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Title:  Systems and methods related to degradation of uremic toxins
United States Patent: 
7,198,785
Issued: 
April 3, 2007

Inventors: 
O'Loughlin; Jill A. (Lincoln, MA), Bruder; Jan Markus (Altena, DE), Lysaght; Michael J. (East Greenwich, RI)
Assignee: 
Brown University (Providence, RI)
Appl. No.:  10/731,877
Filed: 
December 9, 2003


 

George Washington University's Healthcare MBA


Abstract

The present invention generally relates to the treatment of uremic toxins in vivo using uremic toxin-treating enzymes, and/or cells capable of producing uremic toxin-treating enzymes or otherwise reacting with uremic toxins. Non-limiting examples of cases where the treatment of uremic toxins is desired include renal disease or dysfunction, gout, subjects receiving chemotherapy, or the like. In one aspect, the treatment includes an oral delivery composition able to reduce the blood concentration of one or more non-protein nitrogen compounds in vivo. The composition, in some cases, may comprise one, two, or more uremic toxin-treating enzymes, such as urease, uricase or creatininase. The oral delivery composition may be able to deliver the uremic toxin-treating enzymes, substantially undigested, to the intestines, where the enzymes can interact with uremic toxins transported to the intestines from the bloodstream. In another aspect, the treatment includes an oral delivery composition comprising a cell able to reduce the concentration of one or more uremic toxins in vivo. In some cases, the cell may be designed to overexpress one, two, or more uremic toxin-treating enzymes, such as urease, uricase or creatininase, for example, by transfecting the cell with a corresponding gene. In some embodiments, a species able to react with or otherwise sequester by-products of the uremic toxin-treating enzyme reactions may be included with the oral delivery composition. For example, if the by-product is ammonium, the species may be a sorbent able to adsorb ammonium, an enzyme able to react with the ammonium, or the like.

SUMMARY OF INVENTION

The present invention generally relates to the treatment of disorders associated with uremic toxins in vivo using uremic toxin-treating enzymes, and/or cells capable of producing uremic toxin-treating enzymes or otherwise reacting with uremic toxins. The subject matter of this invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, the present invention is an article. The article, in one set of embodiments, includes an oral delivery composition. In one embodiment, the oral delivery composition includes at least one of isolated uricase and isolated creatininase. In another embodiment, the oral delivery composition includes at least one cell transfected with at least one of a uricase gene and a creatininase gene. In yet another embodiment, the oral delivery composition includes at least one cell designed to overexpress at least one of uricase and creatininase. The oral delivery composition includes, in still another embodiment, at least one cell transfected with at least one of a urease gene, a uricase gene, and a creatininase gene, where the at least one cell is not E. coli. In yet another embodiment, the oral delivery composition includes at least one cell able to reduce a blood concentration of at least one non-protein nitrogen compound in a subject when the oral delivery composition is ingested by the subject, where the at least one cell is not E. coli. In still another embodiment, the oral delivery composition includes at least two isolated uremic enzymes. In some cases, the oral delivery composition may include a capsule. The capsule, in some embodiments, may include one or more of the above-described compositions.

In another aspect, the present invention defines a method. In one set of embodiments, the method includes administering, to a subject, an oral delivery composition comprising at least one of uricase and creatininase. The method, in another set of embodiments, includes administering, to a subject, an oral delivery composition comprising at least one cell transfected with at least one of a uricase gene and a creatininase gene. The method, in yet another set of embodiments, includes administering, to a subject, an oral delivery composition comprising at least one cell designed to overexpress at least one of uricase and creatininase. In still another set of embodiments, the method includes administering, to a subject, an oral delivery composition comprising at least one cell transfected with at least one of a urease gene, a uricase gene, and a creatininase gene, where the at least one cell is not E. coli. The method, in yet another set of embodiments, includes administering, to a subject, an oral delivery composition comprising at least one cell able to reduce a blood concentration of at least one non-protein nitrogen compound in the subject when the oral delivery composition is ingested by the subject, where the at least one cell is not E. coli. In another set of embodiments, the method includes administering at least one of isolated uricase and isolated creatininase to an intestine of a subject. The method includes, in still another set of embodiments, administering, to a subject, an oral delivery composition comprising at least two isolated uremic enzymes.

In another aspect, the present invention is directed to a method of making one or more of the embodiments described herein, for example, an oral delivery capsule. In yet another aspect, the present invention is directed to a method of using one or more of the embodiments described herein, for example, an oral delivery capsule. In still another aspect, the present invention is directed to a method of promoting one or more of the embodiments described herein, for example, an oral delivery capsule.

Other advantages and novel features of the invention will become apparent from the following detailed description of the various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to the treatment of disorders associated with uremic toxins in vivo using uremic toxin-treating enzymes, and/or cells capable of producing uremic toxin-treating enzymes or otherwise reacting with uremic toxins to reduce or eliminate the toxic activity of the uremic toxins. Non-limiting examples of (disorders associated with uremic toxins include renal disease or dysfunction, gout, subjects receiving chemotherapy, or the like. In one aspect, the treatment includes an oral delivery composition able to reduce the blood concentration of one or more non-protein nitrogen compounds in vivo. The composition, in some cases, may comprise one, two, or more uremic toxin-treating enzymes, such as urease, uricase or creatininase. The oral delivery composition may be able to deliver the uremic toxin-treating enzymes, substantially undigested, to the intestines, where the enzymes can interact with uremic toxins transported to the intestines from the bloodstream. In another aspect, the treatment includes an oral delivery composition comprising a cell able to reduce the concentration of one or more uremic toxins in vivo. In some cases, the cell may be designed to overexpress one, two, or more uremic toxin-treating enzymes, such as urease, uricase or creatininase, for example, by transfecting the cell with a corresponding gene. In some embodiments, a species able to react with or otherwise sequester by-products of the uremic toxin-treating enzyme reactions may be included with the oral delivery composition. For example, if the by-product is ammonium, the species may be a sorbent able to adsorb ammonium, an enzyme able to react with the ammonium, or the like.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

As used herein, "or" is understood to mean inclusively or, i.e., the inclusion of at least one, but including more than one, of a number or list of elements. Only terms clearly indicated to the contrary, such as "exclusively or" or "exactly one of," will refer to the inclusion of exactly one element of a number or list of elements.

The term "patient" or "subject" as used herein includes mammals such as humans, as well as non-human mammals such as non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, rabbits, or rodents such as mice or rats.

As used herein, a "uremic toxin" is given its ordinary meaning as used in the art, e.g., one or more compounds containing nitrogen produced by the body as waste products, e.g., from the breakdown of proteins, nucleic acids, or the like. Typically, uremic toxins are not proteins. Non-limiting examples of uremic toxins include urea, uric acid, creatinine, and beta-2 (.beta..sub.2) microglobulin (e.g., see FIG. 1). In healthy individuals, uremic toxins are usually excreted from the body through the urine. However, in certain individuals, uremic toxins are not removed from the body at a sufficiently fast rate, leading to uremic toxicity, i.e., a disease or condition characterized by elevated levels of at least one uremic toxin with respect to physiologically normal levels of the uremic toxin. Non-limiting examples of such diseases include renal disease or dysfunction, impaired or partial kidney function, gout, subjects receiving chemotherapy, or the like. Subjects receiving chemotherapy or other treatments may experience significant amounts of necrosis of cell populations, which can cause the releases of purines which are metabolized to uric acid. "Renal disease" includes early renal disease states (i.e., the kidneys do not perform at physiologically normal levels, but are able to process and remove some uremic toxins from the bloodstream), as well as end stage renal disease ("ESRD"), where the kidneys are substantially nonfunctional. Certain uremic toxins are transported between the bloodstream and the intestine, for example, urea, uric acid, or creatinine. As used herein, "transport" refers to any process in which a substance is moved from one location to another, for example, through diffusion (passive transport), facilitated diffusion, convection, transport proteins or other active transport systems, etc.

One aspect of the present invention involves delivering one, two, or more uremic toxin-treating enzymes to a subject, typically to the intestines. Preferably, the enzymes are delivered in a substantially undigested state. In some cases, one or more of the enzymes are isolated (e.g., as described below). As used herein, a "uremic toxin-treating enzyme," or a "uremic enzyme," is an enzyme able to react with a uremic toxin as a substrate, for example, the uremic toxic-treating enzyme may be an enzyme able to react with urea as a substrate, with uric acid as a substrate, or with creatinine as a substrate. Uremic enzymes can be determined in vitro, for example, by allowing the enzyme to react with a uremic toxin in solution and measuring a decrease in the concentration of the uremic toxin. Examples of uremic toxin-treating enzymes include, but are not limited to, ureases (which reacts with urea), uricases (which reacts with uric acid), or creatininases (which reacts with creatinine). FIG. 1 illustrates enzymatic reactions that typically occur with these enzymes. In some cases, each enzyme independently may originate from a different species (i.e., heterologous). In some cases, the enzyme is commercially available, for example, isolated and purified from other sources. A specific non-limiting example of a urease is urease from Canavalia ensiformis, having a sequence SEQ ID NO.: 1 (GenBank Accession number URJB GI:418642). A specific non-limiting example of a uricase is uricase from Schizosaccharomyces pombe, having a sequence SEQ ID NO.: 2 (GenBank Accession number T40869 GI:7493586). A specific non-limiting example of a creatininase is creatininase from Arthrobacter sp., having a sequence SEQ ID NO.: 3 (GenBank Accession number BAA25929.1 GI:3116224). Those of ordinary skill in the art will know of other suitable uremic toxin-treating enzymes. Additionally, minor changes to such enzymes (for example, through chemical changes or modifications, such as the addition of reporting groups, linkage to a physical surface, changes or substitutions in bases in the amino acid sequence of the enzyme, etc.) that do not alter the ability of the enzyme to recognize and react with its substrate are also included herein as uremic toxin-treating enzymes. For example, a urease, uricase, or creatininase may be covalently bound to a surface, for instance, in a microarray or an ELISA.

One or more uremic toxin-treating enzyme described herein may be isolated in certain cases. An "isolated" molecule (e.g., an enzyme), as used herein, is a molecule that is substantially pure and is free of other substances with which it is ordinarily found in nature or in vivo systems to an extent practical and appropriate for its intended use. For instance, the molecular species may be sufficiently pure or sufficiently free from substances such as biological constituents with which it is normally found in vivo so as to be useful in, for example, producing pharmaceutical preparations, or sequencing if the molecular species is a nucleic acid, peptide, enzyme, or polysaccharide. Because an isolated molecular species of the invention may be admixed with a pharmaceutically-acceptable carrier in a pharmaceutical preparation, and/or other physiologically-active agents (e.g., as described below), the molecular species may comprise only a small percentage by weight of the preparation. The molecular species is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems. As examples, a uremic toxin-treating enzyme, such as urease, uricase, and/or creatininase may be associated with other molecules, such as a pharmaceutically acceptable carrier, a sorbent, a capsule (e.g., comprising alginate), etc.

Any suitable system or method may be used to orally deliver the uremic enzyme in a substantially undigested state. As used herein, an "oral delivery composition" is a composition that is designed to be delivered orally to a subject, i.e., the composition has been formulated in such a way that it is designed to be taken orally by a subject in a therapeutically effective amount without substantially adverse effects. For example, an enzyme may be delivered to a subject in an oral delivery composition that is a capsule, a sustained release pill, a controlled release formulation, a liposome, etc. As used herein, a "substantially undigested" enzyme (or other such substance) is an enzyme (or other substance) that enters the gastrointestinal system of a subject, and is not degraded or digested by the gastrointestinal system until at least reaching the site of delivery (e.g., the intestines), and/or is partially degraded or digested, but such that a therapeutically effective amount of the enzyme or other substance is able to reach the site of delivery. Degradation or digestion by the gastrointestinal system of the enzyme (or other substance) can occur, for example, through the action of pH, gastric acid, mechanical action, hydrolysis, digestive enzymes such as pepsin, trypsin, chymotrypsin, etc. In some cases, the enzyme is not degraded or digested by the gastrointestinal system and is excreted substantially intact.

In some cases, an enzyme is included in a formulation that is not substantially susceptible to degradation or digestion by the gastrointestinal system, i.e., a formulation that is able to deliver the enzyme to the site of delivery in a substantially undigested state. For example, the enzyme may be encapsulated in a formulation that resists degradation or digestion, the enzyme may be included in a formulation that is surrounded by a coating at least partially resistant to degradation or digestion, or the like. In certain instances, the formulation may be at least partially susceptible to degradation or digestion, but over time scales greater than the time it takes for the formulation to pass through the gastrointestinal system; thus, the formulation is still able to deliver the enzyme to the site of delivery in a substantially undigested state, even though some degradation or digestion of the enzyme may occur. As used herein, "substantially undigested state" refers to a level of degradation or digestion of the enzyme that does not impede the ability of the enzyme to recognize and react with its substrate. In some cases, it is preferred that the formulation be designed so as to not substantially release the uremic toxin-treating enzyme externally of the capsule, i.e., into the gastrointestinal system. That is, the formulation may be designed such that any release of the uremic toxin-treating enzyme externally of the capsule does not prevent the uremic toxin-treating enzyme remaining within the capsule from being able to react with uremic toxins found in the gastrointestinal system at a therapeutically effective rate.

In certain embodiments, the formulation may be chosen or designed to allow sufficient mass transport of uremic toxins into the formulation to occur such that the enzyme is able to react with uremic toxins found in the gastrointestinal system at a therapeutically effective rate. As used herein, "mass transport" is given its ordinary meaning as used in the art, i.e., the physical movement of a substance from location to another, using processes such as diffusion, convection, osmosis, etc. In some cases, the formulation can be designed such that it does not substantially impede mass transport of the uremic toxin into the formulation, i.e., where "substantially impede" refers to the ability of the uremic toxin to reach the site of the uremic toxin-treating enzyme without being significantly rate-limited, for example, such that the reaction of the uremic enzyme occurs over a time scale comparable to the time scale of mass transport of the uremic toxin from external the formulation to the enzyme. For example, a capsule and/or an enteric coating may allow diffusion to occur therethrough to a uremic enzyme at rates similar to rates of reaction of free enzyme to the uremic toxin.

In one set of embodiments, a formulation of the invention, such as a capsule, may contain one uremic toxin-treating enzyme, or more than one uremic toxin-treating enzyme (i.e., a "combination" formulation). More than one type of formulation may be given to a subject. For example, a subject may be given a first capsule containing a first enzyme and a second capsule containing a second enzyme, e.g., serially or simultaneously.

As one example, a formulation of the invention may contain one, two, or more uremic toxin-treating enzymes encapsulated within a capsule. The capsule may comprise alginate or other suitable polymers or materials able to at least partially resist degradation or digestion in the gastrointestinal system. Alginates are salts of alginic acid, a carbohydrate biopolymer that can be extracted from brown algae or other sources. Typically, alginates include monomers such as mannuronic acid or guluronic acid, although other monomers may be included as well. Other examples of suitable materials include lactic acid, glycolic acid, lysine, and hydroxyapatite, as well as mixtures or copolymers of these and/or other materials, e.g., poly(lactic-co-glycolic acid).

The capsules may be produced by any suitable technique known to those of ordinary skill in the art. For example, one or more uremic enzymes may be placed within a pre-polymeric solution that, upon solidification or polymerization, forms a capsule embedding the enzymes. Other compounds, such as sorbents, stabilizers, buffers, or the like may also be included within the capsule, e.g., as further described below. In some cases, post-processing steps may also be performed, for example, forming an enteric coating around the capsule.

As used herein, an "enteric coating" is given its ordinary meaning as used in the art, i.e., a coating that at least is partially resistant to degradation or digestion within the gastrointestinal system or at least a portion thereof, such as within the stomach. Those of ordinary skill in the art will know of suitable materials useful for enteric coatings. Non-limiting examples include enteric polymers such as cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, ethylhydroxycellulose phthalate, polyvinylacetatephthalate, polyvinylbutyrate acetate, vinyl acetate-maleic anhydride copolymer, styrene-maleic mono-ester copolymer, methyl acrylate-methacrylic acid copolymer, methacrylate-methacrylic acid-octyl acrylate copolymer, etc. These may be used either alone or in combination, or together with other polymers than those mentioned above. The enteric coating may also include insoluble substances which are neither decomposed nor solubilized in living bodies, such as alkyl cellulose derivatives such as ethyl cellulose, crosslinked polymers such as styrene-divinylbenzene copolymer, polysaccharides having hydroxyl groups such as dextran, cellulose derivatives which are treated with bifunctional crosslinking agents such as epichlorohydrin, dichlorohydrin, a diepoxybutane, etc. The enteric coating may also include starch and/or dextrin. In some embodiments, a formulation of the invention may include a species able to react with or otherwise sequester (isolate) one or more by-products of the uremic toxin-treating enzyme reactions. For example, in cases where ammonia (NH.sub.3) is produced as a by-product, the species may react with or sequester ammonia, and that species may be referred to as an "ammonium uptake species." In some instances, the species may sequester the by-product through physical means, such as through physisorption mechanisms, i.e., by use of a sorbent such as zirconium phosphate, carbon, or oxystarch. In other instances, an enzyme able to react with a by-product may be used to react the by-products, for example, into a metabolically neutral form, into a form that can is useful (e.g., an amino acid), into a form that is non-toxic, etc. As an example, in cases where ammonia is produced as a by-product, an enzyme able to react with ammonia may be used, such as glutamine synthetase. As yet another example, a cell able to react with one or more by-products of the reaction may be included in the formulation. Also, combinations of such techniques may also be used in some cases, for example, a sorbent and an enzyme may be included in the capsule or other formulation.

Other active compounds may be added to the formulation as well, for example, other cells, enzymes, chemicals, drugs, reporting agents, etc. For example, bacteria and/or enzymes targeted toward other molecules elevated in uremia, such as beta-2 (.beta..sub.2) microglobulin may be identified and added to the formulation. In another embodiment, bacteria capable of recycling ammonia into amino acid precursors may be used, which may, in some cases, counteract the malnutrition which often accompanies renal failure.

Another aspect of the invention involves using cells designed to overexpress a uremic toxin-treating enzyme. Such cells may be delivered to a subject in an oral delivery composition, such as those previously described (for example, encapsulated), optionally in combination with other cells, enzymes such as uremic toxin-treating enzymes, sorbents, or other species able to react with or otherwise sequester one or more reaction by-products, etc. As used herein, a cell that is "designed" to overexpress a uremic enzyme is intentionally chosen or selected to "overexpress" the uremic enzyme, i.e., to express the uremic enzyme at expression levels significantly greater than the expression level of the enzyme for that cell type (which can include zero or negligible expression levels). For example, such a cell may be artificially selected through natural selection processes to overexpress the uremic toxin-treating enzyme, the cells may be stimulated (e.g., with a hormone to overexpress the uremic toxin-treating enzyme, the cell may be transfected to overexpress the uremic toxin-treating enzyme, or the like.

The cell may be any cell able to overexpress the uremic enzyme at levels that are therapeutically effective. For example, the cell may be a bacterium or a mammalian cell. Bacteria may be advantages in some cases. For example, bacteria can grow and expand during their passage through the gastrointestinal system, thus increasing the effectiveness of this form of treatment. In some cases, the bacteria can metabolize some of the breakdown products from the enzymatic reaction, e.g., preventing their resorption. In certain cases, the bacteria efficiency (e.g. in terms of weight/degradation power) may be higher than that of isolated enzymes or encapsulated enzymes. Bacteria can also be relatively easy to grow quickly in large amounts, and are often less expensive than enzymes. Some bacteria can also metabolize uremic toxins intracellularly, such that the uremic enzymes stay well-protected from the environment of the gastrointestinal system. In one embodiment, the bacteria is not E. coli.

In some cases, the cell may be transfected, e.g., with one, two, or more genes for urease, uricase, creatininase, or other uremic toxin-treating enzymes. Those of ordinary skill in the art will know of suitable ways of transfecting cells. For example, some techniques for transformation (micro-injection, electroporation, calcium phosphate method, etc.) are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y. (1989).

In one embodiment, a gene for urease, uricase, creatininase, or other uremic toxin-treating enzymes may be transfected into a cell using a DNA vector. The vector may be a vector in which the gene is functionally linked to one or more control sequences which allows expression of the corresponding enzymes. These include plasmids which can be replicated and/or expressed in prokaryotes or bacteria such as E. coli and/or in eukaryotic systems such as yeasts or mammalian cell lines.

Expression in prokaryotes may be carried out using techniques known in the art. The gene may be expressed as fusion proteins or as intact, native proteins. In some cases, fusion proteins may be produced in large quantities. The fusion proteins are generally more stable than the native polypeptide and are easy to purify. The expression of these fusion proteins can be controlled by normal host DNA sequences.

Producing intact native polypeptides using bacteria such as E. coli may require, in some cases, a strong, regulatable promoter and an effective ribosome binding site. Promoters which may be used for this purpose include, but are not limited to, the temperature sensitive bacteriophage .lamda.p.sub.L-promoter, the tac-promoter inducible with IPTG or the T7-promoter. Numerous plasmids with suitable promoter structures and efficient ribosome binding sites have been described, such as for example pKC30 (.lamda.p.sub.L; Shimatake and Rosenberg, Nature, 292:128 (1981), pKK173-3 (tac, Amann and Brosius, Gene, 40:183 (1985)) or pET-3 (T7-promoter (Studier and Moffat, J. Mol. Biol., 189:113 (1986)). A number of other suitable vector systems for expressing the DNA according to the invention in bacteria are known from the prior art and are described, for example, in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y. (1989).

Suitable bacterial strains which are specifically tailored to a particular expression vector are known to those skilled in the art (Sambrook, et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, N.Y. (1989)). The experimental performance of the cloning experiments, the expression of the polypeptides in bacteria and the working up and purification of the polypeptides are known.

In addition to prokaryotes, eukaryotic microorganisms such as yeast may also be used in some cases. For expression in yeast, the plasmid YRp7 (Stinchcomb et al. Nature, 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschumper et al., Gene, 10:157 (1980)) and the plasmid YEp13 (Bwach et al., Gene, 8:121 133 (1979)) are used, for example. The plasmid YRp7 contains the TRP1-gene which provides a selection marker for a yeast mutant (e.g., ATCC No. 44076) which is incapable of growing in tryptophan-free medium. The presence of the TRP1 defect as a characteristic of the yeast strain used then constitutes an effective aid to detecting transformation when cultivation is carried out without tryptophan. The same is true with the plasmid YEp 13, which contains the yeast gene LEU-2, which can be used to complete a LEU-2-minus mutant.

Other suitable marker genes for yeast include, for example, the URA3- and HIS3-gene. Preferably, yeast hybrid vectors also contain a replication start and a marker gene for a bacterial host, so that the construction and cloning of the hybrid vectors and their precursors can be carried out in a bacterial host. Other expression control sequences suitable for expression in yeast include, for example, those of PHO3- or PHO5-gene.

Other suitable promoter sequences for yeast vectors contain the 5'-flanking region of the genes of ADH I (Ammerer, Methods of Enzymology, 101: 192 210 (1983)), 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Kawaski and Fraenkel, Biochem. Biophys. Res. Comm., 108:1107 1112 (1982)) such as enolase, glycerinaldehyde-3-phosphate-dehydrogenase, hexokinase, pyruvate-decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose-isomerase and glucokinase. When constructing suitable expression plasmids, the termination sequences associated with these genes may also be inserted in the expression vector at the 3'-end of the sequence to be expressed, in order to enable polyadenylation and termination of the mRNA.

Generally, any vector which contains a yeast-compatible promoter and origin replication and termination sequences is suitable. Thus, hybrid vectors which contain sequences homologous to the yeast 2.mu. plasmid DNA may also be used. Such hybrid vectors are incorporated by recombination within the cells of existing 2.mu.-plasmids or replicate autonomously.

The genetic constructs may generally contain one or more suitable regulatory elements (such as one or more suitable promoters, enhancers, terminators, etc.), 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation. These and other suitable elements for such genetic constructs will be clear to those of ordinary skill in the art, and may, for instance, depend upon the type of construct used, the intended host cell or host organism; the manner in which the nucleotide sequences of the invention of interest are to be expressed (e.g. via constitutive, transient or inducible expression); and/or the transformation technique to be used.

In some cases, one or more elements may be "operably linked" to the above-described genes and/or to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered "operably linked" to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being "under the control of" said promoter). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required. In some cases, the optional further elements of the genetic construct(s) used in the invention may be such that they are capable of providing their intended biological function in the intended host cell or host organism. For instance, a promoter, enhancer or terminator may be "operable" in the intended host cell or host organism, by which is meant that (for example) the promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence (e.g. a coding sequence) to which it is operably linked (as defined above). Such a promoter may be a constitutive promoter or an inducible promoter, and may also be such that it (only) provides for expression in a specific stage of development of the host cell or host organism, and/or such that it (only) provides for expression in a specific cell, tissue, organ or part of a multicellular host organism.

A selection marker may be chosen such that it allows (e.g., under appropriate selection conditions) host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.

A leader sequence may be chosen such that, in the intended host cell or host organism, it allows for the desired post-translational modifications, and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell. A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism.

An expression marker or reporter gene may be chosen such that, in the host cell or host organism, it allows for detection of the expression of (a gene or nucleotide sequence present on) the genetic construct. An expression marker may optionally also allow for the localization of the expressed product, e.g. in a specific part or organelle of a cell and/or in (a) specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism. Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP.

The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al., mentioned above.

Often, the genetic constructs will be obtained by inserting a nucleotide sequence in a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors include: vectors for expression in bacterial cells: pET vectors (Novagen) and pQE vectors (Qiagen); and vectors for expression in yeast or other fungal cells: pYES2 (Invitrogen) and Pichia expression vectors (Invitrogen).

The nucleotide sequences and/or genetic constructs may be used to transform a host cell. The host cell may be any suitable (prokaryotic or eukaryotic) cell or cell line, for example: a bacterial strain, including, but not limited to, E. coli, Bacillus. Streptomyces and Pseudomonas; and a yeast cell, including, but not limited to, Kluyveromyces or Saccharomyces.

In one aspect, a formulation of the invention may be used to control uremic toxins within the subject at an acceptable level. The formulation may be used to treat a subject having or at risk for uremic toxicity, as previously described. In some cases, the formulation may used independently. For example, a formulation of the invention may be given to a subject in lieu of dialysis, or before a subject has reached a state where dialysis is required. For instance, in a subject having or at risk for renal failure, a formulation of the invention may be given to the subject to control uremic toxin levels within the subject, to reduce the need for dialysis or other forms of treatment, etc. As another example, in a subject being treated using chemotherapy, a formulation of the invention may be given to the subject to control uremic toxin levels within the subject, for example, to prevent or at least control uremic toxicity symptoms, or to supplement normal kidney function. In other cases, the formulation may be used in combination with other treatments or strategies for controlling uremic toxins, such as dialysis, e.g., to supplement and/or enhance such treatments. For example, the formulation may be given simultaneously with dialysis, before and/or after dialysis, interspersed with dialysis, etc. For instance, on days where no dialysis is performed, a subject may be given a formulation of the invention, once a day, twice a day, once every other day, or at any other suitable frequency, for example, three times a week, four times a week, or five times a week. As a specific example, in a subject where dialysis is to be performed three times a week, a formulation of the invention may be given to the subject on the four days of the week where no dialysis is performed.

As one example, a formulation of the invention may be used in a subject as a replacement of dialysis (e.g., kidney dialysis), or as a supplement to dialysis. Those of ordinary skill in the art will be able to identify suitable types of dialysis. For example, in kidney dialysis, blood is typically pumped from a subject through a semiporous membrane that allows urea and salt transport across the membrane to occur, but does not allow passage of red blood cells, white blood cells, and other important blood components therethrough. Examples of dialysis techniques include hemodialysis and peritoneal dialysis, for instance, continuous ambulatory peritoneal dialysis ("CAPD"). Dialysis can be performed, for example, using external machines or portable devices. By supplying the subject with the compositions of the invention, the time between dialysis treatments may be extended in some cases. For instance, the subject may be able to prolong the time between dialysis treatments to at least three days, at least four days, at least five days, at least seven days, or at least ten days or more in some cases.

As another example, a formulation of the invention may be used in combination with other small-molecule drugs, and/or other enzymes such as urate oxidase. As yet another example, a formulation of the invention may be used in combination with treatments that allow inhibition of uric acid synthesis, increased uric acid excretion, and/or enzymatic degradation. For instance, for the treatment of gout, a form of inflammatory arthritis in which urate deposits are common in and around the joints and characterizable by elevated levels of uric acid in the blood, the most often used drugs include allopurinol and probenecid. Allopurinol can interfere with uric acid synthesis by inhibiting xanthine oxidase, an enzyme which is required in the formation of uric acid, and probenecid can increase uric acid excretion by inhibiting the reabsorption of urate in the renal tubules. Rasburicase, a form of recombinant urate oxidase cloned from Aspergillus flavus fungi, is an example of a treatment for chemotherapy-induced hyperuricaemia. This enzyme, which may be given by intravenous injection, can degrade the uric acid via conversion of uric acid to allantoin, which is 5 10 times more soluble than uric acid.

Another aspect of the present invention provides a method of orally administering any of the above-described formulations to a subject. After oral delivery, the formulation may stay within the gastrointestinal system until being eliminated by the subject, typically after roughly twenty-four hours after administration. The formulation may be active during part or all of its transit through the gastrointestinal system, for example, within the large and/or small intestine.

When administered, the formulations of the invention are applied in a therapeutically effective, pharmaceutically acceptable amount as a pharmaceutically acceptable formulation. As used herein, the term "pharmaceutically acceptable" is given its ordinary meaning. Pharmaceutically acceptable formulations are generally compatible with other materials of the formulation and are not generally deleterious to the subject. Any of the formulations of the present invention may be administered to the subject in a therapeutically effective dose. A "therapeutically effective" or an "effective" as used herein means that amount necessary to at least partially decrease the concentrations of one or more uremic toxins within the bloodstream of the subject. When administered to a subject, effective amounts will depend on the particular condition being treated and the desired outcome. A therapeutically effective dose may be determined by those of ordinary skill in the art, for instance, employing factors such as those further described below and using no more than routine experimentation.

In administering the formulations of the invention to a subject, dosing amounts, dosing schedules, routes of administration, and the like may be selected so as to affect known activities of these formulations. Dosages may be estimated based on the results of experimental models, optionally in combination with the results of assays of formulations of the present invention. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. The doses may be given in one or several administrations per day. In the event that the response of a particular subject is insufficient at such doses, even higher doses may be employed to the extent that subject tolerance permits. Multiple doses per day are also contemplated in some cases.

The dose of the formulations to the subject may be such that a therapeutically effective amount of the active compound (enzyme and/or cell, etc.) reaches the intestines of the subject. The dosage may be given in some cases at the maximum amount while avoiding or minimizing any potentially detrimental side effects within the subject. The dosage of the formulation that is actually administered is dependent upon factors such as the final concentration desired, the efficacy of the formulation, the longevity of the formulation within the subject, the timing of administration, the effect of concurrent treatments (e.g., as in a cocktail, or in conjunction with dialysis), etc. The dose delivered may also depend on conditions associated with the subject, and can vary from subject to subject in some cases. For example, the age, sex, weight, size, environment, physical conditions, or current state of health of the subject may also influence the dose required. Variations in dosing may occur between different individuals or even within the same individual on different days. It may be preferred that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. Preferably, the dosage form is such that it does not substantially deleteriously affect the subject.

Formulations suitable for oral administration may be presented as discrete units such as hard or soft capsules, pills, cachettes, tablets, troches, or lozenges. Other oral formulations suitable for use with the invention include solutions or suspensions in aqueous or non-aqueous liquids such as a syrup, an elixir, or an emulsion. In another set of embodiments, the formulation may be used to fortify a food or a beverage.

In certain embodiments of the invention, a formulation can be combined with a suitable pharmaceutically acceptable carrier, for example, as incorporated into a liposome, incorporated into a polymer release system, or suspended in a liquid, e.g., in a dissolved form or a colloidal form. In general, pharmaceutically acceptable carriers suitable for use in the invention are well-known to those of ordinary skill in the art. As used herein, a "pharmaceutically acceptable carrier" refers to a non-toxic material that does not significantly interfere with the effectiveness of the biological activity of the active compound(s) to be administered (e.g., enzymes, cells, etc.), but is used as a formulation ingredient, for example, to stabilize or protect the active compound(s) within the formulation before use. The term "carrier" denotes an organic or inorganic ingredient, which may be natural or synthetic, with which one or more active compounds of the invention are combined to facilitate the application of the formulation. The carrier may be co-mingled or otherwise mixed with one or more enzymes and/or cells, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The carrier may be either soluble or insoluble, depending on the application. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble. Those skilled in the art will know of other suitable carriers, or will be able to ascertain such, using only routine experimentation.

In some embodiments, the formulations of the invention include pharmaceutically acceptable carriers with formulation ingredients such as salts, carriers, buffering agents, emulsifiers, diluents, excipients, chelating agents, fillers, drying agents, antioxidants, antimicrobials, preservatives, binding agents, bulking agents, silicas, solubilizers, or stabilizers that may be used with the active compound. For example, if the formulation is a liquid, the carrier may be a solvent, partial solvent, or non-solvent, and may be aqueous or organically based. Examples of suitable formulation ingredients include diluents such as calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin or acacia; lubricating agents such as magnesium stearate, stearic acid, or talc; time-delay materials such as glycerol monostearate or glycerol distearate; suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone; dispersing or wetting agents such as lecithin or other naturally-occurring phosphatides; thickening agents such as cetyl alcohol or beeswax; buffering agents such as acetic acid and salts thereof, citric acid and salts thereof, boric acid and salts thereof, or phosphoric acid and salts thereof; or preservatives such as benzalkonium chloride, chlorobutanol, parabens, or thimerosal. Suitable carrier concentrations can be determined by those of ordinary skill in the art, using no more than routine experimentation. Those of ordinary skill in the art will know of other suitable formulation ingredients, or will be able to ascertain such, using only routine experimentation.

Preparations include sterile aqueous or nonaqueous solutions, suspensions and emulsions Aqueous carriers include water, alcoholic/aqueous solutions, or emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like. Those of skill in the art can readily determine the various parameters for preparing and formulating the formulations of the invention without resort to undue experimentation.

In some embodiments, the present invention includes the step of bringing a formulation of the invention into association or contact with a suitable carrier, which may constitute one or more accessory ingredients. The final formulations may be prepared by any suitable technique, for example, by uniformly and intimately bringing the formulation into association with a liquid carrier, a finely divided solid carrier or both, optionally with one or more formulation ingredients as previously described, and then, if necessary, shaping the product.

In some embodiments, the formulations of the present invention may be present as a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salts" includes salts of the formulation, prepared in combination with, for example, acids or bases, depending on the particular compounds found within the formulation and the treatment modality desired. Pharmaceutically acceptable salts can be prepared as alkaline metal salts, such as lithium, sodium, or potassium salts; or as alkaline earth salts, such as beryllium, magnesium or calcium salts. Examples of suitable bases that may be used to form salts include ammonium, or mineral bases such as sodium hydroxide, lithium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and the like. Examples of suitable acids that may be used to form salts include inorganic or mineral acids such as hydrochloric, hydrobromic, hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, phosphorous acids and the like. Other suitable acids include organic acids, for example, acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, glucuronic, galacturonic, salicylic, formic, naphthalene-2-sulfonic, and the like. Still other suitable acids include amino acids such as arginate, aspartate, glutamate, and the like.

The present invention also provides any of the above-mentioned formulations useful for the treatment of uremic toxins in a subject, optionally including instructions for use of the formulation for such treatments. That is, the kit can include a description of use of the formulation for participation in any biological or chemical mechanism disclosed herein associated with uremic toxicity. The kit can include a description of use of the formulations as discussed herein. The kit can also include instructions for use of a combination of two or more formulations of the invention. Instructions also may be provided for administering the drug by any suitable technique, as described above. A "kit," as used herein, defines a package including any one or a combination of the formulations of the invention, and/or homologs, analogs, derivatives, enantiomers and functionally equivalent formulations thereof, and may also include instructions of any form that are provided in connection with the formulation in a manner such that a clinical professional will clearly recognize that the instructions are to be associated with the specific formulation, for example, as described above. The kits described herein may also contain, in some cases, one or more containers, which can contain formulations such as those described above. The kits also may contain instructions for mixing, diluting, and/or administrating the formulation. The kits also can include other containers with one or more solvents, surfactants, preservative and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting or administering the formulation to the subject.

The formulations of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders. When the formulation provided is a dry powder, the formulation may be reconstituted by the addition of a suitable solvent, which may also be provided. In embodiments where liquid forms of the formulation are used, the liquid form may be concentrated or ready to use. The solvent will depend on the formulation and the mode of use or administration. Suitable solvents for drug formulations are well known and are available in the literature.

The kit, in one set of embodiments, may comprise a carrier that is compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the compartments comprising one of the separate elements to be used in the method. For example, one of the compartments may comprise a positive control for an assay. Additionally, the kit may include containers for other components of the formulations, for example, buffers useful in the assay.

The invention also involves, in some embodiments, the promotion of the treatment of uremic toxins in a subject according to any of the techniques and formulations described herein. As used herein, "promoted" includes all methods of doing business including, but not limited to, methods of selling, advertising, assigning, licensing, contracting, instructing, educating, researching, importing, exporting, negotiating, financing, loaning, trading, vending, reselling, distributing, replacing, or the like that can be associated with the methods and formulations of the invention, e.g., as discussed herein. Promoting may also include, in some cases, seeking approval from a government agency to sell a formulation of the invention for medicinal purposes. Methods of promotion can be performed by any party including, but not limited to, businesses (public or private), contractual or sub-contractual agencies, educational institutions such as colleges and universities, research institutions, hospitals or other clinical institutions, governmental agencies, etc. Promotional activities may include instructions or communications of any form (e.g., written, oral, and/or electronic communications, such as, but not limited to, e-mail, telephonic, facsimile, Internet, Web-based, etc.) that are clearly associated with the invention. As used herein, "instructions" can define a component of instructional utility (e.g., directions, guides, warnings, labels, notes, FAQs ("frequently asked questions"), etc., and typically involve written instructions on or associated with the formulation and/or with the packaging of the formulation, for example, use or administration of the formulation, e.g., in the treatment of uremic toxins in a subject. Instructions can also include instructional communications in any form (e.g., oral, electronic, digital, optical, visual, etc.), provided in any manner such that a user will clearly recognize that the instructions are to be associated with the formulation, e.g., as discussed herein.
 


Claim 1 of 7 Claims

1. An article, comprising: an oral delivery composition, comprising a capsule and a pharmaceutically acceptable carrier, wherein the capsule comprises at least one cell transfected with both a uricase gene and a creatininase gene.

 

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