoni (Weiner et al., 1987a,b). However, glucocorticoids have no effect on transcription of type I collagen gene in cultured fibroblasts (Hamalainen et al., 1985; Raghow et al., 1986). In this latter system, glucocorticoids decrease mRNA stability. Glucocorticoids selectively inhibit the synthesis of hydroxylated collagen peptides by decreasing the activity of prolyl- and lysyl-hydroxylases (Newman and Cutroneo, 1978). Although for several years it was considered that prolyl-4-hydroxylase is not a limiting enzyme and thus plays no role in regulation of collagen synthesis, this concept most be reevaluated. Many of the previous studies were performed with antibodies raised against the fl-subunit of prolyl-4-hydroxylase. It has been recently established that the /3-subunit of prolyl-4-hydroxylase is identical with the chaperon protein disulfide isomerase (Koivu et al., 1987). Accordingly, measurements of prolyl-4-hydroxylase by immunoassays, most likely overestimated the actual enzyme concentration. Therefore, inhibitors of prolyl-4-hydroxylase may have antifibrogenic potential (see below). In bone tissue, glucocorticoids enhance the expression of some bone-specific genes and decrease the expression of collagen genes (Kasugai et al., 1991; Leboy et al., 1991). Whether these bone alterations contribute to the severe osteoporosis produced by glucocorticoids is not known. While glucocorticoids have strong antiinflammatory activity (Oikarinen et al., 1986) and prevent the expression of collagen genes (Hamalainen et al., 1985; Oikarinen et al., 1988; Raghow et al., 1986;

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Weiner et al., 1987) they also inhibit the expression of interstitial collagenase, stromelysin, type IV collagenase and TIMP (Shapiro et al., 1991). It has been also shown that glucocorticoids inhibit the induction of collagenase by phorbol esters (Jonat et al., 1990) and IL-1 (Di Battista et al., 1991). Recently, the mechanism of this inhibition has been investigated. It was found that the glucocorticoid receptor binds to c-fos and c-jun (Jonat et al., 1990; Verma and Evans, 1990; Yang-Yen et al., 1990), proteins involved in transcriptional activation of the collagenase gene (Chiu et al., 1988). Because of the negative effects on collagenase production, the severe cosmetic effects and the metabolic disturbances induced by steroids, the use of glucocorticoids should be restricted only for conditions in which treatment of inflammation or immunosuppression are required to control the illness. Once excess collagen deposition has occurred, glucocorticoids are of no value in the treatment of cirrhosis. 5.2. COLCHICINE Colchicine has been used for centuries for the treatment of gout. It was initially conceived as a drug with potential antifibrogenic activity because of two of its pharmacological activities discovered in the late 1960s. Colchicine inhibited microtubule assembly and thus prevented the transport and secretion of various proteins including collagen (Diegelmann and Peterkofsky, 1972; Ehrlich et al., 1974). In addition, colchicine was a powerful inducer of collagenase activity by cultured synovial cells (Harris and Krane, 1971). In preliminary studies performed in CCl4-cirrhotic rats and in a non-controlled trial of decompensated cirrhotic patients, colchicine was shown to ameliorate fibrosis and to improve some clinical and biochemical parameters (Rojkind et al., 1973). The results indicated that improvement in liver function was not related to changes in fibrosis. While in the rats with cirrhosis colchicine prevented fibrosis (Rojkind et al., 1973; Rojkind and Kershenobich, 1975), in patients treated for one year, only improvement in liver function was observed (Rojkind et al., 1973). Among the first parameters to improve was serum albumin, which returned to normal values in several of the treated patients. The results also indicated that colchicine had a more complex mechanism of action irrespective of whether these effects were mediated by its antimicrotubular activity. It has become evident over the past 20 years that colchicine has a variety of activities that we have artificially divided into two classes: Those that affect various parameters of liver function (Table 2) and those that will reduce inflammation and fibrosis (Table 3). Among the most interesting effects of colchicine are those related to its effect on lipid composition of the plasma membrane in general and of cholesterol in particular. We have previously reported that several membrane enzymes are altered in CC14-cirrhotic liver and that their activity returns to normal after colchicine treatment (Yahuaca et al., 1985). These effects were seen even when colchicine was administered to rats that already had advanced liver cirrhosis and occurred within the first week of therapy. One of the plasma membrane enzymes investigated was TABLE2. Some Pharmacological Properties of Colchicine Liver Function Modifies membranefluidity Improves membraneenzymes Increases glycogenlevels Increments serum albumin TABLE3. Some Pharmacological Properties of colchicine Fibrosis and inflammation Inhibits lipoperoxidation Inhibits galactosamine-inducedliver damage Inhibits chemotacticmigration of inflammatorycells into the liver Inhibits the release of growth factors by Kupffercells and macrophages Inhibits production of IL-1 by peripheral blood monocytes Inhibits collagen synthesisand secretion Enhances collagenaseactivity Inhibits the acute phase response

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Na +, K+-ATPase. Although ATPase activity was close to zero in rats treated for 10 weeks with CC14, the enzyme was present in the membrane. The lack of activity was associated with a change in phospholipid composition of the membrane and in alterations in the cholesterol/phospholipid ratio (Yahuaca et al., 1985). When plasma membranes from cirrhotic rats, that were deficient in ATPase activity, were fused with liposomes containing phosphatidyl serine, a normal ATPase activity was observed (Yahuaca et al., 1985). On the other hand, when plasma membranes from normal rats were enriched in cholesterol, ATPase activity was lost (Yahuaca et al., 1985). Analysis of the lipid composition of the plasma membrane of cirrhotic animals revealed an increase in cholesterol content and a decrease in the cholesterol/phospholipid ratio. In colchicine-treated rats, both lipid values were within normal limits (Yahuaca et al., 1985). Another hepatocyte plasma membrane enzyme which is altered in cirrhotic rats is adenyl cyclase. Alterations in this enzyme activity are accompanied by increased levels of cAMP and the absence of liver glycogen (Mourelle et al., 1981). Upon colchicine treatment, both cAMP and glycogen levels return to normal values. It appears from these data, that the functional improvement of the liver induced by colchicine is indeed related to restoration of membrane cholesterol values and normalization of the cholesterol/phospholipid ratio. The mechanism by which this occurs is unknown. However, it has been shown that colchicine inhibits the enterohepatic circulation of cholesterol thus increasing the excretion of cholesterol in feces (Wallace et al., 1970). Unpublished results from our laboratory (M. Mourelle and M. Rojkind) suggested that colchicine can remove membrane cholesterol in vitro perhaps by direct interaction of colchicine with the plasma membrane. These colchicine effects need further evaluation. Changes induced in lipid composition of the plasma membrane may improve membrane fluidity. This pharmacological activity of colchicine may be pertinent in understanding how the drug improves serum albumin levels in spite of its antimicrotubular effect. Our studies in rats treated with CC14 demonstrated that albumin synthesis in livers of cirrhotic rats was increased (Rojkind and Kershenobich, 1975). These results were confirmed by others who found normal or increased levels of albumin mRNA in cirrhotic animals (Panduro et al., 1988). However, the animals had low serum albumin levels perhaps due to increased retention of albumin within the hepatocytes (Rojkind and Kershenobich, 1975). In cirrhotic rats treated with colchicine, serum albumin levels and albumin synthesis returned to normal. Albumin retained within the liver decreased to normal values (Rojkind and Kershenobich, 1975). The changes in lipid composition of the plasma membrane described above may be responsible for the increased retention of albumin within the hepatocytes. They may occur secondary to alterations in lipid metabolism or to increased lipoperoxidation induced by CC14 treatment. However, they may also represent a compensatory mechanism to protect hepatocytes from increased portal pressure. Accordingly, an increase in membrane fluidity and/or a decrease in portal pressure induced by colchicine could contribute to the improvement in serum albumin levels. The other pharmacological activities of colchicine are related to its antiinflammatory and antifibrogenic action. When rats are treated with CC14, the number of liver non-parenchymal cells increases and reaches its maximum after 48 hr. The increase in cells is due both to arrival of inflammatory cells into the liver and to proliferation of liver non-parenchymal cells (Rojkind and Valadez, 1985). Colchicine administration to CCl4-treated rats inhibits the arrival of inflammatory cells into the liver (Rojkind and Valadez, 1985). Accordingly, if inflammatory cells produce mediators that enhance fat-storing cell proliferation and collagen production, a lesser number of inflammatory cells will produce a lesser degree of fibrosis. Other studies have also shown that colchicine prevents the release of fat-storing cell and fibroblast growth factors from rat Kupffer cells (Armendariz-Borunda et al., 1989) and human monocytes respectively (Kershenobich et al., 1990). In addition, colehieine inhibits the release of interleukin-1 by human blood monocytes (Kershenobich et al., 1990) and inhibits the acute phase reaction induced by turpentine (Greenwel et al., 1991b). These effects of colchicine could also result in amelioration of liver fibrosis since it has been shown that simultaneous induction of an acute phase reaction to rats with the administration of CC14 results in exacerbation of liver fibrosis (Van Gool et al., 1986a,b). Similarly, the acute phase response also enhances pulmonary

90

M. ROJKIND TABLE 4. Clinical Trials o f Colchicine in the Treatment o f Liver Cirrhosis Author

1983

Etiology

No. Patients

Results

Rojkind et al. Kershenobich et al. Nicolaescu et al. Galambos and Rieppe Frysak et al. Tapalaga et al. Kaplan et al. Reinhardt et al. Warnes et al. Bodenheimer et al. Akriviadis et al. Kershenobich et al. Warnes et al. Trinchet et al. Akriviadis et al. Kershenobich et al. Lindor et al. Zifroni and Schaffner

1973 1979 1983 1984 1985 1986 1986 1986 1987 1988 1988 1988 1988 1989 1990 1990 1991 1991

Mixed Mixed Mixed ALD Mixed Mixed PBC Mixed PBC PBC ALD Mixed PBC ALD ALD PBC PSC PBC

7 43 55 22 21 22 60 74 64 57 74 100 61 33 74 8 12 57

+ + + + _ + + _ + + + + __+ +

ALD = Alcohol liver disease. PBC = Primary biliary cirrhosis. PSC = Primary sclerosing cholangitis.

fibrosis in rats treated with bleomycin (Van Gool et al., 1988). These effects of colchicine on cytokines and other mediators of inflammation and fibrosis may be quite relevant to its antifibrogenic potential. However, more work is needed to determine which components of the acute phase reaction are beneficial or detrimental for each specific form of liver injury. In a double blind controlled-randomized trial performed by Kershenobich et al. (1979, 1988) it was shown that colchicine significantly improves survival of treated patients, it improves various clinical and biochemical parameters of liver disease and ameliorates inflammation and fibrosis (Kershenobich et al., 1988). It was shown that in 9 patients on colchicine and in none of the placebo-treated patients liver histology improved and in 3 patients the biopsy was read as normal or as having mild fibrosis. Although needle liver biopsies are subject to sampling error, nonetheless several of the patients studied had from 2-8 biopsies and readings were obtained blindly and were confirmed in more than one biopsy. With a few exceptions, mainly those studies in which colchicine was used for the treatment of alcoholic hepatitis (see Table 4), several laboratories have reported beneficial effects of the treatment. Except for the expected diarrhea, no other significant side reactions have been reported. There are some isolated reports of myositis (Kuncl et al., 1987) and of bone marrow alterations (Wallace and Singer, 1988) which return to normal upon discontinuation of the drug. Colchicine has been used in children with biliary athresia (Collins et al., 1991) and no side reactions have been reported. Similar to other drugs described in this chapter, only close to 50% of the treated patients respond to colchicine. Because of these results and the fact that UDCA, interferons and colchicine may act by different mechanisms, further animal studies using combinations of the aforementioned drugs are needed to determine whether they give better results than single drug therapy. The therapeutic dose of colchicine is yet to be determined. Pharmacokinetic studies have suggested that steady-state levels of colchicine in a normal individual that takes 0.6 mg/12 hr is approximately 1.12 ng/ml. However, in cirrhotic patients, clearance of the drug is impaired and the steady-state levels of colchicine increase to 2.82 ng/ml (Leighton et aL, 1991). Other studies have suggested that colchicine is excreted in feces and urine and that only 2% of the administered drug stays in the liver. When liposomes containing colchicine are administered intradermally to rats, slightly higher levels of the drug reach the liver and they remain elevated for several days (Cerbon et al., 1986). Colchicine has been used for the treatment of rats with CCl4-induced liver cirrhosis and for mice and rabbits with schistosomiasis. Except for one study with schistosomiasis that reported negative results, all other studies have reported improvement in the degree of fibrosis (see Table 5).

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TABLE5. Use of Colchicine in Experimental Liver Cirrhosis Author

Year

Etiology

Animal

Results

Rojkind et al. Tanner et al. Yahuaea et al. Mansour et al. Andrade et al. Jiang et al.

1973 1981 1985 1988 1990 1990

CCI 4

rat

CC14 CCh Schisto Sehisto Schisto

rat

+ + + + +

rat mice mice rabbit

5.3. PROGESTERONE Progesterone competes with glucocorticoids for receptor binding (Tsai et al., 1990) and therefore may share some of the antiinflammatory properties of steroids. However, progesterone may also inhibit the glucocorticoid effect when it binds to the steroid receptor (Xu et al., 1990). It has been shown that progesterone inhibits the acute phase reaction induced with turpentine in rats (Cunningham and Rojkind, 1989) and inhibits liver cirrhosis induced in rabbits with estrogens (Kremer et al., 1985; Stenback et al., 1989). Because of these effects, further experimental studies with progesterone or its analogs are warranted. 5.4. METHOTREXATE

Methotrexate is an inhibitor of folic acid synthesis that has immunosuppressor activity (Segal et al., 1990). It has been used for the treatment of PBC and PSC with some degree of success (Kaplan, 1989; Knox and Kaplan, 1991; Weber et al., 1991). However, methotrexate is hepatotoxic (Banerjee et al., 1988; Clegg et al., 1989; Flowers et al., 1990; Gilbert et al., 1990; Hall et al., 1991; Kaito et al., 1990; Kujala et al., 1990; Mitchell et al., 1990; Risteli et al., 1988; Zachariae et al.,

1989) and can induce cirrhosis. It has been suggested that liver injury that occurs in patients with psoriasis that receive methotrexate is associated with alcohol abuse (Ashton et al., 1982). Although this may be the case with some patients, there are reports in the literature of hypersensitive reactions to the drug and in few instances, patients with rheumatoid arthritis that received methotrexate have developed cirrhosis and required liver transplantation. 5.5. CYCLOSPORIN

Cyclosporin is the immunosuppressor that has been used to prevent graft rejection in patients with liver transplantation (De Groen, 1989). Cyclosporin has been used with some degree of success in the treatment of PBC and PSC (Minuk, 1988; Wiesner et al., 1990). However, it is nephrotoxic and should be used selectively when immunosuppression is specifically indicated.

6. ANTIFIBROGENIC THERAPY Liver fibrosis is an important component of cirrhosis. Although liver collagen content only increases 7-fold, its presence in the wrong locations, its chaotic distribution and organization and the formation of septa may be responsible in part for some of the complications observed in cirrhotic patients. Capillarization of the space of Disse transforms the fenestrated sinusoids into real basement membranes and confronts the bepatocytes with a permeability barrier. The retraction of the scar formed around perivenular or periportal regions may significantly contribute to the increase in portal pressure observed in cirrhotic patients. In addition, the formation of septa that surround nodules of apparently regenerating bepatocytes, isolates hepatocytes within the nodule from the hepatocytes in other nodules (Rojkind and Greenwel, 1988; Rojkind and Mourelle, 1988; Tsukamoto et al., 1990). Therefore, if one could prevent the accumulation of collagen, one may prevent complications of liver cirrhosis (Rojkind, 1980). The mechanisms involved in regulation of collagen gene expression are quite complex and are not the subject of this review (Bornstein and Byers, 1980). However, collagen undergoes several cotranslational and posttranslational modifications that are initiated in the endoplasmic reticulum, continue throughout the Golgi and secretory vesicles and end when the protein has been secreted

92

M. ROJKIND TABLE6. Biosynthesis of Collagen (1) Transcription of the gene (2) Translation (3) Post-translational modifications (a) Hydroxylationof prolyl and lysyl residues (b) Glycosylation (c) Chain alignment (d) Triple helix formation (e) Secretion into the extracellular space (f) Removalof propeptides (g) Formation of cross-links

and assembled into tissue fibers (see Table 6). With a few exceptions, each of the steps requires a specific enzyme which could be a target for pharmacological intervention. However, similar to the hepatoprotector therapy described above, successful treatment of one of the accompanying alterations of liver cirrhosis, does not necessarily imply a successful treatment of the disease. Several drugs are available that modify one or more cotranslational and posttranslational modification of collagen. As indicated previously, in this section we shall describe only those agents that modify one or more steps of collagen biosynthesis and maturation. It is also important to indicate, that although individual agents may modify primarily one step of the biosynthetic pathway, the actual mechanism of action may be shared by more than one agent. For example, proline analogs to be described below, compete with proline for transport, acylation of tRNA and incorporation into proteins (Takeuchi and Prockop, 1969; Takeuchi et al., 1969). The introduction of a single residue of the analog within the protein induces a severe conformational change in the protein that alters some or all of the subsequent steps. Prolyl hydroxylation will be inhibited, helix formation may be delayed or abnormal, secretion will be delayed and collagen will be susceptible to intracellular degradation. Accordingly, the net effect of proline analogs will be similar to that of prolyl-4hydroxylase inhibitors. However, the effects of the latter will be more specific.

6.1. THE PROLINEANALOGS Several years ago it was suggested that the free pool of proline regulated the production of liver collagen (Rojkind and Diaz de Leon, 1970). It was shown that liver proline pool is increased in various models of cirrhosis (Dunn et al., 1977; Rojkind and Diaz de Leon, 1970) including alcoholic liver cirrhosis (Kershenobich et al., 1970) and that linear rates of collagen synthesis by liver slices are obtained only when the pool is 0.45 #M or higher (Dunn et al., 1977). In human alcoholic cirrhosis, increased proline pools are accompanied by increase serum proline and blood lactic acid (Kershenobich et al., 1981). Since lactic acid is a potent inhibitor of prolyl-oxidase (Kowaloff et al., 1977), the mitochondrial enzyme responsible for the oxidation of proline, it was suggested that changes in proline are related to lactic acid accumulation (Kershenobich et al., 1981). The latter was found to increase irrespective of blood alcohol levels. The exact mechanism by which proline regulates the production of collagen is unknown. However, since collagen contains 20% of proline and hydroxyproline residues it was suggested that inhibiting the proline pool with a proline analog would facilitate the incorporation of the analog into the protein. As indicated above, this will disturb the helix and induce alterations in the chain of cotranslational and posttranslational modifications required for collagen secretion, organization and maturation. Several studies with cultured fibroblasts have shown that indeed, proline analogs inhibit collagen synthesis and increase intracellular degradation (Takeuchi and Prockop, 1969; Takeuchi et al., 1969). The administration of one of these analogs, L-azetidine-2-carboxylic acid (AZC) to rats during the induction of liver cirrhosis with CC14, prevented the accumulation of liver collagen (Rojkind, 1973). AZC prevented alterations in albumin biosynthesis in CC14-cirrhotic rats without producing changes in the incorporation of labeled proline into total non-collagenous proteins (Kershenobich and Rojkind, 1973). Most proteins contain proline and, therefore, the analog is incorporated into many proteins (Fowden and Richmond, 1963). However, the selective effect of the analogs on collagen synthesis and deposition could be due to the abundance of proline in collagen and to the critical role of this iminoacid in protein conformation and triple helix

Fibrogenesis in cirrhosis

93

formation. Proline analogs also eliminate fibroblasts from mixed cultures (Kao and Prockop, 1977). Very little is known about the pharmacology and long-term toxicity of proline analogs and, therefore, their potential use in clinical trials has been hampered. 6.2. INHIBITORSOF PROLYLHYDROXYLASE

Collagens contains the hydroxyproline isomers 3-hydroxyproline (3-hypro) and 4-hydroxyproline (4-hypro) and two distinct enzymes are involved in the hydroxylation of proline to form the isomers. 4-hypro is the most abundant of the two and it usually occurs in the third position of triplets containing gly-X-4hypro. 3-hypro is the predominant isomer in type IV collagen and it occurs in the second position of the triplet gly-3-hypro-4-hypro. Both prolylhydroxylases as well as lysylhydroxylases have the same requirements. They use 2-oxoglutarate as a co-substrate which is stoichiometrically decarboxylated to succinate during the formation of hydroxyproline. They use ferrous ion as a cofactor, they require molecular oxygen and a reducing agent such as ascorbic acid (Cardinale and Udenfriend, 1974). Several inhibitors of prolyl-4-hydroxylase have been developed (Gunzler et al., 1988; Karvonen et al., 1990; Majamaa et al., 1984; Sasaki et al., 1987; Tschank et al., 1987). They include among others, peptides that contain the aminoacid oxaproline (Gunzler et al., 1988; Karvonen et al., 1990) and various competitive inhibitors of 2-oxoglutarate. The most potent ones are pyridine 2,4-dicarboxylate (Tschank et al., 1991) and 3,4-dihydroxybenzoate (Sasaki et al., 1987). Both compounds have a carboxyl group and a chelating moiety that correspond to domains I and II of 2-oxoglutarate (Ng et al., 1991). 6.3. PEPTIDESCONTAININGOXAPROLINE

The peptides benzyloxylcarbonyl-Phe-Opr-Gly-benzyl ester and Benzyloxycarbonyl-Phe-OprGly-ethyl ester inactivate prolyl-4-hydroxylase in cultured fibroblasts. The ethyl ester was twice as potent as the benzyl ester and decreased prolyl-4-hydroxylase activity by 50% when cells were cultured with 20-40/~M concentrations. The inactivation of prolyl-4-hydroxylase is highly specific since neither the other enzymes involved in collagen biosynthesis, nor the steady-state levels of type I and type III collagen or prolyl-4-hydroxylase mRNAs were affected. Similarly, accumulation of non-collagenous proteins after pulse labeling with [14C]leucine was not modified by the inhibitor (Karvonen et al., 1990). 6.4. 2-OxoGLUTARATEINI-IIBITORS

The inhibitory effect of pyridine 2,4-dicarboxylate and its diethyl-ester derivative were studied in chick embryo calvaria (Tschank et al., 1991). The diester lacks the free carboxyl groups and therefore it is inactive. However, once it enters the ceils and the ester linkages are hydrolyzed, the molecule becomes activated. As expected, the diester is inactive against prolyl-4-hydroxylase in vitro even when added at a 1 mM concentration. However, the inhibitory activity was manifested at concentrations as low as 10 #M when the inhibitor was added to intact calvaria (Tschank et al., 1991). This finding indicates that hydrolysis of the diester occurs in calvaria. In rico half-maximal inhibition of tissue hydroxyproline formation was achieved with 650 #M of pyridine 2,4-dicarboxylate. The aforementioned results suggest that the diester derivative of pyridine 2,4-dicarboxylate is the prototype of a pro-drug (Tschank et al., 1991), whose effects will be expressed only in cells that hydrolyze the ester linkages to liberate the free carboxyl groups (Tsehank et al., 1991). Collagen produced in the presence of the inhibitors has a lower melting temperature than normal hydroxylated collagen (Tschank et al., 1991). The protein is largely retained within the cells and accumulates in phagolysosomes. In addition, procollagen rather than collagen accumulates in the tissue, thus suggesting that procollagen made in the presence of the analogs either inhibits procollagen N-peptidase or is resistant to the peptidase attack (Tschank et al., 1991). A possible problem with the prolyl-4-hydroxylase inhibitors that compete with 2-oxoglutarate for its binding site on the enzyme is that several other hydroxylases require 2-oxoglutarate (Ng et al., 1991). Therefore, for toxicity studies of the pyridine-2,4-carboxylate derivatives, special attention should be given to their effect on other hydroxylases. JPT 53/I--G

94

M. ROJKIND 6.5. LYSYLOXIDASEINHIBITORS

Collagen maturation occurs in the extracellular space once the protein is secreted, the propetides from the amino and carboxyl-terminal domain are cleaved and the protein assembles with other collagen molecules to form fibrils. At this stage, aldehydes are formed from lysine or hydroxylysine residues located in the first 10-15 amino acids from the amino terminal end of the protein (Yamauchi and Mechanic, 1988). This peptide precedes the beginning of the typical triple helical domains of the collagen molecule. Aldehydes are formed via oxidation by the copper-containing enzyme lysyloxidase (Siegel, 1979). These reactive aldehydes condense with similar aldehydes of other chains or other molecules or with the epsilon amino group of other lysine or hydroxylysine residues to form intramolecular and intermolecular cross-links (Yamauchi and Mechanic, 1988). This cross-links are necessary to stabilize tissue collagen. When cross-linking formation is inhibited by copper-deficient diets (Foster et al., 1975), by the administration of E-amino propionitrile (fl-APN) (Rojkind and Juarez, 1966) or penicillamine (Siegel, 1977), the collagen produced is deficient in cross-links and therefore it is more soluble than normal mature collagen (Rojkind and Juarez, 1966). It was suggested several years ago, that cross-linked collagen was less susceptible to collagenase attack (Wooley et al., 1975). Therefore, inhibitors of collagen cross-links may be of some benefit when properly applied. As indicated above, they do not inhibit collagen synthesis, except when given in high dose, therefore they will only prevent maturation of collagen. This effect of lysyloxidase inhibitors could be used advantageously in conditions in which collagen synthesis and accumulation are needed but inhibition of collagen maturation will be helpful. For example, in patients that develop strictures (vascular, urethral, esophageal, etc.), it would be important to enhance scar formation to prevent ulceration. However, if dilatation of the stricture will improve the diameter of the duct, a collagen with a lesser degree of cross-links will be easier to remodel than old, cross-linked collagen (Chvapil, 1988). 6.6. PENICILLAMINEAND fl-APN Penicillamine and fl-APN are two of the various inhibitors of collagen crosslinks described in the literature (Yamauchi and Mechanic, 1988). Penicillamine has been used unsuccessfully for the treatment of PBC and other chronic liver diseases (Epstein et al., 1979). In addition, its high toxicity precludes its use, except for the treatment of Wilson's disease. In the latter, penicillamine is used for its chelating properties and not for its capacity to inhibit collagen cross-linking (Sternlieb, 1990).

7. COLLAGENASE INDUCERS In this group we can include colchicine, prostaglandins and polyunsaturated lecithins (PUL). Colchicine has been shown to induce collagenase activity in cultured synovial fibroblasts. However, it is not known whether colchicine has the same effect on liver fat-storing ceils. Prostaglandins inhibits collagenase production in various mesenchymal cells via the induction of cAMP levels (Knudsen et al., 1986; Wahl et al., 1977). 7.1. POLYUNSATURATEDLECITHINS Lieber et al. (1990) performed a long-term study to determine the effect of polyunsaturated lecithin (4.1 mg/Kcal) on the development of alcoholic cirrhosis in baboons fed a liquid diet that contained 50% of the total calories derived from ethanol. In control baboons 50% of the caloric intake was derived from carbohydrates. They compared the results with those obtained in baboons fed an equivalent amount of the same diet with or without ethanol. Although both groups of animals accumulated equal amounts of lipids, striking differences were observed in the degree of fibrosis. While a considerable number of animals fed alcohol without lecithin developed fibrosis, those supplemented with PUL only reached the stage of perivenular fibrosis (Lieber et al., 1990). Liver fat-storing cells were also different among both groups. While livers of baboons that received the ethanol liquid diet only contained many transitional cells, those with a PUL supplemented diet contained fewer activated ceils. However, when baboons in the PUL supplemented group were

Fibrogenesis in cirrhosis

95

taken off PUL and continued on the ethanol liquid diet alone, they develop cirrhosis within 18 months. This is approximately half the time required to induce cirrhosis in alcoholic baboons that receive no PUL supplementation in their diet. The effect of PUL appears to be specific since supplementation with choline has no effect on the development of cirrhosis (Lieber et al., 1990). These results suggest that PUL detains the process of fibrosis without inhibiting the basic mechanisms leading to excess collagen accumulation. Therefore, it was important to further investigate the effect of PUL on collagen gene expression by cultured fat-storing cells (Li et al., 1992). Experiments performed in vitro using early passages of rat liver fat-storing cells showed that 175/aM acetaldehyde induced the synthesis of type I collagen (Moshage et al., 1990) as well as the expression of type I collagen and fibronectin mRNAs (Casini et al., 1991). The addition of PUL to cultured cells that received acetaldehyde did not improve collagen synthesis but significantly increased the collagenolytic activity (Li et al., 1992). Accordingly, these results suggest that PUL inhibits collagen deposition by increasing its degradation. Whether this effect of PUL occurs in other animal models of liver cirrhosis, or is relevant to human alcoholic liver disease will have to be further evaluated.

8. MISCELLANEOUS Collagen propeptides and retinoic acid are of potential use in the treatment of fibrosis. It has been shown that propeptides excised during conversion of procollagen to collagen inhibit the expression of collagen (Aycock et al., 1986, Katayama et al., 1991; Wu, C. H. et al., 1991). Several peptides with sequences identical to those present in the propeptides have been synthesized and tested (Aycock et al., 1986; Katayama et al., 1991). However, more experimental work is needed to determine whether they have any use in the treatment of liver cirrhosis in animals. With regard to retinoids there are conflicting results. While it is well known that excess vitamin A may induce cirrhosis (Hruban et al., 1974), the administration of retinoids to cultured fibroblasts or synovial cells inhibits the expression of type I collagen and collagenase mRNAs, respectively (Brinckerhoff, 1990, Oikarinen et aL, 1985). It is also known, that transformation of fat-storing cells into myofibroblasts is accompanied by the decrease in vitamin A and the vitamin A binding protein (Weiner et al., 1990b). However, this may be a result of the transformation of fat-storing cells rather than its cause. As can be seen from this review, there are many reasons to be optimistic about the future of therapy of liver fibrosis and cirrhosis. However, as indicated before, there are some problems that have to be properly solved. Firstly, we have to develop proper diagnostic methods to detect subjects at risk. Secondly, we must develop, sensitive and reliable non-invasive procedures to follow fibrogenesis. Thirdly, we have to learn how to target the drugs to the proper cells and thus avoid side reactions. Fourthly we have to learn how to distinguish responders from non-responders. Fifthly, we need to perform more animal studies to determine which combinations of the available drugs will provide a better therapeutic tool than the individual components. Finally, we must learn to have patience to perform long-term studies and to combine efforts with other groups in order to obtain sufficient patients and generate significant data. Studies that are prematurely analyzed will not provide answers to the questions raised. Acknowledgments--The author is indebted to Patricia Greenwel for her invaluable help in the preparation of

this manuscript. This manuscript was prepared in part with support from grants NIH DK 41918, a Biomedical Research Grant from NIH to Albert Einstein College of Medicine and by a Grant from Alcoholic Beverage Medical Research Foundation.

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