D-GLYCERO-D-MANNO-HEPTOPYRANOSE 1-PHOSPHATE FOR MEDICAL USE

Description

FIELD OF THE INVENTION

[0001] The present invention relates generally to phosphorylated heptose compounds. More specifically, the present invention relates to use of heptopyranose phosphates in modulating an immune response in a subject.BACKGROUND OF THE INVENTION

[0002] An ability to modulate the immune system is becoming more and more critical as we strive to improve the immune response of individuals in order to generate a protective response, e.g. in immunocompromised individuals including cancer patients. This is outlined for example in WO 2016 / 054745 entitled "Methods of modulating immune system responses."

[0003] Pathogen associated molecular patterns (PAMPs) are molecules produced by pathogens that are specifically recognised by the human immune system in order to generate innate and adaptive immune responses to keep foreign pathogens at bay. The ability to synthesise PAMP's will enable the specific modulation of the immune system to improve the immune response and generate protection.

[0004] Only a limited number of PAMPs have been identified, e.g. lipopolysaccharide (LPS), DNA and flagellin. This limits the opportunity to investigate the immunomodulatory properties of these molecules. In most cases PAMPs are difficult to synthesise or isolate and thus precludes an opportunity to specifically address how these PAMP's interact with the immune system in order to exploit this relationship as a pure, fully characterised supply of the PAMPs is unavailable.

[0005] The inventors are also aware of the documents [1] to [5].

[0006] Zamyatina et al. [6] disclose the efficient chemical synthesis of both anomers of ADP L-glycero- and D-glycero-D-manno-heptopyranose. Malott et al. [5] report that Neisseria gonorrhoeae-derived heptose elicits an innate immune response and drives HIV-1 expression.

[0007] There is a need to identify novel PAMP molecules.SUMMARY OF THE INVENTION

[0008] The inventors have identified phosphorylated heptose compounds useful in modulating an immune response in a subject. Also, the compounds are useful as adjuvants.

[0009] More specifically, in accordance with aspects of the invention, there is provided the following: (1) D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use in treating Human Immunodeficiency Virus (HIV) infection in a subject. (2) D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use according to (1), wherein HMP-β induces HIV gene expression from latently infected cells. (3) D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use in treating cancer in a subject. (4) D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use as an adjuvant in the treatment or prevention of a medical condition in a subject. (5) D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) and an immunogen for use according to (4). (6) D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) and an immunogen for use according to (5), wherein the immunogen is in a vaccine composition. (7) D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use according to (5) or (6), wherein the immunogen is an antigen derived from a bacterium, virus or pathogen. (8) D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use according to (4), in combination with a therapeutic agent for the medical condition.

[0010] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In the appended drawings: FIG. 1: A) 1< H NMR of D-glycero-D-manno-heptopyranose 1β-phosphate (JS7 or HMP-β) B) 13< C NMR of compound JS7 or HMP-β C) 31< P NMR of compound JS7 or HMP-β. FIG. 2: A) 1< H NMR of D-glycero-D-manno-heptopyranose 1α-phosphate (JS8 or HMP-α). FIG. 3: A) 1< H NMR of D-mannose 1β-phosphate (JS9 or Man-1β-P). FIG. 4: Purity of HBP JS7 or HMP-β. Chromatogram of JS7 or HMP-β. Detector: PAD, Column: Carbopac ™< Solvent A:NaOH, 0.1M, Solvent B: AcONa, 1M and NaOH 0.05M, Conditions: 0-100% B in 30 minutes and 100% solvent B for 5 minutes. FIG.5: Purity of JS8 or HMP-α. Chromatogram of JS8 or HMP-α. Detector: PAD, Column: Carbopac ™< Solvent A:NaOH, 0.1M, Solvent B: AcONa, 1M and NaOH 0.05M, Conditions: 0-100% B in 30 minutes. FIG 6: Purity of JS9 or Man-1β-P. Chromatogram of JS9 or Man-1β-P. Detector: PAD, Column: Carbopac ™< Solvent A:NaOH, 0.1M, Solvent B: AcONa, 1M and NaOH 0.05M, Conditions: 0-100% B in 30 minutes. FIG. 7: Effects of compounds / products according to the invention on HEK 293T cells encoding an NF-κB-driven luciferase reporter gene. HEK 293T cells were transfected with a plasmid encoding an NF-κB-driven luciferase reporter. After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 µg / mL digitonin) in the presence of culture supernatant from N. meningitidis mutants with (gmhB) or without (hldA) HBP or 20 µg / mL of synthetic compound according to the invention. Treatment was removed; cells were washed and incubated for 3.5 hours in complete medium. A luciferase assay was then performed. The results are mean of technical triplicates. FIG. 8: Stimulation of human colonic epithelial cells by compounds / products according to the invention. Human colonic epithelial cells (HCT 116) that were either wild type (WT) or deficient in TIFA protein expression (knockout, KO) were stimulated for 20 minutes in permeabilization buffer (5 µg / mL digitonin) in the presence of culture supernatant from N. meningitidis mutants with (gmhB) or without (hldA) HBP or 10 µg / mL of synthetic compound according to the invention. Treatment was removed, cells were washed, and cells were incubated for 6 hours in complete media and IL-8 levels in culture supernatants was measured by ELISA. The results are mean of technical duplicates. FIG. 9: Stimulation of HIV proviral expression in Jurkat CD4+ T cells. Jurkat CD4+ T cell line containing latent recombinant HIV encoding mCherry fluorescent protein were exposed to the cytokine TNFα, culture supernatant from N. meningitidis mutants with (gmhB) or without (hldA) HBP, or 20 µg / mL of synthetic compound according to the invention. Expression of mCherry fluorescence was detected by flow cytometry immediately (Day 0) or after 1, 2 or 3 days, as indicated. Mock samples were exposed to buffer alone without added stimulatory agents. FIG. 10: Stimulation of HIV proviral expression in Jurkat CD4+ T cells. Jurkat CD4+ T cell line containing latent recombinant HIV encoding dsRed fluorescent protein were exposed to 150µM or 300µM of synthetic compound according to the invention for 24 hours. Expression of dsRed fluorescence was detected by flow cytometry immediately (Day 0) or after 24 hours. Untreated sample was exposed to buffer alone without added stimulatory agents. FIG 11: Stimulation of human macrophages by compounds / products according to the invention. Human macrophage cells (THP-1) were stimulated for 20 minutes in permeabilization buffer (5 µg / mL digitonin) in the presence of water, 39.8µM of the Nod1 agonist C12-iE-DAP (which stimulates in a TIFA-independent manner), or either 30µM or 150µM of synthetic compound according to the invention. Treatment was removed, cells were washed, and cells were incubated for 6 hours in complete media before the IL-8 levels in culture supernatants were measured by ELISA. The results are the mean and standard error of the mean of three technical replicates. Nod1 agonist: C12-iE-DAP (20 µg / mL, 39.8 µM); JS-7: JS-7 :D-glycero-β-D-manno-heptose-phosphate. FIG 12: In vivo administration of HMP-β (JS7) compared to Man-1β-P (JS9). Administered PBS (vehicle control), HMP, or Man-1β-P intraperitoneally, 500 µg / mouse (HMP and MP) in 0.5 mL, assessed serum KC levels 3h post-injection. Data are presented as box and whisker plots, min / max. Kruskal-Wallis (1way ANOVA, non-parametric), compare against PB. FIG 13: In vivo administration of HMP-β (JS7) compared to Man-1β-P (JS9). Administered PBS (vehicle control), HMP, or Man-1β-P intravenously, 500 µg / mouse (HMP and MP) in 0.1 mL, assessed serum KC levels 1h post-injection. Data are presented as box and whisker plots, min / max. Kruskal-Wallis (1way ANOVA, non-parametric), compare against PBS. FIG 14: Cancer tumor cell targeting. BALB / c mice were injected subcutaneously with 10 5< cells CT26 cells (colon carcinoma from BALB / c mice) in the flank in a volume of 100 µL. Fig 14a. Tumours were measured daily over time. Fig 14b. Tumor sizes were established on D10 post CT26 injection. Fig 14c. Mice were injected intraperitoneally with either LPS (1x at 3 mg / kg), HMP (200 µg per day for 3 days in a row at D10, D11, D12), or PBS (1x per day for 3 days in a row). Changes in tumour volume were measured daily relative to day 10 as displayed. Data was analysed by 2-way ANOVA with Dunnett's multiple comparison. FIG 15: 6-week-old male C57BL / 6NCrl mice were immunized with TbpB originating from group B N. meningitidis, purified from recombinant E. coli. All groups were immunized with 25 µg of TbpB with or without adjuvant, in a total volume of 30 µL intramuscularly: TbpB alone, TbpB + alum, and TbpB + HMP-β JS-7 (200 µg). Three doses were given: D0, D21, and D28. Serum was collected at D0 prior to immunization, D14, D28, and D35 and then examined by ELISA for IgG titers to TbpB ( Fig 15a). Mice were challenged on D36 with 5x10 7< of N. meningitidis strain expressing the homologous TbpB. Mice were injected with human transferrin (200 µL of 8 mg / mL) as this is critical for the development of sepsis in this model. Mice were monitored at the 1h, 12h, 18h, 24h, and 36h time points. At 1h, blood was collected to enumerate CFUs ( Fig 15b). Clinical scores were collected at 12h post challenge ( Fig 15c). DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0012] Before the present invention is further described, it is to be understood that the invention is not limited to the particular embodiments described below, as variations of these embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

[0013] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

[0014] In general, the methods of the present disclosure may be used to therapeutically or prophylactically treat any subjects for which increased activation of the immune system or an altered immune response would be beneficial. This includes, but is not restricted to a subject suffering from a condition which deleteriously affects the immune system, including any subjects at a heightened risk of infection or actually infected, for example due to surgery or imminent surgery, injury, illness, radiation or chemotherapy, and any subjects suffering from autoimmune diseases, inflammatory disorders, cancers, and diseases which cause the normal metabolic immune response to be compromised, such as HIV (AIDS).

[0015] As used herein, the term "phosphorylated heptose compound" refers a monosaccharide with seven carbon atoms, wherein at least one hydroxyl group is replaced by a group comprising a phosphorus atom. For example, the term "mono-phosphorylated heptose compound" refers a monosaccharide with seven carbon atoms, wherein one hydroxyl group is replaced by a group comprising a phosphorus atom. The term also refers to a derivative or an analogue of such compound.

[0016] As used herein, the term "modulate" in connection with an immune or inflammatory response refers to a qualitative or quantitative alteration in the immune or inflammatory response in a subject.

[0017] As used herein, the term "vaccine" or "vaccine composition" refers to a pharmaceutical composition containing an immunogen. The composition may be used for modulating an immune response in a subject. The term also refers to subunit vaccines, i.e., vaccine compositions containing immunogens which are separate and discrete from a whole organism with which the immunogen is associated in nature.

[0018] The term "adjuvant" as used herein in relation to the compound of the invention, refers to the compound serving as enhancer of the effectiveness of a medical treatment and / or enhancer of the immune response to an antigen, in a subject.

[0019] As used herein, the term "effective amount" refers to the amount of a compound or reaction product sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications in a therapeutic intervention comprising the administration of said compound or reaction product. An effective amount for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject.

[0020] As used herein, the term "subject" is understood as being any mammal including a human being treated with a compound of the invention.

[0021] As used herein the terms "treatment" and "treating" mean the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such administration of the active compounds to alleviate the symptoms or complications, to delay the progression of the condition, and / or to cure or eliminate the condition. The subject to be treated is preferably a mammal, in particular a human being.

[0022] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more.

[0023] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0024] The inventors have identified phosphorylated heptose compounds useful in modulating an immune response in a subject. Also, the compounds are useful as adjuvants.

[0025] The present invention is illustrated in further details by the following non-limiting examples.

[0026] D-glycero-D-manno-heptopyranose 1β-phosphate (JS7 or HMP-β), chemical structure below, was synthesized as described by Zamyatina et al. [6]. The spectra obtained ( FIG. 1) are in accordance with the literature data. The sole difference in the synthesis of the compound was that the compound was dissolved in brine and eluted through a G-15 column to exchange the trimethylamine salt for sodium.

[0027] An HPLC analysis shows that compound JS7 or HMP-β is pure; see FIG. 4.

[0028] D-glycero-D-manno-heptopyranose 1α-phosphate (JS8 or HMP-α), chemical structure below, was synthesized as described by Zamyatina et al. [6]. The spectrum obtained ( FIG. 2) is in accordance with the literature data. The sole difference in the synthesis of the compound was that the compound was dissolved in brine and eluted through a G-15 column to exchange the trimethylamine salt for sodium.

[0029] An HPLC analysis shows that compound JS8 or HMP-α is pure; see FIG. 5.

[0030] D-mannose 1β-phosphate (JS9 or Man-1β-P), chemical structure below, was synthesized as described by Zamyatina et al. [6], however starting from acetylated D-mannose instead of acetylated heptose and purifying in brine and eluting through a G-15 column to exchange the trimethylamine salt for sodium. The spectrum obtained (FIG. 3) is in accordance with the literature data [7].

[0031] An HPLC analysis shows that compound JS9 or Man-1β-P is pure; see FIG. 6. Biological experiments

[0032] Example 1: HMP-β JS-7 can immunomodulate via NF-κB stimulation in vitro. Effects of compounds / products according to the invention on HEK 293T cells encoding an NF-κB-driven luciferase reporter gene. HEK 293T cells were transfected with a plasmid encoding an NF-κB-driven luciferase reporter. After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 µg / mL digitonin) in the presence of culture supernatant from N. meningitidis mutants with (gmhB) or without (hldA) HBP or 20 µg / mL of synthetic compound according to the invention. Treatment was removed; cells were washed and incubated for 3.5 hours in complete medium. A luciferase assay was then performed. The results obtained are illustrated in FIG. 7; they are mean of technical triplicates.

[0033] Example 2: HMP-β JS-7 can drive cytokine expression in vitro. Stimulation of human colonic epithelial cells by compounds / products according to the invention. Human colonic epithelial cells (HCT 116) that were either wild type (WT) or deficient in TIFA protein expression (knockout, KO) were stimulated for 20 minutes in permeabilization buffer (5 µg / mL digitonin) in the presence of culture supernatant from N. meningitidis mutants with (gmhB) or without (hldA) HBP or 10 µg / mL of synthetic compound according to the invention. Treatment was removed, cells were washed, and cells were incubated for 6 hours in complete media and IL-8 levels in culture supernatants was measured by ELISA. The results obtained are illustrated in FIG. 8; they are mean of technical duplicates.

[0034] Example 3: Stimulation of HIV proviral expression in Jurkat CD4+ T cells. Jurkat CD4+ T cell line containing latent recombinant HIV encoding mCherry fluorescent protein were exposed to the cytokine TNFα, culture supernatant from N. meningitidis mutants with (gmhB) or without (hldA) HBP, or 20 µg / mL of synthetic compound according to the invention. Expression of mCherry fluorescence was detected by flow cytometry immediately (Day 0) or after 1, 2 or 3 days, as indicated. Mock samples were exposed to buffer alone without added stimulatory agents. The results obtained are illustrated in FIG. 9.

[0035] Example 4: HMP-β JS-7 can drive HIV out of latency in vitro. Stimulation of HIV proviral expression in Jurkat CD4+ T cells. Jurkat CD4+ T cell line containing latent recombinant HIV encoding dsRed fluorescent protein were exposed to 150 or 300µM of HMP-β JS-7. Expression of dsRed fluorescence was detected by flow cytometry immediately at 24 hours. Untreated samples were exposed to buffer alone without added stimulatory agents. The results obtained are illustrated in FIG. 10.

[0036] Example 5: HMP-β JS-7 can drive cytokine expression in vitro. Stimulation of human macrophages by compounds / products according to the invention. Human macrophage cells (THP-1) were stimulated for 20 minutes in permeabilization buffer (5 µg / mL digitonin) in the presence of water, 39.8µM of the Nod1 agonist C12-iE-DAP (which stimulates in a TIFA-independent manner), or either 30µM or 150µM of synthetic compound according to the invention. Treatment was removed, cells were washed, and cells were incubated for 6 hours in complete media before the IL-8 levels in culture supernatants were measured by ELISA (FIG 11). The results are the mean and standard error of the mean of three technical replicates. Nod1 agonist: C12-iE-DAP (20 µg / mL, 39.8µM); JS-7: JS-7:D-glycero-β-D-manno-heptose-phosphate.

[0037] Example 6: HMP-β JS7 alone induces cytokine production in vivo. In vivo administration of HMP-β JS7 (500 µg) via IP ( FIG 12) and IV ( FIG 13) routes induced serum KC levels when compared to the vehicle PBS and the control JS9 Man-1β-P (500 µg).

[0038] Example 7: HMP can reduce increasing tumour volumes in a cancer model. BALB / c mice were injected subcutaneously with 10 5< cells CT26 cells (colon carcinoma from BALB / c mice) in the flank in a volume of 100 µL. Tumours were measured daily over time. Once all mice within the group grew a measurable tumour ( FIG 14a), they were injected intraperitoneally with either LPS (1x at 3 mg / kg), HMP (200 µg per day for 3 days in a row at D10, D11, D12), or PBS (1x per day for 3 days in a row). One mouse from the LPS group was removed since no tumour ever became apparent. Prior to treatment, tumour sizes were not statistically different across mice allocated to different treatment groups ( FIG 14b). The change in tumour volume relative to day 10 was measured for each of the treatment groups and it was found that LPS (p<0.05) and HMP-β JS-7 (p<0.01) significantly reduced the rate of tumour growth over an 11 day period ( FIG 14c).

[0039] Example 8: HMP-β JS-7 can act as an adjuvant. 6-week-old male C57BL / 6NCrl mice were immunized with TbpB originating from group B N. meningitidis, purified from recombinant E. coli. All groups were immunized with 25 µg of TbpB with or without adjuvant, in a total volume of 30 µL intramuscularly TbpB alone, TbpB + alum, and TbpB + HMP-β JS-7 (200 µg). Three doses were given: D0, D21, D28. Serum was collected at D0 prior to immunization, D14, D28, and D35 and then examined by ELISA for IgG titers to TbpB. HMP-β JS-7 coadministration with the antigen resulted in titers that were significantly higher than administration of TbpB alone and greater than observed with alum as the adjuvant ( FIG 15a). Mice were challenged on D36 with 5x10 7< of N. meningitidis strain expressing the homologous TbpB. Mice were injected with human transferrin (200 µL of 8 mg / mL) as this is critical for the development of sepsis in this model. Mice were monitored at the 1h, 12h, 18h, 24h, and 36h time points. At 1h, blood was collected to enumerate CFUs. Clinical scores and weights for mice were collected at all time points. Bacterial burden was reduced and clinical scores were lower for mice that received TbpB antigen along with alum or HMP-β JS-7, consistent with the elevated anti-TbpB titers. FIG 15b bacterial burden CFU in blood and FIG 15c clinical scores 12h post challenge.

[0040] The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.REFERENCES

[0041] 1. Medzhitov R. Immunity (2009) 30, 766-775. 2. Medzhitov R. Nature (2007) 449, 819-826. 3. Robinson J.A. and Moehle K. Pure Appl. Chem. (2014) 86(10), 1483-1538. 4. Gaudet R.G. et al. Science (2015) 348(6240), 1251-1255. 5. Malott R.J. PNAS (2013) 110(25), 10234-10239. 6. Zamyatina et al. Carbohydr. Res. (2003) 338, 2571-2589.

Claims

1. D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use in treating Human Immunodeficiency Virus (HIV) infection in a subject.

2. D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use according to claim 1, wherein HMP-β induces HIV gene expression from latently infected cells.

3. D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use in treating cancer in a subject.

4. D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use as an adjuvant in the treatment or prevention of a medical condition in a subject.

5. D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) and an immunogen for use according to claim 4.

6. D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use according to claim 5, wherein the immunogen is in a vaccine composition.

7. D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use according to claim 5 or 6, wherein the immunogen is an antigen derived from a bacterium, virus or pathogen.

8. D-glycero-D-manno-heptopyranose 1β-phosphate (HMP-β) for use according to claim 4, in combination with a therapeutic agent for the medical condition.