Patent Application
20220184220
The present application claims priority from United Kingdom Patent Application No. GB1904758.8, filed Apr. 4, 2019, which is incorporated herein in its entirety.
The present invention relates to synthetic prodrugs. In particular, the present invention relates to synthetic prodrugs comprising a chemically conjugated carbohydrate, and the use of the prodrugs for treatment of disorders, such as of enteric inflammatory diseases (EIDs).
The corticosteroids prednisolone (PRED) and budesonide (BUDE) have been used in the treatment of inflammatory bowel disease (IBD) for several decades and their pharmacology has been well studied. Their hydrophobicity allows relative ease of passage through cell membranes into the cytoplasm, where they bind to glucocorticoid receptors responsible for modulating genes controlling the inflammation process. The bound complexes may then be transported into the nucleus, where they induce the upregulation of anti-inflammatory protein expression.
5-aminosalicylic acid (5-ASA) is a member of the aminosalicylate class of IBD drugs that operate with a different mechanism hitherto unknown, although it is postulated that these molecules inhibit the function of cyclooxygenases and consequently the production of prostaglandins, which are responsible for the inflammation process in the colon.
These drugs suffer from nonspecific systemic uptake, with oral doses absorbed predominantly in the small intestine instead of the colon where the affects of IBD most commonly manifest. This negates much of the therapeutic effectiveness of these drugs, as they are most potent when applied directly to the site of inflammation. Furthermore, several undesirable side effects can arise as a result of the nonspecific uptake, including, for example, convulsions, hypertension, blood pressure increase, swelling of the stomach lining and an increased risk of stomach ulcers. Another drawback of these drugs is their low solubility, which leads to low bioavailabilities and poor pharmacodynamics.
In light of these problems, there is a continued interest in developing targeted drug delivery systems to the distal large intestine and colon. These systems may be considered broadly in two classes: formulations and prodrugs. In the former, the active drugs are compounded with various excipients and non-pharmaceutically active ingredients into pill or capsule forms having physical properties that allow a pH-dependent or time-delayed release. The latter involves chemically modifying the drug with a carrier molecule to inactivate it, and on reaching the target area, it is regenerated to its active form in situ spontaneously or by the enzymatic action of the local microflora.
Prodrugs of 5-aminosalicylic acid (5-ASA) have been widely reported. The principal example is sulfasalazine, an azo-based derivative of 5-ASA that was developed in the 1950s. On passage into the colon, sulfasalazine is metabolized into active 5-ASA by the azoreductase activity of the local microflora. While the prodrug is generally effective in treating IBD, the carrier molecule sulfapyridine released as the metabolic product gives rise to undesirable side effects which include malaise, drug hypersensitivity and impotency amongst others. A more recent 5-ASA prodrug in which it is chemically linked to taurine exhibited a markedly improved efficiency of colonic delivery where it is readily converted to 5-ASA.
Glycosylation is another strategy for prodrug synthesis and involves the chemical ligation of the drug to a carbohydrate carrier via β-glycosidic bonds. These conjugates are recognized as substrates by β-glycosidase enzymes produced by the gut microbiota and consequently broken down to release the active drug. Seminal work in this area was conducted by Friend and Chang who reported the synthesis of the β-glucosides of the corticosteroids prednisolone and dexamethasone. Oral doses were administered to rats and drug release was studied. The results showed increases in the delivery of the prodrugs to the cecum (pre-colon) as compared to the free drugs when administered as controls. No colonic delivery was detected however, and this was attributed to the limited testing time window before the animals were euthanized. Subsequently, a library of corticosteroid β-glycosides was prepared and kinetic studies were conducted on their hydrolysis in vitro by rat gastrointestinal tract contents. The results showed unequivocally higher rates of hydrolysis in the cecum compared to the stomach and small intestine. The therapeutic efficacy of these prodrugs was then tested on artificially induced IBD in different animal models with positive results.
β-glucosides of several 5-ASA derivatives have also been reported and demonstrated anti-inflammatory effects in the same order of magnitude as the parent drug on chemically induced ulceritis in mice. No studies on the site specificity of drug release were conducted. More recently, the β-gluco- and galactosides of 5-ASA have been reported with improved solubility, pharmacodynamics and specificity of colonic release in rat models.
In spite of advances in targeted drug delivery as described above, to date there is no known method for the delivery of an anti-inflammatory therapeutic agent to the colon without hydrolysis or metabolism in the proximal gastrointestinal tract, that causes minimal side effects and that can be administered over the long term.
The above information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.
An object of the present application is to overcome the drawbacks associated with current colonic drug delivery by providing a therapeutic agent that is resistant to hydrolysis or metabolism in the proximal gastrointestinal tract (stomach, small and large intestines), but allows the release of an active drug, for example, for IBD treatment, in the colon, and that is safe for long term administration.
In accordance with an aspect of the present application, there is provided xyloglucan-containing prodrugs, referred to herein as glyco-caged prodrugs, and methods of manufacture and use thereof.
In accordance with one aspect of the present application, there is provided a prodrug compound of formula (I) or (II)
xyloglucan-β-O—RD (I)
xyloglucan-β-O-LSI-RD (II)
wherein β-O represents a β-glycosidic bond, RD is a drug moiety and LSI is a self-immolative linker, or a pharmaceutically acceptable salt or solvate thereof, wherein cleavage of the β-glycosidic bond connecting the xyloglucan to RD or LSI results in formation of a drug from the drug moiety.
In accordance with another aspect, there is provided a composition comprising a prodrug compound of formula (I) or (II).
In accordance with another aspect, there is provided method of treating a gastrointestinal disorder by administering a prodrug compound of formula (I) or (II), or a composition comprising of formula (I) or (II), to a subject in need thereof.
In accordance with another aspect, there is provided a use of a prodrug compound of formula (I) or (II), or a composition comprising of formula (I) or (II) for treating a gastrointestinal disorder in a subject in need thereof.
In accordance with another aspect, there is provided a method for producing a prodrug compound of formula (I)
xyloglucan-β-O—RD (I)
wherein β-O represents a β-glycosidic bond and RD is a drug moiety, or a pharmaceutically acceptable salt or solvate thereof, said method comprising: (a) protecting the hydroxyl groups of a xyloglucan to form a protected xyloglucan; (b) performing a glycosylation reaction to form a glycosidic bond between the protected xyloglucan and a drug or a drug derivative; and (c) deprotecting the product of step (b).
In accordance with another aspect, there is provided a method for producing a prodrug compound of formula (II)
xyloglucan-β-O-LSI-RD (II)
wherein β-O represents a β-glycosidic bond, RD is a drug moiety and LSI is a self-immolative linker moiety, or a pharmaceutically acceptable salt or solvate thereof, said method comprising: (a) protecting the hydroxyl groups of a xyloglucan to form a protected xyloglucan; (b) reacting the protected xyloglucan to form a glycosyl halide; (c) performing a glycosylation reaction to form a glycosidic bond between the protected xyloglucan and a linker molecule to form a compound of formula (III)
xyloglucan-β-O-LSI (III)
and
(d) attaching a drug or a drug derivative to LSI of the compound of formula (III).
For a better understanding of the application as described herein, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
The term “comprising” as used herein will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and/or ingredient(s) as appropriate.
As used herein, the term “BoGH5A” refers to glycoside hydrolase 5A, which is an endo-xyloglucanase enzyme, produced by Bacteroides ovatus that recognizes and digests xyloglucan by cleaving internal glycosidic bonds at the non-substituted glucose moiety of the xyloglucan backbone.
As used herein, the term “enteric” indicates a relation to all or a portion of the intestine or the intestinal tract, which includes the small intestine (i.e., the duodenum, the jejunum, and the ileum) and the large intestine (i.e., the cecum, the colon and the rectum).
As used herein, the terms “IBD” and “inflammatory bowel disease” are used interchangeably to describe a group of inflammatory conditions of the colon and small intestine. IBD includes, but is not limited to, the conditions of ulcerative colitis (UC) and Crohn's disease (CD). Both conditions are characterized by varying degrees of erosive inflammation and ulceration predominantly in the distal large intestine and colon.
As used herein, the term “in need thereof” is used to refer to a judgment made by a physician or other caregiver (e.g., a veterinarian) that a subject requires or will benefit from treatment or preventative care. This judgment is made based on a variety of factors that are in the realm of the physician's or caregiver's expertise.
As used herein, “pharmaceutically acceptable” means approved or approvable by a regulatory agency of a federal or a state/provincial government or the corresponding agency in countries other than the United States or Canada, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, including in humans.
As used herein, the term “prodrug” will be understood to refer to a compound that, following administration to a subject, is metabolized into a pharmacologically active drug. The term “decaged” is used herein to reference the metabolism of glyco-caged prodrugs to release pharmacologically acid drug.
As used herein, the term “subject” refers to a human or a non-human animal (e.g., a mammal or a bird).
The present application provides prodrugs that are designed to treat, prevent or ameliorate the symptoms of enteric diseases and disorders, such as inflammatory bowel disease (IBD). The prodrugs of the present application comprise a drug portion and a carbohydrate portion that is cleaved following administration to a subject to release the pharmaceutically active drug. In particular, the prodrugs of the present application target release of an active drug in the intestinal tract through the incorporation of a xyloglucan moiety, which is specifically hydrolyzed by symbiotic bacteria found in the lower gastrointestinal tract. The prodrugs provided herein are referred to as “glyco-caged prodrugs” or “caged prodrugs”, due to the incorporation of the xyloglucan moiety.
An example of the general process of pharmacologically active drug release from a glyco-caged prodrug is depicted in
The glyco-caged prodrugs of the present application are specifically hydrolysed by endo-xyloglucanases, for example those found in Bacteroidetes, such as Bacteroides ovatus or Bacteroides uniformis, for example those found in Bacteroidetes, such as Bacteroides species having a Xyloglucan Utilization Locus that is homologous to that found in Bacteroides ovatus. For example, the glyco-caged prodrugs can be specifically hydrolysed by endo-xyloglucanases found in Bacteroides ovatus, Bacteroides uniformis, Bacteroides cellulosyliticus, Bacteroides faecis, Bacteroides xylanisolvens, Bacteroides fluxus, Bacteroides coprocola and Bacteroides salanitronis, which are found in the native flora of the subject's lower gastrointestinal tract, including in mammalian (e.g., human or swine) or avian lower gastrointestinal tract. Other bacteria having an endo-xyloglucanase that can effectively hydrolyse xyloglucans, and therefore, the glyco-caged prodrugs of the present application can include, but are not limited to, Bacillus parlicheniformis, Paeniclostridium sordellii, Parabacteroides distansonis, Prevotella sp., Salmonella enterica, Streptococcus gallolyticus (e.g., subsp. gallolyticus). Consequently, the glyco-caged prodrugs can be effectively targeted for release of the pharmaceutically active drug in the lower gastrointestinal tract of a subject, such as a mammalian subject (e.g., a human or a swine) or an avian subject (e.g., a chicken).
Glyco-Caged Prodrugs
In accordance with certain exemplary embodiments there is provided a glyco-caged prodrug compound of formula (I) or (II)
xyloglucan-β-O—RD (I)
xyloglucan-β-O-LSI-RD tm (II)
wherein β-O represents a β-glycosidic bond, RD is a drug moiety and LSI is a self-immolative linker moiety, or a pharmaceutically acceptable salt or solvate thereof, wherein cleavage of the β-glycosidic bond connecting the xyloglucan to RD or LSI results in formation and release of a drug from the drug moiety.
Xyloglucan is the term used to refer to a class of linear polysaccharides consisting of (β1→4)-linked d-glucan substituted with xylose. In particular, the backbone of xyloglucan is (β1→4)-linked d-glucan, which is the same as cellulose, and three of the four glucose residues are substituted with α-d-xylose at the O6 position. This is the common structure for all the xyloglucan oligosaccharides, but additional residues are attached to xylose, depending on the source of xyloglucan. Typically, xyloglucans are not homopolymers, but a mix of these oligosaccharide substructures. A description of xyloglucan side chain types is provided in Pauly M. and Keegstra K. (2016) “Biosynthesis of the Plant Cell Wall Matrix Polysaccharide Xyloglucan.” Ann. Rev. Plant Bio., Vol 67: 235-259, which is incorporated herein by reference in its entirety. The structures of four types of xyloglucan oligosaccharides, namely XXXG, XLXG, XLLG and XXLG types are shown below:
The xyloglucan portion of the glyco-caged prodrug of the present application can be any xyloglucan or a xyloglucan oligosaccharide. In certain embodiments, the glyco-caged prodrug comprises a xyloglucan that is or comprises an XXXG-, XXLG-, XLXG-, or XLLG-type xyloglucan, or a mixture of two or more of XXXG-, XXLG-, XLXG-, or XLLG-type xyloglucans. In a specific embodiment, the xyloglucan is a xyloglucan oligosaccharide, for example, an XXXG-type xyloglucan oligosaccharide.
Xyloglucan can be obtained from plant material, such as described in, for example, Hoffman, M. et al. (2005) “Structural analysis of xyloglucans in the primary cell walls of plants in the subclass Asteridae.” Carbohydr. Res., Vol 340 (11): 1826-1840, and Buckeridge M. S. et al. (1997) “A new family of oligosaccharides from the xyloglucan of Hymenaea courbaril L. (Leguminosae) cotyledons.” Carbohydr. Res., Vol 303 (2): 233-237, which are incorporated by reference herein in their entirety. In one specific example, the XXXG xyloglucan is prepared from xyloglucan extracted from tamarind seed kernels. The tamarind XyG, which is composed of a mix of oligosaccharide substructures, is extracted. then liberated with an endo-xyloglucanase, and treated to remove the galactose on the XLLG, XXLG, and XLXG variants and treated with galactosidase to reduce the mixture to XXXG.
The drug moiety of the glyco-caged prodrug is selected based on the particular disorder condition, or disease to be treated. The attachment of the drug moiety to the xyloglucan moiety will vary depending on the chemical structure of the drug. However, in each case the structure of the prodrug comprises β-glycosidic bond that is cleavable by endo-xyloglucanases, such as those found in Bacteroidetes.
The drug moiety can be based on, for example, a small molecule drug or a therapeutic peptide. Examples of suitable small molecule drugs include, but are not limited to: aminosalicylates, such as, 5-aminosalicylic acid (5-ASA, or mesalazine) or methyl mesalazine; corticosteroids, such as, prednisolone, budesonide, dexamethasone or isofluprednone; non-steroidal anti-inflammatory drugs, such as, meloxicam, flunixin or ketoprofen, or cannabinoids, endo-cannabinoids, or analogues thereof. The drug moiety can also be a natural product with anti-inflammatory and/or anti-microbial properties (e.g., thymol, methol and the like). Non-limiting examples of therapeutic peptides that can be incorporated in a glyco-caged prodrug are larazotide and cathelicidin KR-12.
In specific examples the glyco-caged prodrug is XXXG-β-O-dexamethasone (XXXG-DEXA), XXXG-β-O-prednisolone (XXXG-PRED), XXXG-β-O-budesonide (BUDE), XXXG-β-O-mesalazineME (XXXG-5ASAME), XXXG-β-O-mesalazine (XXXG-5ASA), or XXXG-β-O-SIL-mesalazine (XXXG-SIL-5ASA).
The diversity in the structure of drugs that are suitable for the treatment of enteric inflammatory diseases and disorders requires the use of various synthetic strategies for attaching the drugs to the xyloglucan moiety. In some embodiments, the drug is attached directly to the xyloglucan moiety via al-glycosidic bond. In other embodiments the drug is attached to the xyloglucan moiety via a self-immolative linker (SIL).
A self-immolative linker, or SIL, useful in the glyco-caged prodrug of the present application, includes β-glycosidic bond that is susceptible to hydrolysis by endo-xyloglucanases found in Bacteroidetes, such as Bacteroides ovatus or Bacteroides uniformis. The hydrolysis triggers a reaction, or a cascade of reactions, resulting in the break down of the self-immolative linker and release of the pharmaceutically active drug moiety. This is illustrated schematically in
As illustrated in
Synthesis of Glyco-Caged Prodrugs
1. Direct Conjugation to Xyloglucan
Provided herein is a method for synthesizing a prodrug compound of formula (I)
xyloglucan-β-O—RD (I)
wherein β-O represents a β-glycosidic bond, RD is a drug moiety, or a pharmaceutically acceptable salt or solvate thereof. The method generally comprises: (a) protecting the hydroxyl groups of a xyloglucan to form a protected xyloglucan; (b) performing a glycosylation reaction to form a glycosidic bond between the protected xyloglucan and a drug or a drug derivative; and (c) deprotecting the product of step (b). Optionally, the step of forming the glycosidic bond between the protected xyloglucan and a drug or drug derivative comprises first reacting the protected xyloglucan to form a glycosyl halide and then performing a substitution reaction with an alcohol-containing drug or drug derivative and the glycosyl halide.
The synthetic scheme provided below summarizes examples of chemoenzmatic methods and chemical methods of synthesizing a prodrug compound of formula (I).
In step i) the β-galactosidase may be omitted to yield a mixture of variably galactosylated oligosaccharides, which may be fractionated, for example by flash chromatography, following per-O-acetylation for further transformation. Note that the standard structural abbreviations used in the above synthetic scheme are G=D-Glcp; X=[α-D-Xylp(1→6)]-Glcp(1→4); L=[β-D-Galp(1→2)-α-D-Xylp(1→6)]-Glcp(1→4).
As shown in the above synthetic scheme the protecting step can comprise acetylation of the hydroxyl groups of the xyloglucan, although other protecting groups/methods can be employed.
In accordance with certain embodiments, the glycosylation reaction of step (b) proceeds according to option iii) shown in the above synthetic scheme. For example, the glycosylation reaction can comprise: reacting the protected xyloglucan to form a glycosyl halide; and performing a substitution reaction with an alcohol-containing drug or drug derivative and the glycosyl halide. The glycosyl halide can be, for example, a glycosyl bromide.
In accordance with another embodiment, the glycosylation reaction comprises: reacting the protected xyloglucan to form a glycosyl halide; treating the glycosyl halide with Ag2CO3 to form a glycosyl alcohol; treating the glycosyl alcohol with trichloroacetonitrile to form an O-glycosyl trichloroacetimidate; and performing a substitution reaction with an alcohol-containing drug or drug derivative and the O-glycosyl trichloroacetimidate.
2. Conjugation to Xyloglucan via a Self-Immolative Linker
As described above, depending on the structure of the drug moiety, it can be useful to incorporate a self-immolative linker in the glyco-caged prodrug in order to facilitate conjugation to the drug moiety. This can be useful, for example, when the conjugation is via hydroxyl or amino groups of the drug moiety.
Self-immolative linker technology has been employed in the past to attach other chemical moieties. Examples of self-immolative linkers that can be successfully incorporated in the present glyco-caged prodrugs include, but are not limited to, carbamate linkers, carbamoyl linkers, and benzyl ester linkers.
One aspect provides a method for producing a prodrug compound of formula (II)
xyloglucan-β-O-LSI-RD (II)
wherein β-O represents a β-glycosidic bond, RD is a drug moiety and LSI is a self-immolative linker moiety, or a pharmaceutically acceptable salt or solvate thereof, which comprises: protecting the hydroxyl groups of a xyloglucan to form a protected xyloglucan; reacting the protected xyloglucan to add an appropriate leaving group moiety; performing a glycosylation reaction to form a glycosidic bond between the protected xyloglucan and a linker molecule to form a compound of formula (III)
xyloglucan-β-O-LSI (III)
and attaching a drug or a drug derivative to LSI of the compound of formula (III). Optionally, the step of adding the appropriate leaving group comprises first reacting the protected xyloglucan to form a glycosyl halide. The next step then comprises performing a substitution reaction with a linker molecule and the glycosyl halide.
The xyloglucan can be an XXXG, XXLG, XLXG, or XLLG-type xyloglucan, or it can be a mixture of two or more of XXXG, XXLG, XLXG, or XLLG-type xyloglucans. In certain embodiments, the xyloglucan is an XXXG-type.
The scheme provided below summarizes a synthetic method for producing phenyl glycoside/carbamate self-immolative linkers. In this synthetic scheme the drug moiety is represented by R or peptide.
The scheme provided below summarizes a synthetic method for producing carbamoyl glycoside self-immolative linkers. In this synthetic scheme the drug moiety is represented by R.
The scheme provided below summarizes a synthetic method for producing alkyl glycoside carbamate self-immolative linkers. In this synthetic scheme the drug moiety is represented by R or Drug.
The scheme provided below summarizes a synthetic method for producing phenyl glycoside-benzyl ester self-immolative linkers. In this synthetic scheme the drug moiety is represented by NSAID as an example, however, other drug types can be incorporated in the glyco-caged prodrug produced using this synthetic method.
Although the above synthetic schemes indicate that the starting material is the per-acetylated XXXG bromide, it should be readily appreciated that other protected xyloglucan derivatives (i.e., in this case, xyloglucans derivatized with an appropriate leaving group), such as protected xyloglucan halides, can be readily employed as starting materials for these processes. Furthermore, the above synthetic schemes are merely illustrative of the methods employed to synthesize the present glyco-caged prodrugs and are not intended to limit the methods for manufacturing these prodrugs.
Pharmaceutical Compositions
In another aspect, the present application provides a pharmaceutical composition comprising a glyco-caged prodrug, optionally in combination with a pharmaceutically acceptable carrier, diluent, excipient or medium, wherein the glyco-caged prodrug is a compound of formula (I) or formula (II):
xyloglucan-β-O—RD (I)
xyloglucan-β-O-LSI-RD (II)
wherein β-O represents β-glycosidic bond, RD is a drug moiety and LSI is a self-immolative linker, or a pharmaceutically acceptable salt or solvate thereof, wherein cleavage of the β-glycosidic bond connecting the xyloglucan to RD or LSI results in formation and release of a drug from the drug moiety
In certain embodiments, the pharmaceutical composition comprises a combination of two or more glyco-caged prodrugs.
In certain embodiments, the pharmaceutical composition comprises one or more of XXXG-β-O-dexamethasone (XXXG-DEXA), XXXG-β-O-prednisolone (XXXG-PRED), XXXG-β-O-budesonide (BUDE), XXXG-β-O-mesalazineME (XXXG-5ASAME), XXXG-β-O-mesalazine (XXXG-5ASA), or XXXG-SIL-β-O-mesalazine (XXXG-SIL-5ASA).
The amount of glyco-caged prodrug in the pharmaceutical composition can be based on the amount of pharmaceutically active drug to be administered at the targetted lower gastrointestinal tract. In some embodiments, the amount of glyco-caged prodrug in the composition is based on an amount of the pharmaceutically active drug that is administered via conventional means since, in certain embodiments, the use of the glyco-cage can reduce the amount of active drug required to have the same pharmaceutical effect. As an example, the dosage range of both the active drug and prodrug forms of dexamethasone currently being investigated in mice is 0.05-2.0 mg/kg, and the range for prednisolone is 0.5-2.2 mg/kg. Dosages used in mice can be extrapolated to ranges for use in humans using standard dose conversion, of example based on FDA guidelines, or allometric scaling (see, for example, Nair A. B. and Jacob S. (2016) “A simple practice guide for dose conversion between animals and human.” J. Basic Clin Pharma., Vol. 7: 27-31, which is incorporated herein by reference in its entirety).
Suitable pharmaceutical compositions of the present application will generally include an amount of the glyco-caged prodrug(s) with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use. The techniques of preparation are generally well known in the art, as exemplified by Remington's Pharmaceutical Sciences (18th Edition, Mack Publishing Company, 1995).
The compositions described herein may be administered systemically or locally, e.g.: orally (e.g., using capsules, powders, solutions, suspensions, tablets, sublingual tablets and the like). Preferably, the compositions are administered orally. In compositions for non-oral administration the glyco-caged prodrug can be formulated with one or more additives to improve uptake and/or bioavailabilty (e.g., DMSO, liposome, lipid, detergent, etc.).
In certain embodiments, the pharmaceutical compositions of the present application comprise a glyco-caged prodrug as the sole active ingredient and one or more other desired pharmaceutically inactive additives, excipients, and/or components (e.g., polymers, sterically hindered primary amines, cations, fillers, binders, carriers, excipients, diluents, disintegrating additives, lubricants, solvents, dispersants, coating additives, absorption promoting additives, controlled release additives, anti-caking additives, anti-microbial additives, preservatives, sweetening additives, colorants, flavors, desiccants, plasticizers, dyes, or the like), and no other active pharmaceutical ingredient(s). In other embodiments, the pharmaceutical composition comprises a combination of two or glyco-caged prodrugs, in combination with one or more other desired pharmaceutically inactive additives, excipients, and/or components.
The glyco-caged prodrug described herein can be combined with any pharmaceutically tolerable carrier or medium, e.g., solvents, dispersants, coatings, absorption promoting agents, controlled release agents, and one or more inert excipients (which include starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, disintegrating agents, and the like), etc.
For treatment of gastrointestinal disorders, the glyco-caged prodrugs described herein are preferably administered orally, e.g., as a tablet, capsule, sachet containing a predetermined amount of the active ingredient pellet, gel, paste, syrup, bolus, electuary, slurry, powder, lyophilized powder, granules, as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, via a liposomal formulation or in some other form. Orally administered compositions can include binders, lubricants, inert diluents, lubricating, surface active or dispersing agents, flavoring agents, and humectants. Orally administered formulations such as tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein.
The glyco-caged prodrug can be co-administered with other agents used to treat gastrointestinal disorders including but not limited to the agents described herein.
In another aspect, suitable pharmaceutical compositions can comprise one or more other therapeutic agents. Such therapeutic agents include, without limitation, analgesic agents; anti-secretory agents; bile acid sequestrants; prokinetic and promotility agents; antibiotics; antiemetics; and mucosal-protecting agents.
As described above, the release of the pharmaceutically active drug from the glyco-caged prodrugs described herein requires the presence of a bacterial endo-xyloglucanase. In most instances, the bacterial endo-xyloglucanase is present in the lower gastrointestinal tract from the microflora that is naturally present in the subject (e.g., a mammallian or avian subject). In particular, the bacterial endo-xyloglucanase can be found in Bacteroidetes, such as Bacteroides ovatus or Bacteroides uniformis, or other Bacteroides species having a Xyloglucan Utilization Locus that is homologous to that found in Bacteroides ovatus, which are found in the native flora of the mammalian lower gastrointestinal tract, including in the swine or human lower gastrointestinal tract, or avian gastrointestinal tract (e.g., chicken).
By way of example, the glyco-caged prodrugs can be specifically hydrolysed by endo-xyloglucanases found in Bacteroides ovatus, Bacteroides uniformis, Bacteroides cellulosyliticus, Bacteroides faecis, Bacteroides xylanisolvens, Bacteroides fluxus, Bacteroides coprocola and Bacteroides salanitronis, which are found in the native flora of the subject's gastrointestinal tract, including in the mammalian (e.g., human) lower gastrointestinal tract. Other bacteria having an endo-xyloglucanase that can effectively hydrolyse xyloglucans, and therefore, the glyco-caged prodrugs of the present application can include, but are not limited to, Bacillus parlicheniformis, Paeniclostridium sordellii, Parabacteroides distansonis, Prevotella sp., Salmonella enterica, Streptococcus gallolyticus (e.g., subsp. gallolyticus).
In some subjects, such as those that have been treated, or are undergoing treatment, with antibiotics, the microflora is reduced such that there is insufficient bacterial endo-xyloglucanase present to effect the release of the pharmaceutically active drug from the glyco-caged prodrugs described herein. Furthermore, even in subjects that have a normal microflora, the release of the pharmaceutically active drug from the glyco-caged prodrugs can be increased or improved with increased amounts of bacterial endo-xyloglucanase in the lower gastrointestinal tract. Accordingly, in some embodiments, the pharmaceutical composition additionally comprises a probiotic to provide additional endo-xyloglucanase. In certain embodiments, the probiotic comprises a Bacteroidetes culture, such as a culture of Bacteriodes, for example B. ovatus or B. uniformis, or a combination thereof. In other embodiments, the probiotic comprises a culture of one of more bacteria that is a Bacteroides species having a Xyloglucan Utilization Locus that is homologous to that found in Bacteroides ovatus. In other embodiments, the probiotic comprises a culture of one or more bacteria selected from Bacteroides ovatus, Bacteroides uniformis, Bacteroides cellulosyliticus, Bacteroides faecis, Bacteroides xylanisolvens, Bacteroides fluxus, Bacteroides coprocola, Bacteroides salanitronis, Bacillus parlicheniformis, Paeniclostridium sordellii, Parabacteroides distansonis, Prevotella sp., Salmonella enterica, Streptococcus gallolyticus (e.g., subsp. gallolyticus).
Alternatively, the pharmaceutical composition is formulated for administration together with a probiotic. Again, the probiotic can comprise a Bacteroidetes culture, such as a culture of B. ovatus or B. uniformis, or a combination thereof. In other embodiments, the probiotic comprises a culture of one of more bacteria that is a Bacteroides species having a Xyloglucan Utilization Locus that is homologous to that found in Bacteroides ovatus. In other embodiments, the probiotic comprises a culture of one or more bacteria selected from Bacteroides ovatus, Bacteroides uniformis, Bacteroides cellulosyliticus, Bacteroides faecis, Bacteroides xylanisolvens, Bacteroides fluxus, Bacteroides coprocola, Bacteroides salanitronis, Bacillus parlicheniformis, Paeniclostridium sordellii, Parabacteroides distansonis, Prevotella sp., Salmonella enterica, Streptococcus gallolyticus (e.g., subsp. gallolyticus).
It should be readily understood that this the above teaching does not provide an exhaustive list of bacteria. Other bacteria that are not listed here, but that have endo-xyloglucanase activity, may be included in a probiotic as described herein.
Method of Treatment
In various embodiments, the glyco-caged prodrugs described herein are useful for the prevention, reduction, alleviation, or treatment of a gastrointestinal disease or disorder. Accordingly, one aspect comprises a method of preventing, reducing, alleviating, or treating a gastrointestinal disease or disorder by administering a glyco-caged prodrug as described herein to a subject in need thereof.
In some embodiments, the gastrointestinal disorder is an enteric disease, such as inflammatory bowel disease. In specific embodiments, the gastrointestinal disorder Crohn's disease or ulcerative colitis.
In some embodiments, the method of preventing, reducing, alleviating, or treating the gastrointestinal disease or disorder additionally comprises administering a probiotic before, during or after administration of the glyco-caged prodrug. In specific embodiments, the probiotic is administered to the subject prior to administration of the glyco-caged prodrug in order to colonize the lower gastrointestinal tract with an endo-xyloglucanase producing bacteria, which may be the same or different from the bacteria present in the subject's microflora. As detailed above, the probiotic is administered in order to increase the amount of bacterial endo-xyloglucanase present in the lower gastrointestinal tract of the subject. In certain embodiments, the probiotic comprises a Bacteroidetes culture, such as a culture of Bacteroides ovatus or Bacteroides uniformis, or a combination thereof. In other embodiments, the probiotic comprises a culture of one of more bacteria that is a Bacteroides species having a Xyloglucan Utilization Locus that is homologous to that found in Bacteroides ovatus. In other embodiments, the probiotic comprises a culture of one or more bacteria selected from Bacteroides ovatus, Bacteroides uniformis, Bacteroides cellulosyliticus, Bacteroides faecis, Bacteroides xylanisolvens, Bacteroides fluxus, Bacteroides coprocola, Bacteroides salanitronis, Bacillus parlicheniformis, Paeniclostridium sordellii, Parabacteroides distansonis, Prevotella sp., Salmonella enterica, Streptococcus gallolyticus (e.g., subsp. gallolyticus).
To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.
Materials
All chemicals were purchased from established vendors and manufacturers.
Chemical-Enzymatic Synthesis of Glyco-Caged Prodrugs
Five glyco-caged prodrugs, XXXG-β-O-dexamethasone (XXXG-DEXA), XXXG-β-O-prednisolone (XXXG-PRED), XXXG-β-O-budesonide (BUDE), XXXG-β-O-mesalazineME (XXXG-5ASAME), and XXXG-β-O-mesalazine (XXXG-5ASA), and (Table 1), have been synthesized via three conjugation methodologies—Koenigs-Knorr, TCA-imidate, and phase transfer. Their syntheses are described in Schemes 1, 2, 3, and 4, beginning from readily available powdered tamarind seed kernel powder. Where problems may arise due to functional group compatibilities to the reagents used, these functional groups were protected with an appropriate protecting group by a known method in advance. These protecting groups were subsequently removed by known methods. The reaction conditions were optimized to increase the combined overall yield and/or to minimize environmental impacts.
Table 1 provides a summary of the glyco-caged prodrugs synthesized in this study.
Synthesis of XXXG
Scheme 1. Step 1
This step is the enzymatic digestion of xyloglucan from powdered tamarind seed kernels by the action of cellulase from the fungus, Trichoderma reesei. This process produces a mixture of several xyloglucan oligosaccharides (XyGOs) that includes XXXG after an average time period of 6 hrs. The mixture is further treated with β-galactosidase enzyme from the fungus, Aspergillus niger, providing XXXG as the major product after about 12-24 hrs. Other enzymes that could be used in the hydrolysis include but are not limited to various broad specificity endo-glucanases/cellulases or specific endo-xyloglucanases (e.g., BoGH5 (Larsbrink J. et al. (2014) “A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes.” Nature, 506, 498-502), CjGH5D (Attia M. A. et al. (2018) “In vitro and in vivo characterization of three Cellvibrio japonicus Glycoside Hydrolase Family 5 members reveals potent xyloglucan backbone-cleaving functions.” Biotechnol. Biofuels, 11, 45), PoGH74 (Arnal G. et al. (2018) “Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 endo-xyloglucanase.” Biochem. J., 475, 3963-3978.), etc. (Attia, M. A. and Brumer, H. (2016) “Recent structural insights into the enzymology of the ubiquitous plant cell wall glycan xyloglucan.” Curr. Opin. Struct. Biol., 40, 43-53; and Eklöf, J. M., Ruda, M., Brumer, H.* (2012) Distinguishing xyloglucanase activity in endo-beta(1-4)glucanases. Method. Enzymol., Vol. 510, Ch. 6, pp. 97-120)) and various beta-galactosidases (e.g. CjBgl35A (Larsbrink J. et al. (2014) “A complex gene locus enables xyloglucan utilization in the model saprophyte Cellvibrio japonicus. Mol. Microbiol., 94, 418-433”), BoGH2 (Larsbrink J. et al. (2014) “A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes.” Nature, 506, 498-502), etc. (Attia, M. A. and Brumer, H. (2016) “Recent structural insights into the enzymology of the ubiquitous plant cell wall glycan xyloglucan.” Curr. Opin. Struct. Biol., 40, 43-53)). These reactions are typically performed in buffered conditions (pH=5 to 6.5) and at temperatures of between 37-58° C. in aqueous solution.
Procedure: De-oiled tamarind kernel powder (20 g) was suspended in 2 L purified Milli-Q water and stirred overnight at 60° C. The suspension was centrifuged and the supernatant decanted into an autoclaved 2 L Erlenmeyer flask. 15 μL of 1 M ammonium acetate buffer and 33 mg of T. reesei cellulase was added, and the mixture was stirred at 45° C. After 6 hrs, 0.31 mL of Aspergillus niger β-galactosidase was added and the mixture was stirred for a further 24 hrs at 45° C. The mixture was then heated at 80° C. for 20 mins, and concentrated in vacuo. The crude material was then lyophilized to provide 13 g of crude XXXG, confirmed by HPLC analysis.
Synthesis of Per-O-Ac-XXXG
Scheme 1, Step 2
This step is the acetylation of all hydroxyl groups present in XXXG (per-O-acetylation) and can be carried out either under acidic or basic conditions with the corresponding catalyst. The most common acetylating agent is acetic anhydride, although more reactive agents e.g. acetyl chloride are sometimes used for more difficult substrates but may lead to side products. The base catalyst is usually 4-DMAP (4-dimethylaminopyridine) and loads typically range from 1-5 mol %. The reaction solvent is usually pyridine although the use of other solvents is not uncommon as long as they are not involved in the reaction. Heating is required in the reaction, and temperatures range from 40-60° C. Reaction times are variable and dependent on the starting material, but range from 3-24 hrs.
Procedure A: 5 g of crude XXXG obtained in Step 1 was suspended in 30 mL of DMF and heated with stirring at 80° C. for 15 to 20 mins. The suspension was centrifuged for 20 mins at 4000 g and decanted into a heat-dried round-bottom flask. 100 mL of acetic anhydride, 100 mL of pyridine and 50 mg of 4-DMAP were then added, and the mixture was stirred at 60° C. for 16 hrs, whereupon it was concentrated in vacuo and the crude product triturated with 50 mL dichloromethane. This solution was then washed with 3×100 mL of water, 100 mL of 5% lithium chloride solution and 100 mL of saturated sodium bicarbonate solution. The washed solution was then dried over anhydrous magnesium sulfate and concentrated in vacuo to provide 3 g of crude material, which was purified with flash column silica gel chromatography using 2:1 toluene:acetone as the eluent to afford 2.4 g of pure Per-O-Ac-XXXG.
Procedure B: 6.0 g of crude XXXG obtained in Step 1 was suspended in 50 mL of AcOH. To this mixture 40 mL of acetic anhydride was added. The stirring was continued for one hr before catalytic amount of sulfuric acid was added dropwise. The progress of the acetylation was monitored via thin layer chromatography. After 4-6 hrs, the acetylation was complete and the reaction was quenched with 1800 mL of water/ice. A heavy white precipitate appeared at this point, and the stirring was continued till all the ice had been melted. The white suspension was filtered. The filter cake was washed with deionized water (30 mL×3) and air dried in a fume hood overnight. Crude Per-O-Ac-XXXG thus obtained (9.86 g) was loaded to a silica gel column. Gradient elution with toluene/acetone (5:1 to 2:1) afforded 8.6 g of pure Per-O-Ac-XXXG as evidenced by thin layer chromatography and MALAI mass spectrometry. TLC single spot Rf=0.25 (toluene/acetone=2:1); m/z (MALDI) 1925.5 (M+Na+, C79H106O53Na requires 1925.5).
Synthesis of Per-O-Ac-XXXG-Br
Scheme 1, Step 3
This step is the anomeric bromination of Per-O-Ac-XXXG by treatment with a solution of hydrogen bromide in acetic acid; a well-known method to access α-anomers. The reaction is usually carried out in the cold (0° C.) and with a large excess of hydrogen bromide, typically 20-30 molar equivalents to ensure complete bromination within reaction times of between 1-3 hrs. Solvents used are typically chlorinated with dichloromethane the most common although others such as chloroform and 1,2-dichloroethane are also used.
Procedure A: To a solution of Per-O-Ac-XXXG (0.5 g, 0.28 mmol) obtained in Step 2 in dichloromethane at 0° C. was added to 2.5 mL of 33 wt % hydrogen bromide in acetic acid, and the mixture was stirred for 2 hrs, whereupon it was poured into 20 g of ice and extracted with 2×30 mL of dichloromethane. The combined dichloromethane extract was then washed with 2×40 mL of cold water, 2×40 mL cold saturated sodium bicarbonate and dried over anhydrous magnesium sulfate. The extract was then concentrated in vacuo to provide 0.48 g of crude material, which was purified with flash column silica gel column chromatography using step gradient elution with 3:1 toluene:acetone to 2:1 toluene acetone to afford 0.35 g of Per-O-Ac-XXXG-Br.
Procedure B: To a solution of Per-O-Ac-XXXG (0.21 g, 0.11 mmol) obtained in Step 2 in 5 mL of dichloromethane/AcOEt (10:1) at room temperature was added 367 mg (1 mmol) of TiBr4 and argon. The mixture was stirred in the dark for 68 hrs, whereupon it was poured into 100 g of ice/water and extracted with 2×30 mL of dichloromethane. The combined dichloromethane extract was then washed with 2×40 mL cold water, 2×40 mL cold saturated sodium bicarbonate and dried over anhydrous magnesium sulfate. The extract was then concentrated in vacuo to provide 0.22 g of crude material, which was purified with flash column silica gel column chromatography using step gradient elution with 3:1 toluene:acetone to 2:1 toluene acetone to afford 0.18 g of pure Per-O-Ac-XXXG-Br as evidenced by TLC and mass spectrometry. TLC single spot Rf=0.26 (toluene/acetone=2:1); m/z (MALDI) 1945.3 (M+Na+, C79H106O53Na requires 1945.4).
Synthesis of Per-O-Ac-XXXG-OH
Scheme 1, Step 4
The freshly made Per-O-Ac-XXXG-Br (1.3 g, 676 mg) was dissolved in 25 mL of acetone, followed by the addition of 5 mL of water. To this mixture was added 0.77 g of AgCO3, and the resultant suspension was stirred in dark overnight. The reaction progress was monitored via thin layer chromatography until the TLC test indicated the selective hydrolysis was complete. The suspension was then filtered and the filtrate was concentrated under reduced pressure. The crude thus obtained was loaded onto a silica gel column. Eluting the column with toluene/acetone (4:1 to 2:1) afforded 1.18 g of pure Per-O-Ac-XXXG-OH.
Synthesis of Per-O-Ac-XXXG-TCA
Scheme 1, Step 5
Under a stream of argon, TCA (trichloroacetonitrile, 0.45 mL, 4.49 mmol) and DBU (1,8-diazabicyclo[5,4,0]-undec-7-ene, 10 μl) were added to a solution of Per-O-Ac-XXXG-OH (0.39 g, 0.20 mmol) in anhydrous dichloromethane (DCM), and the reaction was stirred for 2 hrs at −30° C. until a TLC test indicated the complete consumption of the starting material. The volatiles were removed under reduced pressure, and the crude thus obtained was purified via silica gel flash chromatography, which afforded 0.3 g of Per-O-Ac-XXXG-TCA.
Synthesis of Per-O-Ac-XXXG-β-O-dexamethasone
Scheme 1, Step 6
Per-O-Ac-XXXG-TCA (307 mg, 0.15 mmol) was dissolved in 12 mL of anhydrous DCM, followed by the addition of 3 Å MS (molecular sieve). The suspension was stirred at room temperature for 0.5 hr before it was cooled down to −30° C. BF3.Et2O (50 μl) was added to this mixture dropwise under argon. The reaction was allowed to proceed for 4-20 hrs until a TLC test indicated the complete consumption of the starting material. Triethylamine (1 mL) was added to the reaction mixture at 30° C. to quench the reaction. DCM (50 mL) was added and the reaction mixture was allowed to warm to room temperature. The suspension was then filtered, washed with DCM (20 mL×3). The filtrate was concentrated to afford 480 mg of crude as a white solid which was purified via silica gel flash chromatography (toluene/acetone 4:1 to 2:1). The resultant Per-O-Ac-XXXG-β-O-dexamethasone (140 mg) was characterized by thin layer chromatography, proton NMR and MALDI mass spectrometry. Rf=0.26 (toluene/acetone 2:1); m/z 2257.6 (M+Na+, C99H131FO56Na requires 2257.7).
Synthesis of XXXG-β-O-dexamethasone
Scheme 1, Step 7
Per-O-Ac-XXXG-β-O-dexamethasone (140 mg, 0.062 mmol) was dissolved in a mixture of methanol and dichloromethane (6 mL, 4:1) at room temperature under stirring. To this mixture 15 μL (0.07 mmol) of a 25% (w/w) solution of sodium methoxide in methanol was added. The reaction was allowed to continue for 17 hrs until a TLC test indicated the complete consumption of the starting material. 200 mg of Amberlite™ IR-120 resin (H+ form) was added and stirred for 10 min, whereupon the suspension was filtered and the filtrate was concentrated under reduced pressure. The crude material was then purified with flash column silica gel chromatography using ethyl acetate:isopropanol:water (3:2:1) as the eluent to afford 65 mg (0.045 mmol) of pure XXXG-β-O-dexamethasone. m/z 1459.4 (M+Na+, C61H93FO37Na requires 1459.5).
Synthesis of XXXG-β-O-prednisolone
The XXXG-β-O-prednisolone was obtained in a manner similar to that described above for the synthesis of XXXG-β-O-dexamethasone. 200 mg of XXXG-β-O-prednisolone was obtained by following the general procedure described in scheme 1, step 1-7.
Synthesis of XXXG-β-O-prednisolone and XXXG-β-O-budesonide
The XXXG-β-O-prednisolone was obtained in a manner similar to that described above for the synthesis of XXXG-β-O-dexamethasone.
Alternative Synthesis of XXXG-β-O-prednisolone and XXXG-β-O-budesonide
Steps 1-3 of scheme 2 were performed in the same manner as described above in relation to Scheme 1.
Scheme 2, Step 4
This step is the glycosylation of the known corticosteroidal IBD drugs prednisolone and budesonide with Per-O-Ac-XXXG-Br prepared in Step 3 via the classical Koenigs-Knorr reaction. The reaction involves activation of the anomeric position by a suitable promoter reagent, typically silver (I) salts such as silver (I) carbonate, silver (I) oxide and silver (I) trifluoromethanesulfonate, to generate a reactive oxocarbenium intermediate. If neighbouring group participation is present, a stereoselective glycosylation usually occurred to afford the β-glycoside. Typical loads for the promoter are between 1-3 molar equivalents with respect to the glycosyl donor. It is advantageous to introduce an excess (1.5-3 molar equivalents) of the glycosyl acceptor to drive consumption of the donor to completion. Common solvents used in this reaction are chlorinated, and include, for example, dichloromethane, chloroform and carbon tetrachloride. Reaction temperatures can range between ambient and reflux. Reaction times vary with the starting material but are usually between 5-20 hrs.
Procedure (for preparation of Per-O-Ac-XXXG-PRED): To a solution of Per-O-Ac-XXXG-Br (170 mg, 0.09 mmol) obtained in Step 3 in anhydrous 1,2-dichloroethane (4 mL) was added prednisolone (48 mg, 0.13 mmol) and 4 A molecular sieves, and the mixture was stirred at room temperature for 30 mins. Silver (I) trifluoromethanesulfonate (34 mg, 0.13 mmol) was then added and the mixture was stirred in the dark at 80° C. for 7 hrs, whereupon it was cooled to room temperature, diluted with dichloromethane and filtered through a Celite™ pad. This solution was washed with 2×40 mL saturated sodium bicarbonate and 40 mL of brine and dried over anhydrous magnesium sulfate, whereupon it was concentrated in vacuo to provide 158 mg of crude material, which was purified with flash column silica gel chromatography using step gradient elution with 99:1-98:2-97:3 dichloromethane:methanol to afford 78.6 mg of a mixture of Per-O-Ac-XXXG-PRED and Per-O-Ac-XXXG-OH, a byproduct arising from the hydrolysis of unreacted intermediate during work-up. It was not possible to separate these two species via column chromatography.
The preparation of Per-O-Ac-XXXG-BUDE was carried out by the same procedure as above, whereby 270.1 mg, 0.14 mmol of Per-O-Ac-XXXG-Br was treated with 90.8 mg, 0.21 mmol of budesonide in the presence of 54.2 mg, 0.21 mmol of silver (I) trifluoromethanesulfonate to provide 287 mg of crude material, which was then purified to give 145.9 mg of a mixture of Per-O-Ac-XXXG-BUDE and Per-O-Ac-XXXG-OH. This mixture was used in the de-acetylation without further purification.
Scheme 2, Step 5
This step is the global deacetylation of the glycosides prepared in Step 4 by the action of sodium methoxide. Only a catalytic amount ranging from 20-50 mol % of sodium methoxide is required as it is continually regenerated from the bulk solvent (methanol). The reaction is usually carried out at ambient temperature and reaction times vary with starting materials, but are typically between 1-20 hrs.
Procedure (for preparation of XXXG-PRED): To the mixture of Per-O-Ac-XXXG-PRED and Per-O-Ac-XXXG-OH obtained in Step 4 (78.6 mg, 0.036 mmol calculated w.r.t. Per-O-Ac-XXXG-PRED) in 10% dichloromethane in methanol (2.4 mL) was added via micropipette 1.5 μL (0.007 mmol) of a 25 wt % solution of sodium methoxide in methanol. The mixture was stirred overnight for 17 hrs. 200 mg of Amberlite™ IR-120 H+ form resin was added and stirred for 10 min, whereupon the solvent was removed in vacuo. The crude material was then purified with flash column silica gel chromatography using 3:2:1 ethyl acetate:isopropanol:water as the eluant to afford 24.3 mg of pure XXXG-PRED.
The preparation of XXXG-BUDE was carried out by the same procedure as above, whereby the mixture of Per-O-Ac-XXXG-BUDE and Per-O-Ac-XXXG-OH obtained in Step 4 (145.9 mg, 0.064 mmol calculated w.r.t. PerOAc-XXXG-BUDE) was treated with 2.7 μl of 25 wt % solution of sodium methoxide in methanol. The crude material obtained on work-up was purified to afford 65.3 mg of pure XXXG-BUDE.
Scheme 3, Step 1
This step is the glycosylation of the known IBD drug methyl 5-aminosalicylate. It is an alternative method to the Koenigs-Knorr reaction for the glycosylation of phenolates. In this reaction, the sodium salt of methyl 5-nitrosalicylate was coupled with Per-O-Ac-XXXG-Br obtained in Step 3 (Scheme 1). The reaction is carried out in a biphasic medium consisting of water and dichloromethane and reactant transfer is facilitated by the use of a phase transfer catalyst. Typical loads for the acceptor are between 1.5-3 molar equivalents with respect to the donor, and 0.5-1 molar equivalent with respect to the donor for the phase transfer catalyst. The reaction is usually carried out at ambient temperature and typical reaction times vary with starting material but is usually in the range of 24-48 hrs.
Procedure: To a solution of Per-O-Ac-XXXG-Br (160 mg, 0.085 mmol) and benzyltributylammonium chloride (27 mg, 0.085 mmol) in dichloromethane (2 mL) was added a solution of methyl 2-nitrosalicylate sodium salt (56 mg, 0.26 mmol) in 7 mL water. The mixture was stirred at room temperature for 48 hrs, whereupon it was diluted with water and dichloromethane. The dichloromethane layer was extracted with 2×20 mL water, washed with brine and dried over anhydrous magnesium sulfate. Concentration of the solvent in vacuo provided 190 mg of crude material, which was purified with flash column silica gel chromatography using step gradient elution with 3:1-2:1 toluene:acetone to afford 130 mg of Per-O-Ac-XXXG-5ASAME-NO2.
Scheme 3, Step 2
This step is the hydrogenation of Per-O-Ac-XXXG-5ASAME-NO2 and involves the reduction of the nitro functional group therein. The reaction is performed in the presence of a metal catalyst in a suitable solvent, usually methanol, under a hydrogen atmosphere of 1 atm and at temperatures ranging from ambient to 100° C. Typical catalyst loads fall between 1-10 wt % and reaction times vary with starting material and temperature, but are usually between 3 to 20 hrs. Examples of suitable catalysts include palladium on carbon, platinum on carbon and platinum dioxide.
Procedure: To a solution of Per-O-Ac-XXXG-5ASAME-NO2 (124 mg, 0.061 mmol) obtained in Step a in anhydrous, degassed methanol (2.3 mL) was added 12.4 mg of 10% palladium on carbon, and the mixture was stirred at 40° C. for 3 hrs. The mixture was suction filtered through a Celite pad and the solvent was removed in vacuo to give 110 mg of crude material, which was subsequently purified with flash column silica gel chromatography (the silica gel was pre-neutralized with a 2% triethylamine in hexane solution) using 2:1 toluene:acetone as the eluant to afford 72 mg of Per-O-Ac-XXXG-5ASAME.
Scheme 3, Step 3
This step involves the global deacetylation of Per-O-Ac-XXXG-5ASAME with catalytic sodium methoxide.
Procedure: To a solution of Per-O-Ac-XXXG-5ASAME obtained in Step 2 above (71.1 mg, 0.035 mmol) in 10% dichloromethane in methanol (2.4 mL) was added via micropipette 3.75 μL (0.0017 mmol) of a 25 wt % solution of sodium methoxide in methanol. The mixture was stirred for 3 hrs, whereupon 200 mg of Amberlite™ IR-120 H+ form resin was added and stirred for 10 min. The solvent was removed in vacuo and the residue redissolved in water. The solution was filtered and lyophilized to afford 34 mg of crude XXXG-5ASAME. The pure XXXG-5ASAME (30 mg) was obtained after subjecting the above crude to reverse phase chromatography.
Scheme 4, Step 1
To the freshly made Per-O-Ac-XXXG-Br (417 mg) were added 10 mL of dry acetonitrile, 67 mg of 5-ASAME-NO2 and 1 g of 3 Å MS. The suspension was stirred under argon for 0.5 h before the addition Ag2O (100 mg) and 2,6-lutidine (0.8 mL). The resultant suspension was stirred in the dark overnight at room temperature. 2 g of silica gel was added and the volatiles were removed under reduced pressure. The resultant solid was loaded onto the top of a prepared silica gel column. Elution with toluene/acetone (4:1 to 2:1) afforded 240 mg of Per-O-AcXXXG-5ASAME-NO2. m/z 2062.6 (M+Na+, C85H109NO56Na requires 2062.6).
Scheme 4, Step 2
Per-O-Ac-XXXG-5ASAME-NO2 (240 mg) was dissolved in a mixture of methanol and DCM (4:1, 6 mL). Catalytic amount of sodium methoxide (2 mg) was added and the resultant suspension was stirred overnight before all volatiles were removed under reduced pressure at 40° C. The crude (180 mg, XXXG-5ASAME-NO2) thus obtained was used in the next step without further purification.
Scheme 4, Step 3
The crude XXXG-5ASAME-NO2 (180 mg) obtained in the previous step was dissolved in 1 N sodium hydroxide (8 mL) and the resultant solution was warmed to 45° C. under stirring. The reaction was allowed to continue at the same conditions until a TLC indicated a complete hydrolysis was accomplished. Pure XXXG-5ASA-NO2 (105 mg) was obtained via reverse phase chromatography.
Scheme 4, Step 4
XXXG-5ASA-NO2 (82 mg) obtained in the previous step was dissolved in 7 mL of methanol/water (1:1). To this mixture was added 10 mg of Pd/C (10%) under stirring. Hydrogen was then introduced after degassing. The reduction was complete in 3 to 12 hrs. Pure product XXXG-5ASA (65 mg) was obtained via reverse phase chromatography (water/methanol, 90/10 to 50/50). The compound was characterized by proton NMR and mass spectrometry. m/z 1220 (M+Na+, C46H71NO35Na requires 1220).
In vitro enzyme kinetic assays were performed on XXXG-5ASAME, XXXG-PRED, and XXXG-BUDE as examples of the glyco-caged prodrugs of the present application.
The hydrolysis of XXXG-PRED, XXXG-BUDE, XXXG-5ASAME and XXG-DEXA with the Bacteroides ovatus GH5 endo-xyloglucanase (Larsbrink J. et al. Nature 2014, see above) was demonstrated in sodium citrate buffer, pH 6.5, using a copper-bicinchoninic acid (BCA) reducing-sugar assay essentially as described in Amal G. et al. (2017) “A low-volume, parallel copper-bicinchoninic acid (BCA) assay for glycoside hydrolases”, Chapter 1 in Protein-Carbohydrate Interactions: Methods and Protocols, Abbott D. W. and Lammerts van Bueren A. (eds.), Methods in Molecular Biology, 1588, 3-14. DOI:10.1007/978-1-4939-6899-2 1.
Results from the assay are summarized in
Study Description
This study was designed to directly compare the efficacy of steroids prednisolone and dexamethasone to their glyco-caged formulations (prepared according to embodiments of the present application) during intestinal inflammation. The bacterium, Citrobacter rodentium was administered to incite acute self-limiting colitis in mice. Germ-free mice were used in order to remove any confounding effects of the intestinal microbiota, and to purely observe the impacts of the steroid formulations during C. rodentium and Bacteroides ovatus co-colonization; B. ovatus colonization was necessary for drug de-caging.
The study was approved by the Animal Care Committee at the Agriculture and Agri-Food Canada (AAFC) Lethbridge Research and Development Centre (LeRDC), (Animal Use Protocol 1814).
The treatment groups represented in this experiment are listed below in Table 2.
Citrobacter
Bacteroides
rodentium
ovatus
aPrednisolone
bDexamethasone
Materials & Methods
Animals
Mice were reared from an germ-free (GF) C57BL/6 breeding colony at AAFC LeRDC, and ten males aged 8-months were used in this experiment. The gnotobiotic mice originally used to establish the GF breeding colony were purchased from the University of North Carolina at Chapel Hill (germ-free status C57BL/6J+). Mice were pair-housed (according to treatment) in sterilized Green Line Individually Ventilated Cage (IVC) Sealsafe PLUS™ mouse cages (Tecniplast) using axenic and containment level 2 procedures (Lange M. E. et al. 2019. “Housing gnotobiotic mice in conventional animal facilities”. Curr. Protoc. Mouse Biol. e59). GF mice exitted the isolators and acclimatized to their environment for 3 days in IVCs with ad libitum access to AIN-93G+casein diet and 0.25% xyloglucan (XG) drinking water. The diet was prepared by Dyets Inc (Bethlehem) and the composition in described in Table 3. The animal room was maintained on a 12-hr light/dark cycle at 22° C.
Inoculations
Following acclimatization, mice were gavage-inoculated with Bacteroides ovatus (ATCC 8483) grown at 34° C. under anaerobic conditions. The single B. ovatus inoculum concentration was 2.0×1010 CFU/100 μL. After 2 days of B. ovatus colonization, mice were inoculated with Citrobacter rodentium (ATCC 51459) through an edible format (delivered on a 1.5% agar medium containing 5% ground diet). Mice were inoculated with C. rodentium, grown at 37° C. under aerobic conditions, over the span of 2 days; 8.1×108 CFU/100 μL as the primary and 4.1×108 CFU/100 μL as the secondary inoculation. The mice were colonized with B. ovatus for a total of 7 days, and had an active C. rodentium infection for 5 of those days.
Steroid Administration
The average weight of the mice (27.9 g) was calculated on the second day of the Citrobacter rodentium infection, and this average weight was used to calculate the steroid concentrations for all treatments. The steroids were dissolved as best as possible in sterile water, and stored at 4° C. until needed. Steroids were administered by gavage in 100 volumes for 3 consecutive days at the same time each morning (24 hrs between treatments) for the final 3 days of the C. rodentium infection. Mice in the control groups (without drug intervention) received water by gavage in 100 μL volumes. Gavage needles for drug administration were coated in sucrose solution (1 g/mL, syringe-filter sterilized) as a refinement procedure. Table 4 summarizes the steroid treatment dosages.
During this study, prednisolone did not fully dissolve in water, and it was necessary to rinse the gavage needle with 50 μL of water and feed it to the mice to ensure they received the full dosage.
Fecal Collections
Fecal collections were necessary to enumerate the two species of bacteria that the mice were inoculated with. Feces were first collected on the day the mice transferred from the germ-free (GF) isolator to individually ventilated cages (IVC), and plated on Columbia agar with 10% blood under aerobic and anaerobic conditions to confirm the GF status of mice. During the experimental period, fecal collections from the cages took place 2 days following the Bacteroides ovatus inoculation, and two days following the Citrobacter rodentium inoculation. Since the mice were pair-housed, fecal samples were pooled. After fecal sampling, mice were transferred to sterile IVCs to ensure fresh samples for the next collection. At the experiment endpoint, digesta was excised from the large bowel so that bacterial densities in individual mice could be determined. The bacterial populations were enumerated using microbiological culturing techniques; feces were serial diluted and plated on either Columbia agar to estimate a colony forming units (CFU)/g concentration of B. ovatus (grown under anaerobic conditions at 37° C.) or plated on MacConkey agar to estimated a CFU/g concentration of C. rodentium (grown under aerobic conditions at 37° C.).
Necropsy
Under isoflurane anesthesia, mice were terminally bled by cardiac puncture and then humanely euthanized by cervical dislocation. All collected blood was separated into serum that was dedicated for steroid detection and optimization of steroid detection procedures. Following euthanasia, a midline laparotomy was made and gross observations of the abdominal regions were photo-documented. Approximately 1 cm sections of the cecum, and the most proximal and distal portions of the colon were divided for histological examination. Cecal and colonic tissues were flash frozen in liquid nitrogen for future immunological protein quantification. Digesta extracted from the colon was stored at −80° C. for future steroid detection, and fresh feces from the cages were used to enumerate Bacteroides ovatus and Citrobacter rodentium by microbiological techniques on the same day as the necropsy.
Histopathology
Tissue samples (cecum, proximal colon, and distal colon) were initially stored for preservation in 10% buffered formalin. Tissue processing, sectioning, and staining with hematoxylin and eosin (H&E) were carried out using standard protocols. Histological scoring of inflammation was performed by a veterinary pathologist blinded by treatment, with scoring criteria from previously described methods (Barthold S. W. et al. 1978. “Transmissible murine colonic hyperplasia”. Vet. Pathol. Vol 15: 223-236; and Wu X. et al. 2008. “Saccharomyces boulardii ameliorates Citrobacter rodentium-induced colitis through actions on bacterial virulence factors”. Am. J. Physiol. Gastrointest. Liver Physiol. Vol 294: G295-306). The specific categories for scoring are presented in Tables 5-7 below. The total pathology scores illustrate the sum of scores for all histopathological categories observed for each mouse. The tissues analyzed for germ-free mice and C. rodentium-infected mice (i.e. without drug intervention) controls were collected from a previously conducted experiment (non-related). The mice were infected with C. rodentium by the same protocol, and these controls were required to visually examine the comparative histological differences in the absence of both Bacteroides ovatus and steroid intervention. It was not possible to conduct the experiment with these control treatments as only a limited number of male germ-free mice were available.
Tissue Cytokine Detection
Cecal and colonic homogenate for each mouse was prepared using the tissues suspended in an immunoprecipitation buffer at a ratio of 1:5 (wt/vol, adapted from Burgos-Ramos E. et al. 2012. “Adipose tissue promotes a serum cytokine profile related to lower insulin sensitivity after chronic central leptin infusion”. PLoS One Vol 7(10): e46893) with the addition of 1% Protease Inhibitor Cocktail (Sigma Aldrich). Frozen tissue sections were immediately homogenized using a Tissue-Tearor® model 398 homogenizer (Biospec Products) and centrifuged (14 000×g for 30 min at 4° C.). Supernatants were collected and used immediately for cytokine profiling. Cecal and colonic cytokine concentrations were measured for each treatment group by enzyme-linked immunosorbent assay (ELISA). Cytokines included in the analysis were interleukin-1 beta (IL-1β); IL-6; IL-10; IL-22; and tumour necrosis factor alpha (TNF-α). All cytokines were quantified using mouse DuoSet ELISA Development kits according to the manufacturer's protocols (R&D Systems). ELISAs were performed using 96-well high-binding half area microplates (Greiner Bio-One), and the optical density of the reactions were determined at wavelength of 450 nm on a Synergy HT™ multi-detection microplate reader (BioTek Instruments Inc.) with Gen5™ analysis software (BioTek Instruments Inc.).
Results
Body Weight, Gross Morphology and Histologic Changes
There were no treatment effects on weight loss in mice acutely infected with Citrobacter rodentium after 5 days (
Histologic examination was performed on cecal (Table 5,
C. rodentium
C. rodentium &
B. ovatus
C. rodentium
C. rodentium &
B. ovatus
C. rodentium
C. rodentium &
B. ovatus
Bacterial Densities
The gastrointestinal tract was initially colonized by Bacteroides ovatus, however, densities were reduced 8 days post-inoculation. Without wishing to be bound by theory, this could have been due to either natural clearance of B. ovatus or competitive exclusion by Citrobacter rodentium, and was not related to treatment. The densities of C. rodentium were considered equivalent in the fecal content regardless of steroid treatment.
Cecal and Colonic Cytokines
Cecal cytokine concentrations were measured from tissue homogenates (
Colonic cytokines were also measured from tissue homogenates (
Conclusions
Histopathologically, XXXG-DEXA and XXXG-PRED were found to ameliorate acute inflammation in the proximal and distal colon of gnotobiotic mice colonized by B. ovatus. XXXG-DEXA exhibited a greater efficacy than uncaged dexamethasone. XXXG-PRED also exhibited a greater efficacy than uncaged prednisolone.
XXXG-DEXA was more effective than XXXG-PRED in ameliorating colitis in the cecum and proximal colon incited by Citrobacter rodentium, and both glyco-caged steroid formulations were equally efficacious in the distal colon.
XXXG-DEXA and XXXG-PRED were more effective than the uncaged drugs in decreasing pro-inflammatory cytokines concentrations (IL-1β and TNF-α), and increasing the concentration of the anti-inflammatory, IL-22 in intestinal tissues. There were differential impacts on IL-6 concentrations in cecal and colonic tissues.
Overall, the results of this study demonstrate that the glyco-caged prodrugs of the present application are effective in providing active drug to a subject and in treatment of an enteric inflammatory disease in a subject, at the same or better efficacy than the corresponding uncaged drug.
The of XXXG-SIL-5ASA (see Table 8 below) is described in Scheme 5, beginning from readily available powdered tamarind seed kernel powder. Where problems may arise due to functional group compatibilities to the reagents used, these functional groups were protected with an appropriate protecting group by a known method in advance. These protecting groups were subsequently removed by known methods. The reaction conditions were optimized to increase the combined overall yield and/or to minimize environmental impacts.
Scheme 5, Step 1
Per-O-Ac-XXXG-Br (154 mg) and 4-hydroxybenzaldehyde (214 mg) was dissolved in 8 mL of dry acetonitrile under stirring, followed by the addition of 0.94 g of 3 Å MS and 250 mg of Ag2O. The suspension was then stirred in the dark for 24 hrs before all the volatiles were removed under reduced pressure. The crude thus obtained was purified with flash silica gel chromatography (toluene/acetone 4:1 to 2:1). The conjugated product Per-O-Ac-XXXG-4′-O-benzaldehyde (75 mg) was obtained as a white powder. m/z 1987 (M+Na+, C84H108NO53Na requires 1987).
Scheme 5, Step 2
Per-O-Ac-XXXG-4′-O-hydroxybenzaldehyde (75 mg), silica gel (0.8 g), and IR-120 resin (H+ form, 16 mg) were suspended in chloroform and IPA (6 mL 5:1) under argon at 0° C. 16 mg of NaBH4 was added over a period of 30 mins. The reaction was allowed to continue until a TLC test indicated a complete reduction. The reaction was quenched with saturated ammonium chloride, followed by the addition of 120 mL of ethyl acetate and water (1:1). The organic phase was separated and washed with water (60 mL×3) and brine (50 mL×2) before it was concentrated under reduced pressure. The crude thus obtained was purified with flash chromatography (toluene/acetone 4:1 to 2:1) and Per-O-Ac-XXXG-4′-O-benzylalcohol (65 mg) was obtained as a pure product as evidenced by proton NMR and MALDI mass spectrometry. m/z 1989 (M+Na+, C84H110NO53Na requires 1989).
Scheme 5, Step 3
Per-O-Ac-XXXG-4′-O-benzylalcohol (33.5 mg) was dissolved in 6 mL of DCM under argon at 0° C., followed by the addition of 42 mg of pyridine and 82 mg of p-nitrophenylchloroformate. The resultant mixture was stirred at the same conditions for 2 hrs before the volatiles were removed under reduced pressure. The crude thus obtained was purified with flash chromatography (toluene/acetone 4:1 to 2:1) and Per-O-Ac-XXXG-4′-nitro-benzylcarbonate (32.1 mg) was obtained as a pure product as evidenced by proton NMR and MALDI mass spectrometry. m/z 2154 (M+Na+, C91H113NO57Na requires 2154).
Scheme 5, Step 4
Per-O-Ac-XXXG-4′-nitro-benzylcarbonate (25.5 mg) was dissolved in 10 mL of DCM at 0° C., followed by the addition of 6 mg of 5-ASA and 55 mg of DMAP. The reaction mixture was stirred for 0.5 hr before it was allowed to warm to room temperature. The reaction mixture was stirred for additional 2 hrs until a TLC indicated a complete reaction. 0.5 g of silica gel added and the resultant suspension was concentred under reduced pressure. The resultant crude was subject to a flash silica gel chromatography (DCM/acetonitrile/triethylamine) and 25 mg of Per-O-Ac-XXXG-SIL-5ASA was obtained. m/z 2168.7 (M+Na+, C92H115NO57Na requires 2168.6).
Scheme 5, Step 5
Per-O-Ac-XXXG-SIL-5ASA (25 mg) was dissolved in 4.5 mL of MeOH/DCM (2:1). Catalytic amount of sodium methoxide (2 mg) was added and the resultant suspension was stirred overnight before all volatiles were removed under reduced pressure at 40° C. The crude thus obtained was subject to reverse phase chromatography. 12 mg of XXXG-SIL-5ASA was obtained as evidenced by proton NMR and mass spectrometry. m/z 1392.3973 (M−H++2 Na+, C54H76NO38Na2 requires 1392.3841).
The synthesis of XXXG-SIL-peptide (see Table 9 below) is described in Scheme 6, beginning from the common intermediate Per-O-Ac-XXXG-4′-nitro-benzylcarbonate. The reaction conditions were optimized to increase the combined overall yield and/or to minimize environmental impacts.
Scheme 6, Step 1
Per-O-Ac-XXXG-4′-nitro-benzylcarbonate (50 mg) was dissolved in 10 mL of DCM at 0° C., followed by the addition of 100 mg of Alanine Tripeptide and 120 mg of DMAP. The reaction mixture was stirred for 0.5 hr before it was allowed to warm to room temperature. The reaction mixture was stirred for additional 2 hrs until a TLC indicated a complete reaction. 0.7 g of silica gel added, and the resultant suspension was concentred under reduced pressure. The resultant crude was subject to a flash silica gel chromatography (DCM/acetonitrile/triethylamine) and 45 mg of Per-O-Ac-XXXG-SIL-Peptide was obtained as evidenced by MALDI mass spectrometry. m/z 2246 (M+Na+, C94H125N3O58Na requires 2246).
Scheme 6, Step 2
Per-O-Ac-XXXG-SIL-Peptide (45 mg) was dissolved in a mixture of methanol and dichloromethane (6 mL, 4:1) at room temperature under stirring. To this mixture 7.5 μL of a 25% (w/w) solution of sodium methoxide in methanol was added and the resultant suspension was stirred for 6 hrs until a TLC test indicated the complete consumption of the starting material. 200 mg of Amberlite™ IR-120 resin (H+ form) and stirred for 10 min, whereupon the suspension was filtered, and the filtrate was concentrated under reduced pressure and a 20 mg of XXXG-SIL-Peptide was obtained as evidenced by thin layer chromatography.
This example demonstrates the successful incorporation of a tri-alanine peptide as a model drug moiety in a glyco-caged prodrug. Although the tri-alanine peptide does not have any known effect on IBD or other intestinal disorders this example clearly shows that N-terminal amines on peptides are readily conjugated to an intermediate useful in the production of the self-immolative linker-containing glyco-caged prodrugs described herein. Accordingly, this example shows that peptides, and polypeptides, having free amine groups (for example in alanine and lysine side chains) can be successfully incorporated into self-immolative linker-containing glyco-caged prodrugs.
The glyco-caged prodrugs herein are “decaged,” or metabolized to release pharmacologically active prodrugs, by the action of xyloglucanases. Such xyloglucanases are found in bacteria present the intestine of animals.
The present example demonstrates that xyloglucanase-active bacteria are present in mice, chickens and in the large intestine of swine.
Bacterial Strains from Pig Intestines
Approval to conduct this project was obtained from the AAFC LeRDC Animal Care Committee (Animal Use Protocol 1512). Castrated male piglets at 6-weeks of age provided a mini-pellet ration diet that was free of antibiotics (Proform Pig Starter 2). Feed was provided daily, and piglets were permitted to eat and drink ad libitum. Half of the piglets were orally inoculated with Salmonella enterica Typhimurium DT104 (SA970934) in buffer to incite enterocolitis, and the remaining half were orally administered buffer alone. At 2, 6, and 10 dpi, randomly designated animals were anesthetized for sample collection from live animals. Piglets were sedated, intubated, and general anesthesia established with isoflurane. From animals under general anesthesia, segments of intestine were collected. To ensure the integrity of the intestinal segment and to minimize release of ingesta, double ligatures were established at the two ends of the segment, and the segment was excised from the intestine by cutting between the two ligatures. Bacteria were isolated using the basic method described in Moote P. E. et al. (2020. “Application of culturomics to characterize diverse anaerobic bacteria from the gastrointestinal tract of broiler chickens in relation to environmental reservoirs”. Can. J. Microbiol. Vol 66: 288-302). Isolated bacteria were identified by sequencing the partial 16S rRNA gene. To screening bacteria for endoxyloglucanase activity, congo red staining was used. Briefly, isolated bacteria were grown on Columbia Agar supplemented with 10 mL/L of 0.05% Hemin and xyloglucan (0.25%) in gridded dishes, and cultures were incubated anaerobically for 24-48 hrs. Cultures were removed from the chamber, and 15 mL of 0.1 M phosphate buffered saline (pH 7) was added, bacterial biomass was scraped from the surface of the medium, and the liquid was removed. Cultures were then stained with 1.0% congo red at room temperature for 20 min, the congo red solution was removed, cultures were flooded with 1 M sodium chloride for 20 min, the salt solution was removed, and the medium was fixed with 1 M HCl to produce the blue stained medium with zones of clearing indicative of xyloglucanase activity. Cultures were photodocumented. for evaluations and documentation.
The following strains of Bacteroidetes bacteria that were capable of growth on xyloglucan and exhibited clearing of the medium indicative of endo-xyloglucanase activity (i.e. using the congo red medium) were isolated from pig intestines:
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Bacteroides uniformis
Parabacteroides merdae
XXXG-resorufin was synthesized and used as a model glyco-caged prodrug substrate used to facilitate monitoring of decaging. In particular, cleavage of the XXXG-resorufin substrate was used to measure the xyloglucanase activity of bacterial populations and communities isolated or extracted from pigs.
A limited number of the additional bacterial taxa isolated from the intestines of pigs exhibited endo-xyloglucanase activity, and are thus likely to be capable of decaging XXXG-resorufin (e.g., Streptococcus gallolyticus). Thus, decaging ability within the intestine is not restricted to Bacteroides species and related bacteria. Importantly, evidence of bacterial abundance indicated that B. uniformis and S. gallolyticus were differentially associated with inflamed intestine, which may further enhance targeting of prodrugs at sites of inflammation (Bescucci D. M. et al. unpublished).
To screen the xyloglucan degrading activity, bacterial isolates were screened and Bacterioides ovatus wild type and the ΔGH5 deletion mutant were used as positive and negative controls, respectively. Briefly, the strains stored in glycerol at −80° C. were recovered by inoculation into rich Columbia medium and cultivated anaerobically at 37° C. for 24 hrs. Next, the cells were pelleted by centrifugation 3,000×g and the supernatant was decanted, followed by washing the cells twice with minimum medium supplemented with chopped meat. The pellets were re-suspended in 1 mL of the minimum medium, and 100 μL of the suspension was transferred to a culture tube pre-filled with 1.5 mL of the minimum medium containing either high purity xyloglucan (Megazyme International Ireland Ltd.), D-glucose (0.5% w/v), or no carbohydrate, followed by anaerobic incubation at 37° C. for 24 hrs. The optical density at a wavelength of 600 nm (0D600) was then measured for each bacterial suspension. Half millilitre of each suspension with (i.e., lysed) or without (i.e., whole cells) pre-incubated with BugBuster™ lysis reagent (Novagen, EMD Chemicals Inc.) was mixed with 0.5 mL of the minimum medium containing 100 μmol/L of XXXG-resorufin followed by incubation of the mixture for 4 hrs at room temperature with tubes fully covered with aluminium foil. After the incubation, the cells were heat-killed at 90° C. for 5 min before centrifuging the tubes and transferring 200 μL of each supernatant to a 96-well plate. Fluorescence signal was collected from the plate on a BioTek Synergy™ HT multi-detection microplate reader (BioTek Instruments) installed with a 530/25 nm excitation filter and a 590/35 nm emission filter, and the data were analyzed using the Gen5 2.04 software. Relative fluorescence unit (RFU) was calculated as the ratio of fluorescence reading (with the subtraction of background fluorescence) to OD600.
The distribution of Bacteroides in the gastro-intestinal tract of pigs is illustrated in
A study was performed using a pig colon bioreactor, to demonstrate that XXXG-resorufin could be decaged under ex vivo conditions to mimic in vivo conditions, with or without the addition of xyloglucan. The intestinal tract of a pig was obtained from a local abattoir, and within 30 min of death, ligatures were established as described above, and segments of the colon were excised thereby ensuring that air did not infiltrate the segments. The segments were transferred to an anaerobic chamber with a N2 predominant atmosphere, and digesta was harvested from the segments within the chamber. The digesta was used to populate bioreactor vessels. The basic bioreactor methodology described by Auchtung J. M. et al. (2016. “MiniBioreactor Arrays (MBRAs) as a tool for studying C. difficile physiology in the presence of a complex community”. Meth. Mol. Biol. Vol 1476: 235-250) was followed. Half of the reactors were fed medium amended with xylogucan (0.33 g/L) and the remaining vessels were fed medium alone. An aliquot (0.5 mL) of the bioreactor contents were centrifuged at 3,000×g to fractionate the pellet from the supernatant. The pellet was resuspended in 0.5 mL of sterile bioreactor medium. Both the supernatant and the pellet solution were incubated with 0.5 mL of 30 μmol/L XXXG-resorufin. The fluorescence assay was performed as described above. As shown in
Bacterial Strains from Chicken Intestines
Approval to conduct this project was obtained from the AAFC LeRDC Animal Care Committee (Animal Use Protocol 1622). Ross 308 broiler chickens were used. Chicks (1-day-old) were obtained from a local hatchery, and were reared within two stainless steel pens (2.23 m2 per pen with 10 birds per pen for a stocking density of 0.22 m2 per bird). Birds were provided free access to a non-medicated starter diet (Hi-Pro Feeds) and water. Ambient room temperature was maintained at 22±2° C., and birds were provided free access to brooders for the first 20 days. At 31-days-of-age, arbitrarily-selected birds were anesthetized with isoflurane and humanely euthanized. Bacteria were isolated from the ceca of birds using a variety of anaerobic isolation methods as described in Moote P. E. et al. (2020. “Application of culturomics to characterize diverse anaerobic bacteria from the gastrointestinal tract of broiler chickens in relation to environmental reservoirs”. Can. J. Microbiol. Vol 66: 288-302). Isolated bacteria were identified by sequencing the partial 16S rRNA gene. Two different isolates of Bacteroides ovatus that were isolated from the ceca of broiler chickens demonstrated their ability to decage XXXG-resorufin to generate fluorescent resorufin.
The two isolates of Bacteroides ovatus (33 and 19) isolated from the ceca of chickens were cultured on glucose or xyloglucan and incubated with XXXG-resorufin. The results are shown in
Xyloglucanase Activity in Mouse Feces
Approval to conduct this project was obtained from the AAFC LeRDC Animal Care Committee (Animal Use Protocol 1924). To demonstrate the presence of naturally occurring bacteria having xyloglucanase activity in mice, feces from C57BL/6 mice maintained on an AIN-93G basal diet were collected. One half of the mice were provided the basal diet (XyG−), and the other half of the mice were maintained on the basal diet amended with 2% xyloglucan (XyG+). Freshly excreted feces were obtained from both groups. A portion of the feces was used to isolate anaerobic bacteria possessing endoxyloglucanase activity, and the remaining portion was used to quantify decaging of XXXG-resorufin. Xyloglucanase positive colony forming units (CFUs) were visualized using the congo red screening method described above, and CFUs that produced a zone of clearing were enumerated and isolated. The clearing ability of isolated bacteria was confirmed as above.
To assess the ability of fecal bacteria to decage XXXG-resorufin, 0.5 mg of feces were homogenized in 5.0 mL of sterile buffer and spun at 3,000×g. The soluble fraction, represented lysed cells and secreted enzymes; and the pellet resuspended in an equal volume of buffer, represented intact cells, fibre associated cells, and microbial biofilms. 0.5 mL of the supernatant and the pellet solution were incubated with 0.5 mL of 30 μmol/L XXXG-resorufin. Xylolglucanase activity was measured as described above. Fecal bacteria from both the enriched diet mice and the control mice exhibited xyloglucanase activity, and more xyloglucase positive CFUs were recovered from the feces of mice fed the xyloglucan-enriched diet indicating that endogenous xyloglucanase activity is increased in the presence of xyloglucan (
Approval to conduct this project was obtained from the AAFC LeRDC Animal Care Committee (Animal Use Protocol 1911). Similar to Example 3, this study was designed to demonstrate the improved efficacy of glyco-caged formulations of prednisolone and dexamethasone over the free forms of prednisolone and dexamethasone during intestinal inflammation. The methods employed were essentially the same as those described in Example 3, except that the Citrobacter rodentium (CR) was administered to incite acute self-limiting colitis in mice prior to colonization with Bacteroides ovatus (BO). Furthermore, the C57B1/6 mice used possessed a normal enteric microbiota.
The treatment groups were as summarized below:
No treatment (CR−/BO+), n=6 mice
No treatment (CR+/BO+), n=6 mice
Prenisolone 1 mg/kg (CR+/BO+), n=6 mice
XXXG-PRED, dose equivalent to prenisolone 1 mg/kg (CR+/BO+), n=6 mice
Dexamethasone 1.5 mg/kg (CR+/BO+), n=6 mice
XXXG-DEXA, dose equivalent to dexamethasone 1.5 mg/kg (CR+/BO+), n=6 mice
Results
Inoculation with CR was again found to successfully incite colitis in mice, as evidenced by internal bleeding from the colon and diarrhea.
As in the study described in Example 3, there were gross histopathological differences in the distal colon tissue obtained from mice in the different treatment groups. Histological scoring of inflammation was performed by a board-certified pathologist blinded to treatment. Results are shown in
Conclusions
Histopathologically, XXXG-DEXA and XXXG-PRED were found to ameliorate colitis incited by Citrobacter rodentium in the colon of mice colonized by Bacteroides ovatus. XXXG-DEXA exhibited a greater efficacy than uncaged dexamethasone histologically. XXXG-PRED also exhibited a greater efficacy than uncaged prednisolone histologically. Molecular metrics of inflammation supported the histopathologic findings.
All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent applications was specifically and individually indicated to be incorporated by reference.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1904758.8 | Apr 2019 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/050447 | 4/3/2020 | WO | 00 |
