Title:  Treatment of liver disease and injury with CXC chemokines

United States Patent:  6,719,969

Issued:  April 13, 2004

Inventors:  Hogaboam; Cory M. (Ann Arbor, MI); Bone-Larson; Cynthia L. (Petersburg, MI); Simpson; Kenneth J. (Edinburgh, GB); Lukacs; Nicholas W. (Brighton, MI); Kunkel; Steven L. (Ann Arbor, MI); Colletti; Lisa M. (Webberville, MI); Strieter; Robert M. (Sherman Oaks, CA)

Assignee:  The Regents of The University of Michigan (Ann Arbor, MI); University Court of The University of Edinburgh (GB)

Appl. No.:  632531

Filed:  August 4, 2000

Abstract

Disclosed is the surprising discovery that CXC chemokines function to induce rapid hepatocyte proliferation and liver regeneration after injury. The invention thus provides a range of compositions and methods for use in treating liver injury, including those suitable for treating acetaminophen overdose outside the therapeutic window for N-acetyl-cysteine treatment, and those that limit the hepatotoxic side-effects of gene therapy regimens.

SUMMARY OF THE INVENTION

The present invention overcomes the foregoing and other drawbacks inherent in the prior art by providing a range of new treatments by which to induce liver regeneration and repair. The invention is broadly based upon the surprising finding that CXC chemokines induce rapid hepatocyte proliferation in vitro and stimulate liver repair and regeneration in vivo. Particularly advantageous uses of the invention are in the treatment and/or prevention of liver injury caused by acetaminophen overdose and associated with gene therapy.

The invention is generally based on the use of components or agents, preferably CXC chemokines or CXC chemokine components, that activate or upregulate the CXC chemokine receptor 1 (CXCR1), or more preferably, that activate or upregulate the CXC chemokine receptor 2 (CXCR2). As used herein, the terms "CXC chemokines and CXC chemokine components" are used generically to indicate that the CXC chemokine may be a protein or nucleic acid that encodes a protein.

Therefore, unless otherwise specifically stated, all CXC chemokine biological agents are included within the term "CXC chemokine". Although human CXC chemokines will be preferred for use in human therapy, CXC chemokines from any species may be used in the invention. For use in other animal species, such as in veterinary embodiments, a species of CXC chemokine matched to the animal being treated will be preferred.

A CXC chemokine "protein", as used herein, refers to a proteinaceous CXC chemokine component that has sufficient biological activity to be biologically effective. Accordingly, "CXC chemokine proteins" include full-length CXC chemokine proteins and polypeptides, including those prior to natural biological processing and, preferably, those subsequent to the type of biological processing that occurs naturally.

CXC chemokine proteins and polypeptides also include CXC chemokine proteins and polypeptides that have been subject to non-native processing or biological modification. Such modifications include truncations, extensions, active domains or fragments, fusion proteins, mutants with substantial or sufficient biological activity, peptidomimetics and the like.

Any form of CXC chemokine protein may be used in the invention, including those isolated from natural sources. CXC chemokines prepared by recombinant expression will often be preferred, i.e., those obtained by expressing a CXC chemokine nucleic acid in a recombinant host cell and collecting the expressed CXC chemokine protein. Further preferred CXC chemokine proteins are those prepared by automated peptide synthesis.

"CXC chemokine nucleic acids" are DNA or RNA coding regions that encode, and under conditions appropriate for expression, encode and express any one or more of the biological active CXC chemokine protein- and polypeptide-based components described above, including full-length proteins and polypeptides, and active variants, fragments and fusions thereof. Recombinant vectors, viral vectors and recombinant viruses are preferred for use in various embodiments, as described in detail herein.

A component "that activates or upregulates" the CXC chemokine receptor 1 (CXCR1), or more preferably, the CXC chemokine receptor 2 (CXCR2), is a chemical or biological component or agent that stimulates cell signaling via the CXCR1 or CXCR2 receptor. "Cell signaling" via the CXCR1 or CXCR2 receptor is indicated by the capacity to "transduce" a signal, i.e., transmit a biological effect, to the intracellular environment by binding of an agent to an extracellular portion of the receptor.

Agents that "stimulate" cell signaling via these receptors may do so directly or indirectly. Preferred agents that directly stimulate or "activate" such receptors, preferably the CXCR2 receptor, are generally the biological ligand counterparts to the receptor. Therefore, ELR-containing CXC chemokines will be preferred for directly activating the CXCR2 receptor, as such components are known to bind to and stimulate this receptor in the natural environment.

The term an "ELR-containing CXC chemokine" means a CXC chemokine that includes the ELR motif, i.e., the amino acid sequence ELR (Glu Leu Arg). The ELR sequence is important in defining the receptor binding and biological properties of these chemokines.

Exemplary ELR-containing CXC chemokines for use in all aspects of the invention are MIP-2 (macrophage inflammatory protein-2), ENA-78 (epithelial neutrophil activating protein-78), IL-8 (interleukin-8), GCP-2 (granulocyte chemotactic protein-2), NAP-2 (neutrophil activating peptide-2), CTAP-III (connective tissue activating protein-III), .beta.TG (.beta.-thromboglobulin) and the GRO (growth related oncogene peptide) chemokines, such as GRO-.alpha., GRO-.beta. and GRO-.gamma.. The amino acid and nucleic acid sequences of all such ELR CXC chemokines are known to those of ordinary skill in the art and are further disclosed herein. Certain preferred ELR-containing CXC chemokines for use in the invention are MIP-2, ENA-78 and IL-8.

Where "indirect" stimulation or activation of these receptors, preferably the CXCR2 receptor, is concerned, agents other than biological ligand counterparts will be effective. Such agents are capable of increasing cell signaling via the CXCR2 receptor without themselves binding to the receptor. Accordingly, these components include accessory and accessory signaling molecules, co-stimulators and the like, and agents that remove, inactivate or downregulate inhibitors.

Preferred components that indirectly stimulate the CXCR2 receptor are agents that stimulate or "upregulate" the expression of the CXCR2 receptor. Such components will therefore increase the amount of the receptor expressed at the cell surface and available for binding to the natural biological ligand counterpart, i.e., the "ELR-containing CXC chemokine". Agents that preferentially or specifically upregulate CXCR2 receptor expression are preferred for use in the invention.

The use of agents that specifically upregulate the CXCR2 receptor is exemplified by the surprising use of the non-ELR CXC chemokine, IP-10 (.gamma.-interferon-inducible protein-10). The amino acid and nucleic acid sequences of this component are again known to those of ordinary skill in the art and are further disclosed herein.

In light of the present discoveries, CXC chemokines, preferably ELR CXC chemokines and the non-ELR CXC chemokine, IP-10, may be used in all in vitro and in vivo methods of stimulating hepatocytes and promoting hepatocytes proliferation. All that is required is to contact a composition comprising hepatocytes with a biologically effective amount of at least a first composition comprising at least a first CXC chemokine component that activates or upregulates the CXC chemokine receptor 2 (CXCR2).

Such methods and uses include the addition of the CXC chemokine composition to hepatocytes in vitro. Accordingly, the invention provides methods and uses in culturing hepatocytes in vitro and in generating artificial liver tissue ex vivo. The methods and uses generally comprise providing a biologically effective amount of at least a first composition comprising at least a first CXC chemokine that activates or upregulates the CXCR2 receptor to an in vitro or ex vivo biological sample that contains a population of hepatocytes.

Preferred methods, uses and medicaments of the invention are those in which the CXC chemokine compositions are provided to hepatocytes in vivo, simply by administering the composition to an animal or patient. The invention thus provides methods and uses of inducing liver growth, stimulating hepatic regeneration and, generally, treating animals and patients with various forms of liver damage and disease.

These methods and uses of the invention comprise providing to an animal or patient at least a first composition that comprises at least a first CXC chemokine that activates or upregulates the CXCR2 receptor. The CXC chemokines are provided in amounts effective to promote hepatocyte proliferation, induce liver growth, stimulate hepatic regeneration and/or to generally treat or prevent liver damage, diseases and/or disorders in the animal or patient. This is the meaning of the terms "biologically and therapeutically effective amounts", as used herein, i.e., amounts effective to promote hepatocyte proliferation, induce liver growth, stimulate hepatic regeneration and/or treat or prevent liver damage when administered to an animal or patient.

The in vivo treatment methods of the invention generally require the administration of pharmaceutically or pharmacologically acceptable formulations of CXC chemokine proteins, nucleic acids, vectors and/or recombinant viruses. Systemic administration, including intravenous administration, is suitable for use in the invention. More localized delivery to the liver is also contemplated, including all forms of intra-hepatic administration.

Where protein administration is concerned, the invention contemplates that the CXC chemokine proteins will be administered to animals or patients in doses of between about 1 and about 500 .mu.g/kg body weight; preferably between about 20 and about 400 .mu.g/kg body weight, preferably between about 50 and about 350 .mu.g/kg body weight, and more preferably, between about 100 and 250 .mu.g/kg body weight, such as at about 200 .mu.g/kg body weight.

All intermediate ranges are included, such as 1 to 10, 20, 50, 100, 200, 300, 400 and 500 .mu.g/kg body weight; 20 to 50, 100, 200, 300, 400 and 500 .mu.g/kg body weight; 50 to 100, 200, 300, 400 and 500 .mu.g/kg body weight; 100 to 200, 300, 400 and 500 .mu.g/kg body weight; 200 to 300, 400 and 500 .mu.g/kg body weight, and such like.

In fact, all doses themselves are included, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 and 500 .mu.g/kg body weight, and such like.

The therapeutic aspects of the invention include methods and uses employing at least a first CXC chemokine nucleic acid that expresses a CXC chemokine protein. In these embodiments, the CXC chemokines are preferably, although not exclusively, expressed in cells within or proximal to the liver of the animal or patient, including the hepatocytes themselves.

Recombinant vectors that include one or more exogenous promoters to express the CXC chemokine nucleic acid(s) will generally be preferred for use in these aspects of the invention. Constitutive promoters may be used, generally to obtain high levels of CXC chemokine expression. Exemplary constitutive promoters include SV40, CMV, RSV and ribosomal (RS) promoters.

Alternatively, the use of liver tissue or liver cell-specific or liver tissue or liver cell-preferential promoters may be preferred. Such promoters will specifically or preferentially direct expression of the CXC chemokine in cells within or proximal to the liver, including in hepatocytes. Suitable examples of such promoters include transphyretin, .alpha.1-antitrypsin, plasminogen activator inhibitor type 1 (PAI-1), apolipoprotein AI and LDL receptor gene promoters, each of which specifically or preferentially direct expression in liver cells and tissue.

Using administration via recombinant adenovirus to exemplify the recombinant CXC chemokine delivery aspects of the invention, the CXC chemokine nucleic acids may be provided by administering a recombinant adenovirus to the animal or patient at a dose of between about 1x10⁸ PFU per animal and about 1x10¹² PFU per animal. Those of ordinary skill in the art will understand that the lower doses are preferably administered by infusion into the hepatic artery, whereas higher doses are suitable for systemic administration. Administration of recombinant adenoviruses that comprise at least a first CXC chemokine nucleic acid at doses of about 1x10⁹, 1x10¹⁰ and 1x10¹¹ PFU per animal are also contemplated.

Whether proteins or nucleic acids are employed in the methods and uses of the invention, the CXC chemokines may be used alone or in combination. Accordingly, the invention encompasses the use of one, two, three, four, five, six or more CXC chemokines. The CXC chemokines may also be used in combination with other therapeutic or hepatoproliferative agents. For example, NAC (N-acetyl-cysteine), HGF (hepatocyte growth factor), stem cell factor (SCF), TNF-.alpha. (tumor necrosis factor-.alpha.) and/or IL-6 (interleukin-6) may be used in combination with the present invention.

A wide range of diseases, disorders and conditions associated with liver damage may be treated by the compositions, kits, formulations, methods, uses and medicaments of the invention. These include liver damage associated with exposure to alcohol, hepatotoxic drugs and combinations thereof. Exemplary damaging agents are anticonvulsants, phenytoin, carbamazepine and phenobarbital, and recreations drugs, such as ecstasy (3,4-methylenedioxymethamphetamine).

Side effects resulting from other therapies may also be treated by the invention, including the liver damage associated with exposure to antituberculosis agents and chemotherapeutic agents, such as isoniazid and rifampicin. Liver damage associated with a reduction in viable liver tissue may also be treated, such as occurs after resecting a carcinoma.

Liver damage resulting from or associated with infectious agents may also be counteracted using the present invention. This includes liver damage associated with bacterial, parasitic, fungal and viral infections. For example, liver damage results from Aspergillus fungal infections, Schistosoma parasitic infections and a variety of viral infections, such as adenovirus, retrovirus, adeno-associated virus (AAV), hepatitis virus A, hepatitis virus B, hepatitis virus C, hepatitis virus E, herpes simplex virus (HSV), Epstein-Barr virus (EBV) and paramyxovirus infections. All of which may be treated hereby.

A particularly important use of the present invention is in the treatment or even prevention of liver damage associated with excess acetaminophen (paracetamol) ingestion. This may occur over a prolonged time period, leading to chronic liver damage; or during a short or immediate time period, leading to acute liver damage. The latter embodiments include deliberate and accidental overdoses, including in both adults and children.

The invention therefore provides methods and uses in treating acetaminophen-induced liver damage, which generally comprise administering to an animal or patient with acetaminophen-induced liver damage a biologically effective amount of at least a first composition comprising at least a first CXC chemokine that activates or upregulates the CXC chemokine receptor 2 (CXCR2).

These methods and uses are suitable for treating an acetaminophen overdose, wherein they comprise administering to an animal or patient suffering from an acetaminophen overdose at least a first composition that comprises at least a first CXC chemokine in an amount effective to activate or upregulate the CXC chemokine receptor 2 (CXCR2), thereby promoting liver cell proliferation and liver regeneration in the animal or patient.

Excess acetaminophen ingestion may be effectively treated by the present invention after more time delay than currently available methods, particularly NAC treatment. Accordingly, the invention provides methods and uses for treating an acetaminophen overdose comprising identifying an animal or patient presenting with acetaminophen overdose outside the therapeutic window for NAC treatment and administering to the animal or patient at least a first composition that comprises an amount of at least a first CXC chemokine effective to overcome the acetaminophen overdose.

The importance of the invention is such that the methods and uses include those for avoiding liver transplantation in an acetaminophen overdose animal or patient. These comprise treating the animal or patient with at least a first CXC chemokine that activates or upregulates the CXC chemokine receptor 2 (CXCR2) in a manner effective to regenerate sufficient operative liver tissue so as to render a liver transplant unnecessary.

Treatment of acute liver damage is not limited to that caused by excess acetaminophen ingestion, but is widely applicable. The invention therefore provides methods and uses for treating acute liver damage wherein at least a first dose of the CXC chemokine composition is administered to an animal or patient within a biologically effective time period after onset of the liver damage.

The "biologically effective time periods" after onset of liver damage are exemplified by time periods of between about ten minutes and about 72 hours; preferably, between about half an hour and about 18 hours; and more preferably, between about an hour and about 10, 12 or 15 hours after onset of liver damage. The invention includes all such ranges and particular times.

For example, administration at between about ten minutes and about 1, 2, 3, 5, 8, 10, 12, 15, 18, 24, 36, 48, 60 and 72 hours; between about half an hour and about 1, 2, 3, 5, 8, 10, 12, 15, 18, 24, 36, 48, 60 and 72 hours; between about an hour and about 2, 3, 5, 8, 10, 12, 15, 18, 24, 36, 48, 60 and 72 hours; between about 2 hours and about 3, 5, 8, 10, 12, 15, 18, 24, 36, 48, 60 and 72 hours; between about 5 hours and about 10, 12, 15, 18, 24, 36, 48, 60 and 72 hours; between about 10 hours and about 12, 15, 18, 24, 36, 48, 60 and 72 hours; between about 11 or 12 hours and about 15, 18, 24, 36, 48, 60 and 72 hours; between about 15 hours and about 18, 24, 36, 48, 60 and 72 hours; between about 18 hours and about 24, 36, 48, 60 and 72 hours; between about 24 hours and about 36, 48, 60 and 72 hours; between about 36 hours and about 48, 60 and 72 hours; and between about 48 hours and about 55, 60 and about 72 hours after onset of liver damage.

Rapid administration is desirable, although not essential. The invention therefore includes administration at about 10, 20, 30, 60 or 90 minutes, and at about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours or so after onset of liver damage.

Effective administration at times unsuitable for other therapies is important. These aspects of the invention include administration at about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 39, 30, 31, 32, 33, 34, 35 and 36 hours or so after onset of liver damage.

Administration of at least a first dose of the CXC chemokine composition at a time between about ten hours and about 72 hours after onset of said liver damage is one of these important, although not limiting, features of the present invention as these effective treatment times are outside the effective window of NAC treatment. Administration at any effective time after about 10 hours after onset of liver damage is therefore important. These includes administration at a time of about 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 and 72 hours after onset of liver damage.

The invention thus provides methods and uses for timely treating animals and patients with acute liver damage, comprising administering to the animal or patient a biologically effective amount of at least a first CXC chemokine composition at a time between about ten hours and about 72 hours after the onset of liver damage; wherein the CXC chemokine composition comprises at least a first CXC chemokine that activates or upregulates the CXC chemokine receptor 2 (CXCR2) in the animal or patient.

A further method and use of timely therapeutic intervention in an animal or patient with acute liver failure comprises the steps of:

identifying an animal or patient presenting with acute liver failure outside the therapeutic window for NAC treatment; and

administering to the animal or patient at least a first CXC chemokine composition that comprises an amount of at least a first CXC chemokine effective to overcome acute liver failure; wherein the CXC chemokine activates or upregulates the CXC chemokine receptor 2 (CXCR2) in the animal or patient.

Still further methods and uses of timely therapeutic intervention in an animal or patient with acute liver failure comprise the steps of:

identifying an animal or patient exposed to a liver damaging agent between about ten hours and about 72 hours before intended therapy; and

administering to the animal or patient an amount of at least a first CXC chemokine composition that comprises at least a first CXC chemokine effective to overcome the acute liver failure in the animal or patient by activating or upregulating the CXC chemokine receptor 2 (CXCR2) in the liver of the animal or patient.

The time benefits of the invention are considerable, although not limiting. Accordingly, the invention includes methods and uses for treating chronic liver damage, wherein the CXC chemokine composition is administered to an animal or patient over an extended biologically effective time period. In such treatment, repeated administrations would likely be required, as may be readily achieved by a number of methods, including adenoviral gene therapy. Accordingly, chronic liver damage may be treated by repeated doses of CXC chemokine compositions administered said animal at about 3 day intervals for about 2 months.

In addition to the foregoing general therapeutic embodiments, the methods, uses and medicaments of the present invention that comprise recombinant viruses expressing CXC chemokines are important in connection with treating and/or preventing adenovirus-mediated liver damage, particularly that associated with gene therapy. In such aspects, the invention provides recombinant adenoviruses that comprise at least a first CXC chemokine nucleic acid in combination with a nucleic acid that expresses a therapeutic protein.

The present invention thus encompasses methods and uses for providing a therapeutic nucleic acid to an animal or patient, comprising administering to the animal or patient a single recombinant adenovirus that comprises a first nucleic acid that expresses a therapeutic protein and a second nucleic acid that expresses a CXC chemokine that activates or upregulates the CXC chemokine receptor 2 (CXCR2) in an amount effective to ameliorate hepatotoxic side effects of the recombinant adenovirus.

Such methods of gene therapy further comprise administering to an animal or patient:

a recombinant adenovirus comprising a recombinant nucleic acid that expresses a therapeutic protein in target cells of the animal or patient; and

at least a first composition that comprises at least a first CXC chemokine that activates or upregulates the CXC chemokine receptor 2 (CXCR2) in an amount effective to attenuate hepatotoxic effects of the recombinant adenovirus.

Although CXC chemokine proteins may be used to attenuate hepatotoxic effects of recombinant adenoviruses, the use of CXC chemokine nucleic acids and recombinant viruses is preferred. Recombinant adenoviruses that express CXC chemokines are particularly preferred, especially where the recombinant adenovirus expresses both the CXC chemokine and the therapeutic nucleic acid (or antisense, etc.) of intended therapy.

The invention thus provides methods and uses for preventing or reducing the hepatotoxicity of adenoviral-mediated gene therapy, comprising combining the administration of an adenoviral gene therapy construct with the administration of at least a first composition that comprises at least a first CXC chemokine that activates or upregulates the CXC chemokine receptor 2 (CXCR2) in an amount effective to reduce hepatotoxicity caused by the adenoviral gene therapy construct.

In further embodiments, therapeutic kits are provided. These comprise at least a first recombinant adenovirus that comprises at least a first nucleic acid segment that expresses a therapeutic protein; and a composition comprising at least a first CXC chemokine that activates or upregulates the CXC chemokine receptor 2 (CXCR2) in an amount effective to reduce hepatotoxic effects of the recombinant adenovirus.

The kits may comprise any of the foregoing CXC chemokine proteins, polypeptides, nucleic acid segments and recombinant viruses, including wherein a single recombinant adenovirus expresses at least a first CXC chemokine and a therapeutic RNA or protein. The kits may also comprise at least a first hepatoproliferative agent other than a CXC chemokine.

Recombinant adenovirus compositions form further aspects of the invention. These comprise a recombinant adenovirus that comprises at least a first nucleic acid segment that expresses a therapeutic protein and at least a second nucleic acid segment that expresses a CXC chemokine that activates or upregulates the CXC chemokine receptor 2 (CXCR2). Packing such recombinant adenoviruses gives rise to the gene therapy formulations of the invention.

All such compositions, kits and gene therapy formulations of the invention may comprise any one or more of the CXC chemokine proteins, polypeptides and nucleic acid segments known in the art and described herein, including ELR-containing CXC chemokines, such as MIP-2, ENA-78, IL-8, and the non-ELR-containing CXC chemokine, IP-10, that upregulates CXCR2 receptor expression.

Finally, the invention provides for the use of the compositions in accordance herewith in the preparation of a variety of medicaments for treating one or more conditions associated with liver damage, including chronic and acute liver damage and liver damage associated with exposure to alcohol, surgical intervention, hepatotoxic drugs and infectious agents, including excess acetaminophen ingestion, adenoviral infection and adenoviral-mediated gene therapy.

Claim 1 of 51 Claims

What is claimed is:

1. A method of stimulating hepatocyte proliferation, comprising providing to a hepatocyte at least a first composition comprising at least a first CXC chemokine that activates the CXC chemokine receptor 2 (CXCR2) in an amount effective to stimulate hepatocyte proliferation.

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