**5. References**

172 Pharmacology

To test whether M6P-IL10 is effective *in vivo*, we now administered (modified-)IL10 to bile duct ligated rats after the initiation of the fibrotic process, i.e. from day 4 till day 7 after BDL. In this time frame, pro-inflammatory activity is high in the liver and fibrosis is initiated (39- 43). In addition, M6P/IGF-II receptor expression on HSC is enhanced at day 4 (Greupink and others 2006), which ensures targeting to this receptor at this time point. During the first week after BDL, enhanced IL10 receptor expression was noted (fig 6), also providing a

Treatment with IL-10 or M6P-IL10 had significant effects on the inflammatory activity within the liver. A reduction in the number of infiltrating cells as reflected by DAB-positive cells was noted. These data indicate that M6P-IL10 is pharmacologically active *in vivo*. Based on inflammatory cell influx, its effect may even be superior to native IL10. Of particular interest is the reduction in IL-10 receptor expression after treatment with IL10, but not after treatment with M6P-IL10. The down regulation of the target receptor during treatment is relevant for IL10-based therapies. This may contribute to the lack of effectiveness of such therapies

(Chadban and others 1997; Colombel and others 2001; Herfarth and Scholmerich 2002).

These results indicate that our modified IL10 is pharmacologically active *in vivo*.

Next to the anti-inflammatory effects, we evaluated the effects of M6P-IL10 on fibrogenesis *in vivo*. The target cell of IL10 is the (activated) HSC, the extracellular matrix producing hepatic cell, and therefore antifibrotic effects are primarily anticipated in HSC and the most important feature in this respect is collagen deposition. Our results showed a clear reduction in collagen deposition in these livers after treatment with M6P-IL10 (figure 7). This reduction was established with various methods. The lack of portal-to-portal bridging was evident in nearly all the livers of the cytokine-treated animals. In addition, IL10 and M6P-IL10 also significantly reduced αSMA staining which reflects a reduction in the activation of HSC in these livers.

Although cytokines are interesting compounds which may yield potent new drugs, so far only a relatively few are approved and clinically used. The number is still disappointing low regarding the large number of endogenous cytokines. The main reasons for this are the poor stability and poor pharmacokinetic profile of cytokines. To overcome these pharmacokinetic problems, we apply drug targeting techniques to selectively deliver the cytokine to a specific (diseased) cell. In the current study, we demonstrate the possibilities of this strategy with successful *in vivo* application of a modified IL10. Recently, we also reported on the cellspecific delivery of another very interesting cytokine with antifibrotic activities, that is interferon-gamma (IFNγ) (Bansal and others 2011). This study shows that HSC-targeted IFNγ, in contrast to unmodified IFNγ, blocked liver fibrogenesis in a chronic CCL4 mice model of liver fibrosis, by specifically acting on the key pathogenic cells within the liver. Furthermore, we clearly demonstrated that the targeted IFNγ was devoid of side effects. In addition, others show beneficial effects of a targeted cytokine by means of coupling receptor specific ligands to the cytokine (Curnis and others 2000; Curnis and others 2005; Fournier, Aigner, Schirrmacher 2011; Jazayeri and Carroll 2008; Nissim and others 2004) often focussing on the treatment of tumours. These approaches may lead to a more optimal use of

In summary, we demonstrated potent pharmacological effectivity of a novel liver-specific form of the cytokine IL10. After conjugation with M6P, the novel cytokine efficiently accumulates in the liver and attenuates the fibrotic process *in vivo.* Further dose-response

rationale for the start of treatment at day 4.

cytokines for therapeutic purposes.


Modification of Interleukin-10 with Mannose-6-Phosphate

57(2):518-25.

814-21.

Res 22(5):505-16.

Groups Yields a Liver-Specific Cytokine with Antifibrotic Activity in Rats 175

Khan AQ, Shen Y, Wu ZQ, Wynn TA, Snapper CM. 2002. Endogenous pro- and anti-

Kitching AR, Tipping PG, Timoshanko JR, Holdsworth SR. 2000. Endogenous interleukin-10

Louis H, Le Moine O, Goldman M, Deviere J. 2003. Modulation of liver injury by

Louis H, Van Laethem JL, Wu W, Quertinmont E, Degraef C, Van den Berg K, Demols A,

Meijer DK, Beljaars L, Molema G, Poelstra K. 2001. Disease-induced drug targeting using

Nelson DR, Lauwers GY, Lau JY, Davis GL. 2000. Interleukin 10 treatment reduces fibrosis

Nissim A, Gofur Y, Vessillier S, Adams G, Chernajovsky Y. 2004. Methods for targeting

Oberholzer A, Oberholzer C, Moldawer LL. 2002. Interleukin-10: A complex role in the

Pinzani M, Rombouts K, Colagrande S. 2005. Fibrosis in chronic liver diseases: Diagnosis

Poelstra K, Hardonk MJ, Koudstaal J, Bakker WW. 1990. Intraglomerular platelet aggregation and experimental glomerulonephritis. Kidney Int 37(6):1500-8. Rachmawati H, Reker-Smit C, Lub-de Hooge MN, van Loenen-Weemaes A, Poelstra K,

Rachmawati H, Beljaars L, Reker-Smit C, Van Loenen-Weemaes AM, Hagens WI, Meijer

Reineke U, Sabat R, Volk HD, Schneider-Mergener J. 1998. Mapping of the interleukin-

Schooltink H and Rose-John S. 2002. Cytokines as therapeutic drugs. J Interferon Cytokine

Schreiber S, Fedorak RN, Nielsen OH, Wild G, Williams CN, Nikolaus S, Jacyna M, Lashner

Schuppan D and Popov Y. 2009. Rationale and targets for antifibrotic therapies.

10/interleukin-10 receptor combining site. Protein Sci 7(4):951-60.

novel peptide-ligand albumins. J Control Release 72(1-3):157-64.

biologicals to specific disease sites. Trends Mol Med 10(6):269-74.

streptococcus pneumoniae. Infect Immun 70(2):749-61.

interleukin-10. Acta Gastroenterol Belg 66(1):7-14.

tetrachloride in mice. Hepatology 28(6):1607-15.

and management. J Hepatol 42 Suppl(1):S22-36.

Gastroenterology 118(4):655-60.

Crit Care Med 30(1 Supp):S58-63.

liver fibrosis. Pharm Res 21(11):2072-8.

study group. Gastroenterology 119(6):1461-72.

Schuppan D and Afdhal NH. 2008. Liver cirrhosis. Lancet 371(9615):838-51.

Gastroenterol Clin Biol 33(10-11):949-57.

inflammatory cytokines differentially regulate an in vivo humoral response to

regulates Th1 responses that induce crescentic glomerulonephritis. Kidney Int

Goldman M, Le Moine O, Geerts A, et al. 1998. Interleukin-10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon

in patients with chronic hepatitis C: A pilot trial of interferon nonresponders.

pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug.

Beljaars L. 2007. Chemical modification of interleukin-10 with mannose 6 phosphate groups yields a liver-selective cytokine. Drug Metab Dispos 35(5):

DK, Poelstra K. 2004. Pharmacokinetic and biodistribution profile of recombinant human interleukin-10 following intravenous administration in rats with extensive

BA, Gangl A, Rutgeerts P, et al. 2000. Safety and efficacy of recombinant human interleukin 10 in chronic active crohn's disease. crohn's disease IL-10 cooperative


Curnis F, Sacchi A, Borgna L, Magni F, Gasparri A, Corti A. 2000. Enhancement of tumor

Cuzzocrea S, Mazzon E, Dugo L, Serraino I, Britti D, De Maio M, Caputi AP. 2001. Absence

de Bleser PJ, Jannes P, van Buul-Offers SC, Hoogerbrugge CM, van Schravendijk CF, Niki T,

Demols A, Van Laethem JL, Quertinmont E, Degraef C, Delhaye M, Geerts A, Deviere J.

Di Marco R, Xiang M, Zaccone P, Leonardi C, Franco S, Meroni P, Nicoletti F. 1999.

Fournier P, Aigner M, Schirrmacher V. 2011. Targeting of IL-2 and GM-CSF

Friedman SL. 2010. Evolving challenges in hepatic fibrosis. Nat Rev Gastroenterol Hepatol

Gloor B, Todd KE, Lane JS, Rigberg DA, Reber HA. 1998. Mechanism of increased lung injury after acute pancreatitis in IL-10 knockout mice. J Surg Res 80(1):110-4. Greupink R, Bakker HI, van Goor H, de Borst MH, Beljaars L, Poelstra K. 2006. Mannose-6-

Herfarth H and Scholmerich J. 2002. IL-10 therapy in crohn's disease: At the crossroads.

Iredale JP. 2007. Models of liver fibrosis: Exploring the dynamic nature of inflammation and

Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, Denk H, Desmet V, Korb G,

Jazayeri JA and Carroll GJ. 2008. Fc-based cytokines : Prospects for engineering superior

Jevsevar S, Kunstelj M, Porekar VG. 2010. PEGylation of therapeutic proteins. Biotechnol J

repair in a solid organ. J Clin Invest 117(3):539-48.

exacerbated by endogenous IL-10 deficiency. Autoimmunity 31(2):75-83. Fedorak RN, Gangl A, Elson CO, Rutgeerts P, Schreiber S, Wild G, Hanauer SB, Kilian A,

to aminopeptidase N (CD13). Nat Biotechnol 18(11):1185-90.

with sinusoidal endothelial cells. Hepatology 21(5):1429-37.

induced arthritis. Eur Cytokine Netw 12(4):568-80.

282(6):G1105-12.

119(6):1473-82.

7(8):425-36.

Gut 50(2):146-7.

Hepatol 22(6):696-9.

5(1):113-28.

therapeutics. BioDrugs 22(1):11-26.

Oncol 38(6):1719-29.

necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery

of endogeneous interleukin-10 enhances the evolution of murine type-II collagen-

Rogiers V, van den Brande JL, Wisse E, Geerts A. 1995. Insulinlike growth factor-II/mannose 6-phosphate receptor is expressed on CCl4-exposed rat fat-storing cells and facilitates activation of latent transforming growth factor-beta in cocultures

2002. Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis. Am J Physiol Gastrointest Liver Physiol

Concanavalin A-induced hepatitis in mice is prevented by interleukin (IL)-10 and

Cohard M, LeBeaut A, et al. 2000. Recombinant human interleukin 10 in the treatment of patients with mild to moderately active crohn's disease. the interleukin 10 inflammatory bowel disease cooperative study group. Gastroenterology

immunocytokines to a tumor vaccine leads to increased anti-tumor activity. Int J

phosphate/insulin-like growth factor-II receptors may represent a target for the selective delivery of mycophenolic acid to fibrogenic cells. Pharm Res 23(8):1827-34.

treatment of crohn's disease with the anti-inflammatory cytokine interleukin 10.

MacSween RN. 1995. Histological grading and staging of chronic hepatitis. J


**Part 3** 

**Pharmacogenetic** 

