Patent Application
20210187064
Not applicable.
Inflammation may be thought of as the immune system's response to an irritant. Inflammation can promote fighting an infection or healing from a wound, and is helpful in those situations. Unfortunately, in some conditions, the body reacts unnecessarily and inflammation creates equally unnecessary discomfort. Diseases such as dermatitis or psoriasis can result in inflammation of portions of the skin. Individual organs or organ systems may have conditions, such as nephritis or asthma, which are exacerbated by inflammation and for which treatment can include corticosteroids or other anti-inflammatory drugs.
As the name implies, inflammatory bowel disease (“IBD”), a group of inflammatory conditions of the gastrointestinal tract, most commonly in the colon and small intestine, are diseases with strong inflammatory components. Crohn's disease (“CD”) and Ulcerative Colitis (“UC”) are the major types of IBD. These diseases are chronic inflammatory conditions of the bowel and cause symptoms such as abdominal pain, diarrhea, vomiting, rectal bleeding, and can cause anemia. Currently, there are no cures for IBD but medication can be prescribed to manage the symptoms and improve quality of life for those with the disease. If symptoms of IBD are not managed, abscesses or fistulae may form in severe cases, necessitating surgical removal of parts of the colon or small intestine. Surgical intervention can significantly impact the patients' quality of life, potentially requiring the use of a colostomy bag.
There are a variety of treatments available for IBD. For mild cases, Non-Steroidal Anti-inflammatory Drugs (NSAIDs) may be prescribed; research has shown, however, that NSAIDs may have an adverse effect on IBD patients, sometimes worsening symptoms by damaging the lining of the intestines. Acetaminophen is another option but lacks effectiveness, except in mild cases.
Aminosalicylates are a family of anti-inflammatory compounds also used to treat IBD. This class of drug is effective in mild to moderate cases of IBD, but may still have side effects such as pancreatitis, headache, vomiting, fever, stomach cramping, and diarrhea.
Steroidal treatments are another class of medication used for IBD that are most useful during IBD flare ups due to their fast acting anti-inflammatory properties. However, these treatments are less effective at disease maintenance and can have significant side-effects from long term use, including weakening the immune system.
Immunomodulators are another class of treatment for IBD. These medications are effective against IBD but cause flu like symptoms and also have the side effect of weakening the patients' immune system reducing their ability to fight infection or other disease. Finally, a few biological treatments, including PDE inhibitors and monoclonal antibodies, such as Infliximab, have been used as a treatment for IBD. These biologics also have risk of side effects, including increased infections, serum sickness, and longer term side effects of decreased effectiveness of the drug, multiple sclerosis, and lymphoma.
Due to these potential short and long term side effects of managing these chronic conditions, there is a need for a treatment for IBD that provides limited short and long term side effects, is highly effective, and has a strong safety profile. Further, it would be desirable to have new agents to reduce symptoms of inflammation in other inflammatory diseases, such as asthma, nephritis, dermatitis, and psoriasis. Surprisingly, the present invention fills these and other needs.
In a first group of embodiments, the invention provides compositions comprising a unit dose of: (a) (i) an E. coli heat labile enterotoxin (“LT”) non-toxic A subunit which inhibits ADP-ribosylation in a cell pretreated with said non-toxic A subunit when said cell is then contacted with E. coli. LT holotoxin, or (ii) a LT non-toxic A1 subunit which inhibits ADP-ribosylation in a cell pretreated with said non-toxic A subunit when said cell is then contacted with E. coli LT holotoxin, or, (iii) a combination of said non-toxic A subunit and of said non-toxic A1 subunit, and (b) a carrier which causes internalization of said non-toxic A subunit or non-toxic A1 subunit, or combination thereof, into cells. In some embodiments, the non-toxic A subunit or the non-toxic A1 subunit, or the combination of the non-toxic A subunit and the non-toxic A1 subunit does not induce intracellular cAMP accumulation in an epithelial cell. In some embodiments, the non-toxic A subunit or the non-toxic A1 subunit, or the combination of the non-toxic A subunit and the non-toxic A1 subunit activates expression and secretion of IL-6 in murine dendritic cells contacted with the non-toxic A subunit or the non-toxic A1 subunit, or the combination of the non-toxic A subunit and the non-toxic A1 subunit in vitro in a medium that supports growth of said murine dendritic cells than the said cells when contacted with LT B subunit. In some embodiments, the carrier which causes internalization of the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a LT B subunit and the unit dose is from 0.5 mg or more of non-toxic A subunit, non-toxic A1 subunit, or combination thereof and the B subunit carrier. In some embodiments, the carrier which causes internalization of the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is cholera toxin B subunit (“CTB”) or cholera A2 subunit/B subunit (“CTA2/B”). In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof is chemically conjugated or recombinantly fused to the LT B subunit. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof is chemically conjugated or recombinantly fused to the CTB or CTA2/B subunit. In some embodiments, the carrier which causes internalization of the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a liposome or encapsulating vesicle. In some embodiments, the liposome or encapsulating vesicle is a liposome. In some embodiments, the liposome has an exterior surface and has an antibody or antigen-binding fragment or derivative thereof disposed on the exterior surface. In some embodiments, the antibody or antigen-binding fragment or derivative binds CD11c. In some embodiments, the carrier which causes internalization of the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a β-glucan. In some embodiments, the unit dose composition does not contain an exogenous antigen. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, is non-toxic A subunit. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, is non-toxic A1 subunit. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, is a combination of non-toxic A subunit and non-toxic A1 subunit. In some embodiments, the composition is lyophilized. In some embodiments, the composition further comprises an excipient, a stabilizer, or both an excipient and a stabilizer. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, has a mutation at position E112, E110, S61, or R25. In some embodiments, the mutation is selected from E112K, E112G, E112D, E110K, E110G, S61F, and R25G. In sonic embodiments, the mutation is of E112K. In some embodiments, the composition is in a base suitable for topical administration. In some embodiments, the composition is in a liquid. In some embodiments, the liquid further comprises a flavoring agent and a sweetener. In some embodiments, the unit dose is from about 1 mg±0.2 to 500 mg of the A subunit, A1 subunit, or combination thereof, and carrier. In some embodiments, the unit dose is from 1 mg±0.2 mg to 30 mg of the A subunit, A1 subunit, or combination thereof and carrier. In some embodiments, the unit dose is from 1 mg±0.2 mg to 20 mg±0.2 mg of the A subunit, A1 subunit, or combination thereof and carrier. In some embodiments, the composition is provided as a pill. In some embodiments, the composition is provided as a suppository.
In a further group of embodiments, the invention provides methods of reducing symptoms of inflammation in a subject in need thereof. In some embodiments, the methods comprise administering to the subject a composition comprising a therapeutically effective amount of: (a) (i) an E. coli heat labile enterotoxin (“LT”) non-toxic A subunit which inhibits ADP-ribosylation in a cell pretreated with said non-toxic A subunit when the cell is then contacted with E. coli LT holotoxin, (ii) a LT non-toxic A1 subunit which inhibits ADP-ribosylation in a cell pretreated with the non-toxic A subunit when the cell is then contacted with E. coli LT holotoxin, or, (iii) a combination of the non-toxic A subunit and the non-toxic A1 subunit, and, (b) a carrier which causes internalization of the non-toxic A subunit or non-toxic A1 subunit, or combination thereof, into cells. In some embodiments, the non-toxic A subunit or non-toxic A1 subunit, or combination of non-toxic A subunit and non-toxic A1 subunit does not induce intracellular cAMP accumulation in an epithelial cell. In some embodiments, the non-toxic A subunit or the non-toxic A1 subunit, or the combination of the non-toxic A subunit and the non-toxic A1 subunit does not activate expression and secretion of IL-6 in murine dendritic cells contacted with the non-toxic A subunit or the non-toxic A1 subunit, or the combination of said non-toxic A subunit and the non-toxic A1 subunit in vitro in a medium that supports growth of the murine dendritic cells, compared to the cells when contacted with LT B subunit. In some embodiments, the carrier which causes internalization of said non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a LT B subunit, In some embodiments, the carrier which causes internalization of the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a cholera toxin B subunit (“CTB”) or cholera A2 domain and B subunit (“CTA2/B”). In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof is chemically conjugated or recombinantly fused to said LT B subunit. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof is chemically conjugated or recombinantly fused to the CTB or the CTA2/B subunit. In some embodiments, the carrier which causes internalization of the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a liposome or encapsulated vesicle. In some embodiments, the carrier is a liposome. In some embodiments, the liposome is targeted to a cell antigen by an antibody or antigen-binding fragment or derivative thereof. In some embodiments, the carrier which causes internalization of the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, into cells is a β-glucan. In some embodiments, the composition does not contain an exogenous antigen. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, is non-toxic A subunit. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, is non-toxic A1 subunit. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, is a combination of non-toxic A subunit and non-toxic A1 subunit. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof, and the carrier is lyophilized. In some embodiments, the composition further comprises an excipient. In some embodiments, the composition further comprises a stabilizer. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof has a mutation in said A subunit, the A1 subunit, or both, selected from the group consisting of E112K, E112G, E112D, E110K, E100G, S61F, or R25G. In some embodiments, the non-toxic A subunit, non-toxic A1 subunit, or combination thereof has an E112K mutation in the A subunit, said A1 subunit, or both. In some embodiments, the composition is administered in water. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered rectally. In some embodiments, the composition is administered intra-nasally. In some embodiments, the composition is administered parenterally. In some embodiments, the parenteral administration is intravenous. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered topically. In some embodiments, the composition is administered transdermally. In some embodiments, the therapeutically effective amount is administered as an induction dose followed by one or more maintenance doses. In some embodiments, the composition is administered daily. In some embodiments, the therapeutically effective amount of said composition is from 500 μg to 500 mg. In some embodiments, the therapeutically effective amount of said composition is from 80 μg to 500 mg. In some embodiments, the therapeutically effective amount of said composition is from 1 mg to 250 mg. In some embodiments, the therapeutically effective amount of said composition is from 1 mg to 100 mg. In some embodiments, the subject in need thereof is a mammal. In some embodiments, the mammal is selected from a primate, feline, canine, bovine, equine, porcine, or ovine. In some embodiments, the primate is a human. In some embodiments, the inflammation is gastrointestinal. In some embodiments, the gastrointestinal inflammation is inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is ulcerative colitis. In some embodiments, the inflammatory bowel disease is Crohn's disease. In some embodiments, the inflammation is of the skin. In some embodiments, the inflammation of the skin is psoriasis or dermatitis. In some embodiments, the inflammation is a form of inflammatory arthritis. In some embodiments, the inflammatory arthritis is rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or juvenile idiopathic arthritis. In some embodiments, the inflammation is of an internal organ other than the small or large intestine, bowel, or colon. In some embodiments, the internal organ is a kidney, pancreas, or liver.
Surprisingly, it has now been discovered that non-toxic A subunits of entertoxic E. coli (“ETEC”) heat-labile toxin (“LT”) that interfere with the function of host cell ADP-ribosylation factor (“ARF”) and inhibit ADP-ribosylation also inhibit the activation of dendritic cells and reduce acute and chronic inflammation, including inflammatory T-cell activity. In studies underlying the present disclosure, an exemplar detoxified A subunit and carrier that allowed it to be internalized into cells was administered to animals in several different models of inflammatory bowel disease (“IBD”). Animals treated with the exemplar construct in the studies exhibited improvements in pathology scores, showed no abnormal loss of weight, and showed normal stool consistency. In view of these results, it is believed that this detoxified A subunit, and other detoxified A subunits that likewise interfere with ART intracellular function and inhibit ADP-riboyslation will also inhibit the activation of dendritic cells, reduce acute and chronic inflammation, including inflammatory T-cell activity, and improve symptoms of IBD. Further, a study underlying the present disclosure showed rapid improvement of barrier permeability in the intestine in a murine chronic colitis model, suggesting the constructs were taken up by mucosal epithelial cells and the tissue resident myeloid cells and macrophages and ameliorated symptoms of colitis due to increased permeability. A further study in a murine model using immunodeficient mice showed that mice treated with an exemplar detoxified A subunit and carrier showed improved fecal consistency and reduced blood in feces, but did not improve weight loss, indicating that some of the detoxified A subunit effects are not T-cell dependent. Finally, studies underlying the present disclosure revealed that non-toxic A subunits have a surprisingly different effect on expression and secretion of inflammatory cytokines than do either the LT B subunit or versions of the ETEC holotoxin that act as adjuvants for antigens.
In view of these results, use of non-toxic A subunits with carriers that can cause uptake and internalization of the A subunits into dendritic cells in persons with IBD are expected to reduce and treat symptoms of IBD, in particular, intestinal or bowel inflammation, diarrhea, and blood in stool. Improvement in any one of these symptoms, or any combination of them, is expected to improve patient quality of life. Additionally, as activation of dendritic cells is a common feature of inflammatory diseases, it is believed that non-toxic A subunits that inhibit ADR-riboyslation provided in a carrier that allows internalization into cells will also reduce inflammation in conditions other than IBD. Thus, the studies underlying the present disclosure represent an important advance both in treating IBD, and more generally, in treating inflammation in other conditions in which inflammation exacerbates the condition, such as asthma, dermatitis, psoriasis, psoriatic or rheumatoid arthritis, and nephritis.
As noted, the detoxified A subunits that inhibit ADR-riboyslation are provided in a carrier that allows internalization into cells. Results of studies underlying the present disclosure indicated that the anti-IBD, anti-inflammatory results seen were due to the detoxified A subunit, not the B subunit. Thus, it is believed that the same anti-IBD, anti-inflammatory effects shown in the studies would be seen if the detoxified A subunit is delivered by other means, such as by using as a carrier the cholera toxin B subunit (“CTB”), cholera toxin A2 domain and CTB (“CTA2/B”), or encapsulating the detoxified A subunit in liposomes or other vesicles, or coupling the detoxified A subunit to other carriers that induce internalization of the detoxified A subunit into cells.
In the studies underlying the present disclosure, the detoxified A subunit (the terms “non-toxic” A subunit and “detoxified” A subunit are used as synonyms in this disclosure) was delivered to cells by the entertoxic E. coli (“ETEC”) LT B subunit. (More precisely, the carrier in these experiments was the pentameric structure formed by five B subunits. As the individual B subunits comprising the pentameric structure are not used individually, but only as part of the pentameric structure formed by the five subunits, for convenience of reference, unless specified otherwise, the term “B subunit” herein refers to the pentameric structure formed by five B subunits of the E. coli LT or to the similar pentameric structure formed by five B subunits of the cholera toxin, where the discussion relates to the cholera toxin B subunit.)
In these studies, the detoxified A subunit comprised both the A1 domain of the A subunit, which is responsible in the native toxin for the toxic effects, and the A2 domain of the A subunit, which serves to non-covalently tether the A1 domain to the LT B subunit. The A2 domain is not needed if a carrier other than the ETEC LT B subunit is used, such as CTB, liposomes, and other encapsulated vesicles. Thus, at the practitioner's choice, in those embodiments, the detoxified A subunit may be just the A1 domain of the A subunit, may comprise the intact A subunit, or may comprise some molecules of the A1 domain and some of the intact A subunit.
Because enterotoxic E. coli heat-labile toxin is clinically important as a cause of traveler's diarrhea, there has been some investigation of ETEC LT A subunits, in combination with the LT B subunit, as vaccines against ETEC-related traveler's diarrhea, and for use as adjuvants. Detoxified A subunit mutants that inhibit ADP-ribosylation are useful as anti-inflammatory agents, but are either not useful as adjuvants to enhance a response to an antigen. With regard to vaccine formulations, one reported Phase 3 clinical trial tested LT holotoxin (intact wild-type toxin) as a vaccine administered transdermally via patch. The patch in the clinical trial contained 37.5 μg E. coli LT. Behrens et al., The Lancet, 2014, 14(3):197-204.
Studies in which cells were pretreated with the exemplar composition E112K and then contacted with native holotoxin did not exhibit ADP-ribosylation. The inference is that the E112K composition binds ADP-ribosylation factor (“ARF”), thereby interfering with or blocking its ADP-ribosylation activity.
As shown in
Finally, in animal models of inflammatory bowel disease underlying the present disclosure, animals administered E12K exhibited less blood in stool, better fecal consistency and, in some studies, less weight loss than did animals not treated with E112K. In some embodiments, a subject with an IBD and exhibiting any one of: less blood in stool, better fecal consistency and less weight loss, when administered one of the inventive compositions or treated according to one of the inventive methods is considered to exhibit reduced symptoms of inflammation from the IBD.
The terms “exogenous antigen” in relation to a non-toxic LT A subunit or non-toxic A1 subunit, or both, and a carrier, means that the composition of (a) the A subunit or A1 subunit or both, and (b) the carrier does not also carry with it as an additional component an antigen which will raise an immune response in the subject to which the composition is administered.
The term “carrier” as used herein in relation to a composition comprising a non-toxic LT A subunit or non-toxic A1 subunit, or both, and a carrier, refers to a molecule which, when associated with, fused to, or conjugated to the A subunit or to the non-toxic A1 subunit, facilitates entry of the A subunit or non-toxic A1 subunit into cells. The entry into the cell can be by any means of uptake, such as endocytosis, vesicle-mediated transport, an active transport pathway, or receptor-mediated uptake, depending on the particular carrier employed.
The phrase “a non-toxic LT A subunit or non-toxic A1 subunit, or both” means a composition can comprise (a) one or more non-toxic A subunits, (b) one or more non-toxic A1 subunits, or (c) one or more non-toxic A subunits and one or more non-toxic A1 subunits.
References to mutated A subunits or A1 subunits herein follow the convention of naming the amino acid residue at a particular position in the designated protein, the number of the position, and then the amino acid residue substituted for the original residue at that position. Letters representing the various amino acid residues also follow art-recognized conventions. Thus, for example, E112K describes the substitution of a lysine, K, for a glutamic acid, E, at position 112 of the LT A subunit.
The terms “effective amount” or “therapeutically effective amount” of a composition, as provided herein, refer to a nontoxic but sufficient amount of the composition to provide the desired therapeutic effect, or an amount sufficient to effect treatment of the subject, as defined below. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular macromolecule of interest, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
The phrase “pharmaceutically acceptable,” in connection with administration of a substance to a human refers to a substance that is generally safe for human pharmaceutical use. In connection with administration to a non-human animal of a particular species, it refers to a substance that is generally safe and acceptable to a non-human animal of the species in question.
As used herein, the terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable vehicle” are interchangeable and refer to a fluid vehicle for containing enterotoxin anti-inflammatories compositions that can be injected into a host without adverse effects or administered to a host by another route of administration without adverse effects, depending on the intended route of administration. Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
According to the website of the Centers for Disease Control and Prevention: “Enterotoxigenic Escherichia coli (E. coli), or ETEC, is an important cause of bacterial diarrheal illness. Infection with ETEC is the leading cause of travelers' diarrhea and a major cause of diarrheal disease in lower-income countries, especially among children.” Certain strains of enterotoxigenic E. coli produce a heat-labile enterotoxin (“LT”). It has been established for many years that LT induces cAMP accumulation through binding to host cell ADP-ribosylation factor (“ARF”) to initiate ADP-ribosylation of Gsα, leading to irreversible activation of adenylate cyclase and increased production of intracellular cAMP, which ultimately leads to secretory diarrhea.
LT has an AB5 structure with non-covalently attached A- and B-subunits. The A-subunit causes the enterotoxic effects of the holotoxin, while the B (“binding”) -subunit is responsible for cellular binding and internalization. A ribbon diagram of a partially-inactivated form of the holotoxin (discussed below) is presented in
The nucleic acid and amino acid sequences of both the A and the B subunits have been known for decades, as exemplified by Yamamoto et al., J. Bacterial 1987, 169:1352-57, which presents the nucleic acid and amino acid sequences of both the A and the B subunits for the form found in human isolates (shown in the paper as “LTh”), as well as the nucleic acid and amino acid sequences of the cholera toxin A and B subunits.
E112K is derived from LT, with a single amino acid substitution of a lysine residue in place of a glutamic acid residue at position 112 in the A1 domain (
As shown in the studies reported in the Examples, below, E112K protein is non-toxic and exhibits no alteration in cell viability of culture treated macrophages or epithelial cells (
The E112K construct used in the Examples was the A subunit with an E112K mutation, tethered through the A2 domain to an ETEC LT B subunit, which acted as the carrier to allow internalization of the A subunit and the B subunit into cells, such as dendritic cells. References to “E112K” in the Examples refer to this construct. References in other sections of the disclosure refer to this construct when referring to the Examples, but in other places refer either to this construct or to an A subunit or A1 subunit, which may be paired either with an ETEC LT B subunit, or with a different carrier, such as a cholera toxin B subunit (“CTB”), A2 domain/B subunit (“CTA2/B”), a liposome, and other carriers described elsewhere in this disclosure.
E112K has (1) no ability to ADP-ribosylate host receptor proteins, (2) no ability to induce intracellular cAMP accumulation in epithelial cells, and (3) no ability to act as a robust adjuvant for co-administered antigens. Further, as shown in the Examples and in
Several other mutated forms of the A1 domain are believed to share these functional properties. These forms are: E112G, E112D, E110K, E110G, E110D, S61F, and R25G. Given these shared functional properties, it is believed that each of these other mutated forms of the LT A subunit and of the A1 domain are non-toxic and will also be unable to induce inflammatory cytokines when internalized into a cell. It is therefore believed that each can be used as anti-inflammatory agents. Since it is the A1 domain of the LT A subunit that is responsible for the toxic effect of LT (as noted, the A2 domain serves as a tether to the B subunit, and is not believed to participate in interference with ARF function or inhibition of ADP-ribosylation), references to mutations in the A1 domain or subunit also refer to mutations in the A subunit comprising both the A1 domain and the A2 domain. Further, for convenience of reference, reference herein to “A1 subunit” when referring to the E. coli LT means the A1 domain of the LT A subunit, or of mutated forms of the LT A1 domain of the A subunit, as required by context.
While the eight mutants described above are exemplary of mutations in the A1 subunit that remove the ability of the A1 subunit to induce diarrhea and to avoid other adverse effects, they are only some of the variations in the A subunit or A1 subunit that are expected to have these effects. For example, in E112K, the acidic amino acid residue E, glutamic acid, which is negatively charged at physiological pH (all references to charge in this section refer to charge while in an aqueous solution at physiologic pH), is replaced with a K, a basic amino acid that is positively charged, while in E112D, the glutamic acid has been replaced with D, aspartic acid, another negatively charged residue. This suggests that E112, which is at the active site of the A1 subunit, is particularly sensitive to mutations and that the toxic properties of A1 would also be abrogated at least by substitution of the E with another positively charged amino acid, arginine (“R”). Similarly, in S61F, the uncharged, polar serine (“S”) residue at position 61 is replaced with F, phenylalanine, an amino acid with a hydrophobic side chain terminating in a phenyl ring, suggesting that substituting the S with a tyrosine (“Y”) residue, whose side chain also terminates in a phenyl ring, but with a hydroxyl attached to the ring, will also result in inactivation of the A1 subunit's enzymatic properties. In the R25G detoxified A1 subunit, the arginine, R has been substituted by a small, uncharged amino acid, G, glycine, suggesting that substituting the positively charged R with either of the negatively charged residues E or D will also result in inactivation of the A1 subunit. Other substitutions will readily suggest themselves to persons of skill in the art and can readily be tested to see if they result in (1) inhibition of ADP-ribosylation of host receptor proteins, (2) no ability to induce cAMP in epithelial cells, and (3) no ability to act as a robust adjuvant for co-administered antigens. In some embodiments, therefore the inventive compositions and methods contemplate use of detoxified forms of the A1 subunit sharing these three properties. Other mutations of residues at the active site of the A subunit's ADP-ribosylation activity that confer these properties are also comprehended.
In some embodiments, the detoxified A1 subunit is E112K. In some embodiments, the detoxified A1 subunit is E112G. in some embodiments, the detoxified A1 subunit is E112D. In some embodiments, the detoxified A1 subunit is E110K. In some embodiments, the detoxified A1 subunit is E110G. In some embodiments, the detoxified A1 subunit is E110D. In some embodiments, the detoxified A1 subunit is S61F. In some embodiments, the detoxified A1 subunit is R25G.
The B-subunit mediates binding of the holotoxin to cells and internalization into them. The B-subunit binds to ganglioside receptors, such as GM1 (monosialotetrahexosylganglioside) on gut epithelial cell membranes and dendritic cells. There are several types of B-subunits, Type I and II A and B, which differ in amino acid sequence and the extent to which they bind different ganglioside receptors. See, e.g., Tinker et al., Infection and Immunity, 2005, 73(6): 3627-55. It is believed that the anti-inflammatory effects seen in the studies underlying the present disclosure are due to the detoxified A1 subunit and not to the B subunit. Thus, it is believed that the potent anti-inflammatory effects shown in the studies reported in the Examples can be achieved by delivering a detoxified A1 subunit to dendritic cells and other cells of interest using means of delivery other than an LT B subunit.
In some embodiments, it is contemplated delivering a detoxified A1 subunit to cells of interest using the pentameric cholera toxin B subunit (“CTB”) or the non-toxic cholera toxin CTA2/B.
Chimeras have been made and have demonstrated the efficacy of CTB as a carrier for antigens for some thirty five years, as exemplified by, e.g., McKenzie and Halsey, J Immunol, 1984, 133 (4) 1818-1824 (horseradish peroxidase (HRP) covalently attached to CTB was shown to raise order of magnitude greater amounts of anti-HRP antibody than HRP or CTB alone), In 1990, a genetic construct of nucleic acid encoding glycosltransferase at the N-terminal of CTB was made and shown to maintain structure and function of CTB. The authors stated the study “demonstrated a complete system for constructing, expressing, and purifying cm chimeras.” Dertzbaugh et al., Infect. Immun. 1990, 58(1):70-79, at p. 78. By 2001, researchers in the area were able to state “It is well established that CTB is a highly efficient carrier molecule for the induction of mucosal antibody responses . . . as well as for the induction of mucosally induced systemic T-cell . . . and systemic B-cell . . . tolerance.” George-Chandy, et al., Infect Immun. 2001, 69(9):5716-25 (“George-Chandy”), at p. 5723. (Citations omitted. George-Chandy reported in their study that antigen chemically conjugated or genetically fused to CTB “dramatically lowers the threshold concentration of antigen required for immune cell activation.” See, Abstract.)
Both CTB and constructs of the A2 domain of cholera toxin (“CTA2”) in combination with CTB (“CTA2/B”), have been shown to deliver exogenous proteins to cells. See, e.g., Li et al., Infect. Immun. 2004, 72:7306-7310; Tinker et al., Toxins 2014, 6(4), 1397-1418 (West Nile Virus DIII-CTA2/B chimera shown to be immunogenic after intranasal delivery). CTB has been used to deliver antigens orally and to serve as an adjuvant for mucosal delivery of antigens intranasally, rectally, and vaginally. See, e.g., Holmgren, et al., Vaccine, 1993, 11(12):1179-84; Hajishengallis et al., J Immunol, 1995, 154(9):4322-4332; Langridge et al., Current Opin Investig Drugs, 2010, 11(8):919-928. George-Chandy, supra, reported that chemically conjugating antigen to CTB or expressing the antigen and CTB as a fusion protein resulted uptake of antigen into cells through the GM1 ganglioside receptor, showing that proteins conjugated or fused to the CTB are taken into the cell.
In short, reports over the past three decades have shown that work has shown that a variety of proteins have been successfully recombinantly fused or chemically conjugated to CTB and CTA2/B and successfully delivered into target cells both in vitro and in vivo. It is therefore expected that non-toxic LT A subunits that bind to ARF can be genetically fused or chemically conjugated to CTB or to CTA2/B by the methods developed over the past three decades. It is further expected that such fusions or conjugates will be delivered into cells just like the numerous fusions and constructs already demonstrated to deliver proteins into cells, as exemplified by the antibodies that have been raised against the proteins. Since the LT A2 subunit serves as a linker to the B subunit, but does not participate in interaction with ARF, it is expected that the A2 portion of the A subunit can be omitted in such chimeras, reducing the size of the LT protein to be fused or conjugated to CTB or CTA2/B.
In some embodiments, the detoxified A subunit can be loaded into liposomes or other encapsulating vesicles. Loading therapeutic agents into liposomes has been known since the 1980s. See, e.g., Woodle and Storm, eds.,
In some embodiments, the liposomes or other encapsulating vesicles can be targeted to cell types of interest by tethering antibodies or fragments (e.g., Fab, F(ab′)2) or variable region fusion proteins (e.g., single chain variable fragments, or “scFv”) of antibodies that bind antigen to the exterior of the liposomes. Methods of targeting liposomes to target cells by tethered antibodies, antigen-binding portions, or fusion protein derivatives thereof, such as Fab, F(ab′)2, and scFvs, have been known since at least the early 2000s, as exemplified by, e.g., U.S. Pat. Nos. 6,210,707 and 6,214,388. In some embodiments, the antibodies or antigen-binding fragments or single chain variable fragments thereof bind CD11c. According to Martin, A., in D. Dabbs, ed.,
It is anticipated that symptoms of many inflammatory diseases and conditions can be ameliorated or treated by the inventive methods and compositions.
As enterotoxic E. coli (ETEC″) and cholera affect the mucosal lining of the intestinal tract, the B subunits of their toxins are particularly good for carrying detoxified A subunits to mucosal surfaces. Thus, embodiments of the inventive compositions and methods are particularly suited for ameliorating inflammation in the gastrointestinal tract. Chronic inflammation can occur at various sites within the gastrointestinal tract. See, e.g., Bamford, K., FEMS Immunology & Medical Microbiology, 1999, 24(2)161-168. It is contemplated that in some embodiments, the inventive compositions and methods can be used transmurally to ameliorate symptoms of inflammation throughout the entire tract, while in other embodiments, they can be used to ameliorate symptoms at particular sites or in particular segments, such as the bowel or colon, depending on the form and route of administration. For example, compositions administered orally in liquid form would he expected to travel, and ameliorate symptoms, along the entire tract, while compositions administered in the form of a suppository would be expected to ameliorate symptoms in the rectum. In some embodiments, an endoscope or similar instrument can be used to deliver the inventive compositions to an affected site within the large or the small intestines, or both. For example, a colonoscope can be used to deliver an inventive composition to the junction of the ileum and the colon.
In some embodiments, the gastrointestinal inflammation to he ameliorated is inflammatory bowel disease (sometimes referred to herein as “IBD”). According to the CDC, in 2015, an estimated 1.3% of U.S. adults, or over 3 million individuals, had been diagnosed with IBD, a 50% increase from the number with IBD in 1999. See also, Dahlhamer, et al., MMWR Morb Mortal Wkly Rep., 2016, 65(42):1166-1169.
Two major forms of IBD are Crohn's disease (sometimes referred to herein as “CD”) and ulcerative colitis (sometimes referred to herein as “UC”). In some cases, it cannot be determined if a patient's IBD is CD or UC. In such cases, the patient may be diagnosed with indeterminate colitis. According to the Crohn's and Colitis Foundation of America (“CCF”), CD can affect any portion of the gastrointestinal tract, but most commonly affects the junction of the ileum and the colon mentioned above. The CCF further states that inflammation due to CD can occur in patches and extend through the entire thickness of the intestinal wall. In contrast, the CCF states that UC occurs only in the colon and rectum and inflammation affects only the inner lining of the tract. Both diseases cause abdominal pain, diarrhea, and a feeling of urgency to empty the bowels, and can cause rectal bleeding. CD can cause fistulas and strictures in the intestines. UC and, less commonly, CD, can cause toxic megacolon, in which severe inflammation causes the colon to enlarge, which can lead to nerve and muscle damage and almost complete paralysis of the affected portion. Both UC and CD can also cause the bowel to perforate.
Both CD and UC have been extensively studied. Baumgart and Sandborn, The Lancet, 2012, 380(9853):1590-1605, provides a review of the etiology, diagnosis, and treatment of CD. See also, Hart an Ng, Medicine, 2015, 43(5):282-290. UC is reviewed in, e.g., Ho et al., Medicine, 2015, 43(5):276-281. UC also occurs in children; pediatric UC is discussed in, for example, Turner, Inflam. Bowel D is, 2011, 17(1):440-49. Extensive information regarding UC and CD are also available in standard texts, such as Friedman and Blumberg, “Inflammatory Bowel Disease,” in Jameson et eds.,
In animal studies underlying the present disclosure, colitis scoring was conducted as described in Chassaing, et al., Curr Protoc Immunol, 2014, 104(1): 15.25.1-15.25.14. There are a number of indices for grading UC activity and severity in humans, which use criteria such as histology, inflammation, and endoscopic examination. D'Haens et al., Gastroenterology, 2007, 132:763-786, present a review of a number of activity indices and efficacy end points for clinical trials for UC in adults. The reference reviews ten indexes for measuring the severity of UC (e.g., the Truelove and Witts Severity Index, the Powell-Tuck Index, the Seo Index), and nine endoscopic measures of disease activity (e.g., the Truelove and Witts Sigmoidoscopic Assessment, the Baron Score, and the Modified Baron Score). A scoring system proposed by Geboes et al., Gut. 2000, 47:404-409, provides a scale for grading inflammation. According to Jauregui-Amezaga, et al., Jr Crohn's and Colitis, 2017, 11(3):305-313, the Geboes score is one of the most commonly used histological scores for grading UC, but is somewhat complicated, and proposed a simplified version. Xie et al., Gastroenterology Report, 2018, 6(1):38-44, compares the Ulcerative Colitis Endoscopic Index of Severity (UCEIS) and the Mayo Endoscopic Score (MES), both of which were developed as objective methods to measure endoscopic severity, as predictors of the need for colectomy. Travis et al., Aliment. Pharmacol. Ther., 2011, 34:113-24, review the different definitions of remission of UC used in various trials and propose a standard definition based on clinical symptoms and endoscopy, with histopathology as a third dimension.
While practitioners have not agreed on a universal system for measuring improvement or remission of UC, all the indices mentioned above are intended to provide measures of improvement or worsening of the condition and all are accepted by some portion of the medical community. For purposes of the present disclosure, it is contemplated that a reduction of activity score in any of the activity indexes noted in the references above, or a reduction of the endoscopic activity score as set forth in any of the indexes set forth in those references indicates an amelioration of symptoms of inflammation due to UC.
In view of the reduction of T cell regulatory activity seen in studies underlying the present disclosure, it is believed that, in addition to IBD, embodiments of the inventive compositions and methods can be used to reduce or ameliorate symptoms of inflammation in a number of other conditions in which relieving symptoms of inflammation would be of benefit to the patient. In some embodiments, the conditions are ones in which other anti-inflammatory agents have been found to be helpful. For example, in some embodiments, the inventive compositions can be administered to persons suffering from asthma, in addition to, or in place of, the inhaled corticosteroids currently used as long-term asthma control medications, or as a quick-relief agent to reduce airway inflammation during severe asthma. It is contemplated that in these uses, the inventive compositions will be administered by inhaler, using devices similar to those used to deliver current asthma medications.
In some embodiments, the inventive compositions and methods can be used to relieve inflammation in joints caused by various forms of inflammatory arthritis, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and juvenile idiopathic arthritis. In these embodiments, it is contemplated that the inventive compositions will be mixed into formulations suitable for carrying the compositions into the skin. Creams and ointments for introducing therapeutic agents into the skin are well known. Other conditions for which topical application is expected to be useful are dermatitis and psoriasis. It should be noted that a transdermal patch containing 37.5 μg E. coli LT as a vaccine was tested in clinical trials and found to shorten episodes of traveler's diarrhea and result in fewer loose stools (Frech et al., Lancet 2008,371(9629):2019-25). A Phase 3 study of a skin patch containing 37.5 μg E. coli LT found that the LT was delivered effectively and was immunogenic. Behrens et al., The Lancet, 2014, 14(4197-204, showing that LT can be delivered through the skin into antigen presenting cells. The inventive compositions and methods are also expected to also be useful in reducing inflammation of internal organs, including conditions including nephritis, pancreatitis, and liver inflammation, as well as systemic inflammatory diseases, such as lupus. In these embodiments, the compositions are conveniently delivered to the organs by intravenous (IV) infusion.
Formulations of the inventive compositions will depend in part on the site of the inflammatory condition whose symptoms are to be treated or ameliorated, and the contemplated route of administration. In the murine studies reported in the Examples, detoxified LT E112K administered in drinking water was shown to be effective in several different murine models of inflammatory bowel disease. Based on these studies, formulations for reducing inflammation in IBD, including Crohn's disease and ulcerative colitis, could be as simple as providing a subject in need thereof water containing a composition of a detoxified A subunit and a carrier of choice. The water preferably contains a small percentage of NaCl or another pharmacologically acceptable salt to maintain conformational stability of the proteins and to maintain the proteins in solution. Preferably, the salt is present at 0.1M. In some formulations, the detoxified A subunit and carrier of choice may be provided in lyophilized form, and reconstituted in water prior to being taken orally by the subject. These formulations may be given orally or, for example, may be administered through an endoscope to a desired site in the gastrointestinal tract.
For oral administration, FDA-approved flavoring ingredients and sweeteners that are compatible with the detoxified A subunit and carrier of choice may be added. It is unlikely that any particular generally used flavoring ingredients or sweeteners are not compatible with administration of the detoxified A subunit and carrier of choice, but any particular combination can be readily tested by performing two parallel studies following the protocol described in Example 5, below, or in Example 6,below, one in which the detoxified A subunit and carrier of choice are provided in water and one in which the detoxified A subunit and carrier of choice are provided in water with the combination of flavoring ingredient and sweetener to be tested, and comparing the results. If the addition of the flavoring ingredient and sweetener to the water with the detoxified A subunit and carrier of choice results in reducing the benefit to the mice seen by use of the detoxified A subunit and carrier of choice in water alone, that combination of flavoring agent and sweetener is not a compatible combination with the detoxified A subunit and carrier of choice. In similar formulations, the composition can be provided as syrups or suspensions.
In some embodiments, the detoxified A subunit and carrier of choice may be administered in a suitable oral dosage form, such as a pill, capsule, tablet, lozenge, pastille, pellet, medicated chewing gum, powder, solution, suspension, wafer, or syrup. Making such oral dosage forms is well known in the art, and it is expected that persons of skill are familiar with the considerable literature and guidance that exists, as exemplified by texts such as Gennaro, A.,
In addition to the detoxified A subunit and carrier of choice, the formulations may include one or more pharmaceutically acceptable excipients, stabilizers, binders, lubricants, tillers, buffers, antioxidants, such as ascorbic acid, preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; or benzalkonium chloride), monosaccharides, disaccharides, and other sugars or carbohydrates, including glucose, mannose, sucrose, mannitol, trehalose or sorbitol, low molecular weight polypeptides, proteins, such as serum albumin or gelatin, hydrophilic polymers such as polyvinylpyrrolidone, chelating agents such as EDTA, and salt-forming counter-ions such as sodium. In some embodiments, the pills, capsules, or tablets may be formulated to be taken bucally (against the cheek) or sublingually (under the tongue) or to be orally disintegrating. In some embodiments, the compositions may be included in a film that dissolves and releases the compositions when administered buccally or sublingually, or in a spray that is so administered to the mouth or to the nasal cavity. In some embodiments, the pill, capsules, or tablets may be formulated as modified release dosage forms, including delayed, extended, sustained, pulsed, or fast release forms.
Formulations intended for application to the skin are typically in an ointment base of a thick oil in a 80% oil to 20% water mixture with a high viscosity. A number of bases, such as beeswax, hydrocarbon bases, such as ceresine, and vegetable oil bases are known and can be selected depending on the particular properties of penetrability, stability, solvent property and release of medicament desired by the practitioner. The inventive compositions and methods can also administered topically in the form of gels or in transdermal patches, such as those used in the clinical trials testing LT as a vaccine for enterotoxic E. coli discussed in the preceding section.
For purposes of the inventive methods, an “effective amount” or a “therapeutically effective amount” of a composition comprising a detoxified LT A subunit refers to an amount that, alone or in combination with further doses, produces the desired response, e.g., alleviation or elimination of inflammation. This may involve decreasing the amount of inflammation only temporarily or only briefly, so long as it is measurable. This can be measured by standard methods, such as the UC activity scores and endoscopy score indices discussed in a preceding section.
The “therapeutically effective amount” will depend on the particular condition being treated, the severity of the condition, patient characteristic such as age, physical condition, size, gender, and weight, the duration of treatment, the nature of concurrent therapy, if any, the specific route of administration, and similar factors. These factors are well known to treating physicians, who are trained to make decisions regarding treatment regimens and dosages based on these and similar criteria. It is generally preferred that the patient be administered the highest safe dose according to clinical judgment, but it is understood, as with many therapeutics, that the patient may wish to take a lower dose for any of a variety of reasons. It is also understood that the practitioner's decision about how much to prescribe to a particular patient will be guided by information obtained during clinical trials of the compositions.
In some embodiments, the practitioner may evaluate a patient for the severity of UC before commencing administration of an induction dose and then reevaluate the patient following the induction dose to determine if there has been a desired clinical effect, as shown by any of endoscopic improvement, endoscopic remission, clinical response, an improvement in scoring under the practitioner's choice of disease activity index or endoscopic index, rectal bleeding subscore, endoscopic subscore, histologic improvement, and combinations thereof.
For topical administration or administration by injection, it is contemplated that the dose range will be from about 1 μg (with “about” here meaning±0.1 μg) to about 5 mg (with “about” here meaning±0.2 mg. All dosages stated herein refer to the amount of detoxified A subunit and carrier.) In some embodiments in which the amount of detoxified A subunit and carrier administered is 100 μg or less, the composition administered does not also contain an exogenous antigen to which the practitioner wishes to raise an immune response. In some embodiments in which the amount of detoxified A subunit and carrier administered is 75 μg or less, the composition administered does not also contain an exogenous antigen to which the practitioner wishes to raise an immune response. In some embodiments in which the amount of detoxified A subunit and carrier administered is 50 μg or less, the composition administered does not also contain an exogenous antigen to which the practitioner wishes to raise an immune response.
For oral administration, it is contemplated that the dose range will be from about 100 μg (with “about” here meaning±5 μg) to about 500 mg of detoxified A subunit and carrier (with “about” here meaning±2 mg.) For some conditions causing the inflammation being treated, compositions comprising the detoxified A subunit and carrier may be administered in a single dose intended to reduce symptoms of inflammation, while in others, the compositions will be administered in multiple doses. In some embodiments, it is contemplated that multiple doses of compositions comprising the detoxified. A subunit and carrier will be administered in an larger “induction dose,” followed by one or more smaller “maintenance doses.” In these embodiments, it is contemplated that the larger induction dose will induce an initial reduction of symptoms of inflammation, while the following, maintenance, doses will be at a lower dosage to keep the inflammatory symptoms in check and, preferably, wholly suppressed.
For amelioration of inflammation due to UC or CD, which present as chronic conditions, maintenance doses may be administered, for example, daily, every other day, biweekly, weekly, or every two weeks, for a period of months, or indefinitely. If maintenance doses alone are not keeping symptoms of inflammation in check, one or more additional induction doses may be administered to reduce or bring symptoms of inflammation under control before returning to, and continuing with, maintenance doses. For example, if the inflammatory condition is UC, and the patient's UC activity score has regressed from one achieved after an induction dose, the patient can administered one or more additional induction doses. The additional doses can be administered daily, biweekly, or weekly until clinical improvement is seen. In some embodiments, the induction doses may be continued until there is a clinical remission as determined, for example, by endoscopic examination or the patient's subjective evaluation of quality of life, including reduction or lack of tenesmus, whereupon the patient can be started on, or returned to, maintenance doses. It is contemplated that induction doses administered for UC, CD, or other IBDs, will be 1 mg to 500 mg, about 2-400 mg, about 2-300 mg, about 5-200 mg, about 5-150 mg, about 5-100 mg, about 5-50 mg, or about 5-25 mg with each succeeding range being more preferred than the one before it and “about” in this context meaning±1 mg. It is further contemplated that maintenance doses will range from 1 mg to 300 mg, 1 mg to 200 mg, about 2-200 mg, about 2-150 mg, about 2-100 mg, about 2-75 mg, about 2-50 mg, about 2-40 mg, about 2-30 mg, about 2-20 mg or about 2-10 mg, with each succeeding range being more preferred than the one before it and “about” in this context meaning±1 mg.
In the animal studies reported in the Examples, mice with different models of IBD showed reduction of symptoms when administered daily detoxified A subunit and carrier in water which contained 0.3% NaCl. Accordingly, in some embodiments, the patient may take daily doses of detoxified A subunit and carrier in a suitable liquid, such as water. In some embodiments, a dose for daily administration may be 1 mg to 500 mg, 1 mg to 300 mg, 1 mg to 200 mg, about 2-200 mg, about 2-150 mg, about 2-100 mg, about 2-75 mg, about 2-50 mg, about 2-40 mg, about 2-30 mg, about 2-20 mg or about 2-10 mg, with each succeeding range being more preferred than the one before it and “about” in this context meaning+1 mg.
In veterinary applications, it is contemplated that the veterinarian will make the dosing decision based on the species, size, gender, age, physical condition, and weight of the animal, the duration of treatment, the nature of concurrent therapy, if any, the specific route of administration, and similar factors, in determining the amount of detoxified A subunit and carrier to administer. The formulations will typically be administered in the animal's drinking water. If the animal is free ranging, or has alternative water sources available and does not appear to like the taste of drinking water containing the therapeutic composition, the detoxified A subunit and carrier can be introduced into the animal by other methods used to administer veterinary drugs, such as by injection, by adding it as a food additive, or by administering the composition as a pill.
This Example sets forth some methods and materials used in the studies discussed below.
E112K was prepared by galactose-affinity chromatography as described previously (Clements and. Finkelstein, Infect Immun. 1979, 24(3):760-9, Cheng et al., Vaccine, 2000, 18:38-49). Briefly, toxins were purified from E. coli expression cultures grown overnight in a 10-liter fermenter. Cells were harvested by centrifugation and lysed in a microfluidizer model M-110L (Miocrofluidics, Newton, Mass.). The cell lysates were dialyzed overnight in TEAN (0.05 M Tris, 0.001 M EDTA, 0.003 M NaN3, 0.2 M NaCl, pH 7.5), clarified by centrifugation, and subjected to chromatography on separate immobilized D-galactose columns (Pierce, Rockford, Ill.). Toxins were eluted with 0.3 M galactose in TEAN and passed through an endotoxin removal column (Pierce, Rockford, Ill.). The composition and purity of each protein was confirmed by SDS-PAGE (
This Example shows that E112K prevents basal cytokine secretion and intracellular cAMP accumulation in intestinal cells.
As shown in
It has been established for many years that LT induces cAMP accumulation through binding to host cell ARF to initiate ADP-ribosylation of Gsα, leading to irreversible activation of adenylate cyclase and increased production of intracellular cAMP (which ultimately leads to secretory diarrhea). E112K maintains the strong cellular binding and internalization properties of its parent molecule. Its A- and B-subunits can be detected inside human intestinal epithelial cells for up to seven days after in vitro treatment (
This Example shows that E112K inhibits intracellular cAMP, ADP-ribosylation, and activation of dendritic cells.
One of the consequences of LT intoxication of cells is ADP-ribosylation of host cell proteins and accumulation of cAMP. in dendritic cells, this leads to activation and upregulation of costimulatory surface markers, such as CD80 and CD86. Dendritic cells are key initiators of adaptive immune responses, linking the traditional innate and adaptive arms of the immune system. E112K is rapidly internalized and its A- and B-subunits detected in treated dendritic cells (
This Example shows that E112K inhibits adaptive immunity.
For decades, LT has been known as a potent mucosal and parenteral adjuvant, inducing immune responses to admixed antigens. Older studies also clearly determined that E112K and related mutants with mutations in the A-subunit (e.g., E110D, S61F) that prevent ADP-ribosylation activity are not oral adjuvants (Lobet et al, Infect Immun. 1991, 59(9):2870-9, Cheng et al., Vaccine, 2000, 18:38-49). Similarly, in our studies, increasing doses of E112K admixed with tetanus toxoid result in a progressive decline in antigen-specific antibody responses (
This Example shows that E112K improves inflammatory bowel disease pathologies in dextran sulphate sodium (DSS) colitis mouse models. The classic DSS chemical injury model of chronic, ulcerative colitis was induced in BALB/c mice by successive waves of 5-day drinking water treatment with 4% DSS, followed by 7-days of water. After the 2nd (day 17) or 3rd (day 29) DSS treatment, some mice were treated with 50 μg E112K by oral gavage. The final weight change (day 36, % original weight) or colon length (day 38, mm) were evaluated for all groups. The results are presented in
The intestines show some moderation of DSS-mediated cellular infiltration (black asterisk) with E112K therapy.
This Example shows that E112K is a sate, efficacious therapy for inflammatory bowel disease pathology in the IL-10-/- colitis mouse model.
This Example discusses and extends the studies reported in Example 6.
As indicated in
However, in piroxicam-treated mice that have observable histopathology, including architectural distortion like hyperplasia of the intestinal mucosa, inflammation, and ulceration (
Finally, we tested E112K for efficacy in a third model of inflammatory bowel disease, the T-cell transfer model of colitis. In this model, CD4+CD45RB-hi T-cells are transferred into Rag1-/- mice, causing unchecked intestinal inflammation and wasting (or weight loss) by 20 weeks. The protocol is presented graphically in
Thus, E112K was shown to be an effective therapy for IBD in three different mouse models of colitis.
This Example shows that the exemplar detoxified A subunit-carrier E112K improved intestinal permeability as soon as 24 hours post-treatment. The chronic colitis model was induced in IL-10-/- mice with 7 day exposure to 200 ppm piroxicam at 6-weeks of age. IL-10-/- mice were exposed to piroxicam for 7 days in rodent chow and then 4 days later either left untreated or treated with E112K in drinking water. Intestinal permeability was tested 1-day after treatment by analyzing serum for fluorescent protein after 3 h oral gavage with 4 kD FITC-Dextran. The results are shown in
This Example shows that the exemplar detoxified A subunit-carrier E112K improved disease activity index in DSS acute model of colitis in immunodeficient mice. Rag1-/- mice that lack functional adaptive immune system were exposed to 3% DSS in drinking water for 7 days. A group of these DSS mice were treated with oral E112K on day 5. Weight changes and disease activity index (evaluated by weight loss, fecal consistency, and blood in feces) were evaluated in animals at day 8. The results are shown in graph form in
1×106 bone-marrow derived mouse dendritic cells were treated in triplicate for 48 h with 0.1 μg/ml test proteins before analysis of CD80 and CD86 co-stimulatory marker levels on CD11c+ gated cells. Assay was performed three independent times.
This Example discusses a possible mechanism of action for detoxified LT mutants such as E112K.
Without wishing to be bound by theory, it is believed that the intact detoxified LT mutant E112K binds to GM1 surface receptors on cells such as epithelial cells through B-subunit binding. It is further believed that the binding results in release of the E112K A1-subunit into the cytosol, that it is the A1-subunit that interferes with function of ADP-ribosylation factor (ARF) in the host cell, and that it is the interference of the A1-subunit with ARF activity that inhibits ongoing vesicular trafficking and innate signaling (e.g., IL-6 cytokine secretion) and decreases cAMP levels over time, ultimately dampening overall levels of inflammation. Thus, it is believed that the A1-subunit interference with ARF activity suppresses the release of inflammatory cytokines, suppresses pro-inflammatory signaling responses, and suppresses inflammation, as shown in cartoon form in
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 62/667,992, filed May 7, 2018, the contents of which are incorporated herein by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/031193 | 5/7/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62667992 | May 2018 | US |
