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Protease inhibitors suppress TGF-β generation by hepatic stellate cells
Copyright C European Association for the Study of the Liver 1998
Journal of Hepatology 1998; 29: 1031–1036 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen
Journal of Hepatology ISSN 0168-8278
Correspondence
Protease inhibitors suppress TGF-b generation by hepatic stellate cells To the Editor: Transforming growth factor-b (TGF-b) is the major cytokine implicated in the pathogenesis of liver fibrosis and cirrhosis (1,2). Recently,
Fig. 1. Suppression of the release and activation of latent TGF-b by protease inhibitors in rat HSC cultures. Primary rat HSCs were incubated with plasmin (A), RA (B) or basic FGF (C) in the absence and presence of various protease inhibitors (A-T). Concentrations of TGF-b in the media were measured using [3H]thymidine incorporation by mink lung epithelial cells. M, Latent TGF-b; H, active TGF-b. A, aprotinin; B, tranexamic acid, C, camostat mesilate; D, FOY251; E, gabexate mesilate; F, ONO3403; G, ONO5046; H, FO011; I, FO015; J, FO106; K, FO209; L, FO349 (C-L were kindly supplied by Ono Pharmaceuticals, Osaka, Japan); M, compound .81; N, compound .82; O, compound .83; P, compound .97; Q, compound .105; R, compound .106; S, cis-AMCHA; and T, AMBOCA (MT were kindly supplied by Daiichi Pharmaceuticals, Tokyo, Japan, see reference 6). Columns and bars represent means and SD (nΩ6). Representative results are shown from three independent experiments. *:p∞0.01 compared to the control and groups A–T.
we demonstrated that in porcine serum-treated rats, hepatic fibrosis is exacerbated following simultaneous administration of retinoic acid (RA) by virtue of its ability to produce TGF-b in hepatic stellate cells (HSCs) (3). RA up-regulates expression of plasminogen activator (PA) through nuclear RA receptor (RAR), and thus increases HSC surface plasmin levels (4). This causes the proteolytic release and activation of latent TGF-b1 constitutively produced by HSCs and stored in the adjacent extracellular matrix. Resultant active TGF-b1 autostimulates TGF-b2 and TGF-b3 expression, and all the TGF-bs exacerbate fibrosis by increasing collagen production in HSCs, as well as by suppressing normal function of surrounding hepatocytes (3,5). This observation strongly suggests that proteolytic release and activation of latent TGF-b1 may be a potential target for suppression of TGF-b-mediated liver fibrosis. This idea allowed us to examine which protease inhibitors, including those currently used for human therapy, would be effective in suppressing TGF-b generation by cultured HSCs in vitro (Fig. 1A). This simple experiment is particularly important for exploring our novel idea in vivo in future. Rat primary HSCs, isolated and pre-cultured for 7 days, were incubated for 12 h with 2 units/ml plasmin (Sigma Co., St. Louis, MO, USA) in the absence and presence of 100 mM each of 20 different protease inhibitors. Thereafter, concentrations of both active and total (active plus latent) TGF-b present in the culture medium were determined by bioassays employing CCL64 mink lung epithelial cells. Interestingly, all protease inhibitors tested significantly suppressed plasmin-induced generation of TGF-b in both active and total levels, suggesting that both release and activation were blocked. Similar results were obtained with HSCs stimulated by either 1 mM RA (Sigma) or 10 ng/ml basic fibroblast growth factor (basic FGF) (Boehringer, Mannheim, Germany), reagents which increase endogenous plasmin levels in the cells (Fig. 1B and 1C). These results insure that a protease inhibitor or a combination of several protease inhibitors may be worth testing for their effect on in vivo generation of TGF-b in the liver and resulting development of hepatic fibrosis. Since several compounds (e.g., compounds C and E) are broad-spectrum inhibitors, the result suggests that these protease inhibitors blocked the activity of plasmin and/or plasmin-activated unknown proteases capable of generating TGF-b. Currently, compounds C and E are used for therapy of pancreatitis, reflux esophagitis, and disseminated intravascular coagulation (DIC). The present data suggest that these compounds might be beneficial also for the therapy of liver fibrosis. The idea of using protease inhibitors against hepatic fibrosis is further supported by the fact that compound B has been used episodically as a cytoprotective agent against liver damage (2). We are now testing our idea employing rat hepatic fibrosis models as well as in cirrhotic patients. Masataka Okuno1 Hisataka Moriwaki1, Yasutoshi Muto1 and Soichi Kojima2 1 First Department of Internal Medicine, Gifu University School of Medicine, Gifu 500-8076, Japan, and 2Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba 305-0074, Japan
References 1. Friedman SL. The cellular basis of hepatic fibrosis. N Engl J Med 1993; 328: 1828–35. 2. Gressner AM. Liver fibrosis: perspectives in pathobiochem-
1031
Correspondence ical research and clinical outlook. Eur J Clin Chem Clin Biochem 1991; 29: 293–311. 3. Okuno M, Moriwaki H, Imai S, Muto Y, Kawada N, Suzuki Y, et al. Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF-b in liver stellate cells. Hepatology 1997; 26: 913–21. 4. Imai S, Okuno M, Moriwaki H, Muto Y, Murakami K, Shudo K, et al. 9,13-di-cis-Retinoic acid induces the production of tPA and activation of latent TGF-b via RARa in a human liver stellate cell line, LI90. FEBS Lett 1997; 411: 102–6.
5. Koda H, Okuno M, Imai S, Moriwaki H, Muto Y, Kawada N, et al. Retinoic acid-stimulated liver stellate cells suppress the production of albumin from parenchymal cells via TGFb. Biochem Biophys Res Commun 1996; 221: 565–9. 6. Okano A, Inaoka M, Funabashi S, Iwamoto M, Isoda S, Moroi R, et al. Medicinal chemical studies on antiplasmin drugs. 4. Chemical modification of trans-4-aminomethylcyclohexanecarboxylic acid and its effect on antiplasmin activity. J Med Chem 1972; 15: 247–55.
Coupling to lactosaminated poly-L-lysine reduces the toxic effects of ribavirin on red blood cells To the Editor: Recent data (1,2) confirm that ribavirin (RIBV) enhances the efficacy of interferon (IFN) in the treatment of chronic hepatitis C, but it causes haemolysis. In order to reduce its haemotoxicity, RIBV was coupled to lactosaminated poly-L-lysine (L-poly(LYS)), a galactosylterminating peptide which selectively enters hepatocytes via the asialoglycoprotein receptor (3,4). We showed that this carrier enables the preparation of high drug load conjugates, which can be administered intramuscularly (i.m.) and are well tolerated in acute and subchronic toxicity studies (5). L-poly(LYS)-RIBV injected i.m. in mice infected with murine hepatitis virus (MHV) is selectively taken up by the liver and RIBV is released in pharmacologically active form (3,4). Coupled RIBV, produced a 50% inhibition of intrahepatic MHV replication at a daily dose 1.7 times lower than that of the free drug (4). We could not address the issue of haemotoxicity in mice as it is observed only in primates. The study was therefore performed in cynomolgus monkeys: two groups of four age- and weight-matched females were treated for 15 consecutive days with the same dose (30 mg/kg per day) of free or coupled RIBV. Both groups of monkeys
developed anaemia, which was significantly more severe in animals treated with free drug (Table 1). As observed in humans (2), RIBV increased platelet counts, which were higher in free-drug-treated monkeys. Thirty-five days after the end of treatment haematological parameters returned to normal in both groups of animals. In addition, we studied the penetration of the conjugate into monkey red blood cells (RBC) and the stability of the bond between RIBV and L-poly(LYS) in monkey blood using two radioactive conjugates, labelled in the lactose and in the RIBV moieties, respectively, as previously described (3). After a 6-h incubation, no penetration of conjugate was measured in RBC and no release of drug from the carrier was observed. These results suggest that the residual toxic effect of L-poly(LYS)RIBV on RBC may be due to a partial release of RIBV (and/or its metabolites) from the hepatocyte into the bloodstream after the drug is set free from the carrier inside the cell. This release was demonstrated in mice injected with L-poly(LYS)-[3H]RIBV (3,4). Assuming that RIBV enhances the efficacy of IFN in HCV infection treatment by acting within hepatocytes, our present and previous
TABLE 1 Effect of free and coupled RIBV on monkey haematological parameters Days 0
15
Free RBC ¿106/ml
Coupled
Free
21 Coupled
Free
49 Coupled
Free
Coupled
5.9∫0.3
6.5∫0.4 pΩ0.275
3.3∫0.4
4.7∫0.3 pΩ0.031
3.3∫0.5
5.1∫0.4 pΩ0.031
5.5∫0.2
6.6∫0.4 pΩ0.049
Hgb (g/dl)
11.8∫0.5
11.7∫0.5 pΩ0.892
6.7∫0.9
8.5∫0.3 pΩ0.107
6.7∫0.8
9.0∫0.2 pΩ0.032
11.1∫0.5
11.5∫0.3 pΩ0.518
Hct (%)
38.4∫1.4
39.1∫1.9 pΩ0.777
22.0∫2.9
29.4∫1.4 pΩ0.061
22.5∫3.1
31.4∫1.8 pΩ0.029
38.2∫1.5
40.0∫0.7 pΩ0.319
Platelets ¿103/ml
471∫52
328∫58 pΩ0.116
683∫70
451∫88 pΩ0.086
744∫77
429∫89 pΩ0.029
399∫22
302∫34 pΩ0.054
Monkeys were maintained in an animal facility at the Istituto Superiore di Sanita` (Rome) according to European Guidelines (EEC, Directive 86–609 Nov. 26, 1986). Drug administration was performed in two daily i.m. injections at 10-h intervals. For each injection the volume administered was 0.4 ml/kg. Drugs were dissolved in NaCl 0.9%. Results are given as mean values∫SE.
1032
Journal of Hepatology 1998; 29: 1031–1036 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen
Journal of Hepatology ISSN 0168-8278
Correspondence
Protease inhibitors suppress TGF-b generation by hepatic stellate cells To the Editor: Transforming growth factor-b (TGF-b) is the major cytokine implicated in the pathogenesis of liver fibrosis and cirrhosis (1,2). Recently,
Fig. 1. Suppression of the release and activation of latent TGF-b by protease inhibitors in rat HSC cultures. Primary rat HSCs were incubated with plasmin (A), RA (B) or basic FGF (C) in the absence and presence of various protease inhibitors (A-T). Concentrations of TGF-b in the media were measured using [3H]thymidine incorporation by mink lung epithelial cells. M, Latent TGF-b; H, active TGF-b. A, aprotinin; B, tranexamic acid, C, camostat mesilate; D, FOY251; E, gabexate mesilate; F, ONO3403; G, ONO5046; H, FO011; I, FO015; J, FO106; K, FO209; L, FO349 (C-L were kindly supplied by Ono Pharmaceuticals, Osaka, Japan); M, compound .81; N, compound .82; O, compound .83; P, compound .97; Q, compound .105; R, compound .106; S, cis-AMCHA; and T, AMBOCA (MT were kindly supplied by Daiichi Pharmaceuticals, Tokyo, Japan, see reference 6). Columns and bars represent means and SD (nΩ6). Representative results are shown from three independent experiments. *:p∞0.01 compared to the control and groups A–T.
we demonstrated that in porcine serum-treated rats, hepatic fibrosis is exacerbated following simultaneous administration of retinoic acid (RA) by virtue of its ability to produce TGF-b in hepatic stellate cells (HSCs) (3). RA up-regulates expression of plasminogen activator (PA) through nuclear RA receptor (RAR), and thus increases HSC surface plasmin levels (4). This causes the proteolytic release and activation of latent TGF-b1 constitutively produced by HSCs and stored in the adjacent extracellular matrix. Resultant active TGF-b1 autostimulates TGF-b2 and TGF-b3 expression, and all the TGF-bs exacerbate fibrosis by increasing collagen production in HSCs, as well as by suppressing normal function of surrounding hepatocytes (3,5). This observation strongly suggests that proteolytic release and activation of latent TGF-b1 may be a potential target for suppression of TGF-b-mediated liver fibrosis. This idea allowed us to examine which protease inhibitors, including those currently used for human therapy, would be effective in suppressing TGF-b generation by cultured HSCs in vitro (Fig. 1A). This simple experiment is particularly important for exploring our novel idea in vivo in future. Rat primary HSCs, isolated and pre-cultured for 7 days, were incubated for 12 h with 2 units/ml plasmin (Sigma Co., St. Louis, MO, USA) in the absence and presence of 100 mM each of 20 different protease inhibitors. Thereafter, concentrations of both active and total (active plus latent) TGF-b present in the culture medium were determined by bioassays employing CCL64 mink lung epithelial cells. Interestingly, all protease inhibitors tested significantly suppressed plasmin-induced generation of TGF-b in both active and total levels, suggesting that both release and activation were blocked. Similar results were obtained with HSCs stimulated by either 1 mM RA (Sigma) or 10 ng/ml basic fibroblast growth factor (basic FGF) (Boehringer, Mannheim, Germany), reagents which increase endogenous plasmin levels in the cells (Fig. 1B and 1C). These results insure that a protease inhibitor or a combination of several protease inhibitors may be worth testing for their effect on in vivo generation of TGF-b in the liver and resulting development of hepatic fibrosis. Since several compounds (e.g., compounds C and E) are broad-spectrum inhibitors, the result suggests that these protease inhibitors blocked the activity of plasmin and/or plasmin-activated unknown proteases capable of generating TGF-b. Currently, compounds C and E are used for therapy of pancreatitis, reflux esophagitis, and disseminated intravascular coagulation (DIC). The present data suggest that these compounds might be beneficial also for the therapy of liver fibrosis. The idea of using protease inhibitors against hepatic fibrosis is further supported by the fact that compound B has been used episodically as a cytoprotective agent against liver damage (2). We are now testing our idea employing rat hepatic fibrosis models as well as in cirrhotic patients. Masataka Okuno1 Hisataka Moriwaki1, Yasutoshi Muto1 and Soichi Kojima2 1 First Department of Internal Medicine, Gifu University School of Medicine, Gifu 500-8076, Japan, and 2Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba 305-0074, Japan
References 1. Friedman SL. The cellular basis of hepatic fibrosis. N Engl J Med 1993; 328: 1828–35. 2. Gressner AM. Liver fibrosis: perspectives in pathobiochem-
1031
Correspondence ical research and clinical outlook. Eur J Clin Chem Clin Biochem 1991; 29: 293–311. 3. Okuno M, Moriwaki H, Imai S, Muto Y, Kawada N, Suzuki Y, et al. Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF-b in liver stellate cells. Hepatology 1997; 26: 913–21. 4. Imai S, Okuno M, Moriwaki H, Muto Y, Murakami K, Shudo K, et al. 9,13-di-cis-Retinoic acid induces the production of tPA and activation of latent TGF-b via RARa in a human liver stellate cell line, LI90. FEBS Lett 1997; 411: 102–6.
5. Koda H, Okuno M, Imai S, Moriwaki H, Muto Y, Kawada N, et al. Retinoic acid-stimulated liver stellate cells suppress the production of albumin from parenchymal cells via TGFb. Biochem Biophys Res Commun 1996; 221: 565–9. 6. Okano A, Inaoka M, Funabashi S, Iwamoto M, Isoda S, Moroi R, et al. Medicinal chemical studies on antiplasmin drugs. 4. Chemical modification of trans-4-aminomethylcyclohexanecarboxylic acid and its effect on antiplasmin activity. J Med Chem 1972; 15: 247–55.
Coupling to lactosaminated poly-L-lysine reduces the toxic effects of ribavirin on red blood cells To the Editor: Recent data (1,2) confirm that ribavirin (RIBV) enhances the efficacy of interferon (IFN) in the treatment of chronic hepatitis C, but it causes haemolysis. In order to reduce its haemotoxicity, RIBV was coupled to lactosaminated poly-L-lysine (L-poly(LYS)), a galactosylterminating peptide which selectively enters hepatocytes via the asialoglycoprotein receptor (3,4). We showed that this carrier enables the preparation of high drug load conjugates, which can be administered intramuscularly (i.m.) and are well tolerated in acute and subchronic toxicity studies (5). L-poly(LYS)-RIBV injected i.m. in mice infected with murine hepatitis virus (MHV) is selectively taken up by the liver and RIBV is released in pharmacologically active form (3,4). Coupled RIBV, produced a 50% inhibition of intrahepatic MHV replication at a daily dose 1.7 times lower than that of the free drug (4). We could not address the issue of haemotoxicity in mice as it is observed only in primates. The study was therefore performed in cynomolgus monkeys: two groups of four age- and weight-matched females were treated for 15 consecutive days with the same dose (30 mg/kg per day) of free or coupled RIBV. Both groups of monkeys
developed anaemia, which was significantly more severe in animals treated with free drug (Table 1). As observed in humans (2), RIBV increased platelet counts, which were higher in free-drug-treated monkeys. Thirty-five days after the end of treatment haematological parameters returned to normal in both groups of animals. In addition, we studied the penetration of the conjugate into monkey red blood cells (RBC) and the stability of the bond between RIBV and L-poly(LYS) in monkey blood using two radioactive conjugates, labelled in the lactose and in the RIBV moieties, respectively, as previously described (3). After a 6-h incubation, no penetration of conjugate was measured in RBC and no release of drug from the carrier was observed. These results suggest that the residual toxic effect of L-poly(LYS)RIBV on RBC may be due to a partial release of RIBV (and/or its metabolites) from the hepatocyte into the bloodstream after the drug is set free from the carrier inside the cell. This release was demonstrated in mice injected with L-poly(LYS)-[3H]RIBV (3,4). Assuming that RIBV enhances the efficacy of IFN in HCV infection treatment by acting within hepatocytes, our present and previous
TABLE 1 Effect of free and coupled RIBV on monkey haematological parameters Days 0
15
Free RBC ¿106/ml
Coupled
Free
21 Coupled
Free
49 Coupled
Free
Coupled
5.9∫0.3
6.5∫0.4 pΩ0.275
3.3∫0.4
4.7∫0.3 pΩ0.031
3.3∫0.5
5.1∫0.4 pΩ0.031
5.5∫0.2
6.6∫0.4 pΩ0.049
Hgb (g/dl)
11.8∫0.5
11.7∫0.5 pΩ0.892
6.7∫0.9
8.5∫0.3 pΩ0.107
6.7∫0.8
9.0∫0.2 pΩ0.032
11.1∫0.5
11.5∫0.3 pΩ0.518
Hct (%)
38.4∫1.4
39.1∫1.9 pΩ0.777
22.0∫2.9
29.4∫1.4 pΩ0.061
22.5∫3.1
31.4∫1.8 pΩ0.029
38.2∫1.5
40.0∫0.7 pΩ0.319
Platelets ¿103/ml
471∫52
328∫58 pΩ0.116
683∫70
451∫88 pΩ0.086
744∫77
429∫89 pΩ0.029
399∫22
302∫34 pΩ0.054
Monkeys were maintained in an animal facility at the Istituto Superiore di Sanita` (Rome) according to European Guidelines (EEC, Directive 86–609 Nov. 26, 1986). Drug administration was performed in two daily i.m. injections at 10-h intervals. For each injection the volume administered was 0.4 ml/kg. Drugs were dissolved in NaCl 0.9%. Results are given as mean values∫SE.
1032