阅读说明：本技术 预防或治疗鼻病毒感染的药物 (Medicine for preventing or treating rhinovirus infection ) 是由 J·斯托克 G·加尔多尼 于 2018-12-28 设计创作，主要内容包括：本发明提供了用于预防或治疗人鼻病毒(HRV)感染的药物组合物。该组合物包含己醛糖,其中己醛糖的碳2处的羟基被H、F、Cl、Br、I、SH、Me、OMe和SMe中的任何一个取代,例如2-脱氧葡萄糖。此外,提供了用于鼻内给药的分配器,例如含有所述药物组合物的鼻喷雾器或滴鼻器。另外,提供了包含所述组合物的吸入装置,例如计量吸入器,干粉吸入器或雾化器。(The present invention provides pharmaceutical compositions for preventing or treating Human Rhinovirus (HRV) infection. The composition comprises an aldohexose wherein the hydroxyl group at carbon 2 of the aldohexose is substituted with any of H, F, Cl, Br, I, SH, Me, OMe and SMe, for example 2-deoxyglucose. Furthermore, a dispenser for intranasal administration, such as a nasal spray or nasal drops, containing the pharmaceutical composition is provided. In addition, inhalation devices, such as metered dose inhalers, dry powder inhalers or nebulizers, comprising the composition are provided.)

1. A pharmaceutical composition for use in the prevention or treatment of Human Rhinovirus (HRV) infection, the composition comprising an aldohexose in which the hydroxyl group at carbon 2 of the aldohexose is replaced by any of H, F, Cl, Br, I, SH, Me, OMe and SMe, preferably the aldohexose is D-aldohexose.

2. The pharmaceutical composition according to claim 1, wherein the aldohexose is glucose, preferably D-glucose, wherein the hydroxyl group at carbon 2 of the aldohexose is replaced by any of H, F, Cl, Br, I, SH, Me, OMe and SMe.

3. A pharmaceutical composition for preventing or treating HRV infection, the composition comprising 2-deoxy-D-glucose.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is in liquid form, preferably wherein the composition is an aqueous solution.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the aldohexose, especially 2-deoxy-D-glucose, is in a concentration of 0.01mM to 1000mM, preferably 0.1mM to 500mM, more preferably 0.25mM to 250mM, even more preferably 0.5mM to 100mM, especially 1mM to 50mM or even 2.5mM to 25 mM.

6. Pharmaceutical composition according to any one of claims 1 to 5, wherein the composition further comprises at least one excipient and/or at least one additional active agent, preferably the additional active agent is selected from the group consisting of a detumescence agent, in particular a norepinephrine releaser, an alpha-adrenergic receptor agonist, and a corticosteroid, and a non-steroidal anti-inflammatory agent.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein a dose of the pharmaceutical composition is administered to a human subject, preferably wherein the total amount of aldohexoses, especially 2-deoxy-D-glucose, in the dose is 0,001 μmol to 100 μmol, preferably 0,01 μmol to 50 μmol, more preferably 0,025 μmol to 25 μmol, even more preferably 0,05 μmol to 5 μmol, especially 0,1 μmol to 2,5 μmol or even 0,2 μmol to 1,25 μmol.

8. The pharmaceutical composition according to claim 7, wherein the dose is at least once every other day, preferably at least once daily, more preferably at least twice daily, in particular at least three times daily, and preferably for a period of 2 to 14 days, more preferably 3 to 10 days, especially 4 to 7 days.

9. A pharmaceutical composition according to any one of claims 7 to 8, wherein the dose is administered to the nostrils of the subject, preferably independently to each nostril.

10. Pharmaceutical composition according to any one of claims 1 to 9, wherein the composition is applied to the mucosa, preferably the mucosa of the respiratory tract, in particular the mucosa of the nasal cavity or the mucosa of the lower respiratory tract.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the composition is for use in the prevention or treatment of the common cold, rhinitis or lower respiratory tract infections, especially in immunosuppressed individuals or individuals with COPD or asthma.

12. A dispenser for intranasal administration of a pharmaceutical composition, said dispenser containing said pharmaceutical composition, wherein the composition comprises an aldohexose in which the hydroxyl group at carbon 2 of the aldohexose is replaced by any of H, F, Cl, Br, I, SH, Me, OMe and SMe, preferably said aldohexose is 2-deoxy-D-glucose;

preferably, the pharmaceutical composition is a pharmaceutical composition according to any one of claims 1 to 11.

13. A dispenser according to claim 12, which is a nasal spray or nasal dropper.

14. An inhalation device for administration of a pharmaceutical composition, preferably for administration of a pharmaceutical composition to the lower respiratory tract, wherein said device is charged with said pharmaceutical composition, wherein the composition comprises an aldohexose, wherein the hydroxyl group at carbon 2 of the aldohexose is replaced by any of H, F, Cl, Br, I, SH, Me, OMe and SMe, preferably said aldohexose is 2-deoxy-D-glucose;

preferably the inhalation device is a metered dose inhaler, a dry powder inhaler or a nebulizer, and/or preferably the pharmaceutical composition is a pharmaceutical composition according to any one of claims 1 to 11.

15. A method of delaying the onset of or treating an HRV infection comprising

-obtaining a pharmaceutically acceptable formulation comprising an aldohexose wherein the hydroxyl group at carbon 2 is replaced by any of H, F, Cl, Br, I, SH, Me, OMe and SMe; and

-administering an effective amount of the formulation to an individual having or at risk of developing an HRV infection;

preferably wherein the aldohexose is as defined in any of claims 2 to 11 or as listed as such.

The effect of 2-deoxy-glucose on enveloped viruses has been described in the prior art. For example, Schnitzer et al (Virology 67(1975)306-309) describe the effect of 2-deoxy-glucose and glucosamine on the growth and function of respiratory syncytial virus and parainfluenza virus type 3. Kilbourne (Nature 183(1959)271-272) discloses the inhibition of influenza virus proliferation by 2-deoxy-glucose. Hodes et al (Virology 63(1975)201-208) describe the inhibitory effect of 2-deoxy-glucose on respiratory syncytial virus, parainfluenza virus type 3 and measles virus. However, prior to the present invention, there was no indication that 2-deoxy-glucose was also effective against non-enveloped RNA viruses.

Arita et al (Carbohydrate Research 62(1978)143-154), which is not related to 2-deoxy-glucose, disclose the synthesis of various analogs and derivatives of phenyl glycosides and their detection of antiviral activity against certain enveloped viruses (influenza and herpes simplex viruses).

For all aspects of the present invention, the modified aldohexose comprised in the pharmaceutical composition (or pharmaceutically acceptable formulation) is preferably one of compounds I-VIII (depicted as fischer projection):

wherein R is any one of H, F, Cl, Br, I, SH, Me, OMe and SMe. In other words, R replaces the hydroxyl group at carbon 2 of the corresponding aldohexose. Compound I (i.e., a D-glucose-based compound) is particularly preferred. 2-deoxy-D-glucose is Compound I when R is H. 2-deoxy-D-glucose is also referred to herein as "2-DG".

Currently, three HRV species are known, rhinovirus A, B and C. An overview on the HRV taxonomy is given, for example, in Palmenberg et al, 2009. Preferably, for all aspects of the invention, the HRV intended to be treated or prevented is rhinovirus a or B.

In the context of the present invention, a "pharmaceutical composition" refers to any composition comprising at least one active agent (e.g. a modified aldohexose), and preferably one or more excipients, which is pharmaceutically acceptable for administration (in particular for topical or intranasal administration) to a subject (in particular a mammal, especially a human). Suitable excipients are known to the person skilled in the art, for example water (in particular water for injection), saline (saline), Ringer's solution, dextran solution (dextrose solution), buffers, Hank's solution (Hank solution), vesicle-forming compounds (for example lipids), fixed oils (fixed oils), ethyl oleate, 5% dextran-saline solution, substances which enhance isotonicity and chemical stability, buffers and preservatives such as benzalkonium chloride. The pharmaceutical compositions according to the invention may be liquid or readily soluble in liquids such as sterile, deionized or distilled water or sterile isotonic Phosphate Buffered Saline (PBS). Preferably, 1000 μ g (dry weight) of such a composition comprises or consists of: 0.1-990 μ g, preferably 1-900 μ g, more preferably 10-200 μ g of modified aldohexose, and optionally 1-500 μ g, preferably 1-100 μ g, more preferably 5-15 μ g (buffered) salt (preferably to produce isotonic buffer in the final volume), and optionally 0.1-999.9 μ g, preferably 100-999.9 μ g, more preferably 200-. Preferably, 100mg of this dry composition is dissolved in sterile deionized/distilled or sterile isotonic Phosphate Buffered Saline (PBS) to a final volume of 0.1-100ml, preferably 0.5-20ml, more preferably 1-10 ml. However, the dosage and method of administration will generally depend on the individual to be treated. Generally, the modified aldohexose may be administered at a dose of 1 μ g/kg to 10mg/kg, more preferably 10 μ g/kg to 5mg/kg, most preferably 0.1 to 2 mg/kg.

According to a particularly preferred embodiment of the invention, the pharmaceutical composition is a liquid, preferably an aqueous solution. In general, liquid compositions are particularly suitable for intranasal administration, and this is the preferred mode of administration.

In another preferred embodiment, the modified aldohexose, especially 2-deoxy-D-glucose, is in a concentration of 0.01mM to 1000mM, preferably 0.1mM to 500mM, more preferably 0.25mM to 250mM, even more preferably 0.5mM to 100mM, especially 1mM to 50mM or even 2.5mM to 25 mM. These concentration ranges are relatively safe and effective in the treatment of the present invention.

The pharmaceutical composition may comprise other active agents, in particular in order to further increase the efficacy or to achieve further symptom relief of the common cold. Thus, according to another preferred embodiment, the pharmaceutical composition further comprises at least one additional active agent. Preferably, the additional active agent is selected from the group consisting of detumescence agents (decongestants), in particular norepinephrine-releasing agents (e.g. pseudoephedrine (pseudoephedrine), ephedrine (ephedrine) and phenylpropanolamine (phenylpropanolamine)), alpha-adrenergic receptor agonists (e.g. oxymetazoline (oxymetazoline) and xylometazoline (xylometazoline)), and corticosteroids (e.g. budesonide), flunisolide (flunisolide) and fluticasone (fluticasone)), and non-steroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid, ibuprofen, diclofenac (diclofenac) and phenylbutazone. Such agents may be expected to act synergistically with the modified aldohexose against rhinovirus infections (e.g. rhinitis or common cold). In contrast, modified aldohexoses such as 2-deoxy-D-glucose may also be the sole active agent in the pharmaceutical composition (preferably in the presence of one or more excipients).

The subject to be treated according to the invention is preferably a human subject, in particular an immunosuppressed subject or a subject suffering from COPD or asthma. According to a preferred embodiment, one dose of the pharmaceutical composition is administered to a human subject, preferably by the intranasal route (in other words: to the nostrils of the subject, preferably independently to each nostril) and/or by the mucosal route, preferably the mucosa of the respiratory tract, in particular the mucosa of the nasal cavity or the mucosa of the lower respiratory tract. Advantageously, the dose is at least once every other day, preferably at least once daily, more preferably at least twice daily, in particular at least three times daily, and preferably over 2 to 14 days, more preferably 3 to 10 days, especially 4 to 7 days.

For a relatively safe and effective treatment, preferably the total amount of modified aldohexose, especially 2-deoxy-D-glucose, in the dose is from 0,001. mu. mol to 100. mu. mol, preferably from 0, 01. mu. mol to 50. mu. mol, more preferably from 0,025. mu. mol to 25. mu. mol, even more preferably from 0, 05. mu. mol to 5. mu. mol, especially from 0, 1. mu. mol to 2, 5. mu. mol or even from 0, 2. mu. mol to 1, 25. mu. mol.

According to a particularly preferred embodiment, the pharmaceutical composition (or pharmaceutically acceptable formulation) of the invention is for use in the prevention or treatment of the common cold, rhinitis or lower respiratory tract infections, especially in immunosuppressed individuals or individuals with COPD or asthma. Herein, the term "prevention" refers to the complete or almost complete or at least to some extent (preferably to a significant extent) cessation of the occurrence of a disease state or disorder in an individual, especially when the individual is predisposed to the risk of such contracting a disease state or disorder.

The dispenser for the above-mentioned intranasally administered pharmaceutical composition is preferably a nasal spray or a nasal dropper. Nasal sprays and nasal drops are known in the art, see for example us 2,577,321, us 6,000,580 or EP0170198 a 2. Herein, the term "nose drop applicator" refers to any dispenser suitable for (and especially intended for) administering nasal drops.

The inhalation device for administering the above-described pharmaceutical composition, preferably for lower respiratory administration, is preferably a metered dose inhaler, a dry powder inhaler or a nebulizer.

Metered dose inhalers are devices that aerosolize a predetermined dose of a pharmaceutical composition (i.e., produce a relatively short burst of aerosol with a prescribed dose), usually by the patient's own self-administration. For example, US 6,260,549B 1 discloses a metered dose inhaler.

Dry powder inhalers are devices that inhale a dry powder formulation into a patient. Such devices are disclosed, for example, in U.S. Pat. nos. 4,995,385 and 4,069,819. Mature dry powder inhalers are for example

And

nebulizers are devices that generate aerosol for inhalation and can generally be inhaled continuously as long as they are turned on or driven by breathing. Mature nebulizer products include, for exampleAnd

document US9,364,618B2 alsoAn atomizer is disclosed.

The invention is further illustrated by, but not limited to, the following figures and examples.

FIG. 1 inhibition of HRV replication and HRV protein expression in vitro by 2-deoxyglucose. Panel A) shows the effect of 2-DG on HRV 14 replication in HeLaOhio cells and primary human fibroblasts. Cells were infected for 1 hour, washed in PBS, then administered with the indicated concentration of the reagent and incubated in complete growth medium for an additional 6 hours. After the incubation period, RNA was obtained, reverse transcribed into cDNA, and viral RNA load was then assessed by qPCR. A summary of 6 independent experiments is shown. Normalized data was subjected to Wilcoxon signed rank test (Wilcoxon signed rank test) to calculate p < 0.05. Panel B) shows the expression of the viral protein VP1-3 in HRV 14-infected HeLa Ohio cells. Cells were infected as above + -2-DG treatment, then lysed and subjected to Western blot analysis. Representative experiments of 2 independent experiments performed in duplicate are shown.

FIG. 2-deoxyglucose is non-toxic to human cells treatment with 10mM 2-deoxyglucose (with or without HRV) had no significant effect on cell viability of HeLa cells. Cells were infected and treated as described above, then loaded with fluorescently labeled viability dye and evaluated by flow cytometry.

FIG. 3 2-deoxyglucose reduces viral load and ameliorates inflammation due to HRV respiratory tract infection in vivo. C57BL/6 mice were infected intranasally with HRV 1B. + -. 50. mu.L 5mM 2-DG/50. mu.L PBS. 24 hours after infection, mice were euthanized, bronchoalveolar lavage (BAL) performed, and tissues were obtained for qPCR and histological analysis. Shown is the presence of HRV1B RNA normalized to hypoxanthine ribosyltransferase (HPRT) -expressed lung tissue, which is indicative of viral load, as well as the count of leukocytes in BAL (total leukocytes ═ CD45+, neutrophil ═ CD45+ Ly6G +, B ═ CD45+ CD19+, dendritic cell ═ CD45+ CD11c +, T helper cell ═ CD45+ CD3+ CD4+, NK cell ═ CD45+ NK1.1+), which is indicative of pulmonary inflammation.

FIG. 4 2-deoxyglucose ameliorates inflammation due to HRV respiratory tract infection in vivo as histologically confirmed. Two representative Hematoxylin and Eosin (HE) stains of lung tissue after HRV-challenged mice were treated with either placebo or 2-DG are depicted. Representative experiments of 2 independent experiments are shown. In each experiment, 10 mice were tested per infected group and 2 mice were tested in the uninfected control group. The placebo-treated mice infiltrated significantly more peripheral bronchial leukocytes than the 2-DG-treated mice.

Example 1 cell culture and in vitro HRV infection

Experiments involving human material were performed according to the Helsinki declaration principles after approval by the ethical Committee of the university of Vienna medical Committee and written informed consent from participants was obtained (vote No. 1149/2011: cells were isolated and cultured from normal human skin biopsy specimens and analyzed).

HeLa cells (cell line: Ohio, Flow laboratory) were cultured in RPMI 1640 (from Gibco Ltd., Paisley, Scotland) supplemented with 2mM l-glutamine (also from Gibco Ltd., Paisley, Scotland), 100U/mL penicillin, 100. mu.g/mL streptomycin (PAA Laboratories, Austria) and 10% FCS (Gibco). For isolation of fibroblasts, including skin and subcutaneous fat (100-²) And used to isolate mast cells, fibroblasts and keratinocytes. The skin is not evident in both clinical examination and histological examination. Removing subcutaneous tissue and reticular dermis, and cutting the remaining separated thickness of skin to 0.5cm²The small blocks were placed in 2.4U/ml neutral protease (dispase) II (Roche, Vienna, Austria) overnight at 4 ℃. After separation of the epidermis, the dermis was digested in collagenase I (Gibco, Vienna, Austria) for 2 hours at 37 ℃. CD117+ mast cells were isolated using magnetic beads (MACS System; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. To increase the purity of the recovered cells, magnetic separation was repeated on CD117+ cells starting from the first round of separation. CD117+ mast cells were then seeded in dmem (gibco) supplemented with 10% FCS, penicillin/streptomycin (all from Biochrom, Berlin, germany) and 100ng/ml recombinant human stem cell factor (PeproTech, Rocky Hill, NY, usa). After isolation of mast cells, CD117 adherent cells (═ fibroblasts) were cultured at fed RPM as described aboveI1640. (Gschwandtner et al, 2017)

HeLa Ohio cells or fibroblasts were plated on polystyrene plates overnight (Corning Incorporated, Corning, new york, usa). On the next day, cells were infected with HRV 14 (belonging to the rhinovirus type B species), each cell being infected with a specified amount of HRV 14 (multiplicity of infection 3.5-10, depending on the experiment) at 50% tissue culture infection dose (TCID 50). 1 hour after infection, cells were washed with 37 ℃ Phosphate Buffered Saline (PBS), incubated for another 6 hours with media supplemented with the indicated reagents at the indicated concentrations, and then further processed. To assess cell viability, cells were stained with an immortable viability dye (65-0865-14; eBioscience, Vienna, Austria).

Western blot analysis HeLa Ohio cells were infected as described above, 7 hours after infection, cells were lysed in 0.5% Triton-X buffer for 5 minutes on ice after lysis, the suspension was centrifuged at 13000 × g for 5 minutes, and the supernatant was used for further analysis Western blot analysis was performed as described previously (Gualdoni et al, 2015), anti-HRV VP1-3 antibody and anti-GAPDH were diluted at 1: 1000 for use

ECL Western blot substrates (Thermo Fisher Scientific, Waltham, Mass.) were tested on LAS-4000(Fujifilm, Tokyo, Japan). Data analysis, quantification and processing were performed with fiji (imagej) image processing software.

As a result:

2-deoxyglucose strongly inhibited HRV proliferation in HeLa cells and primary human fibroblasts (see FIG. 1A). To verify this effect on proliferation, viral replication was also analyzed at a protein level mirrored by expression of Viral Proteins (VP)1-3 in HeLa cells, and the results were similar to those at RNA level (fig. 1A). 2-deoxyglucose this agent had no measurable effect on cell viability at the doses used (FIG. 2).