sign in sign up
<<
Home , Mannoheptulose, Sedoheptulose

(19) TZZ _ __T

(11) EP 2 781 219 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 24.09.2014 Bulletin 2014/39 A61K 31/7004 (2006.01) A61P 29/00 (2006.01)

(21) Application number: 13160443.1

(22) Date of filing: 21.03.2013

(84) Designated Contracting States: • Wagner, Oswald AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 1090 Vienna (AT) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • Nagy, Csörsz PL PT RO RS SE SI SK SM TR 1090 Vienna (AT) Designated Extension States: • Marculescu, Rodrig BA ME 1090 Vienna (AT)

(71) Applicant: Medizinische Universität Wien (74) Representative: Sonn & Partner Patentanwälte 1090 Vienna (AT) Riemergasse 14 1010 Wien (AT) (72) Inventors: • Haschemi, Arvand 1090 Vienna (AT)

(54) Use of for prevention or treatment of inflammation

(57) The invention discloses sedoheptulose for use in the prevention or treatment of inflammation. EP 2 781 219 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 781 219 A1 2

Description ourful fruits and vegetables, oily fish (which contain high- er levels of omega-3 fatty acids), nuts, seeds and certain [0001] The technical field of the present invention is spices, such as ginger. Extra-virgin olive oil contains the the prevention or treatment of inflammation. chemical oleocanthal that acts similarly to ibuprofen. [0002] Inflammation is part of the complex biological 5 Omega-3 fatty acids have been shown to disrupt inflam- response of vascular tissues to harmful stimuli, such as mation cell signaling pathways by binding to the GPR120 pathogens, damaged cells, or irritants. The classical receptor. signs of acute inflammation are pain, heat, redness, [0007] However, there is still a need for prevention and swelling and loss of function. Inflammation is a protective treatment of inflammation, especially chronic inflamma- attempt by the organism to remove the injurious stimuli 10 tion. Such prevention and treatment should be provided and to initiate the healing process. Inflammation is a ster- without severe side effects or, preferably, without the risk eotyped response, and therefore it is considered as a for long term adverse developments, such as addiction, mechanism of innate immunity, as compared to adaptive metabolic disorders, declining efficacy, etc.. immunity, which is specific for each pathogen. [0008] Therefore, the present invention provides se- [0003] Inflammation can be classified as either acute 15 doheptulose for use in the prevention or treatment of in- or chronic. Acute inflammation is the initial response of flammation. the body to harmful stimuli and is achieved by the in- [0009] Sedoheptulose (CAS number: 3019-74-7; creased movement of plasma and leukocytes (especially PubChem: 5459879; ChemSpider: 4573620; MeSH: se- granulocytes) from the blood into the injured tissues. A doheptulose; ChEBI: CHEBI:16802) or D-altro-heptu- cascade of biochemical events propagates and matures 20 lose is a ketoheptose - a with seven the inflammatory response, involving the local vascular atoms and a functional group. It is one of system, the immune system and various cells within the the few found in nature. Sedoheptulose is pro- injured tissue. Prolonged inflammation, known as chronic ducible by extraction from natural sources or by chemical inflammation, leads to a progressive shift in the type of synthesis. It can be purified to high purity degrees (e.g. cells present at the site of inflammation and is character- 25 > 90 % purity, preferably > 95 % purity, more preferred ized by simultaneous destruction and healing of the tis- > 99 % purity, especially > 99.9 % purity). sue from the inflammatory process. [0010] Sedoheptulose is a relatively unknown metab- [0004] Inflammatory abnormalities (inflammatory dis- olite compared to its phosphorylated form, sedoheptu- orders) are a large group of disorders which underlie a lose-7-phosphate (S7P), which is recognised as an in- vast variety of human diseases. The immune system is 30 termediate molecule of primary metabolism. The often involved with inflammatory disorders, demonstrat- natural existence of sedoheptulose, a ketoheptose, was ed in both allergic reactions and some myopathies, with first reported in plants and was subsequently identified many immune system disorders resulting in abnormal in human urine, blood spots and recently, within mouse inflammation. Non-immune diseases with etiological or- cells. S7P is derived from the transketolase-catalysed igins in inflammatory processes include cancer, athero- 35 conversion of -5-phosphate (R5P) and -5- sclerosis and ischaemic heart disease. Examples of dis- phosphate (X5P). This reaction occurs in the non-oxida- orders associated with inflammation include acne vul- tive arm of the phosphate pathway and gener- garis, asthma, autoimmune diseases, celiac disease, ates -3-phosphate (G3P), a key glycolytic chronic prostatitis, glomerulonephritis, hypersensitivi- intermediate, in addition to S7P. G3P and S7P are also ties, inflammatory bowel diseases, pelvic inflammatory 40 produced by transaldolase using -6-phosphate disease, reperfusion injury, rheumatoid arthritis, sar- (F6P) and -4-phosphate (E4P) as substrates. coidosis, transplant rejection, vasculitis, interstitial cysti- S7P is thus a crucial intermediate in the non-oxidative tis, atherosclerosis, allergy, myopathies, leucocyte de- PPP and S7P bioavailability therefore contributes to cel- fects and cancer. lular carbon flux. Recently, data from several groups [0005] Medical treatment of acute and chronic infec- 45 have indicated that sedoheptulose kinase may also pro- tions has been performed with corticosteroids or non- duce S7P by direct phosphorylation of sedoheptulose steroidal anti-inflammatory drugs (NSAIDs). (Haschemi et al., Cell Metab. 15 (2012), 813-826). This [0006] Also anti-inflammatory foods have been sug- finding demonstrated the unexpected existence of an ad- gested to treat or prevent inflammation. Prostaglandins ditional carbon source, sedoheptulose, which actively are hormone-like substances that affect the body in va- 50 participates in cellular carbon metabolism. riety of ways, also regulating inflammatory mediation. An [0011] Free sedoheptulose can either be diet-derived anti-inflammatory diet includes less food that create in- from fruits and vegetables or formed enzymatically by flammation-causing prostaglandins (PGE2) in the body, the dephosphorylation of endogenously produced S7P. and more foods that create anti-inflammatory prostag- Thus far, no specific sedoheptulose transporter or S7P- landins (PGE1 and PGE3). Suggested diets to prevent 55 specfic phosphatase has been reported. Other bio-active inflammation include those rich in vegetables and low in heptoses include the phenolic compound 7-O-Galloyl- simple and fats, such as saturated fats sedoheptulose (GS), and glycohep- and trans-fats. Anti-inflammatory foods include most col- tose. GS was reported to be protective in diabetic injury

2 3 EP 2 781 219 A1 4 of the kidney by alleviating inflammatory responses. els were not changed by CARKL perturbation. The S7P Mannoheptulose (various patents pending) is an isomer levels were not changed by overexpression but de- of sedoheptulose and a potent inhibitor com- creasedsignificantly by CARKL loss,indicating sedohep- monly found in avocados. Glycoheptose, although no tulose phosphorylation as a rate-limiting factor for glyc- chemical nature of this compound remains unidentified, 5 olysis-derived G3P distribution. Therefore, the regulation has been shown to serve as accessible of S7P availability might be the mechanism by which source in rabbits. Sedoheptulosan is the anhydride of CARKL or excessive amounts of sedoheptulose modu- sedoheptulose and might also serve as source for free late cellular metabolism. sedoheptulose. [0016] The present invention is therefore directed to [0012] Free sedoheptulose can be isolated, e.g. from 10 use of C7, e.g. to balance C6-(over)consumption, obes- the plant sedum spectabile and has been previously re- ity, diabetes, immune function and - generally - verte- ported to contain high amounts of -carbohy- brate’s energy utilization. The C7 supplement therefore drates. also provides an effective strategy to manage physiolog- [0013] Sedoheptulose kinase phosphorylates free se- ical redox-regulation and thereby also related disorders. doheptulose, which enables cells to directly route sedo- 15 With the present invention, it turned out that, both, high heptulose to carbohydrate metabolism, similar to hexoki- sedoheptulose and high sedoheptulose kinase results in nase and glucose. According to the present invention, enhanced sedoheptulose turn-over. Moreover, tissue ex- sedoheptulose is a direct carbon source that feeds pri- pression of sedoheptulose kinase reveals that metabolic mary carbohydrate catabolism and anabolism, whereup- organs such as liver or adipose-tissue have the capability on CARKL constitutes its entry point. Sedoheptulose can 20 to consume sedoheptulose. Additionally, brown-adipose therefore surprisingly be compared to glucose and fruc- tissue (burns high amounts of lipids) express significantly tose because these compounds all exist as free carbo- more (∼2,5 x) sedoheptulose kinase than white-adipose hydrates and phosphorylated forms. To enter metabo- tissue. lism, free carbohydrates are energetically activated by [0017] Sedoheptulose (and/or other heptoses), admin- an initial phosphorylation event. Hexokinase phosphor- 25 istered to patients having inflammation or being at risk ylates glucose to form G6P and is a key determinant of of suffering from inflammation, minimizes the glycemic- cellular glucose flux. Fructose is phosphorylated by ke- load and positively contributes to prevent e.g. diet-in- tohexokinase to form fructose-1-phosphate (F1P), which duced obesity and/or diabetes. Furthermore, data from must first be converted by aldolase to glyceraldehyde transgenic sedoheptulose kinase animals - as model for and phosphate (DHAP). DHAP can 30 enhanced sedoheptulose metabolism - show that high then directly enter the carbon-cycle, whereas glyceral- sedoheptulose metabolism increases lipid-oxidation (in- dehyde must be further phosphorylated. The formation dicated by lower RQ values). This is a beneficial effect of S7P from sedoheptulose by sedoheptulose kinase, in of enhanced sedoheptulose turnover. Also energy ex- a manner analogous to G6P formation from glucose by penditure is lower in animals with high sedoheptulose hexokinase, enables free sedoheptulose to readily enter 35 turnover. This again shows that sedoheptulose metabo- the catabolic system. It was also reported that a compet- lism is an effective mean to regulate the metabolic-phe- itive inhibition of F6P phosphorylation in the presence of notype in vertebrates. high S7P concentrations. Interestingly, fractions contain- [0018] With the present invention, specific and surpris- ing fructose 1,6-bisphosphatase (FBPase) were also re- ing anti-inflammatory effects could be shown to be ported to possess sedoheptulose 1,7-bisphosphatase 40 caused by high sedoheptulose turnover: Enhanced in- (SBPase) activity. flammation plays an important role in the development [0014] The present invention therefore is also based of diseases, such as obesity related disorders like insulin on a coexistence of the - and heptose-(bis)phos- resistance. In a functional kinase screen for novel regu- phate shunts. Notably, high glucose levels or incubation lators of macrophages activation, sedoheptulose kinase with F2,6bP augmented the S1, 7bP formation in per- 45 was found to repress the lipopolysaccharide (LPS) in- fused rat liver and in rat liver cytosol, respectively. Glu- duced tumour necrosis factor α secretion. In the same cagon, in contrast to high glucose, favoured S1, 7bP de- screen, overexpression of hexokinase, ketohexokinase phosphorylation and therefore S7P formation. These re- and phosphofructokinase (high glucose/fructose metab- sults show that sedoheptulose metabolism is sensitive olism) had the opposite effect. This showed opposite ef- to hormonal control. 50 fects of hexose and heptose-carbohydrate metabolism: [0015] Sedoheptulose metabolism participates in met- boost inflammation and heptose inhibit inflam- abolic regulation; in fact, sedoheptulose directs metabol- mation. Moreover, increased CARKL expression re- ic fluxes by providing an S7P supply independently from pressed LPS-induced macrophage activation and result- glucose. Increased CARKL expression (and therefore in- ed in blunted intracellular superoxide formation, whereas creased sedoheptulose consumption) in a mouse mac- 55 loss of CARKL (mimics reduced sedoheptulose metab- rophage cell line resulted in reduced G3P, X5P and R5P olism) enhanced the inflammatory response of those steady state levels, whereas CARKL knockdown showed cells. the reverse effect. Notably, the basal sedoheptulose lev- [0019] In addition to these effects, it was shown in the

3 5 EP 2 781 219 A1 6 course of establishing the present invention that cytokine or ankylosing spondylitis and other rheumatic disorders production is decreased after inflammation (elicited by including the entire spectrum of autoimmune connective LPS injections) in CARKL overexpressing animals com- tissue disorders and myositis, vasculitis of any etiology pared to wild-type controls. This showed that administra- and manifestation, age-related macular degeneration tion of sedoheptulose dampens inflammation and there- 5 (AMD) chronic inflammatory skin disorders like psoriasis fore positively affects healthcare-related aspects of pa- and atopic dermatitis, periodontitis, major depressive dis- tients having or being at risk for inflammatory diseases, order and cancer. especially chronic inflammatory diseases. Chronic in- [0023] Another preferred inflammation according to flammatory diseases according to the present invention the present invention is wherein the inflammation is in- are defined as persistent inflammation, local or systemic, 10 volved in the pathogenesis of cancer including cancer of any etiology, known (like for instance persistent infec- causation, progression, metastasis, resistance to thera- tion) or currently unknown (like for instance sarcoidosis py, relapse after therapy and cancer manifestations, es- or most cases of chronic rhinosinusitis). pecially cancer-related cachexia, anemia, fatigue and [0020] Preferred inflammations to be prevented or depression. Given the universal involvement of inflam- treated according to the present invention are systemic 15 mation in the pathogenesis of cancer the present inven- inflammatory response syndrome (SIRS), sepsis, meta- tion is applicable to all cancer entities regardless of tissue inflammation, inflammaging, inflammation in the patho- origin, differentiation or localization including all types of genesis of an inflammation-related disorder, inflamma- carcinoma and sarcoma, melanoma and other tumors of tion in the pathogenesis and inflammation in the course neural crest-derived cells, tumors of the central nervous of acute or chronic transplant rejection. Accordingly, a 20 system, leukemia and lymphoma. preferred inflammation according to the present inven- [0024] Another preferred inflammation according to tion is wherein the inflammation is SIRS or sepsis, de- the present invention is wherein the inflammation is in- fined as massive systemic inflammation, especially in re- volved in acute or chronic transplant rejection, especially sponse to polytrauma or infection, including all the organ- rejection of allogeneic lung, liver, kidney, heart, intestine specific manifestations, especially acute respiratory dis- 25 or pancreatic islet cell transplants or, conversely, in acute tress syndrome (ARDS) or multiple organ dysfunction or chronic graft-versus-host disease following autolo- syndrome (MODS). gous, syngeneic or allogeneic bone marrow, peripheral [0021] Another preferred inflammation according to blood stem cell (PBSC) or cord blood transplantation. the present invention is wherein the inflammation is meta- [0025] Other preferred inflammations according to the inflammation or inflammaging, defined as chronic (low 30 present invention is selected from the group consisting grade) systemic inflammation as reflected by increased of an inflammation of adipose tissue, an inflammation of levels of pro-inflammatory cytokines (like for instance tu- the nervous system (neuroinflammation), an inflamma- mor necrosis factor alpha or interleukin-6) or other in- tion of blood vessels (vascular inflammation) and an in- flammation markers (like for instance c-reactive protein flammation of the peritoneum especially as a conse- or serum amyloid alpha) in peripheral blood and involved 35 quence of peritoneal dialysis as used for kidney replace- in aging, adiposity, atherosclerosis, impaired glucose tol- ment therapy. erance and type 2 diabetes, neurodegenerative disor- [0026] Sedoheptulose is not toxic; it is a natural source ders, like dementia, major depressive disorder (MDD) of carbohydrates for human metabolism. It may therefore and cancer. be administered in virtually all medically reasonable ways [0022] Another preferred inflammation according to 40 without risking unexpected side effects or adverse reac- the present invention is wherein the inflammation is in- tions. According to preferred administration dosages, es- volved, causally or otherwise, in the pathogenesis of an pecially single dosage forms, of the present invention, inflammation-related disorder, especially adiposity, im- sedoheptulose is administered in an amount of 0.001 to paired glucose tolerance, diabetes mellitus and diabetic 300 g per daily dosage, preferably in an amount of 0.5 complications, metabolic syndrome, atherosclerosis, in- 45 to 200 g per daily dosage, especially in an amount of 1 flammatory bowel disease (Crohn’s disease, colitis ulce- to 100 g per daily dosage. rosa and rarer entities), chronic inflammatory liver dis- [0027] Preferred administration of sedoheptulose is ease including chronic viral hepatitis, autoimmune hep- performed parentally, subcutaneously, by intravenous, atitis, primary sclerosing cholangitis and fatty liver dis- intratumoural subcutaneous, intramuscular or intraperi- ease in all stages, chronic obstructive pulmonary disease 50 toneal injection, especially wherein sedoheptulose is ad- (COPD) and asthma bronciale, inflammatory diseases ministered orally. of the central nervous system including multiple sclero- [0028] Sedoheptulose may virtually be included in al- sis, Alzheimer’s and Parkinson’s disease or peripheral most any pharmaceutical formulation form, including nervous system including diabetic polyneuropathy, Guil- pharmaceutically acceptable excipients. Preferably, se- lain-Barre and related syndromes and neuritides/neu- 55 doheptulose is administered as a powder, granulate, tab- ropathies associated with autoimmune collagen tissue let, pellet, capsule or syrup containing sedoheptulose. diseases or chronic infection like for instance Lyme dis- [0029] According to a preferred embodiment, sedo- ease, osteoarthritis, arthritides like rheumatoid arthritis heptulose is administered in an amount of 0.001 to 300

4 7 EP 2 781 219 A1 8 g, preferably in an amount of 0.5 to 200 g, especially in before and after food uptake to investigate if diet- an amount of 1 to 100 g. These administration dosages derived C7 can enter the human blood-stream. We are e.g. for a normal 70 to 80 kg human individual but compared sedoheptulose and glucose level of four can be adapted easily based on sex, inflammation de- overnight fastened (fastened) individuals to levels gree (or risk), other therapeutic regimen, physiological 5 measured two hours after a meal (diet-fed) contain- status/fitness of the patient, etc.. ing mainly carrots (∼700g) with some olive-oil, salt [0030] According to a preferred embodiment, sedo- and pepper. The change in serum carbohydrate lev- heptulose is administered in combination with hexoki- els was expressed as mean fold change (n=4, +/- nase inhibitors, preferably mannoheptulose or 2-deoxy- S.D.). D-glucose. Hexokinase inhibitors are well known in the 10 Figure 2: Sedoheptulose kinase (CARKL) mRNA art and can be applied in the present invention in the levels were measured in a mouse cDNA tissue li- amounts and dosages already known or suggested in brary. Data represents normalized (to β-actin) mean the prior art to the patient. For example, the inhibitor can CARKL expression relative to thymus CARKL ex- be added in an amount of 0.0001 % to 50 % w/w, espe- pression in fold change (all tissues n=3, S.D.). cially 0.001 % to 1 %, relative to the sedoheptulose con- 15 Figure 3: (A) Extracellular acidification rates (ECAR) tent (e.g. 0.0001 to 50 mg, if 100 mg sedoheptulose is and (B) cellular oxygen consumption rates (OCR) administered). were recorded after addition (indicated by red dotted [0031] According to another embodiment, the present line) of glucose, sedoheptulose or glucoheptose ( all inventionrelates to a method for storage ofhuman organs at 0.1g/L) to pre-starved (cultivated for 1hr without for transplantation purposes wherein the organ to be20 carbon sources) mouse macrophage cell line transplanted to a patient is stored in a storage solution (RAW264.7, ATCC). Changes due to carbohydrate comprising sedoheptulose. This aspect is based on the addition in ECAR and OCR were expressed as % anti-inflammatory capacity of sedoheptulose and serves change to basal rates before addition of carbohy- the same aim of reducing inflammation in the transplant drates (mean, n=4-6). (C) To functionally test the patient (to whom sedoheptulose may be applied as well): 25 impact of sedoheptulose on glucose metabolism we The anti-inflammatory property of the sedoheptulose compared mean OCR of RAW264.7 cells either storage solution that is used for impregnating the organ treated with glucose (1mM) alone or in combination to be transplanted also safeguards the reduction or pre- with sedoheptulose (1mM) (n=8-9). vention of inflammatory processes in the isolated organ Figure 4: In an in vitro kinase assay, which employs itself during storage and also after transplantation so that 30 ADP formation as activity readout, the sedoheptu- not only anti-inflammatory protection is present in the pa- lose turnover rate at constant ATP (150pM) concen- tient, but also in the organ that has been transplanted. tration could be enhanced either by (A) increasing [0032] In a preferred embodiment of this method, the sedoheptulose concentration or (B) by increasing storage solution comprises sedoheptulose in a concen- the amount of the rate-limiting enzyme sedoheptu- tration of 0.001 to 500 mg/ml, preferably in a concentra- 35 lose kinase (CARKL). (C, D) Our recently developed tion of 0.01 to 300 mg/ml, especially in a concentration sedoheptulose kinase overexpressing transgenic of 0.1 to 150 mg/ml, defined as the concentration of all mice were used an in vivo model of systemically in- liquid material attached to or covering the organ to be creased sedoheptulose metabolism. As metabolic transplanted (i.e. the solution that remains in the organ indicators we determined (C) the respiratory quotient storage containment after removing the organ from the 40 (RQ) and (D) energy expenditure (EE, containment). kcal/day/kg^0.75) per activity by indirect calorimetry [0033] According to another aspect, the present inven- and compared CARKL to wild-type (WT) littermates tion also relates to a peritoneal dialysis solution contain- during day (8am to 4pm) and night (8pm to 4am). ing sedoheptulose in a concentration of 0.001 to 500 Data represents mean values, n=3, +/- SD. mg/ml, preferably in a concentration of 0.01 to 300 mg/ml, 45 Figure 5: Reduced and oxidized nicotinamide ade- especially in a concentration of 0.1 to 150 mg/ml. nine dinucleotide levels of cells with perturbed [0034] The present invention is further described in the CARKL expression were calculated from previously following examples and the figures, yet without being re- recorded metabolomics data (Haschemi et al., 15 stricted thereto. (2012), 813-826). RAW264.7 cell line either (A) ex- 50 pressing high CARKL levels by overexpression or Figure 1: (A) Pooled samples from carrots or leaves (B) low CARKL levels by shRNAmir expression were of indoor grown Sedum Spectabile plants were an- compared to individual control cell lines to illustrate alyzed by gas-chromatography coupled to mass CARKL mediatednicotinamide adenine dinucleotide spectrometry to specifically measure relative glu- regulation. Data represents mean fold change of cose (C6) and sedoheptulose (C7) levels. The ratio 55 three individual experiments +/- SEM. of C6/C7 was calculated by dividing their respective Figure 6: The immune system of sedoheptulose ki- peak area. (B) We further assessed sedoheptulose nase overexpressing mice (CARKL) and control lit- and glucose levels by GC-MS in serum of individuals termates was challenged with a sub-lethal lipopoly-

5 9 EP 2 781 219 A1 10

saccharide injection (LPS, 7mg/kg) and measured a glucose free media and incubated for one hour in carbon panel of 19 cytokines 24hrs after immune activation free media before adding individual carbohydrates to the in serum. Data represents relative mean fluores- well per automatic injection. ECAR and OCR were nor- cence intensities (MFI) of individual cytokines meas- malized to respective rates before carbohydrate addition ured in wild type (WT=100%) and CARKL serum by 5 (100%). Changes induced by the different carbohydrate milliplex map mouse cytokine profiler (n=3, +/- SD). sources were expressed as relative change in % to illus- trate the effect of each carbon source. Examples Sedoheptulose kinase tissue expression Materials and Methods 10 [0038] We used a previously established mouse cDNA Carbohydrates library generated from C57/BL6 mice. Briefly, total RNA was extracted and reverse-transcribed using commercial [0035] Glucose was purchased from Sigma Aldrich kits (QIAGEN, Applied Biosystems). Quantitative real- and glucoheptose from Carbosynth. Sedoheptulose was 15 time PCRs were performed on an AbiPRISM 7900HT previously isolated from the plant sedum Spectabile real-time cycler using iTaq SYBR Green Supermix with (Haschemi et al., Cell Metab. 15 (2012), 813-826). ROX (BioRad) to measure CARKL and beta-actin ex- pression as previously described (Haschemi et al., Cell Glucose and sedoheptulose measurements Metab. 15 (2012), 813-826). Expression of CARKL was 20 normalized to beta-actin expression. Relative expression [0036] Fresh carrots with an Austrian "organic-certifi- of CARKL was calculated to thymus expression by the cate" were sliced into small pieces and snap-frozen in delta-delta CT method. liquid nitrogen. The same procedure was applied to leaves freshly harvested from indoor-grown Sedum Sedoheptulose Kinase assay Spectabile plants. Three samples of each tissue were 25 pooled and homogenized to powder by glass-beads. The [0039] We used the ADP Quest Assay (DiscoveRx) to carbohydrate rich fractions of the samples were isolated indirectly measure S7P formation by ADP accumulation by 20% H2O and 80% MeOH extraction solution spiked over time according to the manufacture instructions. Re- with 13C standards and processed for standard gas- combinant sedoheptulose kinase was previously pro- chromatography coupled to mass spectrometry (GC-30 duced in E.coli and purified by affinity purification MS). Glucose and sedoheptulose were identified by their (Haschemi et al., Cell Metab. 15 (2012), 813-826). individual masses and their relative amount was derived from the peak area of each individual analyte normalized Indirect calorimetry to 13C content. Glucose and sedoheptulose were also measured in equal volumes of serum from overnight fast- 35 [0040] The respiratory quotient (VCO2 production / ed or carrot fed individuals. The carrots were steamed VO2 consumption) and energy expenditure (EE, and some olive-oil, salt and pepper were used for flavour. kcal/day/kg^0.75) per activity of mice were measured in Two hours before and two hours after the carrot contain- metabolic cages (Harvard Apperatus) by indirect calor- ing meal human serum was prepared in VACUETTE ® Z imetry. We housed either one CARKL transgenic mouse Serum Sep tubes from blood. Serum samples were proc- 40 or a wild-type littermate, per cage for one day before we essed as previously described and also analyzed by GC- started to record mean respiratory quotient and energy MS as detailed above and in Ruiz-Matute AI et al., Chro- expenditure per activity during day (8am to 4pm) and matogr B Analyt Technol Biomed Life Sci. (2011) May night (8pm to 4am). 15 879(17-18). 45 Immunechallenge and cytokine response profiling invivo Extracellular acidification rates and oxygen consumption rates [0041] For sublethal murine in vivo endotoxemia, we injected 7 mg/kg LPS (Sigma Aldrich) intraperitoneally in [0037] We used the XF-Analyzer (Seahorse Bio.) to male CARKL transgenic or wild-type littermates. Serum measure changes in cellular metabolism induced by dif- 50 was isolated by VACUETTE® mini Z Serum Sep tubes ferent carbohydrates. Extracellular acidification rates from whole blood. Cytokine were measured by milliplex (ECAR, mpH/min) and oxygen consumption rates (OCR, map mouse cytokine profiler according to manufactures pmoles/min) were recorded in RAW264.7 mouse cell line instructions (Millipore). All animal experiments were car- according to the manufacture instructions. Briefly, cells ried out according to an ethical animal license protocol were seeded in seahorse cell plates at a density of 10^5 55 and contract approved by the Medical University Vienna cells per well one day before the experiment in normal (BMWF-66.009/0140-II/10b/2010). DMEM growth media containing 10% FBS and antibiot- ics. At the day of experiments the cells were washed with

6 11 EP 2 781 219 A1 12

Results fined C6/C6 ratios over time will be established by ana- lytical methods like GC-MS or HPLC and authentic ana- Diet-derived sedoheptulose uptake in humans lytical standards to establish the C6/C7 ratio also for hu- man bio-fluids (serum and urine). [0042] To determine if nutritional sedoheptulose is ab- 5 sorbed from diets via the digestive tract into the human Sedoheptulose kinase is highly expressed in metaboli- blood, which is a critical step for sedoheptulose to be- cally active tissue. come a metabolic active carbohydrate, we measured glu- cose (C6) and sedoheptulose (C7) levels in carrots and [0046] In order to understand which mammalian tissue in human serum. To determine the C6/C7 ratio of carrots 10 is able to consume nutritional sedoheptulose we meas- we homogenized plant tissue, extracted the small mole- ured sedoheptulose kinase (CARKL) mRNA levels in a cule fractions and measured simultaneously relative glu- mouse cDNA tissue library (Figure 2). Sedoheptulose cose and sedoheptulose amounts by GC-MS (Figure kinase is the rate-limiting enzyme of sedoheptulose me- 1A). Leaves of Sedum Spectabile, a succulent plant tabolism and is highly expressed in liver, kidney, in brown which contains high sedoheptulose levels, were ana- 15 and white adipose tissue (BAT and WAT), digestive or- lyzed in parallel as positive control. The leaves of Sedum gans, in glands, in the male reproductive system as well Spectabile contained glucose and approximately twice as in the brain. These results demonstrate that diet-de- as much sedoheptulose, which resulted in a C6/C7 ratio rived sedoheptulose can become a systemic carbohy- of approximately 0.5. In fresh food grade organic carrots drate source for many tissues but especially for metabol- we detected almost equal quantities of glucose and se- 20 ically active organs such as the liver, kidney and adipose doheptulose and thereby a C6/C7 ratio of around 1. Po- tissue. tatoes, in contrast to carrots, were reported to contain no sedoheptulose (Kardon et al., FEBS Lett. (2008) Oct 15, Cellular sedoheptulose metabolism 582(23-24)) and are therefore expected to possess a very high (or even infinite) C6/C7 ratio. Next we ad-25 [0047] To investigate the metabolic consequences of dressed if diet-derived sedoheptulose, originating from sedoheptulose in cells we measured cellular oxygen con- carrots, reaches the human blood by monitoring serum sumption rates (OCR) and extracellular acidification level of glucose and sedoheptulose before and after a rates (ECAR) as indicators for mitochondrial respiration meal containing mainly steamed carrots ( ∼700g per male and lactic acid fermentation, respectively. Addition of glu- adult) with some olive-oil, salt and pepper. Two hours 30 cose to pre-starved (for 1hr no carbon source in cell cul- after the meal, we observed an approximately two-fold ture media) mouse macrophage cell line (RAW264.7, increase in sedoheptulose serum levels in humans. This ATCC) increased ECAR (Figure 3A), indicating that glu- result clearly indicated that nutritional sedoheptulose is cose is largely used by non-oxidative metabolism, com- absorbed by the human digestive tract and that it enters monly found in muscle cells, activated immune cells and the blood to become available as a carbohydrate for sub- 35 in cancer cells. In response to glucose, cellular oxygen sequent metabolism. Serum glucose levels normalized consumption rates (OCR) dropped within the first hour already within two hours after food uptake and thereby and only moderately increased over time (Figure 3B). suggest that humans metabolize sedoheptulose and glu- Addition of sedoheptulose had the opposite effect of glu- cose differently. cose, as it induced cellular OCR with time while ECAR [0043] These results indicate that carrots, which are 40 showed only a moderate increase. This cellular metabol- considered as healthy vegetables and constituents of di- ic-signature of sedoheptulose was observed in response ets around the world, are a natural sedoheptulose source to sedoheptulose, but was lost if sedoheptulose was sub- for humans. It is interesting to mention that carrots, al- stituted with the rare heptose glucoheptose. though they contain carbohydrates such as glucose, are [0048] In order to functionally test the impact of in- suitable and well tolerated by diabetics and by providing 45 creasedsedoheptulose levels on glucosemetabolism we β-carotene even protects people with common genetic compared the cellular oxygen consumption rates after risk factors for type-2 diabetes (Patel CJ et al., Hum glucose addition with or without the addition of equimolar Genet. (2013) Jan 20, [Epub ahead of print]. amounts of sedoheptulose to pre starved RAW264.7 [0044] The C6/C7 index will be extended to other com- cells. Glucose alone induced a rapid decrease in OCR mon food products (common vegetables and diet con- 50 and returned to baseline after one hour (Figure 3C). Ad- stituents, instant foods and fast foods) and beverages dition of glucose in combination with sedoheptulose (soft-drinks, energy-drinks, sport-drinks and fresh juices counter-balanced glucose-induced OCR inhibition and prepared from fruits) by measuring glucose, fructose, se- also recovered OCR faster to baseline levels if compared doheptulose and mannoheptulose by analytical methods to glucose treatment alone. like GC-MS or HPLC and authentic analytical standards. 55 [0049] These results demonstrate that sedoheptulose [0045] Absolute concentrations of sedoheptulose, is consumed by cells and that its metabolic value is dif- mannoheptulose, glucose and fructose in human serum ferent from glucose. Glucose is primarily consumed by and urine samples after various standard-diets with de- non-oxidative metabolism, whereas sedoheptulose in-

7 13 EP 2 781 219 A1 14 duced oxidative metabolism. Furthermore, the results in- corn syrup ad libitum. The following parameters will be dicate that sedoheptulose alters glucose metabolism by addressed: nutritional intake, calorimetry, body weight balancing cellular oxygen-consumption. and composition, insulin sensitivity, glucose tolerance, [0050] The metabolic signature (ECAR and OCR) of blood lipid profiles, inflammatory markers, redox-regula- primary cells isolated from different organs (including liv- 5 tion and physical activity by standard assays. We will er, adipose tissue and pancreas to name but a few) in also test supplementation of diets with pure C7 (sedo- response to C6 (glucose, fructose and ) and C7 heptulose with and without mannoheptulose). (sedoheptulose and mannoheptulose) carbohydrates in various concentrations will be measured by the XF-An- Improved physiological redox-regulation by enhanced alyzer to provide further support from non-transformed 10 sedoheptulose turnover cells, specifically, a short time-exposure (0 - 6 hrs) and long term exposure (days - weeks). [0054] Increased cellular CARKL expression resulted in approximately 4 fold induction of redox-factors like re- Sedoheptulose metabolism in vivo duced nicotinamide adenine dinucleotide (NADH) and 15 also glutathione (GSH) level increase in CARKL overex- [0051] In order to test sedoheptulose metabolism in pressing RAW264.7 cells (Figure 5A and Haschemi et vivo, we generated transgenic mice overexpressing se- al., Cell Metab. 15 (2012), 813-826). However, deficiency doheptulose kinase ubiquitously in the entire body. We of CARKL and therefore also lack of sedoheptulose turn- hypothesized that increased sedoheptulose metabolism over showed the opposite effect (Figure 5B). can either be achieved by increasing the concentration 20 [0055] Kaneko et al. demonstrated that increased nico- of the substrate sedoheptulose, or by elevating the level tinamide adenine dinucleotide levels protected axonal of its rate-limiting enzyme sedoheptulose kinasedegeneration and improved behavioral deficits in exper- (CARKL). In an vitro kinase assay, we incubated a con- imental autoimmune encephalomyelitis (EAE), a multiple stant amount of recombinant CARKL protein with in- sclerosis (MS) model (Kaneko et al., 2006, The Journal creased doses of sedoheptulose. This resulted in en- 25 of Neuroscience, 20 September 2006, hanced sedoheptulose turnover rates as measured by 26(38):9794-9804). Therefore, the protective effects of ADP formation (Figure 4A). The same effect was ob- increased sedoheptulose turnover in CARKL overex- served by increasingCARKL protein concentration, while pressing mice and wild-type littermates will be compared sedoheptulose concentration was kept constant (Figure in a well-established the EAE model, quantified by dis- 4B). These results demonstrate that CARKL overexpres- 30 ease progression and by the MS scoring system. sionis a validmodel to investigate the effectsof increased [0056] Physical performance of CARKL overexpress- sedoheptulose metabolism. To investigate the metabolic ing and control animals by will be measured by exercise effect of increased sedoheptulose turnover to an entire models (i.e. wheel-running testes) to assess the impact organism we compared the respiratory quotient (RQ; RQ of high sedoheptulose consumption on endurance and 35 = exhaled CO2/oxygen consumption), as well as energy related metabolic-parameters (i.e. lactate formation, expenditure per activity of control and sedoheptulose ki- heartbeat, oxygen consumption to name but a few). nase transgenic mice, measured by indirect calorimetry [0057] As CARKL is strongly expressed in the male during day and night as systemic indicators of metabo- reproductive tract (Figure 2) and also the cellular redox- lism (Figure 4A and 4B). Sedoheptulose kinase overex- setting was associated with fertility (Geva E et al., Fertil pressing animals (CARKL) have lower RQ values com- 40 Steril. (1996) Sep;66(3):430-4.), we will also record off- pared to wild type (WT) littermates. CARKL mice showed spring distribution between CARKL transgenic and con- increased oxygen consumption and therefore lower RQ trol animals to test if sedoheptulose kinase positively in- values and thereby mirrored the effects of sedoheptulose fluences on sperm viability and fertility, on reproductive on cells (Figure 3). Furthermore, CARKL mice also tend cycles and reproduction rates of mice. to have a lower EE per activity if compared to control45 mice. Sedoheptulose metabolism and the immune response [0052] These experiments showed that increased se- of animals doheptulose metabolism does not only alter metabolism in vitro but also in vivo. The lower RQ values observed [0058] We present data of CARKL overexpression and in CARKL mice combined with the by trend lower EE per 50 its effects on the mouse immune-system in a model of activity is indicative for a more efficient and sustainable acute immune activation. In CARKL mice inflammation metabolic program, which can be induced by increasing elicited by a sub-lethal lipopolysaccharide (LPS) injection sedoheptulose metabolism. resulted in a suppressed cytokine response compared [0053] CARKL transgenic and control mice in metabol- to wild-type controls (Figure 6). We measured a panel of ic-cages (Harvard Apparatus), will be fed with various 55 19 cytokines in WT and CARKL mouse serum, which foods products with defined C6/C7 values (i.e. vegeta- was isolated 24hrs after LPS injection (7mg/kg, IP), by bles, normal chow and a "western" diet rich in fat and mouse cytokine profiler. Twelve cytokines reached the sucrose)and providewater with and without high fructose threshold of 50% mean change, which we defined as cut-

8 15 EP 2 781 219 A1 16 off to highlight cytokines regulated by CARKL overex- cols to measure their bone resorptive activity. pression in vivo. IL-13, MIP1 α, RNATES, IL-12 (p70), IL- 2, TNF-α, MCP1, KC, GM-CSF, IL-6, IL-17 and IL-15 were decreased by at least 50% mean change in CARKL Claims mice compared to wild-type littermates. Cytokines IL-6, 5 IL-17 and IL-15 even reached a threshold of 75% mean 1. Sedoheptulose for use in the prevention or treatment change. This indicates that a nutritional supplement con- of inflammation. taining sedoheptulose might dampen inflammation and therefore positively affect healthcare-related aspects of 2. Sedoheptulose according to claim 1, wherein the in- nutrition with a low C6/C7 ratio. 10 flammation is a chronic inflammation. [0059] CARKL overexpressing mice in standard mod- els for rheumatoid arthritis (i.e. joint-swelling) and athero- 3. Sedoheptulose according to claim 1, wherein the in- sclerosis (i.e. ApoE-/- mice on high fat diet) can reveal flammation is a systemic inflammatory response that increased sedoheptulose turnover is beneficial (i.e. syndrome (SIRS) or sepsis, defined as massive sys- reduces swelling of the joints or in the case of athero- 15 temic inflammation, especially in response to poly- sclerosis based on atherosclerotic plaque formation and trauma or infection, including all the organ-specific staability) as effective mean to reduce chronic-inflamma- manifestations, especially acute respiratory distress tion measured by cytokine profiling. syndrome(ARDS) or multipleorgan dysfunction syn- [0060] CARKL overexpressing mice in animal models drome (MODS). of diet-induced low-grade inflammation ("meta-inflam- 20 mation") compare the transgenic and control mice in met- 4. Sedoheptulose according to claim 1 or 2, wherein abolic-cages (Harvard Apparatus), feed them various the inflammation is meta-inflammation or inflammag- foods products with defined C6/C7 values (i.e. vegeta- ing, defined as chronic (low grade) systemic inflam- bles, normal chow and a "western" diet rich in fat and mation as reflected by increased levels of pro-inflam- sucrose)and providewater with and without high fructose 25 matory cytokines, especially tumor necrosis factor corn syrup ad libitum. The following parameters will be alpha or interleukin-6, or other inflammation mark- addressed: nutritional intake, calorimetry, body weight ers, especially c-reactive protein or serum amyloid and composition, insulin sensitivity, glucose tolerance, alpha, in peripheral blood and involved in aging, ad- blood lipid profiles, inflammatory markers, redox-regula- iposity, atherosclerosis, impaired glucose tolerance tion and physical activity by standard assays. 30 and type 2 diabetes, neurodegenerative disorders, [0061] We will also test our CARKL mice in in vivo mod- especially dementia, major depressive disorder els of multiple sclerosis (i.e. EAE) as effective mean to (MDD) and cancer. reduce neurological disease development, determined by an improved the MS scoring system, by reducing in- 5. Sedoheptulose according to any one of claims 1 to flammation as measured by cytokine profiling. 35 4, wherein the inflammation is involved, causally or otherwise, in the pathogenesis of an inflammation- Sedoheptulose metabolism in macrophages, osteo- related disorder, preferably adiposity, impaired glu- clasts and cancer cells cose tolerance, diabetes mellitus and diabetic com- plications, metabolic syndrome, atherosclerosis, in- [0062] Both, metabolism and the immune-system play 40 flammatory bowel disease, especially Crohn’s dis- an important role in maintaining bone- and cancer-tissue. ease, colitis ulcerosa and rarer entities, chronic in- For example, estrogen dependent breast cancer cells flammatory liver disease including chronic viral hep- decrease CARKL expression in response to growth fac- atitis, autoimmune hepatitis, primary sclerosing tor estrogen (public GeneChip repository data available cholangitis and fatty liver disease in all stages, chron- at http://www.ncbi.nlm.nih.gov/geoprofiles/; 45 ID: ic obstructive pulmonary disease (COPD) and asth- 53372898; GDS3285 / 219713_at / SHPK). CARKL is ma bronciale, inflammatory diseases of the central also decreased in macrophages after LPS-induced acti- nervoussystem, especially multiple sclerosis, Alzhe- vation (Haschemi et al., Cell Metab. 15 (2012), 813-826). imer’s and Parkinson’s disease, or peripheral nerv- We plan to test breast cancer cells (i.e primary tumor ous system, especially diabetic polyneuropathy, cells or MCF-7 cell line), primary macrophages and os- 50 Guillain-Barre and related syndromes, and neu- teoclasts cultivated in the presence of various C6 carbo- ritides/neuropathies associated with autoimmune hydrates(glucose, fructoseor sucrose), C7 carbohydrate collagen tissue diseases or chronic infection, espe- (sedoheptulose with or without mannoheptulose) or with cially Lyme disease, osteoarthritis, arthritides, espe- the combination of both, C6- and C7- carbohydrates as cially rheumatoid arthritis or ankylosing spondylitis exclusive carbon source and examine for cancer cells 55 and other rheumatic disorders, especially autoim- proliferation, invasion and migration, and for macrophag- mune connective tissue disorders and myositis, vas- es cytokine secretion in response to immune-activation. culitis of any etiology and manifestation, age-related Osteoclast function will be assessed by standard proto- macular degeneration (AMD) chronic inflammatory

9 17 EP 2 781 219 A1 18

skin disorders, especially psoriasis and atopic der- 14. A method according to claim 13, wherein the storage matitis, periodontitis, major depressive disorder and solution comprises sedoheptulose in a concentration cancer. of 0.001 to 500 mg/ml, preferably in a concentration of 0.01 to 300 mg/ml, especially in a concentration 6. Sedoheptulose according to any one of claims 1, 2, 5 of 0.1 to 150 mg/ml, defined as the concentration of 4 and 5, wherein the inflammation is involved in the all liquid material attached to or covering the organ pathogenesis of cancer including cancer causation, to be transplanted. progression, metastasis, resistance to therapy, re- lapse after therapy and cancer manifestations, es- 15. A peritoneal dialysis solution containing sedoheptu- pecially cancer-related cachexia, anemia, fatigue 10 lose in a concentration of 0.001 to 500 mg/ml, pref- and depression. erably in a concentration of 0.01 to 300 mg/ml, es- pecially in a concentration of 0.1 to 150 mg/ml. 7. Sedoheptulose according to claim 1, wherein the in- flammation is involved in acute or chronic transplant rejection, especially rejection of allogeneic lung, liv- 15 er, kidney, heart, intestine or pancreatic islet cell transplants or, conversely, in acute or chronic graft- versus-host disease following autologous, syn- geneic or allogeneic bone marrow, peripheral blood stem cell (PBSC) or cord blood transplantation. 20

8. Sedoheptulose according to claim 1, wherein the in- flammation is selected from the group consisting of an inflammation of adipose tissue, an inflammation of the nervous system, an inflammation of blood ves- 25 sels and an inflammation of the peritoneum espe- cially as a consequence of peritoneal dialysis as used for kidney replacement therapy.

9. Sedoheptulose according to any one of claims 1 to 30 8, wherein the sedoheptulose is administered in an amountof 0.001 to 300 gper dailydosage, preferably in an amount of 0.5 to 200 g per daily dosage, es- pecially in an amount of 1 to 100 g per daily dosage. 35 10. Sedoheptulose according to any one of claims 1 to 9, wherein sedoheptulose is administered parental- ly, subcutaneously, by intravenous, intratumoral, subcutaneous, intramuscular or intraperitoneal in- jections, especially wherein sedoheptulose is admin- 40 istered orally.

11. Sedoheptulose according to any one of claims 1 to 10, wherein sedoheptulose is administered as a powder, granulate, tablet, pellet, capsule or syrup 45 containing sedoheptulose in an amount of 0.001 to 300 g, preferably in an amount of 0.5 to 200 g, es- pecially in an amount of 1 to 100 g.

12. Sedoheptulose according to any one of claims 1 to 50 11, wherein sedoheptulose is administered in com- bination with hexokinase inhibitors, preferably man- noheptose or 2-deoxy-D-glucose.

13. A method for storage of organs for transplantation 55 purposes wherein the organ to be transplanted to a patient is stored in a storage solution comprising se- doheptulose.

10 EP 2 781 219 A1

11 EP 2 781 219 A1

12 EP 2 781 219 A1

13 EP 2 781 219 A1

14 EP 2 781 219 A1

15 EP 2 781 219 A1

16 EP 2 781 219 A1

5

10

15

20

25

30

35

40

45

50

55

17 EP 2 781 219 A1

5

10

15

20

25

30

35

40

45

50

55

18 EP 2 781 219 A1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description

• HASCHEMI et al. Cell Metab., 2012, vol. 15, 813-826 • PATEL CJ et al. Hum Genet., 20 January 2013 [0010] [0035] [0038] [0039] [0054] [0062] [0043] • RUIZ-MATUTE AI et al. Chromatogr B Analyt Tech- • KANEKO et al. The Journal of Neuroscience, 20 nol Biomed Life Sci., 15 May 2011, vol. 879, 17-18 September 2006, vol. 26 (38), 9794-9804 [0055] [0036] • GEVA E et al. Fertil Steril., September 1996, vol. 66 • KARDON et al. FEBS Lett., 15 October 2008, vol. (3), 430-4 [0057] 582, 23-24 [0042]

19