SEMINARS IN LIVER DISEASE-VOL.
10, NO. I , 1990
Therapy for Hepatic Fibrosis
A hallmark of cirrhosis is the increased deposition of connective tissue proteins. Fibrogenesis (that is, an increased rate of synthesis of extracellular matrix molecules) plays an important role in this accumulation of extracellular tissue and results in a severalfold increase in hepatic collagen content. Other extracellular matrix proteins, including laminin, fibronectin, and proteoglycans, are also found in greater abundance. One result of such matrix protein accumulation is increased hepatic vascular resistance and consequent portal hypertension. This leads to the well-known complications of cirrhosis, which are often difficult to manage and may be fatal. In some liver diseases the stimuli leading to cirrhosis have been identified and specific therapies designed to counteract these stimuli. For instance, the iron overload of hemochromatosis is treated with phlebotomy or chelation, hepatitis B may be treated with antiviral agents, autoimmune chronic active hepatitis may be treated with immunosuppressive therapy, and copper overload of Indian childhood cirrhosis and Wilson's disease is treated with copper chelation. This review will not discuss these specific therapies but will instead address potential therapeutic interventions that might inhibit the ongoing process of hepatic fibrogenesis regardless of etiology. After briefly introducing some aspects of matrix production and assessment, we will discuss the agents most used or most heralded in the treatment of hepatic fibrosis. Since collagen is the major constituent of hepatic fibrous tissue, many therapies have been designed to interfere specifically with its biosynthesis. Although 12 separate collagens have been identified, the large fibrillar types I and I11 predominate in the fibrous tissue of cirrhosis. For this reason, many strategies designed to limit production of this abnormal fibrous tissue have concentrated on suppressing the secretion and accumulation of collagen types I and 111 in particular. However, it remains a possibility that strategically positioned but less abundant molecules, that is, collagen IV or laminin, deposited in a membrane covering the fenestrations of the sinusoidal endothelium, may be the critical factor in
the hemodynamic alterations that are the consequence of hepatic fibrosis. This should be borne in mind when evaluating therapies principally devoted to the reduction in collagen types I and 111. The metabolism of collagen is a complex process providing many opportunities for pharmacologic intervention (Fig. 1). In brief, the collagen genes are transcribed by DNA polymerase 11, and the resulting collagen transcripts are processed and transported to the rough endoplasmic reticulum where they -are translated as prepropeptides. Nascent polypeptide chains then undergo extensive post-translational modifications, including hydroxylation of select lysyl and prolyl residues and glycosylation of hydroxylysyl residues.' Three polypeptide chains spontaneously form a stable triple helix through noncovalent interactions. The triple helix is secreted extracellularly with cleavage of the amino terminal and carboxy terminal propeptides. The collagen fibrils are formed by aggregation of the triple helices with covalent cross-linking between the triple helices. The collagen peptides and the cleaved propeptides can undergo degradation either intracellularly4 or extracelluAny of these steps might be blocked by therapeutic agents in order to prevent collagen synthesis and secretion. In general, agents enhancing matrix degradation and remodeling have not been exploited for therapeutic effect. A primary obstacle to the evaluation of all therapies for hepatic fibrogenesis has been the lack of a safe means of assessing therapeutic effect. Liver biopsy has wellknown hazards as well as unavoidable sampling . - error. A noninvasive measure of fibrotic matrix accumulation is urgently needed. One proposed measure has been the levels of circulating collagen propeptides, particularly the carboxy terminal propeptide of collagen I11 (PIIIP).' However, there are conceptual difficulties with the use of this measurement7 (see article by Maher in this issue of Seminars).
THERAPEUTIC AGENTS Colchicine
From the Department of Medicine and Center for Molecular Genetics, University of California, and VA Medical Center, San Diego, California. Reprint requests: Dr. Brenner, Department of Medicine, "niversity of California, San Diego school of Medicine, La Jolla, CA 92093.
The rationale for the use of colchicine in fibrotic diseases is that colchicine inhibits the polymerization of to be required microtubules, a process that is for collagen secretion. In cultured fibroblasts and hepatocytes, colchicine transiently inhibits the secretion of
Copyright O 1990 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, N Y 10016. All rights reserved.
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DAVID A. BRENNER, M.D., and JOSEPH M. ALCORN, M.D.
SEMINARS IN LIVER DISEASE-VOLUME
10, NUMBER 1, 1990
Proposed mechanisms of inhibitors
Collagen Biosynthesis Procollagen gene
I
transcription
i
translation
b--,--
propeptides
Procollagen polypeptide
I I
post translational modification
I
gamma-interferon glucocorticoids
mRNA
chain alignment
prolyl 4-hydroxylase inhibitors proline analogs
Triple Helix
1 4
secretion
Extracellular procollagen
+ NxQ%zQs - -
colchicine
Removal of propeptide
-
C
1
procollagen-to-collagen inhibitors
Collagen Covalent cross-linking
I
Fibrils
FIG. 1. A simplified pathway for the biosynthesis of collagen. Some of the agents that inhibit collagen synthesis, as discussed in the text, are shown at the sites of their proposed actions.
collagen into the medium for the first 2 to 3 hours8,' but collagen secretion returns to normal levels after prolonged i n c u b a t i ~ n .Other ~ actions of colchicine that might inhibit fibrosis include inhibition of mitosis,1° stimulation of collagenase secretion," and anti-inflammatory effects, including the inhibition of polymorphonuclear leukocyte functions.12 Two research groups have demonstrated that colchicine decreases collagen content in the carbon tetrachloride (CCl,) model of hepatic fibrosis in rats.I3.l4This effect is not mediated by inhibition of prolyl hydroxylase activity,I4 a critical enzyme in the biosynthesis of collagen. Colchicine has been used in clinical trials in several types of cirrhosis. In primary biliary cirrhosis, one clinical trialI5 showed initial improvement in some biochemical liver tests of patients treated with colchicine, whereas a second trial demonstrated a more sustained improvement.16 Neither of these two clinical trials dem-
onstrated improvement in the histology of the liver biopsies nor in the mortality of patients treated with colchicine. However, the small number of patients in these trials make results subject to type I1 error. In a much discussed double-blind placebo-controlled trial, patients with cirrhosis of all etiologies were treated with colchicine or placebo.'' Treatment with colchicine resulted in sustained improvement in survival as well as improvement in liver biopsy histologic patterns. Criticisms of this study include a disproportionate number of women and a higher mean albumin level in the colchicine group.I8 In addition, there was no monitoring of compliance with medication, and a large number of patients were lost to follow-up. In all of the trials mentioned, colchicine had a low incidence of side effects, which consisted of diarrhea, nausea, and epigastric distress. The much rarer complications of colchicine, including agranulocytosis, aplastic anemia, epistaxis, and myopathy, were not reported in these small studies. A
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quired for the formation and stability of the collagen triple helix. The enzyme prolyl 4-hydroxylase requires ferrous iron, molecular oxygen, 2-oxyglutrate, and ascorbate to catalyze the stereospecific hydroxylation of proline residues in the polypeptide chain with the miniGlucocorticoids mum required sequence of X-Pro-Gly. The reaction consists of the decarboxylation of 2-oxyglutrate with Glucocorticoids such as dexamethasone selectively concomitant Fe + generation and the subsequent hydrodecrease collagen synthesis by several different mechaxylation of the prolyl residues30 (Fig. 2). Elegant studies nisms in a variety of experimental models.19 Glucocoron the structure and function of the purified prolyl 4ticoids markedly decrease mRNA levels of type I collagen in primary cultures of adult rat h e p a t o ~ y t e s ~and ~ , ~ ' hydroxylase enzyme led to the identification of compounds that inhibit the enzyme's activity by competing in murine hepatic schistos~miasis.~~ This effect is in part the result of decreased transcriptional a ~ t i v i t y . ~Fur' . ~ ~ with 2-oxyglutarate for binding." Two compounds, pyridine-2,4-dicarboxylate (2,4thermore, a regulatory element in the collagen a2(I) proPDCA) and its isomer pyridine-2,5-dicarboxylate(2,smoter region that is specifically inhibited by the presence PDCA) were identified as potent inhibitors of purified of glucocorticoids has been identified.21 On the other prolyl4-hydroxylase in vitro. In addition, in a variety of hand, in primary fibroblast cultures, glucocorticoids decultured cells, 2,4-PDCA and 2,5-PDCA decrease the crease type I collagen mRNA level^^'-^^ and type I colgeneration of hydroxyproline and the secretion of collalagen mRNA ~ t a b i l i t y without ~ ~ , ~ ~ any change in the gen into the media.3' This is a selective effect of these Glucocortitranscriptional rate of the collagen compounds in that there is a minimal effect on total procoids also decrease the post-translational modifications tein synthesis or on the secretion of nonhydroxyproline of the procollagen peptides by decreasing the activities proteins. of prolyl hydroxylase and lysyl hydroxyla~e.~~ However, A separate group of prolyl hydroxylase inhibitors the activities of these enzymes do not appear to be rate that are analogues of both a-ketoglutarate and ascorbate limiting in the biosynthesis of collagen, and therefore has been developed. Ethyl 3-4-dihydroxy benzoate, a glucocorticoids do not appear to inhibit collagen synthemodel of this type of prolyl hydroxylase inhibitor, was sis at the level of post-translational modification^.'^ extensively studied in cultured human fibrobla~ts.~~ This Glucocorticoids have been used as specific therapy compound decreases cellular prolyl hydroxylase activity in the treatment of chronic liver disease, including priin the cells, decreases the incorporation of hydroxypromary biliary cirrhosis27and autoimmune chronic hepatiline into proteins, decreases the stability of the collagen ti^.^^ There is some evidence that glucocorticoids detriple helix, and decreases the synthesis and secretion of crease collagen synthesis in patients,29 which might type I and 111 collagens. The inhibitor has no effect on reflect the mechanisms of action discussed in addition to collagen mRNA levels. Ethyl 3-4-dihydroxy benzoate, the anti-inflammatory effects of glucocorticoids. Unforwhich is hydrophobic and therefore able to cross the cell tunately, the detrimental systemic effects of long-term membrane, specifically inhibits the production of collaglucocorticoid therapy, including severe osteoporosis in genous proteins and is nontoxic. patients with primary biliary cirrhosis,27preclude its agMore recently, hepatocyte-targeted lipophilic proingressive use as a general treatment of fibrogenic liver hibitors have been developed that may specifically supdisease. press hepatocyte collagen hydroxylation and synthesis in vivo (Hanauske-Abel HM: Personal communication). Prolyl CHydroxylase Inhibitors These exciting compounds require further evaluation with respect to their mechanisms, effectiveness, and toxThe post-translational modification of certain proicity in animal models. Such further evaluation may reline residues in collagen to trans-4-hydroxyproline is re-
0
rk!
3
0
s
g 8
d
+-
2 cn
c 0 .
.-0C 0
2i & 0
Ci. prolyl 4-hydroxylase
-C-N
YH H I I P CH-CH2
..
C y ,CH-C-N-.
.,.
H
II
0 Proline
4-Hydroxyproline
FIG. 2. The substrates and products of the reaction catalyzed by prolyl Chydroxylase.
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theoretical concern is that the inhibition of normal healing by colchicine might lead to worsening of esophageal variceal hemorrhage.18
SEMINARS IN LIVER DISEASE-VOLUME
veal additional steps in which they suppress collagen synthesis and secretion.
Proline Analogues Several analogues of the amino acid proline are recognized by prolyl tRNA and incorporated in the place of proline into nascent polypeptide chains. These analogues alter the structure and function of the resulting proteins.33 Azetidine carboxylic acid, an example of a proline analogue, has been studied in cultured fibroblast^.^^ Incubation with azetidine carboxylic acid results in the synthesis of a nontriple helical form of collagen that is subject to degradation by proteases to which the triple helix of collagen is resistant. The resulting collagen is also subject to degradation by endogenous proteases with production of a dialyzable form of hydroxyproline. In addition, fibroblast proliferation decreases, which might also be the result of decreased secretion of extracellular matrix proteins. Using the CC1,-induced cirrhotic model in rats, one study demonstrated that azetidine carboxylic acid inhibits liver f i b r ~ s i s . The ~ ' rats treated with azetidine carboxylic acid have decreased hepatic collagen histologically and decreased incorporation of proline into the hydroxyproline of collagen. The agent does not appear toxic, since the incorporation of radioactivity into noncollagenous proteins was unchanged.
Procollagen-to-Collagen Inhibitors The removal of the amino terminal and carboxy terminal propeptides is required for the proper alignment of the collagen triple helices to form fibrils and for subsequent formulation of covalent cross-links to stabilize the fibrils. If the propeptides are not removed, then the stability of the collagen is decreased. Leung et aP6 first demonstrated that arginine and its analogue concavanine inhibit the removal of the carboxy propeptide of type I procollagen. Subsequent experiments demonstrated that several amino acids and their analogues, polyamines, and dipeptides inhibit the removal of the carboxy propeptide of type I1 collagen. The removal of the amino ' . ~one ~ study, cadaverine propeptide is not a f f e ~ t e d . ~ In (a polyamine) and lysine were the most effective inhibitors tested.37In general, a free amino group is necessary but not sufficient to inhibit the conversion of carboxy procollagen to collagen. The application of these agents to the treatment of models of fibrosis or clinical fibrotic diseases has not yet been reported.
Prostaglandins Prostaglandins have diverse intracellular metabolic effects. The prostaglandin analogue, 16,16-dimethyl prostaglandin E, (DMPG) decreases fibrosis and fat accumulation induced by the choline-deficient diet model of hepatic fibrosis in rats.39Several possible mechanisms might explain the beneficial effects of prostaglandins in hepatic fibrosis. Prostaglandins increase intracellular cyclic adenosine monophosphate levels, which increase the intracellular degradation of collagen in cultured fi-
10, NUMBER 1, 1990
b r o b l a s t ~ . ~Alternatively, ~,~' DMPG may have a hepatrophic effect and prevent fibrosis at an earlier stage of injury. DMPG decreases hepatic injury from the acute toxicity of CC1,4, and from hepatocellular necrosis induced by the murine hepatitis virus in vivo and in ~ i t r o . ~ ~ Other potentially beneficial effects of prostaglandins include increased hepatic blood alteration of membrane fluidity, changes in systemic levels of hormone, including glucagon and and inhibition of the release of inflammatory factors by macro phage^.,^ To date, prostaglandins have not been used in the treatment of hepatic fibrosis in humans.
Retinoids Certain retinoids have been used for the treatment of hyperproliferative epidermal diseases. Retinoids, including all trans-retinoic acid and 13 cis-retinoic acid, selectively decrease type I collagen synthesis and type I collagen mRNA levels in cultured f i b r ~ b l a s t s . ~ Wthe n other hand, these retinoids decrease collagenase production by m o n o c y t e ~These . ~ ~ in vitro results cannot be extrapolated to using retinoids in the treatment of hepatic fibrosis because of the presence of hepatic Ito cells. In vivo, retinoids are largely stored as retinol palmitate in the hepatic Ito cells. When Ito cells are cultured on type I collagen matrix, the addition of retinol leads to an increase in type I collagen production. However, when Ito cells are cultured on a type IV collagen matrix, the addition of retinol produces an increase in type I11 collagen but a decrease in type I ~ o l l a g e n(see ~ ~ .also ~ ~ article by Friedman in this issue of Seminars). Therefore the regulation of collagen production by Ito cells represents a complex interaction between the extracellular matrix and the intracellular vitamin A c ~ n c e n t r a t i o n . The ~ ' ~ ~thera~ peutic effectiveness of vitamin A in hepatic fibrosis is unknown. Indeed, a number of studies suggest that prolonged exposure to elevated levels of retinoids may result in hepatic fibro~is.'~
Malotilate Malotilate (di-isopropyl 1,3-dithiol-2-ylidenemalonate) is a water-insoluble dithiolane derivative developed by the Nihon Nohyaku Company as a nontoxic inducer of hepatic protein p r o d ~ c t i o n .It~ 'is absorbed from the gastrointestinal tract and undergoes extensive hepatic firstpass m e t a b ~ l i s m . Its ~ ~pharmacologic ,~~ actions include an increase in hepatic protein to DNA and RNA to DNA ratios, induction of cytochrome b, (without significant effect on cytochrome P450), an increase in reduced nicotinamide-adenine dinucleotide phosphate cytochrome C reductase activity, and induction of glucose-6-phosphate dehydrogenase activity.52 Primarily because of its effect on hepatic protein production, malotilate has been considered for potential use in liver disease. Although a number of studies have now been performed evaluating the effects of this agent on acute hepatic injury from a variety of compounds, few studies specifically address fibrogenesis. Dumont et a15, demonstrated histologic protection from both inflammation and fibrogenesis when maloti-
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chelation of inappropriately elevated copper is well late was administered to rats treated twice weekly with known.7' In addition, penicillamine scavenges immune intraperitoneal CCI,. Malotilate also suppressed CCI, complexes,72interferes with leukocyte c h e m o t a x i ~and ,~~ induction of collagen synthesis and accumulation as % n copper-penicillamine d inhibits T-cell f ~ n c t i o n , ~ ~ . ~ the assessed by hepatic collagen content and proline incorporation. In another study of CC1,-induced fibrosis, ' inhibicomplex has superoxide-dismutase a ~ t i v i t y . ~An Schuppan et aIs5 reported that malotilate maintained tion of intra- and intermolecular collagen cross-linking and leukotreine D, catabohas also been de~cribed,~',~' nearly normal serum PIIIP (collagen I11 amino propeplism has recently been shown to be inhibited.7yFinally, tide) levels while attenuating collagen accumulation. Of interest, the pulmonary fibrosis induced by intraperitoD-penicillamine may increase the solubility of secreted neal CCI, is also attenuated by the use of m a l ~ t i l a t e . ~ ~ collagen.x0 .~~ One or more or these actions may explain the Siegers et alSxproduced cirrhosis in rats with oral CCI, beneficial effects of penicillamine on Indian childhood and 5% ethanol instead of drinking water. In this model ~ i r r h o s i s .However, ~' the largest clinical experience with penicillamine in fibrosis in liver disease has been with malotilate, but not colchicine, penicillamine, or cianiprimary biliary cirrhosis, for which its use has been disdanol, completely protected the-animal from histologic app~inting,~' perhaps in part because of a failure to stratfibrosis and was associated with a nearly normal liver ify patients according to the severity of their disease. hydroxyproline content. Steatosis, attenuated by maloThese clinical trials of penicillamine coincided with tilate following acute CCI, intoxication in other studin vitro assessment of its effect on matrix proteins and i e ~ , ~was " ~not affected in this model of fibrosis. Polargely preceded evidence of its efficacy in animal tentially important in this model could be malotilate models of fibrosis. A small number of trials of penicilsuppression of acetaldehyde p r o d u ~ t i o n , ~a 'known prolamine in animal models of hepatic fibrosis have been moter of collagen synthesis.62 Partial protection against reported. In general, they demonstrate little or no effect fibrosis has also been reported in a dimethyl nitrosamine of penicillamine on fibr~genesis.'~.~" In vitro effects at model of hepatic fibrosish' and in immunologic models, high doses include decreased fibroblast proliferation and including egg yolk sensitization.@ ~ ~ .proteoglycan ~~ synthesis has collagen p r o d u c t i ~ n ,but Although malotilate does not seem to interfere with been reported to be increasedx" and decreasedXJin differCC1, uptake, it is possible that it inhibits the metabolism ent systems. Ultimately, the paucity of data leaves the of CCI, to toxic mediators of inflammation or interferes question of a specific effect of penicillamine on hepatic . ~ ~ evidence of generally with free radical g e n e r a t i ~ nThe fibrogenesis unanswered. decreased tissue injury and inflammation in these models is consistent with a primary effect on inflammatory meGamma-Interferon diators of fibrogenesis. Of interest in this regard is a recent report that malotilate specifically inhibits 5-lipoxyThe first clinical trials of interferon began in 1972 genase-dependent leukotriene p r o d u c t i ~ n . ~ ~ ~ ~ ~ with the treatment of osteosarcoma. More than a decade Experiments designed to clarify the mechanism of of use in a variety of malignancies passed before pure action of this agent have been ambiguous. In studies on preparations were available and before gamma-interthe hydroxyproline content of tissues from healthy growferon, the 17 kD interferon of T-lymphocyte origin, was ing rats, malotilate had no effect when compared with purified and cloned (reviewed in by Bonnem and placebo.h7Similarly, no effect was found on the synthe01dhamx5 and Boccix6). Although a-interferon has had sis or secretion of hydroxyproline-labeled proteins or on more extensive clinical use (with clinical efficacy depro a 1(I) RNA in cultured fibroblasts. However, another monstrable in patients with hairy cell leukemia, chronic group reported inhibition of collagen synthesis in culgranulocytic leukemia, Kaposi's sarcoma, and condytured fibroblasts, although they could demonstrate this loma acuminatum), gamma-interferon shared penicillaonly in cells that had been recently trypsinized and remine's legacy of clinical Phase I and I1 trials well before plated.68 Thus, although animal studies demonstrate an its actions were fully elucidated in vitro or in animal inhibitory effect of malotilate on fibrogenesis in several models. 87 models, it remains unclear whether this effect is directly The antiproliferative effect of gamma-interferon on or inon collagen production, collagenolytic a~tivity,~' many cell types, including fibroblasts, was among its flammation. first characterized functions. Because inflammatory cells Clinical trials of malotilate in chronic liver disease and lymphocytes are often found in proximity to fibrous are ongoing in Europe. To date, the clinical experience tissue in hepatic fibrosis, gamma-interferon was invesreported in humans is largely in the Japanese literature. tigated and found to inhibit collagen production in fibroSummaries of this work suggest an effect of the agent on blasts independent of its effect on cell p r o l i f e r a t i ~ n .A~ ~ serum albumin and total protein in human cirrhotics, but variety of other cell types and culture conditions have evidence of an effect on fibrogenesis per se has not been been used to verify this effect, although whether or not ~btained.~'-'~ the suppression of collagen production represents a purely transcriptional regulatory effect remains controv e r ~ i a I . ' ~ - Gamma-interferon ~' has also been observed to have an effect on the expression of fibronectin, preA use for D-penicillamine in general conditions of sumably mediated through the gamma-interferon conaccelerated hepatic fibrogenesis has been suggested sensus sequence identified in the rat fibronectin 5' based on several properties of the drug. The efficacy of untranslated region. However, both increased and depenicillamine in treatment of Wilson's disease through
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SEMINARS IN LIVER DISEASE-VOLUME
creased fibronectin secretion has been attributed to gamma-interferon's effects in cultured fibroblast^.^^-^^ Although gamma-interferon has been used to decrease collagen scar tissue in mice93and has an antifibrogenic effect in a schistosome model of murine liver fib r ~ s i s , 'the ~ extrapolation of its in vivo effects from in vitro studies is problematic because of the complex interaction of gamma-interferon with other cytokines. Thus, gamma-interferon priming of macrophages may modulate their production of cytokines such as interleukin 1 (IL-I) and tumor necrosis factor a (TNFa), which are capable of up-regulatingy5 or down-regulatingy6 collagen expression, respectively. Gamma-interferonstimulated cells may produce prostaglandin E,, also an inhibitor of collagen production in some culture syst e m ~ Furthermore, .~~ collagen and fibronectin exposure may induce interferon production from circulating lymphoc ytes. " Finally, decreased synthesis of collagen in gammainterferon-stimulated cells may be accompanied by an increased efficiency of deposition in the extracellular matrix.98 It is apparent that a very complicated interaction of immune-function cells and matrix-producing cells may occur so that the net effect of gamma-interferon in vivo may vary with such conditions as macrophage number and state of activation, local prostaglandin levels, and the presence or absence of other matrix proteins. Nevertheless, the consistent effects of gamma-interferon on collagen production in cultured fibroblasts and murine liver encourage further in vivo study.
Collagen Propeptides It has been known for 10 years that both the collagen amino terminal propeptide known as Col- I, and the collagen 111 amino propeptide inhibit procollagen synthesis in cultured fibroblast^.^^ Additionally, the ColI propeptide inhibits procollagen mRNA translation in a cell-free system.Io0 These effects seem to be specific to collagen and the transcriptional effects occur at the level of polypeptide chain elongation or termination.lOl Subsequently, evidence has appeared for dose-dependent pretranslational inhibition of collagen synthesis by the carboxy terminal propeptide of collagen IIo2and for posttranslational regulation of collagen by a synthetic homologue of the carboxy terminal propeptide.Io3 Of particular interest was the demonstration that fibronectin, another primary constituent of the abnormal extracellular matrix found in cirrhosis, was also inhibited. Circulating propeptides themselves undergo extensive clearance in the liver with initial uptake in endothelial cells.lo4 Collagen I propeptides are also bound and taken up by fibroblasts in vitro.lo5 Thus, the potential exists for propeptides to play a physiologic role in collagen regulation by virtue of intracellular concentrations established by the amount of propeptide retained at the time of initial cleavage, and perhaps by propeptide actively taken up from the circulating pool. The complex kinetics of these propeptides are the subject of evaluation, but as yet no attempts to make therapeutic use of them have been published.
10, NUMBER 1, 1990
SUMMARY Although there is no established therapy for the fibrogenesis of hepatic cirrhosis, many potential therapies are now emerging. The requirements for the "perfect therapy" for hepatic fibrosis can be listed: (1) the pharmacologic agent should be active only in the liver; (2) its effect should be specific for collagen (or another critical extracellular matrix component); and (3) it should not be toxic. To date no agents fulfill these criteria. Of the agents we reviewed, only colchicine appears sufficiently safe for use outside of controlled clinical trials for cirrhotic patients whose underlying disease is not otherwise treatable. However, confirmation of the efficacy of colchicine in additional well-controlled clinical trials is still required. Agents such as collagen propeptides require extensive in vitro development, while trials in animal models are required for prolyl 4-hydroxylase inhibitors, proline analogues, and prostaglandins. For more developed agents, such as malotilate and gammainterferon, there is now a need for well-designed longterm clinical trials.
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Rojkind M, Kershenobich D: Effect of colchicine on collagen, albumin, and transferrin synthesis by cirrhotic rat liver slices. Biochim Biophys Acta 348:415-423, 1975. Tanner MS, Jackson D, Mowat AP: Hepatic collagen synthesis in a rat model of cirrhosis, and its modification by colchicine. J Pathol 135: 179-187, 1981. Warnes TW, Smith A, Lee FI, et al: A controlled trial of colchicine in primary biliary cirrhosis: Trial design and preliminary report. J Hepatol 5:l-7, 1987. Kaplan MM, Alling DW, Zimmerman HJ, et al: A prospective trial of colchicine for primary biliary cirrhosis. N Engl J Med 315:1448-1454, 1986. Kershenobich D, Vargas F, Garcia-Tsao G , et al: Colchicine in the treatment of cirrhosis of the liver. N Engl J Med 318:1709-1713, 1988. Poralla T.Colchizintherapie bei Leberzirrhose. Z Gastroenterol 27:lO-12, 1989. Oikarinen AI, Uitto J, Oikarinen J: Glucocorticoid action on connective tissue: From molecular mechanisms to clinical practice. Med Biol 64:221-230, 1986. Jefferson DM, Reid LM, Giambrone M-A, et al: Effects of dexamethasone on albumin and collagen gene expression in primary cultures of adult rat hepatocytes. Hepatology 5: 1420, 1985. Weiner FR, Czaja MJ, Jefferson DM, et al: The effects of dexamethasone on in vitro collagen gene expression. J Biol Chem 262:6955-6958, 1987. Weiner FR, Czaja MJ, Giambrone M-A, et al: Transcriptional and posttranscriptional effects of dexamethasone on albumin and procollagen messenger RNAs in murine schistosomiasis. Biochemistry 26: 1557-1562, 1987. Hamalainen L, Oikarinen J , Kivirikko KI: Synthesis and degradation of type I procollagen mRNAs in cultured human skin fibroblasts and the effect of cortisol. J Biol Chem 260:720725, 1985. Raghow R, Gossage D, Kang AH: Pretranslational regulation of type I collagen, fibronectin, and a 50-kilodalton noncollagenous extracellular protein by dexamethasone in rat fibroblasts. J Biol Chem 261:46774684, 1986. Oikarinen AI, Vuorio EI, Zaragoza EJ, et al: Modulation of collagen metabolism by glucocorticoids: Receptor-mediated effects of dexamethasone on collagen biosynthesis in chick embryo fibroblasts and chondrocytes. Biochem Pharmacol 37:1451-1462, 1988. Newman RA, Cutroneo KR: Glucocorticoids selectively decrease the synthesis of hydroxylated collagen peptides. Mol Pharmacol 14:185-198, 1978. Matloff DC, Kaplan M M , Neer RM, et al: Osteoporosis in primary biliary cirrhosis: Effects of 25-hydroxy vitamin D3 treatment. Gastroenterology 83:97-102, 1982. Kirk AP, Jain S, Pocock S, et al: Late results of the Royal Free Hospital prospective controlled trial of prednisolone therapy in hepatitis B surface antigen negative chronic active hepatitis. Gut 21:78-83, 1980. Ballardini G, Faccani A, Bianchi FB, et al: Steroid treatment lowers hepatic fibroplasia, as explored by serum aminoterminal procollagen 111 peptide, in chronic liver disease. Liver 4:348-352, 1984. Majamaa K, Hanauske-Abel HM, Giinzler V, et al: The 2oxoglutarate binding site of prolyl 4-hydroxylase: Identification of distinct subsites and evidence for 2-oxoglutarate decarboxylation in a ligand reaction at the enzyme-bound ferrous ion. Eur J Biochem 138:239-245, 1984. Tschank G, Raghunath M, Giinzler V, et al: Pyridinedicarboxylates, the first mechanism-derived inhibitors for prolyl 4-hyrodxylase, selectively suppress cellular hydroxyprolyl biosynthesis: Decrease in interstitial collagen and C l q secretion in cell culture. Biochem J 248:625-633, 1987.
81 Sasaki T, Majamaa K, Uitto J: Reduction of collagen production in keloid fibroblast cultures by ethyl-3,4-dihyroxybenzoate: Inhibition of prolyl hydroxylase activity as a mechanism of action. J Biol Chem 262:9397-9403, 1987. Uitto J, Ryhanen L, Tan EML, et al: Pharmacological inhibition of excessive collagen deposition in fibrotic diseases. Fed Proc 43:28 15-2820, 1984. Tan EML, Ryhanen L, Uitto J: Proline analogues inhibit human skin fibroblast growth and collagen production in culture. J Invest Dermatol 80:261-267, 1983. Rojkind M: Inhibition of liver fibrosis by L-azetidine-2-carboxylic acid in rats treated with carbon tetrachloride. J Clin Invest 52:245 1-2456, 1973. Leung MK, Fessel KK, Greenberg DB, et al: Separate amino and carboxyl procollagen peptides in chicken embryo tendon. J Biol Chem 254:224-232, 1979. Ryhanen L, Tan EML, Rantala-Ryhanen, et al: Conversion of type I1 procollagen to collagen in vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids, polyamines, and structurally related compounds. Arch Biochem Biophys 215:230-236, 1982. Zaragoza EJ, Ryhanen L, Oikarinen AI, et al: Inhibition of type I1 procollagen to collagen conversion by lysine derivatives and related compounds. Mapping of the inhibitory structural features. Biochem Pharmacol 35:532-535, 1986. Ruwart MJ, Rush BD, Snyder KF, et al: 16.16-Dimethyl prostaglandin El delays collagen formation in nutritional injury in rat liver. Hepatology 8%-64, 1988. Berg RA, Moss J, Baum BJ, et al: Regulation of collagen production by the P-adrenergic system. J Clin Invest 67: 1457-1462, 1981. Baum BJ, Moss J, Breul SD, et al: Effect of cyclic AMP on the intracellular degradation of newly synthesized collagen. J Biol Chem 255:2843-2847, 1980. Stachura J, Tarnowski A, Ivey KJ, et al: Prostaglandin protection of carbon tetrachloride-induced liver cell necrosis in the rat. Gastroenterology 8 1:21 1-217, 198 1. Abecassis M, Falk JA, Makowka L, et al: 16.16-Dimethyl prostaglandin E, prevents the development of fulminant hepatitis and blocks the induction of monocytelmacrophage procoagulant activity after murine hepatitis virus strain 3 infection. J Clin Invest 80:881-889, 1987. Richardson PD, Withrington RG: The vasodilator actions of isoprenaline, histamine, prostaglandin E,. glucagon and secretin on the hepatic arterial vascular bed of the dog. Br J Pharmacol 57:581-588, 1976. Robert A, Ruwart MJ: The effects of prostaglandins on the digestive system. In: Lee JB (Ed): Prostaglandins. New York, ElsevierINorth Holland, 1982. Oikarinen H, Oikarinen AI, Tan EML, et al: Modulation of procollagen gene expression by retinoids: Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures. J Clin Invest 75:1545-1553, 1985. Ohta A, Louise JS, Uitto J: Retinoid modulation of collagenase production by adherent human mononuclear cells in culture. Ann Rheum Dis 46:357-362, 1987. Davis BH, Pratt BM, Madri JA: Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels. J Biol Chem 262:10280-10286, 1987. Davis BH: Transforming growth factor P responsiveness is modulated by the extracellular collagen matrix during hepatic Ito cell culture. J Cell Physiol 136:547-553, 1988. Russell RM, Boyer JL, Bagheri SA, Hruban Z: Hepatic injury from chronic hypervitaminosis A resulting in portal hypertension and ascites. N Engl J Med 291:435440, 1974.
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Kahari V-M, Heino J, Vuorio T, Vuorio E: Interferon-a and gamma-interferon reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture. Biochim Biophys Acta 968:45-50, 1988. Melin M, Harmann DJ, Magliore H, et al: Human recombinant gamma-interferon stimulates proliferation and inhibits collagen and fibronectin production by human dental pulp fibroblasts. Cell Mol Biol 35:97-110, 1989. Granstein RD, Murphy GF, Margolis RJ, et al: Gamma-interferon inhibits collagen synthesis in vivo in the mouse. J Clin Invest 79: 1254-1258, 1987. Czaja MJ, Weiner FR, Giambrone M-A, et al: Antifibrogenic effects of gamma-interferon in murine schistosomiasis. Hepatology 7: 1047, 1987. Goldring MB, Krane SM: Modulation by recombinant interleukin 1 of synthesis of types I and I11 collagens and associated procollagen mRNA levels in cultured human cells. J Biol Chem 262: 16724-16729, 1987. Solis-Herruzo JA, Brenner DA, Chojkier M: Tumor necrosis factor a inhibits collagen gene transcription and collagen synthesis in cultured human fibroblasts. J Biol Chem 263:5841-5845, 1988. Ofosu-Appiah W, Warrington RJ, Morgan K, Wilkins JA: Lymphocyte extracellular matrix interactions: Induction of interferon by connective tissue components. Scand J Immuno1 29:517-525, 1989. Clark JG, Dedon TF, Wayner EA, Carter WG: Effects of gamma-interferon on expression of cell surface receptors for
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collagen and deposition of newly synthesized collagen by cultured human lung fibroblasts. J Clin Invest 83: 1505-15 11, 1989. Wiestner M, Kreig T, Horlein D, et al: Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures. J Biol Chem 254:7016-7023, 1979. Paglia LM, Wiestner M, Duchene M, et al: Effects of procollagen peptides on the translation of type I1 collagen mRNA and on collagen biosynthesis in chondrocytes. Biochemistry 20:3523-3527, 1981. Horlein, D. McPherson J, Goh SH, et al: Regulation of protein synthesis: Translational control by procollagen-derived fragments. Proc Natl Acad Sci USA 78:6163-6167, 1981. Wu CH, Donovan CB, Wu GY: Evidence for pretranslational regulation of collagen synthesis by procollagen propeptides. J Biol Chem 261: 10482-10484, 1986. Aycock RS, Raghow R, Stricklin GP, et al: Post-transcriptional inhibition of collagen and fibronectin synthesis by a synthetic homolog of a portion of the carboxyl-terminal propeptide of human type I collagen. J Biol Chem 261:1435514360, 1986. Smedsrod B: Aminoterminal peptide of type 111 procollagen is cleared from the circulation by receptor-mediated endocytosis in liver endothelial cells. Coll Relat Res 8:375-388, 1988. Schlumberger W, Thie M, Volmer H, et al: Binding and uptake of Col ](I), a peptide capable of inhibiting collagen synthesis in fibroblasts. Eur J Cell Biol 46:244-252, 1988.
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THERAPY FOR HEPATIC FIBROSIS-BRENNER.
10, NO. I , 1990
Therapy for Hepatic Fibrosis
A hallmark of cirrhosis is the increased deposition of connective tissue proteins. Fibrogenesis (that is, an increased rate of synthesis of extracellular matrix molecules) plays an important role in this accumulation of extracellular tissue and results in a severalfold increase in hepatic collagen content. Other extracellular matrix proteins, including laminin, fibronectin, and proteoglycans, are also found in greater abundance. One result of such matrix protein accumulation is increased hepatic vascular resistance and consequent portal hypertension. This leads to the well-known complications of cirrhosis, which are often difficult to manage and may be fatal. In some liver diseases the stimuli leading to cirrhosis have been identified and specific therapies designed to counteract these stimuli. For instance, the iron overload of hemochromatosis is treated with phlebotomy or chelation, hepatitis B may be treated with antiviral agents, autoimmune chronic active hepatitis may be treated with immunosuppressive therapy, and copper overload of Indian childhood cirrhosis and Wilson's disease is treated with copper chelation. This review will not discuss these specific therapies but will instead address potential therapeutic interventions that might inhibit the ongoing process of hepatic fibrogenesis regardless of etiology. After briefly introducing some aspects of matrix production and assessment, we will discuss the agents most used or most heralded in the treatment of hepatic fibrosis. Since collagen is the major constituent of hepatic fibrous tissue, many therapies have been designed to interfere specifically with its biosynthesis. Although 12 separate collagens have been identified, the large fibrillar types I and I11 predominate in the fibrous tissue of cirrhosis. For this reason, many strategies designed to limit production of this abnormal fibrous tissue have concentrated on suppressing the secretion and accumulation of collagen types I and 111 in particular. However, it remains a possibility that strategically positioned but less abundant molecules, that is, collagen IV or laminin, deposited in a membrane covering the fenestrations of the sinusoidal endothelium, may be the critical factor in
the hemodynamic alterations that are the consequence of hepatic fibrosis. This should be borne in mind when evaluating therapies principally devoted to the reduction in collagen types I and 111. The metabolism of collagen is a complex process providing many opportunities for pharmacologic intervention (Fig. 1). In brief, the collagen genes are transcribed by DNA polymerase 11, and the resulting collagen transcripts are processed and transported to the rough endoplasmic reticulum where they -are translated as prepropeptides. Nascent polypeptide chains then undergo extensive post-translational modifications, including hydroxylation of select lysyl and prolyl residues and glycosylation of hydroxylysyl residues.' Three polypeptide chains spontaneously form a stable triple helix through noncovalent interactions. The triple helix is secreted extracellularly with cleavage of the amino terminal and carboxy terminal propeptides. The collagen fibrils are formed by aggregation of the triple helices with covalent cross-linking between the triple helices. The collagen peptides and the cleaved propeptides can undergo degradation either intracellularly4 or extracelluAny of these steps might be blocked by therapeutic agents in order to prevent collagen synthesis and secretion. In general, agents enhancing matrix degradation and remodeling have not been exploited for therapeutic effect. A primary obstacle to the evaluation of all therapies for hepatic fibrogenesis has been the lack of a safe means of assessing therapeutic effect. Liver biopsy has wellknown hazards as well as unavoidable sampling . - error. A noninvasive measure of fibrotic matrix accumulation is urgently needed. One proposed measure has been the levels of circulating collagen propeptides, particularly the carboxy terminal propeptide of collagen I11 (PIIIP).' However, there are conceptual difficulties with the use of this measurement7 (see article by Maher in this issue of Seminars).
THERAPEUTIC AGENTS Colchicine
From the Department of Medicine and Center for Molecular Genetics, University of California, and VA Medical Center, San Diego, California. Reprint requests: Dr. Brenner, Department of Medicine, "niversity of California, San Diego school of Medicine, La Jolla, CA 92093.
The rationale for the use of colchicine in fibrotic diseases is that colchicine inhibits the polymerization of to be required microtubules, a process that is for collagen secretion. In cultured fibroblasts and hepatocytes, colchicine transiently inhibits the secretion of
Copyright O 1990 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, N Y 10016. All rights reserved.
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DAVID A. BRENNER, M.D., and JOSEPH M. ALCORN, M.D.
SEMINARS IN LIVER DISEASE-VOLUME
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Proposed mechanisms of inhibitors
Collagen Biosynthesis Procollagen gene
I
transcription
i
translation
b--,--
propeptides
Procollagen polypeptide
I I
post translational modification
I
gamma-interferon glucocorticoids
mRNA
chain alignment
prolyl 4-hydroxylase inhibitors proline analogs
Triple Helix
1 4
secretion
Extracellular procollagen
+ NxQ%zQs - -
colchicine
Removal of propeptide
-
C
1
procollagen-to-collagen inhibitors
Collagen Covalent cross-linking
I
Fibrils
FIG. 1. A simplified pathway for the biosynthesis of collagen. Some of the agents that inhibit collagen synthesis, as discussed in the text, are shown at the sites of their proposed actions.
collagen into the medium for the first 2 to 3 hours8,' but collagen secretion returns to normal levels after prolonged i n c u b a t i ~ n .Other ~ actions of colchicine that might inhibit fibrosis include inhibition of mitosis,1° stimulation of collagenase secretion," and anti-inflammatory effects, including the inhibition of polymorphonuclear leukocyte functions.12 Two research groups have demonstrated that colchicine decreases collagen content in the carbon tetrachloride (CCl,) model of hepatic fibrosis in rats.I3.l4This effect is not mediated by inhibition of prolyl hydroxylase activity,I4 a critical enzyme in the biosynthesis of collagen. Colchicine has been used in clinical trials in several types of cirrhosis. In primary biliary cirrhosis, one clinical trialI5 showed initial improvement in some biochemical liver tests of patients treated with colchicine, whereas a second trial demonstrated a more sustained improvement.16 Neither of these two clinical trials dem-
onstrated improvement in the histology of the liver biopsies nor in the mortality of patients treated with colchicine. However, the small number of patients in these trials make results subject to type I1 error. In a much discussed double-blind placebo-controlled trial, patients with cirrhosis of all etiologies were treated with colchicine or placebo.'' Treatment with colchicine resulted in sustained improvement in survival as well as improvement in liver biopsy histologic patterns. Criticisms of this study include a disproportionate number of women and a higher mean albumin level in the colchicine group.I8 In addition, there was no monitoring of compliance with medication, and a large number of patients were lost to follow-up. In all of the trials mentioned, colchicine had a low incidence of side effects, which consisted of diarrhea, nausea, and epigastric distress. The much rarer complications of colchicine, including agranulocytosis, aplastic anemia, epistaxis, and myopathy, were not reported in these small studies. A
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quired for the formation and stability of the collagen triple helix. The enzyme prolyl 4-hydroxylase requires ferrous iron, molecular oxygen, 2-oxyglutrate, and ascorbate to catalyze the stereospecific hydroxylation of proline residues in the polypeptide chain with the miniGlucocorticoids mum required sequence of X-Pro-Gly. The reaction consists of the decarboxylation of 2-oxyglutrate with Glucocorticoids such as dexamethasone selectively concomitant Fe + generation and the subsequent hydrodecrease collagen synthesis by several different mechaxylation of the prolyl residues30 (Fig. 2). Elegant studies nisms in a variety of experimental models.19 Glucocoron the structure and function of the purified prolyl 4ticoids markedly decrease mRNA levels of type I collagen in primary cultures of adult rat h e p a t o ~ y t e s ~and ~ , ~ ' hydroxylase enzyme led to the identification of compounds that inhibit the enzyme's activity by competing in murine hepatic schistos~miasis.~~ This effect is in part the result of decreased transcriptional a ~ t i v i t y . ~Fur' . ~ ~ with 2-oxyglutarate for binding." Two compounds, pyridine-2,4-dicarboxylate (2,4thermore, a regulatory element in the collagen a2(I) proPDCA) and its isomer pyridine-2,5-dicarboxylate(2,smoter region that is specifically inhibited by the presence PDCA) were identified as potent inhibitors of purified of glucocorticoids has been identified.21 On the other prolyl4-hydroxylase in vitro. In addition, in a variety of hand, in primary fibroblast cultures, glucocorticoids decultured cells, 2,4-PDCA and 2,5-PDCA decrease the crease type I collagen mRNA level^^'-^^ and type I colgeneration of hydroxyproline and the secretion of collalagen mRNA ~ t a b i l i t y without ~ ~ , ~ ~ any change in the gen into the media.3' This is a selective effect of these Glucocortitranscriptional rate of the collagen compounds in that there is a minimal effect on total procoids also decrease the post-translational modifications tein synthesis or on the secretion of nonhydroxyproline of the procollagen peptides by decreasing the activities proteins. of prolyl hydroxylase and lysyl hydroxyla~e.~~ However, A separate group of prolyl hydroxylase inhibitors the activities of these enzymes do not appear to be rate that are analogues of both a-ketoglutarate and ascorbate limiting in the biosynthesis of collagen, and therefore has been developed. Ethyl 3-4-dihydroxy benzoate, a glucocorticoids do not appear to inhibit collagen synthemodel of this type of prolyl hydroxylase inhibitor, was sis at the level of post-translational modification^.'^ extensively studied in cultured human fibrobla~ts.~~ This Glucocorticoids have been used as specific therapy compound decreases cellular prolyl hydroxylase activity in the treatment of chronic liver disease, including priin the cells, decreases the incorporation of hydroxypromary biliary cirrhosis27and autoimmune chronic hepatiline into proteins, decreases the stability of the collagen ti^.^^ There is some evidence that glucocorticoids detriple helix, and decreases the synthesis and secretion of crease collagen synthesis in patients,29 which might type I and 111 collagens. The inhibitor has no effect on reflect the mechanisms of action discussed in addition to collagen mRNA levels. Ethyl 3-4-dihydroxy benzoate, the anti-inflammatory effects of glucocorticoids. Unforwhich is hydrophobic and therefore able to cross the cell tunately, the detrimental systemic effects of long-term membrane, specifically inhibits the production of collaglucocorticoid therapy, including severe osteoporosis in genous proteins and is nontoxic. patients with primary biliary cirrhosis,27preclude its agMore recently, hepatocyte-targeted lipophilic proingressive use as a general treatment of fibrogenic liver hibitors have been developed that may specifically supdisease. press hepatocyte collagen hydroxylation and synthesis in vivo (Hanauske-Abel HM: Personal communication). Prolyl CHydroxylase Inhibitors These exciting compounds require further evaluation with respect to their mechanisms, effectiveness, and toxThe post-translational modification of certain proicity in animal models. Such further evaluation may reline residues in collagen to trans-4-hydroxyproline is re-
0
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.-0C 0
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-C-N
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..
C y ,CH-C-N-.
.,.
H
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4-Hydroxyproline
FIG. 2. The substrates and products of the reaction catalyzed by prolyl Chydroxylase.
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theoretical concern is that the inhibition of normal healing by colchicine might lead to worsening of esophageal variceal hemorrhage.18
SEMINARS IN LIVER DISEASE-VOLUME
veal additional steps in which they suppress collagen synthesis and secretion.
Proline Analogues Several analogues of the amino acid proline are recognized by prolyl tRNA and incorporated in the place of proline into nascent polypeptide chains. These analogues alter the structure and function of the resulting proteins.33 Azetidine carboxylic acid, an example of a proline analogue, has been studied in cultured fibroblast^.^^ Incubation with azetidine carboxylic acid results in the synthesis of a nontriple helical form of collagen that is subject to degradation by proteases to which the triple helix of collagen is resistant. The resulting collagen is also subject to degradation by endogenous proteases with production of a dialyzable form of hydroxyproline. In addition, fibroblast proliferation decreases, which might also be the result of decreased secretion of extracellular matrix proteins. Using the CC1,-induced cirrhotic model in rats, one study demonstrated that azetidine carboxylic acid inhibits liver f i b r ~ s i s . The ~ ' rats treated with azetidine carboxylic acid have decreased hepatic collagen histologically and decreased incorporation of proline into the hydroxyproline of collagen. The agent does not appear toxic, since the incorporation of radioactivity into noncollagenous proteins was unchanged.
Procollagen-to-Collagen Inhibitors The removal of the amino terminal and carboxy terminal propeptides is required for the proper alignment of the collagen triple helices to form fibrils and for subsequent formulation of covalent cross-links to stabilize the fibrils. If the propeptides are not removed, then the stability of the collagen is decreased. Leung et aP6 first demonstrated that arginine and its analogue concavanine inhibit the removal of the carboxy propeptide of type I procollagen. Subsequent experiments demonstrated that several amino acids and their analogues, polyamines, and dipeptides inhibit the removal of the carboxy propeptide of type I1 collagen. The removal of the amino ' . ~one ~ study, cadaverine propeptide is not a f f e ~ t e d . ~ In (a polyamine) and lysine were the most effective inhibitors tested.37In general, a free amino group is necessary but not sufficient to inhibit the conversion of carboxy procollagen to collagen. The application of these agents to the treatment of models of fibrosis or clinical fibrotic diseases has not yet been reported.
Prostaglandins Prostaglandins have diverse intracellular metabolic effects. The prostaglandin analogue, 16,16-dimethyl prostaglandin E, (DMPG) decreases fibrosis and fat accumulation induced by the choline-deficient diet model of hepatic fibrosis in rats.39Several possible mechanisms might explain the beneficial effects of prostaglandins in hepatic fibrosis. Prostaglandins increase intracellular cyclic adenosine monophosphate levels, which increase the intracellular degradation of collagen in cultured fi-
10, NUMBER 1, 1990
b r o b l a s t ~ . ~Alternatively, ~,~' DMPG may have a hepatrophic effect and prevent fibrosis at an earlier stage of injury. DMPG decreases hepatic injury from the acute toxicity of CC1,4, and from hepatocellular necrosis induced by the murine hepatitis virus in vivo and in ~ i t r o . ~ ~ Other potentially beneficial effects of prostaglandins include increased hepatic blood alteration of membrane fluidity, changes in systemic levels of hormone, including glucagon and and inhibition of the release of inflammatory factors by macro phage^.,^ To date, prostaglandins have not been used in the treatment of hepatic fibrosis in humans.
Retinoids Certain retinoids have been used for the treatment of hyperproliferative epidermal diseases. Retinoids, including all trans-retinoic acid and 13 cis-retinoic acid, selectively decrease type I collagen synthesis and type I collagen mRNA levels in cultured f i b r ~ b l a s t s . ~ Wthe n other hand, these retinoids decrease collagenase production by m o n o c y t e ~These . ~ ~ in vitro results cannot be extrapolated to using retinoids in the treatment of hepatic fibrosis because of the presence of hepatic Ito cells. In vivo, retinoids are largely stored as retinol palmitate in the hepatic Ito cells. When Ito cells are cultured on type I collagen matrix, the addition of retinol leads to an increase in type I collagen production. However, when Ito cells are cultured on a type IV collagen matrix, the addition of retinol produces an increase in type I11 collagen but a decrease in type I ~ o l l a g e n(see ~ ~ .also ~ ~ article by Friedman in this issue of Seminars). Therefore the regulation of collagen production by Ito cells represents a complex interaction between the extracellular matrix and the intracellular vitamin A c ~ n c e n t r a t i o n . The ~ ' ~ ~thera~ peutic effectiveness of vitamin A in hepatic fibrosis is unknown. Indeed, a number of studies suggest that prolonged exposure to elevated levels of retinoids may result in hepatic fibro~is.'~
Malotilate Malotilate (di-isopropyl 1,3-dithiol-2-ylidenemalonate) is a water-insoluble dithiolane derivative developed by the Nihon Nohyaku Company as a nontoxic inducer of hepatic protein p r o d ~ c t i o n .It~ 'is absorbed from the gastrointestinal tract and undergoes extensive hepatic firstpass m e t a b ~ l i s m . Its ~ ~pharmacologic ,~~ actions include an increase in hepatic protein to DNA and RNA to DNA ratios, induction of cytochrome b, (without significant effect on cytochrome P450), an increase in reduced nicotinamide-adenine dinucleotide phosphate cytochrome C reductase activity, and induction of glucose-6-phosphate dehydrogenase activity.52 Primarily because of its effect on hepatic protein production, malotilate has been considered for potential use in liver disease. Although a number of studies have now been performed evaluating the effects of this agent on acute hepatic injury from a variety of compounds, few studies specifically address fibrogenesis. Dumont et a15, demonstrated histologic protection from both inflammation and fibrogenesis when maloti-
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ALCORN
chelation of inappropriately elevated copper is well late was administered to rats treated twice weekly with known.7' In addition, penicillamine scavenges immune intraperitoneal CCI,. Malotilate also suppressed CCI, complexes,72interferes with leukocyte c h e m o t a x i ~and ,~~ induction of collagen synthesis and accumulation as % n copper-penicillamine d inhibits T-cell f ~ n c t i o n , ~ ~ . ~ the assessed by hepatic collagen content and proline incorporation. In another study of CC1,-induced fibrosis, ' inhibicomplex has superoxide-dismutase a ~ t i v i t y . ~An Schuppan et aIs5 reported that malotilate maintained tion of intra- and intermolecular collagen cross-linking and leukotreine D, catabohas also been de~cribed,~',~' nearly normal serum PIIIP (collagen I11 amino propeplism has recently been shown to be inhibited.7yFinally, tide) levels while attenuating collagen accumulation. Of interest, the pulmonary fibrosis induced by intraperitoD-penicillamine may increase the solubility of secreted neal CCI, is also attenuated by the use of m a l ~ t i l a t e . ~ ~ collagen.x0 .~~ One or more or these actions may explain the Siegers et alSxproduced cirrhosis in rats with oral CCI, beneficial effects of penicillamine on Indian childhood and 5% ethanol instead of drinking water. In this model ~ i r r h o s i s .However, ~' the largest clinical experience with penicillamine in fibrosis in liver disease has been with malotilate, but not colchicine, penicillamine, or cianiprimary biliary cirrhosis, for which its use has been disdanol, completely protected the-animal from histologic app~inting,~' perhaps in part because of a failure to stratfibrosis and was associated with a nearly normal liver ify patients according to the severity of their disease. hydroxyproline content. Steatosis, attenuated by maloThese clinical trials of penicillamine coincided with tilate following acute CCI, intoxication in other studin vitro assessment of its effect on matrix proteins and i e ~ , ~was " ~not affected in this model of fibrosis. Polargely preceded evidence of its efficacy in animal tentially important in this model could be malotilate models of fibrosis. A small number of trials of penicilsuppression of acetaldehyde p r o d u ~ t i o n , ~a 'known prolamine in animal models of hepatic fibrosis have been moter of collagen synthesis.62 Partial protection against reported. In general, they demonstrate little or no effect fibrosis has also been reported in a dimethyl nitrosamine of penicillamine on fibr~genesis.'~.~" In vitro effects at model of hepatic fibrosish' and in immunologic models, high doses include decreased fibroblast proliferation and including egg yolk sensitization.@ ~ ~ .proteoglycan ~~ synthesis has collagen p r o d u c t i ~ n ,but Although malotilate does not seem to interfere with been reported to be increasedx" and decreasedXJin differCC1, uptake, it is possible that it inhibits the metabolism ent systems. Ultimately, the paucity of data leaves the of CCI, to toxic mediators of inflammation or interferes question of a specific effect of penicillamine on hepatic . ~ ~ evidence of generally with free radical g e n e r a t i ~ nThe fibrogenesis unanswered. decreased tissue injury and inflammation in these models is consistent with a primary effect on inflammatory meGamma-Interferon diators of fibrogenesis. Of interest in this regard is a recent report that malotilate specifically inhibits 5-lipoxyThe first clinical trials of interferon began in 1972 genase-dependent leukotriene p r o d u c t i ~ n . ~ ~ ~ ~ ~ with the treatment of osteosarcoma. More than a decade Experiments designed to clarify the mechanism of of use in a variety of malignancies passed before pure action of this agent have been ambiguous. In studies on preparations were available and before gamma-interthe hydroxyproline content of tissues from healthy growferon, the 17 kD interferon of T-lymphocyte origin, was ing rats, malotilate had no effect when compared with purified and cloned (reviewed in by Bonnem and placebo.h7Similarly, no effect was found on the synthe01dhamx5 and Boccix6). Although a-interferon has had sis or secretion of hydroxyproline-labeled proteins or on more extensive clinical use (with clinical efficacy depro a 1(I) RNA in cultured fibroblasts. However, another monstrable in patients with hairy cell leukemia, chronic group reported inhibition of collagen synthesis in culgranulocytic leukemia, Kaposi's sarcoma, and condytured fibroblasts, although they could demonstrate this loma acuminatum), gamma-interferon shared penicillaonly in cells that had been recently trypsinized and remine's legacy of clinical Phase I and I1 trials well before plated.68 Thus, although animal studies demonstrate an its actions were fully elucidated in vitro or in animal inhibitory effect of malotilate on fibrogenesis in several models. 87 models, it remains unclear whether this effect is directly The antiproliferative effect of gamma-interferon on or inon collagen production, collagenolytic a~tivity,~' many cell types, including fibroblasts, was among its flammation. first characterized functions. Because inflammatory cells Clinical trials of malotilate in chronic liver disease and lymphocytes are often found in proximity to fibrous are ongoing in Europe. To date, the clinical experience tissue in hepatic fibrosis, gamma-interferon was invesreported in humans is largely in the Japanese literature. tigated and found to inhibit collagen production in fibroSummaries of this work suggest an effect of the agent on blasts independent of its effect on cell p r o l i f e r a t i ~ n .A~ ~ serum albumin and total protein in human cirrhotics, but variety of other cell types and culture conditions have evidence of an effect on fibrogenesis per se has not been been used to verify this effect, although whether or not ~btained.~'-'~ the suppression of collagen production represents a purely transcriptional regulatory effect remains controv e r ~ i a I . ' ~ - Gamma-interferon ~' has also been observed to have an effect on the expression of fibronectin, preA use for D-penicillamine in general conditions of sumably mediated through the gamma-interferon conaccelerated hepatic fibrogenesis has been suggested sensus sequence identified in the rat fibronectin 5' based on several properties of the drug. The efficacy of untranslated region. However, both increased and depenicillamine in treatment of Wilson's disease through
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SEMINARS IN LIVER DISEASE-VOLUME
creased fibronectin secretion has been attributed to gamma-interferon's effects in cultured fibroblast^.^^-^^ Although gamma-interferon has been used to decrease collagen scar tissue in mice93and has an antifibrogenic effect in a schistosome model of murine liver fib r ~ s i s , 'the ~ extrapolation of its in vivo effects from in vitro studies is problematic because of the complex interaction of gamma-interferon with other cytokines. Thus, gamma-interferon priming of macrophages may modulate their production of cytokines such as interleukin 1 (IL-I) and tumor necrosis factor a (TNFa), which are capable of up-regulatingy5 or down-regulatingy6 collagen expression, respectively. Gamma-interferonstimulated cells may produce prostaglandin E,, also an inhibitor of collagen production in some culture syst e m ~ Furthermore, .~~ collagen and fibronectin exposure may induce interferon production from circulating lymphoc ytes. " Finally, decreased synthesis of collagen in gammainterferon-stimulated cells may be accompanied by an increased efficiency of deposition in the extracellular matrix.98 It is apparent that a very complicated interaction of immune-function cells and matrix-producing cells may occur so that the net effect of gamma-interferon in vivo may vary with such conditions as macrophage number and state of activation, local prostaglandin levels, and the presence or absence of other matrix proteins. Nevertheless, the consistent effects of gamma-interferon on collagen production in cultured fibroblasts and murine liver encourage further in vivo study.
Collagen Propeptides It has been known for 10 years that both the collagen amino terminal propeptide known as Col- I, and the collagen 111 amino propeptide inhibit procollagen synthesis in cultured fibroblast^.^^ Additionally, the ColI propeptide inhibits procollagen mRNA translation in a cell-free system.Io0 These effects seem to be specific to collagen and the transcriptional effects occur at the level of polypeptide chain elongation or termination.lOl Subsequently, evidence has appeared for dose-dependent pretranslational inhibition of collagen synthesis by the carboxy terminal propeptide of collagen IIo2and for posttranslational regulation of collagen by a synthetic homologue of the carboxy terminal propeptide.Io3 Of particular interest was the demonstration that fibronectin, another primary constituent of the abnormal extracellular matrix found in cirrhosis, was also inhibited. Circulating propeptides themselves undergo extensive clearance in the liver with initial uptake in endothelial cells.lo4 Collagen I propeptides are also bound and taken up by fibroblasts in vitro.lo5 Thus, the potential exists for propeptides to play a physiologic role in collagen regulation by virtue of intracellular concentrations established by the amount of propeptide retained at the time of initial cleavage, and perhaps by propeptide actively taken up from the circulating pool. The complex kinetics of these propeptides are the subject of evaluation, but as yet no attempts to make therapeutic use of them have been published.
10, NUMBER 1, 1990
SUMMARY Although there is no established therapy for the fibrogenesis of hepatic cirrhosis, many potential therapies are now emerging. The requirements for the "perfect therapy" for hepatic fibrosis can be listed: (1) the pharmacologic agent should be active only in the liver; (2) its effect should be specific for collagen (or another critical extracellular matrix component); and (3) it should not be toxic. To date no agents fulfill these criteria. Of the agents we reviewed, only colchicine appears sufficiently safe for use outside of controlled clinical trials for cirrhotic patients whose underlying disease is not otherwise treatable. However, confirmation of the efficacy of colchicine in additional well-controlled clinical trials is still required. Agents such as collagen propeptides require extensive in vitro development, while trials in animal models are required for prolyl 4-hydroxylase inhibitors, proline analogues, and prostaglandins. For more developed agents, such as malotilate and gammainterferon, there is now a need for well-designed longterm clinical trials.
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