WO2014068320A1 - Ketone body inhibitors for use in the treatment of gastrointestinal tract mucosa impairment - Google Patents

Ketone body inhibitors for use in the treatment of gastrointestinal tract mucosa impairment Download PDF

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Publication number
WO2014068320A1
WO2014068320A1 PCT/GB2013/052850 GB2013052850W WO2014068320A1 WO 2014068320 A1 WO2014068320 A1 WO 2014068320A1 GB 2013052850 W GB2013052850 W GB 2013052850W WO 2014068320 A1 WO2014068320 A1 WO 2014068320A1
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ketone body
inhibitor
body inhibitor
use according
spp
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PCT/GB2013/052850
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French (fr)
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Lars Fändriks
Ville Wallenius
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Benf Ab
Smith, Stephen Edward
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1783—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/64—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • proinflammatory cytokines e.g., tumour necrosis factor-a and IL- 1 ⁇
  • the inflamed, and therefore "leaky,” gut may then allow passage of bacteria and/or bacterial components across the intestinal barrier.
  • TLR Toll-like receptor
  • lymph node lymphoid cells mainly omental and mesenteric fat
  • bacterial stimuli may lead to local activation of peroxisome proliferator-activated receptor- ⁇ and therefore induce adipocyte proliferation and differentiation in the mesenteric fat depot, although the underlying mechanisms remain largely unclear.
  • inflammation impairs gut barrier function and results in the leakage of microbial antigens.
  • the present inventors have surprisingly found that the barrier properties of the Gl mucosa are regulated by local (gastrointestinal) ketogenesis and, consequently, that barrier impairment of the Gl tract can be regulated by modifying Gl ketogenesis, Gl ketones and/or effects induced by Gl ketones.
  • Cytosolic HMG-CoA synthase contains sterol regulatory elements that modulate transcriptional activity by sterols, mediated by sterol regulatory element binding proteins (SREBP)-1 and -2 [9, 10], which have not been observed in the promoter of the mitochondrial HMG-CoA synthase.
  • SREBP sterol regulatory element binding proteins
  • PPRE peroxisome proliferator regulatory element
  • the wall of the Gl tract has a general structure. Most of the tube's luminal surface is highly convoluted, a feature that greatly increases the surface area available for absorption. From the stomach on, this surface is covered by a single layer of epithelial cells linked together along the edges of the luminal surfaces by tight junctions.
  • E. cloacae, E. faecalis and E. faecium Escherichia spp. (such as E. coli), Flavobacterium spp., Hafnia spp., Klebsiella spp., Mycobacteria spp., Mycoplasma spp., Peptococcus spp., Plesiomonas shigelloides, Propionibacterium spp (such as P. acnes), Proteus spp., Providencia spp., Pseudomonas spp (such as P. aeruginosa), Ruminococcus spp (such as R.
  • Flavobacterium spp. Hafnia spp., Klebsiella spp., Mycobacteria spp., Mycoplasma spp., Peptococcus spp., Plesiomonas shigello
  • the gastrointestinal mucosa barrier impairment may be associated with increased paracellular or transcellular permeability of the gastrointestinal mucosa.
  • the gastrointestinal mucosa barrier impairment is associated with increased paracellular permeability of the gastrointestinal mucosa.
  • the TJ forms a continuous, circumferential, belt-like structure at the luminal end of the intercellular space, where it serves as a gatekeeper of the paracellular pathway.
  • the TJ plaque is the site of a growing number of TJ-associated protein complexes.
  • the cell membranes of adjacent epithelial cells are brought into intimate focal contact sites in which the intercellular space is obliterated.
  • the TJ In addition to serving as a regulated gate/barrier in the paracellular pathway, the TJ also functions as a fence in the plane of the plasma membrane, where it contributes to the maintenance of asymmetrically distributed integral membrane proteins and lipids.
  • the gastrointestinal tract mucosa barrier impairment is associated with ketogenesis in one or more sites selected from the group consisting of the stomach, duodenum, jejunum, ileum, cecum and colon.
  • the gastrointestinal tract mucosa barrier impairment may not be associated with ketogenesis in one or more sites selected from the group consisting of the stomach, duodenum, jejunum, ileum, cecum and colon; for example, the barrier impairment may not be associated with (i.e., occur independently of) ketogenesis of the colon.
  • the gastrointestinal tract mucosal barrier impairment is present in one or more site selected from the group consisting of the stomach, duodenum, ileum, cecum and colon.
  • the gastrointestinal tract mucosal barrier impairment may be absent from one or more site selected from the group consisting of the stomach, duodenum, ileum, cecum and colon.
  • ketoone body inhibitor we include agents capable of (i) reducing or preventing ketogenesis; (ii) reducing or abolishing ketones themselves; and/or (iii) reducing or preventing ketone-induced effects.
  • the ketone body inhibitor is capable of inhibiting ketone bodies in non-gastrointestinal mucosa tissues by no more than 25%, for example, no more than 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 % or no more than 0%.
  • the ketone body inhibitor may be formulated to reduce or prevent its exposure to non-gastrointestinal mucosa tissue. For example, exposure of the ketone body inhibitor to non-gastrointestinal mucosa tissue may reduced by at least 10%, for example, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100%.
  • the ketone body inhibitor may be an inhibitor of a ketogenic enzyme.
  • HMG-CoA synthase also known as 3-hydroxy-3-methylglutaryl- Coenzyme A synthase 2 (mitochondrial), H GCS2, );
  • BC044217.1 (version BC044217.1) .
  • HMGCS2 (transcript variant 2) ; GenBank Accession No. :
  • An antibody of desired specificity may alternatively be generated using well-known molecular biology methods, including selection from a molecular library of recombinant antibodies, or grafting or shuffling of complementarity-determining regions (CDRs) onto appropriate framework regions.
  • Human antibodies may be selected from recombinant libraries and/or generated by grafting CDRs from non-human antibodies onto human framework regions using well-known molecular biology techniques.
  • an expression vector containing a nucleotide sequence as described in the second aspect of the invention In a third aspect of the invention there is provided an expression vector containing a nucleotide sequence as described in the second aspect of the invention.
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
  • An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, NJ, USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • a further method uses synthetic molecules called linkers and adaptors.
  • DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3' termini and fill in recessed 3' ends.
  • Synthetic linkers, pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini.
  • Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.
  • a desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
  • the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • a host cell comprising a nucleotide sequence or expression vector as described in the second and third aspects of the invention.
  • DNA encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
  • Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters and the promoters of retroviral LTRs. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will desirably also contain sites for transcription initiation and termination, and in the transcribed region, a ribosome binding site for translation. (WO 98/16643)
  • the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector and it will therefore be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence marker, with any necessary control elements, that codes for a selectable trait in the transformed cell.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracyclin, kanamycin or ampicillin resistance genes for culturing in E.coli and other bacteria.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell. Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • bacteria eg. E.coli and Bacillus subtilis
  • yeasts eg. Saccaromyces cerevisiae
  • insect cell systems transformed with, for example, viral expression vectors (eg. baculovirus)
  • plant cell systems transfected with, for example viral or bacterial expression vectors
  • animal cell systems transfected with, for example, adenovirus expression vectors.
  • a pharmaceutical composition comprising a ketone body inhibitor as described in the first aspect of the invention and a pharmaceutically acceptable excipient, diluent or carrier.
  • pharmaceutically acceptable is included that the formulation is sterile and pyrogen free.
  • Suitable pharmaceutical carriers are well known in the art of pharmacy. The carriers) must be “acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used.
  • pharmaceutically acceptable carrier and pharmaceutically acceptable excipient includes any compound(s) used in forming a part of the formulation that is intended to act merely as a carrier, i.e., not intended to have biological activity itself.
  • the pharmaceutically acceptable carrier or excipient is generally safe, non-toxic, and neither biologically nor otherwise undesirable.
  • a pharmaceutically acceptable carrier or excipient as used herein includes both one and more than one such carrier or excipient.
  • the medicaments and agents will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19 th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA, which is incorporated herein by reference).
  • the medicaments and agents can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • the medicaments and agents may also be administered via intracavernosal injection.
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the medicaments and agents of the invention can also be administered parenterally, for example, intravenously, intra-articularly, intra-arterially, intraperitoneally, intra- thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • the daily dosage level of the medicaments and agents will usually be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses.
  • the medicaments and agents can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
  • the medicaments and agents can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder.
  • the compounds of the invention may also be transdermal ⁇ administered, for example, by the use of a skin patch. They may also be administered by the ocular route.
  • the medicaments and agents can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • a sustained-release drug delivery system such as a microsphere. These are designed specifically to reduce the frequency of injections.
  • a sustained-release drug delivery system such as a microsphere.
  • Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
  • Sustained-release immunoglobulin compositions also include liposomally entrapped immunoglobulin.
  • Liposomes containing the immunoglobulin are prepared by methods known per se. See, for example Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688- 92 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4 (1980); U.S. Patent Nos. 4,485,045; 4,544, 545; 6,139,869; and 6,027,726.
  • the liposomes are of the small (about 200 to about 800 Angstroms), unilamellar type in which the lipid content is greater than about 30 mole percent (mol. %) cholesterol; the selected proportion being adjusted for the optimal immunoglobulin therapy.
  • polypeptide medicaments and agents can be administered by a surgically implanted device that releases the drug directly to the required site.
  • Electroporation therapy (EPT) systems can also be employed for the administration of proteins and polypeptides.
  • EPT Electroporation therapy
  • a device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
  • Proteins and polypeptides can also be delivered by electroincorporation (El).
  • El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin.
  • the particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
  • Protein and polypeptide pharmaceuticals can also be delivered orally.
  • One such system employs a natural process for oral uptake of vitamin B12 in the body to co- deliver proteins and polypeptides. By riding the vitamin B12 uptake system, the protein or polypeptide can move through the intestinal wall.
  • Complexes are produced between vitamin B12 analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B12 portion of the complex and significant bioactivity of the drug portion of the complex.
  • IF intrinsic factor
  • the skilled person will appreciate that the most appropriate formulation will depend on a number of factors including route of administration, patient type (e.g. patient age, weight/size).
  • Roux-en-Y Gastric Bypass results in reduced incidence and long-term remission of obesity associated metabolic comorbidities: insulin resistance/type-2 diabetes, hypertension and dyslipidemia. Its effect on type-2 diabetes does not seem to depend only on weight loss and is not to a large extent considered to be caused by food restriction or malabsorption. The mechanisms of action of GBP on type-2 diabetes are considered by the scientific community to be largely unresolved. It is also known that GBP reduces endotoxinemia and the signs on a systemic inflammatory reaction (Monte et a/., Surgery. 2012 Apr;151(4):587-93). Also the mechanisms to this are principally unknown.
  • the high-fat-fed jejunal mucosa produces ketone bodies upon exposure to fat and fatty acids
  • the level of ketone-bodies in the blood leaving the small intestine increased upon luminal exposure to a mixture of emulsified lipids (Intralipid, 0.1 ml) in mice on HFD with confirmed jejunal H GCS2 expression.
  • standardized mucosal scrapings dissolved in saline from the HFD mice contained more ketone bodies than when obtained from LFD-mice (2.06 ⁇ 0.7 versus 1.2 ⁇ 0.4 mM).
  • obesity is associated with expression of the ketogenic enzyme HMGCS2 the small intestinal mucosa; fat and/or fatty acids in the intestinal lumen result in intestinal mucosal ketogenesis and that this response is mediated by H GCS2; and

Abstract

The present invention provides a ketone body inhibitor for use in the treatment or prevention of: gastrointestinal tract (Gl tract) barrier impairment; and/or a disease or condition associated with gastrointestinal tract barrier impairment. Related aspects of the invention provide pharmaceutical compositions of the invention, together with methods or identifying and using the same.

Description

KETONE BODY INHIBITORS FOR USE IN THE TREATMENT OF
GASTROINTESTINAL TRACT MUCOSA IMPAIRMENT
FIELD OF INVENTION The present invention provides ketone body inhibitor for use in the treatment or prevention of: gastrointestinal tract (Gl tract) mucosa barrier impairment; and/or a disease or condition associated with gastrointestinal tract barrier impairment. Related aspects of the invention provide pharmaceutical compositions comprising a polypeptide of the invention, together with methods or identifying and using the same.
INTRODUCTION
The constellation of metabolic abnormalities including centrally distributed obesity, decreased high-density lipoprotein cholesterol (HDL-C), elevated triglycerides, elevated blood pressure (BP), and hyperglycaemia is known as the metabolic syndrome. Associated with a 3-fold and 2-fold increase in type 2 diabetes and cardiovascular disease (CVD), respectively, it is thought to be a driver of the modern day epidemics of diabetes and CVD and has become a major public health challenge around the world.
The prevalence of obesity and the associated factors of the metabolic syndrome is dramatically increasing. For years it has been known that metabolic syndrome is followed by a mild to moderate systemic inflammatory response, for example manifested with an elevated CRP value (and several other so called acute phase factors). The association to obesity is strong because also a moderate weight reduction (independent on method; i.e., dieting, pharmacology, surgery) improves the 'inflammation' and the associated metabolic aberrations, for example insulin resistance. Distinct gene expression patterns, with both proinflammatory and complement genes over-represented in the omental as compared to subcutaneous fat, implicate a depot- specific role of adipose tissue in the innate immune system. Despite the increased production of anti-inflammatory cytokines, the state of low-grade inflammation in obesity suggests a dominant effect of the proinflammatory cytokines on whole body metabolism. More importantly, the association between the elevated circulating levels of inflammatory mediators and the prevalence of obesity-related metabolic complications including insulin resistance, type 2 diabetes, and cardiovascular disease indicates a role of adipose tissue-derived cytokines in the pathogenesis of metabolic disorders in obesity. The deleterious nature of inflammation is further highlighted by the proinflammatory state of omental fat, in which the increased release of proinflammatory cytokines including tumour necrosis factor-a, IL-6, IL-8, and plasminogen activator inhibitor-1 as compared to subcutaneous fat has been implicated as a key determinant of the depot-specific metabolic effect of adipose tissue. Why is an obese person with the metabolic syndrome in an inflammatory state?
The reasons for biochemical signs of inflammation in obesity are obscure but have been ascribed to that fat depots exert proinflammatory endocrine signalling (cf. adipokines etc).
Around 2007 data emerged demonstrating that intestinal bacterial endotoxin (lipopolysacharide; LPS) was present in the plasma of these patients. One plausible explanation to this finding is that bacterial components from the microbiota of the intestinal lumen translocate into the blood stream or lymphatic drainage of the intestines. Once in the circulation (i.e., the state of endotoxinemia), and probably also already in the sub-epithelial compartment of the Gl mucosa, the toxic/antigenic properties of LPS elicit an inflammatory response, possibly with sequential cascade effects in distant tissues like the adipose tissue, which responds with pro-inflammatory signalling.
There is an increasing body of evidence of a role for the gut in visceral fat hypertrophy and dysfunction. First, a key recent observation by Gummesson and colleagues has been made of a highly significant relationship between gut "leakiness" (i.e., gastrointestinal tract barrier impairment) at the level of the lower gastrointestinal tract and increased visceral adiposity in women who were normal to mildly overweight but otherwise healthy. Second, patients with Crohn's disease, a condition characterized by inflammation of the intestine, have a higher ratio of intra-abdominal to total abdominal fat compared to healthy controls and the prevalence of "fat wrapping" (excess adipose tissue extension from the mesenteric attachment) is correlated with the biochemical and clinical activity of the disease. A causal relationship between gut inflammation and mesenteric fat dysfunction has been demonstrated in animal models. Rats with experimental colitis have 35% more mesenteric fat mass than controls, with no difference between groups in the other fat depots. In addition to the expansion of the fat depot, mesenteric fat alterations associated with gut inflammation are also characterized by an increase in macrophage infiltration and the release of proinflammatory cytokines. The effect of inflammation on gut barrier function is a key factor mediating the gut-visceral fat interactions. Inflammation increases gut permeability, as evident by the reduction in the thickness of the intestinal mucous layer with increased severity of inflammation. At the cellular level, proinflammatory cytokines (e.g., tumour necrosis factor-a and IL- 1 β) increase intestinal tight junction permeability by inducing the expression and activation of myosin light chain kinase, which results in a contraction of perijunctional actin-myosin filaments and the subsequent opening of the intestinal epithelial tight junction barrier. The inflamed, and therefore "leaky," gut may then allow passage of bacteria and/or bacterial components across the intestinal barrier. This phenomenon is demonstrated in a study by Cenac and coworkers, in which experimental colonic inflammation in mice resulted in an increase in paracellular permeability of the colon and the subsequent translocation of bacteria into the peritoneal organs and, presumably, the adjacent mesenteric fat. Exposure to gut microbiota and their metabolites alters mesenteric adipose tissue metabolism in a number of ways. First, lipopolysaccharide (LPS) derived from Gram-negative intestinal microbiota triggers an inflammatory response in adipocytes that, together with macrophage colony- stimulating factor infiltrated from the intestine, promotes adipose tissue macrophage (ATM) recruitment in the mesenteric fat depot. LPS also interacts with Toll-like receptor (TLR)-4, which is upstream of the nuclear factor-κΒ pathway, to induce the transcription of proinflammatory genes in ATMs. Second, immune cells in the mesenteric fat are under prolonged stimulation by bacterial antigens, leading to the activation of lymph node lymphoid cells and the subsequent enlargement of lymph node-associated fat depots (mainly omental and mesenteric fat). Third, bacterial stimuli may lead to local activation of peroxisome proliferator-activated receptor-γ and therefore induce adipocyte proliferation and differentiation in the mesenteric fat depot, although the underlying mechanisms remain largely unclear. Taken together, inflammation impairs gut barrier function and results in the leakage of microbial antigens. Mesenteric fat hypertrophy and/or hyperplasia associated with gut inflammation may have a defensive role in trapping the luminal bacteria and their products to prevent further inflammation in the peritoneal cavity. The metabolic consequences of impaired gut barrier function are not confined to mesenteric fat dysfunction, but also extend to a more general effect on whole-body metabolism. Endotoxins derived from gut bacteria are normally detoxified in the liver. In the case of an impaired gut barrier function, increased flux of endotoxin (predominantly LPS) may exceed the capacity of Kupffer cells in the liver so that the endotoxin enters the systemic circulation. In active Crohn's disease and ulcerative colitis patients, the circulating endotoxin level is 40-60% higher as compared to healthy controls and correlates with disease activity. LPS forms a complex with LPS-binding protein which then binds to CD 14 (a protein expressed mainly in macrophages and to a lesser extent in neutrophils, monocytes, and liver) to initiate an acute immune response via the TLR4 and nuclear factor-κΒ pathways. An increase in circulating LPS leads to "metabolic endotoxemia," a condition characterised by a low-grade proinflammatory state, insulin resistance and increased cardiovascular risk. This accords with the elevated plasma level of LPS-binding protein, a marker of subclinical endotoxemia, in overweight/obese individuals. Furthermore, intervention studies have revealed a direct effect of endotoxemia on whole-body metabolism. A chronic subcutaneous infusion of LPS increased fasting plasma glucose and insulin levels and reduced insulin sensitivity in mice, whereas experimental endotoxemia induced a transient inflammatory response (as evident by the increases in circulating inflammatory markers and mRNA expression of proinflammatory cytokines in subcutaneous fat) and insulin resistance in healthy humans. How is LPS transported through the intestinal mucosa?
The gastrointestinal lumen harbours a high number of microbes, particularly in the distal small intestine and in the colon. The gastrointestinal mucosa acts normally as a dynamic barrier between the luminal contents (ingested meals undergoing digestion to absorbable nutrients and the microbiota) and the tissues of the organism. It follows that a crucial factor related to occurrence of bacterial components in the blood is that the barrier properties is altered allowing permeation, and ultimately translocation, of large molecules like endotoxin from lumen to the tissue and its circulation. The condition of a decreased barrier capability is commonly termed 'the leaky epithelium' but the mechanisms behind this condition are not known. The leaky epithelium is suspected to occur in predisposed individuals exposed to certain environmental influences (dietary pattern, type of macronutrients, endogenous digestive aggressors, stress and perhaps others). Furthermore, it cannot be excluded that certain luminal microbes are better disposed than others for eliciting obesity- related inflammation ('obesogenic microbiota'). The intestinal mucosal epithelium
The surface epithelium of the intestinal mucosa is a very dynamic tissue with high cellular turnover rate. The mucosa is organised in folds, the epithelium in villi (small intestine only) and crypts, and the enterocyte's luminal (apical) cell membrane has microvilli altogether enlarging the luminal surface to a total area of about 180 m2. The epithelium consists of a single layer with polarised columnar cells with a limited number of phenotypes (mostly enterocytes, but also specialised cells like Goblet cells, Paneth cells, enteroendocrine cells, antigen presenting M-cells). Stem cells are situated in the crypts and replicated cells move during continuous differentiation towards the tips of the villus where they eventually are shedded into the luminal contents and undergo digestion. This continuous differentiation means that a single cell changes shape during the crypt to villus transport. Consequently, the functional properties of the crypt epithelium can differ considerably compared to villus. The crypt to villus transport of a single enterocyte takes only 2 to 4 days, with the highest rate in the proximal small intestine, and most of the intestinal epithelium is therefore turned over within the same time.
Transepithelial passage of large molecules
While transepithelial transport of water and small solutes (e.g., NaCI, nutrient monomers and oligomers) is quite well characterised, the passage of larger molecules is not. Generally there are two routes available; transcellular and paracellular transport. The transcellular transport can occur as part of the antigen presentation in the enteric immune system: M-cells and dendritic cells absorb large molecules (including endotoxin) and even whole microbes by endocytosis and transport these to the sub-epithelial lymphoid tissues ('Peyer's patches') for 'immunological identification' as part of the process of immune defence or tolerance. Larger molecules (e.g., maternal immunoglobulins during the suckling period) can also be absorbed and delivered into the subepithelium by the non-specialised enterocytes.
Paracellular transport The endocytotic transcellular active transport of large molecules is commonly called 'transcytosis' and is an energy-demanding process. From a quantitative perspective transport between the epithelial cells (the paracellular route) is potentially more powerful because it can be driven by concentration gradients. However, normally the paracellular transport is restricted by intercellular junction protein complexes (i.e., tight junctions, adherence junctions and desmosomes) creating small pores (<8A) allowing charge selective permeation of small molecules (-ions) only. In addition to the small pores it has also been postulated that there exists a larger pore population allowing passage of molecules of a molecular weight in the order of <600Da.
The state of the permeability of the intestinal epithelium is not constant. The intercellular junctional complex is linked to the cytoskeleton of the enterocyte involving also intracellular contractile actomyosin elements. The paracellular permeability can be regulated by the type of junctional and cytoskeletal proteins being expressed, and also by influencing the conformational state of these proteins.
How does endotoxin penetrate the intestinal epithelium?
Bacterial endotoxin has a molecular weight in the order of 30kDa meaning it is impossible for it to pass between the epithelial cells, at least during the condition defined by the present state of knowledge. The transcellular antigenic presentation route is one alternative and can initiate proinflammatory signalling for example via activation of subepithelial toll like receptors (TLRs), but the quantity of endotoxin transported this way is too low to result in measurable endotoxinemia.
Studies link the absorption of endotoxin from the gastrointestinal tract into the circulation with the weight gain and insulin resistance induced by diet. A diet enriched in energy, either a high-fat or high-carbohydrate diet, induces an increase in plasma endotoxin levels in mice. Moreover, the high-fat diet resulted in a much greater increase in plasma endotoxin levels than either the control diet or the high-carbohydrate diet (2-3-fold increase). Similarly, in humans, there is a positive correlation between plasma endotoxin levels and energy or fat intake (correlations were observed for saturated, monounsaturated, and polyunsaturated fat intake). Circulating endotoxin levels are also increased in patients with type 2 diabetes. Finally, in humans a single high-fat meal acutely increased plasma endotoxin levels. Taken together, these results indicate that a high-fat diet produces an increase in circulating endotoxin levels. Animal studies have shown that chronic endotoxinemia induces obesity, insulin resistance, and diabetes. The ability of enteral fat to sustain the gut barrier and decrease the translocation of macromolecules raises questions regarding the mechanism accounting for how dietary fat leads to increased circulating endotoxin and what the consequences are. The paper of Ghoshal et al. in the Journal of Lipid Research addresses these important questions. Using both animal models and polarized, cultured gut cells, they show that endotoxin, which is internalized into gut cells, is secreted into the circulation during the formation and secretion of chylomicrons. Intragastric lavage with triolein (which forms chylomicrons) increases plasma endotoxin, whereas gavage with tributyrin (whose fatty acids enter the circulation without chylomicron formation) does not increase endotoxinemia. Polarized CaCo-2 cells secrete endocytosed endotoxin when incubated with oleate, which forms chylomicrons in those cells, but not when incubated with butyrate, which does not. Importantly, Pluronic L-81, an inhibitor of chylomicron formation, blocked the effect of oleate. Thus, endotoxin is transported into the circulation in conjunction with chylomicron formation and secretion, not just translocated due to breakdown of the intestinal barrier.
Ghoshal et al., also provide data that are important to our understanding of the inflammatory response. They show that mesenteric lymph nodes are activated by the endotoxin on chylomicrons. Mesenteric lymph nodes likely play an important role of scavenging the loosely attached endotoxin and decreasing the amount that reaches the systemic circulation. But scavenging means activating the cells in the lymph nodes to secrete cytokines, hence inducing systemic inflammation. What happens when the mesenteric lymph nodes are defective, for example in a disease such as in HIV infection where mesenteric lymph nodes are depleted early in the disease and do not return to normal at the time of repletion of circulating CD4 cells? The likely answer is more endotoxin makes it into the circulation even in healthy patients with HIV infection, with ensuing inflammation and metabolic disturbances. Is endotoxin transport linked to intestinal lipid absorption a pathological process?
The literature is so far not conclusive. On one hand, the binding of endotoxin to chylomicron (one of the five major groups of lipoproteins, along with VLDL, IDL, LDL and HDL) may be protective by reducing direct exposure of the liver to toxin (chylomicron-endotoxin complex would mainly enter the circulation via the lymphatic drainage and not via the portal blood). On the other hand, iterated activation of the innate immunity in the subepithelial lamina propria (a thin layer of loose connective tissue which lies beneath the epithelium and together with the epithelium constitutes the mucosa) and the regional lymph nodes resulting in an inflammatory cascade that, although controlled, may in the long run be deleterious due to induction of insulin resistance/T2DM and the initiation of atherosclerosis. In some individuals LPS/microbial interaction with the epithelium may for unknown reasons (genetic, environmental..?) cause pronounced (uncontrolled) inflammatory reactions in the intestinal mucosa resulting in the clinical picture of inflammatory bowel disease; IBD.
Linking the Gut. Visceral Obesity, and related metabolic disorders
Given the role of the gut in mesenteric fat inflammation and deposition and energy homeostasis, it is tempting to postulate that the gut is an important driving force for the pathophysiology of obesity and its related metabolic disorders, in particular fatty liver diseases - thanks to the portal system. Similar gut-liver interactions have been implicated as an important underlying mechanism by which alcoholic liver disease occurs. The process may start with, or is perpetuated by, gut inflammation. As a consequence of alterations of gut microbiota composition and/or other external factors, gut mucosal barrier function is impaired and results in a "leaky gut." The infiltration of microbial products into the lamina propria of the mucosa and subsequently to the mesenteric fat triggers an innate immune response and subsequently induces the production of proinflammatory cytokines. The local fat depot may expand as a protective mechanism to prevent the microbial antigens from further infiltrating the peritoneal cavity. Together this results in an increase flux of free fatty acids and proinflammatory factors, originating from both gut microbiota and the visceral fat depot, to the liver via the portal circulation. When the availability of free- fatty acids exceeds the capacity of both fat oxidation and triglyceride export as very- low-density lipoprotein, an excess of lipid accumulates in the liver. Subsequently, the increase in lipid derivatives (e.g., ceramide and diacylglycerol), together with proinflammatory factors from the portal circulation, activate inflammatory pathways in the liver. These events lead to common obesity-associated liver diseases including non-alcoholic fatty liver disease and hepatic insulin resistance. The diseased liver, of course, leads to further metabolic abnormalities. Hepatic insulin resistance impairs the suppression of glucose production in the liver. Together with increased lipogenesis, the liver contributes to the elevated circulating levels of glucose and fatty acids that induce insulin secretion, elicit peripheral insulin resistance and eventually lead to a vicious cycle of metabolic dysfunction. Inflammation has been implicated as an important contributor to the pathogenesis of obesity-related metabolic disorders. Understanding the genesis of that inflammation is critical. The rapidly growing body of literature summarized here invites consideration of a link between gut inflammation/permeability and mesenteric fat dysfunction and then on to liver inflammation and hepatic and systemic insulin resistance. To date the origin of gut inflammation is not entirely clear and it appears to be multifactorial, shown to be at least host genotype- and diet-dependent. Elucidating the mechanisms underlying gut inflammation will be critical to develop gut specific approaches for the treatment of obesity-related metabolic disorders. There is a dramatically growing literature in gut health which is beyond the scope of this review. However, the reader is directed to a number of recent articles which focus on modifying the gut microbial community using inter alia, resistant starches, prebiotics and/ or prebiotics, which seem to provide promising approaches in improving gut function and metabolic variables.
To summarise; inflammation has been implicated as an important contributor to the pathogenesis of obesity-related metabolic disorders. The rapidly growing body of literature links high-fat diet, gut inflammation/permeability and mesenteric fat dysfunction to liver inflammation and hepatic and systemic insulin resistance. To date the origin of gut inflammation is not entirely clear and it appears to be multifactorial, shown to be at least host genotype- and diet-dependent. Elucidating the mechanisms underlying gut inflammation will be critical to develop gut-specific approaches for the treatment of obesity-related metabolic disorders. There is a dramatically growing literature in gut health. The majority of recent articles on the topic focus on modifying the gut microbial community using inter alia, resistant starches, prebiotics and/or prebiotics, which seem to provide promising approaches in improving gut function and metabolic variables.
SUMMARY OF THE INVENTION
The present inventors have surprisingly found that the barrier properties of the Gl mucosa are regulated by local (gastrointestinal) ketogenesis and, consequently, that barrier impairment of the Gl tract can be regulated by modifying Gl ketogenesis, Gl ketones and/or effects induced by Gl ketones.
The endogenous ketone bodies are acetone, acetoacetic acid, and beta- hydroxybutyric acid. Other ketone bodies such as beta-ketopentanoate and beta- hydroxypentanoate may be created as a result of the metabolism of synthetic triglycerides such as triheptanoin.
Hence, by "ketone body" we include any ketone body found in humans, preferably, acetone, acetoacetic acid, and beta-hydroxybutyric acid.
Gastric bypass (GBP) surgery is a powerful method for achieving sustained weight reduction in obese subjects. This gastrointestinal 'reconstruction' improves and usually resolves obesity-associated metabolic aberrations (i.e. the metabolic syndrome: obesity, insulin resistance, hypertension, dyslipidemia) partly in a weight-independent fashion.
In order to obtain new information for the development of non-surgical alternatives, the present inventors explored the mechanisms behind the metabolic effects of GBP. A global proteomic analysis of the human small intestinal mucosa before surgery and after 6 to 8 months was performed. Using very conservative criteria with the individual as own control they have discovered that a limited number of mucosal proteins are consistently regulated by the GBP-procedure. Most were related to the cytoskeleton and the intercellular junctional complexes of the mucosal epithelium. In addition, a surprisingly high expression of the ketogenic enzyme HMGCS2 was present in the preoperative (obese) situation and was markedly down-regulated after GBP.
It was hypothesised that obesity and the metabolic syndrome is caused by an increased intestinal mucosal ketogenesis that, when activated, induces transepithelial paracellular passage of endotoxin (and the in turn, induction of associated pathophysiological inflammation).
Indeed, after a number of different experiments (human, mice, Caco-2 cell lines) we can confirm that ketone bodies are produced in intestinal mucosae upon a brief exposure to fat (i.e. FFA). This response demands that HMGCS2 is present and such expression is induced by high fat diet over some time (in mice <1week). Furthermore, activation of intestinal epithelial ketogenesis is associated with increased transepithelial permeability. Furthermore, pharmacological interference with HMGCS2 by use of the specific inhibitor hymeglusin reduced such fat-induced increase in epithelial permeability. When taken together, the findings indicate a fundamental property of the intestinal mucosa of an obese individual: the mucosa responds with HMGCS2-mediated ketogenesis when it is exposed to an energy-rich fatty diet. This response pattern and the underlying cellular structure may be part of an adaptation to fat as the principal source for dietary energy (thus resembling the situation of the neonate). The fat-adapted mucosa with ketogenic enzyme expression has a high capacity for formation of ketone bodies after food intake (thus the opposite to what is generally described in the texts: ketogenesis occurs following fasting/starvation). The meal induced mucosal ketone bodies in turn increase the permeability to large molecular passage by influencing the cytoskeleton and the intercellular junctional proteins. It seems plausible that the fat-induced structural changes of the cytoskeletal and intercellular junctional proteins contribute to this response pattern, but the fundamental mechanism is the expression of ketogenic enzyme HMGCS2 and the signalling properties of ketone bodies themselves.
The two 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthases
The enzyme HMG-CoA synthase (EC 4.1.3.5) catalyses the condensation of acetoacetyl-CoA and acetyl-CoA to formHMGCoA plus free CoA. HMG-CoA synthase activity is located in two different compartments: the cytosol and the mitochondria. The HMG-CoA produced by the cytosolic HMG-CoA synthase is transformed into mevalonate by the action of HMG-CoA reductase. This starts the isoprenoid pathway which, in addition to cholesterol as the main end-product, produces several important products, such as ubiquinone, dolichol, isopentenyl adenosine and farnesyl groups, which covalently modify proteins. The HMG-CoA produced inside the mitochondria by the mitochondrial HMG-CoA synthase is transformed into acetoacetate by the action of HMG-CoA lyase. Acetoacetate is transformed into hydroxybutyrate and acetone; all of these are known as ketone bodies.
Structural and functional comparisons of the promoter regions of the two synthases [5,7] has indeed shown that the two promoters are very different. Cytosolic HMG-CoA synthase contains sterol regulatory elements that modulate transcriptional activity by sterols, mediated by sterol regulatory element binding proteins (SREBP)-1 and -2 [9, 10], which have not been observed in the promoter of the mitochondrial HMG-CoA synthase. Conversely, the peroxisome proliferator regulatory element (PPRE) is present in the mitochondrial HMG-CoA synthase promoter, but has not been detected in the promoter of the cytosoiic synthase [11]. These lines of evidence emphasize that the promoter of each gene is responsible for the control of one of the two different pathways: the cytosoiic HMG-CoA synthase is a control site of the isoprenoid biosynthetic pathway, and the mitochondrial HMG-CoA synthase is an important control site of the ketogenic pathway.
Accordingly, a first aspect of the invention provides a ketone body inhibitor for use in the treatment or prevention of: gastrointestinal (Gl tract) mucosal barrier impairment; and/or a disease or condition associated with gastrointestinal tract mucosal barrier impairment. By "gastrointestinal tract' or "Gl tract" we include the stomach, large intestine and small intestine.
From the mid-oesophagus to the anus, the wall of the Gl tract has a general structure. Most of the tube's luminal surface is highly convoluted, a feature that greatly increases the surface area available for absorption. From the stomach on, this surface is covered by a single layer of epithelial cells linked together along the edges of the luminal surfaces by tight junctions.
Included in this epithelial layer are exocrine cells that secrete mucus into the lumen of the tract and endocrine cells that release hormones into the blood. Invaginations of the epithelium into the underlying tissue form exocrine glands that secrete acid, enzymes, water and ions, as well as mucus.
Just below the epithelium is a layer of connective tissue, the lamina propria, through which pass small blood vessels, nerve fibres and lymphatic ducts. The lamina propria is separated from underlying tissues by a thin layer of smooth muscle, the muscularis mucosa. The combination of these three layers - the epithelium, lamina propria and muscularis mucosa - is called the mucosa. In addition to contributing to the digestion and absorption of nutrients and water, the mucosa has barrier properties preventing luminal potentially noxious agents (e g. toxins and antigens including microbiota) from penetrating into the tissues of the body. By "mucosal barrier impairment" we include that the permeability of the Gl tract mucosa is increased compared to healthy, non-obese individuals having a normal diet. In particular, we include that the permeability of the Gl tract mucosa to potentially noxious agents (e.g., toxins and antigens, including microbiota) increased compared to healthy, non-obese individuals having a normal diet. Preferably, we include that the permeability of the Gl tract mucosa to endotoxin is increased compared to healthy, non-obese individuals having a normal diet.
Permeability of the Gl tract can be measured by suitable means known in the art, for example:
Epithelial permeability to defined molecular probes as described in Gummesson A, Carlsson LM, Storlien LH, Backhed F, Lundin P, Lofgren L, Stenlof K, Lam YY, Fagerberg B, Carlsson B. Intestinal permeability is associated with visceral adiposity in healthy women. Obesity (Silver Spring).
2011 Nov; 19(11):2280-2.
Epithelial permeability and epithelial electrical characteristics. These measurements are usually performed in-vitro utilising Ussing chambers, as described in Fromm M, Krug SM, Zeissig S, Richter JF, Rosenthal R,
Schulzke JD, Gunzel D. High-resolution analysis of barrier function. Ann N Y Acad Sci. 2009 May; 1165:74-81 or Butt, Jones & Abbott, Journal of Physiology (1990), Electrical resistance across the blood-brain barrier in anaesthetized rats: A developmental study , 429, pp. 47-62. This method is often validated by assessing the permeability of specific macromolecules of interest.
Endotoxinemia as a sign of barrier dysfunction, as described in Creely SJ, McTernan PG, Kusminski CM, Fisher M, Da Silva NF, Khanolkar M, Evans M, Harte AL, Kumar S. Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes.Am J Physiol Endocrinol Metab. 2007 Mar;292(3):E740-7. Translocation of endotoxin from an luminal intestinal compartment to the tissue-side can also be studied in-vitro using cultured cell layer sheets that can act as good models, as described in Laugerette F, Vors C, Geloen A, Chauvin MA,
Soulage C, Lambert-Porcheron S, Peretti N, Alligier M, Burcelin R, Laville M, Vidal H, Michalski MC. Emulsified lipids increase endotoxemia: possible role in early postprandial low-grade inflammation. J Nutr Biochem. 2011 Jan;22(1):53-9.
By "normal diet" we include diets that are of recommended calories and fat levels to sustain normal weight and activity.
Hence, the first aspect of the invention may be for use in the treatment or prevention of gastrointestinal tract mucosal barrier impairment. Alternatively/additionally, it may be for use in the treatment or prevention of a disease or condition associated with gastrointestinal tract mucosal barrier impairment.
The disease or condition associated with gastrointestinal tract mucosal barrier impairment may be selected from the group consisting of:
(i) metabolic syndrome (also known as metabolic syndrome X, cardiometabolic syndrome, syndrome X, insulin resistance syndrome, eaven's syndrome and CHAOS),
(ii) obesity,
(iii) insulin resistance,
(iv) type I diabetes mellitus,
(v) type II diabetes mellitus,
(vi) hypertension,
(vii) dyslipidemia,
(viii) inflammatory bowel disease,
(ix) irritable bowel syndrome (also known as irritable bowel disease),
(x) Crohn's disease,
(xi) ulcerative colitis,
(xii) collagenous colitis,
(xiii) lymphocytic colitis,
(xiv) ischaemic colitis,
(xv) diversion colitis,
(xvi) Behcet's disease,
(xvii) indeterminate colitis,
(xviii) coeliac disease,
(xix) systemic T cell activation,
(xx) inflammation of the gastrointestinal tract,
(xxi) graft-versus-host disease, (xxii) stroke,
(xxiii) myocardial infarction,
(xxiv) cancer,
(xxv) non-alcoholic steato-hepatitis (NASH),
(xxvi) liver cirrhosis,
(xxvii) allergy (especially food-allergies e.g., milk-protein allergy),
(xxviii) asthma,
(xxix) atherosclerosis, and
(xxx) pancreatitis.
Accordingly, the disease or condition associated with gastrointestinal tract mucosal barrier impairment may be pancreatitis. Pancreatitis is associated with increased intestinal permeability, which may be a driving mechanism for severe pancreatitis and associated sepsis (Trop Gastroenterol. 2012 Jan-Mar;33(1):45-50. Alterations in intestinal permeability and endotoxemia in severe acute pancreatitis. Sharma M, Sachdev V, Singh N, Bhardwaj P, Pal A, Kapur S, Saraya A.)
Accordingly, the disease or condition associated with gastrointestinal tract mucosal barrier impairment may be metabolic syndrome. As mentioned above, metabolic syndrome is a constellation of interrelated risk factors of metabolic origin (metabolic risk factors). The most widely recognized of the metabolic risk factors are atherogenic dyslipidemia, elevated blood pressure, and elevated plasma glucose. Individuals with these characteristics commonly manifest a prothrombotic state and a proinflammatory state as well. Atherogenic dyslipidemia consists of an aggregation of lipoprotein abnormalities including elevated serum triglyceride and apolipoprotein B (apoB), increased small LDL particles, and a reduced level of HDL cholesterol (HDL-C). There are various criteria for diagnosing metabolic syndrome, discussed in Grundy et al., 2005, "Diagnosis and Management of the Metabolic Syndrome: An American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement", 2005, Circulation. 112:2735-2752.
Preferably, the following criteria are used for the diagnosis of metabolic syndrome:
• Elevated waist circumference:≥102 cm (>40 inches) in men≥88 cm (≥35 inches) in women;
• Elevated triglycerides≥150 mg/dL (1.7 mmol/L) or on drug treatment for
elevated triglycerides; • Reduced HDL-C <40 mg/dL (1.03 mmol/L) in men <50 mg/dL (1.3 mmol/L) in women or on drug treatment for reduced HDL-C;
• Elevated blood pressure≥130 mm Hg systolic blood pressure or≥85 mm Hg diastolic blood pressure or on antihypertensive drug treatment in a patient with a history of hypertension;
• Elevated fasting glucose≥100 mg/dL or on drug treatment for elevated.
The disease or condition associated with gastrointestinal tract mucosal barrier impairment may be associated with infection, for example, an infection selected from the group consisting of bacterial infection, viral infection, protozoal infection, fungal infection and helminth infection.
Thus, the infection may be associated with bacterial infection. The bacterial infection may comprise or consist of a Gram positive bacterial infection, a Gram negative bacterial infection, or a mixed infection of Gram positive and Gram negative bacteria.
Hence, the bacterial infection may be an infection with one or more of the group of bacteria consisting of Achromobacter spp., Acidaminococcus fermentans, Acinetobacter cacoaceticus, Aeromonas spp., Alcaligenes faecalis, Arizona spp., Bacillus spp., Bacteroides fragilis, Bifidobacterium spp., Butyriviberio fibrosolvens., Campylobacter spp (such as C. coli), Citrobacter spp., Clostridium spp (such as C. difficile and C. sordeliii), Edwardsiella spp., Eikenella corrodens., Enterobacter spp. (such as E. cloacae, E. faecalis and E. faecium), Escherichia spp. (such as E. coli), Flavobacterium spp., Hafnia spp., Klebsiella spp., Mycobacteria spp., Mycoplasma spp., Peptococcus spp., Plesiomonas shigelloides, Propionibacterium spp (such as P. acnes), Proteus spp., Providencia spp., Pseudomonas spp (such as P. aeruginosa), Ruminococcus spp (such as R. bromii), Salmonella spp., Sarcina spp., Serratia spp., Shigella spp., Staphylococcus aureus, Streptococcus anginosus, Streptococcus viridans, Vibrio spp., Yersinia spp (such as S. enterocolitica), Yersinia enterocolitica, Veillonella spp., Chryseomonas spp., Pseudomonas luteola, Lactobacillus spp., Enterococcus spp., Bacteroides spp., Streptococcus spp., Corynebacterium spp., Nocordia spp., Rhodococcus spp., Fusobacterium spp., Helicobacter spp., (such as Helicobacter pylori).
Alternatively or additionally, the infection may be a viral infection (such as enteroviruses). Virus infection (translocation) has been proposed to be involved in the activating an immuneresponse contributing to type 1 diabetes (autoimmune beta cell destruction). For example, see de Kort S, Kesztheiyi D, Masclee AA. Leaky gut and diabetes mellitus: what is the link? Obes Rev. 2011 Jun;12(6):449-58 andRichardson SJ, Willcox A, Bone AJ, Foulis AK, Morgan NG. The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes. Diabetologia 2009; 52: 1 143-1151 which are incorporated by reference herein.
In an alternative or additional embodiment, the ketone body inhibitor of the first aspect of the invention the gastrointestinal mucosa barrier impairment comprises or consists of barrier impairment of one or more of the group consisting of: the epithelium, lamina propria and muscularis muscosae.
Hence, the gastrointestinal mucosa barrier impairment may comprise or consist of barrier impairment of the epithelium.
The gastrointestinal mucosa barrier impairment may be associated with increased paracellular or transcellular permeability of the gastrointestinal mucosa. Preferably, the gastrointestinal mucosa barrier impairment is associated with increased paracellular permeability of the gastrointestinal mucosa.
The integrity of the epithelial cell layer(s) that protects multicellular organisms from the external environment is maintained by intercellular junctional complexes composed of tight junctions (TJ), adherens junctions, and desmosomes, whereas gap junctions provide for intercellular communication. The transmembrane proteins constituting these junctions are linked to components of the cytoskeleton, thereby establishing connections to other cell-cell and cell-substratum adhesion sites.
As the apical member of the junctional complex, the TJ forms a continuous, circumferential, belt-like structure at the luminal end of the intercellular space, where it serves as a gatekeeper of the paracellular pathway. On the cytoplasmic side of the TJ, the TJ plaque is the site of a growing number of TJ-associated protein complexes. Within the confines of the TJ, the cell membranes of adjacent epithelial cells are brought into intimate focal contact sites in which the intercellular space is obliterated. The ability of epithelia to create a diffusion barrier between cellular compartments of very different fluid and solute composition is controlled by essentially two pathways: 1) the transcellular pathway, which is governed by energy-dependent transporters and channels that are asymmetrically distributed on the apical and basolateral cell membranes; and 2) the paracellular pathway, in which integral TJ proteins span the apical intercellular space and regulate the passive diffusion of ions and small noncharged solutes via the paracellular space.
Although the mechanisms involved in transcellular transport are well studied, it is only relatively recently that those controlling the paracellular route have begun to be elucidated. In addition to serving as a regulated gate/barrier in the paracellular pathway, the TJ also functions as a fence in the plane of the plasma membrane, where it contributes to the maintenance of asymmetrically distributed integral membrane proteins and lipids.
Thus, the gastrointestinal mucosa barrier impairment may be associated with epithelial intracellular junction relaxation, which may be associated with tight junction 'relaxation'.
In one embodiment of the first aspect of the invention gastrointestinal tract mucosal barrier impairment is present in one or more sites selected from the group consisting of the stomach, duodenum, jejunum, ileum, cecum and colon. Hence, gastrointestinal tract mucosa barrier impairment may be absent from one or more sites selected from the group consisting of the stomach, duodenum, jejunum ileum, cecum and colon. For example, gastrointestinal mucosa tract barrier impairment may be absent from the colon. In one embodiment the Gl tract mucosa barrier impairment is not associated with ketogenesis. However, in an alternative embodiment, the Gl tract mucosa barrier impairment is associated with ketogenesis. By "associated with ketogenesis" we include that the Gl tract mucosa barrier impairment is caused or augmented by ketogenesis or occurs concurrently with it (e.g., due to the resultant increase in levels of ketone bodies).
In one embodiment, the gastrointestinal tract mucosa barrier impairment is associated with ketogenesis in one or more sites selected from the group consisting of the stomach, duodenum, jejunum, ileum, cecum and colon. Hence, the gastrointestinal tract mucosa barrier impairment may not be associated with ketogenesis in one or more sites selected from the group consisting of the stomach, duodenum, jejunum, ileum, cecum and colon; for example, the barrier impairment may not be associated with (i.e., occur independently of) ketogenesis of the colon.
The ketogenesis may be acute but is preferably chronic or recurrent. By "chronic" we include that the Gl tract ketogenesis has been present for at least one month. By "recurrent", we include that the Gl tract ketogenesis occurs intermittently, but that each period has a duration of less than a month. The ketogenesis is may be meal- induced, preferably under a high fat diet. By "high fat diet" we include a diet having greater than or equal to a 30% total energy content derived from fat (equal to approximately 18.5% w/w). Hence, the high fat diet may have greater than or equal to 35% total energy content derived from fat, e.g., greater than or equal to 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% total energy content derived from fat. Preferably, the high fat diet has a carbohydrate content of less than or equal to 40% w/w, e.g., 35%, 30% w/w, 25% w/w, 20% w/w, 15% w/w, 10 or 5% w/w carbohydrate content. For example, in humans the diet has less than or equal to 20 g/day of carbohydrate, e.g., less than or equal to 19g/day, 18g/day, 17g/day, 16g/day, 15g/day, 14g/day, 13g/day, 12g/day, 1 1g/day, 10g/day, 9g/day, 8g/day, 7g/day, 6g/day or 5g/day of carbohydrate. Most preferably, the high-fat diet is a ketogenic diet (e.g., see Freeman J, State-of- the-Art review: The ketogenic diet: one decade later, Pediatrics 2007, which is incorporated by reference herein).
In one embodiment, the gastrointestinal tract mucosal barrier impairment is present in one or more site selected from the group consisting of the stomach, duodenum, ileum, cecum and colon. Hence, the gastrointestinal tract mucosal barrier impairment may be absent from one or more site selected from the group consisting of the stomach, duodenum, ileum, cecum and colon. Preferably, the ketone body inhibitor for use is capable of reducing gastrointestinal tract mucosa barrier impairment by at least 5%, for example, at least, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100%. The disease or condition associated with gastrointestinal tract mucosa barrier impairment may be selected from the group consisting of gastritis, enteritis, colitis, gastroenteritis and enterocolitis. In one embodiment, gastrointestinal tract barrier impairment or the disease or condition associated with gastrointestinal tract mucosa barrier impairment is associated with inflammation. The inflammation may be chronic or acute, but is preferably chronic. The inflammation may comprise or consist of inflammation of the gastrointestinal mucosa (i.e., local inflammation). In one embodiment Inflammation of the gastrointestinal mucosa is present in one or more sites selected from the group consisting of the stomach, duodenum, ileum, cecum and colon. Hence, inflammation may be absent from one or more sites selected from the group consisting of the stomach, duodenum, ileum, cecum and colon. For example, inflammation of the gastrointestinal mucosa is absent from the colon.
Alternatively or additionally, the inflammation may comprise or consist of inflammation non-gastrointestinal mucosa tissue (i.e., remote focal inflammation). Preferably, the inflammation comprises or consists of systemic inflammation (i.e., a generalised inflammatory state).
The ketone body inhibitor may be capable of reducing inflammation as assessed by, for example C-reactive protein (CRP) or other acute phase proteins, preferably by at least 5%, for example, at least, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100%.
By "ketone body inhibitor" we include agents capable of (i) reducing or preventing ketogenesis; (ii) reducing or abolishing ketones themselves; and/or (iii) reducing or preventing ketone-induced effects.
Hence, the "ketone body inhibitor" may be a ketogenesis inhibitor.
The "ketone body inhibitor" may be capable of reducing or abolishing ketones (i.e., ketolytic agents or ketone sequestering agents). The "ketone body inhibitor" may be an inhibitor of ketone-induced effects. For example, the G protein-coupled receptor 81 (also known as GPR81 , HCAR1 ; GPR104; GPR81 ; HCA1 ; LACR1 and TA-GPCR), which is expressed in the intestinal epithelium, mediates the effects induced/precipitated by ketone bodies (3- hydroxyoctanoate) such as anti-lipolytic effects, as does Niacin receptor 2 (also known as NIACR2, HCAR3; GPR109B; HCA3; HM74; PUMAG and Puma-g), which is expressed in the colonic epithelium (see Blad et al., 2012, Nature reviews: Drug Discovery, 11 :603-619). Hence, without wishing to be bound by theory, it is envisaged that the ketone body inhibitors of the present invention may include inhibitors of ketone-regulated proteins such as HCA2 and HCA3.
Preferably, the ketone body inhibitor is capable of selective inhibition of the gastrointestinal tract. By 'selective' we mean that the ketone body inhibitor inhibits said biological activity to a greater extent than it modulates the activity of other tissues/sites.
The ketone body inhibitor may be capable of inhibiting one or more layer of the gastrointestinal tract selected from the group consisting of the mucosa, submucosa, muscularis externa and serosa.
The ketone body inhibitor may be capable of selectively inhibition in one or more sites selected from the group consisting of the stomach, duodenum, jejunum, ileum, cecum and colon. Preferably, the ketone body inhibitor is capable of selective gastric inhibition. Preferably, the ketone body inhibitor is capable of selective jejunal inhibition. Preferably, the ketone body inhibitor is capable of selective ileal inhibition. Preferably, ketone body inhibitor is capable of selective cecal inhibition. Preferably, the ketone body inhibitor is capable of selective duodenal inhibition. The ketone body inhibitor may be capable of selective inhibition of: the duodenum, jejunum and ileum; or the cecum and colon; or the colon.
The ketone body inhibitor may be capable of inhibiting ketone bodies of the gastrointestinal mucosa by at least 5%, for example, at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 600%, 700%, 800%, 900% or at least 1000%. Quantification of ketone bodies in biological fluids have for long been used in clinical settings, particularly related to diabetes care with the aim to avoiding ketoacidosis. For example, dipsticks assessing occurrence of urinary acetoacetate using the Legal reaction have been frequenly used. However, this technique is now principally abandonded due to concerns about reliability. Instead plasma/serum ketone analyses based on enzymatic assays and spectrophotometry are today preferred assessments in clinical chemistry. These technologies are very accurate but demand specialised laboratories and trained laborants resulting in long delay from sampling to analysis. To improve time resolution and with still acceptable accuracy ambulatory tests kits have been developed. The enzymatic assays have been incorpotated into test cards that after exposure to a biological fluid of interest can be immediately analysed with regard to ketone bodies by use of portable analysers (Muso-Veloso 2004). A normal level of ketone bodies in the circulation is defined as <0,5 mM whereas hyperketonemia is defined as above 1 ,0 mM. This level can be physiological during fasting and may reach as high as around 6 mM. Hyperketonemia can also be associated to pathological conditions such as diabetic ketoacidosis, severe liver disease etc (Laffel 1999).
Preferably, the ketone body inhibitor is capable of inhibiting ketone bodies of the gastrointestinal mucosa by at least 10% more than in non-gastrointestinal mucosa tissues, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 600%, 700%, 800%, 900% or at least 1000% more than in non-gastrointestinal mucosa tissues.
Preferably, the ketone body inhibitor is capable of inhibiting ketone bodies in non-gastrointestinal mucosa tissues by no more than 25%, for example, no more than 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 % or no more than 0%.
Preferably, the ketone body inhibitor is incapable (in at least in some circumstances e.g., when used correctly) of inhibiting ketogenesis in non-gastrointestinal mucosa tissues. Preferably, the ketone body inhibitor comprises or consists of a pharmaceutical agent that is inherently capable of selectively inhibiting ketogenesis of the gastrointestinal mucosa. Alternatively, the ketone body inhibitor may comprise or consist of a pharmaceutical agent that is not inherently capable of selectively inhibiting ketogenesis of a specific tissue type or location (such as the gastrointestinal mucosa). Such ketone body inhibitors are preferably formulated for selective inhibition of ketogenesis of the gastrointestinal mucosa.
The ketone body inhibitor may be formulated to reduce or prevent its exposure to non-gastrointestinal mucosa tissue. For example, exposure of the ketone body inhibitor to non-gastrointestinal mucosa tissue may reduced by at least 10%, for example, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100%. The ketone body inhibitor may be an inhibitor of a ketogenic enzyme. The ketogenic enzyme may be selected from the group consisting of: acetoacetyl-CoA thiolase (also known as acetyl-CoA C-acetyltransferase, acetyl-CoA.acetyl-CoA C-acetyltransferase, beta-acetoacetyl coenzyme A thiolase, 2-methylacetoacetyl-CoA thiolase, 3-oxothiolase, acetyl coenzyme A thiolase, acetyl-CoA acetyltransferase, acetyl-CoA.N-acetyltransferase, ACAT1 , and thiolase II;
(ii) HMG-CoA synthase (also known as 3-hydroxy-3-methylglutaryl- Coenzyme A synthase 2 (mitochondrial), H GCS2, );
(iii) HMG-CoA lyase (also known as Hydroxymethylglutaryl-CoA lyase, 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase
(hydroxymethylglutaricaciduria), and HMGCL); and jS-hydroxybutyrate dehydrogenase (also known as (R)-3- hydroxybutanoate:NAD+ oxidoreductase, NAD+-beta- hydroxybutyrate dehydrogenase, hydroxybutyrate oxidoreductase, beta-hydroxybutyrate dehydrogenase, D-beta-hydroxybutyrate dehydrogenase, D-3-hydroxybutyrate dehydrogenase, D-(-)-3- hydroxybutyrate dehydrogenase, beta-hydroxybutyric acid dehydrogenase, 3-D-hydroxybutyrate dehydrogenase, and beta- hydroxybutyric dehydrogenase). Preferably, the ketone body inhibitor is a HMG-CoA synthase inhibitor. The H G- CoA synthase may be selected from the group of proteins defined by database accession numbers: CAG33131 , NM_005518, NM_001 166107 and BC044217. For example, the HMG-CoA synthase may be selected from the group of proteins defined by database accession numbers: CAG33131.1 , NM_005518.3, NM_001 166107.1 and BC044217.1.
In one embodiment, the ketone body inhibitor is a direct inhibitor. The ketone body inhibitor may be transcriptional inhibitor, a post-transcriptional inhibitor, a translational inhibitor, a post-translational inhibitor or a functional inhibitor (i.e., an inhibitor of protein activity).
The ketone body inhibitor is capable of binding to one or more of the following amino acid sequences:
(i) HMGCS2 ; GenBank Accession No.: CAG33131 (version CAG33131.1)
MQ LLTPVKRILQLTRAVQETSLTPARLLPVAHQRFSTASAVPLA TDT WPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLGQTRMGFCSV QEDINSLCLTWQRLMERIQLPWDSVGRLEVGTETIIDKSKAVKTVLME LFQDSGNTDIEGIDTTNACYGGTASLFNAANWMESSSWDGR AMWCGD IAVYPSGNARPTGGAGAVAMLIGPKAPLALERGLRGTYMENVYDFYKPN LASEYPIVDGKLSIQCYLRALDRCYTSYRKKIQNQWKQAGSDRPFTLDD LQYMIFHTPFCK VQKSLARLMFNDFLSASSDTQTSLYKGLEAFGGLKL EDTYTNKDLDKALLKASQDMFD KTKASLYLSTHNGNMYTSTLYGCLAS LLSHHSAQELAGSRIGAFSYGSGLAASFFSFRVSQDAAPGSPLDKLVSS TSDLP RLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTNSLFPGTW YLERVDEQHRRKYARRPV
[SEQ ID NO: 1]
(ii) HMGCS2 (complete cds) ; GenBank Accession No. :
BC044217.1 (version BC044217.1) QRLLTPVKRILQLTRAVQETSL PARLLPVAHQRFSTASAVPLAKTDT PKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLGQTR GFCSV QEDINSLCLTWQRLMERIQLPWDSVGRLEVGTETIIDKSKAVKTVLME LFQDSGNTDIEGIDTTNACYGGTASLFNAANWMESSSWDGRYAMWCGD IAVYPSGNARPTGGAGAVAMLIGPKAPLALERGLRGTHMENVYDFY PN LASEYPIVDG LSIQCYLRALDRCYTSYRKKIQNQWKQAGSDRPFTLDD LQYMIFHTPFCKMVQKSLARLMFNDFLSASSDTQTSLYKGLEAFGGLKL EDTYTNKDLDKALLKASQDMFDKKTKASLYLSTHNG YTSSLYGCLAS LLSHHSAQELAGSRIGAFSYGSGLAASFFSFRVSQDAAPGSPLDKLVSS TSDLPKRLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTNSLFPGTW YLERVDEQHRRKYARRPV
[SEQ ID NO: 2]
(iii) HMGCS2 (transcript variant 1) ; GenBank Accession No.: NM_005518 (version NM_005518.3 ) QRLLTPVKRILQLTRAVQETSLTPARLLPVAHQRFSTASAVPLAKTDT WPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLGQTR GFCSV QEDINSLCLTWQRLMERIQLPWDSVGRLEVGTETIIDKS AVKTVLME LFQDSGNTDIEGIDTTNACYGGTASLFNAANWMESSSWDGRYAMWCGD IAVYPSGNARPTGGAGAVAMLIGPKAPLALERGLRGTHMENVYDFYKPN LASEYPIVDGKLSIQCYLRALDRCYTSYRKKIQNQWKQAGSDRPFTLDD LQYMIFHTPFCKMVQ SLARLMFNDFLSASSDTQTSLYKGLEAFGGLKL EDTYTNKDLD ALLKASQDMFDKKTKASLYLSTHNGNMYTSSLYGCLAS LLSHHSAQELAGSRIGAFSYGSGIJAASFFSFRVSQDAAPGSPLDKLVSS TSDLPKRLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTNSLFPGTW YLERVDEQHRRKYARRPV
[SEQ ID NO: 3]
HMGCS2 (transcript variant 2) ; GenBank Accession No.: NM 001166107 (version NM 001166107.1)
MQRLLTPVKRILQLTRAVQETSLTPARLLPVAHQRFSTASAVPLAKTDT WPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLGQTRMGFCSV QEDINSLCLTWQRLMERIQLPWDS GRLEVGTE IIDKSKAVKTVL E LFQDSGNTDIEGIDTTNACYGGTASLFNAANWMESSSWDGLRGTHMENV YDFYKPNLASEYPIVDG LSIQCYLRALDRCYTSYRKKIQNQWKQAGSD RPFTLDDLQYMIFHTPFCKMVQKSLARLMFNDFLSASSDTQTSLYKGLE AFGGLKLEDTYTNKDLD ALLKASQDMFDKKTKASLYLSTHNGNMYTSS LYGCLASLLSHHSAQELAGSRIGAFSYGSGLAASFFSFRVSQDAAPGSP LDKLVSSTSDLPKRLASRKCVSPEEFTEIMNQREQFYHKV FSPPGD N SLFPGT YLERVDEQHRRKYARRPV
The ketone body inhibitor may be capable of binding to one or more of the following nucleic acid sequences:
(i) HMGCS2 (complete cds) ; GenBank Accession No. :
BC044217.1 (version BC044217.1) .
(ii) HMGCS2 (complete gene, Ch 1) ; Database Accession No. :
NG_013348 (version NG_013348.1)
(ill) HMGCS2 (transcript variant 1) ; GenBank Accession No. :
NM_005518 (version NM_005518.3)
(iv) HMGCS2 (transcript variant 2) ; GenBank Accession No. :
NM_001166107 (version NM_001166107.1)
The ketone body inhibitor may be capable of binding to one or more of the following positions of the HMGCS2 gene sequence:
(i) TATAAA (TATA sequence at position -28);
(ii) GGCGGG (Sp1 sequence at -54);
(iii) AGACCTTTGGCCC (NRRE sequence at -130);
(iv) TGATGTTTTC (IRE sequence at position -130);
(v) TGGCA (CTF-NF1 sequence at positions -520 and 836);
(vi) GTGCGTCA (CRE sequence at position -546);
(vii) AGTCAAAAG (C-EBP sequence at position -778);
(viii) G CTACAG GTTGTG CT (GRE sequence at position -995);
(ix) TGGCA (NFI-like sequence at positions -520, -836, -885, -1140);
(x) TGTGTCA (c-Jun sequence at position -249);
(xi) TGCGTCA (c-Jun sequence at position -554); and
(xii) TGACTCC (c-Jun sequence at position -781 ).
In an alternative embodiment, the ketone body inhibitor may be an indirect inhibitor, for example an indirect HMG-CoA synthase inhibitor. The indirect HMG-CoA synthase inhibitor may be an inhibitor (i.e., antagonist or inverse agonist) of a HMG-CoA synthase activator selected from the group consisting of: CREB (CRE binding protein; cAMP response element-binding protein [Homo sapiens]), PPARa (peroxisome-proliferator-activated receptor, alpha [Homo sapiens]), RXR (c/s-retinoid receptor), Sp1 (Specificity Protein 1) and COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1/also known as NR2F1 (Nuclear Receptor subfamily 2, group F, member 1)). Alternatively, the indirect HMG-CoA synthase inhibitor may be an enhancer (i.e., agonist) of an HMG-CoA synthase repressor selected from the group consisting of: Arp-1 (apo A1 regulatory protein-1), COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1/also known as NR2F1 (Nuclear Receptor subfamily 2, group F, member 1)), HNF-4 (hepatocyte nuclear factor-4), FXR (Farnesoid X receptor), c-myc (myc proto-oncogene protein) and Miz-1.
Exemplary sequences of the above indirect HMG-CoA synthase inhibitors follows:
AAA52072 (AAA52072.1) - CREB
AAB32649 (AAB32649.1) - PPAR a
AAA86429 (AAA86429.1) - arp-1
NP_005645 (NP_005645.1) - COUP transcription factor 1
CAA54248 (CAA54248.1) - hepatocyte nuclear factor-4
BAH02290 (BAH02290.1) - FXR
NP_002458 (NP_002458.2) - c-myc
CAA70889 (CAA70889.1) - Miz-1
There are 3 RXRs, RXR alpha, beta, gamma. These heterodimerize with RAR (retinoic acid receptors) alpha, beta and gamma.
NP_002948 (NP_002948.1) - RXR alpha
P28702 (P28702.1) - RXR beta
AAA80681 (AAA80681.1 ) - RXR gamma
NP_001 138773 (NP_001138773.1) - RAR alpha isoform 1
NP_001019980 (NP_001019980.1 ) - RAR alpha isoform 2
NP_00 138774 (NP_001138774.1 ) - RAR alpha isoform 4
NP_000956 (NP_000956.2) - RAR beta isoform 1
NP_057236 (NP_057236.1 ) - RAR beta isoform 2
NP_000957 (NP_000957.1) - RAR gamma isoform 1 NP_001036193 (NP_001036193.1) - RAR gamma isoform 2
NP_001230661 (NP_001230661.1) - RAR gamma isoform 3
NP_00 230659 (NP_001230659.1 ) - RAR gamma isoform 4
NP_612482 (NP_612482.1)- transcription factor Sp1 isoform a
NP_003100 (NP_003100.1)- transcription factor Sp1 isoform b
NP_001238754 (NP_001238754.1 ) - transcription factor Sp1 isoform c NP_005645 (NP_005645.1)- COUP transcription factor 1
Hence, the ketone body inhibitor may comprise or consist of a pharmaceutical agent selected from the group consisting of:
(i) a small molecule;
(ii) an antibody or antigen-binding fragment thereof, or a variant, fusion or derivative thereof which retains the ability to bind antigen;
(iii) an antibody mimic; and
(iv) a nucleic acid molecule (including an affibody).
In one embodiment the ketone body inhibitor comprises or consists of a small molecule.
The ketone body inhibitor may be a HMG-CoA synthase inhibitor selected from the group consisting of:
(i) succinyl-CoA;
(ii) hymeglusin (also known as (2R,3R)-p-lactone, 1233A and L-659699), and
(iii) Ceestatin (see Peng et a/., 2011 , J. Infect. Dis. 15;204(4):609-16).
The ketone body inhibitor is a PPARa (peroxisome-proliferator-activated receptor, alpha) inhibitor (for example MK886).
The ketone body inhibitor may be a RXR (c/'s-retinoid receptor) inhibitor, an Sp1 (Specificity Protein 1) inhibitor, a COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1) inhibitor, an Arp-1 (apo A1 regulatory protein-1) enhancer a COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1) enhancer, an HNF-4 (hepatocyte nuclear factor-4) enhancer or an HMG-CoA lyase inhibitor.
The ketone body inhibitor may be a /S-hydroxybutyrate dehydrogenase inhibitor. Preferably, the ketone body inhibitor reduces the presence or absorbance of luminal fatty acids. For example, luminal fatty acid presence or absorbance may be reduced by at least 5%, for example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100%.
Preferably, the ketogenesis is a pancreatic lipase inhibitor, such as lipstatin or orlistat. The ketone body inhibitor may reduce the presence of bile salts or be an enterocyte uptake inhibitor.
In one embodiment ketone body inhibitor comprises or consists of an antibody or antigen-binding fragment thereof, or a variant, fusion or derivative thereof which retains the ability to bind antigen.
Antibodies comprise two identical polypeptides of Mr 50,000-70,000 (termed "heavy chains") that are linked together by a disulphide bond, each of which is linked to one of an identical pair of polypeptides of Mr 25,000 (termed "light chains"). There is considerable sequence variability between individual N-termini of heavy chains of different antibody molecules and between individual light chains of different antibody molecules and these regions have hence been termed "variable domains". Conversely, there is considerable sequence similarity between individual C-termini of heavy chains of different antibody molecules and between individual light chains of different antibody molecules and these regions have hence been termed "constant domains".
The antigen-binding site is formed from hyper-variable regions in the variable domains of a pair of heavy and light chains. The hyper-variable regions are also known as complementarity-determining regions (CDRs) and determine the specificity of the antibody for a ligand. The variable domains of the heavy chain (VH) and light chain (VL) typically comprise three CDRs, each of which is flanked by sequence with less variation, which are known as framework regions (FRs). The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison er a/., 1984, Proc. Natl. Acad. Sci. USA, 81 , 6851-6855). That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al., 1988, Science, 240:1041). Fv molecules (Skerra ef al., 1988, Science, 240, 1038); single-chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird ef a/., 1988, Science 242:423; Huston ef al., 1988, Proc. Natl. Acad. Sci. USA, 85:5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward ef al., 1989 Nature 341 , 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter ef al., 1991 , Nature, 349, 293-299.
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants that can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).
The term "monoclonal antibody" (mAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
The antibody may be a human or humanised antibody or fragment thereof. The antibody may be a fragment including scFv molecules or Fab molecules.
By "ScFv molecules" we mean molecules wherein the VH and VL partner domains are linked via a flexible oligopeptide. The advantages of using antibody fragments which have antigen-binding activity, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from Escherichia coli (E. coli), thus allowing the facile production of large amounts of the said fragments.
Whole antibodies, and F(ab')2 fragments are "bivalent". By "bivalent" we mean that the said antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site. Methods for generating, isolating and using antibodies for a desired antigen or epitope are well known to those skilled in the relevant art. For example, an antibody may be raised in a suitable host animal (such as, for example, a mouse, rabbit or goat) using standard methods known in the art and either used as crude antisera or purified, for example by affinity purification. An antibody of desired specificity may alternatively be generated using well-known molecular biology methods, including selection from a molecular library of recombinant antibodies, or grafting or shuffling of complementarity-determining regions (CDRs) onto appropriate framework regions. Human antibodies may be selected from recombinant libraries and/or generated by grafting CDRs from non-human antibodies onto human framework regions using well-known molecular biology techniques.
Hence, the ketone body inhibitor may comprise or consist of an intact antibody, for example, a monoclonal antibody. Alternatively, the ketone body inhibitor may comprise or consist of an antigen-binding fragment selected from the group consisting of Fv fragments (e.g. single chain Fv, disulphide-bonded Fv and domain antibodies), and Fab-like fragments (e.g., Fab fragments, Fab' fragments and F(ab)2 fragments), preferably an scFv. In one embodiment the ketone body inhibitor comprises or consists of a domain antibody (as discussed above). The domain antibody maybe selected from the group consisting of single domain antibodies from cameloids, single domain antibodies from sharks and isolated VH or VL domains from humans.
The ketone body inhibitor may be an IgG antibody, for example an lgG1 , lgG2, lgG3 or lgG4 antibody. Preferably the antibody or antigen binding fragment is human or humanised.
Alternatively, the ketogenesis may be an antibody mimic such as the antibody mimics selected from the group consisting of affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains and affilins.
Alternatively, the ketone body inhibitor may be an interfering nucleic acid molecule. The interfering nucleic acid molecule may be selected from the group consisting of siRNA, antisense RNA, dsRNA, DNA aptamers, RNA aptamers and XNA aptamers. Hence, the interfering nucleic molecule may be complementary to the nucleotide sequence encoding HMG-CoA synthase or fragments or variants thereof such as an antisense polynucleotide which is capable of hybridising to the nucleotide sequence encoding HMG-CoA synthase or fragments or variants thereof.
A second aspect of the invention provides a nucleotide sequence encoding a peptide or nucleic acid ketone body inhibitor as described in the first aspect of the invention.
"oligonucleotide" are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double- stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material. In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U). It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil). Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
In a third aspect of the invention there is provided an expression vector containing a nucleotide sequence as described in the second aspect of the invention.
Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA, USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, NJ, USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, CA 92037, USA).
A typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, NJ, USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps). Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls. One such method involves ligation via homopolymer tails. Homopolymer polydA (or polydC) tails are added to exposed 3' OH groups on the DNA fragment to be cloned by terminal deoxynucleotidyl transferases. The fragment is then capable of annealing to the polydT (or polydG) tails added to the ends of a linearised plasmid vector. Gaps left following annealing can be filled by DNA polymerase and the free ends joined by DNA ligase.
Another method involves ligation via cohesive ends. Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
A further method uses synthetic molecules called linkers and adaptors. DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3' termini and fill in recessed 3' ends. Synthetic linkers, pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini. Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA. A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
In a fourth aspect of the invention there is provided a host cell comprising a nucleotide sequence or expression vector as described in the second and third aspects of the invention.
The DNA is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention. Thus, the DNA encoding the polypeptide constituting the compound of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter ef al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crowl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7 July 1987 to Goeddel, 4,704,362 issued 3 November 1987 to Itakura et al, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr. ef al, 4,766,075 issued 23 August 1988 to Goeddel ef al and 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference.
The DNA encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. Thus, the DNA insert may be operatively linked to an appropriate promoter. Bacterial promoters include the E.coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the phage λ PR and PL promoters, the phoA promoter and the f/p promoter. Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters and the promoters of retroviral LTRs. Other suitable promoters will be known to the skilled artisan. The expression constructs will desirably also contain sites for transcription initiation and termination, and in the transcribed region, a ribosome binding site for translation. (WO 98/16643) The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector and it will therefore be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence marker, with any necessary control elements, that codes for a selectable trait in the transformed cell. These markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracyclin, kanamycin or ampicillin resistance genes for culturing in E.coli and other bacteria. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell. Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered. The polypeptide of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
Many expression systems are known, including (but not limited to) systems employing: bacteria (eg. E.coli and Bacillus subtilis) transformed with, for example, recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeasts (eg. Saccaromyces cerevisiae) transformed with, for example, yeast expression vectors; insect cell systems transformed with, for example, viral expression vectors (eg. baculovirus) ; plant cell systems transfected with, for example viral or bacterial expression vectors; animal cell systems transfected with, for example, adenovirus expression vectors.
The vectors can include a prokaryotic replicon, such as the Col E1 ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E.coli, transformed therewith. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. The term "subject" means all animals including humans. Examples of subjects include humans, cows, dogs, cats, goats, sheep, and pigs. The term "patient" means a subject having a disorder in need of treatment.
In a fifth aspect of the invention there is provided a pharmaceutical composition comprising a ketone body inhibitor as described in the first aspect of the invention and a pharmaceutically acceptable excipient, diluent or carrier.
By "pharmaceutically acceptable" is included that the formulation is sterile and pyrogen free. Suitable pharmaceutical carriers are well known in the art of pharmacy. The carriers) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used. Thus, "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" includes any compound(s) used in forming a part of the formulation that is intended to act merely as a carrier, i.e., not intended to have biological activity itself. The pharmaceutically acceptable carrier or excipient is generally safe, non-toxic, and neither biologically nor otherwise undesirable. A pharmaceutically acceptable carrier or excipient as used herein includes both one and more than one such carrier or excipient.
The ketone body inhibitors of the invention can be formulated at various concentrations, depending on the efficacy/toxicity of the compound being used. Preferably, the formulation comprises the agent of the invention at a concentration of between 0.1 μΜ and 1 mM, more preferably between 1 μΜ and 100 μΜ, between 5 μΜ and 50 μΜ, between 10 μΜ and 50 μΜ, between 20 μΜ and 40 μΜ and most preferably about 30 μΜ. For in vitro applications, formulations may comprise a lower concentration of a compound of the invention, for example between 0.0025 μΜ and 1 μΜ.
It will be appreciated by persons skilled in the art that the medicaments and agents (;.e. polypeptides) will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA, which is incorporated herein by reference).
For example, the medicaments and agents can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The medicaments and agents may also be administered via intracavernosal injection.
Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof. The medicaments and agents of the invention can also be administered parenterally, for example, intravenously, intra-articularly, intra-arterially, intraperitoneally, intra- thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
For oral and parenteral administration to human patients, the daily dosage level of the medicaments and agents will usually be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses.
The medicaments and agents can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
Aerosol or dry powder formulations are preferably arranged so that each metered dose or 'puff contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
Alternatively, the medicaments and agents can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The compounds of the invention may also be transdermal^ administered, for example, by the use of a skin patch. They may also be administered by the ocular route.
For application topically to the skin, the medicaments and agents can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Where the medicament or agent is a polypeptide, it may be preferable to use a sustained-release drug delivery system, such as a microsphere. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
Sustained-release immunoglobulin compositions also include liposomally entrapped immunoglobulin. Liposomes containing the immunoglobulin are prepared by methods known per se. See, for example Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688- 92 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4 (1980); U.S. Patent Nos. 4,485,045; 4,544, 545; 6,139,869; and 6,027,726. Ordinarily, the liposomes are of the small (about 200 to about 800 Angstroms), unilamellar type in which the lipid content is greater than about 30 mole percent (mol. %) cholesterol; the selected proportion being adjusted for the optimal immunoglobulin therapy.
Alternatively, polypeptide medicaments and agents can be administered by a surgically implanted device that releases the drug directly to the required site.
Electroporation therapy (EPT) systems can also be employed for the administration of proteins and polypeptides. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
Proteins and polypeptides can also be delivered by electroincorporation (El). El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
An alternative method of protein and polypeptide delivery is the thermo-sensitive ReGel injectable. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.
Protein and polypeptide pharmaceuticals can also be delivered orally. One such system employs a natural process for oral uptake of vitamin B12 in the body to co- deliver proteins and polypeptides. By riding the vitamin B12 uptake system, the protein or polypeptide can move through the intestinal wall. Complexes are produced between vitamin B12 analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B12 portion of the complex and significant bioactivity of the drug portion of the complex. The skilled person will appreciate that the most appropriate formulation will depend on a number of factors including route of administration, patient type (e.g. patient age, weight/size).
A sixth aspect of the invention provides the use of a ketone body inhibitor as defined in the first aspect of the invention in the treatment or prevention of gastrointestinal tract (Gl tract) mucosa barrier impairment; and/or a disease or condition associated with gastrointestinal tract mucosa barrier impairment as defined in the first aspect of the invention. A seventh aspect of the invention provides a method of treating or preventing gastrointestinal tract (Gl tract) barrier impairment; and/or a disease or condition associated with gastrointestinal tract barrier impairment as defined in the first aspect of the invention comprising providing to a patient in need thereof a therapeutically effective amount of ketone body inhibitor as defined in the first aspect of the invention..
The terms "treating", and "treatment", and the like are used herein to generally mean obtaining a desired pharmacological and physiological effect. Further, it refers to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving the mammal's condition, directly or indirectly. Thus, treatment includes both therapeutic and prophylactic use.
The polypeptide or pharmaceutical composition of the invention is administered to the patient in an effective amount. A 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides inhibition of a biological activity of complement 5a (C5a) and/or the N- formyl-peptide, fMLP. This is a predetermined quantity of active material calculated to produce the desired therapeutic effect. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
A eighth aspect of the invention provides the use of a ketone body inhibitor as defined in the first aspect of the invention in the manufacture of a medicament for the in the treatment or prevention of gastrointestinal tract (Gl tract) mucosa barrier impairment; and/or a disease or condition associated with gastrointestinal tract mucosa barrier impairment as defined in the first aspect of the invention.
A ninth aspect of the invention provides a ketone body inhibitor as defined in the first aspect of the invention for use in medicine. A tenth aspect of the invention provides a kit of parts comprising:
(i) a ketone body inhibitor as defined in the first aspect of the invention or a pharmaceutical composition defined in the second aspect of the invention;
(ii) (optionally) apparatus for administering the biological inhibitor or pharmaceutical composition; and
(iii) (optionally) instructions for use.
An eleventh aspect of the invention provides a method of identifying agents for use in the treatment of gastrointestinal tract barrier impairment and/or diseases or conditions associated with gastrointestinal tract barrier impairment comprising measuring the ability of the test agent to inhibit ketogenesis, wherein the ability to inhibit ketogenesis is indicative of utility in the treatment of gastrointestinal tract barrier impairment and/or diseases or conditions associated with gastrointestinal tract barrier impairment.
A twelfth aspect of the invention provides a ketone body inhibitor obtained or obtainable by the method defined in the eleventh aspect of the invention. A thirteenth aspect of the invention provides a method of measuring the efficacy of a gastrointestinal tract barrier impairment treatment or prevention strategy comprising measuring intestinal ketogenesis. The method may comprise measuring intestinal ketogenesis in vitro or in vivo. The method may comprise measuring intestinal ketogenesis of intestinal epithelial cells. The intestinal cells may be derived from or present in the stomach, duodenum, ileum, cecum or colon.
Exemplary embodiments of the invention are described in the following non-limiting examples, with reference to the following figures:
Figure 1
(A) Western blot of HMGCS2 protein expression in mouse (C57BL/6) jejunal mucosa with low-fat diet (LFD, n=5) or high-fat diet (HFD, n=5) for 3 weeks. GAPDH was used as a control for equal loading of wells. PC=positive control. (B) Epithelial electrical resistance in Ussing chamber experiments with jejunal mucosa of HFD mice treated with vehicle or Hymeglusin, a specific inhibitor of HMGCS2. * P<.05, *** P< 001 by Student's t-test. Figure 2
Western blot quantification of HMGCS2 protein expression in human jejunal mucosa before and after GBP surgery. Data show means ± SEM, n=6 in each group.
Figure 3
Beta-hydroxy butyrate levels in portal blood in response to peroral Intralipid gavage (0.1 ml) to mice on low-fat diet (LFD) or high-fat diet (HFD). 1 mg hymeglusin or vehicle was given simultaneously with Intralipid. Data show means ± SEM, n=7 in each group. Figure 4
Beta-hydroxy butyrate levels in cell culture medium of Caco-2 cells treated with 10 mM butyrate, or butyrate + 1 μΜ hymeglusin. Data show means ± SEM, n=7 in each group. Figure 5
Ussing chamber measurements of epithelial electrical resistance (Rep) in the presence of butyrate alone or in combination with the HMGCS2 inhibitor hymeglusin, compared to time control. Data shown are means of ≥5 Ussing preparations each.
EXAMPLES
Small intestinal expression of the ketogenic enzyme HMGCS2 was induced by feeding adult C57BL/6 mice a high fat diet (HFD, n=5) over 7 days. Mice fed a conventional low fat diet (LFD, n=5) were used as time controls. After euthanesia the jejunal mucosa was dissected and Western blotting confirmed an increased epithelial protein expression of HMGCS2 in the HFD mice compared to LFD (Fig 1A). Furthermore, standardized mucosal scrapings dissolved in saline from the HFD mice contained more ketone bodies than when obtained from LFD-mice (2.06±0.7 versus 1.2± 0.4 mM).
Mucosal specimens were mounted in oxygenated Ussing chambers and the mucosal permeability was reflected by assessing epithelial electrical resistance (Rep). Over a 60 min study period the mucosae of the HFD fed mice gradually lost 40% of baseline Rep (Fig 1 B). Administration of the specific HMGCS2 inhibitor hymeglusin (1 uM) significantly reduced this spontaneous decrease in Rep (Fig 1B). In other words, presence of the compound hymeglusin was associated with a better maintained epithelial resistance, i.e., lower mucosal permeability. 1. Background
Roux-en-Y Gastric Bypass (GBP) results in reduced incidence and long-term remission of obesity associated metabolic comorbidities: insulin resistance/type-2 diabetes, hypertension and dyslipidemia. Its effect on type-2 diabetes does not seem to depend only on weight loss and is not to a large extent considered to be caused by food restriction or malabsorption. The mechanisms of action of GBP on type-2 diabetes are considered by the scientific community to be largely unresolved. It is also known that GBP reduces endotoxinemia and the signs on a systemic inflammatory reaction (Monte et a/., Surgery. 2012 Apr;151(4):587-93). Also the mechanisms to this are principally unknown.
2. The first discovery
To identify the mechanisms of action for GBP surgery on proinflammatory signalling as well as diabetes prevention and remission we examined small intestinal mucosal samples taken from the jejunum in 7 patients. The samples were obtained during GBP procedure (thus in the obese state) and 6-month after the procedure (the patient now in the weight-loosing phase). These samples were analysed using 2D- gel electrophoresis and intra-individual comparative proteomics. Statistically significant protein changes with each patient as own control, of at least 50%, occurring unanimously in all patients were considered for further analysis and peptide sequence identification by mass spectrometry. After GBP surgery a total of 27 protein spots corresponding to 11 unique proteins were found to be consistently regulated. The protein that changed the most (a 70% postoperative decrease) was mitochondrial HMG-CoA synthase (HMGCS2). This finding was repeatedly verified by Western blot analysis (Figure 2).
3. HMGCS2 - background art
HMGCS2 is the rate limiting ketogenic enzyme which is known to be expressed in the liver of adults and is activated during prolonged fasting (starvation). HMGCS2 is constitutively present in differentiated human colon-cells (Camarero N et al, Mol Cancer Res, 2006) but only to a very small extent in the small intestine of adult slim subjects (own observations). Small intestinal HMGCS2 expression has been observed in suckling rats but disappears after weaning (Hegardt FG, Biochem J, 1999). On the other hand a high-fat diet (≥ 35% of the dietary energy intake being fat) can induce HMGCS2 gene expression also in the adult small intestinal mucosa (de Wit et al 2011). So, the novel observation that jejunal mucosal HMGCS2 in obese people is reduced (or actually normalised) by GBP surgery can be explained as the result of reduced fat exposure and fat digestion in the jejunal lumen. This is due to the fact that the altered anatomy following the GBP procedure releases a considerable part of the small intestine from fat exposure, digestion and absorption, and also to the fact that the preference for fatty foods are much lower after this type of surgery (le Roux CW et al, Am J Physiol Regul Integr Comp Physiol. 2011 Oct;301 (4):R1057-66.).
4. A high fat diet induces HMGCS2 preferably in the jejunum
As a concept test we treated C57BL/6 mice with a high fat diet (HFD) over 1 week. Compared to controls on normal low fat diet, this treatment did only marginally influence body weight but changed the appearance of the intestinal mucosa (numerous fat vacuoles appeared in the epithelial cells). In addition, we observed that the HMGCS2 protein was induced in the jejunal mucosa of the HFD treated mice and to a much lesser degree (<10% of jejunal protein expression) in the duodenum and ileum (Figure 1A).
5. The high-fat-fed jejunal mucosa produces ketone bodies upon exposure to fat and fatty acids The level of ketone-bodies in the blood leaving the small intestine (the portal blood) increased upon luminal exposure to a mixture of emulsified lipids (Intralipid, 0.1 ml) in mice on HFD with confirmed jejunal H GCS2 expression. The increased portal vein ketone body concentration in HFD fed mice, compared to LFD fed mice, was completely blocked by the selective HMGCS2 inhibitor Hymeglusin (Figure 3). Furthermore, standardized mucosal scrapings dissolved in saline from the HFD mice contained more ketone bodies than when obtained from LFD-mice (2.06±0.7 versus 1.2± 0.4 mM). Also cultured enterocyte-like Caco-2 cells that constitutively express H GCS2 responded promptly with liberation of ketone bodies into the culturing medium when exposed to the short chain fatty acid butyrate. This ketone body production was completely abolished by treatment with Hymeglusin (Figure 4).
7. Intestinal ketoqenesis was associated to high transepithelial permeability
In order to confirm enterocyte ketogenesis in human jejunal mucosal specimens we investigated tissue samples obtained during gastric bypass surgery. The patients (n=5) under study had not been treated with pre-operative low-calory-diet in order to maintain a pronounced HMGCS2 level in the mucosa. The mucosae were dissected from the wall musculature and then immediately mounted in oxygenated Ussing chambers. One mucosal sample was sufficient to 4 to 6 Ussing preparations. Baseline data were recorded whereafter butyrate (10 mM) was added to the luminal chamber of all preparations. Untreated preparations (time controls) served as reference to the effect of butyrate. In half of the butyrate treated preparations the HMGCS2 inhibitor hymeglusin was added to reach a concentration of 1 μΜ. During the measurements of ketone body production, we surprisingly found that the electrical properties of the specimens were changed. Particularly, the mucosal permeability as reflected by epithelial electrical resistance (Rep) was decreased by butyrate addition and increased in the presence of hymeglusin. A high Rep represents low permeability to large sized molecular probes as previously shown with this set up (Bjorkman E et al, Scand J Gastroenterol, in press 20 2). It follows, that the butyrate-induced time-dependent decrease of the Rep, indicated increased transepithelial permeability. In the preparations with the HMGCS2 inhibitor hymeglusin the situation was reversed: butyrate now increased Rep (ie reduced the permeability) compared to time control (Fig 5).
In summary, these biological examples demonstrate that:
• obesity is associated with expression of the ketogenic enzyme HMGCS2 the small intestinal mucosa; fat and/or fatty acids in the intestinal lumen result in intestinal mucosal ketogenesis and that this response is mediated by H GCS2; and
an intestinal epithelium harbouring HMGCS2 responds with an increased permeability upon exposure with a fatty acid and this response is absent in presence of an inhibitor of intestinal ketogenesis.

Claims

A ketone body inhibitor for use in the treatment or prevention of: gastrointestinal tract (Gl tract) mucosa barrier impairment; and/or a disease or condition associated with gastrointestinal tract mucosa barrier impairment.
The ketone body inhibitor for use according to Claim 1 wherein the ketone body inhibitor is for use in the treatment or prevention of gastrointestinal tract mucosa barrier impairment.
The ketone body inhibitor for use according to Claim 1 or 2 wherein the ketone body inhibitor is for use in the treatment or prevention of a disease or condition associated with gastrointestinal tract mucosa barrier impairment.
The ketone body inhibitor for use according to any one of Claims 1 to 3 wherein the disease or condition associated with gastrointestinal tract mucosa barrier impairment is selected from the group consisting of:
(i) metabolic syndrome (also known as metabolic syndrome X, cardiometabolic syndrome, syndrome X, insulin resistance syndrome, Reaven's syndrome and CHAOS),
(ii) obesity,
(iii) insulin resistance,
(iv) type I diabetes mellitus,
(v) type II diabetes mellitus,
(vi) hypertension,
(vii) dyslipidemia,
(viii) inflammatory bowel disease,
(ix) irritable bowel syndrome (also known as irritable bowel disease),
(x) Crohn's disease,
(xi) ulcerative colitis,
(xii) collagenous colitis,
(xiii) lymphocytic colitis,
(xiv) ischaemic colitis, (XV) diversion colitis,
(xvi) Behcet's disease,
(xvii) indeterminate colitis,
(xviii) coeliac disease,
(xix) systemic T cell activation,
(XX) inflammation of the gastrointestinal tract,
(xxi) graft-versus-host disease,
(xxii) stroke,
(xxiii) myocardial infarction,
(xxiv) cancer,
(xxv) non-alcoholic steato-hepatitis (NASH),
(xxvi) liver cirrhosis,
(xxvii) allergy (especially food-allergies e.g., milk-protein allergy),
(xxviii) asthma, and
(xxix) atherosclerosis.
The ketone body inhibitor for use according to any one of Claims 1 to 4 wherein the disease or condition associated with gastrointestinal tract mucosa barrier impairment is metabolic syndrome.
The ketone body inhibitor for use according to any one of Claims 1 to 5 wherein the disease or condition is associated with infection.
The ketone body inhibitor for use according to Claim 6 wherein the infection is selected from the group consisting of bacterial infection, viral infection, protozoal infection, fungal infection and helminth infection.
The ketone body inhibitor for use according to Claim 7 wherein the infection is bacterial infection.
The ketone body inhibitor for use according to Claim 7 wherein the infection comprises or consists of infection with a Gram positive bacterial infection.
The ketone body inhibitor for use according to Claim 7 wherein the infection comprises or consists of infection with a Gram negative bacterial infection. The ketone body inhibitor for use according to Claim 10 wherein the infection is a bacterial infection with one or more of the group consisting of Achromobacter spp., Acidaminococcus fermentans, Acinetobacter cacoaceticus, Aeromonas spp., Alcaligenes faecalis, Arizona spp., Bacillus spp., Bacteroides fragilis, Bifidobacterium spp., Butyriviberio fibrosolvens., Campylobacter spp (such as C. coli), Citrobacter spp., Clostridium spp (such as C. difficile and C. sordellii), Edwardsiella spp., Eikenella corrodens., Enterobacter spp. (such as E. cloacae, E. faecalis and E. faecium), Escherichia spp. (such as E. coli), Flavobacterium spp., Hafnia spp., Klebsiella spp., Mycobacteria spp., Mycoplasma spp., Peptococcus spp., Plesiomonas shigelloides, Propionibacterium spp (such as P. acnes), Proteus spp., Providencia spp., Pseudomonas spp (such as P. aeruginosa), Ruminococcus spp (such as . bromii), Salmonella spp., Sarcina spp., Serratia spp., Shigella spp., Staphylococcus aureus, Streptococcus anginosus, Streptococcus viridans, Vibrio spp., Yersinia spp (such as S. enterocolitica), Yersinia enterocolitica, Veiflonella spp., Chryseomonas spp., Pseudomonas luteola, Lactobacillus spp., Enterococcus spp., Bacteroides spp., Streptococcus spp., Corynebacterium spp., Nocordia spp., Rhodococcus spp., Fusobacterium spp., Helicobacter spp., (such as Helicobacter pylori).
The ketone body inhibitor for use according to Claim 7 wherein the infection viral infection.
The ketone body inhibitor for use according to any one of the preceding claims wherein the gastrointestinal mucosa barrier impairment comprises or consists of barrier impairment of one or more of the group consisting of: the epithelium, lamina propria and muscularis muscosae.
The ketone body inhibitor for use according to Claim 13 wherein the gastrointestinal mucosa barrier impairment comprises or consists of barrier impairment of the epithelium.
The ketone body inhibitor for use according to any one of the preceding claims wherein the gastrointestinal tract barrier impairment is associated with increased paracellular or transcellular permeability of the gastrointestinal mucosa. The ketone body inhibitor for use according to any one of the preceding claims wherein the gastrointestinal tract barrier impairment is associated with increased paracellular permeability of the gastrointestinal mucosa.
The ketone body inhibitor for use according to Claim 16 wherein the increased paracellular permeability is associated with epithelial intracellular junction relaxation.
The ketone body inhibitor for use according to Claim 16 wherein the increased paracellular permeability is associated with tight junction relaxation.
The ketone body inhibitor for use according to any one of the preceding claims wherein gastrointestinal tract barrier impairment is present in one or more sites selected from the group consisting of the stomach, duodenum, jejunum, ileum, cecum and colon.
The ketone body inhibitor for use according to any one of the preceding claims wherein the gastrointestinal tract barrier impairment is absent from one or more sites selected from the group consisting of the stomach, duodenum, ileum, cecum and colon.
The ketone body inhibitor for use according to any one of the preceding claims wherein the gastrointestinal tract barrier impairment is absent from the colon. 22. The ketone body inhibitor for use according to any one of the preceding claims wherein the barrier impairment is not associated with ketogenesis.
23. The ketone body inhibitor for use according to any one of Claims 1 to 21 wherein the barrier impairment is associated with ketogenesis.
24. The ketone body inhibitor for use according to Claim 23 wherein the gastrointestinal tract barrier impairment is associated with ketogenesis in one or more sites selected from the group consisting of the stomach, duodenum, ileum, cecum and colon.
25. The ketone body inhibitor for use according to Claim 24 wherein the gastrointestinal tract barrier impairment is not associated with ketogenesis in one or more sites selected from the group consisting of the stomach, duodenum, ileum, cecum and colon.
The ketone body inhibitor for use according to Claim 24 wherein the gastrointestinal tract barrier impairment is not associated with ketogenesis of the colon.
27. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketogenesis is chronic.
28. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketogenesis is meal-induced.
29. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketogenesis is high fat diet-induced.
30. The ketone body inhibitor for use according to any one of the preceding claims wherein the gastrointestinal tract barrier impairment present in one or more sites selected from the group consisting of the stomach, duodenum, ileum, cecum and colon.
The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is capable of reducing gastrointestinal tract barrier impairment by at least 5%, for example, at least, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100%.
The ketone body inhibitor for use according to Claim 4 wherein the disease or condition is inflammation of the gastrointestinal tract selected from the group consisting of gastritis, enteritis, colitis, gastroenteritis and enterocolitis.
The ketone body inhibitor for use according to any one of the preceding claims wherein the gastrointestinal tract barrier impairment and/or disease or condition associated with gastrointestinal tract barrier impairment is associated with inflammation.
34. The ketone body inhibitor for use according to Claim 33 wherein the inflammation is chronic or acute.
35. The ketone body inhibitor for use according to Claim 33 or 34 wherein the inflammation is chronic.
36. The ketone body inhibitor for use according to any one of Claims 33 to 35 wherein the inflammation comprises or consists of inflammation of the gastrointestinal mucosa (i.e., local inflammation).
37. The ketone body inhibitor for use according to any one of Claims 33 to 36 wherein inflammation of the gastrointestinal mucosa is present in one or more sites selected from the group consisting of the stomach, duodenum, ileum, cecum and colon.
38. The ketone body inhibitor for use according to any one of Claims 33 to 36 wherein inflammation of the gastrointestinal mucosa is absent from one or more sites selected from the group consisting of the stomach, duodenum, ileum, cecum and colon.
39. The ketone body inhibitor for use according to any one of Claims 33 to 36 wherein inflammation of the gastrointestinal mucosa is absent from the colon.
The ketone body inhibitor for use according to any one of Claims 33 to 35 wherein the inflammation comprises or consists of inflammation non- gastrointestinal mucosa tissue (i.e., remote focal inflammation).
The ketone body inhibitor for use according to any one of Claims 33 to 35 wherein the inflammation comprises or consists of systemic inflammation.
The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is capable of reducing inflammation by at least 5%, for example, at least, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100%
43. The ketone body inhibitor for use according to any one of the preceding claims the ketone body inhibitor is capable of selectively inhibiting ketogenesis of the gastrointestinal tract. 44. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is capable of inhibiting ketone bodies of one or more layer of the gastrointestinal tract selected from the group consisting of the mucosa, submucosa, muscularis externa and serosa.
The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is capable of inhibiting ketone bodies of one or more layer of the gastrointestinal tract selected from the group consisting of: epithelium, lamina propria, muscularis mucosae, submucus nerve plexus, circular muscle, myenteric nerve plexus and longitudinal muscle.
The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is capable of selectively inhibiting ketogenesis in one or more sites selected from the group consisting of the stomach, duodenum, jejunum, ileum, cecum and colon.
The ketone body inhibitor for use according to any one of Claims 1 to 46 the preceding claims wherein the ketone body inhibitor is capable of selectively inhibiting gastric ketogenesis. 48. The ketone body inhibitor for use according to any one of Claims 1 to 46 wherein the ketone body inhibitor is capable of selectively inhibiting ketogenesis duodenal ketogenesis.
49. The ketone body inhibitor for use according to any one of Claims 1 to 46 wherein the ketone body inhibitor is capable of selectively inhibiting ketogenesis jejunal ketogenesis.
50. The ketone body inhibitor for use according to any one of Claims 1 to 46 wherein the ketone body inhibitor is capable of selectively inhibiting ileal ketogenesis.
51. The ketone body inhibitor for use according to any one of Claims 1 to 46 wherein the ketone body inhibitor is capable of selectively inhibiting ketogenesis duodenal, jejunal and ileal ketogenesis
The ketone body inhibitor for use according to any one of Claims 1 to 46 wherein the ketone body inhibitor is capable of selectively inhibiting cecal ketogenesis.
The ketone body inhibitor for use according to any one of Claims 1 to 46 wherein the ketone body inhibitor is capable of selectively inhibiting cecal and colonic ketogenesis
The ketone body inhibitor for use according to any one of Claims 1 to 46 wherein the ketone body inhibitor is capable of selectively inhibiting colonic ketogenesis.
The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is capable of inhibiting ketone bodies of the gastrointestinal mucosa by at least 10%, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 600%, 700%, 800%, 900% or at least 1000%.
56. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is capable of inhibiting ketone bodies of the gastrointestinal mucosa by at least 10% more than in non-gastrointestinal mucosa tissues, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 600%, 700%, 800%, 900% or at least 1000% more than in non- gastrointestinal mucosa tissues.
57. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is capable of inhibiting ketone bodies in non- gastrointestinal mucosa tissues by no more than 25%, for example, no more than 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or no more than 0%.
58. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is incapable of inhibiting ketone bodies in non-gastrointestinal mucosa tissues.
59. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor comprises or consists of a pharmaceutical agent that is inherently capable of selectively inhibiting ketogenesis of the gastrointestinal mucosa.
60. The ketone body inhibitor for use according to any one of Claims 1 to 58 wherein the ketone body inhibitor comprises or consists of a pharmaceutical agent that is not inherently capable of selectively inhibiting ketogenesis of a specific tissue type or location (such as the gastrointestinal mucosa).
61. The ketone body inhibitor for use according to Claim 60 wherein the ketone body inhibitor is formulated for selective inhibition of ketogenesis of the gastrointestinal mucosa.
62. The ketone body inhibitor for use according to Claim 61 wherein the ketone body inhibitor is formulated to reduce or prevent its exposure to non- gastrointestinal mucosa tissue.
63. The ketone body inhibitor for use according to Claim 62 wherein exposure of the ketosis inhibitor to non-gastrointestinal mucosa tissue is reduced by at least 10%, for example, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or at least 100%.
64. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is an inhibitor of a ketogenic enzyme. The ketone body inhibitor for use according to Claim 64 wherein the ketone body inhibitor is an inhibitor of a ketogenic enzyme selected from the group consisting of: (i) acetoacetyl-CoA thiolase (also known as acetyl-CoA
C-acetyltransferase, acetyl-CoA:acetyi-CoA C-acetyltransferase, beta-acetoacetyl coenzyme A thiolase, 2-methylacetoacetyl-CoA thiolase, 3-oxothiolase, acetyl coenzyme A thiolase, acetyl-CoA acetyltransferase, acetyl-CoA:N-acetyltransferase, ACAT1 , and thiolase II;
(ii) HMG-CoA synthase (also known as 3-hydroxy-3-methylglutaryl- Coenzyme A synthase 2 (mitochondrial), HMGCS2, );
(iii) HMG-CoA lyase (also known as Hydroxymethylglutaryl-CoA lyase, 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria), and HMGCL); and
(iv) jS-hydroxybutyrate dehydrogenase (also known as (R)-3- hydroxybutanoate:NAD+ oxidoreductase, NAD+-beta- hydroxybutyrate dehydrogenase, hydroxybutyrate oxidoreductase, beta-hydroxybutyrate dehydrogenase, D-beta-hydroxybutyrate dehydrogenase, D-3-hydroxybutyrate dehydrogenase, D-(-)-3- hydroxybutyrate dehydrogenase, beta-hydroxybutyric acid dehydrogenase, 3-D-hydroxybutyrate dehydrogenase, and beta- hydroxybutyric dehydrogenase).
The ketone body inhibitor for use according to Claim 65 wherein the ketone body inhibitor is a HMG-CoA synthase inhibitor.
The ketone body inhibitor for use according to Claim 65 or 66 wherein the HMG-CoA synthase is selected from the group of proteins defined by database accession numbers: CAG33131 , NM_005518, NM_001166107 and BC044217.
The ketone body inhibitor for use according to Claim 65, 66 or 67 wherein the HMG-CoA synthase is selected from the group of proteins defined by database accession numbers: CAG33131.1 , NM_005518.3, NM_001166107.1 and BC044217.1. The ketone body inhibitor for use according to any one of the preceding claims wherein the ketone body inhibitor is a direct inhibitor. wherein the ketone body inhibitor is capable of binding to one or more of the following amino acid sequences:
(v) HMGCS2; GenBank Accession No.: CAG33131 (version CAG33131.1)
MQRLLTPVKRILQLTRAVQETSLTPARLLPVAHQRFSTASAVPLA
KTDTWPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLG
QTRMGFCSVQEDINSLCLTWQRLMERIQLPWDSVGRLEVGTET
11 D KS KAVKTVLM E LFQ DS G NTDI EG I DTTNAC YG GTAS LFN AAN
WMESSSWDGRYAMWCGDIAVYPSGNARPTGGAGAVAMLIGP
KAPLALERGLRGTYMENVYDFYKPNLASEYPIVDGKLSIQCYLRA
LDRCYTSYRKKIQNQWKQAGSDRPFTLDDLQYMIFHTPFCKMVQ
KSLARLMFNDFLSASSDTQTSLYKGLEAFGGLKLEDTYTNKDLDK
ALLKASQDMFDKKTKASLYLSTHNGNMYTSTLYGCLASLLSHHS
AQELAGSRIGAFSYGSGLAASFFSFRVSQDAAPGSPLDKLVSST
SDLPKRLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTNSLF
PGTWYLERVDEQHRRKYARRPV
[SEQ ID NO: 1]
(vi) HMGCS2 (complete cds); GenBank Accession No.:
BC044217.1 (version BC044217.1)
MQRLLTPVKRILQLTRAVQETSLTPARLLPVAHQRFSTASAVPLA
KTDTWPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLG
QTRMGFCSVQEDINSLCLTWQRLMERIQLPWDSVGRLEVGTET
IIDKSKAVKTVLMELFQDSGNTDIEGIDTTNACYGGTASLFNAAN
WMESSSWDGRYAMWCGDIAVYPSGNARPTGGAGAVAMLIGP
KAPLALERGLRGTHMENVYDFYKPNLASEYPIVDGKLSIQCYLRA
LDRCYTSYRKKIQNQWKQAGSDRPFTLDDLQYMIFHTPFCKMVQ
KSLARLMFNDFLSASSDTQTSLYKGLEAFGGLKLEDTYTNKDLDK
ALLKASQDMFDKKTKASLYLSTHNGNMYTSSLYGCLASLLSHHS
AQELAGSRIGAFSYGSGLAASFFSFRVSQDAAPGSPLDKLVSST SDLPKRLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTNSLF PGTWYLERVDEQHRRKYARRPV
[SEQ ID NO: 2]
HMGCS2 (transcript variant 1); GenBank Accession No.: NM_005518(version NM_005518.3)
MQRLLTPVKRILQLTRAVQETSLTPARLLPVAHQRFSTASAVPLA
KTDTWPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLG
QTRMGFCSVQEDINSLCLTWQRLMERIQLPWDSVGRLEVGTET
IIDKSKAVKTVLMELFQDSGNTDIEGIDTTNACYGGTASLFNAAN
WMESSSWDGRYAMWCGDIAVYPSGNARPTGGAGAVAMLIGP
KAPLALERGLRGTHMENVYDFYKPNLASEYPIVDGKLSIQCYLRA
LDRCYTSYRKKIQNQWKQAGSDRPFTLDDLQY IFHTPFCK VQ
KSLARLMFNDFLSASSDTQTSLYKGLEAFGGLKLEDTYTNKDLDK
ALLKASQDMFDKKTKASLYLSTHNGNMYTSSLYGCLASLLSHHS
AQELAGSRIGAFSYGSGI-AASFFSFRVSQDAAPGSPLDKLVSST
SDLPKRLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTNSLF
PGTWYLERVDEQHRRKYARRPV
[SEQ ID NO: 3]
HMGCS2 (transcript variant 2); GenBank Accession No.: NM_001166107(version NM_001166107.1 )
MQRLLTPVKRILQLTRAVQETSLTPARLLPVAHQRFSTASAVPLA
KTDTWPKDVGILALEVYFPAQYVDQTDLEKYNNVEAGKYTVGLG
QTRMGFCSVQEDINSLCLTWQRL ERIQLPWDSVGRLEVGTET
IIDKSKAVKTVLMELFQDSGNTDIEGIDTTNACYGGTASLFNAAN
WMESSSWDGLRGTHMENVYDFYKPNLASEYPIVDGKLSIQCYL
RALDRCYTSYRKKIQNQWKQAGSDRPFTLDDLQYMIFHTPFCKM
VQKSLARLMFNDFLSASSDTQTSLYKGLEAFGGLKLEDTYTNKD
LDKALLKASQDMFDKKTKASLYLSTHNGNMYTSSLYGCLASLLS
HHSAQELAGSRIGAFSYGSGLAASFFSFRVSQDAAPGSPLDKLV
SSTSDLPKRLASRKCVSPEEFTEIMNQREQFYHKVNFSPPGDTN
SLFPGTWYLERVDEQHRRKYARRPV
[SEQ ID NO: 4] wherein the ketone body inhibitor is capable of binding to one or more of the following nucleic acid sequences:
(ix) HMGCS2 (complete cds); GenBank Accession No.:
BC044217.1 (version BC044217.1)
(x) HMGCS2 (complete gene, Ch 1); Database Accession No.:
NG_013348 (version NG_013348.1)
(xi) HMGCS2 (transcript variant 1); GenBank Accession No.:
NM_005518(version NM_005518.3)
(xii) HMGCS2 (transcript variant 2); GenBank Accession No.:
N M_001166107 (version N _001166107.1 ) wherein the ketone body inhibitor is capable of binding to one or more of the following positions of the HMGCS2 gene sequence:
(xiii) TATAAA (TATA sequence at position -28);
(xiv) GGCGGG (Sp1 sequence at -54);
(xv) AGACCTTTGGCCC (NRRE sequence at -130);
(xvi) TGATG I I I I C (IRE sequence at position -130);
(xvii) TGGCA (CTF-NF1 sequence at positions -520 and 836);
(xviii) GTGCGTCA (CRE sequence at position -546);
(xix) AGTCAAAAG (C-EBP sequence at position -778);
(xx) GCTACAGGTTGTGCT (GRE sequence at position -995);
(xxi) TGGCA (NFI-like sequence at positions -520, -836, -885, -1140);
(xxii) TGTGTCA (c-Jun sequence at position -249);
(xxiii) TGCGTCA (c-Jun sequence at position -554); and
(xxiv) TGACTCC (c-Jun sequence at position -781 ).
The ketone body inhibitor for use according to any one of Claims 1 to 68 wherein the ketone body inhibitor is an indirect inhibitor.
The ketone body inhibitor for use according to Claim 73 wherein the indirect inhibitor is an HMG-CoA synthase inhibitor. The ketone body inhibitor for use according to Claim 73 or 74 wherein the indirect H G-CoA synthase inhibitor is an inhibitor (i.e., antagonist or inverse agonist) of a HMG-CoA synthase activator selected from the group consisting of: CREB (CRE binding protein), PPARa (peroxisome-proliferator-activated receptor, alpha), RXR (c/s-retinoid receptor), Sp1 (Specificity Protein 1) and COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1/also known as NR2F1 (Nuclear Receptor subfamily 2, group F, member 1)).
The ketone body inhibitor for use according to Claim 73 or 74 wherein the indirect HMG-CoA synthase inhibitor is an enhancer (i.e., agonist) of a HMG-
CoA synthase repressor selected from the group consisting of: Arp-1 (apo A1 regulatory protein-1), COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1/also known as NR2F1 (Nuclear Receptor subfamily 2, group F, member 1)), and HNF-4 (hepatocyte nuclear factor-4).
76. The ketone body inhibitor for use according to any one of the preceding Claims wherein the ketone body inhibitor comprises or consists of a pharmaceutical agent selected from the group consisting of:
(i) a small molecule;
(ii) an antibody or antigen-binding fragment thereof, or a variant, fusion or derivative thereof which retains the ability to bind antigen;
(iii) an antibody mimic; and
(iv) a nucleic acid molecule (including an affibody).
The ketone body inhibitor for use according to Claim 77 wherein the ketone body inhibitor comprises or consists of a small molecule.
The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor is a acetoacetyl-CoA thiolase inhibitor, selected from the group consisting of:
The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor is a HMG-CoA synthase inhibitor selected from the group consisting of: succinyl-CoA; (vi) hymeglusin (also known as (2R,3R)-p-lactone, 1233A and L-659699); and
(vii) Ceestatin. 80. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the HMG-CoA synthase inhibitor is CREB (CRE binding protein) inhibitor.
The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the HMG-CoA synthase inhibitor is PPARa (peroxisome-proliferator- activated receptor, alpha), for example, MK886.
82. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the HMG-CoA synthase inhibitor is RXR (c s-retinoid receptor) inhibitor.
83. The ketone body inhibitor for use according to any one of Claims 1 to 63wherein the HMG-CoA synthase inhibitor is Sp1 (Specificity Protein 1) inhibitor.
84. The ketone body inhibitor for use according to any one of Claims 1 to 63wherein the HMG-CoA synthase inhibitor is COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1) inhibitor. 85. The ketone body inhibitor for use according to any one of Claims 1 to 63wherein the HMG-CoA synthase inhibitor is Arp-1 (apo A1 regulatory protein-1) enhancer.
86. The ketone body inhibitor for use according to any one of Claims 1 to 65 wherein the HMG-CoA synthase inhibitor is COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1) enhancer.
87. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the HMG-CoA synthase inhibitor is HNF-4 (hepatocyte nuclear factor- 4) enhancer.
88. The ketone body inhibitor for use according to any one of Claims wherein the ketone body inhibitor is a HMG-CoA lyase inhibitor.
89. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor is a 3-hydroxybutyrate dehydrogenase inhibitor.
90. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor reduces the presence or absorbance of luminal fatty acid.
91. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor is a pancreatic lipase inhibitor, such as lipstatin and orlistat.
92. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor is reduces the presence of bile salts.
93. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor is an enterocyte uptake inhibitor.
94. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor comprises or consists of an antibody or antigen-binding fragment thereof, or a variant, fusion or derivative thereof which retains the ability to bind antigen.
95. The ketone body inhibitor for use according to Claim 94 wherein the ketone body inhibitor comprises or consists of an intact antibody, for example, a monoclonal antibody.
96. The ketone body inhibitor for use according to Claim 94 wherein the ketone body inhibitor comprises or consists of an antigen-binding fragment selected from the group consisting of Fv fragments (e.g. single chain Fv, disulphide- bonded Fv and domain antibodies), and Fab-like fragments (e.g., Fab fragments, Fab' fragments and F(ab)2 fragments).
97. The ketone body inhibitor for use according Claim 96 wherein the antigen- binding fragment is an scFv.
98. The ketone body inhibitor for use according to Claim 96 wherein the ketone body inhibitor comprises or consists of a domain antibody.
99. The ketone body inhibitor for use according to Claim 95 wherein the domain is selected from the group consisting of single domain antibodies from cameloids, single domain antibodies from sharks and isolated VH or VL domains from humans.
100. The ketone body inhibitor for use according to Claim 94 wherein the antibody is an IgG antibody.
101. The ketone body inhibitor for use according to Claim 94 wherein the IgG antibody is an lgG1, lgG2, lgG3 or lgG4 antibody.
102. The ketone body inhibitor for use according to Claim 94 wherein the antibody is human or humanised.
103. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor is an antibody mimic.
104. The ketone body inhibitor for use according to Claim 103 wherein the antibody mimic is selected from the group consisting of affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains and affilins.
105. The ketone body inhibitor for use according to any one of Claims 1 to 63 wherein the ketone body inhibitor is an interfering nucleic acid molecule.
106. The ketone body inhibitor for use according to Claim 105 wherein the interfering nucleic acid molecule molecule is selected from the group consisting of siRNA, antisense RNA, dsRNA, DNA aptamers, RNA aptamers and XNA aptamers.
107. The ketone body inhibitor for use according to Claim 105 or 106 wherein the interfering nucleic molecule is complementary to the nucleotide sequence encoding HMG-CoA synthase or fragments or variants thereof. 108. The ketone body inhibitor for use according to Claim 105 or 106 wherein the interfering nucleic acid is an antisense polynucleotide which is capable of hybridising to the nucleotide sequence encoding HMG-CoA synthase or fragments or variants thereof. 109. A nucleotide sequence encoding a ketone body inhibitor as claimed in any one of Claims 94 to 102 or 106 to 108.
110. An expression vector containing a nucleotide sequence as claimed in Claim 109.
111. A host cell comprising a nucleotide sequence or expression vector as claimed in Claims 109 or 1 10.
112. A pharmaceutical composition comprising a ketone body inhibitor as defined in any one of Claims 1 to 108, a nucleotide sequence as defined in Claim 109, an expression vector as defined in Claim 110 or a host cell as defined in Claim 111 and a pharmaceutically acceptable excipient, diluent or carrier.
113. Use of a ketone body inhibitor as defined in any one of Claims 1 to 108 in the treatment or prevention of gastrointestinal tract (Gl TRACT) barrier impairment; and/or a disease or condition associated with gastrointestinal tract barrier impairment.
1 14. A method of treating or preventing gastrointestinal tract (Gl TRACT) barrier impairment; and/or a disease or condition associated with gastrointestinal tract barrier impairment comprising providing to a patient in need thereof a ketone body inhibitor as defined in any one of Claims 1 to 108.
115. Use of a ketone body inhibitor as defined in any one of Claims 1 to 108 in the manufacture of a medicament for the treatment or prevention of gastrointestinal tract (Gl TRACT) barrier impairment; and/or a disease or condition associated with gastrointestinal tract barrier impairment.
1 16. A ketone body inhibitor as defined in any one of Claims 43 to 54 for use in medicine (limit to gastrointestinal tract limited).
117. A ketone body inhibitor as defined in any one of Claims 43 to 54 for use in medicine (limit to gastrointestinal tract limited).
118. A kit of parts comprising:
(viii) a ketone body inhibitor as claimed in any one of Claims 1 to 108 or a pharmaceutical composition as claimed in Claim 112;
(ix) (optionally) apparatus for administering the biological inhibitor or pharmaceutical composition; and
(x) (optionally) instructions for use.
1 9. A method of identifying agents for use in the treatment of gastrointestinal tract barrier impairment and/or diseases or conditions associated with gastrointestinal tract barrier impairment comprising measuring the ability of the test agent to inhibit ketogenesis, wherein the ability to inhibit ketogenesis is indicative of utility in the treatment of gastrointestinal tract barrier impairment and/or diseases or conditions associated with gastrointestinal tract barrier impairment.
120. A ketone body inhibitor obtained by the method defined in Claim 119.
A method of measuring the efficacy of a gastrointestinal tract barrier impairment treatment or prevention strategy comprising measuring intestinal ketogenesis. 122. The method according to Claim 121 comprising measuring intestinal ketogenesis in vitro.
123. The method according to Claim 121 comprising measuring intestinal ketogenesis in vivo.
124. The method according to any one of Claims 121 to 123 comprising measuring intestinal ketogenesis in intestinal epithelial cells. The method according to any one of Claims 121 to 124 comprising measuring intestinal ketogenesis in intestinal epithelial cells derived from or present in the stomach, duodenum, ileum, cecum or colon.
PCT/GB2013/052850 2012-11-01 2013-10-31 Ketone body inhibitors for use in the treatment of gastrointestinal tract mucosa impairment WO2014068320A1 (en)

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WO2015079254A1 (en) * 2013-11-29 2015-06-04 University Of East London Leukotriene receptor antagonists and their derivatives for use as antibacterial agents
CN105510261A (en) * 2015-11-30 2016-04-20 山东博科生物产业有限公司 High-sensitivity D3-hydroxybutyric acid (D3H) detection reagent and detection method thereof
CN110179782A (en) * 2019-06-19 2019-08-30 曾辉 A kind of drug that treating AML and application
CN110420228A (en) * 2019-09-16 2019-11-08 山东大学齐鲁医院 Application of the bacteroides fragilis YCH46 in the drug of preparation treatment or assisting in treating hypertension
CN116059406A (en) * 2022-10-21 2023-05-05 西南医科大学附属医院 Bdh1 gene medicine for treating diabetes kidney disease

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015079254A1 (en) * 2013-11-29 2015-06-04 University Of East London Leukotriene receptor antagonists and their derivatives for use as antibacterial agents
CN105510261A (en) * 2015-11-30 2016-04-20 山东博科生物产业有限公司 High-sensitivity D3-hydroxybutyric acid (D3H) detection reagent and detection method thereof
CN110179782A (en) * 2019-06-19 2019-08-30 曾辉 A kind of drug that treating AML and application
CN110420228A (en) * 2019-09-16 2019-11-08 山东大学齐鲁医院 Application of the bacteroides fragilis YCH46 in the drug of preparation treatment or assisting in treating hypertension
CN116059406A (en) * 2022-10-21 2023-05-05 西南医科大学附属医院 Bdh1 gene medicine for treating diabetes kidney disease

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