WO2008156638A2 - Method of treating disorders that would benefit from increased levels of bilirubin in a subject - Google Patents

Abstract

Methods of treating diseases or disorders that would benefit from increased levels of bilirubin are described.

Description

METHODS OF TREATING DISORDERS THAT WOULD BENEFIT FROM INCREASED LEVELS OF BILIRUBIN IN A SUBJECT

Related Applications

This application claims priority to U.S. Application Serial No. 60/934686, entitled "METHODS OF TREATING DISORDERS THAT WOULD BENEFIT FROM INCREASED LEVELS OF BILIRUBIN IN A SUBJECT", filed on June 15, 2007, the contents of which are hereby incorporated by reference.

Background of the Invention

The degradation products of heme, Le., bilirubin and biliverdin, have shown promise in the amelioration of the symptoms of certain diseases and disorders. For example, bilirubin and biliverdin have been found to attenuate inflammation and to suppress tissue damage associated with ischemia. Humans with relatively higher bilirubin levels have atherosclerosis-like disorders less frequently than do those with lower levels of bilirubin. The identification of new methods of increasing the concentration of bilirubin in a subject would be of great benefit.

Summary of the Invention

The invention is based, at least in part, on the discovery that β-glucuronidase can be used to increase the level of bilirubin in a subject. The oral administration of β- glucuronidase to a subject results in higher bilirubin levels and, thereby, ameliorates the symptoms of diseases and/or disorders that would benefit from the presence of increased levels of bilirubin. Accordingly, the instant invention pertains to methods of increasing endogenous levels of bilirubin by administration of β-glucuronidase.

In one embodiment, β-glucuronidase is administered to a subject already suffering from a symptom of a disease or disorder that would benefit from the presence of increased levels of bilirubin. In another embodiment, β-glucuronidase is administered to a subject not yet suffering from a symptom of a disease or disorder that would benefit from the presence of increased levels of bilirubin, β-glucuronidase may be administered for acute therapy as well as for chronic treatment of a disorder or the symptoms thereof. In one embodiment, the subject methods may be used in combination with one or more additional means of increasing levels of endogenous or exogenous bilirubin and/or biliverdin. In another embodiment, the subject methods may be used in combination with art recognized methods for treating a disorder or disease. In one aspect, the invention is directed to a method of treating a disease or disorder that would benefit from increased levels of bilirubin in a subject, comprising orally administering to said subject an effective amount of a β-glucuronidase polypeptide, such that the disease or disorder is treated in said subject. In one embodiment, the composition is selected such that the β-glucuronidase polypeptide is released in the small intestine. In one embodiment, the composition comprises an enteric coating.

In one embodiment, the composition is selected such that the β-glucoronidase polypeptide is released in the duodenum. In one embodiment, the composition is formulated in microcrystalline form, powder, or tablet.

In one embodiment, the subject's stomach acid has been suppressed prior to administration of the β-glucoronidase polypeptide.

In one embodiment, the effective amount is effective to increase bilirubin levels in said subject.

In one embodiment, the bilirubin levels are increased at least about 25%. In one embodiment, the bilirubin levels are increased at least about 50%. In one embodiment,, the bilirubin levels are increased at least about 100%. In one embodiment, the bilirubin levels are increased at least about 2000% or greater. In one embodiment, the method further comprises administering a bilirubin oxidase polypeptide in combination with said β-glucuronidase polypeptide.

In one embodiment, the bilirubin oxidase polypeptide is conjugated to said β- glucuronidase polypeptide.

In one embodiment, the bilirubin oxidase polypeptide and β-glucuronidase polypeptide are administered in an amount effective to increase biliverdin levels in said subject.

In one embodiment, the biliverdin levels are increased at least about 10%. In one embodiment, the biliverdin levels are increased at least about 50%. In one embodiment, the biliverdin levels are increased at least about 2000%. In one embodiment, the subject is suffering or at risk of suffering from an inflammatory disorder.

In one embodiment, the inflammatory disorder is asthma, adult respiratory distress syndrome, interstitial pulmonary fibrosis, pulmonary emboli, chronic obstructive pulmonary disease, primary pulmonary hypertension, chronic pulmonary emphysema, congestive heart failure, peripheral vascular disease, stroke, atherosclerosis, ischemia- reperfusioή injury, heart attacks, glomerulonephritis, conditions involving inflammation of the kidney, infection of the genitourinary tract, viral and toxic hepatitis, cirrhosis, ileus, necrotizing enterocolitis, specific and non-specific inflammatory bowel disease, rheumatoid arthritis, deficient wound healing, graft versus host disease, and hemorrhagic, septic, or anaphylactic shock..

In one embodiment, the said subject is a human. In one aspect, the for increasing the level of bilirubin in a subject, comprising orally administering to said subject an effective amount of a β-glucuronidase polypeptide, such that the bilirubin level in said subject are increased.

In one embodiment, the bilirubin levels are increased at least about 25%. In one embodiment, the bilirubin levels are increased at least about 50%. In one embodiment, the bilirubin levels are increased at least about 100%. In one embodiment, the said bilirubin levels are increased at least about 2000%.

In one embodiment, the bilirubin levels are increased by an amount effective to treat inflammation. In one embodiment, the inflammation is chronic. In one embodiment, the inflammation is acute. In another aspect, the invention pertains to a method for increasing the level of biliverdin in a subject, comprising orally administering to said subject an effective amount of a β-glucuronidase polypeptide in combination with a bilirubin oxidase polypeptide, such that the biliverdin level in said subject are increased.

In one embodiment, the biliverdin levels are increased at least about 10%. In one embodiment, the biliverdin levels are increased at least about 50%. In one embodiment, the biliverdin levels are increased at least about 2000%.

In one embodiment, the biliverdin levels are increased by an amount effective to treat inflammation. In one embodiment, the inflammation is chronic. In one embodiment, the inflammation is acute. The pharmaceutical composition comprising β-glucuronidase formulated for oral administration.

In one embodiment, the composition comprises an enteric coating. In one embodiment, the enteric coating is designed to release the β-glucuronidase polypeptide in the duodenum. In one embodiment, the composition is formulated as a tablet, micro crystalline form, powder, or tablet.

In one embodiment, the composition comprises an effective amount of a bilirubin oxidase polypeptide. In one embodiment, the bilirubin oxidase compound is conjugated to said β-glucuronidase polypeptide. In one embodiment, the amount is effective to treat an inflammatory disorder.

In one embodiment, the inflammatory disorder is chronic. In one embodiment, the inflammatory disorder is acute. Detailed Description of the Invention

The invention is based, at least in part, on the discovery that β -glucuronidase can be used to increase the level of bilirubin in a subject.

In normal adults, up to approximately 400 mg of bilirubin is produced daily. Approximately 70-80% of daily bilirubin is derived from degradation of the heme moiety of hemoglobin. The rest is mostly derived from the hepatic turnover of heme proteins, such as myoglobin, cytochromes, and catalase. In the circulation unconjugated bilirubin is tightly bound to albumin and the bilirubin-albumin conjugates are readily taken up by the liver.

Bilirubin is hydrophobic and poorly soluble in water at physiologic pH because intramolecular hydrogen bonding shields the hydrophilic sites of the bilirubin molecule. Hepatic conjugation of the propionic acid side chains of bilirubin with glucuronic acid makes the molecule water-soluble and excretable in bile. Bilirubin is primarily excreted in normal human bile as a mono or diglucuronide. Bilirubin conjugates secreted into bile are poorly reabsorbed in the intestine; there is no active or facilitated transport system for these molecules in the gut. In the ileum and colon, anaerobic bacteria produce enzymes which cleave the glucuronic acid to produce unconjugated bilirubin, but bilirubin is not soluble at the pH of the large intestine, therefore it is not efficiently absorbed, but instead is converted to urobilinoids.

While most of the bilirubin is eliminated in the feces via the mechanism outlined above, a small proportion is passively reabsorbed by the liver via the enterohepatic circulation. By using β -glucuronidase to facilitate the removal of glucuronic acid from bilirubin in the small intestine, the uptake of unconjugated bilirubin by the enterohepatic circulation can be increased, thereby increasing the level of bilirubin present in the systemic circulation of a subject. Accordingly, the methods of the invention include administering an effective amount of a β-glucuronidase polypeptide to a subject to increase levels of bilirubin in the systemic circulation of the subject. β-glucuronidase (GUS) activity is found in many bacterial species. It is also common in all tissues of vertebrates, and is present in organisms of various invertebrate taxa. β-glucuronidase from prokaryotes or eukaryotes may be used in connection with the instant invention. I. Definitions

Preferably, the term "β-glucuronidase polypeptide" refers to a β-glucuronidase polypeptide having a pH optimum of about 6-7 so that it functions well in the duodenum. Exemplary β-glucuronidases include prokaryotic and eukaryotic enzymes, e.g., bacterial, fungal, mammalian, and human enzymes. A "β-glucuronidase polypeptide" can be from the same or different species as the subject to which it is to be administered.

Amino acid sequences of β-glucuronidase polypeptides that can be used in the invention are known in the art. The language "β-glucuronidase polypeptide" also includes variant proteins which vary in sequence from wild-type β-glucuronidase polypeptides, yet have sufficient amino acid identity with β-glucuronidase such that they maintain β-glucuronidase activity. Such variants can be naturally or non-naturally occurring. In one embodiment, a β-glucuronidase polypeptide is a biologically active fragment of a β-glucuronidase polypeptide. In one embodiment, a β-glucuronidase polypeptide comprises additional amino acids or non-amino acid moieties that impart desired properties to the molecule.

Preferably, the term "bilirubin oxidase polypeptide" refers to human bilirubin oxidase, however, bilirubin oxidase polypeptides of other species are also included in the term. A "bilirubin oxidase polypeptide" can be from the same or different species as the subject to which they are to be administered. For example, "bilirubin oxidase polypeptides" include those of human origin as well as orthologs of non-human origin. Amino acid sequences of bilirubin oxidase polypeptides that can be used in the invention are known in the art. The language "bilirubin oxidase" also includes variant proteins which vary in sequence from wild-type bilirubin oxidase polypeptides, yet have sufficient amino acid identity with bilirubin oxidase such that they maintain bilirubin oxidase activity. Such variants can be naturally or non-naturally occurring. In one embodiment, a bilirubin oxidase polypeptide is a biologically active fragment. In another embodiment, a bilirubin oxidase polypeptide comprises additional amino acids or non-amino acid moieties that impart desired properties to the molecule. As used herein, the term "increased levels" includes increased concentrations of bilirubin in a subject or in a particular portion of a subject, e.g., the blood, serum, or plasma of a particular subject. The increased concentration of these molecules may be expressed in terms of concentration or in terms of a percent increase in the level of the molecule. In one embodiment, the levels of bilirubin are increased at least by about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared with the levels prior to administration of b-glucuronidase. In another embodiment, levels of bilirubin are increased by at least about 200%, 250%, 300%, 400%, 500%, 600%, 750%, 1000%, 1500%, 2000%, 2500%, 3000%, 3500%, 4000%, 4500%, or 5000%.

In a further embodiment, levels of bilirubin are at least about 0.01 mg/dl, about 0.1 mg/dl, 0.25 mg/dl, about 0.5 mg/dl, about 0.75 mg/dl, about 1 mg/dl, about 5 mg/dl, about 10 mg/dl, about 20 mg/dl, about 30 mg/dl, about 40 mg/dl, about 50 mg/dl, about 120mg/dl, about 130 mg/dl, about 140 mg/dl, about 150 mg/dl, about 160 mg/dl, about 200 mg/dl or greater. In a further embodiment, the term includes levels of bilirubin in a subject in the range of from about 0.01 to about 300 mg/dl, e.g., about 0.1 to about 200 mg/dl, or about 1 to about 100 mg/dl, or about 1-10 mg/dl.

π. Preferred β-Glucuronidase Molecules In one embodiment, a β-glucuronidase molecule of the invention comprises the complete amino acid sequence of a β-glucuronidase polypeptide and, optionally additional amino acid sequences and/or non-amino acid moieties that improve, e.g., activity, stability, bioavilability, and/or provide other improved properties to the molecule. In another embodiment, a β-glucuronidase molecule of the invention comprises a biologically active portion of the molecule, e.g., the active site of the molecule and, optionally additional amino acid sequences and/or non-amino acid moieties as above. β-glucuronidase for use in the instant invention may be purchased commercially of may be produced recombinantly, e.g., in a cell (such as a bacterial, animal, or plant cell) or in an organism (a transgenic organism e.g., a goat, a cow, or a plant). Exemplary sequences for β-glucuronidase from different sources are known in the art.

Exemplary β-glucuronidase molecule have been crystallized (see, e.g., Jain, S., et al. (1996) Nature Struct. Biol, 3, 375-381) and important amino acid molecules in the active site have been identified (JBC 274:23451-23455). Accordingly, active portions of the molecule will be easily identifiable to one of ordinary skill in the art.

While, in preferred embodiments, the peptides of this invention utilize naturally- occurring amino acids or D forms of naturally occurring amino acids, substitutions with non-naturally occurring amino acids (e.g., methionine sulfoxide, methionine methylsulfonium, norleucine, episilon-aminocaproic acid, 4-aminobutanoic acid, tetrahydroisoquinoline-3-carboxylic acid, 8-aminocaprylic acid, 4-aminobutyric acid, Lys(N(epsilon)-trifluoroacetyl), alpha-aminoisobutyric acid, and the like) are also contemplated. In one embodiment, peptidomimetics of β-glucuronidase may also be used. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics" (Fauchere (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem. 30: 1229) and are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.

In another embodiment, a cell or organism that produces sufficient levels of β- glucuronidase (either endogenous or exogenous β-glucuronidase) may be used to increase β-glucuronidase in the duodenum of a subject.

A. Optional Modifications to Polypeptides

In one embodiment, a β-glucuronidase polypeptide or β-glucuronidase molecule of the invention may be modified to improve its properties. Exemplary modifications are set forth below.

1. D-Form Amino Acids

In one embodiment, a β-glucuronidase polypeptide of the invention comprises one or more D-form amino acids. In certain embodiments, every amino acid (e.g. every enantiomeric amino acid) is a D-form amino acid.

D-amino acids are incorporated at one or more positions in the peptide simply by using a D-form derivatized amino acid residue in the chemical synthesis. D-form residues for solid phase peptide synthesis are commercially available from a number of suppliers (see, e.g., Advanced Chem Tech, Louisville; Nova Biochem, San Diego;

Sigma, St Louis; Bachem California Inc., Torrance, etc.). The D-form amino acids can be incorporated at any position in the peptide as desired. Thus, for example, in one embodiment, the peptide can comprise a single D-amino acid, while in other embodiments, the peptide comprises at least two, generally at least three, more generally at least four, most generally at least five, preferably at least six, more preferably at least seven and most preferably at least eight D amino acids. In particularly preferred embodiments, essentially every other (enantiomeric) amino acid is a D-form amino acid. In certain embodiments at least 90%, more preferably at least 95% of the enantiomeric amino acids are D-form amino acids. In one particularly preferred embodiment, essentially every enantiomeric amino acid is a D-form amino acid. 2. Protecting Groups

In certain embodiments, one or more R-groups on the constituent amino acids and/or the terminal amino acids are blocked with one or more protecting groups.

A wide number of protecting groups are suitable for this purpose. Such groups include, but are not limited to acetyl, amide, and alkyl groups with acetyl and alkyl groups being particularly preferred for N-terminal protection and amide groups being preferred for carboxyl terminal protection. In certain particularly preferred embodiments, the protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others. Particularly preferred carboxyl protecting groups include amides, esters, and ether-forming protecting groups. In one preferred embodiment, an acetyl group is used to protect the amino terminus and an amide group is used to protect the carboxyl terminus. These blocking groups enhance the helix- forming tendencies of the peptides. Certain particularly preferred blocking groups include alkyl groups of various lengths, e.g. groups having the formula: CH3~(CH₂)_n~ CO-- where n ranges from about 1 to about 20, preferably from about 1 to about 16 or 18, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.

In certain particularly preferred embodiments, the protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others. Particularly preferred carboxyl protecting groups include amides, esters, and ether- forming protecting groups. In one preferred embodiment, an acetyl group is used to protect the amino terminus and an amide group is used to protect the carboxyl terminus. These blocking groups enhance the helix-forming tendencies of the peptides. Certain particularly preferred blocking groups include alkyl groups of various lengths, e.g. groups having the formula: CH3~(CH₂)_n~CO~ where n ranges from about 3 to about 20, preferably from about 3 to about 16, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.

Other protecting groups include, but are not limited to Fmoc, t-butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl- benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5, 7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzI), 4-methoxybenzyl (MeOBzI), Benzyloxy (BzIO), Benzyl (BzI), Benzoyl (Bz), 3-nitro 2-pyridinesulphenyl (Npys), l-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2- bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy (cHxO), t- butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

Protecting/blocking groups are well known to those of skill as are methods of coupling such groups to the appropriate residue(s) comprising the peptides of this invention (see, e.g. Greene et al., (1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc. Somerset, N.J.). In one preferred embodiment, for example, acetylation is accomplished during the synthesis when the peptide is on the resin using acetic anhydride. Amide protection can be achieved by the selection of a proper resin for the synthesis. During the synthesis of the peptides described herein in the examples, rink amide resin was used. After the completion of the synthesis, the semipermanent protecting groups on acidic bifunctional amino acids such as Asp and GIu and basic amino acid Lys, hydroxyl of Tyr are all simultaneously removed. The peptides released from such a resin using acidic treatment comes out with the n-terminal protected as acetyl and the carboxyl protected as NH₂ and with the simultaneous removal of all of the other protecting groups.

III. Preparation of β-glucuronidase Polypeptides of the Invention

A. Chemical Synthesis In one embodiment, the β-glucuronidase polypeptides used in this invention are chemically synthesized using standard chemical peptide synthesis techniques or, particularly where the peptide does not comprise "D" amino acid residues, are recombinantly expressed. Where the polypeptides are recombinantly expressed and comprises D amino acids, a host organism (e.g. bacteria, plant, fungal cells, etc.) is cultured in an environment where one or more of the amino acids is provided to the organism exclusively in a D form. Recombinantly expressed peptides in such a system then incorporate those D amino acids.

In one embodiment, the peptides are chemically synthesized by any of a number of fluid or solid phase peptide synthesis techniques known to those of skill in the art. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the polypeptides of this invention. Techniques for solid phase synthesis are well known to those of skill in the art and are described, for example, by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3 284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem. Soc. 85: 2149 2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce

Chem. Co., Rockford, 111.

In another embodiment, the peptides are synthesized by the solid phase peptide synthesis procedure using a benzhyderylamine resin (Beckman Bioproducts, 0.59 mmol of NH₂/g of resin) as the solid support. The COOH terminal amino acid (e.g., t- butylcarbonyl-Phe)is attached to the solid support through a 4-(oxymethyl)phenacetyl group. This is a more stable linkage than the conventional benzyl ester linkage, yet the finished peptide can still be cleaved by hydrogenation. Transfer hydrogenation using formic acid as the hydrogen donor is used for this purpose. Detailed protocols used for peptide synthesis and analysis of synthesized peptides are describe in a miniprint supplement accompanying Anantharamaiah et al. (1985) J. Biol. Chem., 260(16): 10248

10255.

It is noted that in the chemical synthesis of peptides, particularly peptides comprising D amino acids, the synthesis usually produces a number of truncated peptides in addition to the desired full-length product. The purification process (e.g.

HPLC) typically results in the loss of a significant amount of the full-length product.

B. Recombinant Expression

In another embodiment of the invention, a β-glucuronidase polypeptide is recombinantly expressed, e.g., in a cell or in an animal, using techniques known in the art. In one embodiment, an expression vector comprising a nucleic acid encoding β- glucuronidase in a form suitable for expression of the nucleic acid in a host cell is used to express β-glucuronidase. Such recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence {e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" includes promoters, enhancers and other expression control elements {e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells {e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acid molecules. Recombinant expression vectors can be designed for expression of β- glucuronidase in prokaryotic or eukaryotic cells. For example, β-glucuronidase can be , expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out mE. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors can serve one or more purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification; 4) to provide an epitope tag to aid in detection and/or purification of the protein; and/or 5) to provide a marker to aid in detection of the protein {e.g., a color marker using β- galactosidase fusions). Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Recombinant proteins also can be expressed in eukaryotic cells as fusion proteins.

Examples of suitable inducible non- fusion E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69:301-315) and pET 1 Id (Studier et al.. Gene Expression Technology: Methods in Enzymology 185. Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave, the recombinant protein (Gottesman, S., Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (\ 992) Nuc. Acids Res. 20:2111 -2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari. et al, (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933- 943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).

Alternatively, β-glucuronidase can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., (1983) MoI. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A, and Summers, M.D., (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pMex-NeoI, pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBOJ. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue- specific regulatory elements are known in the art. Non- limiting examples of suitable tissue-specific promoters include lympho id-specific promoters (Calame and Eaton

(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (\9S9) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) CeIl 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), the albumin promoter (liver-specific; Pinkert etal. (1987) Genes Dev. 1:268-277), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-9161 and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264, 166). Developmentally- regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546). Moreover, inducible regulatory systems for use in mammalian cells are known in the art, for example systems in which gene expression is regulated by heavy metal ions (see e.g., Mayo et al. (1982) Cell 29:99-108; Brinster et al. (1982) Nature 296:39-42: Searle et al. (1985) MoI. Cell. Biol. 5:1480-1489), heat shock (see e.g., Nouer et al. (1991) in Heat Shock Response, e.d. Nouer, L. , CRC, Boca Raton , FL, pp 167-220), hormones (see e.g., Lee et al. (1981) Nature 294:228-232; Hynes et al (1981) Proc. Natl. Acad Sci. USA 78:2038-2042; Klock et al. (1987) Nature 329:734-736; Israel & Kaufman (1989) Nucl. Acids Res. 17:2589-2604; and PCT Publication No. WO 93/23431), FK506-related molecules (see e.g., PCT Publication No. WO 94/18317) or tetracyclines (Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad Sci. USA 89:5547- 5551; Gossen, M. et al. (1995) Science 268:1766-1769; PCT Publication No. WO 94/29442; and PCT Publication No. WO 96/01313). Accordingly, in another embodiment, the invention provides a recombinant expression vector in which T-bet DNA is operatively linked to an inducible eukaryotic promoter, thereby allowing for inducible expression of b-glucuronidase protein in eukaryotic cells. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker may be introduced into a host cell on the same vector as that encoding b-glucuronidase or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

IV. Pharmaceutical Formulations of β-glucuronidase The β-glucuronidase polypeptides of the invention are generally administered in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological saline solution, but other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. The language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and uptake enhancers such as lipids, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers. The use of such media and agents for pharmaceutically active substances is well known in the art.

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound depends, for example, on the route of administration of the active agent(s) and on the particular physio-chemical characteristics of the active agent(s).

The excipients are preferably sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well-known sterilization techniques. Supplementary active compounds can also be incorporated into the compositions. It may also be desirable that a suitable buffer be present in the composition. Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection. The primary solvent can be aqueous or alternatively non-aqueous. The β-glucuronidase polypeptides can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requiring treatment.

The carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier. Such excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion.

In order to carry out the methods of the invention, one or more of the β- glucuronidase polypeptides of this invention are administered, e.g. to an individual diagnosed as having one or more symptoms of a disease or disorder that would benefit from increased concentration of a heme degredation product. The β-glucuronidase polypeptides can be administered in the "native" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrugs or derivative is suitable pharmacologically, i.e., effective in the present method. Salts, esters, amides, prodrugs and other derivatives of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N. Y. Wiley-Interscience.

For example, acid addition salts are prepared from the free base using conventional methodology, that typically involves reaction with a suitable acid. Generally, the base form of the b-glucuronidase polypeptide is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt either precipitates or may be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g:, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. An acid addition salt may be reconverted to the free base by treatment with a suitable base. Particularly preferred acid addition salts of the b-glucuronidase polypeptides are halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of the b-glucuronidase polypeptides are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. Particularly preferred basic salts include alkali metal salts, e.g., the sodium salt, and copper salts.

Preparation of esters typically involves functionalization of hydroxyl and/or carboxyl groups which may be present within the molecular structure of the b- glucuronidase polypeptides. The esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties that are derived from carboxylic acids of the formula RCOOH where R is alkyl, and preferably is lower alkyl. Esters can be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.

Amides and prodrugs may also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine. Prodrugs are typically prepared by covalent attachment of a moiety that results in a compound that is therapeutically inactive until modified by an individual's metabolic system.

The β-glucuronidase polypeptides are preferably formulated for enteral administration. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories.

In preferred embodiments, the β-glucuronidase polypeptides of this invention are administered orally (e.g. via a tablet) in accordance with standard methods well known to those ofskill in the art.

In another embodiment, one or more components of the solution can be provided as a "concentrate", e.g., in a storage container (e.g., in a premeasured volume) ready for dilution, or in a soluble capsule ready for addition to a volume of water. Additional pharmacologically active agents may be delivered along with the primary active agents, e.g., the β-glucuronidase polypeptides of this invention. The β-glucuronidase polypeptides can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. For example, the β-glucuronidase polypeptides.

V. Oral Formulations of β-glucuronidase

In one embodiment, the β-glucuronidase polypeptides of this invention can be administered orally without protection against proteolysis by stomach acid, etc. Nevertheless, in certain embodiments, peptide delivery can be enhanced, e.g.,by the use of protective excipients. This is typically accomplished either by complexing the polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the polypeptide in an appropriately resistant carrier such as a liposome. Exemplary means of protecting polypeptides for oral delivery are well known in the art (see, e.g., U.S. Pat. No. 5,391,377 describing lipid compositions for oral delivery of therapeutic agents) and are set forth below.

A. Enteric Coatings

In one embodiment, a β-glucuronidase polypeptide is protected so that it traverses the stomach and can act in the duodenum, releasing the polypeptide prior to the descending duodenum. Exemplary coatings are described below.

1. Tablets and Capsules

For enteral application, particularly suitable are tablets, dragees or capsules having talc and/or carbohydrate carrier binder or the like, the carrier preferably being lactose and/or corn starch and/or potato starch. Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.

Enteric coatings (which generally do not substantially dissolve in solutions with a pH lower than about 5.5) may delay release of the β-glucuronidase polypeptide compounds until delivery to the intestinal tract. Examples of enteric coatings include, but are not limited to, coatings made from methacrylic acid copolymers, cellulose acetate (and its succinate and phthalate versions), styrol maleic acid copolymers, polymethacrylic acid/acrylic acid copolymer, hydroxypropyl methyl cellulose phthalate, polyvinyl acetate phthalate, hydroxyethyl ethyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, cellulose acetate tetrahydrophtalate, acrylic resin, timellitate, and shellac, and combinations thereof. 2. Encapsulation Technologies

Encapsulation, such as that described in U.S. Pat. Nos. 4,579,779 and 5,500,223, may be used to formulate the β-glucuronidase polypeptides of the invention. This process can be performed, for example, by forming an emulsion by the high-shear mixing of an aqueous dispersion of silica with the β-glucuronidase polypeptides of the invention, and then gelling the emulsion. Conveniently, the gelling is generally accomplished by the addition of a gelling agent, which may comprise an acidifying agent and/or a salt and/or a positively charged surfactant. It may be sufficient to perform the gelling step using an appropriate gelling agent, but otherwise the gelling step can be followed with a further treatment.

The gelling step may form or stabilise a shell-like structure of silica particles around individual droplets of the hydrophobic material (e.g., β-glucuronidase polypeptides) in the emulsion. The resulting capsules have sufficient stability to be used generally as such. It is found, furthermore, that it is possible to give such shell-like structures significant strength and a degree of imperviousness in relation to the encapsulated material.

The shell-like encapsulating structures formed are also found to be capable of holding relatively high loadings of hydrophobic material. In one embodiment, the invention pertains to a micro-encapsulate comprising an outer silica layer and containing a the b-glucuronidase polypeptides of the invention in which the loading of the polypeptides to the silica is at least 1.5: 1 and is preferably greater than 2:1.

If an appropriate gelling agent is needed, the gelling step can be carried out in a coacervation process using a positively charged compound such as a positively charged polymer or a gum or a silicone which, as it attaches itself to the silica shell, is able to form an outer layer on the capsule.

Suitable silica materials include colloidal silica and fumed silica. Colloidal silica is supplied commercially as an alkaline solution (pH>9) and a hydrophobicizing agent may be added to help produce the aqueous dispersion. Suitable hydrophobicizing agents include positively charged surfactants such as cetyltrimethylammonium bromide (CTAB). Fumed silica forms an acidic emulsion (pH 3-5) which requires no hydrophobicizing agent.

In another form of treatment, the capsules can be given an outer coating by blending the gelled material with a suitable material such as a liquid crystal forming surfactant or a polymer. Suitable surfactants may be selected from any of the major classes (ie. non-ionic, cationic, anionic and zwitterionic) and where appropriate their ability to form liquid crystal structures may be assisted by the inclusion of structuring aids such as steroids. Examples of suitable surfactants include polyethoxylated fatty alcohols (eg. POE(2) cetyl alcohol), sorbitan esters (e.g., sorbitan monostearate), glycerol esters (e.g., glycerol monostearate), phospholipids (e.g., phosphatidylcholine), fatty alcohols (e.g., cetyl alcohol), quaternaries (e.g., dimethylditallowammonium chloride). Suitable polymers may include starch, modified starch, other polysaccharides (eg. gums), derivatised polysaccharides, and synthetic polymers such as silicones, polyacrylics (eg. polyacrylamide) and polyvinyl pyrrolidones.

B. Controlled Release Controlled release in the pharmaceutical field has been addressed by various means and can be used to administer the β-glucuronidase polypeptides of the invention. U.S. Pat. No. 5,569,467 refers to the use of sustained release microparticles comprising a biocompatible polymer and a pharmaceutical agent, which is released as the polymer degrades. U.S. Pat. No. 5,603,956 refers to solid, slow release pharmaceutical dosage units comprising crosslinked amylase, alpha amylase and a pharmaceutical agent. U.S. Pat. No. 4,606,909 refers to oral, controlled-release multiple unit formulations in which homogeneous cores containing particles of sparingly soluble active ingredients are coated with a pH-sensitive erodable coating. U.S. Pat. No. 5,593,697 refers to pharmaceutical or veterinary implants comprising a biologically active material, an excipient comprising at least one water soluble material and at least one water insoluble material and a polymer film coating adapted to rupture at a predetermined period of time after implant.

C. Microcrystalline Formulation The β-glucuronidase family of polypeptides of this invention can be administered as a microcrystalline formulation for controlled release delivery. One of the advantages of a microcrystalline formulation is that additional components are not required in the formulation. Micro crystallization has been used, for example, in the controlled release pulmonary delivery of insulin (Kwon et al. (2004) Eur. J. Pharm. Sci. 22 (2-3): 107-116.). Microcrystals of the β-glucuronidase polypeptides of this invention can be produced using a seed zone method or any other method that produces microcrystals of high yield and efficiency.

D. Lipid Containing Formulations In certain embodiments, the β-glucuronidase polypeptides of this invention are administered in conjunction with one or more lipids. The lipids can be formulated as an excipient to protect and/or enhance transport/uptake of the β-glucuronidase polypeptides or they can be administered separately.

The lipids can be formed into liposomes that encapsulate the β-glucuronidase polypeptides of this invention and/or they can be simply complexed/admixed with the polypeptides. Methods of making liposomes and encapsulating reagents are well known to those of skill in the art (see, e.g.,, Martin and Papahadjopoulos (1982) J. Biol. Chem., 257: 286 288; Papahadjopoulos et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 11460 11464: Huang et al. (1992) Cancer Res., 52:6774 6781; Lasic et al. (1992) FEBS Lett., 312: 255 258., and the like). In one embodiment, the lipids are phospholipids. Preferred phospholipids for use in these methods have fatty acids ranging from about 4 carbons to about 24 carbons in the sn-1 and sn-2 positions. In certain preferred embodiments, the fatty acids are saturated. In other preferred embodiments, the fatty acids can be unsaturated.

Furthermore, the β-glucuronidase polypeptide of the invention can be in a composition which aids in delivery into the cytosol of a cell. For example, the β- glucuronidase polypeptide may be conjugated with a carrier moiety such as a liposome that is capable of delivering the peptide into the cytosol of a cell. Such methods are well known in the art (for example see Amselem et al, 1993, Chem Phys Lipids 64: 219-237, which is incorporated by reference). Alternatively, the β-glucuronidase polypeptide can be modified to include specific transit peptides or fused to such transit peptides which are capable of delivering the b-glucuronidase polypeptide into a cell. In addition, the b- ghicuronidase polypeptide can be delivered directly into a cell by microinjection.

E. Crosslinked Polypeptide Crystals In another embodiment, the formulations of the invention include crosslinked polypeptide crystals of the β-glucuronidase polypeptide of the invention. This approach includes crosslinked enzyme crystal ("CLEC™) technology (N. L. St. Clair and M. A., Navia, J. Am. Chem. Soc, 11.4, pp. 4314-16 (1992)) and is described in U.S. Patent Application Publ. No. 20040202643. Crosslinked enzyme crystals may retain their activity in environments that are normally incompatible with enzyme function. Such environments include prolonged exposure to proteases and other protein digestion agents, high temperature or extreme pH. In such environments, crosslinked enzyme crystals remain insoluble and stable.

Protein crystals are grown by controlled crystallization of protein out of aqueous solution or aqueous solution-containing organic solvents. Conditions to be controlled include, for example, the rate of evaporation of solvent, the presence of appropriate co- solutes and buffers, pH and temperature. A comprehensive review of the various factors affecting the crystallization of proteins has been published by McPherson, Methods Enzymol., 114, pp. 112-20 (1985).

McPherson and Gilliland, J. Crystal Growth, 90, pp. 51-59 (1988) have compiled comprehensive lists of proteins and nucleic acids that have been crystallized, as well as the conditions under which they were crystallized. A compendium of crystals and crystallization recipes, as, well as a repository of coordinates of solved protein and nucleic acid structures, is maintained by the Protein Data Bank at the Brookhaven National Laboratory [http/Λvww. pdb.bnl.gov; Bernstein et al., J. MoI. Biol., 112, pp. 535-42 (1977)]. These references can be used to determine the conditions necessary for crystallization of a protein, as a prelude to the formation of an appropriate crosslinked protein crystal, and can guide the crystallization strategy for other proteins. Alternatively, an intelligent trial and error search strategy can, in most instances, produce suitable crystallization conditions for many proteins, provided that an acceptable level of purity can be achieved for them [see, e.g., C. W. Carter, Jr. and C. W. Carter, J. Biol. Chem., 254, pp. 12219-23 (1979)].

In general, crystals are produced by combining the protein to be crystallized with an appropriate aqueous solvent or aqueous solvent containing appropriate crystallization agents, such as salts or organic solvents. The solvent is combined with the protein and subjected to agitation at a temperature determined experimentally to be appropriate for the induction of crystallization and acceptable for the maintenance of protein activity and stability. The solvent can optionally include co-solutes, such as divalent cations, co- factors or chaotropes, as well as buffer species to control pH. The need for co-solutes and their concentrations are determined experimentally to facilitate crystallization. In an industrial-scale process, the controlled precipitation leading to crystallization can best be carried out by the simple combination of protein, precipitant, co-solutes and, optionally, buffers in a batch process. Alternative laboratory crystallization methods, such as dialysis or vapor diffusion, can also be adopted. McPherson, supra and Gilliland, supra, include a comprehensive list of suitable conditions in their reviews of the crystallization literature. Occasionally, incompatibility between the crosslinking agent and the crystallization medium might require exchanging the crystals into a more suitable solvent system.

Many of the proteins for which crystallization conditions have already been described, may be used to prepare crosslinked protein crystals according to this invention. It should be noted, however, that the conditions reported in most of the above-cited references have been optimized to yield, in most instances, a few large, diffraction quality crystals. Accordingly, it will be appreciated by those of skill in the art that some degree of adjustment of these conditions to provide a high yielding process for the large scale production of the smaller crystals used in making crosslinked protein crystals may be necessary.

Once protein crystals have been grown in a suitable medium they can be crosslinked. Crosslinking results in stabilization of the crystal lattice by introducing covalent links between the constituent protein molecules of the crystal. This makes possible transfer of.the protein into an alternate environment that might otherwise be incompatible with the existence of the crystal lattice or even with the existence of intact protein.

Advantageously, crosslinking is carried out in such a way that, under conditions of storage, the crosslinking interactions prevent the constituent protein molecules in the crystal from going back into solution, effectively insolubilizing or immobilizing the protein molecules into microcrystalline particles. Upon exposure to a trigger in the environment surrounding the crosslinked protein crystals, such as under conditions of use rather than storage, the protein molecules dissolve, releasing their protein activity. The rate of dissolution is controlled by one or more of the following factors: the degree of crosslinking, the length of time of exposure of protein crystals to the crosslinking agent, the rate of addition of crosslinking agent to the protein crystals, the nature of the cross linker, the chain length of the crosslinker, the surface area of the crosslinked protein crystals, the size of the crosslinked protein crystals, the shape of the crosslinked protein crystals and combinations thereof.

Crosslinking can be achieved using one or a combination of a wide variety of multifunctional reagents, at the same time (in parallel) or in sequence, including bifunctional reagents. Upon exposure to a trigger in the surrounding environment, or over a given period of time, the crosslinks between protein crystals crosslinked with such multifunctional crosslinking agents lessen or weaken, leading to protein dissolution or release of activity. Alternatively, the crosslinks may break at the point of attachment, leading to protein dissolution or release of activity. Such crosslinking agents include glutaraldehyde, succinaldehyde, octanedialdehyde and glyoxal. Additional multifunctional crosslinking agents include halo-triazines, e.g., cyanuric chloride; halo- pyrimidines, e.g., 2,4,6-trichloro/bromo-pyrimidine; anhydrides or halides of aliphatic or aromatic mono- or di-carboxylic acids, e.g., maleic anhydride, (meth)acryloyl chloride, chloroacetyl chloride; N-methylol compounds, e.g., N-methylol-chloro acetamide; di- isocyanates or di-isothiocyanates, e.g., phenylene-l,4-di-isocyanate and aziridines. Other crosslinking agents include epoxides, such as, for example, di-epoxides, tri- epoxides and tetra-epoxides. According to a preferred embodiment of this invention, the crosslinking agent is glutaraldehyde, used alone or in sequence with an epoxide. For a representative listing of other available crosslinking reagents see, for example, the 1996 catalog of the Pierce Chemical Company. Such multifunctional crosshnking agents may also be used, at the same time (in parallel) or in sequence, with reversible crosslinking agents, such as those described below.

According to an alternate embodiment of this invention, crosslinking may be carried out using reversible crosslinkers, in parallel or in sequence. Examples of reversible crosslinkers are described in T. W. Green, Protective Groups in Organic ,_; Synthesis, John Wiley & Sons (Eds.) (1981). Any variety of strategies used for reversible protecting groups can be incorporated into a crosslinker suitable for producing crosslinked protein crystals capable of reversible, controlled solubilization. Various approaches are listed, in Waldmann's review of this subject, in Anaewante Chemie InI. Ed. Engl., 35, p. 2056 (1996).

Other types of reversible crosslinkers are disulfide bond-containing crosslinkers. The trigger breaking crosslinks formed by such crosslinkers is the addition of reducing agent, such as cysteine, to the environment of the crosslinked protein crystals. Disulfide crosslinkers are described in the Pierce Catalog and Handbook (1994-1995). Examples of such crosslinkers include: Homobifunctional (Symmetric); DSS~Dithiobis (succinimidylpropionate), also know as Lomant's Reagent; DTSSP--3-3¹- Dithiobis(sulfosuccinimidylpropionate), water soluble version of DSP; DTBP—Dimethyl 3,3'-dithiobispropionimidate.HCl; BASED~Bis-(β-[4-azidosalicylamido]ethyl)disulfide; DPDPB-- 1 ,4-Di-(3'-[2^l-pyridyldithio]-propionamido)butane. Heterobifunctional (Asymmetric) crosslinkers include: SPDP~N-Succinimidyl-3-(2-pyridyldithio) propionate; LC-SPDP~Succinimidyl-6-(3 -[2-pyridyldithio]propionate)hexanoate; Sulfo- LC-SPDP~Sulfosuccinimidyl-6-(3 -[2-pyridyldlthio]propionate)h- exanoate, water soluble version of LC-SPDP; APDP~N-(4-[p-azidosalicylamido]butyl)-3'-(2'- pyridyldithio)propion- amide; SADP~N-Succinimidyl(4-azidophenyl)l,3'- dithiopropionate; Sulfo-S ADP~Sulfosuccinimidyl(4-azidophenyl) 1 ,3'-dithiopropionate, water soluble version of SADP; SAED~Sulfosuccinimidyl-2-(7-azido-4- methycoumarin-3-acetamide)ethy-l-l,3'dichiopropionate; SAND~Sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido)ethyl-l,3'-dith- iopropionate; SASD~Sulfosuccinimidyl-2- (p-azidosalicylamido)ethyl-l,3'-dithiopro- pionate; SMPB--Succinimidyl-4-(p- maleimidophenyl)butyrate; Sulfo-SMPB~Sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate; SMPT- 4-Succinimidyloxycarbonyl-methyl-α-(2-pyridylthio)toluene; Sulfo- LC-SMPT~Sulfosuccinimidyl-6-(α-methyl-α-(2-pyridylthio)toluamido)hexanoate.

F. Gastrointestinal Site Directed Formulations

In addition to the enteric coatings and other formulations described above, in one embodiment, the oral formulation of the β-glucuronidase polypeptides of the invention for gastrointestinal delivery may comprises an adhesion site-controlling layer for attaching the formulation to a selected site in the digestive tract, a polypeptide-carrying layer for containing the polypeptide and an adhesive and a protecting layer for protecting the polypeptide in the polypeptide-carrying layer. These types of formulations are referred to herein as "Gastrointestinal Site Directed Formulations" (or "GISD formulations"). In GISD formulations of the present invention, the polypeptidercarrying layer is located between the protecting layer and the adhesion site-controlling layer. Furthermore, the adhesion site-controlling layer may attach directly to the protecting layer. These types of formulations are described in general detail in U.S. Patent Application Pub. No. 20050069591.

When the GISD formulations are orally administered, the adhesion site- controlling layer dissolves at an unique site in the digestive tract. The site of the mucosal membrane of the digestive tract, to which the polypeptide-carrying layer attaches, is controlled by such dissolution of the adhesion site-controlling layer at an unique site in the digestive tract. After the oral administration of the GISD formulation, the protecting layer prevents digestive juice from permeating into the polypeptide- carrying layer and the polypeptide -carrying layer from releasing the polypeptide, and also prevents digestive juice and digestive enzymes from permeating into the polypeptide -carrying layer after the formulation attaches to the mucosal membrane of the digestive tract. The bioavailability of the polypeptide orally administered is improved by the protection of the polypeptides from the attack of digestive enzymes and the retention of the concentration gradient over a long period of time.

The adhesion site-controlling layer of the GISD formulation comprises a substance which prevents the drug from burst-releasing in the early phase and dissolves at a specific patch (adhesion) site selected in the digestive tract. Typically, the adhesion site-controlling layer is made of a pH dependent enteric polymer such as hydroxypropyl methylcellulose phathalate (HP-55^®), methacrylic copolymer (Eudragit^® L), methacrylic copolymer-LD (Eudragit^® LD) and methacrylic copolymer-S (Eudragi^® S).

The polypeptide-carrying layer is an intermediate layer which exists between the protecting layer and the adhesion site-controlling layer. The polypeptide-carrying layer contains a polypeptide and an adhesive. The adhesive is used for attaching the polypeptide -carrying layer to the mucosal membrane of the digestive tract when the adhesion site-controlling layer dissolves at a site selected in the digestive tract. The adhesive for attaching the polypeptide-carrying layer to the mucosal membrane of the digestive tract may be prepared by mixing a plasticizer to polymers or gums such as carboxyvinyl polymer, acrylate/octyl acrylate copolymer, 2-ethylhexyl acrylate/vinylpyrrolidone copolymer, acrylate silkbroin copolymer resin, methyl acrylate/2-ethylhexyl acrylate copolymer resin, gum arabic, poly(vinyl alcohol), polyvinylpyrrolidone, methylcellulose, polyisoprene, polyacrylate, and sodium polyacrylate, followed by kneading the mixture with water. For example, an adhesive may be prepared by kneading 0.8 g of Hiviswako^® 103, 250 ul of PEG 400 and 2 ml of 5 purified water. • ...., . When a support is used as the polypeptide-carrying layer, examples of the support include porous substrate soaked with a polypeptide such as polyester fiber, thin cloth, tissue paper, and synthetic paper or film made of a synthetic cellulose polymer or an enteric polymer. When a polypeptide layer is used as the polypeptide -carrying layer,0 the gel layer may be prepared by mixing an aqueous solution of a polypeptide, powders of a polypeptide, or a solid dispersion of a polypeptide, or a micro- or nano-encapsuled drug with a concentrated solution of a gel-forming polymer such as carboxyvinyl polymer. Instead of the gel layer, there may be used a hydrophilic wax layer prepared by adding a polypeptide and an adhesive such as Hiviswako 103^® to a hydrophilic wax 5 such as polyethylene glycol 400 (PEG 400). When nano- or micro-encapsulation is applied to the polypeptide, a use of hydroxypropyl methylcellulose phthalate (HP-55¹^ as a nano- or microcapsule wall gives rapid release of the polypeptide after attaching to the gastrointestinal wall.

In addition, by formulating the absorption promoter such as polyoxyethylated0 castor oil derivatives, capric acid and ursodeoxycholic acid etc. into the polypeptide - carrying layer, the bioavailability of the polypeptide can be further improved. Alternatively, by formulating protease inhibitors such as aprotinin in the polypeptide - carrying layer, hydro lytic degradation of the polypeptide can be effectively inhibited and bioavailability of the polypeptide can also be improved. 5 As explained below, the polypeptide-carrying layer used in the GISD formulations of the polypeptides of the present invention may be in the form of film or may exist in the inner space of the hemispherical form made of the protecting layer.

The protecting layer (backing layer) functions for protecting the polypeptide in the polypeptide -carrying layer and is in the form of a film or a wall, for example a0 hemispheric form, which is made of a water-insoluble polymer, a wax, or a mixture thereof to inhibit permeation of the polypeptide into the polypeptide -carrying layer and digestive enzymes. The protecting layer may be prepared, for example, by using a water-insoluble pharmaceutical polymer such as ethylcellulose, aminoalkylmethacrylate copolymer (Eudragit^® RS), cellulose acetate, chitin and chitosan, or a wax such as5 stearic acid, stearyl alcohol, white beeswax, cacao butter, hard fat, purified shellac, polyoxyl 40 stearate, cetanol and polyoxyethyl lauryl ether. According to the present invention, the polypeptide-carrying layer exists between the protecting layer and the adhesion site-controlling layer. Examples of those wherein the polypeptide -carrying layer exists between the protecting layer and the adhesion site- controlling layer include (1) where the adhesion site-controlling layer, the polypeptide - carrying layer and the protecting layer are in the form of film respectively, and these three layers are laminated in order (hereinafter referred to as the formulation according to the first embodiment) and (2) where the protecting layer is in the hemispherical form, the polypeptide -carrying layer exists in the inner space of the hemispherical form, and the adhesion site-controlling layer covers the opening part of the hemisphere (hereinafter referred to as the formulation according to the second embodiment).

In order to prepare the GISD formulations, a film for the protecting layer is formed by using a water-insoluble polymer or a wax mentioned above. More specifically, a water-insoluble polymer or wax is dissolved in an organic solvent such as ethanol, the resulting solution is cast in a Teflon flame, and the solvent is evaporated. For example, 550 mg of ethylcellulose and 150 μl, of triethyl citrate are dissolved in 5 ml of a mixture of methylene chloride and methanol (4: 1), and the resulting solution is cast on a Teflon plate.

Next, as explained above, the polypeptide-carrying layer is formed on the protecting layer. An adhesive may be applied on the protecting layer and then the support containing a polypeptide is attached thereon to form the polypeptide -carrying layer, or alternatively, a polypeptide and an adhesive (such as gel forming polymers) may be mixed and then applied on the protecting layer.

As the adhesion site-controlling layer, a film from having from 20 to 100 μm thickness that is made of an enteric polymer as explained above may be used. For example, 225 mg of HP-55^R (Shin-etsu Chemical Ind. Co. Ltd.) and 25 μl of triethyl citrate are dissolved in 5 ml of a mixture of methylene chloride and methanol (4: 1), and the resulting solution is cast on a Teflon plate to form a film. As another example, a film may be used which is prepared by dissolving 225 mg of Eudragit^® SlOO or Eudragit LlOO and 150 μl of triethyl citrate in 5 ml of a mixture of methylene chloride and methanol (4: 1) and casting the resulting solution on a Teflon plate. One GISD formulation of the invention may be prepared by attaching such a film on the polypeptide-carrying layer with an adhesive and the like and cutting the three-layered film into an appropriate size. In addition, in order to prevent a leak of a polypeptide from the edges of the polypeptide -carrying layer between the protecting layer and the adhesion site-controlling layer, the formulation may be sealed by sprinkling a water- insoluble substance such as stearic acid fine powders. The size of the film patch GISD formulations of the invention are not limited so far that the formulation can be filled into a gelatin capsule or an enteric capsule made of an enteric polymer. For example, the size may be a square of 3x3 mm and a circle of 5 mm in diameter. In order to prevent adhesion or sticking of the films to each other when filled into a capsule in piles, the lubrication treatment is preferably applied by sprinkling magnesium silicate powders. . .

In order to prepare other GISD formulations of the invention, the protecting layer in the hemispherical form may be prepared by cutting minicapsules or microcapsules made of a water-insoluble polymer or a wax into half. The minicapsules or microcapsules are prepared by using a water-insoluble polymer such as ethylcellulose or Eudragit^® RSlOO in a conventional method, and then the resulting capsules are cut at the center into two pieces, and the inside is scraped off to prepare hollow half-minicapsules or half-microcapsules in the hemispherical (bowl) form, which serves as the protecting layer. For preparing the polypeptide -carrying layer, a polypeptide and a gel forming polymer are mixed and filled into the half-minicapsules or half-microcapsules. The adhesion site-controlling layer may be formed by attaching a film made of an enteric polymer on the upper part of the half-minicapsules or half-microcapsules using a gel forming polymer glue so as to cap the half-capsules. Alternatively, the minicapsules or microcapsules which contain a polypeptide and an adhesive polymer, may be prepared by using a water-insoluble polymer such as ethylcellulose or Eudragit^® RSlOO in a conventional method, and the resulting capsules are cut at the center into two pieces. Then, the adhesion site-controlling layer may be formed by attaching a film made of an enteric polymer on the upper part of the half-minicapsules or half-microcapsules using a gel forming polymer glue so as to cap the half-capsules. Alternatively, in order to prepare the GISD formulations of the invention in a large amount, a film is formed by using a water-insoluble polymer or a wax in a similar manner to that in preparing the protecting layer used in the formulations in the first embodiment. The resulting film is put on a thorny object having many projections of the micron order regularly arranged. A metal mold prepared by micro-machine techniques may also be used as an object on which the film is put. The film is allowed to stand under heating to a high temperature for a few hours, and then cooled to prepare a film with many cavities in the form of a micro-container having the depth of from 50 to 500 μm and the caliber of from 20 to 800 μm.

For filling a polypeptide into the micro-container-like cavities formed on the film, two ways of the solid-phase method and the liquid-phase method may be used. In the solid-phase method, a polypeptide and an adhesive are mixed and then the resulting mixture is filled into the micro-container-like cavities at a fixed amount under the solid condition. In the liquid-phase method, an adhesive is injected into the cavities by means of a solid-phase method, and a polypeptide solution is injected into the cavities by a microinjector.

Then, the adhesion site-controlling layer (a film made of an enteric polymer) is provided so as to cover the upper part of the micro-container- like cavities. The adhesion site-controlling layer and the film having micro-container-like cavities may be adhered to each other by previously applying an adhesive on the adhesion site-controlling layer, and applying to the film having micro-container-like cavities filled with the polypeptide. The adhesion site-controlling layer may be adhered to the film having micro-container- like cavities filled with the polypeptide, by heating press methods, when the polypeptide is heat-resistant.

IX. Methods of Administration of the β-glucuronidase β-glucuronidase may be administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo or on a surface to treat inflammation. By "biologically compatible form suitable for administration in vivo" is meant a form of the polypeptide to be administered in which any toxic effects are outweighed by the therapeutic effects of the polypeptide. Administration of an agent as described herein can be in any pharmacological form including a therapeutically active amount of a an agent alone or in combination with a pharmaceutically acceptable carrier. Oral administration of a composition comprising β-glucuronidase is preferred. In certain embodiments, e.g., for oral administration, the subject's stomach acid may be neutralized or suppressed using methods known in the art prior to administration of an agent of the invention. In such cases, the agent may be formulated in microcrystalline form or as a powder.

Stomach acid may be supressed prior to, concurrently with, or after administration of β-glucuronidase using methods known to those of ordinary skill in the art. These methods include the administration of drugs that result in the suppression of stomach acid (e.g. a proton pump inhibitor (PPI) and famotidine, omeprazole magnesium, a histamine-2 receptor antagonists (H2RAs or H2-blockers)).

The term "subject" includes animals, human or non-human, rodent or non- rodent, to whom treatment according to the methods of the present invention is provided. The term includes but is not limited to birds, reptiles, amphibians, and mammals, e.g., humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep, bears, and goats. Preferred subjects are humans, farm animals, and domestic pets such as cats and dogs. The term "treated," "treating" or "treatment" includes therapeutic and/or prophylactic treatment. The treatment includes the diminishment or alleviation of at least one symptom associated or caused by inflammation, the state, disorder or disease being treated. For example, treatment can be diminishment of one or several symptoms of a inflammation or complete eradication of the inflammation or other disorder treatable by the compositions of the invention. Persons of ordinary skill in the art will appreciate that a subject can be diagnosed by a physician (or veterinarian, as appropriate for the patient being diagnosed) as suffering from or at risk for a condition described herein by any method known in the art, e.g., by assessing the subject's medical history, performing diagnostic tests, and/or by employing imaging techniques. The compositions described herein can be administered (and/or administration can be supervised) by any person, e.g., a health-care professional, veterinarian, or caretaker (e.g., an animal (e.g., dog or cat) owner), depending upon the subject to be treated, and/or by the subject him/herself, if the subject is capable of self-administration. Administration of a therapeutically active amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result, e.g., sufficient to increase bilirubin or biliverdin levels or to treat one or more symptoms of a disease or disorder. For example, a therapeutically active amount of an agent may vary according to factors such as the disease state, age, sex, reproductive state, and weight of the individual, and the ability of an agent to elicit a desired response in the individual. Dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The language "effective amount" of an agent or other compound of the invention is that amount necessary or sufficient to treat or prevent inflammation or treat another disorder described, e.g. prevent the various morphological and somatic symptoms of the particular disorder. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular an agent. For example, the choice of the an agent can affect what constitutes an "effective amount". One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of an agent without undue experimentation.

The regimen of administration can affect what constitutes an effective amount. The agent can be administered to the subject either prior to or after the onset of inflammation. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. In one embodiment, a composition comprising b-glucuronidase is administered about 4-6 times per day. Further, the dosages of an agent can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. In a further embodiment, the effective amount an agent is effective to increase bilirubin levels, treat inflammation, or treat other disorders treatable by the elevation of heme degradation compounds, such as bilirubin and biliverdin. The levels of the bilirubin may be increased in any portion of the subject which results in the treatment of inflammation, as compared with bilirubin levels prior to administration of the β- glucuronidase polypeptide.

In a further embodiment, the agent can be administered to a subject to generate serum levels of bilirubin of at least about 0.01 mg/dl, about 0.1 mg/dl, 0.25 mg/dl, about 0.5 mg/dl, about 0.75 mg/dl, about 1 mg/dl, about 5 mg/dl, about 10 mg/dl, about 20 mg/dl, about 30 mg/dl, about 40 mg/dl, about 50 mg/dl, about 120mg/dl, about 130 mg/dl, about 140 mg/dl, about 150 mg/dl, about 160 mg/dl, about 200 mg/dl or greater. In a further embodiment, the term includes levels of bilirubin in a subject in the range of from about 0.01 to about 300 mg/dl, e.g., about 0.1 to about 200 mg/dl, or about 1 to about 100 mg/dl, or about 1 to about 10 mg/dl.

X. Methods of Treatment Using the Agents of the Invention

The invention also pertains, at least in part, to therapeutic applications of the agents of the invention polypeptide and compositions comprising the peptide. Increased levels of bilirubin are of benefit in treating diseases or disorders of organs or tissues other than the brain. In a further embodiment, the invention pertains to a method for increasing the level of bilirubin in a subject, by administering to the subject an effective amount of an agent of the invention or a pharmaceutical composition of the invention comprising an agent. In yet another embodiment, the invention pertains to administering to the subject an effective amount of an agent of the invention or a pharmaceutical composition of the invention such that endogenous levels of bilirubin are increased. In yet another embodiment, the invention pertains to administering to the subject an effective amount of an agent of the invention or a pharmaceutical composition of the invention such that at least one symptom of a disease or disorder that would benefit from increased levels of bilirubin is ameliorated.

In therapeutic methods of the invention, the compositions of the inventions (which comprise an agent of the invention) are administered to a subject suffering from one or more symptoms of a disease or disorder that would benefit from increased levels of bilirubin and/or biliverdin (or at risk for developing such a disease or disorder) in an amount sufficient to ameliorate at least one symptom of the disease or disorder, that would cure the disease or disorder, or that would at least partially prevent or arrest the development of the disease or disorder. An amount adequate to accomplish this is defined as a "therapeutically effective dose" or "prophylactically effective dose." Effective amounts will depend upon the severity of the disease and the general state of the subject's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agent of this invention to effectively treat (ameliorate one or more symptoms) or prevent/arrest development of symptoms in the subject.

The concentration of the agent can vary widely, and can easily be selected by one of skill in the art based on the subject's needs. Agents may be administerd once per day or on multiple occasions during the day. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 or 1 mg/kg/day to about 500 ^■ mg/kg/day and sometimes higher. Typical dosages of half bilirubin molecules range from about 5 mg/kg/day to about 10 mg/kg/day, preferably from about 25 mg/kg/day to about 50 mg/kg/day, more preferably from about 50 mg/kg/day to about 100 mg/kg/day, and most preferably from about 100 mg/kg/day to about 500 mg/kg/day. In certain preferred embodiments, dosages range from about 100 mg/kg/day to about 200 mg/kg/day. It will be appreciated that such dosages may be varied to optimize a therapeutic regimen in a particular subject or group of subjects.

It should be noted that the the agent may be administered in a regime sufficient to treat a chronic or acute condition. If the condition is a chronic condition, the agent may be administered over a long period of time to amelerate or treat the disorder. If the condition is acute, the agent maybe administered until the symptoms and/or condition is ameliorated. This may occur over a short period of time (e.g., a day or a week) or a longer period of time (e.g., several weeks to several years or longer).

Furthermore, in one embodiment, the compositions of the invention may be advantageously administered at the time of a meal. Not to be limited by theory, but it is believed that administration after a meal may maximize the hydrolytic effects on bilirubin conjugates following gall bladder contraction.

The agent may be administered to the subject in a dosage unit form. The term "dosage unit form" includes physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of the polypeptide calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a polypeptide for the treatment of sensitivity in individuals. The specific dose can be readily calculated by one of ordinary skill in the art, e.g., according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in assay preparations of target cells. Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration.

Toxicity and therapeutic efficacy of such compositions of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions which exhibit large therapeutic indices are preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the method for the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. XI. Combination Therapy

In a further embodiment, β-glucuronidase is administered in combination with a bilirubin oxidase polypeptide or with another agent, e.g., one capable of converting bilirubin into biliverdin. Bilirubin oxidase may be formulated into the same pharmaceutical composition as the agent, or administered consecutively or simultaneously in different pharmaceutical compositions. The bilirubin oxidase polypeptide may also be linked chemically (e.g., through a covalent, ionic, hydrophobic or through other suitable linkages) to the agent. In a further embodiment, the bilirubin oxidase polypeptide is conjugated covalently to the agent. The bilirubin oxidase can be formulated in the same or different formulation as the agent of the invention. In one embodiment, the both the agent and the bilirubin oxidase are formulated as crosslinked proteins. In another embodiment, the proteins are encapsulated using encapsultation technology or combined in an enterically coated formulation. The invention contemplates the any combination of possible formulations of the bilirubin oxidase and the agent.

In another further embodiment, the invention also pertains, at least in part, to a method for increasing the level of biliverdin in a subject. The method includes administering to the subject an effective amount of an agent of the invention in combination with a bilirubin oxidase polypeptide, e.g., bilirubin oxidase. In another embodiment, the invention pertains to administering to a subject an effective amount of an agent of the invention in combination with biliverdin or bilirubin.

In a further embodiment, the effective amount of the agent is effective to increase biliverdin levels and/or treat inflammation, as compared with biliverdin levels prior to administration of the agent and bilirubin oxidase polypeptide. In another embodiment, β glucuronidase may be administered in combination with an art recognized agent useful for treatment of a particular disease or disorder that would benefit from increased bilirubin levels, .e.g, an anti-inflammatory agent.

In yet another embodiment, β glucuronidase is administered in combination with an agent that increases hemoxygenase-1 (HO-I) using methods known in the art.

Xn. Measurement of Bilirubin/Biliverdin Levels

The levels of bilin compounds in a subject can be determined using methods known in the art. Techniques which can be used to quatitate the amount of bilirubin and biliverdin include, but are not limited to, spectroscopy (including, but not limited, UV, visual, IR, NMR, mass, etc.) and chromatography (gas, HPLC, etc). Bilirubin levels also may be tested using commercial methods. The bilirubin/biliverdin levels may be measured at any point after administration of the compound, agent or composition of the invention. For example, the level of bilirubin/biliverdine may be measured about 30 minutes, about one hour, about two hours, about three hours, about four hours, about five hours, about six hours, about seven hours, about eight hours, about nine hours or about ten hours after administration.

XHL Diseases and Disorders

The present invention is useful for the treatment of diseases and/or disorders that would benefit from the presence of increased levels of biliverdin and/or bilirubin. Examples of such disorders include, but are not limited to, inflammatory disorders, respiratory disorders, cardiovascular disorders, renal disorders, hepatobiliary disorders, reproductive disorders, gastrointestinal disorders, shock, cellular proliferative disorders, cellular differentiative disorders, and for the reducution of the effects of ischemia; and to aid in wound healing. In another embodiment, the present invention is useful for the treatment of diseases or disorders in which apoptosis plays a role.

Examples of such diseases and disorders which are treatable using the methods and compositions of the invention include:

A. Inflammation

The methods and compositions of the present invention can be used to treat inflammatory disorders. The terms "inflammatory disorder(s)" and "inflammation" are used to describe the fundamental pathological process consisting of a dynamic complex of reactions (which can be recognized based on cytologic and histologic studies and in other ways) that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical or biologic agent, including the local reactions and resulting morphologic changes, the destruction or removal of the injurious material, and the responses that lead to repair and healing. Inflammation is characterized in some instances by the infiltration of immune cells (e.g., neutrophils, monocytes/macrophages, natural killer cells, lymphocytes (e.g., B and T lymphocytes)). In addition, inflamed tissue may contain cytokines and chemokines that are produced by the cells that have infiltrated into the area. Often, inflammation is accompanied by thrombosis, including both coagulation and platelet aggregation. The term inflammation includes various types of inflammation such as acute, chronic, allergic (including conditions involving mast cells), degenerative, atrophic, catarrhal (most frequently in the respiratory tract), croupous, fibrinopurulent, fibrinous, immune, hyperplastic or proliferative, subacute, serous and serofibrinous inflammation. Inflammation localized in the gastrointestinal tract, or any portion thereof, liver, heart, skin, spleen, brain, kidney, pulmonary tract, and the lungs can be treated with the methods of the present invention. Inflammation associated with shock, e.g., septic shock, hemorrhagic shock caused by any type of trauma, and anaphylactic shock can also be treated. Further, it is contemplated that the methods of the present invention could be used to treat rheumatoid arthritis, lupus, and other inflammatory and/or autoimmune.., diseases; heightened inflammatory states due to immunodeficiency, e.g., due to infection with HIV; and hypersensitivities.

The inflammation can be associated with a condition selected from the following group: asthma, adult respiratory distress syndrome, interstitial pulmonary fibrosis, pulmonary emboli, chronic obstructive pulmonary disease, primary pulmonary hypertension, chronic pulmonary emphysema, congestive heart failure, peripheral vascular disease, stroke, atherosclerosis, ischemia-reperfusion injury, heart attacks, glomerulonephritis, conditions involving inflammation of the kidney, infection of the genitourinary tract, viral and toxic hepatitis, cirrhosis, ileus, necrotizing enterocolitis, specific and non-specific inflammatory bowel disease, rheumatoid arthritis, deficient wound healing, graft versus host disease, and hemorrhagic, septic, or anaphylactic shock.

Furthermore, the inflammation of the invention includes inflammation of the heart, lung, liver, pancreas, joints, eye, bronchi, spleen, skin, and/or kidney. The inflammation can also be an inflammatory condition localized in the gastrointestinal tract, e.g., amoebic dysentery, bacillary dysentery, schistosomiasis, Campylobacter enterocolitis, yersinia enterocolitis, enterobius vermicularis, radiation enterocolitis, ischaemic colitis, eosinophilic gastroenteritis, ulcerative colitis, indeterminate colitis, and Crohn's disease. Alternatively, it can be a systemic inflammation.

The methods of the invention may be also used to promote wound healing (e.g., in transplanted, lacerated (e.g., due to surgery), or burned skin). The compounds of the invention may be, for example, applied locally to the wound (e.g., as a wound dressing, lotion, or ointment) or delivered systemically. Inflammation associated with certain reproductive disorders, e.g., impotence and/or inflammation associated with sexually transmitted diseases may also be treated using the methods of the invention. The methods of the invention may be used to prevent premature uterine contractions, and may be used to prevent premature deliveries and menstrual cramps.

B. Tissue Damage Resulting From Transplantation In another embodiment, the invention features a method of transplanting an organ, tissue, or cells, which includes administering to a donor (or to an organ of the donor in situ) a pharmaceutical composition comprising an agent the invention, and transplanting an organ tissue or cells of the donor into a recipient, wherein the agent is administered in an amount sufficient to enhance survival or function of the transplant after.transplantation into the recipient.

The invention also features a method of transplanting an organ, tissue, or cells, which includes (a) providing an organ, tissue, or cells of a donor; (b) administering to the organ, tissue, or cells ex vivo a pharmaceutical composition comprising the agent; and (c) transplanting the organ, tissue, or cells into a recipient, wherein the agent is administered in an amount sufficient to enhance survival or function of the transplant after transplantation. In one embodiment, the donor is treated in addition to or in lieu of treating the cells to be transplanted.

Further, the invention features a method of transplanting an organ, tissue, or cells, which includes providing an organ, tissue or cells from a donor, transplanting the organ, tissue or cells into a recipient, and before, during, or after step the transplanting step, administering to the recipient a pharmaceutical composition comprising an agent of the inention; wherein the agent is administered in an amount sufficient to enhance survival or function of the organ after transplantation of the organ to the recipient.

C. Tissue Damage or Injury Associated with Angioplasty

The invention also provides a method of performing angioplasty on a subject, which includes performing angioplasty on the patient; and before, during, or after the performing step, administering to the patient a pharmaceutical composition comprising an agent of the invention. The agent is administered in an amount sufficient to reduce (e.g., prevent) intimal hyperplasia in the subject. The angioplasty can be any angioplasty procedure, e.g., balloon angioplasty; laser angioplasty; artherectomy, e.g., directional atherectomy, rotational atherectomy, or extraction atherectomy; and/or any angioplasty procedure using a stent, or any combination of such procedures.

D. Treatment of Restenosis or Intimal Hyperplasia

The invention also provides a method of treating (e.g., preventing or decreasing) restenosis or intimal hyperplasia in a subject. The method includes administering to a subject diagnosed as suffering from or at risk for restenosis a pharmaceutical composition comprising an agent of the invention. The intimal hyperplasia or restenosis can be caused by balloon angioplasty; laser angioplasty; artherectomy, e.g., directional atherectomy, rotational atherectomy, or extraction atherectomy; and/or any angioplasty procedure using a stent, or any combination of such procedures.

£. Tissue Damage Associated with Surgery The invention also features a method of performing surgery (e.g., other than

-transplant surgery) e.g., vascular and/or abdominal surgery, on a subject, .which includes performing surgery on the subject; and before, during, and/or after performing the surgery, administering to the subject a pharmaceutical composition comprising an agent of the invention.

F. Treatment of a Cellular Proliferative and/or Differentiative Disorder The invention features a method of treating a cellular proliferative and/or differentiative disorder (e.g., naturally arising cancer) in a subject, which includes identifying a subject suffering from or at risk for a cellular proliferative and/or differentiative disorder (e.g., naturally arising cancer); and administering to the subject a pharmaceutical composition comprising the agent of the invention, in an amount sufficient to treat the cellular proliferative and/or differentiative disorder.

Examples of cellular proliferative and/or differentiative disorders include, but are not limited to, carcinoma, sarcoma, metastatic disorders, and hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

G. Treatment of Cancer Any type of cancer can be treated using the methods described herein. The cancer can be cancer found in any part(s) of the subject's body, e.g., cancer of the stomach, small intestine, colon, rectum, mouth/pharynx, esophagus, larynx, liver, pancreas, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder, skin, kidney, central nervous system, head, neck, throat, bone, or any combination thereof. The cancer may also be a hematopoietic disorder, such as leukemia.

The term "cancer" refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. Cancer that is "naturally arising" is any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen(s), cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation. The term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

Cancers that may be treated using the methods and compositions of the present invention include, for example, cancers of the stomach, colon, rectum, mouth/pharynx, esophagus, larynx, liver, pancreas, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder, skin, kidney, central nervous system, head, neck and throat; Hodgkins disease, non-Hodgkins leukemia, sarcomas, choriocarcinoma, and lymphoma, among others. For cancer treatment, the methods can be used alone or in combination with other methods for treating cancer in patients. Accordingly, in another embodiment, the methods described herein can include treating the subject using surgery (e.g., to remove a tumor or portion thereof), chemotherapy, immunotherapy, gene therapy, and/or radiation therapy. Treatments described herein can be administered to a patient at any point, e.g., before, during, and/or after the surgery, chemotherapy, immunotherapy, gene therapy, and/or radiation therapy.

H. Reduction of Angiogenesis

The methods of the present invention can also be used to inhibit unwanted (e.g., detrimental) angiogenesis in a patient and to treat angiogenesis dependent/associated conditions associated therewith. The term "angiogenesis" includes the generation of new blood vessels in a tissue or organ. An "angiogenesis dependent/associated condition" includes any process or condition that is dependent upon or associated with angiogenesis. The term includes conditions that involve cancer, as well as those that do not. Angiogenesis dependent/associated conditions can be associated with (e.g., arise from) unwanted angiogenesis, as well as with wanted (e.g., beneficial) angiogenesis. The term includes, e.g., solid tumors; tumor metastasis; benign tumors, e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis, lupus, and other connective tissue disorders; psoriasis; rosacea; ocular angiogenic diseases, e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation. Other processes in which angiogenesis is involved include reproduction and wound healing. In another aspect, the invention features a method of treating unwanted angiogenesis in a subject. The method includes administering to a subject diagnosed as suffering from or at risk for unwanted angiogenesis a pharmaceutical composition comprising a agent in an amount sufficient to treat unwanted angiogenesis.

I. Treatment of Hepatitis

The invention also features a method of treating hepatitis in a subject. The method includes administering to a subject diagnosed as suffering from or at risk for hepatitis: (i) a pharmaceutical composition comprising a agent in an amount sufficient to treat hepatitis. The hepatitis can be the result of, or a subject may be considered at risk for hepatitis because of, any of a number of factors, e.g., infections, e.g., viral infections, e.g., infection with hepatitis A, B, C, D, E and/or G virus; alcohol use (e.g., alcoholism); drug use (e.g., acetaminophen, anesthetics, anti-tuberculosis drugs, antifungal agents, antidiabetic drugs, neuroleptic agents, and drugs used to treat HIV infection and ADDS); autoimmune conditions (e.g., autoimmune hepatitis); and/or surgical procedures.

J. Reduction of the Effects of Ischemia

In still another aspect, the invention features a method of reducing the effects of ischemia in a patient, which includes identifying a patient suffering from or at risk for ischemia; and administering to the patient a pharmaceutical composition comprising nitric oxide, in combination with administering at least one treatment selected'from: inducing HO-I or ferritin in the recipient, expressing HO-I or ferritin in the patient, and administering a pharmaceutical composition comprising CO, HO-I, bilirubin, biliverdin, ferritin, DFO, SIH, iron dextran, or apoferritin to the patient, in amounts sufficient to reduce the effects of ischemia.

K. Respiratory Disorders

Examples of respiratory conditions include, but are not limited to asthma; Acute Respiratory Distress Syndrome (ARDS), e.g., ARDS caused by peritonitis, pneumonia (bacterial or viral), or trauma; idiopathic pulmonary diseases; interstitial lung diseases, e.g., Interstitial Pulmonary Fibrosis (IPF); pulmonary emboli; Chronic Obstructive Pulmonary Disease (COPD); emphysema; bronchitis; cystic fibrosis; lung cancer of any type; lung injury, e.g., hyperoxic lung injury; Primary Pulmonary Hypertension (PPH); secondary pulmonary hypertension; and sleep-related respiratory disorders, e.g., sleep apnea.

L. Cardiovascular Disorders

Cardiovascular disorders include disorders involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused, for example, by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include congestive heart failure, peripheral vascular disease, pulmonary vascular thrombotic diseases such as pulmonary embolism, stroke, ischemia-reperfusion (I/R) injury to the heart, atherosclerosis, and heart attacks.

M. Renal Disorders Disorders involving the kidney include but are not limited to pathologies of glomerular injury such as in situ immune complex deposition and cell-mediated immunity in glomerulonephritis, damage caused by activation of alternative complement pathway, epithelial cell injury, and pathologies involving mediators of glomerular injury including cellular and soluble mediators, acute glomerulonephritis, such as acute proliferative (poststreptococcal, postinfectious) glomerulonephritis, e.g., poststreptococcal glomerulonephritis and nonstreptococcal acute glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis (membranous nephropathy), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, IgA nephropathy (Berger disease), focal proliferative and necrotizing glomerulonephritis (focal glomerulonephritis) and chronic glomerulonephritis. Disorders of the kidney also include infections of the genitourinary tract.

N. Hepatobiliary Disorders Disorders involving the liver include but are not limited hepatitis, cirrhosis and infectious disorders. Causative agents of hepatitis include, for example, infections, e.g., infection with specific hepatitis viruses, e.g., hepatitis A, B, C, D, E, and G viruses; or hepatotoxic agents, e.g., hepatotoxic drugs (e.g., isoniazid, methyldopa, acetaminophen, amiodarone, and nitrofurantoin), and toxins (e.g., endotoxin or environmental toxins). Hepatitis may occur postoperatively in liver transplantation patients. Further examples of drugs and toxins that may cause hepatitis (i.e., hepatotoxic agents) are described in Feldman: Sleisenger & Fordtran's Gastrointestinal and Liver Disease, 7th ed., Chapter 17 (Liver Disease Caused by Drugs, Anesthetics, and Toxins), the contents of which are expressly incorporated herein by reference in their entirety. Such examples include, but are not limited to, methyldopa and phenytoin, barbiturates, e.g., phenobarbital; sulfonamides (e.g., in combination drugs such as co-trimoxazole (sulfamethoxazole and trimethoprim); sulfasalazine; salicylates; disulfiram; .beta. -adrenergic blocking agents e.g., acebutolol, labetalol, and metoprolol); calcium channel blockers, e.g., nifedipine, verapamil, and diltiazem; synthetic retinoids, e.g., etretinate; gastric acid suppression drugs e.g., oxmetidine, ebrotidine, cimetidine, ranitidine, omeprazole and famotidine; leukotriene receptor antagonists, e.g., zafirlukast; anti-tuberculosis drugs, e.g., rifampicin and pyrazinamide; antifungal agents, e.g., ketoconazole, terbinafϊne, fluconazole, and itraconazole; antidiabetic drugs, e.g., thiazolidinediones, e.g., troglitazone and rosiglitazone; drugs used in neurologic disorders, e.g., neuroleptic agents, antidepressants (e.g., fluoxetine, paroxetine, venlafaxine, trazodone, tolcapone, and nefazodone), hypnotics (e.g., alp idem, Zolpidem, and bentazepam), and other drugs, e.g., tacrine, dantrolene, riluzole, tizanidine, and alverine; nonsteroidal anti- inflammatory drugs, e.g., bromfenac; COX-2 inhibitors; cyproterone acetate; leflunomide; antiviral agents, e.g., fialuridine, didanosine, zalcitabine, stavudine, lamivudine, zidovudine, abacavir; anticancer drugs, e.g., tamoxifen and methotrexate; recreational drugs, e.g., cocaine, phencyclidine, and 5-methoxy-3,4- methylenedioxymethamphetamine; L-asparaginase; amodiaquine; hycanthone; anesthetic agents; e.g., halothane, enflurane, and isoflurane; vitamins e.g., vitamin A; and dietary and/or environmental toxins, e.g., pyrrolizidine alkaloids, toxin from Amanita phalloides or other toxic mushrooms, aflatoxin, arsenic, Bordeaux mixture (copper salts and lime), vinyl chloride monomer; carbon tetrachloride, beryllium, dimethylformamide, dimethylnitrosamine, methylenedianiline, phosphorus, chlordecone (Kepone), 2,3,7, 8-tetrachloro-dibenzo p-dioxin (TCDD), tetrachloroethane, tetrachloroethylene, 2,4,5-trinitrotoluene, 1,1,1-trichloro ethane, toluene, and xylene, and known "herbal remedies," e.g., ephedrine and eugenol.

O. Gastrointestinal Disorders

Gastrointestinal disorders include, but are not limited to, ileus (of any portion of the gastrointestinal tract, e.g., the large or small intestine), inflammatory bowel disease, e.g., specific inflammatory bowel disease, e.g., infective specific inflammatory bowel disease, e.g., amoebic or bacillary dysentery, schistosomiasis, Campylobacter enterocolitis, yersinia enterocolitis, or enterobius vermicularis; non-infective specific inflammatory bowel disease, e.g., radiation enterocolitis, ischaemic colitis, or eosinophilic gastroenteritis; and non-specific bowel disease, e.g., ulcerative colitis, indeterminate colitis, and Crohn's disease; necrotizing enterocolitis (NEC), and pancreatitis.

P. Neurological Disorders

The methods and compositions of the present invention can also be used to treat neurological disorders. Further, the methods may be used to treat pain disorders. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain. Also included in this category are seizure disorders, e.g., epilepsy.

Q. Wound Healing

Based on the anti- inflammatory properties of heme degradation products, the present invention contemplates that the methods described herein can be used to promote wound healing (e.g., in transplanted, lacerated (e.g., due to surgery), or burned skin). The compositions of the invention would typically be applied locally to the wound (e.g., as a wound dressing, lotion, or ointment), but could be delivered systemically as well.

R. Reproductive Disorders

The methods described herein can be used to treat or prevent certain reproductive disorders, e.g., impotence and/or inflammation associated with sexually transmitted diseases. Further, the methods of the present invention can be used to prevent premature uterine contractions, and may be used to prevent premature deliveries and menstrual cramps.

XL Kits

In another embodiment this invention provides kits for amelioration of one or more symptoms of a disease or disorder treatable by the compositions or the methods of the invention. The kits preferably comprise a container containing one or more of the agents of this invention. The agent may be provided in a unit dosage formulation (e.g. suppository, tablet, cap let, patch, etc.) and/or may be optionally combined with one or more pharmaceutically acceptable excipients.

In addition, the kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the practice of the methods or use of the "therapeutics" or "prophylactics" of this invention. Preferred instructional materials ^• describe the use of one or more polypeptides of this invention to mitigate one or more symptoms of a disease or disorder treatable by the compositions or the methods of the invention. The instructional materials may also, optionally, teach preferred dosages/therapeutic regiment, counter indications and the like.

While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

Claims

1. A method of treating a disease or disorder that would benefit from increased levels of bilirubin in a subject, comprising orally administering to said subject an effective amount of a β -glucuronidase polypeptide, such that the disease or disorder is treated in said subject. . . .

2. The method of claim 1, wherein said composition is selected such that the β- glucuronidase polypeptide is released in the small intestine.

3. The method of claim 1, wherein said composition comprises an enteric coating.

4. The method of claim 1, wherein said composition is selected such that the β- glucoronidase polypeptide is released in the duodenum.

5. The method of claim 1, wherein said composition is formulated in microcrystalline form, powder, or tablet.

6. The method of claim 1, wherein said subject's stomach acid has been suppressed prior to administration of the β-glucoronidase polypeptide.

7. The method of any one of claims 1-6, wherein said effective amount is effective to increase bilirubin levels in said subject.

8. The method of claim 7, wherein said bilirubin levels are increased at least about 25%.

9. The method of claim 7, wherein said bilirubin levels are increased at least about 50%.

10. The method of claim 7, wherein said bilirubin levels are increased at least about 100%.

11. The method of claim 7, wherein said bilirubin levels are increased at least about 2000% or greater.

12. The method of any one of claims 1-11, further comprising administering a bilirubin oxidase polypeptide in combination with said β-glucuronidase polypeptide.

13. The method of claim 12, wherein said bilirubin oxidase polypeptide is conjugated to said β-glucuronidase polypeptide.

14. The method of any one of claims 12-13, wherein said bilirubin oxidase polypeptide and β-glucuronidase polypeptide are administered in an amount effective to increase biliverdin levels in said subject.

15. The method of claim 14, wherein said biliverdin levels are increased at least about 10%.

16. The method of claim 14, wherein said biliverdin levels are increased at least about 50%.

17. The method of claim 14, wherein said biliverdin levels are increased at least about 2000%.

18. The method of any one of claims 1-17, wherein said subject is suffering or at risk of suffering from an inflammatory disorder.

19. The method of claim 18, wherein said inflammatory disorder is asthma, adult respiratory distress syndrome, interstitial pulmonary fibrosis, pulmonary emboli, chronic obstructive pulmonary disease, primary pulmonary hypertension, chronic pulmonary emphysema, congestive heart failure, peripheral vascular disease, stroke, atherosclerosis, ischemia-reperfusion injury, heart attacks, glomerulonephritis, conditions involving inflammation of the kidney, infection of the genitourinary tract, viral and toxic hepatitis, cirrhosis, ileus, necrotizing enterocolitis, specific and non-specific inflammatory bowel disease, rheumatoid arthritis, deficient wound healing, graft versus host disease, and hemorrhagic, septic, or anaphylactic shock..

20. The method of any one of claims 1-19, wherein said subject is a human.

21. A method for increasing the level of bilirubin in a subject, comprising orally administering to said subject an effective amount of a β-glucuronidase polypeptide, such that the bilirubin level in said subject are increased.

22. The method of claim 21, wherein said bilirubin levels are increased at least about 25%.

23. The method of claim 21, wherein said bilirubin levels are increased at least about 50%. , .

24. The method of claim 21, wherein said bilirubin levels are increased at least about 100%.

25. The method of claim 21, wherein said bilirubin levels are increased at least about 2000%.

26. The method of claim 21, wherein said bilirubin levels are increased by an amount effective to treat inflammation.

27. The method of claim 26, wherein said inflammation is chronic.

28. The method of claim 26, wherein said inflammation is acute.

29. A method for increasing the level of biliverdin in a subject, comprising orally administering to said subject an effective amount of a β-glucuronidase polypeptide in combination with a bilirubin oxidase polypeptide, such that the biliverdin level in said subject are increased.

30. The method of any one of claim 29 wherein said biliverdin levels are increased at least about 10%.

31. The method of claim 29, wherein said biliverdin levels are increased at least about 50%.

32. The method of claim 29, wherein said biliverdin levels are increased at least about 2000%.

33. The method of claim 29, wherein said biliverdin levels are increased by an amount effective to treat inflammation.

34. The method of claim 33, wherein said inflammation is chronic.

35. The method of claim 33, wherein said inflammation is acute.

36. The pharmaceutical composition comprising β-glucuronidase formulated for oral administration. ,

37. The pharmaceutical composition of claim 36, wherein said composition comprises an enteric coating.

38. The pharmaceutical composition of claim 36, wherein said enteric coating is designed to release the β-glucuronidase polypeptide in the duodenum.

39. The pharmaceutical composition of claim 36, wherein said composition is formulated as a tablet, micro crystalline form, powder, or tablet.

40. The pharmacuetical composition of any one of claims 36-39, further comprising an effective amount of a bilirubin oxidase polypeptide.

41. The pharmaceutical composition of claim 40, wherein said bilirubin oxidase compound is conjugated to said β-glucuronidase polypeptide.

42. The pharmaceutical composition of any one of claims 36-41, wherein said effective amount is effective to treat an inflammatory disorder.

43. The pharmaceutical composition of claim 42, wherein said inflammatory disorder is chronic.

44. The pharmaceutical composition of claim 42, wherein said inflammatory disorder is acute.