**Modification of Interleukin-10 with Mannose-6-Phosphate Groups Yields a Liver-Specific Cytokine with Antifibrotic Activity in Rats**

Heni Rachmawati1,2, Adriana Mattos2, Catharina Reker-Smit2, Klaas Poelstra2 and Leonie Beljaars2 *1Pharmaceutics, Bandung Institute of Technology, Bandung 2University of Groningen, Dept. of Pharmacokinetics, Toxicology and Targeting, 1Indonesia 2The Netherlands* 

#### **1. Introduction**

160 Pharmacology

Lerch,M. & Pichler, W. J. (2004). The immunological and clinical spectrum of delayed drug-

Luznik, L. & Fusch, E. J. (2010). High-dose post-transplantation cyclophosphamide to

Marcinkiewicz, J. Bryniarski, K. & Ptak, W. (1994). Cyclophosphamide uncovers two

different patterns of monokine production. *Cytokine*. Vol. 6 (5), pp. 472-477. Posadas, S. J. & Pichler, W. J. (2007). Delayed drug hypersensitivity reactions – new

Snowden, J. A. Angel, C. A. Winfield, D. A. Pringle, J. H. West, K. P. (1997). Angiotropic

Suzuki, R. (2010). Treatment of advanced NK/T cell lymphoma: Nasal type and aggressive

Szczepanik, M. Bryniarski, K. Pryjma, J. & Ptak, W. (1993). Distinct population of antigen-

Vitolo, V. Ferreri, A. J. M. & Zucca, E. (2008). Primary testicular lymphoma. *Critical Review in* 

Weiner, H. L. and Cohen, J. A. (2002). Treatment of multiple sclerosis with

Zhao, J. Cao, Y. Lei, Z. Yang, Z. Zhang, B. & Huang B. (2010). Selective depletion of

concepts. *Clinical and Experimental Allergy*. Vol. 37, pp. 989-999.

NK-cell leukemia. *Int J Hematol*. Vol. 92, pp. 697-701.

*Oncology/Hematology*.Vol.65, pp. 183-189.

*Sclerosis.* Vol. 8, pp. 142 –154.

*Immunology*. Vol. 34 pp.336-344.

*Immunological Research*. Vol. 47 (1-3), pp. 65-77.

pp. 411-419.

Vol. 50, pp. 67-70.

320-326.

immunotherapy of established tumors to be curative. *European Journal of* 

induced exanthems. *Current Opinion in in Allergy and Clinical Immunology.* Vol. 4,

promote graft-host tolerance after allogenic hematopoetic stem cell transplantation.

separate macrophage subpopulations with opposite immunogenic potential and

lymphoma: report of a case with histiocytic features. *Journal of Clinical Pathology* 

presenting macrophages are required for induction of effector and regulatory cells in contact sensitivity response in mice. *Journal of Leukocyte Biology* Vol. 53 (3), pp.

cyclophosphamide: critical review of clinical and immunologic effects. *Multiple* 

CD4+CD25+FoxP3+ regulatory T cells by low-dose cyclophosphamide is explained by reduced intracellular ATP levels. *Cancer Research*. Vol. 70, (12), pp. 4850-4858.

Cytokines and other biological compounds are considered as future drugs and they are of particular interest for the treatment of chronic diseases. These endogenous compounds, that normally mediate local cellular communications, are very promising candidates to generate new drugs because of their high potency (pM-nM concentrations) and their fundamental roles in pathological processes. However, the therapeutic application of cytokines is limited, because several problems are encountered with their application *in* vivo (Schooltink and Rose-John 2002; Standiford 2000; Vilcek and Feldmann 2004). For instance, some cytokines are efficiently degraded in plasma by various enzymes and cytokines are rapidly excreted by the kidneys. Consequently their residence time in the body and thus the exposure to the diseased cells is short (plasma half life is often minutes), which does not favour an optimal biological efficacy. Another major problem is the occurrence of side effects. Because cytokine receptors are ubiquitously expressed in all organs, unusual high plasma concentrations of the cytokine can lead to (unwanted) effects in various organs.

To overcome these problems, we use drug targeting techniques to selectively deliver the cytokine to a specific (diseased) cell (Allen and Cullis 2004; Beljaars, Meijer, Poelstra 2002). The challenge is to improve its distribution within the body and direct the cytokine to a cell of interest, while maintaining the biological activity of that particular cytokine after chemical modification. A conventional way to modify proteins is conjugation with polyethylene glycol (PEG) (Jevsevar, Kunstelj, Porekar 2010). The attachment of PEG moieties improves the pharmacokinetics. That is, PEG substitution prevents rapid renal elimination which results in compounds with prolonged plasma concentrations, thereby making a reduced number of doses possible. For instance, PEGasys (PEGylated interferon 2a), an example of a PEGylated cytokine that is now commonly used to treat patients infected with viral hepatitis, is dosed once a week while the unmodified interferon is dosed daily. This leads to an improvement in the compliance and quality of life in patients with chronic diseases. The side effects, however, are not diminished after PEGylation.

Modification of Interleukin-10 with Mannose-6-Phosphate

create biological effects within relevant cells.

**2. Results** 

the cytokine.

**2.1 Synthesis of M6P-IL10** 

Groups Yields a Liver-Specific Cytokine with Antifibrotic Activity in Rats 163

2001). Liver-selective delivery of this cytokine may prevent these clinical problems and

Upregulation of mannose-6-phosphate/IGF-II receptors during liver injury on HSC (de Bleser and others 1995) offers an excellent target for receptor-mediated antifibrotic drug delivery as shown with albumin substituted with mannose-6-phosphate groups (Beljaars and others 1999). We therefore modified IL10 with the sugar mannose-6-phosphate (M6P) to selectively deliver this cytokine to HSC in fibrotic livers (Rachmawati and others 2007). We showed that after modification of IL10 with mannose 6-phosphate (M6P) a compound was generated that binds to the M6P/IGF-II receptor which is highly present on activated HSC. Our chemically engineered cytokine, M6P-IL10, displayed good pharmacological activity in vitro in freshly isolated HSC. We now performed biodistribution studies of radiolabeled-M6P-modified IL10 using gamma-camera techniques to examine the active delivery of this compound to the diseased tissue. Furthermore, we studied the pharmacological activities of this conjugate in rats at an early stage of liver fibrosis and compared this to the effects of unmodified IL10.

Mannose-6-phosphate-residues were coupled to the amino acid lysine in recombinant human IL10 as described (Rachmawati and others 2007). The product was first characterized by Western Blotting techniques using a rabbit polyclonal anti-IL10 antibody (Santa Cruz Biotechnology, USA). Western Blot analysis of unmodified IL10 yielded two bands corresponding to a molecular weight (MW) of 18.5 and 37 kD (fig.1) representing the monomeric and the homodimeric form of IL10 (Reineke and others 1998). The prepared conjugate M6P-IL10 revealed a shift in these bands: both bands had a higher MW (resp. approximately 20 and 40 kD) than unmodified IL10 indicating covalent binding of M6P to

Mw marker IL-10 M6PIL-10

Fig. 1. Western blot analysis of M6P-IL10 and IL10. Note the increase in molecular weight of the monomeric (± 20 kD) and the homodimeric (± 40 kD) forms of M6P-IL10 compared with the monomeric and homodimeric forms of native IL10 (respectively, 18.5 kD and 37 kD).

20 kD

40 kD

Our strategy of active drug targeting, in contrast to the abovementioned PEGylation approach, aims to improve pharmacokinetics and efficacy while simultaneously avoiding side-effects by cell-specific delivery of the cytokine to the diseased cell via receptormediated interaction. To that end, the cytokine is modified with homing devices that recognize receptors present on the diseased (target) cells. In the past, we designed sugars and receptor-recognizing peptides that interact with hepatic stellate cells (HSC) (Beljaars and others 1999; Beljaars and others 2000; Beljaars and others 2003). These cells play the central role in liver cirrhosis (Bataller and Brenner 2005; Friedman 2010; Schuppan and Afdhal 2008). Our newly designed homing devices displayed affinity for the mannose 6 phosphate/insulin-like growth factor (M6P/IGF) II receptor, platelet derived growth factor (PDGF)- or collagen type VI receptors, which are all essential during stellate cell functioning in fibrogenesis, and upregulate in the diseased liver.

Currently, it is accepted that liver cirrhosis is a fibrotic disease that is reversible (Iredale 2007). However, to date, no drug is marketed that is able to reverse the fibrotic process in patients (Pinzani, Rombouts, Colagrande 2005). The only treatment that is applied to these patients deals with the treatment of complications and with eradication of the cause (for instance removal of the hepatitis virus in case of HCV-induced cirrhosis). However, fibrosis often progresses to end-stage liver failure leaving a liver transplantation as the only available option. Therefore, worldwide research focuses on the identification of compounds that are able to reverse the disease, but unfortunately many potential interventions fail in clinical trials (Pinzani, Rombouts, Colagrande 2005). We hypothesize that this failure may be due to an inadequate pharmacokinetic profile of the potential drugs or due to the occurrence of side-effects of these drugs preventing the administration of effective doses, which may be solved by applying drug targeting techniques.

The selective delivery of cytokines to the cells that control pathological processes is quite relevant. PEGylation of cytokines like interferon α, TNFα, and IL-2 has provided substantial benefits, but in that approach cytokines are not actively delivered to the site of action. In the present study, we will show an example of this second approach using the cytokine interleukin-10 (IL10). IL10 has potent immunosuppressive and anti-inflammatory effects (Di Marco and others 1999; Khan and others 2002; Kitching and others 2000; Oberholzer, Oberholzer, Moldawer 2002) and also direct antifibrotic properties in HSC (Cuzzocrea and others 2001; Demols and others 2002; Gloor and others 1998; Louis and others 1998; Louis and others 2003; Thompson and others 1998; Wang and others 1998). Several of these studies showed beneficial effects of IL10 therapies in animal models and clinical trials during various diseases. However, other studies demonstrated only a limited effect of IL10 or even showed disappointing results (Chadban and others 1997; Colombel and others 2001; Herfarth and Scholmerich 2002). This variable efficacy might be due to the low concentration of IL10 at the target sites. Recombinant IL10 is a low molecular weight protein that is rapidly cleared from the circulation through glomerular filtration. The plasma halflife of IL10 is only 2 min (Rachmawati and others 2004). The ultimate concentrations at the site of action therefore could be too low to result in clear effects. Dose escalation of systemically administered IL10 leads to adverse effects due to its inherent biological actions (Fedorak and others 2000; Schreiber and others 2000). In accordance with this, clinical studies reported beneficial effects of long-term IL10 therapy to treat HCV-associated liver fibrosis but this was accompanied by an immunosuppressive action, as noted in a flare-up of the viral burden, and low therapeutic efficacy (Nelson and others 2000)(Meijer and others 2001). Liver-selective delivery of this cytokine may prevent these clinical problems and create biological effects within relevant cells.

Upregulation of mannose-6-phosphate/IGF-II receptors during liver injury on HSC (de Bleser and others 1995) offers an excellent target for receptor-mediated antifibrotic drug delivery as shown with albumin substituted with mannose-6-phosphate groups (Beljaars and others 1999). We therefore modified IL10 with the sugar mannose-6-phosphate (M6P) to selectively deliver this cytokine to HSC in fibrotic livers (Rachmawati and others 2007). We showed that after modification of IL10 with mannose 6-phosphate (M6P) a compound was generated that binds to the M6P/IGF-II receptor which is highly present on activated HSC. Our chemically engineered cytokine, M6P-IL10, displayed good pharmacological activity in vitro in freshly isolated HSC. We now performed biodistribution studies of radiolabeled-M6P-modified IL10 using gamma-camera techniques to examine the active delivery of this compound to the diseased tissue. Furthermore, we studied the pharmacological activities of this conjugate in rats at an early stage of liver fibrosis and compared this to the effects of unmodified IL10.
