Title of Invention: IL-6/SIL-6R complex for promotion of liver functions
Abstract: A method for treating an injury of a liver of a subject with a composition featuring a pharmaceutically acceptable amount of an IL-6/sIL-6R complex, preferably Hyper-IL-6. The composition is administered to the subject such that the injury to the liver is treated.
Detailed Description: Part PAC: DETAILED DESCRIPTION OF THE PRESENT INVENTION
Part PAR: The present invention is drawn towards novel methods of use of the IL-6/sIL-6R complex, such as Hyper-IL-6. The IL-6/sIL-6R complex has now been shown to promote liver regeneration and the restoration of liver functions in subjects suffering from liver injury (see Example 1). Furthermore, administration of the IL-6/sIL-6R complex has now been shown to increase the lifespan of these subjects, as compared to untreated subjects (see Example 2). Thus, the IL-6/sIL-6R complex has significant utility for the treatment of many different types of liver diseases.
Part PAR_2: The principles and operation of the methods of treatment which feature IL-6/sIL-6R complex according to the present invention may be better understood with reference to the non-limiting illustrative examples below.
Part PAC_2: EXAMPLE 1
Part PAC_3: Effects of the IL-6/sIL-6R Complex in a Mouse Experimental Model
Part PAR_3: The effects of Hyper-IL-6 were examined in a mouse experimental model of liver damage. Two groups of mice underwent partial hepatectomy, in which a portion of the liver was removed, thereby seriously reducing overall liver functions. One group of mice received Hyper-IL-6, while a second, control group did not. The mice treated with Hyper-IL-6 showed and increased rate of liver regeneration and of a substantial restoration of liver functions. By contrast, the control mice showed a much slower rate of restoration of liver functions through liver regeneration. The experimental methods were as follows.
Part PAR_4: First, recombinant human IL-6.sup.39 and Hyper-IL-6.sup.19 were prepared as described in these references (39 and 19, respectively).
Part PAR_5: For partial hepatectomy, C57xBL/6 mice of 8 weeks old were obtained from the animal facility of the University of Mainz, Germany. In an initial experiment the relation between total body weight and liver weight was established in 20 mice. The mean total body weight was 27.45.+-.1.25 g. The mean liver weight was 1.2.+-.0.056 g. The mean ratio of liver weight (LW)/mean total body weight (BW) was 0.045.
Part PAR_6: Partial hepatectomy was performed as described by Higgins and Anderson.sup.40. Briefly, at the day of the operation, food was withdrawn at 8 AM in the morning and the surgery was carried out between 6 and 8 PM. Animals were anesthetized by an intraperitoneal injection of 2.5% avertin (mixture of 10 grams of tribromomethyl alcohol and 10 ml tertiary amyl alcohol). The total body weight of each mouse was recorded. The mice were then subjected to midventral laparotomy with an approximately 50% liver resection (left lateral and left half of medal lobes), slightly modified according to the procedure originally described by Higgins and Anderson.sup.40. The weight of the removed liver lobes was recorded. The weight of the residual liver lobes left behind was calculated by application of the formula 0.045=LW/BW. This weight was designated as "liver weight 1 at time 0". After the operation, the peritoneum was sutured and the skin was closed with wound clips.
Part PAR_7: Immediately after surgery, three groups, each including four to six mice, was subjected to one of three treatments: 2 .mu.g Hyper-IL-6, 20 .mu.g IL-6, or no treatment (control). All treatments were administered by injection; the control mice received an injection of physiological saline. After different time points as indicated in the figures, the mice were killed by cervical dislocation. The residual enlarged lobes were totally removed and their weight was recorded. This weight was designated as "liver weight 2 at time x". The percentage of the change, increase or decrease, in the weight of the liver after a defined period of time was calculated by subtracting liver weight 1 from liver weight 2.
Part PAR_8: In order to label the tissues with BrdU (5-bromo-2'-deoxyuridine), the animals were injected intraperitoneally with BrdU (50 mg/kg) (0.2% solution in PBS) one hour before the remnant liver was harvested and fixed. BrdU is a thymidine-analogue which is incorporated during the S-phase of the cell cycle into DNA. Applying immunhistochemical analyses using anti-BrdU-antibodies, S-phase-nuclei can be specifically detected. BrdU had been previously shown to be incorporated in S-phase-nuclei to the same extent as 3H!-Thymidine.sup.20. One hour after injection, the liver was then removed and immediately fixed in 4% paraformaldehyde (pH 7.2) at 4.degree. C. An automated tissue processor was used to embed the livers in paraffin. Tissue sections (5 microns) were cut on a microtome and adhered to poly-lysine-coated glass slides. Staining of fixed tissue samples was carried out using an antibody to BrdU (Boehringer Mannheim) enabling proliferating cells (red nuclei) to be distinguished from quiescent ones (blue nuclei). The immunhistochemical study was performed as suggested by the manufacturer (BrdU labelling and detection kit by Boehringer Mannheim) and as described previously.sup.41.
Part PAR_9: For the determination of haptoglobin protein determination in the serum, one microliter of murine serum was loaded on a 12.5 SDS gel and was subjected to SDS-PAGE gel analysis. The gel was blotted onto a nitrocellulose membrane and was subjected to Western blot analysis using a rabbit anti-human haptoglobin antibody (Dako, Glostrup, Denmark).
Part PAL: Results
Part PAR_10: The results demonstrated that Hyper-IL-6 causes an accelerated reconstitution of the liver weight following partial hepatectomy when compared to control mice and mice treated with IL-6, as shown in FIG. 1. Both untreated and IL-6-treated mice had a comparable increase of their liver weight. At 36 and 72 hours post surgery, IL-6-treated mice had slightly higher liver weights compared to untreated mice, which was not statistically significant. In Hyper-IL-6-treated mice, however, there was a dramatic increase of the liver weight at 72 and 120 hours post surgery (p&lt;0.005). At 168 hours, the liver weights of all treatment groups had reached their baseline weight. These data demonstrate for the first time that the presence of the IL-6/sIL-6R-complex is able to significantly increase the liver weight restoration following partial removal of the liver. Most remarkably, IL-6 alone did not improve the rate of liver weight increase in mice in this experimental model.
Part PAR_11: FIG. 2 demonstrates that the increased rate of weight gain found in mice receiving Hyper-IL-6 treatment is caused by the significant acceleration of liver proliferation in these mice, when compared to control mice and mice receiving IL-6 treatment. Control and IL-6-treated animals showed a peak of BrdU-labelled cells at 72 and 120 hours post surgery. By contrast, in Hyper-IL-6 treated mice, the maximal percentage of BrdU-positive cells was detected as early as 24 and 36 hours post surgery. The difference was highly statistically significant. The results demonstrate that the presence of the IL-6/sIL-6R-complex markedly accelerates liver proliferation which results in the fast restoration of the liver weight.
Part PAR_12: FIG. 3 shows representative immunohistochemical studies after 50% partial hepatectomy (untreated (A-C), treated with 20 .mu.g IL-6/mouse (A'-C'), or treated with 2 .mu.g Hyper-IL-6 (A"-C"); sacrificed 24 hours (A, A', A"), 36 hours (B, B', B"), or 120 hours (C, C', C") following surgery). The bars represent 100 .mu.m. As shown, control mice and IL-6-treated mice do not have any BrdU-positive cells detectable at 24 and 36 hours post surgery. However, at these time points, in Hyper-IL-6-treated mice, there is a high number of BrdU-labelled cells.
Part PAR_13: FIG. 4 shows that the hepatic acute phase protein production is intact after partial hepatectomy, as determined by analysis of the serum haptogiobin concentration by Western blot analysis in blood samples. First, the serum haptoglobin concentrations were measured in serum samples from mice which did not undergo partial hepatectomy. These mice were injected with saline, IL-6 alone (20 .mu.g), or Hyper-IL-6 (2 .mu.g). The upper panel of FIG. 4 shows that 24 hours after injection, IL-6 treatment alone leads to some slight haptoglobin protein increase in the serum, whereas the treatment with Hyper-IL-6 resulted in a marked elevation of the haptoglobin concentration in the serum. The haptoglobin mRNA concentration in the liver corresponds with the protein data shown in FIG. 4 (data not shown).
Part PAR_14: When the haptoglobin serum concentration was determined in hepatectomized mice 24 hours following the operation, mice receiving saline injections also had a slight haptoglobin protein elevation in their serum when compared to mice which did not undergo hepatectomy. The treatment with IL-6 alone and with Hyper-IL-6 resulted in a comparable serum haptoglobin concentration when compared to mice which did not undergo hepatectomy (FIG. 4, lower panel). These data show that the regenerating liver is capable of mounting a normal acute phase protein response.
Part PAR_15: These data have demonstrated for the first time that in the presence of IL-6 and its soluble receptor, sIL-6R, liver regeneration in mice following partial hepatectomy is greatly accelerated. Moreover, the IL-6/sIL-6R complex has now been shown to rapidly cause hepatocyte proliferation of the liver following partial hepatectomy in mice. IL-6 alone at a ten-fold higher dosage than the designer cytokine Hyper-IL-6 did not cause accelerated liver regeneration or hepatocyte proliferation as compared to untreated animals. Thus, only Hyper-IL-6 was able to induce liver regeneration and the restoration of liver functions in mice which had undergone partial hepatectomy.
Part PAC_4: EXAMPLE 2
Part PAC_5: Survival of Rats with Hepatatic Failure
Part PAR_16: In order to assess the ability of Hyper-IL-6 to treat fulminant hepatatic failure (FHF), FHF was induced in four rats. Two also received Hyper-IL-6, one received hepatocytes and one received human IL-6 alone. The rats which received Hyper-IL-6 survived for over one month, while the other rats died within 24-72 hours. The experimental method was as follows.
Part PAR_17: Four male Sprague-Dawley rats were deprived of food, but not of drinking water, for 12 hours. Next, the rats were injected i.p. with D-Galactosamine (1.4 g/kg, pH=6.8). After 24 hours of D-Galactosamine treatment, fulminant hepatic failure was induced. Two rats then received Hyper-IL-6 (7 micrograms, i.p.). One rat received human IL-6 (80 micrograms, i.p.). One rat received a transplantation of syngeneic hepatocytes (2.times.10.sup.5 cells).
Part PAR_18: The rat which received human IL-6 died 24 hours after treatment, while the rat which received the hepatocyte transplant died within 72 hours. Only the animals treated with Hyper-IL-6 survived for over one month before being sacrificed. Thus, Hyper-IL-6 was clearly able to prolong the life-span of rats suffering from hepatic failure.
Part PAC_6: EXAMPLE 3
Part PAC_7: Compositions and Methods of Treatment with Hyper-IL-6
Part PAR_19: As described previously in the section entitled "Summary of the Invention", the term "IL-6/sIL-6R complex" refers to a bimolecular protein complex which features both the IL-6 polypeptide and sIL-6R, the soluble IL-6 receptor protein; and to a unimolecular protein which includes the bioactive portions of IL-6 and sIL-6R connected with a flexible linker, as previously described in PCT Patent Application No. PCT/DE97/00458, and in Fischer, M. et al., Nature Biotech. 15, 142-145 (1997), as well as pharmaceutically acceptable salts thereof.
Part PAR_20: The composition containing the IL-6/sIL-6R complex, and in particular Hyper-IL-6, can be administered to a subject in a number of ways, which are well known in the art. Hereinafter, the term "subject" refers to the human or lower animal to whom Halofuginone was administered. For example, administration may be done topically (including ophtalmically, vaginally, rectally, intranasally and by inhalation), orally, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, or intramuscular injection.
Part PAR_21: Particularly preferred routes of administration include parenteral, intranasal and by inhalation.
Part PAR_22: Formulations for topical administration may include but are not limited to lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Part PAR_23: Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.
Part PAR_24: Formulations for parenteral administration may include but are not limited to sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
Part PAR_25: Dosing is dependent on the severity of the symptoms and on the responsiveness of the subject to the IL-6/sIL-6R complex, as well as on the particular embodiment administered. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates.
Part PAR_26: As noted previously, the compositions found to be useful in the methods of the present invention include the IL-6/sIL-6R complex. The methods of the present invention are useful for the treatment of injury to the liver. The following example is an illustration only of a method of treating such an injury to the liver, and is not intended to be limiting in any way.
Part PAR_27: The method includes the step of administering the composition including the IL-6/sIL-6R complex, in a pharmaceutically acceptable carrier as described above, to a subject to be treated. The composition preferably features Hyper-IL-6 as the embodiment of the IL-6/sIL-6R complex. The composition is administered according to an effective dosing methodology, preferably until a predefined endpoint is reached, which could include one or more of the following: a normalized level of coagulation factors 5 or 7; normalized prothrombin time; the absence of hepatic encephalopathy; normalized levels of liver enzymes such as aspartate aminotransferase and alanine aminotransferase; and normalized ammonia levels.
Part PAR_28: In a preferred embodiment of the method of the present invention, the composition including the IL-6/sIL-6R complex is administered to a subject before, during or after liver transplantation, or a combination of these timepoints of administration thereof, in order to promote growth and regeneration of the transplanted liver.
Part PAR_29: Examples of injuries to the liver for which such a method of treatment would be suitable include but are not limited to liver damage caused by toxic substances, including alcoholic hepatitis and drug induced hepatopathy; damage caused by mechanical disruption or trauma; damage caused by a malignancy, whether primary or metastasizing from another body tissue; damage caused by an autoimmune or other genetically-related pathological process; and damage caused by a pathogen such as any of the group of Hepatitis viruses, including dominant viral hepatitis. The term "injury to the liver" also encompasses acute or chronic liver failure, including fulminant hepatic failure, as well as conditions in which liver failure has not occured, including any condition featuring a reduction of liver functions from a substantially normal level.
Part PAR_30: The term "treating" includes ameliorating, alleviating or substantially eliminating a liver injury, as well as substantially preventing a liver injury.
Part PAR_31: While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made.


{'bibliographic_information': "{'Patent Number': '059197633', 'Series Code': '9', 'Application Number': '0877964', 'Application Type': '1', 'Art unit': '164', 'Application Filing Date': '19980601', 'Title of Invention': 'IL-6/SIL-6R complex for promotion of liver functions', 'Issue Date': '19990706', 'Number of Claims': '7', 'Exemplary Claim Number(s)': '1', 'Assistant Examiner': 'Delacroix-Muirheid; C.', 'Primary Examiner': 'Tsang; Cecilia J.', 'Number of Drawing Sheets': '5', 'Number of figures': '4'}", 'source_file': './grant/1999/pftaps19990706_wk27.txt', 'source_data': 'USPTO-Grant'}