Patent Application
20200397805
The present invention relates to pharmaceutical compositions for use in prevention or treatment of a human rhinovirus (HRV) infection, such as the common cold.
HRV is an ssRNA non-enveloped virus of the family of the Picornaviridae. It causes the common cold, a condition responsible for several billion dollars of healthcare costs (Bertino, 2002), and is the causative agent of lower respiratory tract infection in the immunosuppressed (Kaiser et al., 2006; Liu et al., 2010) and both chronic obstructive pulmonary disease (COPD) and asthma exacerbations (Papi et al., 2006; Steinke and Borish, 2016).
To date, therapeutic treatment options effective against HRV are very limited. For instance, the antiviral pleconaril may have a marginal treatment effect against HRV, which is however outweighed by the risk of its side effects. Accordingly, the United States Food and Drug Administration (FDA) rejected approval of pleconaril for treatment of the common cold (Fleischer and Laessig, 2003).
It is thus an object of the present invention to provide a prevention or treatment which is relatively safe and effective against an HRV infection, such as the common cold or a lower respiratory tract infection, especially in an immunosuppressed individual or in an individual having COPD or asthma.
Therefore, the present invention provides a pharmaceutical composition for use in prevention or treatment of an HRV infection. The composition comprises an aldohexose (in particular a D-aldohexose) such as glucose (preferably D-glucose) wherein the hydroxyl group at carbon 2 of the aldohexose is replaced by any one of the following substituents: H, F, Cl, Br, I, SH, methyl (Me), methoxy (OMe) and methylmercapto (SMe). Herein, an aldohexose compound according to this definition is also called “the modified aldohexose”. In an especially preferred embodiment, the modified aldohexose is 2-deoxy-glucose.
In a further aspect, the present invention relates to a dispenser for intranasal administration such as a nasal spray, containing a pharmaceutical composition comprising an aldohexose wherein the hydroxyl group at carbon 2 of the aldohexose is replaced by any one of H, F, Cl, Br, I, SH, Me, OMe and SMe, in particular 2-deoxy-D-glucose.
In another aspect, the present invention relates to an inhalation device, such as a metered-dose inhaler, a dry-powder inhaler or a nebuliser, for administration of a pharmaceutical composition preferably to the lower respiratory tract, comprising a pharmaceutical composition comprising an aldohexose, wherein the hydroxyl group at carbon 2 of the aldohexose is replaced by any one of H, F, Cl, Br, I, SH, Me, OMe and SMe, preferably 2-deoxy-D-glucose.
In yet another aspect of the present invention, a method for delaying the onset of or treating an HRV infection, comprising
In the course of the present invention, it was surprisingly found that treatment with the modified aldohexose effectively inhibits HRV reproduction in-vitro (cf. example 1 and
While 2-deoxy-glucose was disclosed in the prior art to be effective against some enveloped viruses such as influenza virus (see e.g. Kilbourne, 1959 and Courtney et al., 1973), the prior art did not suggest at all that 2-deoxy-glucose is effective against HRV infection. Specifically, influenza virus and HRV are both very distinct in their structure and pathophysiology. Influenza virus belongs to the unrelated family of orthomyxoviridae, is enveloped and binds to sialic acid expressing receptors for infection of epithelial cells (see Sun and Whittaker, 2013). In contrast, HRV is non-enveloped and most subtypes bind to ICAM-1 for host cell entry (Blaas and Fuchs, 2016). Apart from these differences, the viruses evolved very different strategies in endosomal escape and translation initiation (see Blaas and Fuchs, 2016, and Watanabe and Kawaoka, 2015). Consequently, the state-of-the art therapy against influenza virus infections, oseltamivir, is not effective against HRV. Moreover, an inhibitory capacity of 2-deoxy-glucose on any human-pathogenic non-enveloped RNA viruses (such as HRV) was not known in the prior art (cf. e.g. Kang and Hwang, 2006), making the effectiveness of the inventive HRV treatment all the more surprising.
Completely unrelated to therapy, let alone prevention or treatment of a HRV infection, Ojima et al. (Annals of nuclear medicine 3 (1989): 143-147) describe the preparation of a fine powder of 2-deoxy-2-(18F)fluoro-D-glucose (18FDG) suitable for inhalation to diagnose non-infectious lung diseases by means of positron emission tomography (PET). Also Venegas et al. (Database CA, Chemical Abstracts Service, Database accession no. 2013:196451) describe the use of 18FDG for PET imaging of the lung, again merely for diagnostic purposes and entirely unrelated to viral infection, let alone HRV infection.
Also unrelated to RNA viruses, DE 197 06 489 A1 discloses the use of 2-deoxy-glucose for the suppression of viral DNA transcription in virus-associated lesions and/or neoplasms, which are caused by DNA viruses (e.g. human papillomavirus, hepatitis B virus, Epstein-Barr virus).
Kang and Hwang describe the role of 2-deoxy-glucose as an anticancer and antiviral therapeutic. It is disclosed that 2-deoxy-glucose can inhibit multiplication of some enveloped viruses (influenza virus, sindbis virus and semliki forest virus, herpes simplex virus, respiratory syncytial virus and measles virus) by inhibiting viral envelope biosynthesis and virion assembly. No inhibitory capacity on any non-enveloped RNA viruses is disclosed.
The effects of 2-deoxy-glucose on enveloped viruses have 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 functions of respiratory syncytial and parainfluenza 3 viruses. Kilbourne (Nature 183 (1959) 271-272) discloses the inhibition of influenza virus multiplication by 2-deoxy-glucose. Hodes et al. (Virology 63 (1975) 201-208) describe the inhibition of respiratory syncytial, parainfluenza 3 and measles viruses by 2-deoxy-glucose. However, prior to the present invention there was no indication that 2-deoxy-glucose could also be effective against non-enveloped RNA viruses.
Unrelated to 2-deoxy-glucose, Arita et al. (Carbohydrate Research 62 (1978) 143-154) disclose the synthesis of a variety of analogues and derivatives of phenyl glycosides and the examination of their antiviral activity against some enveloped viruses (influenza virus and Herpes simplex virus).
In connection with all aspects of the present invention, the modified aldohexose contained in the pharmaceutical composition (or pharmaceutically acceptable formulation) preferably is one of compounds I-VIII (depicted as Fischer projections):
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 respective aldohexose. Compound I (i.e. the compound based on D-glucose) is particularly preferred. 2-deoxy-D-glucose is compound I wherein R is H. 2-deoxy-D-glucose is also called “2-DG” herein.
At present, three HRV species are known, namely Rhinovirus A, B and C. An overview over HRV taxonomy is for instance given in Palmenberg et al., 2009. Preferably, in connection with all aspects of the present invention, the HRV to be treated or prevented is a Rhinovirus A or B.
In the context of the present invention, the expression “pharmaceutical composition” refers to any composition comprising at least one active agent (e.g. the modified aldohexose), and preferably one or more excipients, which is pharmaceutically acceptable for administration (in particular for topical administration or intranasal administration) to an individual, especially a mammal, in particular a human. Suitable excipients are known to the person skilled in the art, for example water (especially water for injection), saline, Ringer's solution, dextrose solution, buffers, Hank solution, vesicle forming compounds (e.g. lipids), fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives, such as benzalkonium chloride. The pharmaceutical composition according to the present invention may be liquid or ready to be dissolved in liquid such as sterile, deionised or distilled water, or sterile isotonic phosphate-buffered saline (PBS). Preferably, 1000 μg (dry-weight) of such a composition comprises or consists of 0.1-990 μg, preferably 1-900 μg, more preferably 10-200 μg modified aldohexose, and optionally 1-500 μg, preferably 1-100 μg, more preferably 5-15 μg (buffer) salts (preferably to yield an isotonic buffer in the final volume), and optionally 0.1-999.9 μg, preferably 100-999.9 μg, more preferably 200-999 μg other excipients. Preferably, 100 mg of such a dry composition is dissolved in sterile, de-ionised/distilled water or sterile isotonic phosphate-buffered saline (PBS) to yield a final volume of 0.1-100 ml, preferably 0.5-20 ml, more preferably 1-10 ml. The dosage and method of administration, however, typically depends on the individual to be treated. In general, the modified aldohexose may be administered at a dose of between 1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg.
According to a particularly preferred embodiment of the present invention, the pharmaceutical composition is liquid and preferably an aqueous solution. In general, liquid compositions are especially suitable for intranasal administration, which is the preferred mode of administration.
In a further preferment, the concentration of the modified aldohexose, in particular 2-deoxy-D-glucose, is 0.01 mM-1000 mM, preferably 0.1 mM-500 mM, more preferably 0.25 mM-250 mM, even more preferably 0.5 mM-100 mM, in particular 1 mM-50 mM or even 2.5 mM-25 mM. These concentration ranges are relatively safe and effective in connection with the inventive treatment.
The pharmaceutical composition may comprise further active agents, in particular to further increase effectiveness or achieve further symptom reduction of the common cold. Thus, according to a further 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 decongestants, in particular norepinephrine releasing agents (such as pseudoephedrine, ephedrine and phenylpropanolamine), α-adrenergic receptor agonists (such as oxymetazoline and xylometazoline), and corticosteroids (such as budesonide, flunisolide, and fluticasone), and nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid, ibuprofen, diclofenac, and phenylbutazone. Such agents can be expected to act synergistically together with the modified aldohexose against a rhinovirus infection such as rhinitis or the common cold. By contrast, the modified aldohexose such as 2-deoxy-D-glucose may also be the single active agent in the pharmaceutical composition (preferably in the presence of one or more excipients).
The individual to be treated according to the present invention is preferably a human individual, in particular an immunosuppressed individual or an individual having COPD or asthma. According to a preferred embodiment, a dose of the pharmaceutical composition is administered to a human individual, preferably intranasally (in other words: into a nostril of the individual, preferably into each nostril independently) and/or to a mucosal membrane, preferably a mucosal membrane of the respiratory tract, in particular a mucosal membrane of the nasal cavity or of the lower respiratory tract. Advantageously, the dose is administered at least once every other day, preferably at least once per day, more preferably at least twice per day, in particular at least thrice per day, and preferably for 2-14 days, more preferably for 3-10 days, in particular for 4-7 days.
For relatively safe and effective treatment, according to a preference, the total amount of the modified aldohexose, in particular 2-deoxy-D-glucose, in the dose is 0.001 μmol-100 mol, preferably 0.01 μmol-50 mol, more preferably 0.025 μmol-25 mol, even more preferably 0.05 μmol-5 mol, in particular 0.1 μmol-2.5 μmol or even 0.2 μmol-1.25 mol.
According to a particularly preferred embodiment, the pharmaceutical composition (or pharmaceutically acceptable formulation) according to the present invention is used for prevention or treatment of the common cold, a rhinitis or a lower respiratory tract infection, especially in an immunosuppressed individual or in an individual having COPD or asthma. The term “preventing” or “prevention” as used herein means to stop a disease state or condition from occurring in an individual completely or almost completely or at least to a (preferably significant) extent, especially when the individual is predisposed to such a risk of contracting a disease state or condition.
The dispenser for intranasal administration of the pharmaceutical composition described hereinabove is preferably a nasal spray or a nose drop applicator. Nasal sprays and nose drop applicators are known in the art and e.g. disclosed in U.S. Pat. Nos. 2,577,321, 6,000,580 or EP 0 170 198 A2. As used herein, the term “nose drop applicator” refers to any dispenser suitable for, especially intended for, the administration of nose drops.
The inhalation device for administration of the pharmaceutical composition described hereinabove (preferably to the lower respiratory tract) is preferably a metered-dose inhaler, a dry-powder inhaler or a nebuliser.
A metered-dose inhaler is a device that aerosolises a pre-defined dose of a pharmaceutical composition (i.e. produces a comparatively short burst of aerosol with a defined dose), usually for self-administration by the patient. A metered-dose inhaler is for instance disclosed in U.S. Pat. No. 6,260,549 B1.
Dry-powder inhalers are devices for inhalation of dry-powder formulations by the patient. Such devices are for instance disclosed in U.S. Pat. Nos. 4,995,385 and 4,069,819. Established dry-powder inhalers are for instance SPINHALER®, ROTAHALER®, FLOWCAPS®, INHALATOR®, DISKHALER® and AEROLIZER®.
Nebulisers are devices that produce aerosols for inhalation, typically continuously as long as they are switched on or breath-actuated. Established nebuliser products are for instance Aeroneb® and Pari®. Document U.S. Pat. No. 9,364,618 B2, e.g., also discloses a nebuliser.
The present invention is further illustrated by the following figures and examples, without being restricted thereto.
Experiments involving human material were carried out according to the Declaration of Helsinki principles after approval by the ethics committee of the Medical University of Vienna and after obtaining written informed consent from the participants (vote number 1149/2011: isolation and culture of cells from and analysis of normal human skin biopsies).
HeLa cells (strain: Ohio, Flow laboratories) were cultivated in RPMI 1640 supplemented with 2 mM 1-glutamine, (both Gibco Ltd., Paisley, Scotland), 100 U/mL penicillin, 100 μg/mL streptomycin (PAA Laboratories, Austria) and 10% FCS (Gibco). For fibroblast isolation, tissue samples including skin and subcutaneous fat (100-300 cm2) were obtained from patients undergoing routinely performed body contouring surgeries and were used for the isolation of mast cells, fibroblasts, and keratinocytes. The skin was inconspicuous upon clinical inspection and in histology. Subcutaneous tissue and reticular dermis were removed and the remaining split thickness skin was cut into 0.5 cm2 pieces and placed overnight at 4° C. in 2.4 U/ml dispase II (Roche, Vienna, Austria). After the separation of the epidermis, dermis was digested in collagenase I (Gibco, Vienna, Austria) at 37° C. for 2 h. 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 recovered cells, magnetic isolation was repeated with CD117+ cells from the first isolation round. CD117+ mast cells were then seeded in DMEM (Gibco) supplemented with 10% FCS, penicillin/streptomycin (both Biochrom, Berlin, Germany), and 100 ng/ml recombinant human stem cell factor (PeproTech, Rocky Hill, N.Y., USA). After the isolation of mast cells, CD117 adherent cells (=fibroblasts) were cultured in supplemented RPMI 1640 as above. (Gschwandtner et al., 2017)
HeLa Ohio cells or fibroblasts were plated on polystyrene plates overnight (Corning Incorporated, Corning, N.Y., USA). On the subsequent day, cells were infected with the indicated amount of 50% Tissue culture Infective Dose (TCID 50) of HRV 14 (belonging to species Rhinovirus B) per cell (multiplicity of infection 3.5-10, depending on the experiment). After 1 h of infection, cells were washed with 37° C. phosphate-buffered saline (PBS) and incubated another 6 h with medium±the indicated agent in the indicated concentration before further processing. For assessment of cell viability, cells were stained with a fixable viability dye (65-0865-14; eBioscience, Vienna, Austria).
Western blot analysis: HeLa Ohio cells were infected as described above. After 7 h of infection, cells were lysed in 0.5% Triton-X buffer for 5 min on ice. After lysis, the suspension was centrifuged for 5 min at 13000×g, and the supernatant was utilized for further analysis. Western blot analysis was performed as described before (Gualdoni et al., 2015). Anti-HRV VP1-3 antibody and anti-GAPDH were used in a dilution of 1:1000. Detection was performed with Pierce® ECL Western blotting substrate (Thermo Fisher Scientific, Waltham, Mass.) on a LAS-4000 (Fujifilm, Tokyo, Japan). Data analysis, quantification, and processing were performed with Fiji (ImageJ) image processing software.
Results:
2-deoxyglucose strongly inhibited HRV reproduction both in HeLa cells and in primary human fibroblasts (see
All animal experimentation protocols were evaluated by the Animal Ethics Committee of the Medical University of Vienna and approved by the Ministry of Economy and Science (BMWFW-66.009/0356_WF/V/3b/2015). Animal husbandry and experimentation was performed according to the Federation of Laboratory Animal Science Association guidelines. Female C57BL/6 J mice aged 6 to 8 wk from in-house breeding (originally obtained from The Jackson Laboratory, Bar Harbor, Me., USA) were used for all experiments. An animal care professional not related to the study performed allocation of mice to the groups randomly.
Experiments were performed according to a published protocol (Bartlett et al., 2008) with minor modifications. Mice were sedated with isoflurane and an inoculum of 5×10{circumflex over ( )}6 TCID50 of HRV1B (belonging to species Rhinovirus A) in PBS was applied intranasally. Either 5 mM 2-DG dissolved in PBS or PBS solo were simultaneously applied intranasally. After 24 h, mice were euthanized and a bronchioalveolar lavage performed. One lung lobe was obtained for PCR analysis and one for histological examination. For PCR analysis, material was homogenized before RNA isolation with the RNEasy kit as above. For histological analysis, lung lobes were fixed in 10% formaldehyde and embedded in paraffin. Lung sections (4 μm) were stained with H&E and evaluated by a pathologist blinded to group allocation.
Results:
In this well-established murine model of rhinovirus airway infection (Bartlett et al., 2008), 2-DG reduced HRV load in infected lung tissue and reduced virus induced lung inflammation (see
A nasal spray as e.g. disclosed in U.S. Pat. No. 6,000,580 is provided containing 10 ml of the following pharmaceutical composition: 10 mM 2-deoxy-D-glucose, 0.9% w/v NaCl dissolved in sterile deionised water.
A nose drop applicator as e.g. disclosed in EP 0 170 198 A2 is provided containing 5 ml of the following pharmaceutical composition: 0.5 mM 2-deoxy-2-fluoro-D-mannose, 0.05% (w/v) oxymetazoline hydrochloride, 0.05% (w/w) benzalkonium chloride, 85% (v/v) glycerol in sterile deionised water.
A nebuliser as e.g. disclosed in U.S. Pat. No. 9,364,618 B2 is provided comprising the following pharmaceutical composition in its fluid reservoir: 35 mM 2-deoxy-D-glucose, 0.9% w/v NaCl dissolved in sterile deionised water. The nebuliser is used to deliver the composition by inhalation as an aerosol to the lower respiratory tract of an immune-suppressed patient suffering from a rhinovirus infection in the lungs.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18154088.1 | Jan 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/097060 | 12/28/2018 | WO | 00 |
