WO2003029281A1 - An sil-6r/il-6 fusion protein cooperates synergistically with hgf to stimulate hepatocyte proliferation in vivo - Google Patents
An sil-6r/il-6 fusion protein cooperates synergistically with hgf to stimulate hepatocyte proliferation in vivo Download PDFInfo
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- WO2003029281A1 WO2003029281A1 PCT/US2002/028150 US0228150W WO03029281A1 WO 2003029281 A1 WO2003029281 A1 WO 2003029281A1 US 0228150 W US0228150 W US 0228150W WO 03029281 A1 WO03029281 A1 WO 03029281A1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/204—IL-6
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- Chemical & Material Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
A composition of two or more cytokine protein components. These components may optionally be present in one of a number of different combinations. For example, the combination may optionally include at least one of IL-6 or IL-6/sIL-6R complex as a first component, and at least one of HGF protein, TNF-α or an engineered derivative there of as a second component. Additionally or alternatively, the combination may include a complex of any member of the IL-6 family and its corresponding receptor or an engineered derivative thereof. The present invention also encompasses methods of use thereof, including for the treatment of a pathological of a subject. The composition may optionally be delivered through gene therapy for such treatment.
Description
AN SIL-6R IL-6 FUSION PROTEIN COOPERATES SYNERGISTICALLY WITH HGF TO STIMULATE HEPATOCYTE PROLIFERATION fN VWO
FIELD OF THE INVENTION The present invention is of a composition of two or more cytokine protein components, such as IL-6/sIL-6R complex and IIGF protein or TNF-α (Tumor Necrosis Factor alpha) or an engineered derivative thereof, and/or a complex of any member of the IL-6 family and its corresponding receptor or an engineered derivative thereof, as well as methods of use thereof, including for the treatment of a pathological condition of a subject. The composition may optionally be delivered through gene therapy for such treatment.
BACKGROUND OF THE INVENTION
Following injury, the liver has a remarkable capacity to replace even substantial tissue loss by regeneration (for reviews see (1-3)). The molecular signaling pathways mediating hepatocyte proliferation during liver regeneration is a complex and highly regulated multistcp process, thought to involve at least two distinct and critical steps: "priming", involving the transition of quiescent hepatocytes into the cell cycle, and "progression", in which (he cells are stimulated beyond the restriction point in the Gl phase of the cycle (2). It has been proposed that these steps are under separate controls, the priming step by TNF-α (tumor necrosis factor alpha) and the IL-6 (Interleukin-6) family cytokines, and progression by growth factors, mainly HGF (hepatocyte growth factor) and TGF-α (transforming growth factor alpha) (2).
IL-6 is a plieotropic cytokine (4), which in the liver mediates the acute phase response, and has both cyto-protective and mitogenic functions (2). IL-6 is a member of a family of cytokines that act via receptor complexes containing at least one subunit of the transmembrane signal transducing protein g l30 (5). On target cells, IL-6 acts by binding to a specific transmembrane cognate receptor (gp80 or IL-6R), which triggers the homodimerization of gpl30, and leads to the activation of the Jak/Stat signaling pathway (5, 6), IL-6 has both mitogenic and anti-apoptotic effects on hepatocytes in liver regeneration (7). IL-6 pretreatmeni can protect the liver from injury induced by warm ischemia/reperfusion (8), conA (9), as well as Fas agonist induced apoptotic injury (10). However, while evidence from experiments in IL-6-defecient mice has shown that while IL- 6 is essential to induce hepatocyte proliferation to proceed at a normal rate following injury
or tissue loss, it is not absolutely required for liver regeneration (7, 11, 12). Thus, like TNF- α, IL-6 is not known in the art to have a mitogenic eflFect oft hepatocytes in vivo (2).
Hyper-IL-6 is a super agonistic designer cytokine consisting of human IL-6 linked by a flexible peptide chain to a soluble form of the IL-6 receptor (sIL-6R) (13, 14). Previous studies by the present inventors have shown that treatment of animals with hyper-ΪL-6, as opposed to treatment with IL-6, dramatically enhanced hepatocyte proliferation and accelerated liver regeneration following the induction of liver injury by the hepatotoxin D- galactosamine (15), or after partial hepatectomy (16). Without wishing to be limited to a single hypothesis, one possible explanation of the remarkable potential of hyper-IL-6 to stimulate liver regeneration is that the superagonistic fusion protein is more efficiently able to enhance the growth stimulating potential of growth factors, such as HGF, that are naturally activated during the process of liver regeneration. Another alternative explanation is that prolonged hyper-stimulation of g l30 may have mitogenic effects. In support of this later hypothesis, it has been observed that double transgenic mice expressing high levels of both human IL-6 and sIL-6R under the control of liver-specific promoters spontaneously develop nodules of hepatocellular hyperplasia around periportal spaces and present signs of sustained hepatocyte proliferation (17, 18). These observations would suggest that gρl30- hyperstimulation by the IL-6/sIL-6R complex is a primary stimulus to hepatocyte proliferation, and under certain conditions can act as a complete mitogen.
SUMMARY OF THE INVENTION
The background art does not teach or suggest a composition of two or more cytokine protein components, in which one component is a member of the IL-6 family. In addition, the background art does not teach or suggest the use of such a combination for the treatment of a pathological condition of a subject, particularly a liver-related condition.
The present invention overcomes these deficiencies of the background art by providing a composition of two or more cytokine protein components. Preferably, these components include at least one combination component, which is a combination of at least a portion of a member of the IL-6 family and at least a portion of the corresponding receptor. Such a combination component is also referred to herein as an "IL-6 family combination component". Such a combination component may optionally and preferably comprise a complex of any member of the IL-6 family and its corresponding receptor or an engineered
derivative thereof. A prefejπred but non-limiting example of such a combination component is a IL-6/sIL-6R complex. As described in greater detail below, a EL-6/sIL-6R complex includes at least a portion of both the IL-6 polypeptide and SΪL-6R, the soluble IL-6 receptor protein as a complex. Other illustrative examples include but are not limited to combinations of at least one of at least a portion of each of IL-11 (interleukin 11) and its respective receptor; OSM (oπcostatin-M) and its respective receptor; CT-1 (car iotrophtn-1) and its respective receptor; LIF (leukemia inhibitory factor) and its respective receptor; NT-1 (neurotrophin-1/B cell stimulating factor 3) and its respective receptor; or CNTF (ciliary neurotrophic factor) and its respective receptor In other words, for each iigand/receptor pair, at least a portion of the ligand and at least a portion of the respective receptor foim the complex.
Also preferably, at least a second component is a liver regenerating factor, which more preferably is a growth factor. Illustrative examples of such growth factors include, but are not limited to, HGF (hepatocyte growth factor), EGF (epidermal growth factor) and TGF-alpha (transforming growth factor alpha). Other illustrative examples of growth factors which may optionally be possible to use include, but are not limited to, KGF (keratinocyte growth factor) (see for example "Effects of keratinocyte and hepatocyte growth factor in vivo; implications for retrovirus-mediated gene transfer to the liver", Bosch et al.. Hum Gene Ther. 1998, vol 9; 1747-54, hereby incorporated by reference as if My set forth herein), A non-limiting, illustrative example of another type of liver regenerating factor is TNF-alpha (tumor necrosis factor alpha).
These components may optionally be present in one of a number of different combinations. For example, the combination may optionally include at least one of IL- 6/sIL-6R complex as a first component, and at least one of HGF protein, TNF-α or an engmeered derivative thereof as a second component. Additionally or alternatively, the combination may include a complex of any member of the IL-6 family and its corresponding receptor or an engineered derivative thereof.
The present invention also encompasses methods of use thereof, including for the treatment of a pathological condition of a subject. The composition may optionally be delivered through gene therapy for such treatment.
Particuiariy preferred combinations according to the present invention include, but are not limited to, a combination of an IL-6/sIL-6R complex and TNF-α or HGF protein. An additional or alternative preferred combination according to the present invention includes
any member of the IL-6 family and its cognate soluble receptor (EL- 11, OSM, CNTF, or any other such receptor), or an engineered derivative thereof,
Hereinafter, the term "IL-6/5IL-6R complex" refers both 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 slL- 6R connected with a flexible linker, substantially as previously described in PCT Patent Applicatio s. PCT/DE97/0045S (WO 97/32891), in US Patent No. 5,919,763 and in Fischer, M. et al, Nature Biotech. 15, 142-145 (1997), all of which are incorporated by reference as if My set forth herein, as well as any biologically active equivalents thereof. The other non-limiting examples of complexes of another member of the IL-6 family and its corresponding receptor or an engineered derivative thereof may optionally be constructed similarly.
Hereinafter, the term "Hyper-IL-6" refers to a unimolecular protein which includes the bioactive portions of IL-6 and SIL-6R connected with a flexible linker, substantially as previously described and shown in PCT Patent Application No. PCT/DE97/004S8 (WO 97/32891; referred to as "H-IL-6" in that reference and also below), for example in Figure 1 or in any portion of that reference, or any biologically active equivalent thereof.
Hereinafter, the term "biologically active" refers to molecules, or complexes thereof, which are capable of exerting an effect in a biological system. Hereinafter, the term "amino acid" refers to both natural and synthetic molecules which are capable of forming a peptidic bond with another such molecule. Hereinafter, the term "natural amino acid" refers to all naturally occurring a ino acids, including both regular and non-regular natural amino acids. Hereinafter, the term "regular natural amino acid" refers to those amino acids which are normally used as components of a protein. Hereinafter, the term "non-regular natural amino acid" refers to naturally occurring amino acids, produced by mammalian or non-mammalian eukaryotes, or by prokaryotes, which are not usually used as a component of a protein by eukaryotes or prokaryotes. Hereinafter, the term "synthetic amino acid" refers to all molecules which are artificially produced and which do not occur naturally in eukaryotes or prokaryotes, but which fulfill the required characteristics of an amino acid as defined above. Hereinafter, the term "peptide" includes both a chain of a sequence of amino acids of substantially any of the above-referenced types of amino acids, and analogues and mimetics having substantially similar or identical functionality thereof.
With regard to the unimolecular protein, such as Hyper-IL-6 for example, and the bimolecular protein complex, the expression "linker" relates to linkers of any kind, which are suitable for the binding of polypeptides. Examples of such linkers include but are not limited to bifinctional, chemical cross-linkers; a disuhide-bridge connecting two amino acids of both polypeptides; and a peptide or polypeptide.
The bimolecular protein complex may optionally include both IL-6 and sIL-6R, and/or another IL-6 family member with its receptor, as well as biologically active portions and variants thereof, connected by a linker. The term "variants" includes any homologous peptide to either IL-6 or sIL-6R, or the equivalent peptides for the other members of this family, for example including any amino acid substitution or substitutions which still maintain the biological activity of the original peptide or a polypeptide which directly stimulates the membrane receptor for the IL-6/sIL-6R complex which is called gp 130 or for any of the other complexes formed by other members of the IL-6 ligand/receptor family. The unimolecular protein can optionally be a fusion polypeptide. For example, polypeptides featuring the bioactive portions of IL-6 and sIL-6R (or of any other family member combination components) can optionally be fused with each other and the linker can be a disulfide-bridge produced by the two polypeptides Preferably the linker is a polypeptide, which connects the two other polypeptides with each other. These fusion polypeptides optionally and preferably include a human slL-6R-polypeptide for the example comprising IL-ό itselζ which is the extracellular subunit of an iπterleukin-6-receptor and a human IL-6-polypeptide, whereby the polypeptides are connected by different polypeptide- linkers with each other. The accession number for IL-6 is M14584 (GenBank Protein Sequences Database), and for the soluble IL-6 receptor is M57230, M20566 and X12830. The accession number for HGF is XI 6323. The accession number for IL-11 is NM00641 ; for OSM is NM020530; for LIF is
NM002309, for CTFl is NM001330; and for CNTF is NM013246.
With regard to any of the above-listed proteins for which an accession number is given, it should be noted that all such accession numbers may optionally be used to obtain the full protein and/or RNA and/or cDNA sequences from the NCBI (National Center for Biotechnology Information) database, and/or from GenBank Protein Sequences Database. NCBI provides access to this database through the Internet (http://www.ncbi. nlm. nih. sov as of September 30 2002). However, variants on, or modifications of, any of the above-listed
proteins may also optionally be used, as such variants are known in the art and may also be listed in the above databases.
With regard to the IL-6/sIL-6R complex specifically, a variation of the unimolecular protein, which includes only amino acids 114-323 inclusive from the sIL-6R-polyρeρtide, is also preferably included. A second optional but preferred variation includes amino acids 113-323 inclusive of the sIL-6R-polypeptide and amino acids 29-212 of the IL-6- polypeptide. Other variations and combinations as previously disclosed in PCT Patent Application No. PCT/DE97/0045S (WO 97/32891), in US Patent No. 5,919,763 and in Fischer, M. et al., Nature Biotech. 15, 142-145 (1997) are also included in the unimolecular protein embodiment of the IL-6/sIL-6R complex.
Hereinafter, the term "treatment" includes both the amelioration or elimination of an existing condition and the prevention of the genesis of a condition.
Hereinafter, the term "injury to the liver" includes but is not limited to liver damage caused by toxic substances, by mechanical disruption or trauma, by a malignancy whether primary or metastasizing from another body tissue, by an autoimmune or other genetically- related pathological process, or by a pathogen such as any of the group of Hepatitis viruses. The term "injury to the fiver" also encompasses acute or chronic liver failure, as well as conditions in which liver failure has not occurred.
Hereinafter, the term "injury to the kidney" includes but is not limited to kidney damage caused by toxic substances, by mechanical disruption or trauma, by a malignancy whether primary or metastasizing from another body tissue, by an autoimmune or other genetically-related pathological process, or by a pathogen. The term "injury to the liver" also encompasses acute or chronic kidney failure, as well as conditions in which kidney failure has not occurred, According to the present invention, there is provided a therapeutic composition, comprising (a) an IL-6 family combination component; and (b) a liver regenerating factor. Preferably, the combination component comprises a IL-6/sϊL-6R complex. More preferably, the IL-6/sIL-ΘR complex comprises a bimolecular protein complex featuring at least a portion of both IL-6 and IL-6 receptor protein. Alternatively, the IL-6/sIL-6R complex comprises a unimolecular protein comprising bioactive portions of IL-6 and sIL-6R connected with a flexible linker. Preferably, the IL- 6/&IL-6R complex comprises Hyper-IL-6.
According to preferred embodiments of the present invention, the combination component comprises a combination of at least one of at least a portion of each of IL-I 1 (interleukin 11) and its respective receptor; OSM (oncostatin-M) and its respective receptor; CT-I (cardiotrophin-1) and its respective receptor; LIF (leukemia inhibitory factor) and its respective receptor; NT-1 (neurotrophin-lZB cell stimulating factor 3) and its respective receptor; or CNTF (ciliary neurotrophic factor) and its respective receptor.
According to other preferred embodiments of the present invention, the liver regenerating factor comprises a growth factor, Preferably, the growth factor comprises HGF (hepatocyte growth factor), EGF (epidermal growth factor) and TGF-alpha (transforming growth factor alpha) or an engineered derivative thereof.
Alternatively or additionally, the liver regenerating factor comprises TNF-alpha (tumor necrosis factor alpha) or an engineered derivative thereof.
Preferably, the combination component comprises a 1L-6 L-6R complex and the liver regenerating factor comprises HGF. Also preferably, the combmation component comprises a IL-6/sIL-6R complex and the liver regenerating factor comprises TNF-alpha.
Preferably, the combination component comprises Hyper-IL-6 and the liver regenerating factor comprises at least one of HGF and TNF-alpha.
Optionally and more preferably, the composition further comprises a pharmaceutically acceptable carrier. Most preferably, the combination component and the liver regenerating factor are present in therapeutically effective amounts,
According to another embodiment of the present invention, there is provided a method for treating an injury to a liver of a subject, comprising administering, to the subject, a pharmaceutically acceptable amount of a therapeutic composition in a pharmaceutically acceptable carrier, the therapeutic composition comprising: (a) an ΪL-6 family combination component; and (b) a liver regenerating factor; such that the injury to the liver is treated. Preferably, the therapeutic composition is administered to the subject parenterally. Optionally and preferably, the injury to the liver is selected from the group consisting of reduction of liver function from a normal level caused by a toxic substance, reduction of liver function from a normal level caused by mechanical trauma, reduction of liver function from a normal level caused by a malignancy, and reduction of liver function from a normal level caused by a pathogen. More preferably, the reduction of liver function from a normal level caused by the toxic substance includes alcoholic hepatitis and drug induced
hepatopathy. Also more preferably, the injury to the liver is selected from the group consisting of acute liver failure and chronic liver failure.
Optionally and preferably, the therapeutic composition is administered to the subject at least one of before, during or after liver transplantation, or a combination thereof. According to other preferred embodiments of the present invention, there is provided a method for treating a subject receiving a fiver transplant, the method comprising administering, to the subject, a pharmaceutically acceptable amount of a therapeutic composition in a pharmaceutically acceptable carrier, the therapeutic composition comprising: (a) an IL-6 family combination component; and (b) a liver regenerating factor; wherein the therapeutic composition is administered to the subject at least one of before, during or after liver transplantation, or a combination thereof.
According to still other preferred embodiments of the present invention, there is provided a method for treating a pathological condition of a subject, comprising administering, to the subject, a pharmaceutically acceptable amount of a therapeutic composition in a pharmaceutically acceptable carrier, the therapeutic composition comprising: (a) an IL-6 family combination component; and (b) a liver regenerating factor; wherein at least one of the IL-6 family combination component and the liver regenerating factor is administered encoded by a plasmid, the plasmid being administered to the subject; such that the pathological condition is treated. Preferably, the therapeutic composition is delivered to the subject through a gene therapy protocol. More preferably, the gene therapy protocol features the delivery of any nucleic acid polymer. Most preferably, the composition is used for enhancement of gene delivery in a gene therapy protocol involving viral vectors requiring or augmented by hepatocyte proliferation. Optionally and preferably, the protocol is retroviral vector or leπtiviral vector based gene therapy.
Preferably, the pathological condition is a liver-related condition. More preferably, the liver-related condition is at least one of liver disease, and liver failure. Most preferably, the liver disease includes at least one of liver fibrosis or cirrhosis. Optionally, the liver failure is acute or fulminant hepatic failure.
Alternatively or additionally and preferably, the pathological condition is a kidney disease. More preferably, the kidney disease includes at least one of renal failure; the effect
of damaging substances on renal tissue and functions; and loss of renal function following surgery.
Optionally and preferably, the pathological condition is treated through regeneration of a tissue in the subject According to yet other preferred embodiments of the present invention, there is provided use of a composition for inducement of tissue growth and/or renewal in vitro, the composition comprising: (a) an IL-6 family combination component; and (b) a liver regenerating factor.
All references listed in this application are hereby incorporated by reference as if fully set forth herein.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, aspects and advantages will be better understood from the following detailed description of a preferred embodiment of the invention with reference to the drawings, wherein.
FIG. 1 is a schematic representation of examples of plasmid DNA constructs utilized for cytokine expression in vivo for the present invention;
FIG. 2 shows the duration and level of in vivo Hyper-IL-6 expression following hydrodynamics based plasmid DNA injection is promoter dependent. C57BL/6 mice were injected i,v, with 10 raicro-grams total plasmid DNA in 1,5 ml saline in 5-8 seconds. Hyper- IL-6 levels in the serum of treated mice were analyzed by using a human IL-6 ELISA Plasmid DNA constructs are identical, with the exception of the promoter: pCI-HIL6 (5 micro-gram) contains the CMV immediate early promoter, pSI-HIL6 (10 micro-grams) the SV40 immediate early promoter, and phAAT-HIL6 (10 micro-grams) contains the human αl-antitiypsin promoter driving the HIL6 cDNA. Treatment conditions were normalized with non-relevant control plasmid DNA such that all mice received a total of 10 μg plasmid DNA, Values represent mean ± standard error mean (π=8),
FIG. 3 demonstrates that combined HGF and FHL-6 treatment leeds to enhanced mitogenesis of hepatocytes in vivo. A histogram of percent of mitotically active hepatocytes in liver sections from mice following treatment with different combinbations of cytokines is shown, Mice were treated with TNF-α protein (0.5μg i,p.), HIL-6 (lOμg of ρhAAT-HIL6 DNA), or HGF (iOμg of phAAT-HGF DNA), either alone, or in combinations as indicated.
Treatment conditions were normalized with an irrelavent control plasmid DNA such that all mice received a total of 20 μg DNA. Histological sections from parafin imbedded livers removed on day 2 post treatment were immunohistochemically stained for PCNA, Positive nuclei were scored from twenty randomly selected fields (-4140 total hepatocyte nuclei) per liver, at a magnification of 400x. Mitotically active hepatocytes appearing following treatment with HGF alone or in combmation with either HIL-6, or TNF- were located mainly in the central-lobular regions. Values represent mean ± standard error mean (n=6) following subtraction of baseline mitogenic activity (1.3+0,2 %) resulting from the injection of control plasmid DNA. TO.OOland **P<0,0005, with respect to HGF alone, Student's t test;
FIG. 4 shows that enhanced hepatocyte itotic acitivity and normal liver morphology following treatment with HGF and hyper-IL-6 revealed by im unohistochemical analysis. Liver sections prepared from animals 2 days following treatment with: (a) control plasmid DNA (b) hyper-IL-6 and TNF-α treated mice, or (c) HGF and hyper-IL-6. Sections were stained with hemotoxylin-eosin (H&E) and anti-PCNA antibodies to reveal mitogenic activity, Positive nuclei appear red, in contrast to nuclei that are not in the S-phase of the cell cycle (blue). Original magnification x200; and
FIG. 5 demonstrates that combined HGF and HIL-6 (Hyper-IL-6), but not IL-6 treatment leeds to enhanced mitogenesis of hepatocytes in vivo. A histogram of percent of mitotically active hepatocytes in liver sections from mice following treatment with different combinations of cytokines is shown. Mice were treated with HIL-6 (10μg of phAAT~HIL6 DNA), D 6 (lOμg of phAAT-IL6 DNA) or HGF (lOμg of phAAT-HGF DNA), either alone, or in combinations as indicated. Treatment conditions were normalized with an irrelevent control plasmid DNA (pCI) such that all mice received a total of 20 μg DNA. Experimental conditions and analysis are as described in Fig. 3. Values represent mean +- standard error mean (n=6) following subtraction of baseline mitogenic activity (4.9±0.S %) resulting from the injection of control plasmid DNA. Mitotic activity in untreated mice was 4.6+1.0 %. *P>0,2 and **P<0.002, with respect to HGF alone, Student's /test.
BRIEF DESCRIPTION OF THE TABLES
The foregoing and other objects, aspects and advantages may also be better understood from the following detailed description of a preferred embodiment of the invention with reference to the supporting tables, wherein;
Table I shows plasmid DNAs utilized in this study for demonstrating the efficacy of the present invention; and
Table π provides data which demonstrate the influence of TNF-α and hyper-IL-6 on hepatocyte mitotic activity in vivo.
DESCRIPTION OF THE PREFERJRED EMBODIMENTS The present invention is of a composition of two or more cytokine protein components. These components may optionally be present in one of a number of different combinations, For example, the combination may optionally include at least one of IL- 6/sIL-6R complex as a first component, and at least one of HGF protein, TNF-α or an engineered derivative thereof as a second component. Additionally or alternatively, the combination may include a complex of any member of the IL-6 family and its corresponding receptor or an engineered derivative thereof.
The present invention also encompasses methods of use thereof, including for the treatment of a pathological condition of a subject, The composition may optionally be delivered through gene therapy for such treatment. Particularly preferred combinations according to the present invention include, but are not limited to, a combination of an TL-6 L-6R complex and TNF-α or HGF protein; and IL-6 and TNF-α or HGF protein. An additional or alternative preferred combination according to the present invention includes any member of the IL-6 family and its cognate soluble receptor (IL-11, OSM, CNTF, or any other such receptor), or an engineered derivative thereof.
Examples of methods of use of the compositions of the present invention include but are not limited to, treatment of a pathological condition of a subject. A preferred example of such a condition is a liver-related condition, including but not related to, liver disease, such as fibrosis and/or cirrhosis for example; and liver failure, including acute and fulminant hepatic failure for example.
Another example of a pathological condition of a subject which may be treated with the compositions of the present invention is treatment of kidney disease and/or failure (see Kawaida et al (1994) (23)). HGF exerts both mitogenic and morphogenic effects on renal
tubular epithelial cells, and blood HGF levels are markedly induced following unilateral nephrectomy and acute renal failure. Kawaida et at (23) have demonstrated that treatment of mice with recombiαant human HGF following induction of severe renal dysfunction by administration of cisplatin or HgCl2 remarkably suppressed increases in blood urea nitrogen and serum creatinine, Moreover, HGF treatment stimulated DNA synthesis in renal tubular cells following HgCl2 induced renal injury or unilateral nephrectomy and induced reconstruction of normal renal tissue structure. As such, it has been suggested that HGF treatment may to be an effective treatment for patients with renal dysfunction (23). The present invention extends this suggestion for treatment with the compositions of the present invention, as such compositions have been shown to have greater (synergistic) efficacy in comparison to the effect of the individual components alone.
In addition or alternatively, the present invention may optionally be used for enhancement, of gene delivery in gene therapy protocols involving viral vectors requiring or augmented by hepatocyte proliferation. Suitable examples of such protocols would include retroviral vector and lentiviral vector based gene therapy.
The efficacy of the present invention has been demonstrated as described in greater detail below, by using the recently described procedure of hydrodynamics-based transfection in mice through systemic administration of plasmid DNA (19, 20) to express the IL-6/sIL- 6R fusion protein in vivo at various levels for durations ranging up to 9 days. This procedure has been used by the present inventors to demonstrate the mitogenic potential of gpl30 stimulation as well as the potential of the hyper-TL-6 to cooperate with TNFα and HGF to simulate hepatocyte proliferation. As described in greater detail below, evidence is provided that prolonged gpl 0 hyper-stimulation is not mitogenic, but. rather enhances hepatocyte proliferation in cooperation with growth factors such as HGF,
MATERIALS AND METHODS Animals
All animal work was conducted in compliance with regulations of the Hebrew University Hadassah Medical School Animal Housing Committee. Male C57BL/6 mice 5 to 6 weeks of age, purchased from Harlan Animal Breeding Center, Jerusalem were housed in cages in a temperature-controlled room with a 12 hour light dark cycle and free access to food and water. Prior to systemic injections animals were heated for 4-5 minutes at approximately 37-41° C under an infra-red lamp. Systemic DNA transfections were
performed by injection of a 1.5 ml saline solution containing a total of 20 μg DNA into the tail vein, within 5 to 8 seconds, as described previously (19). Recombinant TNF-α (PeproTech, Rocky Hill, NX, USA) was reconstituted in water to 100 micro-gram/ml and diluted in physiological saline prior to administration, TNF-α treated animals were administered a dose of 0,5 micro-gram by intraperitoneal injection immediately following systemic injection of plasmid DNAs.
Plasmid DNAs
The plasmid DNAs utilized in this study are listed in Table T, pCI-HIL6 was constructed using a human hyper-IL-6 cDNA gene coding the human SD -6R (amino acid residues 1-323) and human TL-6 (amino acid residues 29-212) fused by a synthetic DNA linker coding for the amino acid sequence Arg-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Val- Glu (13). The cDNA gene was removed from the plasmid pDCM8-H-IL-6 by digestion on one side with H dHI, which had been made blunt-ended by filling-in with T4 DNA polymerase, and subsequently digested with Notl The agarose gel electrophoresis purified DNA fragment was then ligated into the S al and Notl sites of the mammalian expression vector pCI (Promega). phAAT-HIL6 was constructed by sequential digestion of pCI-HIL6 with Ilindlll, filling-in with T4 DNA polymerase and digestion with BglH to release and remove the CMV promoter. The remaining hyper-IL-6 encoding vector DNA fragment was ligated with the human αl-antitrypsin (hAAT) promoter element, which had been removed from the plasmid phAAT-FX (a kind gift from Katherine Ponder) as a 400bp Notl-Bglϋ fragment, made blunt-ended at the Notl site with T4 DNA polymerase. The plasmid, pSI- HIL6, was constructed by removal of the CMV promoter of pCl-HIL6 with Bgiπ and Mlul and ligation with a 695 bp BgUI-Mluϊ fragment containing the SV40 promoter derived from the plasmid pSI (Promega). phAAT-HGF was constructed in two steps involving Hgation of a 2.3 kb Xbal-Sall fragment encoding the HGF cDNA from the plasmid pBS-7, (kindly provided by T. Nakamura, Osaka Medical School) into the same restriction sites of pCI, and ligated into the same sites of phAAT-HUL6 from which the HEL6 sequences had been removed by restriction enzyme digestion. The human IL-6 expression plasmid, pCI-IL6 was constructed by removal of the IL-6 cDNA from the plasmid pCDM-h-IL-6 (Schiel X, Rose-John S, Dufhues G, Schooltink H, Gross V, Heinrich PC, Eur J Immunol 1990 Aρr;20(4);8S3-7) by digestion on one side with Hindm, which had been made blunt-ended by filling-in with T4 DNA polymerase, and
subsequently digested with Notl, The agarose gel electrophoresis purified DNA fragment was then ligated into the S al and Notl sites of the mammalian expression vector pCI (Promega). The expression vector ρhAAT-IL6 was generated by removal of the cmv promoter and adjacent intron by restriction with the restriction enzymes Bglπ and Mlul, and re-ligated with a similar Bgiπ Mlul DNA fragment containing the hAAT promoter and adjacent intron isolated from the plasmid phAAT-H3L6.
All plasmid DNAs were transfected into E. coli strains DH5α or JM109 by electroporation. A schematic diagram of the plasmid DNAs is shown in Fig. 1.
Endotoxin free plasmid DNAs used for systemic injection experiments were prepared using either the Wizard PureFection Plasmid DNA purification system (Promega), or the Endotoxin Free Maxi plasmid purification Kit (QIAGEN).
Serum biochemistry and enzyme-linked immunosarbent assays (ET.JSA)
Serum alanine a inotransferase (ALT) and aspartate aminotransferase (AST) levels were determined using a Reflotron (Roche) and Reflotron test strip reagents, Normal physiological saline was used to dilute samples with transaminase levels in excess of the assay linear range. Hyper-IL-6 and human IL-6 levels in mouse serum were determined using a Pelikine Compact™ human IL-6 ELISA kit (CLB, Amsterdam), which is specific for human IL-6. Serum HGF levels were determined using a human HGF Cytoscreen Immunoassay Kit (Biosource International, Inc., Camarillo, CA).
Histological, immunohistochenύstry staining and DNA labeling
Paraffin-embedded sections were stained with haematoxylin-eosin (H&E). For immunohistochemical staining, sections were incubated in 0.01 M citrate buffer, pH 6.0, and heated by microwave (750 W) to boiling for 2 minutes and cooked at 20% frill power for a further 20 minutes. The samples were left in the heated buffer for 20 minutes, then rinsed with distilled water and blocked with a solution of 1% BSA in PBS containing 0.5% Triton X-100. The sections were stained with an anti-PCNA (PC10) mouse monoclonal antibody (Santa Cniz Biotechnology, Santa Cruz, CA) and detected using the EnVision™+ HRP.Mouse (DAKO, Copenhagen) kit developed with 3-amino-9-ethylcarbazole (AEC), and counter-stained with haematoxylin, Hepatocyte mitotic activity was assayed by scoring PCNA positive nuclei in immunohistochemically stained liver sections in twenty randomly selected, uninterrupted microscopic fields, at a magnification of x400. Twenty fields were
estimated to contain on average 4140 hepatocytes. Labeling of SI phase hepatic nuclei with 5-bromo-2'-deoxyuridine (BrdU) was performed by injection (i.p.) of mice twice daily with 1,25 micro-gram BrdU (Sigma) in PBS, with the last injection administered 2 hours before sacrificing. Immunohistochemical staining for BrdU in liver sections was performed using a BrdU Cell proliferation kit (Amersham Pharmacia Biotech, UK).
RESULTS
Extended gplSO hyper-stimulation is not mitogenic in the liver.
Three different plasmid DNAs encoding the hyper-IL-6 gene under the transcriptional control of three different promoters were constructed and tested for the ability to express the hyper-IL-6 in vivo using the hydrodynamics-based procedure for transgene expression in mice by systemic administration of plasmid DNA (19). The plasmid constructs (Fig. 1) were identical in all respects with the exception of the promoters, which were selected in order to achieve varying levels and duration of hyρer-IL-6 expression in vivo. The promoters tested were the cytomegalovirus immediate-early promoter (CMV), the SV40 early promoter (SV40), and the fiver specific human αi-antitrypsin gene promoter (hAAT). Following rapid systemic injection of the plasmid DNAs into the mice, substantial but transient expression of hyper-IL-6 protein was detected in the serum by human IL-6 ELISA showing distinct patterns of the hyper-IL-6 transgene expression which depended on the promoter utilized (Fig. 2).
Maximum serum hyper-IL-6 levels were observed on day 1 following injection, regardless of the promoter; however the pattern of expression differed significantly thereafter. The CMV promoter driven gene construct (pCI-HIL6) produced the highest levels of hyper-IL-6 protein (-7000 pg ml), followed by the hAAT promoter (-1200 pg ml), and the SV40 promoter (-400 pg/ml). The duration of transgene expression was also significantly different. The two viral derived promoters produced maximum hyper-IL-6 levels on day 1 that diminished by 1.5 to 2 orders of magnitude by day 2, and were nearly undetectable by day 6. In contrast, serum hyper-IL-6 levels produced by phAAT-HEL6 transfection declined by only one-half between days 1 and 3, and one-tenth of maximal levels by day 6. It has been observed that following partial hepatectomy in mice, the expression levels of endogenous TL-6 reach approximately 3,500 pg/ml (21). Because the hyper-IL-6 protein is a super-agonist of gpl30, that is 100 to 1000 fold more potent than IL-
6, the cytokine levels produced by phAAT-HIL6 transfection, at least initially following transfection, are significantly above equivalent IL-6 physiological levels.
High level hyper-IL-6 expression is lethal in transfected mice ivith no evidence of liver damage
Systemic injection of 10 micro-gram pCl-HIL6 to the mice led to serum hyper-IL-6 levels exceeding 1000 ng'ml. As a result of this extremely high level of expression, the treated animals displayed considerable morbidity for a period of 1 to 2 days manifest by cessation of eating and grooming behavior and the assumption of immobile hunched postures, followed by mortality that reached 100 percent in most experiments. However, transfection of mice with 5 micro-gram pCI-HIL6, or up to 20 micro-gram of phAAT-HIL6 or ρSI-HIL6, which produced substantially lower serum hyper-IL-6 levels, did not produce any apparent morbidity.
In order to understand the nature of this lethal effect in the mice, tissue samples, including liver, kidney, heart, lung, pancreas, spleen, and brain, were removed from mice that were either moribund or deceased by day 2 post transfection, and subjected to bistopathological analysis, The results of this analysis showed that, surprisingly, with the exception of the spleens, all tissues examined displayed normal morphological structure (data not shown). The spleens of most of the treated mice were reactive, with significant macrophage infiltration evident. However, no evidence of necrosis was observed in any of the tissue specimens examined. In addition, serum biochemistries from the moribund mice demonstrated normal ALT and glucose levels, confirming the absence of liver and pancreatic injury.
gpl30 hyper-sύmulation is not mitogenic in normal mice
In order to assess the mitogenic potential of hyper-IL-6 mediated g l30 stimulation on normal hepatocytes in vivo, mice were treated by systemic injection with pCT-HIL6 (5 micro-gram), ρhAAT-HIL6 (10 micro-gram), or control plasmid DNA (pGEM) and analyzed 3 to 9 days later for increased hepatocyte mitotic activity as indicated by either BrdU incorporation, or by expression of proliferating cell nuclear antigen (PCNA), Immunohistochemical staining for PCNA in liver sections from mice sacrificed 2 days post transfection indicated that expression of hyper-IL-6 had at best only a mild mitotic effect in comparison to injection of control plasmid (Table H and Figs. 3 & 4a). Furthermore, this
effect was not consistently evident between experiments, suggesting that factors other than hyper-IL-6 expression may be involved. Even following lethal doses of ρCI-HΪL6, with serum hyper-TL-6 levels in excess of 1000 ng/ml, no substantial hepatocyte mitotic activity was evident by PCNA staining (data not shown). In contrast, on day 2 following systemic injection of a plasmid DNA encoding a hAAT promoter driven HGF gene (phAAT-HGF), which directed expression of serum human HGF levels of approximately 284±95 pg/mL, 24 hours following transfection, produced substantial mitotic activity in liver hepatocytes was evident by PCNA staining (Figs. 3 & 4c). To eliminate the possibility that the mitogenic effect of gpl30 hyper-stimulation may be a delayed effect, plasmid DNA transfected mice were administered BrdU by twice daily injections over a period of 9 days and assessed for BrdU incorporation by immunohistochemical staining of liver sections. However, in this analysis as well, no substantial mitotic activity was observed in mice expressing hyper-IL-6 (data not shown). Thus, no evidence of substantially increased hepatocyte mitotic activity resulting from hyper-IL-6 expression was observed regardless of the level or duration of expression.
Hyper-EL-6 cooperates synergistically with HGF to induce hepatocyte mitosis in vivo Without wishing to be limited by a single hypothesis, these results suggest that previous observations by the present inventors, of the ability of hyper-IL-6, but not IL-6 treatment, to enhance hepatocyte replication and liver regeneration following D-gal induced liver damage (15), may have been due to the combined effects of gρl30 hyper-stimulation together with endogenous factors that are naturally induced or released upon induction of liver injury. TNF-α and HGF are released during the process of D-gal induced liver injury, and have been shown to have substantial roles in the cytokine mediated mechanism of liver regeneration (2), HGF is the most potent known itogen of hepatocytes in vitro and is able to induce liver proliferation as well (1, 2, 21). TNF-α, hile being a major initiator of programmed cell death mediated liver injury, is not mitogenic by itself, but rather functions as a priming agent to stimulate hepatocyte proliferation by growth factors such as HGF, at the start liver regeneration (3, 22). To test whether hyρer-IL-6 mediated gpl30 stimulation may cooperate with either of these factors to stimulate hepatocyte proliferation, mice were treated with hyper-IL-6, HGF or TNF-α either alone or in combinations of two.
In these experiments, the results of which are shown in Figs, 3 & 4, both hyper-IL-6 and HGF were administered as transgenes expressed under the control of the hAAT promoter, and TNF-α was administered by a single intraperitoneal injection of recombiπant protein following systemic injection of control plasmid DNA, PCNA immunohistochemical analysis of liver sections obtained from the treated mice on day 2 post transfection showed that, as expected, TNF-α and hyper-IL-6 treatments induced a small and inconsistent mitotic effect ranging from 0-1.5%, above the background index of PCNA positive nuclei observed in control plasmid DNA treated mice. As expected, HGF treatment alone raised the labeling index to approximately 4% A combined treatment of TNF-α with HIL-6 also increased the labeling index to approximately 4%, equivalent to that of HGF alone. It should be noted that the effective treatment of HΪL-6 with TNF-alpha, administered as a protein, or with HGF, administered as a plasmid, clearly indicates the utility and efficacy of administering these Substances as proteins or as plasmids,
However, treatment with HGF combined with either hyper-IL-6 or TNF-α raised the labeling index even further to 9% and 12%, respectively. Positively stained hepatocytes, regardless of treatment, tended to be located in the central lobular regions, close to the central vein rather than the triad. As shown by the histopathological analysis in the H&E Stained panels (Fig. 4), these treatments did not produce any damage to the liver parenchyma
HGF induced mitosis is not enhanced by IL-6 treatment
In order to determine whether the synergistic mitogenic effect of combined HGF and hyper-IL-6 treatment was dependent on the presence of the sIL-6R moiety of the hyper-IL-6 fusion protein, a control experiment was performed to compare the mitogenic effect of HGF in the presence or absence of TL-6 or hyper-IL-6 (Fig. 5), As in previous experiments, the cytokines were administered to mice via high-pressure plasmid DNA transfection to the liver, The results of this analysis revealed that while hyper-IL-6 treatment significantly enhanced the mitogenic effect of HGF treatment alone, IL-6 treatment had no apparent effect on the mitogenic effect of HGF alone As in previous experiments, neither hyper-IL-6 nor IL-6 plasmid treatments significantly increased hepatocyte mitosis above background levels induced by injection of the control plasmid DNA, which in itself was similar to that observed in untreated animals,
METHODS AND COMPOSITIONS FOR TREATING A PATHOLOGICAL CONDITION IN A SUBJECT
The present invention also includes methods of use of the compositions of the present invention for treating, e.g., inhibiting, preventing or delaying the onset of a pathological condition in a subject, by administering a composition according to the present invention to the subject. The subject may optionally and preferably be mammalian, e.g., a human, a rodent such as a mouse or rat, or a dog or cat,
A preferred example of such a condition is a liver-related condition, including but not related to, liver disease, such as fibrosis and/or cirrhosis for example; and fiver failure, including acute and fulminant hepatic failure for example.
Another example of a pathological condition of a subject which may be treated with the compositions of the present invention is treatment of kidney disease, as previously described (see Kawaida et al (1994) (23)). HGF exerts both mitogenic and morphogenic affects on renal tubular epithelial cells and blood HGF levels are markedly induced following unilateral nephrectomy and acute renal failure. As such, it has been suggested that HGF treatment may to be an effective treatment for patients with renal dysfunction (23). The present invention extends this suggestion for treatment with the compositions of the present invention, as such compositions have been shown to have greater (synergistic) efficacy in comparison to the effect of the individual components alone, In addition or alternatively, the present invention may optionally be used for enhancement of gene delivery in gene therapy protocols involving viral vectors requiring or augmented by hepatocyte proliferation. Suitable examples of such protocols would include retroviral vector and lentiviral vector based gene therapy.
Methods of administration of compositions according to the present invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, vaginal, rectal, parenteral and oral routes. The compositions of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal rnucosa, etc.) and may optionally be administered together with other biologically-active agents. Administration can optionally be systemic or local. In addition, it may be advantageous to administer the composition according to the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be
facilitated by an intraventricular catheter attached to a reservoir (e.g., an Ommaya reservoir). Intra-arterial infusion to the fiver may optionally be employed, for example by infusion pump, intravenous drip, or intraperitoneal, subcutaneous or intramuscular injection. Pulmonary administration may also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
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. 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.
Formulations for parenteral administration may include but are not limited to sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. It may also optionally, additionally or alternatively, be desirable to administer the composition according to the present invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, In a specific embodiment, administration may be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
Various delivery systems arc known and can be used to administer a composition according to the present invention including but not limited to, e.g.: (j) encapsulation in liposomes, microparticles, microcapsules; (ii) recombinant cells capable of expressing at least the cytokine-related components of the composition; (iii) receptor-mediated endocytosis (See, e.g., Wu and Wu, 1987, J Biol Chem 262:4429-4432); (iv) construction of a nucleic acid as part of a retroviral or other vector, and the like.
In one embodiment of the present invention, the composition may be delivered in a vesicle, in particular a liposome. In a liposome, the protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin,
phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill b the art, as disclosed, for example, in U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which are incorporated herein by reference.
In yet another embodiment, the composition according to the present invention may optionally be delivered in a controlled release system including, e.g.: a delivery pump (See, e.g., Saudek, et al., 1989, NewBnglJMed 321:574 and a semi-permeable polymeric material (See, e.g., Howard, et al, 1989, J Neurosurg 71:105). Additionally or alternatively, the controlled release system may optionally be placed in proximity of the therapeutic target (e.g., the brain), thus requiring only a fraction of the systemic dose. See, e.g., Goodson, In: Medical Applications of Controlled Release 1984. (CRC Press, BoccaRatoη, FL).
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition according to the present invention that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amehoration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions, When applied to an individual active ingredient, administered alone, the terra refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. The amount of the composition according to the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques by those of average skill within the art, In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
Ultimately, the attending physician decides the amount of the composition according to the present invention with which to treat each individual patient (subject). Initially, the attending physician may optionally administer low doses of the composition according to the present invention and observe the patient's response. Larger doses of the composition according to the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. Effective doses
may be extrapolated from dose-response curves derived from in vitro or animal model test systems,
The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
The methods of the present invention are useful for the treatment of injury to the liver and to the kidney, 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.
The method includes the step of administering the composition in a pharmaceutically acceptable carrier as described above, to a subject to be treated, The composition is administered according to an effective dosing methodology, preferably until a predefined endpoint is reached, which could optionally 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. Of course, another endpoint or endpoint(s) could be used in addition to, or in place of, one or more of the previous endpoint(s), In a preferred embodiment of the method of the present invention, the composition 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.
Cells may also be cultured ex vivo (in vitro) in the presence of component(s) of the composition according to the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then optionally be introduced in vivo for therapeutic purposes.
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.
Tables
Table 1: Plasmid DNAs, Sources and Properties.
References Cited
1. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997; 276:60-6.
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8. Camargo CA, Jr., Madden JF, Gao W, Selvan RS, Clavien PA. Intcrleukin-6 protects liver against warm ischemia reperfusion injury and promotes hepatocyte proliferation in the rodent. Hepatology 1997; 26 : 1513-20.
9. Mizuhara H, O'Neill E, Seki N, Ogawa T, Kusunold C, Otsuka K, Satoh S, Niwa M, Senoh H, Fujiwara H. T eel] activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6. JExp Med 1 94; 179 :1529-37. 10. Kovalovich K, Li W, DeAngelis R, Greenbau LE, Ciliberto G, Taub R.
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14. Peters M, Blinn G, Solem F, Fischer M, Meyer zu Buschenfelde KH, Rose- John S. In vivo and in vitro activities of the gpl 30-stimulating designer cytokine Hyper-IL-6. J Immunol 1998; 161 :3575-81. 15. Galun E, Zcira E, Pappo 0, Peters M, Rose- John S. Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury. Faseb J 2000; 14 :1979-87.
16. Peters M, Blinn G, Jostock T, Schumacher P, Meyer Zum Buschenfelde KH, Galle PR, Rose- John S. Combined Interlcukiπ 6 and Soluble Interleukin 6 Receptor Accelerates Murine Liver Regeneration. Gastroenterology 2000; 119
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18. Maione D, Di Carlo E, Li W, Musiani P, Modesti A, Peters M, Rose-John S, Delia Rocca C, Tripodi M, Lazzaro D, Taub R, Savino R, Ciliberto G, Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver. EmboJ 1998; 17 :5588-97. 19. Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid D A. Gene Ther 1999; 6 :1258-66. 20. Zhang G, Budker V, Wolff JA. High l vels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DΝΛ. Hum Gene Ther 1999; 10 :1735-7. 21. Patijn GA, Lieber A, Schowalter DB, Schwall R, Kay MA. Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral- mediated gene transfer in mice. Hepatology 1998; 28 :707-1 .
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Claims
1. A therapeutic composition, comprising:
(a) an TL-6 family combination component; and
(b) a liver regenerating factor.
2. The composition of claim 1, wherein said combination component comprises a IL-6/slL-6R complex.
3. The composition of claim 2, wherein said IL-6/sTL-6R complex comprises a bimolecular protein complex featuring at least a portion of both IL-6 and IL-6 receptor protein.
4. The composition of claim 2, wherein said !L-6/sIL-6R complex comprises a unimolecular protein comprising bioactive portions of IL-6 and sIL-6R connected with a flexible linker.
5. The composition of claim 4, wherein said lL-6/sTL-6R complex comprises Hyper-IL-6.
6. The composition of claim 1, wherein said combination component comprises a combmation of at least one of at least a portion of each of IL-11 (interleukin 11) and its respective receptor, OSM (oncostatin-M) and its respective receptor; CT-1 (cardiotrophin-1) and its respective receptor; LIF (leukemia inhibitory factor) and its respective receptor; NT-t (neurotrophin-1/B cell stimulating factor 3) and its respective receptor; or CNTF (ciliary ncurotrophic factor) and its respective receptor.
1, The composition of any of claims 1-6, wherein said liver regenerating factor comprises a growth factor.
8. The composition of claim 7, wherein said grovrth factor comprises I IGF (hepatocyte growth factor), EGF (epidermal growth factor) and TGF-alpha (transforming growth factor alpha) or an engineered derivative thereof.
9. The composition of any of claims 1-6, wherein said liver regenerating factor comprises ITsIF-alpha (tumor necrosis factor alpha) or an engineered derivative thereof.
10. The composition of claim 1 , wherein said combination component comprises a IL-6/sIL-6R complex and said liver regenerating factor comprises HGF.
11. The composition of claim 1, wherein said combination component comprises a IL-6/sIL-6R complex and said liver regenerating factor comprises TNF-alpha.
12. The composition of claim 1, wherein said combination component comprises Hyper-IL-6 and said liver regenerating factor comprises at least one of HGF and TNF-alpba.
13. The composition of any of claims 1-12, further comprising a pharmaceutically acceptable carrier.
14. The composition of any of claims 1-13, wherein said combination component and said liver regenerating factor are present in therapeutically effective amounts.
15. A method for treating an injury to a liver of a subject, comprising administering, to the subject, a pharmaceutically acceptable amoimt of a therapeutic composition in a pharmaceutically acceptable carrier, said therapeutic composition comprising:
(a) an TI^-6 family combination component; and
(b) a liver regenerating factor; such that the injury to the liver is treated.
16. The method of claim 1 , wherein said combination component comprises a IL-6/sIL-6R complex.
1 . The method of claim 1 , wherein said IL-6/sTL-6R complex comprises a bimolecular protein complex featuring at least a portion of both IL-6 and IL-6 receptor protein.
18. The method of claim 1 , wherein said IL-6/sIL-6R complex comprises a unimolecular protein comprising bioactive portions of IL-6 and sIL-6R connected with a flexible linker.
19. The method of claim 18, wherein said IL-6/sIL-6R complex comprises Hyper-IL-6.
20. The method of claim 15, wherein said combination component comprises a combination of at least one of at least a portion of each or IL-11 (interleukin 11) and its respective receptor; OSM (oncostatin-M) and its respective receptor; CT-1 (cardiotroρhin-1 ) and its respective receptor; LIF (leukemia inhibitory factor) and Its respective receptor; NT-1 (neur lrophin-1/B cell stimulating factor 3) and its respective receptor; or CNTF (ciliary neurotrophic factor) and its respective receptor.
21. The method of any of claims 15-20*, wherein said liver regenerating factor comprises a growth factor.
22. The method of claim 21, wherein said growth factor comprises HGF (hepatocyte growth factor), EGF (epidermal growth factor) and TGF-alpha (transforming growth factor alpha) or an engineered derivative thereof.
23. The method of any of claims 15-22, wherein said liver regenerating factor comprises TNF-alpha (tumor necrosis factor alpha) or an engineered derivative thereof.
24. The method of claim 15, wherein said combination component comprises a lL-6/sIL-6R complex and said liver regenerating factor comprises HGF.
25. The method of clai 15, wherein said combination component comprises a IL-6/sIL-6R complex and said liver regenerating factor comprises TNF-alpha.
26. The method of claim 15, wherein said combmation component comprises Hyper-IL"6 and said liver regenerating factor comprises at least one of HGF and TNF-alpha.
27. The method of any of claims 15-26, wherein said therapeutic composition is administered to the subject parenterally.
28 The method of any of claims 15-27, wherein the injury to the liver is selected from the group consisting of reduction of liver function from a normal level caused by a toxic substance, reduction of liver function from a normal level caused by mechanical trauma, reduction of liver function from a normal level caused by a malignancy, and reduction of liver function from a normal level caused by a pathogen.
29. The method of claim 28, wherein said reduction of liver function from a normal level caused by said toxic substance includes alcoholic hepatitis and drug induced hepatopathy.
30. The method of claim 28, wherein the injury to the liver is selected from the group consisting of acute liver failure and chronic liver failure.
31. The method of any of claims 15-30, wherein said therapeutic composition is administered to the subject at least one of before, during or after liver transplantation, or a combination thereof.
32. A method for treating a subject receiving a liver transplant, the method comprising administering, to the subject, a pharmaceutically acceptable amomit of a therapeutic composition in a pharmaceutically acceptable carrier, said therapeutic composition comprising:
(a) an IL-6 family combination component; and
(b) a liver regenerating factor; wherein said therapeutic composition is administered to the subject at least one of before, during or after liver transplantation, or a combination thereof.
33. A method for treating a pathological condition of a subject, comprising administering, to the subject, a pharmaceutically acceptable amount of a therapeutic composition in a pharmaceutically acceptable carrier, said therapeutic composition comprising:
(a) an _Q 6 family combination component; and
(b) a liver regenerating factor; wherein at least one of said IL-6 family combmation component and said liver regenerating factor is administered encoded by a plasmid, said plasmid being administered to the subject; such that the pathological condition is treated.
34. The method of claim 33, wherein said therapeutic composition is delivered to the subject through a gene therapy protocol.
35. The method of claim 34, wherein said gene therapy protocol features the delivery of any nucleic acid polymer.
36. The method of claims 34 or 35, wherein the composition is used for enhancement of gene delivery in a gene therapy protocol involving viral vectors requiring or augmented by hepatocyte proliferation.
37. The method of any of claims 34-36, wherein said protocol is rctroviral vector or lentivirai vector based gene therapy.
38. The method of any of claims 33-37, wherein the pathological condition is a liver-rclatcd condition.
39. The method of claim 38, wherein said liver-related condition is at least one of liver disease, and liver failure.
40. The method of claim 39, wherein said liver disease includes at least one of liver fibrosis or cirrhosis.
41. The method of claim 40, wherein said liver failure is aeute or fulminant hepatic failure.
42. The method of any of claims 33-37, wherein the pathological condition is a kidney disease.
43. The method of claim 42, wherein said kidney disease includes at least one of renal failure; the effect of damaging substances on renal tissue and functions; and loss of renal function following surgery.
44. The method of any of claims 33-37, wherein the pathological condition is treated through regeneration of a tissue in the subject.
45. Use of a composition for inducement of tissue growth and/or renewal in vitro, the composition comprising:
(a) an IL-6 family combination component; and
(b) a liver regenerating factor.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US32520701P | 2001-09-28 | 2001-09-28 | |
| US60/325,207 | 2001-09-28 |
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| Publication Number | Publication Date |
|---|---|
| WO2003029281A1 true WO2003029281A1 (en) | 2003-04-10 |
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ID=23266895
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2002/028150 WO2003029281A1 (en) | 2001-09-28 | 2002-09-30 | An sil-6r/il-6 fusion protein cooperates synergistically with hgf to stimulate hepatocyte proliferation in vivo |
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| Country | Link |
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| WO (1) | WO2003029281A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008546681A (en) * | 2005-06-16 | 2008-12-25 | ハダジット メディカル リサーチ サービシズ アンド ディベラップメント リミテッド | Methods for treating renal failure |
| US8372389B2 (en) | 2007-11-28 | 2013-02-12 | Hadasit Medical Research Services And Development Ltd. | Methods for the treatment of radiation or chemotherapy-induced tissue damage |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919763A (en) * | 1998-06-01 | 1999-07-06 | Hadasit Medical Research Services And Development Company Ltd. | IL-6/SIL-6R complex for promotion of liver functions |
-
2002
- 2002-09-30 WO PCT/US2002/028150 patent/WO2003029281A1/en not_active Application Discontinuation
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919763A (en) * | 1998-06-01 | 1999-07-06 | Hadasit Medical Research Services And Development Company Ltd. | IL-6/SIL-6R complex for promotion of liver functions |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008546681A (en) * | 2005-06-16 | 2008-12-25 | ハダジット メディカル リサーチ サービシズ アンド ディベラップメント リミテッド | Methods for treating renal failure |
| US7897145B2 (en) | 2005-06-16 | 2011-03-01 | Hadasit Medical Research Services And Development Ltd. | Methods for the treatment of renal failure |
| EP2335714A1 (en) | 2005-06-16 | 2011-06-22 | Hadasit Medical Research Services And Development Ltd. | A complex comprising IL-6 and soluble IL-6 receptor for use in the treatment of renal failure |
| US8372389B2 (en) | 2007-11-28 | 2013-02-12 | Hadasit Medical Research Services And Development Ltd. | Methods for the treatment of radiation or chemotherapy-induced tissue damage |
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