The present invention relates to the prevention and treatment of injury and diseases to the liver, biliary tract, bile ducts, gall bladder and related hepatobiliary system. Specifically, the present invention relates to methods for decreasing the action of the RON receptor tyrosine kinase in liver physiology. More specifically, the present invention relates to the use of analogs and antagonists and antibodies for inhibiting the action of the RON receptor tyrosine kinase for the prevention and treatment of liver injury or damage in acute and chronic clinical conditions.

STATEMENT OF THE INVENTION

The present invention provides for the prevention and treatment of injury and diseases to the liver, biliary tract, bile ducts, gall bladder and related hepatobiliary system. Specifically, the present invention relates to methods for decreasing the action of the RON receptor tyrosine kinase in liver physiology. More specifically, the present invention relates to the use of analogs and antagonists, antibodies and nucleic acid modifiers (e.g., ribozymes) for inhibiting the action of the RON receptor tyrosine kinase for the prevention and treatment of liver injury or damage in acute and chronic clinical conditions.

This invention concerns the use of 1) whole or truncated HGFL protein analogs, which are readily available, 2) HGFL blocking antibodies, 3) RON receptor blocking antibodies, 4) peptide fragments based on the sequences of the HGFL or RON receptor obtained from any species, 5) nonspecific or specific RON receptor tyrosine kinase activity inhibitors or 6) Ron receptor or HGFL nucleic acid inhibitors, e.g., single stranded DNA or RNA antisense molecules designed to interfere with the stability or translation of HGFL or RON receptor mRNA, or with the transcription from HGFL or RON receptor genomic DNA sequences, in pharmaceutical compositions for the treatment of any kind of impairment or degeneration of the hepatobiliary system of inflammatory, infectious or drug/toxin-induced origin, including but not limited to pathologic states leading to apoptosis of hepatocytes. The use of these agents, either alone or in conjunction with nonspecific or liver-specific nitric oxide donors, may lead to improved outcomes in patients being treated for these pathologies, as measured by magnitude and/or duration of abnormal aminotransferase elevation, duration of treatment regimens, liver graft function or survival, or mortality.

The present invention is based on the experimental finding that inhibition of the RON tyrosine kinase receptor provides effective protection in an experimental model of acute liver injury, based on assessment of liver tissue necrosis and elevated serum transaminase levels, both indicative of liver damage.

The present invention relates to methods for the prevention or treatment of the progression of liver damage in a patient at risk of developing or having been diagnosed with liver damage comprising administering to the patient a preventatively effective amount of a RON receptor-inhibiting compound. The patient preferably is mammalian, more preferably human.

In another embodiment, the invention relates to a method for the treatment of a patient with a hepatoprotective therapeutic agent effective in the prevention or treatment of a disorder or pathophysiological condition comprising (a) administering to said patient simultaneously or in optional order (1) a biologically effective dose of said therapeutic agent and (2) a preventatively effective amount of RON receptor inhibiting agent and (b) monitoring said patient for indication of liver damage and (c) continuing said treatment until the disorder or condition is eliminated or until liver damage is improved.

In another embodiment, the present invention relates to a method for the prevention of the establishment of liver damage in a patient at risk for developing liver damage comprising administering to said patient a liver damage preventative amount of RON receptor inhibiting compound.

In one embodiment, the RON receptor-inhibiting compound is an HGFL analog. In another embodiment, the RON receptor-blocking compound is a RON receptor tyrosine kinase antagonist. In another embodiment, the RON receptor-blocking agent is an antibody directed towards the RON receptor in which the binding of the antibody with the RON receptor prevents normal activation of the RON receptor.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the RON receptor inhibitor is a protein that comprises an amino acid sequence with least about 65% sequence homology, and more preferably at least about 75% sequence homology with the amino acid sequence coded for by one or more of the oligonucleotide sequences shown in the sequences of SEQ ID NO: 1 and SEQ ID NO: 3. In another embodiment, the RON receptor inhibitor is a protein that comprises an amino acid sequence with least about 65% sequence homology, and more preferably at least about 75% sequence homology with the amino acid sequences shown in the sequences of SEQ ID NO: 2 and SEQ ID NO: 4.

In another embodiment, the RON receptor inhibitor comprises HGFL variants and fragments without substantial HGFL biological activity and RON receptor variants and fragments which interfere with the action of HGFL wherein the inhibitor is a polypeptide comprising an amino acid sequence at least 6 amino acids in length that includes at least 4 contiguous amino acids of a sequence coded for by one or more of the oligonucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3, and retains its abilities as a RON receptor inhibitor. Preferable, the sequence at least 10 amino acids in length, more preferably at least 20 amino acids in length. Preferably, the sequence contains at least 6 contiguous amino acids, more preferably at least 9 contiguous amino acids of a sequence coded for by one or more of the oligonucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3. Generally, the sequence is no more than 200 amino acids in length, preferably no more than 100 amino acids in length.

In another embodiment, the RON receptor inhibitor comprises HGFL variants and fragments without substantial HGFL biological activity and Ron receptor variants and fragments which inhibit HGFL biological activity wherein the inhibitor is a polypeptide comprising an amino acid sequence at least 6 amino acids in length that includes at least 4 contiguous amino acids of a polypeptide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4, which retains its abilities as a RON receptor inhibitor.

In another embodiment, the RON receptor inhibitor comprises HGFL variants and fragments without substantial HGFL biological activity wherein the inhibitor is a polypeptide comprising an amino acid sequence at least 6 amino acids in length that includes at least 4 contiguous amino acids of a sequence coded for by one or more of the mutant HGFL oligonucleotide sequences selected from the group consisting of .DELTA.PAP, .DELTA.K1, .DELTA.K2, .DELTA.K, .DELTA.K4, .DELTA.K1K2, .DELTA.L, K1K2, Glu, Xa, IIa, and 48G, which retains its abilities as a RON receptor inhibitor.

One aspect of the invention pertains to isolated HGFL proteins, and portions thereof, or derivatives, fragments, analogs or homologs thereof which interfere with the RON receptor activity. The present invention also provides for fusion polypeptides, comprising HGFL polypeptides and fragments. Homologous polypeptides may be fusions between two or more HGFL polypeptide sequences or between the sequences of HGFL and a related protein. Fusion partners include immunoglobulins, bacterial beta -galactosidase, trpE, protein A, .beta.-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor.

Fragments provided herein are defined as sequences of at least 4 (contiguous) amino acids, a length sufficient to allow for specific recognition of an epitope and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of an amino acid sequence of choice. Derivatives are amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are amino acid sequences that have a structure similar to, but not identical to, the native compound but differ from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.

Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified amino acid, as described below. Derivatives or analogs of the proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the proteins of the invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80 99%) over an amino acid sequence of identical size.

The HGFL analogs and antagonists useful in the practice of the present invention can be prepared in a number of ways. For instance, the HGFL antagonists can be prepared using an isolated or purified form of HGFL antagonists. Methods of isolating and purifying HGFL antagonists are known in the art. HGFL antagonists can be chemically synthesized and prepared using recombinant DNA techniques known in the art.

The HGFL antagonists may be from human or any non-human species. For instance, a mammal may have administered HGFL fragments from a different mammalian species (e.g., mice can be treated with human HGFL antagonists). There is substantial homology (about 81% amino acid identity) between mouse HGFL and human HGFL, and thus, it is expected that HGFL antagonists from different mammalian species can be employed. Preferably, however, the mammal is treated with homologous HGFL antagonists (e.g., humans are treated with human HGFL antagonists) to avoid potential immune reactions to the HGFL antagonists.

In a preferred embodiment of the invention, the RON receptor antagonists are provided. Non-limiting examples of RON receptor antagonists include antibodies, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like.

In another embodiment of the invention, the RON receptor inhibitors include Ron receptor or HGFL nucleic acid inhibitors. Such Ron receptor or HGFL nucleic acid inhibitors include single stranded DNA or RNA antisense molecules designed to interfere with the stability or translation of HGFL or RON receptor mRNA, or with the transcription from HGFL or RON receptor genomic DNA sequences. Examples of such nucleic acid inhibitors include, but are not limited to, ribozymes (RNA species which serve as sequence-specific molecular scissors) or single-strand DNA species which may selectively inhibit the HGFL- or RON receptor-biological activity. Design of such molecules is familiar to those skilled in the art. (e.g. Bock L. et al. (1992) Nature 355, 564 566).

In another embodiment, the RON receptor antagonists of the invention are RON receptor antibodies. For instance, the antagonistic antibodies may be polyclonal antibodies. Methods of preparing polyclonal antibodies are known to those skilled in the art. One can raise polyclonal antibodies in a mammal, for example, or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins which may be employed include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. An aggregating agent such as alum may also be employed to enhance the mammal's immune response. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). One skilled in the art, without undue experimentation, may select the immunization protocol. The mammal can then be bled, and the serum assayed for RON receptor antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus.

The antagonistic antibodies of the invention may, alternatively, be monoclonal antibodies. Antagonistic monoclonal antibodies of the invention may be prepared using hybridoma methods well known in the art. In a hybridoma method, a mouse or other appropriate host animal, is typically immunized (such as described above) with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent may alternatively comprise a fragment or portion of HGFL or a RON receptor having one or more amino acid residues that participate in the binding of HGFL to its receptor.

Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.

Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, incorporated by reference in its entirety. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for a RON receptor and another antigen-combining site having specificity for a different antigen.

It is believed, however, that monovalent antibodies capable of binding to a RON receptor will be especially useful as RON receptor antagonists. Such monovalent antibodies may be directed against the HGFL binding site of the receptor or may otherwise be capable of interfering with HGFL, its fragments or its variants binding to the RON receptor, such as by sterically hindering HGFL, its fragments or its variants access to the receptor, or by binding HGFL itself. Alternatively, the monovalent antibodies may be capable of sterically preventing RON receptor dimerization.

Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In a preferred embodiment of the invention, the antagonists comprise Fab fragments of monoclonal antibodies specific for the RON receptor.

In a preferred embodiment of the invention, the RON receptor inhibitor, e.g., an antagonist, monoclonal antibody or fragment or analog thereof, will inhibit at least one of (a) RON receptor activation, (b) binding of HGFL, its fragments or its variants, or (c) HGFL biological activity at least by about 50%, preferably, greater than about 80%, and more preferably, greater than about 90%.

In addition to the antagonistic antibodies described above, it is contemplated that chimeric or hybrid antagonistic antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. The antagonistic antibodies of the invention may further comprise humanized antibodies or human antibodies. Methods for humanizing non-human antibodies are well known in the art. See U.S. Pat. No. 4,816,567), incorporated herein by reference in its entirety.

In another embodiment of the invention, methods for treating liver damage are provided. In the methods, RON receptor antagonist is administered to a mammal diagnosed as having liver damage. While the term "liver damage" as used herein is not limited to any one specific form of the disease, it is believed that the methods will be particularly effective for the treatment of pathologies of the hepatobiliary system of inflammatory, infectious, toxin, or liver graft preservation-induced origin. It is of course contemplated that the methods of the invention can be employed in combination with still other therapeutic techniques such as surgery and chemotherapy.

The RON receptor inhibitor is preferably administered to a mammal in a pharmaceutically acceptable carrier. Suitable carriers and their formulations are well known in the art. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically acceptable carrier include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.8. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of RON receptor inhibitor being administered.

The RON receptor inhibitor or HGFL antagonist may be administered to a subject mammal, preferably human, via any of the accepted modes of administration for agents that exhibit such activity. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Effective dosages and schedules for administering the antagonist may be determined empirically, and making such determinations is within the skill in the art. Interspecies scaling of dosages can be performed in a manner known in the art. It is understood by those skilled in the art that the dose of RON receptor inhibitor that must be administered will vary depending on, for example, the mammal which will receive the RON receptor inhibitor, the nature of the medical condition or therapy believed to be responsible for liver injury or damage, the extent of damage to the tissues, the route of administration, and the identity of any other drugs being administered to the mammal. It is also understood that it may be necessary to give more than one dose of RON receptor inhibitor. Generally, multiple doses of RON receptor inhibitor will be required for administration. Administration of RON receptor inhibitor should be continued until acceptable liver function levels in the mammal are attained. Guidance in selecting appropriate doses for antibody antagonists is well known in the art.

The effect of the RON receptor inhibitors on hepatocyte growth and prevention of liver damage can also be tested in vivo in transgenic animal models such as described in U.S. Pat. No.5,087,571, incorporated herein by reference in its entirety.

In the aforementioned methods, the RON receptor inhibitor can alternatively be administered in combination with one or more biologically or chemically active agents. The skilled medical practitioner can determine the appropriate doses of each agent useful herein, generally reducing the normal dose when RON receptor inhibitor is combined with any of these agents. The RON receptor inhibitor can be administered in the same formulation as the other agent(s) or separate administration of RON receptor inhibitor and the other agent(s) can occur. The other agents are administered in modes, routes, and schedules appropriate for the particular agent.

A typical daily dosage of the RON receptor inhibitor used alone will range from about 0.01 .mu.g/kg to about 1000 mg/kg of body weight per day, depending on the factors mentioned above. Preferably, the daily dosage of the antagonist used alone will be from about 0.1 .mu.g/kg to about 100 mg/kg of body weight per day.

In an alternate embodiment, the present invention provides for a method of treating acute or chronic liver failure comprising administering to the patient a therapeutically effective amount of an inhibitor of phosphatidylinositol 3-kinase ("PI(3)") (for example, wortmannin, LY294002 (Affiniti, Exeter, UK), viridin, viridiol, demethoxyviridin, demethoxyviridiol, and analogs and derivatives thereof) either alone, or in conjunction with a RON receptor inhibitor, e.g., an HGFL truncated protein analog, and/or HGFL or RON receptor blocking antibodies as described above.

Physiologically effective levels of wortmannin range from about 10 to 1000 nM, usually from about 100 to 500 nM, and optimally at about 200 nM. Physiologically effective levels of LY294002 range from about 1 to 500 .mu.M, usually from about 25 to 100 .mu.M, and optimally at about 50 .mu.M. The inhibitors are administered at a dose sufficient to provide for these concentrations in the target tissue.

Other inhibitors of PI(3) kinase include anti-sense reagents that are specific for PI(3) kinase. Of particular interest are anti-sense molecules derived from the human PI(3) kinase sequence, particularly the catalytic p110 subunit, using the publicly available sequence. Alternatively, antibodies, antibody fragments and analogs or other blocking agents are used to bind to the PI(3) kinase in order to reduce the activity.

Representative United States patents that teach the preparation of phosphatidylinositol 3-kinase inhibiting agents include, but are not limited to, U.S. Pat. Nos.: 6,245,754, 5,504,103, 5,480,906, 5,378,725, each of which is herein incorporated by reference.

In another embodiment, the invention relates to a method for the treatment of a patient with a hepatotoxic therapeutic agent effective in the prevention or treatment of a disorder or pathophysiological condition comprising (a) administering to said patient simultaneously or in optional order (1) a biologically effective dose of said therapeutic agent and (2) a preventatively effective amount of a phosphatidylinositol 3-kinase inhibiting agent and (b) monitoring said patient for indication of liver damage and (c) continuing said treatment until the disorder or condition is eliminated or until liver damage is ameliorated. In optional embodiment, the invention comprises in step (a) above, administering to said patient simultaneously or in optional order (1) a biologically effective dose of said therapeutic agent and (2) a preventatively effective amount of a phosphatidylinositol 3-kinase inhibiting agent and (3) a preventatively effective amount of RON receptor inhibiting agent.

In another embodiment, the present invention relates to a method for the prevention of the establishment of liver damage in a patient at risk for developing liver damage comprising administering to said patient a liver damage preventative amount of a phosphatidylinositol 3-kinase inhibiting agent. In a further embodiment, the method includes administering to the patient simultaneously or in optional order a liver damage preventative amount of a RON receptor inhibiting compound. The patient preferably is mammalian, more preferably human.

In the methods of the present invention, the RON receptor inhibiting agent may be administered sequentially or concurrently with the one or more other therapeutic agents. Therapeutic agents contemplated include chemotherapeutics, amino acids, vitamins, immunoadjuvants, growth factors, proteins with growth factor-like activities, such as cytokines or cytokine antagonists, tissue plasminogen activator, antioxidants, nitric oxide donors and compounds capable of inducing nitric oxide generation or other therapeutics.

In another embodiment, the RON receptor inhibiting agent or antagonist may be administered in a composition further comprising an anti-oxidant selected from the group consisting of one or more antioxidants such as methionine, choline, N-acetylcysteine, vitamins (e.g., B complex, vitamin K vitamin E, vitamin A, vitamin C and their derivatives), gluthathione, cysteine, and 2-mercaptoethanol.

In another embodiment, the RON receptor inhibitor may be combined with a vasodilator such as nifedipine, felodipine, verapamil, debrisoquine, clonidine, doxazosin, pazosin, labetalol, irbesartan, lydrallazine, minoxidil, amladipine and nitroglycerine.

In one preferred embodiment, a composition containing the RON receptor inhibitor is administered to a subject mammal alone according to the present invention, or combined with other therapies effective in the prevention or treatment of liver damage wherein the composition further comprises one or more growth factors.

Also included in the invention are "polypeptide growth factors," which possess one or more of the biological functions or activities of the growth factors described herein. Alternatively, polypeptide growth factors useful in the invention can consist of active fragments of the factors. By "active fragment," as used herein in reference to polypeptide growth factors, is meant any portion of a polypeptide that is capable of invoking the same activity as the full-length polypeptide. The active fragment will produce at least 40%, preferably at least 50%, more preferably at least 70%, and most preferably at least 90% (including up to 100%) of the activity of the full-length polypeptide. The activity of any given fragment can be readily determined in any number of ways. For example, a fragment of bFGF that, when administered according to the methods of the invention described herein, is shown to produce performance in functional tests that is comparable to the performance that is produced by administration of the full-length bFGF polypeptide, would be an "active fragment" of bFGF. It is well within the abilities of skilled artisans to determine whether a polypeptide growth factor, regardless of size, retains the functional activity of a full length, wild type polypeptide growth factor.

In another embodiment, the additional therapeutic agent is a nitric oxide (NO) stimulator, a nitric oxide synthase substrate, or a combination thereof. The use of NO donors is known for decreasing blood pressure in the treatment of angina, ischemic diseases, congestive heart failure, impotence in males, hypertension, arteriosclerosis, cerebral vasospasm, and coronary vasospasm (U.S. Pat. Nos. 4,954,526; 5,278,192, incorporated herein by reference in their entirety).

NO therapy is generally achieved by administering drugs, such as nitroglycerine, that donate NO once inside the cell. The rest of the molecule (or NO degradation products) may be metabolically active, thus further complicating the problem of delineating the specific effect of NO on hypertension.

It is known that some NO donors exhibit varying degrees of tolerance that may necessitate intermittent administration of such compounds. For this reason, it may be beneficial to employ organic nitrites that induce less tolerance. Alternatively, it may be preferable to co-administer other agents that help to alleviate the tolerance problem, such as sulfhydryl donors, or to employ agents that stimulate NOS production in vivo, or serve as a substrate for NOS. Such agents include those that stimulate NOS, and those that inhibit the catabolism of NO or feedback inhibition of NOS.

In a further embodiment of the invention, the present invention provides for a method for preventing hepatobiliary damage in a subject due to exposure to a hepatotoxic agent. Such method comprises administering to a subject in need of such treatment a therapeutically effective amount of a composition comprising a RON receptor tyrosine kinase inhibitor wherein the RON receptor inhibitor effectively ameliorates liver damage due to exposure to the hepatotoxic agent. In an additional embodiment, the hepatotoxic agent is one or more of anesthetics, neuropsychotropics, anticonvulsants, analgesics, hormones, antimicrobials, cardiovascular drugs, immunosuppressives, radiation, and antineoplastics. In a preferred embodiment, the administration of RON receptor inhibitor is initiated within 24 hours after exposure to the one or more hepatotoxic agents.

In a further embodiment of the invention, there are provided articles of manufacture and kits containing materials and compositions for inhibiting the RON receptor useful for the prevention and treatment of liver damage or detecting.

The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for the prevention and treatment of liver damage or for detecting or purifying RON receptor. The active agent in the composition is generally a RON receptor antagonist and preferably, comprises Fab fragments of monoclonal antibodies specific for the RON receptor.

The kit of the invention comprises the container described above and a second container comprising a buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos.: 5,547,932; 5,583,020; 5,591,721; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

The RON receptor inhibitors of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesi red toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine. As used herein, a "pharmaceutical addition salt" includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids.

For HGFL antagonists, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

The present invention also includes pharmaceutical compositions and formulations that include the RON receptor inhibitors of the invention. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

A "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more RON receptor inhibitors to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a RON receptor inhibitor and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration that do not deleteriously react with RON receptor inhibitors can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of RON receptor inhibitors may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the RON receptor inhibitors in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not deleteriously react with RON receptor inhibitors can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the RON receptor inhibitor(s) of the formulation. Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more RON receptor inhibitors and (b) one or more other chemotherapeutic agents that function by a non-RON receptor mechanism. Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Other non-RON receptor chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual HGFL antagonists, and can generally be estimated based on EC.sub.50 found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 .mu.g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 .mu.g to 100 g per kg of body weight, once or more daily, to once every 20 years.

 

Claim 1 of 20 Claims

1. A method for the treatment of hepatobiliary damage in a subject comprising (a) identifying a subject in need of treatment for hepatobiliary damage; (b) administering to the subject a therapeutically effective amount of a composition comprising a RON receptor tyrosine kinase inhibitor wherein the RON receptor tyrosine kinase inhibitor is an HGFL protein antagonist and wherein the HGFL protein antagonist comprises a polypeptide sequence of at least the C-terminal serine protease-like domain of an MSP beta-chain protein (SEQ ID NO:2); wherein the polypeptide comprises at least 9 contiguous amino acids of SEQ ID NO:2 and is less than 100 amino acids in length; wherein the polypeptide sequence is lacking at least one kringle region selected from the group comprising the 2.sup.nd (.DELTA.K2), 3.sup.rd (.DELTA.K3) or 4.sup.th (.DELTA.K4) kringle domains; and wherein the polypeptide is capable of binding the RON receptor and preventing activation of the RON receptor by HGFL; and (c) continuing the administration of the composition for a time sufficient to treat hepatobiliary damage in the subject.

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