Patent Application
20080025945
The invention is based on the finding that treatment with a low dose of IL-6 is efficient in a model of liver resection of cirrhotic liver. The beneficial results obtained with a low dose of IL-6, ranging from 0.1 to 10 mcg/kg, under such severe liver cirrhotic conditions were unexpected.
The invention therefore, relates to the use of a low dose of IL-6, in the range of 0.1 to 10 mcg/kg, or of a mutein, isoform, fused protein, functional derivative, active fraction, circularly permutated derivative or salt thereof for the manufacture of a medicament for the treatment and/or prevention of liver injury e.g. liver cirrhosis.
The terms “treating” and “preventing” as used herein should be understood as preventing, inhibiting, attenuating, ameliorating or reversing any or all symptoms or cause(s) of liver cirrhosis, as well as symptoms or diseases accompanying liver cirrhosis, and in particular the neuroanantomical and behavioral changes associated with the disease.
The term “liver cirrhosis” as used herein, is also called liver injury. The disease, as well as its causes, have been described in detail in the “Background of the Invention”.
The term “dose” relates to the quantity to be administered at one time, such as a specified amount of medication.
The term “dosage” relates to the determination and regulation of the size, frequency, and number of doses.
The invention provides for a new possibility of treating and/or preventing liver injury such as liver cirrhosis. At present, the treatment of cirrhosis includes withdrawing toxic agents such as alcohol, receiving proper nutrition including supplemental vitamins, and treating complications as they arise. Liver transplantation is presently the only cure and may help a person with advanced cirrhosis. Thus, the present invention presents a substantial progress, namely, a low dose of IL-6, in the range of 0.1 to 10 mcg/kg, exhibit a significant beneficial effect in experimental liver cirrhosis. As shown in the examples below, a low dose of IL-6, in the range of 0.1 to 10 mcg/kg, exhibited an effect which was significant with regard to amelioration of the aberrations tested in an established animal model of liver cirrhosis.
The term “resection” refers to the excision of a portion or all of an organ or other structure.
The invention relates to the treatment of liver damage caused by a wide range of hepatotoxic agents and gene therapy vectors by administration of low dose of IL-6 allowing rapid hepatocyte proliferation.
The results obtained clearly show that when in presence of a functionally compensated cirrhosis and resection, the intraperitoneal injection of IL-6: does not increase the postoperative mortality after major hepatic resections and enhances the physiological response to a major parenchymal loss by inducing a massive proliferation of mature hepatocytes when using both high and low doses.
In addition to STAT-3 and AP-1, it was found that NF-κB has a role in decreasing apoptosis in hepatic cells following treatment with IL-6 suggesting that the IL-6 induced liver mass expansion in cirrhotic liver may be mediated also by an anti-apoptotic effect of IL-6.
In another embodiment of the present invention it was found that in cirrhotic animals, the expression of gp80 significantly increased in cells from both total liver and isolated hepatocytes in comparison to normal controls (
The results obtained do not indicate an increased risk of neoplasm onset in the IL-6 treated rats versus control cirrhotic non-treated rats. In particular, the incidence of severe dysplasia or hepatocellular-carcinoma (HCC) was 8% in control animals (2/25) and 3.3 in IL-6 animals (1/30);
Among the treated animals the study of angiogenesis in regenerating cirrhotic parenchyma did not show an abnormal increase of the micro-vessels density inside the cirrhotic nodules, unpaired arteriolas or undifferentiated endothelia, data correlated to HCC onset.
As used herein the term “muteins” refers to analogs of an IL-6, in which one or more of the amino acid residues of the naturally occurring components of IL-6 are replaced by different amino acid residues, or are deleted, or one or more amino acid residues are added to the original sequence of an IL-6, without changing considerably the activity of the resulting products as compared with the original IL-6. These muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefor.
Muteins in accordance with the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA, which encodes an IL-6, in accordance with the present invention, under stringent conditions. The term “stringent conditions” refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as “stringent”. See Ausubel et al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., §§6.3 and 6.4 (1987, 1992), and Sambrook et al.(Sambrook, J. C., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Without limitation, examples of stringent conditions include washing conditions 12-20° C. below the calculated Tm of the hybrid under study in, e.g., 2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15 minutes; 0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then, a 0.1×SSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10 -40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
Any such mutein preferably has a sequence of amino acids sufficiently duplicative of that of an IL-6, such as to have substantially similar, or even better, activity to IL-6.
Characteristic activity of IL-6 is the capability of binding to the gp80 portion of the IL-6 receptor and/or capability of inducing hepatocyte proliferation. As long as the mutein has substantial capability of binding to the gp80 portion of the IL-6 receptor and/or capability of inducing hepatocyte proliferation, it can be considered to have substantially similar activity to IL-6. Thus, it can be determined whether any given mutein has at least substantially the same activity as IL-6 by means of routine experimentation comprising subjecting hepatocytes to such mutein, and to determine whether or not it induces hepatocyte proliferation e.g. by measuring BrdU or labelled methionine uptake or just by counting the cells the cells vis-a-vis non treated control cells and cells treated with WT IL-6. An ELISA type assay for measuring the binding of IL-6R/IL-6 chimera to gp130 has been described in detail in example 7 on page 39 of WO 99/02552, which is fully incorporated by reference herein. The person skilled in the art will appreciate that a similar ELISA type assay can be developed for the binding of IL-6 to gp80.
As long as the mutant has substantial binding activity to its binding region of GP80 it can be considered to have substantially similar activity to IL-6.
Thus it can be determined whether any given mutant has at least substantially the same activity as IL-6 by means of routine experimentation comprising subjecting such mutant e.g. to a simple sandwich binding assay to determine whether or not it binds to an immobilized gp80 or soluble gp80 (extracellular fragment of gp80) as described in example 7 of WO 99/02552.
In a preferred embodiment, any such mutein has at least 40% identity or homology with the sequence of mature IL-6. More preferably, it has at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity or homology thereto.
Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotides or two polypeptide sequences, respectively, over the length of the sequences being compared.
For sequences where there is not an exact correspondence, a “% identity” may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting “gaps” in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
Methods for comparing the identity and homology of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % homology between two polypeptide sequences. BESTFIT uses the “local homology” algorithm of Smith and Waterman (1981) and finds the best single region of similarity between two sequences. Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, 1990; Pearson 1988).
Muteins of IL-6, which can be used in accordance with the present invention, or nucleic acid coding therefor, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
Preferred changes for muteins in accordance with the present invention are what are known as “conservative” substitutions. Conservative amino acid substitutions of IL-6 may include synonymous amino acids within a group which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule (Grantham, 1974). It is clear that insertions and deletions of amino acids may also be made in the above-defined sequences without altering their function, particularly if the insertions or deletions only involve a few amino acids, e.g., under thirty, and preferably under ten, and do not remove or displace amino acids which are critical to a functional conformation, e.g., cysteine residues. Proteins and muteins produced by such deletions and/or insertions come within the purview of the present invention.
Preferably, the synonymous amino acid groups are those defined in Table A. More preferably, the synonymous amino acid groups are those defined in Table B; and most preferably the synonymous amino acid groups are those defined in Table C.
Examples of production of amino acid substitutions in proteins which can be used for obtaining muteins of IL-6 polypeptides, for use in the present invention include any known method steps, such as presented in U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, to Mark et al; 5,116,943 to Koths et al., 4,965,195 to Namen et al; 4,879,111 to Chong et al; and 5,017,691 to Lee et al; and lysine substituted proteins presented in U.S. Pat. No. 4,904,584 (Shaw et al).
Specific muteins of IL-6 which are useful in connection with the present invention have been described (WO9403492A1). Furthermore, EP667872B1 describes mutant IL-6 with improved biological activity over wild type IL-6. In addition to this, EP656711B1 describes methods to isolate superagonists of IL-6. The mutants or superagonists may be used according to the invention.
The term “fused protein” refers to a polypeptide comprising an IL-6, or a mutein or fragment thereof, fused with another protein, which, e.g., has an extended residence time in body fluids. An IL-6, may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.
“Functional derivatives” as used herein cover derivatives of IL-6, and their muteins and fused proteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein which is substantially similar to the activity of IL-6, and do not confer toxic properties on compositions containing it.
These derivatives may, for example, include polyethylene glycol side-chains, which may mask antigenic sites and extend the residence of an IL-6 in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example that of seryl or threonyl residues) formed with acyl moieties.
An “active fraction” according to the present invention may e.g. be a fragment of IL-6. The term fragment refers to any subset of the molecule, that is, a shorter peptide which retains the desired biological activity. Fragments may readily be prepared by removing amino acids from either end of the IL-6 molecule and testing the resultant fragment for its properties to bind to gp80 and or inducing hepatocyte proliferation. Proteases for removing one amino acid at a time from either the N-terminal or the C-terminal of a polypeptide are known, and so determining fragments which retain the desired biological activity involves only routine experimentation.
As active fractions of an IL-6, muteins and fused proteins thereof, the present invention further covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has substantially similar activity to IL-6 e.g. bind to the IL-6 binding site of gp80 and/or induce hepatocyte proliferation.
The term “salts” herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the IL-6 molecule or analogs thereof. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Of course, any such salts must retain the biological activity of IL-6, e.g. the ability to induce hepatocyte proliferation and or the ability to bind the IL-6 binding site of gp80.
The term “circularly permuted derivatives” as used herein refers to a linear molecule in which the termini have been joined together, either directly or through a linker, to produce a circular molecule, and then the circular molecule is opened at another location to produce a new linear molecule with termini different from the termini in the original molecule. Circular permutations include those molecules whose structure is equivalent to a molecule that has been circularized and then opened. Thus, a circularly permuted molecule may be synthesized de novo as a linear molecule and never go through a circularization and opening step. The preparation of circularly permutated derivatives is described in WO95/27732.
In a preferred embodiment of the invention, the substance of the invention is glycosylated at one or more sites.
A glycosylated form of an IL6R/IL6 chimera has been described in WO 99/02552 (PCT/IL98/00321), which is the chimeric molecule highly preferred according to the invention. The IL6R/IL6 chimera described therein is a recombinant glycoprotein, which was obtained fusing the entire coding sequence of the naturally occurring soluble IL-6 receptor δ-Val (Novick et al., 1990) to the entire coding sequence of mature naturally occurring IL-6, both from human origin. The person skilled in the art will appreciate that the glycosylated IL-6 can be produced by recombinant means as well, i.e. by expression in eukaryotic expression systems.
The IL-6 according to the invention may be produced in any adequate eukaryotic or procaryotic cell type, like yeast cells, insect cells, bacteria, and the like. It is preferably produced in mammalian cells, most preferably in genetically engineered CHO cells as described in WO 99/02552.
In a further embodiment of the invention, the substance of the invention is not glycosylated. Advantageously, the molecule can then be produced in bacterial cells, which are not capable of synthesizing glycosyl residues, but usually have a high yield of produced recombinant protein. The production of non-glycosylated IL-6 has been described in detail in EP504751B1, for example.
In yet a further embodiment, the substance according to the invention comprises an immunoglobulin fusion, i.e. the molecules according to the invention are fused to all or a portion of an immunoglobulin. Methods for making immunoglobulin fusion proteins are well known in the art, such as the ones described in WO 01/03737, for example. The person skilled in the art will understand that the resulting fusion protein of the invention retains the biological activity of the IL-6. The resulting fusion protein ideally has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or facilitated purification of the fusion protein.
Preferably, the substance according to the invention is fused to the constant region of an Ig molecule. It may be fused to heavy chain regions, like the CH2 and CH3 domains of human IgG1, for example. Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric.
Functional derivatives of the substance according to the invention may be conjugated to polymers in order to improve the properties of the protein, such as the stability, half-life, bioavailability, tolerance by the human body, or immunogenicity.
Therefore, a preferred embodiment of the invention relates to a functional derivative of the substance according to the invention comprising at least one moiety attached to one or more functional groups which occur as one or more side chains on the amino acid residues.
A highly preferred embodiment relates to a substance of the invention linked to Polyethlyenglycol (PEG). PEGylation may be carried out by known methods, such as the ones described in WO 92/13095, for example.
Preferably, the substance of the invention is used in an amount ranging from 0.1 to 10 mcg/kg. In a preferred embodiment of the invention the substance is administered daily. In a further preferred embodiment, the substance is administered three times per week. In yet a further preferred embodiment, the substance is administered once a week.
Low dose of IL-6, or a mutein, isoform, fused protein, functional derivative, active fraction, circularly permutated derivative or salt thereof can be used, according to the invention, in liver injury such as:
A—end stage liver insufficiency to chronic liver disease may be due to decompensated liver cirrhosis of viral (HBV, HCV, other hepatitis) aethiology or exotoxic (alcoholic)aethiology,
B—liver insufficiency after resective liver surgery caused e.g. after liver resection of hepatocellular carcinoma with liver remnant, and
C—acute live insufficiency due to viral infection (HCV), toxic (alcohol, paracetamol, abuse, mushroom poisoning) aethiology and traumatic aethiology.
Treatment with a low dose of IL-6, or a mutein, isoform, fused protein, functional derivative, active fraction, circularly permutated derivative or salt thereof, in accordance to the invention, may be beneficial for the expansion of liver mass to improve survival and to delay liver transplant for patients e.g. having end stage liver insufficiency to chronic liver disease, waiting for liver transplant, in the absence of matched donors.
Treatment with a low dose of IL-6, or a mutein, isoform, fused protein, functional derivative, active fraction, circularly permutated derivative or salt thereof, in accordance with the invention, can be beneficial in patients showing unexpected postoperative liver failure or in patients exhibiting postoperative liver insufficiencies. For example, in patients in which liver has been resected due to secondary neoplasms.
Treatment with low dose of IL-6 or a mutein, isoform, fused protein, functional derivative, active fraction, circularly permutated derivative or salt thereof, in accordance with the invention, could help for the regeneration of cirrhotic liver and for prevention of surgery.
It is a further object of the present invention to provide a pharmaceutical composition comprising IL-6, a mutein, fused protein, functional derivative, active fraction, circularly permutated derivative or salt thereof, optionally together with one or more pharmaceutically acceptable carriers, diluents or excipients, for the treatment and/or prevention of liver injury such as liver cirrhosis.
The definition of “pharmaceutically acceptable” is meant to encompass any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. For example, for parenteral administration, IL-6 may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
The IL-6 can be administered to a patient in need of administration thereof in a variety of ways. The routes of administration include intraliver, intradermal, transdermal (e.g. in slow release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, topical, and intranasal routes. Any other therapeutically efficacious route of administration can be used, for example absorption through epithelial or endothelial tissues or by gene therapy wherein a DNA molecule encoding the IL-6 is administered to the patient (e.g. via a vector) which causes the IL-6 to be expressed and secreted in vivo. In addition the IL-6 can be administered together with other components of biologically active agents such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles.
For parenteral (e.g. intravenous, subcutaneous, intramuscular) administration, IL-6 can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (e.g. water, saline, dextrose solution) and additives that maintain isotonicity (e.g. mannitol) or chemical stability (e.g. preservatives and buffers). The formulation is sterilized by commonly used techniques.
It is a further object of the present invention to provide for a method for treating and/or preventing liver injury such as liver cirrhosis, comprising administering to a patient in need thereof an effective amount/dose of IL-6, a mutein, fused protein, functional derivative, active fraction, circularly permutated derivative or salt thereof optionally together with a pharmaceutically acceptable carrier.
An “effective amount” refers to an amount of the active ingredients that is sufficient to affect the course and the severity of the diseases described above, leading to the reduction or remission of such pathology. The effective amount will depend on the route of administration and the condition of the patient.
The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factor, including IL-6 pharmacokinetic properties, the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled.
The invention relates to the use a of low dose of IL-6 or a mutein, fused protein, active fraction or circularly permutated derivative in the manufacture of a medicament for the treatment of liver injured acutely and severely (as in submassive necrosis with hepatitis).
The invention relates also to the use of a low dose of IL-6 or a mutein, fused protein, active fraction or circularly permutated derivative thereof, or comprising administering to a patient in need thereof an expression vector comprising the coding sequence of IL-6, a mutein, fused protein, active fraction or circularly permutated derivative thereof in the manufacture of a medicament for the treatment of severe conditions of the liver e.g. for the treatment of cirrhotic liver including a resection and/or engraftment treatment.
A method for treating a liver injury such as cirrhosis, comprising administering to a patient in need thereof an effective low dose/amount of IL-6, a mutein, fused protein, active fraction or circularly permutated derivative thereof, or comprising administering to a patient in need thereof an expression vector comprising the coding sequence of IL-6, a mutein, fused protein, active fraction or circularly permutated derivative thereof, are further objects of the present invention.
In a preferred embodiment of the invention, the amount of IL-6 administered in the range of 0.1 to 10 mcg/kg is highly preferred.
A method for treating injury of the liver such as e.g. liver cirrhosis and including a resection and/or engraftment treatment, comprising administering to a patient in need thereof an effective low amount of IL-6, a mutein, fused protein, active fraction or circularly permutated derivative thereof, or comprising administering to a patient in need thereof an expression vector comprising the coding sequence of IL-6, a mutein, fused protein, active fraction or circularly permutated derivative thereof, are further objects of the present invention.
The low dose of IL-6 can be administered before during and/or after transplantation. The low dose of IL-6 can be administered before during and/or after resection treatment in both acceptor and/or donor of liver tissue.
The term “transplantation” referred herein as the grafting of tissues or cells taken from the patients own body or from another (donor).
In a preferred embodiment of the invention, the amount of IL-6 administered in the range of 0.1 to 10 mcg/kg is highly preferred.
The present invention will now be described in more detail in the following non-limiting examples and the accompanying drawings.
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation.
While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.
All references cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent application, issued U.S. or foreign patents or any other references, are entirely incorporated by reference herein, including all data, tables, figures and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.
Reference to known method steps, conventional methods steps, known methods or conventional methods is not any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various application such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning an range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
Experiments were structured in order to evaluate the main biological effect of IL-6 treatment in the expansion of the functioning liver mass of “basal” cirrhotic liver, before any acute surgical stress, and in the increased regenerative response of cirrhotic liver after a major surgical resection.
The study was carried on male Sprague Dowley rats weighting 150-175 gr. The animals were maintained on a 12-hour light/dark cycle with free access to standard rat food and water.
Experimental centro-lobular cirrhosis was induced by carbon tetrachloride (CC14) and phenobarbital. Rats chronically received Phenobarbital (0.35 g/L) in the drinking water from the beginning until the end of the 10th CC14 treatment in order to enhance the hepato-toxic effect of CC14. CC14 (diluted 1:9 in olive oil) was administered once a week for 10 weeks by intragastric route (gavage). The starting dose of 0.2 ml/kg varied ±0.2 ml/kg according to daily weight change of each animal observed during the 7 days following each gavage. Half the theoretical CC14 dose was administered at gavage n°9 and n°10. Any treatment stopped after the 10th gavage and the rats had a 5 days resting period.
Anaesthesia was given by ether inhalation and the surgical procedure performed through a midline laparotomy. Hepatic ligaments were sectioned taking care to haemostasis of collateral circles due to portal hypertension. The hepatic pedicle was prepared and sectioned between ligatures; then the left lateral and median hepatic lobes were resected after separate ligation of the hepatic veins. Wall closure was performed by non resorbable running suture.
a. Presence or absence of neurological impairment due to porto-systemic encephalopathy;
b. presence or absence of ascites.
These parameters were considered both at the time of operation and of sacrifice and allowed to class each animal as bearing a functionally compensated or decompensated cirrhosis. In the following experiment only animals bearing a compensated cirrhosis at the time of operation, showing “diffuse cirrhosis” or “incomplete cirrhosis with porto-portal fibrosis” (respectively cirrhotic alterations involving 80-100% and 50-79% of hepatic lobules, associated to porto-portal fibrosis in non cirrhotic parenchyma) were considered excluding systematically those showing only minor cirrhotic modifications.
The hepato-cell proliferation was measured by BrdU uptake. BrdU uptake was detected by anti-BrdU antibodies and the percentage of BrdU-stained hepatocytes evaluated in 20 high-power fields (40×).
When hepatocellular proliferation was to be evaluated by BrdU uptake, rats received BrdU in drinking water (1 g/L) from the first IL-6 injection until hepatic resection (IL-6 effects on “basal” cirrhotic liver) or from the time of operation until sacrifice (IL-6 effects on regenerating cirrhotic liver).
Cirrhotic rats receiving IL-6 at doses of 500, 100, 10, 1 or 0.1 mcg/kg formed the treated groups. IL-6, IL-6 100, IL-6 10, IL-6 1 and IL-6 0.1 will indicate respectively: IL-6 treated animals, rats treated with 100 mcg/kg, 10 mcg/kg, mcg/kg and 0.1 mcg/kg of IL-6. The doses 500 mcg/kg and 100 mcg/kg were considered high doses while doses ranging from 10 mcg/kg through 0.1 mcg/kg were considered low doses.
Evaluations of IL-6 effects on cirrhotic liver considered:
1. the operative mortality;
Evaluations of IL-6 effects on regenerating hepatocirrhotic parenchyma after hepatic resection considered:
1. the postoperative mortality;
2. the liver function at sacrifice;
3. the hepatic regeneration determined as the percentage of regenerated liver mass and calculated by the following equation:
Hepatic regeneration rate (%)=100×[C−(A−B)]/A
where: A is the estimated liver weight at the time of liver resection [this data was checked and validated on 15 controls and 24 IL-6 treated animals (data not shown)],
B is the weight of resected liver,
C is the weight of the regenerated liver at sacrifice;
After induction of cirrhosis the left-lateral and median hepatic lobes were resected and IL-6 pharmacological effects on regenerating hepatocirrhotic parenchyma after liver resection was explored. The following parameters were monitored: 1. post-operative mortality, 2. Assessment of liver function at the time of sacrifice 3. Hepatic regeneration, and 4. BrdU uptake at sacrifice.
After induction of cirrhosis the left-lateral and median hepatic lobes were resected. The results obtained point out that when in presence of a functionally compensated cirrhosis, the intraperitoneal injection of IL-6 does not increase the operative mortality of major hepatic resections;
The IL-6 pharmacological effects on regenerating hepatocirrhotic parenchyma after liver resection were explored. The following parameters were monitored: 1. post-operative mortality, 2. Assessment of liver function at the time of sacrifice 3. Hepatic regeneration, and 4. BrdU uptake at sacrifice.
A 7.1% of mortality in control animals (2/28) versus a 2.8% (2/70) in IL-6 treated rats was observed. One control and 1 treated rat died for hepatic insufficiency, the other 2 for unclear reasons. It should be noted that in the treated group all deaths occurred in the high dose groups.
a. neurological impairment due to porto-systemic encephalopathy was never observed;
b. ascites were observed in 4 rats: 1/26 controls (4.1%) and 3/26 IL-6 high doses animals (11.5%).
a. among controls a mean of 16.5±10.1, median 16.3;
b. among IL-6 0.1 animals a mean of 25.5±15.7, median 27.5;
c. among IL-6 1 animals a mean of 38.4±10.8, median 40.4;
d. among IL-6 10 animals a mean of 29.0±13.4, median 25.9;
e. among IL-6 100 animals a mean of 40.2±9.9 median 42.1;
f. among IL-6 500 animals a mean of 32.2±12.1, median 31.3.
Seven control, 9 IL-6 0.1, 8 IL-6 1, 7 IL-6 10, 9 IL-6 100 and 7 IL-6 500 rats received BrdU after hepatic resection. The count of BrdU stained hepatocytes showed (
a. among controls a mean of 52.2±14.9 median 53;
b. among IL-6 0.1 animals a mean of 65.3±8.2, median 65.3;
c. among IL-6 1 animals a mean of 68.4±11.9, median 65.5;
d. among IL-6 10 animals a mean of 60.8±10.0, median 60.0;
e. among IL-6 100 animals a mean of 79.4±8.1, median 78.4;
f. among IL-6 500 animals a mean of 80.9±10.7, median 83.7.
In this case differences between means had a statistical significance when comparing controls to IL-6 0.1, 1, 100 and 500 animals (t Dunnett<0.05), A statistical significance was not evident when considering the subgroup of animals treated with the dose 10 mcg/kg.
does not increase the postoperative mortality after major hepatic resections;
enhances the physiological response to a major parenchymal loss by inducing a massive proliferation of mature hepatocytes when both high and low-doses (except 10 mcg/kg) are employed.
The involvement of transcriptional factors, which are directly activated by IL-6, such as STAT-3 and AP-1, on the action of IL-6 on hepatocellular regeneration in cirrhotic liver was explored. The possible involvement of NF-κB activation on the proliferative hepatocellular response observed after IL-6 treatment in conditions of functionally compensated hepatocirrhosis was also explored.
First, liver fragments obtained from cirrhotic rats killed at different time-points after IL-6 injection were examined. Rats were treated with a dose of 100 mcg/kg of IL-6 accordingly to the injection protocol already described.
In addition it was observed by shift assay that IL-6 intra peritoneal injection evoked a transient activation of NF-κB, detectable after 1 and 4 hours, showing a peak after 2 hours (
1—In control animals the liver resection induced a modest STAT-3 activation which was evident at 1 hour and further increased 2 hours after the surgical procedure. IL-6 intra peritoneal injection at the end of the surgical procedure induced earlier and enhanced STAT-3 activation (
2—In control animals the liver resection induced a modest AP-1 activation above basal levels which was observed at 2 hours after surgery. IL-6 intra peritoneal injection at the end of the surgical procedure induced earlier and enhanced AP-1 activation (
3—In control animals the liver resection did not induce a significant NF-κB activation above basal levels. On the contrary, IL-6 intra peritoneal injection at the end of the surgical procedure induced NF-κB activation (
Five hundred mg of liver were homogenized in a homogeneization buffer (10 mM Hepes/KOH pH 7.6, 25 mM KCl, 1 mM EDTA, 2M Sucrose, 10% glycerol) containing a protease inhibitor cocktail (Sigma) and centrifuged at 100.000 g for 50 minutes to obtain a nuclear pellet. Nuclei were lysed in a lysis buffer (20 mM Hepes-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 dithiothreitol and protease inhibitors cocktail) and stored at −80° C. Eight micrograms of extracts were incubated with radiolabeled double stranded oligonucleotide (5 ng) for 20 min at 25° C. in binding buffer (10 mM Tris pH 7.8, 5% glycerol, 1 mM EDTA, 0.5 mM dithiothreitol, 0.5 ug of poly dI-dC) and electrophoresed on non-denaturating 5% polyacrilamide gel in TBE 1×. The gels were dried and exposed to radiograph film. The probes used were high-performance liquid chromatography-purified oligonucleotides of the following base sequences:
Each probe was end labelled with T4 polynucleotide kinase and g-32P-ATP. For competition experiments, a 100 fold excess of specific unlabelled probe was incubated with extracts for 20 minutes before the addition of the radiolabeled probe. Supershift assays were performed by incubating 30 minutes the nuclear extracts with 1 μg of an anti-Stat3 antibody (Santa Crutz Biotech). To perform loading controls, the nuclear extracts were analyzed also for DNA-binding activity vs octamer-1 whose site is present in many house-keeping genes, using the following probe: 5′-GATCGAATGCAAATCACTAGCT-3′.
It has been reported that different physio-pathological conditions may modify the expression of IL-6 receptors thereby modulating cellular responsiveness to IL-6. The literature reports as physiologic an increased amount of IL-6 in the liver, in conditions of chronic cirrhosis. It was thus investigated whether this increase in IL-6 is also associated to an increased expression of the specific membrane receptors.
The experiments were carried on liver fragments obtained at liver resection. Liver from normal, non cirrhotic, animals was used as control.
Since in cirrhotic liver there is an increase in cell of lineage different from hepatocytes (Ito, myofibroblast, oval cells and inflammatory cells), both total liver homogenate and hepatocytes purified from cirrhotic liver were analyzed . Analogous procedure was performed on normal liver.
Hepatocytes were obtained by an innovative procedure that uses mechanical tissue dissociation which avoids enzymatic treatment, such as collagenase, which may destroy membrane structures particularly receptors (Dr Giovanna Mazzoleni, Dept Biomedical Sciences and Biotechnology, Viale Europa 11, 25100 Brescia, methodological procedure in press).
100 mg of liver or 16 mg of hepatocytes were homogenized in 1 ml of lysis buffer (150 mM NaCl2, 10 mM tris/HCl pH 7.4, 1% Igepal, 1% desossicholate, 0.1% SDS, 1 mM Na3VO4) containing a protease inhibitor cocktail (Sigma), incubated at 4° C. for 20 min and centrifuged at 12.000 g for 30 min. Forty micrograms were loaded on a 10% SDS-PAGE, transferred to a PVDF membrane, probed with polyclonal antibodies against gp80 and gp130 (Santa Crutz) and developed with ECL.
In cirrhotic animals the expression of gp130 significantly decreased in cellular extracts from total liver compared to normal controls. On the contrary in cirrhotic animals, the expression of gp130 significantly increased in total cellular extracts from purified hepatocytes compared to normal controls (
Literature data indicate that in hepatic cells the responsiveness to IL-6 depends on the presence of the specific gp80 subunit of the IL-6 receptor complex. In addition it has been demonstrated that an increase in gp80 or in both gp80 and gp130 subunits, induces an increase in cellular responsiveness to IL-6.
It was further investigated whether the hepatocellular proliferation induced by IL-6 is associated with an increased incidence of hepatocellular carcinoma in treated versus untreated cirrhotic rats. Consequently the regenerating liver of hepatocirrhotic rats was submitted to histological and immunohistochemical investigations, under blind conditions, in order to directly evidence the presence of neoplasia or dysplasia but also those alterations of parenchymal vascularization considered as markers of an increased risk of development of hepatocell carcinoma. This morphologic evaluation was finally completed by immunohistochemical evaluation of different oncogenes.
All histological evaluations were carried out on a minimum of 2 hematoxilin-eosin stained specimens per animal. In particular the presence of neoplasia and dysplasia was carefully investigated.
Several immunohistochemical evaluations were performed on a minimum of 2 specimens per animal per immunohistochemical reaction:
The angiogenesis in the regenerating cirrhotic parenchyma was investigated. This included the study of sinusoidal capillarization expressed as micro-vessels density in the hepatocirrhotic lobule, the study of arteriolas not associated to biliary structures (unpaired arteriola) and the evaluation of the endothelial differentiation.
This study was carried out by means of:
anti factor VIII antibodies for demonstration of capillary vessels;
anti smooth muscle specific actin antibodies for demonstration of arteriolas;
ULEX lectin for investigation of endothelial differentiation.
The study of oncogene expression in the regenerating cirrhotic parenchyma was investigated evaluating the expression of:
P 53;
MIBI 1;
Bcl-2
The 3 animals showing dysplasia or HCC at the histological analysis were also positive at the research for unpaired arteriolas; furthermore the 2 controls were also positive to the study of the micro-vessels density and 1 to the evaluation of the endothelial differentiation.
All the remnant specimens clearly showed that sinusoidal capillarization was directly related to the cirrhosis severity and diffusion and differences in micro-vessels density between controls and IL-6 treated animals were not observed. Furthermore in these cases we did not observe arteriolas not associated to biliary structures in control or IL-6 treated animals and in all cases the ULEX lectin showed a normal differentiation of the regenerating endothelia.
The study of P 53 and MIBI 1 were always negative. In contrast thereto, in 4 cases (1 control, 1 IL-6 500 and 2 IL-6 100) Bcl-2 was focally positive. It should be noted that in these 4 animals the histological analysis and the study of angiogenesis were absolutely negative, the 3 animals showing dysplasia or HCC at the histological analysis did not present an over-expression of the studied oncogenes.
The direct and indirect data obtained do not indicate an increased risk of neoplasm onset. In particular:
Among the treated animals the study of angiogenesis in regenerating cirrhotic parenchyma did not show an abnormal increase of the micro-vessels density inside the cirrhotic nodules, unpaired arteriolas or undifferentiated endothelia, data correlated to HCC onset.
The oncogene expression was almost completely negative. Only Bcl-2 showed a focal positivity rate of 4% and 13.3% respectively in control and IL-6 animals. These results indicate that high doses of IL-6 do not induce significant events related with hepatocellular carcinoma onset.
Object of the experiments are those rats that, from experiments of Example 1 which, developed a functionally decompensated cirrhosis with evidence of ascites at the moment of laparotomy or IL-6 injection, together with a small number of animals submitted to a longer CC14 treatment in order to deliberately achieve a decompensated liver function. Included in the experiment were 32 rats, 13 control and 19 IL-6. Among the IL-6 group, 11 animals received a dose of 100 mcg/kg, 3 a dose of 500 mcg/kg, 2 a dose of 10 mcg/kg, 2 a dose of 1 mcg/kg and 1 a dose of 0.1 mcg/kg.
A 46.2% of mortality among controls (6/13) versus a 10.5% (2/19) among IL-6 rats was observed. In one case mortality was related to acute intraoperative haemorrhage, 7 rats (5 controls and 2 IL-6) died of cardio-respiratory failure.
The ratio: (resected liver/rat weight)×100, extrapolated from 13 control animals and 19 IL-6 rats showed (
among controls a mean of 19.44±5.19, median 18.7;
among IL-6 animals a mean of 23.6±7.1, median 23.8.
Considering those 14 rats treated with high doses (H.D.) of IL-6 (500 and 100 mcg/kg) the result was:
Five control and 5 IL-6 rats (3 IL-6 100) received BrdU before hepatic resection. The count of BrdU stained hepatocytes showed (
among controls a mean of 17.4±8.6, median 17.2;
among IL-6 animals a mean of 34.3±14.8, median 42.1.
We observed a 42.8% of mortality in control (3/7) versus a 53% (9/17) in IL-6 treated rats. In all but 1 case the animals died of hepatic insufficiency (1 IL-6 animal died of evisceration).
a. among controls a mean of 8.6±9.8, median 7.1;
b. among IL-6 animals a mean of 22.5±11.4, median 22.3.
One control and 4 IL-6 rats (3 IL-6 100 and 1 IL-6 10) received BrdU after hepatic resection. The count of BrdU stained hepatocytes showed:
g. a 12.9% of BrdU stained hepatocites in the control rat;
h. a 89.1%, 87.6%, 33.9% and 61.4% respectively in the 3 IL-6 100 and IL-6 10 rats.
These data suggest that when in the presence of functionally decompensated cirrhosis, the intraperitoneal injection of IL-6 triggers the same biological effects detected when in presence of a functionally compensated cirrhosis, leading to a clear reduction of the operative mortality among IL-6 animals.
| Number | Date | Country | Kind |
|---|---|---|---|
| 159558 | Dec 2003 | IL | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IL04/01158 | 12/23/2004 | WO | 00 | 6/18/2007 |
