EP4247350A1 - Composition for use in a method for the prevention and treatment of diseases of the respiratory system - Google Patents

Composition for use in a method for the prevention and treatment of diseases of the respiratory system

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Publication number
EP4247350A1
EP4247350A1 EP21830494.7A EP21830494A EP4247350A1 EP 4247350 A1 EP4247350 A1 EP 4247350A1 EP 21830494 A EP21830494 A EP 21830494A EP 4247350 A1 EP4247350 A1 EP 4247350A1
Authority
EP
European Patent Office
Prior art keywords
composition
amount comprised
msm
chlorhexidine
nac
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21830494.7A
Other languages
German (de)
French (fr)
Inventor
Umberto DI MAIO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Neilos SRL
Original Assignee
Neilos SRL
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Filing date
Publication date
Application filed by Neilos SRL filed Critical Neilos SRL
Publication of EP4247350A1 publication Critical patent/EP4247350A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/04—Drugs for disorders of the respiratory system for throat disorders

Definitions

  • composition for use in a method for the prevention and treatment of diseases of the respiratory system “Composition for use in a method for the prevention and treatment of diseases of the respiratory system”
  • the present invention relates to a composition comprising N-acetylcysteine (NAG), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM).
  • the present invention also relates to a composition comprising N-acetylcysteine (NAG), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM) useful in the prevention and/or in the treatment of diseases associated with the respiratory system.
  • the combination is particularly effective thanks to the synergistic action of the components thereof.
  • the respiratory system is the set of organs and tissues that allow the breathing process.
  • Respiration consists of assimilating the oxygen inspired with air and the simultaneous ejection of the carbon dioxide generated by cellular activity and representing a waste product.
  • the main anatomical elements of the respiratory system are: the nose with the cavities thereof, the mouth, the pharynx, the nasopharynx, the larynx, the trachea, the bronchi, the bronchioles, the lungs and the muscles of respiration, muscles of the diaphragm and intercostal muscles.
  • Diseases affecting the respiratory system may be of the following types: obstructive, restrictive, vascular or infectious/environmental.
  • the respiratory system can be divided into two main components: the upper respiratory tract (or upper airways) and the lower respiratory tract (or lower airways).
  • the upper respiratory tract includes the nose with the nasal cavities thereof, the mouth, the pharynx, the nasopharynx and the larynx; the lower respiratory tract instead includes the trachea, the bronchi, the bronchioles, the lungs and the muscles of respiration, muscles of the diaphragm and intercostal muscles.
  • Cold is a viral disease, the most common agents are rhinovirus, human parainfluenza virus, respiratory syncytial virus, adenovirus, echovirus and coxsackievirus. Agents vary depending on the age of the host and the time of year. Symptoms include nasal secretion, nasal obstruction and throat irritation, usually associated with low-intensity fever, malaise, sneezing and nasal secretions that could become purulent.
  • Symptoms are not specific and not characteristic of any agent.
  • the standard duration of symptoms is about 7 days.
  • the common cold in children resolves alone in most cases.
  • the use of antibiotics is potentially harmful, because it increases the risk of colonisation with resistant organisms. This can lead to subsequent antibiotic-resistant bacterial infections
  • Cough occurs in 60-80% of common colds, symptoms usually last 2-7 days. Patients usually improve within 10 days, but sometimes cough and nasal secretions may persist for 2 weeks or more.
  • Sinusitis consists of inflammation of the mucosal lining of one or more paranasal sinuses. It can be caused by infections or non-infectious causes such as allergies. In children it manifests itself as complication of a common cold where there is a congestion of the nasal mucous membranes and sinusitis, leading to rhinosinusitis.
  • Viral rhinosinusitis is 20 -200 times more common than bacterial sinusitis. Sinusitis is classified according to the duration of symptoms in acute sinusitis (up to 3 weeks), subacute sinusitis (3-10 weeks) and chronic sinusitis (>10 weeks).
  • Sinusitis manifests itself when a viral agent in the upper respiratory airways produces inflammation of the mucous membranes which leads to obstruction of the sinuses. This in turn leads to the entrapment of liquids into the sinus cavities and to the proliferation of the normal bacterial flora of the upper respiratory airways.
  • the most common organisms that cause sinusitis are S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus and S. pyogenes J
  • most of the rhinosinusitises are viral and, therefore, about 60% resolve spontaneously without antimicrobial therapy.
  • the symptoms can be sinus morbidity, pain in the teeth, headache and high fever.
  • Rhinorrhoea is mostly purulent, but it can also be serous or aqueous. Other indicators of sinusitis are the presence of severe symptoms during an episode of respiratory infection. These include high fever > 39 ⁇ , persistent fever, periorbital swelling and facial pain.
  • Pharyngitis is an inflammatory disease of the mucous membranes of the throat and the underlying structures.
  • Pharyngitis is divided into two categories: symptomatic nasal disease (nasopharyngitis) and disease without nasal involvement (pharyngitis or tonsillopharyngitis).
  • Nasopharyngitis is almost always of viral aetiology, while pharyngitis without nasal signs has various aetiology signs including bacteria, viruses, fungi and other infectious agents.
  • Adenoviruses are the most common agents that trigger the nasopharyngitis; other viruses are flu, parainfluenza and enteroviruses.
  • Bacterial agents that cause pharyngitis include Streptococcus pyogenes, H. influenzae, C. diphtheriae and N. meningitidis.
  • Group A streptococci cause about 15% of bacterial pharyngitis and about 1-2 out of 10 patients with sore throat. Infection in most of the other cases can therefore be attributed to viruses.
  • Symptoms of pharyngitis comprise: inflammation of the pharynx, characterised by erythema, exudate or ulcer, pharyngeal pain, dysphagia and fever, while rhinorrhoea, cough, raucousness, conjunctivitis and diarrhoea suggest a viral aetiology.
  • Pharyngitis in paediatric age is of viral type, whereas in pharyngitis of bacterial type it is often due to streptococcus.
  • the published document WO 2019/064255 A1 discloses a composition comprising or, alternatively, consisting of an effective amount of a mixture comprising or consisting of at least one Pelargonium sidoides extract, an Adhatoda vasica extract and N-acetylcysteine for use in a method for the preventive or therapeutic treatment of at least one disorder of the respiratory tract in a subject.
  • the combination subject of the present invention consists of a combination of hyaluronic N- acetylcysteine (NAC), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM).
  • NAC hyaluronic N- acetylcysteine
  • MSM methylsulfonylmethane
  • NAC N-acetylcysteine
  • N-acetylcysteine has various activities, among which the antioxidant action stands out.
  • NAC can eliminate ROSs, increase glutathione levels, undergo autoxidation and act as a reducing agent.
  • NF-kB Activation of NF-kB in response to a variety of signals (IL-1, TNF, H2O2) may be inhibited by NAC, showing that ROSs are signalling modulators.
  • NAC may interfere with cell adhesion, oxidative stress, stability of atherosclerotic plaques at risk of rupture in the cardiovascular system and reduce lung inflammation, fibrosis and smoking-related changes.
  • arthritis In arthritis it can reduce inflammation, synovial invasion and cartilage damage.
  • this agent inhibits angiogenesis, reduces smoking-induced carcinogenesis, induces selective apoptosis of transformed cells, interferes with the cell cycle, and it has anti-invasive and anti- metastatic effects.
  • chondrocytes, neurons it promotes cell growth and survival.
  • NAC decreases oxidative stress from various agents, and it provides considerable protection against fibrosis, viral infections, and toxic agents.
  • NAC reduces TNF-induced NF-kB activation, and it also reduces TNF and RANTES-induced p38 MARK production.
  • NAC may also exert the antioxidant effect thereof indirectly facilitating the biosynthesis and the availability of GSH.
  • N-acetylcysteine has been shown to inhibit expression, in human endothelial cells of VCAM-1, resulting in blocking of monocyte adhesion in response to stimuli such as tumour necrosis factor a (TNF-a). This effect is demonstrated by the suppression of NF-KB activation.
  • NAC has a marked mucolytic activity, thanks to the ability to break the disulfide bridges of mucus protein complexes, depolymerizing mucin molecules.
  • the immunomodulating activity of NAC was assayed by means of in vivo tests.
  • a 0.1 g/kg solution of buthionme sulfoximme (BSO) was injected into a group of rats and after 2 hours LPS was injected in a dose of 0.1 mg/kg in 0.2 ml of pH saline solution.
  • the animals were subjected to blood sampling and then sacrificed to remove liver and pancreas in order to test glutathione activity and TNF concentration.
  • the results of the test show that in the group treated with NAG the levels of TNF are significantly reduced at a minimum effective dose of 0.5 g/kg.
  • the TNF-inhibitory effect was compared to treatment with GSH, with which it showed comparable activity, at the dose of 1g/kg.
  • the efficacy of NAG was also evaluated by administering BSO which inhibits GSH increasing TNF.
  • NAG antagonised BSO.
  • the cavies were divided into groups. 2 treated groups in which 0.6M of NAG was administered for 5 minutes to one through the inhalation route, while another treated group received a 20 mg/kg NAG tablet, while the control groups were treated with saline solution using the same routes of administration.
  • the animals were placed in a chamber in which they inhaled citric acid for 3 minutes at a concentration of 0.3 mol/L. The improvement of the airflow following coughing was evaluated using a pneumotachograph and the sounds and movements accompanying this respiratory response were analysed.
  • Patient were divided into control group and treated group.
  • the treated group was administered a 600 mg NAC tablet 2 times a day for 6 months.
  • the test result showed that the incidence of flu in the treated group was 29% compared to 51%. This incidence was also analysed for each month of treatment, showing that during the period of the year where the frequency of infection is higher, the treated group showed a markedly lower portion of infected patients with respect to placebo, 13% with respect to 30%.
  • NAC neurodegenerative disease 2019
  • symptoms related to flu episodes including symptoms in the respiratory system such as rhinorrhoea, sore throat, catarrh and cough, and general symptoms such as headache and myalgia-arthralgia.
  • protective effects do not depend simply on mucolytic action, but they are likely to be further linked to other mechanisms, such as antioxidant and immunomodulant effects.
  • NAC acts by inhibiting cytokines.
  • the test result showed a 41% reduction in disease exacerbation in the group treated with NAC. Furthermore, the number of patients with two or more exacerbations in the NAC group is 29%, with respect to the control group where it is 49%. The number of days of disease in the NAC group is 82 days, with respect to 155 days in the control group. Furthermore, a reduction in exacerbation severity in the treated group with respect to the control group was observed.
  • Chlorhexidine is a biguamde, consisting of two chloro-guanidine chains linked by hexamethylene chains.
  • the substance is colourless, odourless, bitter-tasting and insoluble in water, it covalently binds the proteins of the skin and of the mucosa, thus ensuring a long duration of action and poor systemic absorption.
  • Chlorhexidine is absorbed by the phosphorylated component of proteins of the bacterial wall. Binding with microbial proteins triggers lysis of the cytoplasmic membrane, at bacteriostatic concentrations, while in bactericidal amounts chlorhexidine forms an irreversible bond with adenosine triphosphate and nucleic acids, precipitating them.
  • chlorhexidine Besides having bacteriostatic, bactericidal, fungicidal activity and activity against some viral species, chlorhexidine also has a greater affinity for the bacterial wall of Gram+ bacteria.
  • Chlorhexidine effectively acts on the dental biofilm which produces enzymes that degrade fibronectin.
  • the advantage in treatment with chlorhexidine lies in the broad spectrum of action thereof, on a wide variety of microbial agents.
  • the antimicrobial activity of chlorhexidine was tested on various cell lines including rabbit kidney cells and human foreskin fibroblasts. Each cell line was associated with a viral strain, particularly flu virus was inoculated into the cell line in the Madin Darby canine kidney, and the human parainfluenza virus on the African green monkey kidney cell line. Antiviral activity was compared to a placebo and monitored over time, monitored between 30 seconds after treatment, up to the subsequent 15 minutes.
  • the test result showed a significant time-dependent decrease in viral growth.
  • the inhibition of human parainfluenza virus changes from 59% after 30 seconds up to 99% after 15 minutes, while the flu virus decreases by 93% after 30 seconds up to 98% after 15 minutes.
  • the trial was conducted on a cohort of patients, randomised and divided into two groups treated with penicillin V and placebo in the form of spray, while the other group was treated with the same penicillin enriched with 0.12% chlorhexidine gluconate and 0.15% benzamide chloride. Both therapies were administered for 10 days, 4 times a day.
  • test result showed a decrease in symptoms, significantly greater in the treated group with respect to the control group, showing the effectiveness thereof in combating this type of infections.
  • Another study tested the bacteriostatic activity of chlorhexidine, showing that the MIC for some bacterial species is: 1 :500000 for Streptococcus pyogenes, 1 :500000 for Corynebacterium diphteriae, 1 :500000 for Diplococcus pneumoniae.
  • the antimicrobial activity of chlorhexidine against some microbial species is also tested in this study.
  • a 0.5% solution of chlorhexidine in 50% of alcohol and subsequently a 1% solution are inoculated into a culture broth containing Micrococcus pyogenes, a bacterium responsible for some respiratory diseases, and compared with a control broth.
  • the test result showed the absence of bacteria in the media treated with chlorhexidine.
  • the test was conducted on a cohort of volunteers, taking the bacterial strain through a pharyngeal swab, in particular from the tonsils.
  • the presence of p-haemolytic streptococcus was evaluated through a swab and subsequently 2 tablets of 0.2% chlorhexidine were administered to one group of patients 5 times a day for one week, while another group received placebo.
  • the result showed the presence of p-haemolytic streptococcus in only a small part of patients treated with chlorhexidine, and showing the efficacy thereof.
  • Methylsulfonylmethane or more simply MSM is an organosulfide, an organic compound containing sulphur.
  • the nutraceutical properties of methylsulfonylmethane derive from the sulphur content thereof in a bioavailable form, thanks to the presence of an organic component which facilitates the absorption thereof.
  • MSM inhibits the transcriptional activity of NF-kB, preventing the degradation of the inhibitor of this molecule. Furthermore, MSM showed to block the phosphorylation of the p65 subunit at serine-536.
  • regulatory cascade of NK-kB is a pro-inflammatory signalling mechanism, responsible for activating gene expression cytokines, chemokines and adhesion molecules.
  • IL-1 interleukin-1
  • IL-6 interleukin-6
  • TNF-o interleukin-1
  • IL-1 and TNF-o are inhibited in a dose-dependent manner.
  • MSM may also decrease the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing NF-KB; thus reducing the production of vasodilator agents such as nitric oxide (NO) and prostanoids. Besides modulating the vascular tone, NO also regulates mast cell activation. Therefore, MSM may indirectly play an inhibitory role on mast cells. Decreasing cytokines and vasodilator agents allows to inhibit the flow and recruitment of immune cells to sites of inflammation. MSM negatively affects the expression of the NLRP3 inflammasome, decreasing the production and transcription of NF-KB and blocking the ROS activation signal.
  • iNOS inducible nitric oxide synthase
  • COX-2 cyclooxygenase-2
  • the MSM affects the activation of at least four types of transcription factors: NF-KB, signal transducers and activators of transcription (STATs), p53 and Nrf2. By mediating these transcription factors, MSM can regulate the balance between ROS and antioxidant enzymes.
  • MSM can inhibit the transcriptional activity of NF-KB and therefore reduce the expression of enzymes and cytokines involved in the production of ROS.
  • the down-regulation of COX-2 and iNOS reduces the amount of superoxide radicals and of (NO), respectively.
  • MSM suppresses expression of cytokines such as TNF-o, which may reduce any ROS generated at mitochondrial level. Decreases in cytokine expression may also be involved in reduced paracrine signalling and activation of other transcription factors.
  • MSM has been shown to suppress the expression or activity of STAT transcription factors in a given number of tumour cell lines in vitro.
  • the janus kinase (Jak)ZSTAT signalling pathway is involved in the regulation of apoptosis, differentiation and proliferation-related genes, which generate ROS as a necessary signalling component.
  • Signalling through the Jak/STAT pathway may also be inhibited by reduced cytokine expression.
  • the reduction of the Jak/STAT pathway may further decrease ROS synthesis by decreasing the expression of oxidase and B cell lymphoma 2 (Bcl-2).
  • p53 shows a dichotomic oxidative function, depending on levels of intracellular ROS, it exerts an antioxidant action at low levels of intracellular ROS and pro-oxidative activities at high levels of ROS.
  • the antioxidant function of p53 stimulates scavenging enzymes such as sestrin, glutathione peroxidase (GPx) and aldehyde dehydrogenase (ALDH).
  • GPx glutathione peroxidase
  • ADH aldehyde dehydrogenase
  • the pro-oxidative function of p53 stimulates oxidase, and at the same time suppresses antioxidant genes.
  • Nrf2 Murine neuroblastoma cells cultured with the human immunodeficiency virus type 1 transactivating the regulatory protein (HIV-1 Tat) showed reduced nuclear translocation of Nrf2; however, inserting into the MSM culture medium allowed to resume the translocation of Nrf2 to the nucleus at regular levels.
  • Nrf2 is well documented for the association thereof with antioxidant enzymes including glutamate-cysteine ligase (GCL), superoxide dismutase (SOD), catalase (CAT), peroxiredoxin (Prdx), GPx, glutathione S-transferase (GST), and others.
  • GCL glutamate-cysteine ligase
  • SOD superoxide dismutase
  • CAT catalase
  • Prdx peroxiredoxin
  • GST glutathione S-transferase
  • Stress can trigger an acute response by the innate immune system and a resulting adaptive immune response if the stress factor is pathogenic.
  • MSM plays a crucial role in supporting the immune response. Through mechanisms such as those mentioned above, MSM modulates the immune response through the link between oxidative stress and inflammation.
  • MSM has been shown to reduce IL-6, which is involved in the maintenance of chronic inflammation.
  • MSM histamine release from mast cells is inhibited by DMSO2; however, the effects of MSM on histamine release remain unknown. Previous studies show that MSM has an inhibitory role on vascular function. Other in vitro studies demonstrate that MSM has the ability to dampen the expression of vasodilating agents such as nitric oxide (NO) and prostanoids. A reduction of NO protects macrophages against NO stimulated apoptosis.
  • NO nitric oxide
  • MSM may provide other immune modulatory effects linked to cell cycle and cell death.
  • MSM does not chemically neutralise ROS in stimulated neutrophils but it instead suppresses mitochondrial generation of superoxide, hydrogen peroxide, and hypochlorous acid. Furthermore, MSM is able to restore the reduced glutathione (GSH)Zoxidised glutathione (GSSG) ratio to normal levels, decrease the production of NO, and reduce the production of neuronal ROS following exposure to HIV-1 Tat.
  • Animal studies using MSM as primary treatment for experimentally induced lesions show reductions in malondialdehyde (MDA), GSSG, myeloperoxidase (MPO), NO, carbon monoxide (CO) and increase in GSH, CAT, SOD and GPx.
  • MDA malondialdehyde
  • MPO myeloperoxidase
  • CO carbon monoxide
  • a clinical trial was conducted to test anti-inflammatory activity in patients with allergic rhinitis.
  • the cohort of patients was treated with capsules of 650 mg of MSM 4 times a day, 2 in the morning and two in the afternoon, for 30 days.
  • the group of patients non-responsive to the therapy continued for another 14 days, doubling the treatment dose.
  • MSM seems to be well tolerated and safe.
  • a series of toxicity studies was conducted on various animal species, including rats, mice and dogs.
  • a preliminary toxicity study report there was reported a single mortality in a female rat to which there was administered an oral aqueous dose of 15.4 g/kg after two days; however, the post-mortem necroscopic examination did not reveal any serious disease.
  • Other technical reports show that mild skin and eye irritation were observed when MSM is applied topically.
  • the FDA recognises MSM as GRAS, considering it safe at doses below 4845.6 mg/day.
  • N-acetylcysteine NAG
  • composition subject of the present invention consists of an association or combination of N- acetylcysteine (NAG), chlorhexidine or a salt thereof and methylsulfonylmethane (MSM) for use in a method for the prevention and/or treatment of diseases of the respiratory system both in humans and in animals.
  • NAG N- acetylcysteine
  • MSM methylsulfonylmethane
  • Such diseases are selected from the group comprising or, alternatively, consisting of; rhinitis, allergic rhinitis, nasopharyngitis or cold, cough, nasal congestion, accumulation of nasal mucus, flu, rhinosinusitis, sinusitis, pharyngitis, epiglottitis, laryngitis, tracheitis, bronchitis, bronchiolitis and bronchiectasis.
  • N-acetylcysteine is present in an amount comprised between 0.01% and 30%, more preferably in an amount comprised between 0.05% and 20%, even more preferably in an amount comprised between 0.1% and 10%, on the total weight or volume of the composition; chlorhexidine or a salt thereof is present in an amount comprised between 0.01% and 40%, more preferably in an amount comprised between 0.05% and 30%, even more preferably in an amount comprised between 0.1% and 20%, on the total weight or volume of the composition; methylsulfonylmethane (MSM) is present in an amount comprised between 0.05% and 35%, more preferably in an amount comprised between 0.1% and 25%, even more preferably between 0.5% and 5%, on the total weight or volume of the composition.
  • NAG N-acetylcysteine
  • the dosage form may be a pharmaceutical composition or a medical device or a cosmetic or a dietary supplement, a food for special medical purposes or a complementary feed including the aforementioned active ingredients mixed to each other.
  • the preferred route of administration is nasal, topical, inhalation, oral.
  • the pharmaceutical forms which can be used according to the present invention are selected from nasal sprays, vials for nasal instillation, vials for inhalation through aerosol, nasal drops, nasal creams, nasal ointments or other pharmaceutical forms suitable for nasal, topical, inhalation, oral administration.
  • composition of the present invention further optionally comprises pharmaceutical or food or cosmetic grade excipients and additives.
  • Suitable excipients may be selected from those usually known in the state of the art and they include, though not limited thereto: diluents (for example dibasic calcium phosphate, lactose, microcrystalline cellulose and cellulose derivatives), thickeners (for example gums, hydroxypropyl methyl cellulose and other cellulose derivatives), sweeteners (for example sorbitols, mannitol and other polyols, acesulfame K, aspartame, cyclamates, saccharin, sucralose), lubricants (for example magnesium stearate, stearic acid, waxes), dispersants, surfactants (for example sodium lauryl sulfate and polysorbate), flavour-enhancement agents, adsorbents (for example silica gel, talc, starch, bentonite, kaolin), glidants and anti-adherents (for example talc, colloidal silica, maize starch, silicon
  • Example 1 spray for nasal application
  • Example 5 solution for inhalation application through aerosol
  • N-acetylcysteine N-acetylcysteine
  • MSM methylsulfonylmethane
  • the experimental groups differ in the group treated with single substance, group treated with combination of substances and control group.
  • the antiviral activity is for example evaluated by means of various types of tests which allow to evaluate the effect of the tested compounds on the plaques of the virus, on a particular effect thereof (cytotoxicity), on some proteins fundamental for the viruses, or on particular phases of the reproduction cycle such as attachment, inlet, uncoating, replication, assembly, release etc.
  • Antimicrobial activity is evaluated on the main bacterial strains belonging to the categories of Gram positive and/or Gram negative and/or other microbial species, such as for example S.aureus (respiratory pathogen model).
  • In vitro assays such as for example DPPH tests, radical scavenging activity on nitric oxide or peroxynitrite radical, TEAC (total radical-trapping antioxidant parameter), FRAP (ferric reducing- antioxidant power), HORAC (hydroxyl radical averting capacity), ORAC (oxygen radical absorbance capacity) tests and the like, are suitable to demonstrate the antioxidant efficacy of the composition according to the present invention.
  • the antioxidant activity can also be evaluated by means of cell tests known to the person skilled in the art.
  • a composition comprising a combination of N-acetylcysteine (NAC), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM).
  • NAC N-acetylcysteine
  • MSM methylsulfonylmethane
  • composition according to FR1 wherein N-acetylcysteine is present in an amount comprised between 0.01% and 30%, more preferably in an amount comprised between 0.05% and 20%, even more preferably in an amount comprised between 0.1% and 10%, on the total weight or volume of the composition.
  • FR4 The composition according to any one of FR1-FR3, wherein the methylsulfonylmethane (MSM) is present in an amount comprised between 0.05% to 35%, more preferably in an amount comprised between 0.1% and 25%, even more preferably in an amount comprised between 0.5% and 15%, on the total weight or volume of the composition.
  • MSM methylsulfonylmethane
  • composition according to any one of FR1-FR4, wherein the composition is a composition for medical device according to regulation EU 2017/745, a cosmetic product, a dietary supplement, a nutraceutical, dietetic and nutritional composition, a food product, a beverage, a nutraceutical product, a medicament, a medicated food, a pharmaceutical composition, a food for special medical purposes, a complementary feed.
  • FR6 The composition according to any one of FR1-FR5, wherein said composition is for use in a method for the prevention and/or treatment of diseases of the respiratory system and of the related symptoms both in humans and in animals.
  • composition for use according to FR6 wherein the diseases associated with the respiratory system are selected from the group comprising or, alternatively, consisting of: rhinitis, allergic rhinitis, nasopharyngitis or cold, cough, nasal congestion, accumulation of nasal mucus, flu, rhinosinusitis, sinusitis, pharyngitis, epiglottitis, laryngitis, tracheitis, bronchitis, bronchiolitis and bronchiectasis.
  • the diseases associated with the respiratory system are selected from the group comprising or, alternatively, consisting of: rhinitis, allergic rhinitis, nasopharyngitis or cold, cough, nasal congestion, accumulation of nasal mucus, flu, rhinosinusitis, sinusitis, pharyngitis, epiglottitis, laryngitis, tracheitis, bronchitis, bronchiolitis and bronchiectasis.

Abstract

The present invention relates to a composition comprising N-acetylcysteine (NAG), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM) useful in the prevention and/or in the treatment of diseases associated with the respiratory system. The combination is particularly effective thanks to the synergistic action of the components thereof.

Description

Industrial invention patent application having the title:
“Composition for use in a method for the prevention and treatment of diseases of the respiratory system”
DESCRIPTION
The present invention relates to a composition comprising N-acetylcysteine (NAG), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM). The present invention also relates to a composition comprising N-acetylcysteine (NAG), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM) useful in the prevention and/or in the treatment of diseases associated with the respiratory system.
The combination is particularly effective thanks to the synergistic action of the components thereof.
Background of the invention
Respiratory system
The respiratory system is the set of organs and tissues that allow the breathing process.
Respiration consists of assimilating the oxygen inspired with air and the simultaneous ejection of the carbon dioxide generated by cellular activity and representing a waste product.
The main anatomical elements of the respiratory system are: the nose with the cavities thereof, the mouth, the pharynx, the nasopharynx, the larynx, the trachea, the bronchi, the bronchioles, the lungs and the muscles of respiration, muscles of the diaphragm and intercostal muscles.
Diseases affecting the respiratory system may be of the following types: obstructive, restrictive, vascular or infectious/environmental.
The respiratory system can be divided into two main components: the upper respiratory tract (or upper airways) and the lower respiratory tract (or lower airways).
The upper respiratory tract includes the nose with the nasal cavities thereof, the mouth, the pharynx, the nasopharynx and the larynx; the lower respiratory tract instead includes the trachea, the bronchi, the bronchioles, the lungs and the muscles of respiration, muscles of the diaphragm and intercostal muscles.
The most common diseases of the airways are cold, sinusitis and pharyngitis.
Cold is a viral disease, the most common agents are rhinovirus, human parainfluenza virus, respiratory syncytial virus, adenovirus, echovirus and coxsackievirus. Agents vary depending on the age of the host and the time of year. Symptoms include nasal secretion, nasal obstruction and throat irritation, usually associated with low-intensity fever, malaise, sneezing and nasal secretions that could become purulent.
Symptoms are not specific and not characteristic of any agent. The standard duration of symptoms is about 7 days. The common cold in children resolves alone in most cases. The use of antibiotics is potentially harmful, because it increases the risk of colonisation with resistant organisms. This can lead to subsequent antibiotic-resistant bacterial infections
Cold has a development that resolves in almost all cases and only sometimes can it lead to complications such as acute otitis media with effusion, tonsillitis, sinusitis and infection of the lower respiratory tract.
Cough occurs in 60-80% of common colds, symptoms usually last 2-7 days. Patients usually improve within 10 days, but sometimes cough and nasal secretions may persist for 2 weeks or more.
Sinusitis consists of inflammation of the mucosal lining of one or more paranasal sinuses. It can be caused by infections or non-infectious causes such as allergies. In children it manifests itself as complication of a common cold where there is a congestion of the nasal mucous membranes and sinusitis, leading to rhinosinusitis.
Viral rhinosinusitis is 20 -200 times more common than bacterial sinusitis. Sinusitis is classified according to the duration of symptoms in acute sinusitis (up to 3 weeks), subacute sinusitis (3-10 weeks) and chronic sinusitis (>10 weeks).
Sinusitis manifests itself when a viral agent in the upper respiratory airways produces inflammation of the mucous membranes which leads to obstruction of the sinuses. This in turn leads to the entrapment of liquids into the sinus cavities and to the proliferation of the normal bacterial flora of the upper respiratory airways. Though the most common organisms that cause sinusitis are S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus and S. pyogenes J, it should be pointed out that most of the rhinosinusitises are viral and, therefore, about 60% resolve spontaneously without antimicrobial therapy. The symptoms can be sinus morbidity, pain in the teeth, headache and high fever.
Rhinorrhoea is mostly purulent, but it can also be serous or aqueous. Other indicators of sinusitis are the presence of severe symptoms during an episode of respiratory infection. These include high fever > 39~, persistent fever, periorbital swelling and facial pain.
Pharyngitis is an inflammatory disease of the mucous membranes of the throat and the underlying structures.
Pharyngitis is divided into two categories: symptomatic nasal disease (nasopharyngitis) and disease without nasal involvement (pharyngitis or tonsillopharyngitis). Nasopharyngitis is almost always of viral aetiology, while pharyngitis without nasal signs has various aetiology signs including bacteria, viruses, fungi and other infectious agents.
Adenoviruses are the most common agents that trigger the nasopharyngitis; other viruses are flu, parainfluenza and enteroviruses. Bacterial agents that cause pharyngitis include Streptococcus pyogenes, H. influenzae, C. diphtheriae and N. meningitidis.
Group A streptococci cause about 15% of bacterial pharyngitis and about 1-2 out of 10 patients with sore throat. Infection in most of the other cases can therefore be attributed to viruses.
Symptoms of pharyngitis comprise: inflammation of the pharynx, characterised by erythema, exudate or ulcer, pharyngeal pain, dysphagia and fever, while rhinorrhoea, cough, raucousness, conjunctivitis and diarrhoea suggest a viral aetiology.
Pharyngitis in paediatric age is of viral type, whereas in pharyngitis of bacterial type it is often due to streptococcus.
The published document WO 2019/064255 A1 discloses a composition comprising or, alternatively, consisting of an effective amount of a mixture comprising or consisting of at least one Pelargonium sidoides extract, an Adhatoda vasica extract and N-acetylcysteine for use in a method for the preventive or therapeutic treatment of at least one disorder of the respiratory tract in a subject.
The published document MORVARID ELAHI HOMAYOUN ELAHI: "The Effects of N-Acetyl Cysteine on Nasal Mucociliary Clearance in Healthy Volunteers: A Randomized, Double-Blind and Placebo- Controlled Stud , OTOLARYNGOLOGY, vol. 05, no. 01,1 January 2015, discloses a study aimed at demonstrating that N-acetylcysteine exerts a measurable effect on nasal mucociliary clearance and that it may therefore be useful in the treatment of disorders related to mucociliary clearance such as rhinitis and sinusitis.
The published document WU YAN ET AL: "The study on Chlorhexidine acetate against pathogens bacteria of acute and chronic pharyngitis in vitro", ZHONGGUO KANGSHENGSU ZAZHI/ CHINESE JOURNAL OF ANTIBIOTICS, SICHUAN, ON, vol. 36, no. 9, 1 September 2011 (2011 -09-01), pages 712-715, is a study conducted to evaluate the antibacterial activity of chlorhexidine acetate against pathogens of pharyngitis.
The published document BARRAGER E. ET AL: "A multicentered, open-label trial on the safety and efficacy of methylsulfonylmethane in the treatment of seasonal allergic rhinitis", JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINE, MARY ANN LIEBERT, NEW YORK, NY, US, vol. 8, no. 2, 1 January 2002 (2002-01-01), pages 167-173 is a study aimed at evaluating the efficacy of methylsulfonylmethane (MSM) in reducing symptoms associated with seasonal allergic rhinitis (SAR).
Having therapeutic and preventive treatments for diseases of the respiratory tract is the main objective of the present patent application.
The combination subject of the present invention consists of a combination of hyaluronic N- acetylcysteine (NAC), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM).
N-acetyicysteine
N-acetylcysteine (NAC) is a thiol, a mucolytic agent and a precursor of L-cysteine and reduced glutathione. NAC is a source of sulfhydryl groups in cells and it is a free radical scavenger given that it interacts with ROS such as OH- e H2O2.
N-acetylcysteine has various activities, among which the antioxidant action stands out.
NAC can eliminate ROSs, increase glutathione levels, undergo autoxidation and act as a reducing agent.
Activation of NF-kB in response to a variety of signals (IL-1, TNF, H2O2) may be inhibited by NAC, showing that ROSs are signalling modulators.
NAC may interfere with cell adhesion, oxidative stress, stability of atherosclerotic plaques at risk of rupture in the cardiovascular system and reduce lung inflammation, fibrosis and smoking-related changes. In arthritis it can reduce inflammation, synovial invasion and cartilage damage. In cancer, this agent inhibits angiogenesis, reduces smoking-induced carcinogenesis, induces selective apoptosis of transformed cells, interferes with the cell cycle, and it has anti-invasive and anti- metastatic effects. In some types of cells (chondrocytes, neurons) it promotes cell growth and survival. In the liver, NAC decreases oxidative stress from various agents, and it provides considerable protection against fibrosis, viral infections, and toxic agents.
At the respiratory level, in bronchial cells, NAC reduces TNF-induced NF-kB activation, and it also reduces TNF and RANTES-induced p38 MARK production.
NAC may also exert the antioxidant effect thereof indirectly facilitating the biosynthesis and the availability of GSH.
N-acetylcysteine has been shown to inhibit expression, in human endothelial cells of VCAM-1, resulting in blocking of monocyte adhesion in response to stimuli such as tumour necrosis factor a (TNF-a). This effect is demonstrated by the suppression of NF-KB activation.
Furthermore, NAC has a marked mucolytic activity, thanks to the ability to break the disulfide bridges of mucus protein complexes, depolymerizing mucin molecules.
The immunomodulating activity of NAC was assayed by means of in vivo tests. A 0.1 g/kg solution of buthionme sulfoximme (BSO) was injected into a group of rats and after 2 hours LPS was injected in a dose of 0.1 mg/kg in 0.2 ml of pH saline solution.
Forty-five minutes prior to treatment with LPS a dose of 1 g/kg of NAG was administered.
The animals were subjected to blood sampling and then sacrificed to remove liver and pancreas in order to test glutathione activity and TNF concentration.
The results of the test show that in the group treated with NAG the levels of TNF are significantly reduced at a minimum effective dose of 0.5 g/kg. The TNF-inhibitory effect was compared to treatment with GSH, with which it showed comparable activity, at the dose of 1g/kg. The efficacy of NAG was also evaluated by administering BSO which inhibits GSH increasing TNF. In the treated group, NAG antagonised BSO.
Furthermore, the mortality of animals in the group treated with NAG, to which a massive dose of LPS (33mg/kg) was administered was tested. The result showed a lower risk of mortality.
A further in vivo study tested the mucolytic and expectorant activity of NAG.
In this study the cavies were divided into groups. 2 treated groups in which 0.6M of NAG was administered for 5 minutes to one through the inhalation route, while another treated group received a 20 mg/kg NAG tablet, while the control groups were treated with saline solution using the same routes of administration. To test the frequency and intensity of the tussive stimuli, the animals were placed in a chamber in which they inhaled citric acid for 3 minutes at a concentration of 0.3 mol/L. The improvement of the airflow following coughing was evaluated using a pneumotachograph and the sounds and movements accompanying this respiratory response were analysed.
The result of the test showed that NAG administered in both modes, both in mono and repeated administration, reduced the frequency of tussive stimuli. Ciliary movement in chronic administration, in both systems, was significantly reduced in the treated groups.
Some clinical studies tested NAC activity at the respiratory level.
Among these, a clinical trial was conducted on a cohort of patients, using NAC as prophylactic therapy for flu symptoms.
Patients were divided into control group and treated group. The treated group was administered a 600 mg NAC tablet 2 times a day for 6 months. The test result showed that the incidence of flu in the treated group was 29% compared to 51%. This incidence was also analysed for each month of treatment, showing that during the period of the year where the frequency of infection is higher, the treated group showed a markedly lower portion of infected patients with respect to placebo, 13% with respect to 30%.
Besides the incidence of flu symptoms, the severity thereof was compared, showing that in the treated group 72% of cases experienced mild symptoms with respect to 48% in the placebo group and that there were only 26% of moderate symptoms in the treated group with respect to 46% in the control group. The development of symptoms reflects the seasonality shown in the incidence of cases.
To evaluate the efficacy of the treatment, part of patients underwent an A/H1N1 seroconversion, showing that only 25% of patients in the treated group developed symptoms, with respect to 79% in the control group.
The trial shows that NAC was able to significantly prevent most of the symptoms related to flu episodes, including symptoms in the respiratory system such as rhinorrhoea, sore throat, catarrh and cough, and general symptoms such as headache and myalgia-arthralgia. Such evidence suggests that protective effects do not depend simply on mucolytic action, but they are likely to be further linked to other mechanisms, such as antioxidant and immunomodulant effects.
Lastly, the trial shows that besides a mechanical activity of ejecting the virus by fluidifying the sputum, NAC acts by inhibiting cytokines.
A further study was conducted in patients with moderate to severe COPD treated with NAC.
Patients were separated into a group subjected to standard therapy plus 600 mg NAC per day for 6 months and patients treated exclusively with standard therapy.
During the treatment period, exacerbation of the disease was evaluated, observing the increase in symptoms such as cough, dyspnoea and sputum.
The test result showed a 41% reduction in disease exacerbation in the group treated with NAC. Furthermore, the number of patients with two or more exacerbations in the NAC group is 29%, with respect to the control group where it is 49%. The number of days of disease in the NAC group is 82 days, with respect to 155 days in the control group. Furthermore, a reduction in exacerbation severity in the treated group with respect to the control group was observed.
In a self-evaluation test, 29% of the control group experienced improvement in symptoms with respect to 65% of the NAC group.
The trial showed no differences in terms of toxicity and linked the reduction in symptoms to the antioxidant effects of NAC.
A meta-analysis was conducted to evaluate studies on the efficacy of the treatment with NAC in COPD patients. A study of several reviews found that out of 100 patients treated with NAC through the oral route at the dose of 400-600 mg per day for 12-24 weeks, 17 did not experience any disease exacerbation and this is not observed in any of the control patients. Furthermore, out of 100 patients, 26 of the treated group experienced improvement in bronchitis-related symptoms, an event not observable in any of the control patients. The result of the review shows that NAC has a therapeutic effect in the treatment of bronchitis and the mechanism appears to be linked to antioxidant activity.
Chlorhexidine Chlorhexidine is a biguamde, consisting of two chloro-guanidine chains linked by hexamethylene chains.
The substance is colourless, odourless, bitter-tasting and insoluble in water, it covalently binds the proteins of the skin and of the mucosa, thus ensuring a long duration of action and poor systemic absorption.
Chlorhexidine is absorbed by the phosphorylated component of proteins of the bacterial wall. Binding with microbial proteins triggers lysis of the cytoplasmic membrane, at bacteriostatic concentrations, while in bactericidal amounts chlorhexidine forms an irreversible bond with adenosine triphosphate and nucleic acids, precipitating them.
Besides having bacteriostatic, bactericidal, fungicidal activity and activity against some viral species, chlorhexidine also has a greater affinity for the bacterial wall of Gram+ bacteria.
The topical application of antiseptic, in the oral cavity, (dentifrices or mouthwashes) exerts the antimicrobial activity thereof through electrostatic binding with the oral mucous membranes.
Chlorhexidine effectively acts on the dental biofilm which produces enzymes that degrade fibronectin. The advantage in treatment with chlorhexidine lies in the broad spectrum of action thereof, on a wide variety of microbial agents.
An in vitro test showed the efficacy of chlorhexidine in eradicating various viral species, including hepatitis B, Herpes virus, human parainfluenza virus and type A flu virus.
The antimicrobial activity of chlorhexidine was tested on various cell lines including rabbit kidney cells and human foreskin fibroblasts. Each cell line was associated with a viral strain, particularly flu virus was inoculated into the cell line in the Madin Darby canine kidney, and the human parainfluenza virus on the African green monkey kidney cell line. Antiviral activity was compared to a placebo and monitored over time, monitored between 30 seconds after treatment, up to the subsequent 15 minutes.
The test result showed a significant time-dependent decrease in viral growth.
The inhibition of human parainfluenza virus changes from 59% after 30 seconds up to 99% after 15 minutes, while the flu virus decreases by 93% after 30 seconds up to 98% after 15 minutes.
A further study showed the efficacy of chlorhexidine, combined with benzamide, in the treatment of pharyngotonsillitis induced by Streptococcus Pyogenes.
The trial was conducted on a cohort of patients, randomised and divided into two groups treated with penicillin V and placebo in the form of spray, while the other group was treated with the same penicillin enriched with 0.12% chlorhexidine gluconate and 0.15% benzamide chloride. Both therapies were administered for 10 days, 4 times a day.
The test result showed a decrease in symptoms, significantly greater in the treated group with respect to the control group, showing the effectiveness thereof in combating this type of infections. Another study tested the bacteriostatic activity of chlorhexidine, showing that the MIC for some bacterial species is: 1 :500000 for Streptococcus pyogenes, 1 :500000 for Corynebacterium diphteriae, 1 :500000 for Diplococcus pneumoniae.
The antimicrobial activity of chlorhexidine against some microbial species is also tested in this study. For example, a 0.5% solution of chlorhexidine in 50% of alcohol and subsequently a 1% solution are inoculated into a culture broth containing Micrococcus pyogenes, a bacterium responsible for some respiratory diseases, and compared with a control broth. The test result showed the absence of bacteria in the media treated with chlorhexidine.
Lastly, the bacteriostatic action against p-haemolytic streptococcus was tested. The test was conducted on a cohort of volunteers, taking the bacterial strain through a pharyngeal swab, in particular from the tonsils. The presence of p-haemolytic streptococcus was evaluated through a swab and subsequently 2 tablets of 0.2% chlorhexidine were administered to one group of patients 5 times a day for one week, while another group received placebo. The result showed the presence of p-haemolytic streptococcus in only a small part of patients treated with chlorhexidine, and showing the efficacy thereof.
Methylsulfonylmethane (MSM)
Methylsulfonylmethane or more simply MSM is an organosulfide, an organic compound containing sulphur.
The nutraceutical properties of methylsulfonylmethane derive from the sulphur content thereof in a bioavailable form, thanks to the presence of an organic component which facilitates the absorption thereof.
It has various pharmacological activities including anti-inflammatory and antioxidant action.
In vitro studies show that MSM inhibits the transcriptional activity of NF-kB, preventing the degradation of the inhibitor of this molecule. Furthermore, MSM showed to block the phosphorylation of the p65 subunit at serine-536. Conventionally, regulatory cascade of NK-kB is a pro-inflammatory signalling mechanism, responsible for activating gene expression cytokines, chemokines and adhesion molecules.
In vitro, the inhibitory effect of MSM on NF-kB results in a decrease in mRNA regulation for interleukin (IL)-1 , IL-6 and TNF-o). Furthermore, IL-1 and TNF-o are inhibited in a dose-dependent manner.
MSM may also decrease the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing NF-KB; thus reducing the production of vasodilator agents such as nitric oxide (NO) and prostanoids. Besides modulating the vascular tone, NO also regulates mast cell activation. Therefore, MSM may indirectly play an inhibitory role on mast cells. Decreasing cytokines and vasodilator agents allows to inhibit the flow and recruitment of immune cells to sites of inflammation. MSM negatively affects the expression of the NLRP3 inflammasome, decreasing the production and transcription of NF-KB and blocking the ROS activation signal.
The antioxidant effect of MSM was observed for the first time when neutrophil stimulated production of ROS was suppressed in vitro but it was not affected in a cell-free system; for this reason it was proposed that the antioxidant mechanism acts on mitochondria rather than at chemical level.
The MSM affects the activation of at least four types of transcription factors: NF-KB, signal transducers and activators of transcription (STATs), p53 and Nrf2. By mediating these transcription factors, MSM can regulate the balance between ROS and antioxidant enzymes.
It should be observed that each of these is also, partly, activated by ROS. As mentioned above, MSM can inhibit the transcriptional activity of NF-KB and therefore reduce the expression of enzymes and cytokines involved in the production of ROS. The down-regulation of COX-2 and iNOS reduces the amount of superoxide radicals and of (NO), respectively.
Furthermore, MSM suppresses expression of cytokines such as TNF-o, which may reduce any ROS generated at mitochondrial level. Decreases in cytokine expression may also be involved in reduced paracrine signalling and activation of other transcription factors.
MSM has been shown to suppress the expression or activity of STAT transcription factors in a given number of tumour cell lines in vitro. The janus kinase (Jak)ZSTAT signalling pathway is involved in the regulation of apoptosis, differentiation and proliferation-related genes, which generate ROS as a necessary signalling component. Signalling through the Jak/STAT pathway may also be inhibited by reduced cytokine expression. The reduction of the Jak/STAT pathway may further decrease ROS synthesis by decreasing the expression of oxidase and B cell lymphoma 2 (Bcl-2).
Pre-treatment with MSM in vitro to decrease the accumulation of redox-sensitive transcription factor p53 was found in macrophage-like cells. p53 shows a dichotomic oxidative function, depending on levels of intracellular ROS, it exerts an antioxidant action at low levels of intracellular ROS and pro-oxidative activities at high levels of ROS. The antioxidant function of p53 stimulates scavenging enzymes such as sestrin, glutathione peroxidase (GPx) and aldehyde dehydrogenase (ALDH). The pro-oxidative function of p53 stimulates oxidase, and at the same time suppresses antioxidant genes.
Murine neuroblastoma cells cultured with the human immunodeficiency virus type 1 transactivating the regulatory protein (HIV-1 Tat) showed reduced nuclear translocation of Nrf2; however, inserting into the MSM culture medium allowed to resume the translocation of Nrf2 to the nucleus at regular levels. Nrf2 is well documented for the association thereof with antioxidant enzymes including glutamate-cysteine ligase (GCL), superoxide dismutase (SOD), catalase (CAT), peroxiredoxin (Prdx), GPx, glutathione S-transferase (GST), and others. Although it is unclear what the direct effect of MSM on Nrf2 which can also be regulated by the expression of p53 and p21 or by the expression - by Jak/STAT - of extra-large B cell lymphoma (Bcl-XL).
Stress can trigger an acute response by the innate immune system and a resulting adaptive immune response if the stress factor is pathogenic.
MSM plays a crucial role in supporting the immune response. Through mechanisms such as those mentioned above, MSM modulates the immune response through the link between oxidative stress and inflammation.
Chronic exposure to stress factors may have harmful effects on the immune system when it becomes desensitised or excessively stressed and unable to elicit a typical immune response. In vitro, MSM has been shown to reduce IL-6, which is involved in the maintenance of chronic inflammation.
Histamine release from mast cells is inhibited by DMSO2; however, the effects of MSM on histamine release remain unknown. Previous studies show that MSM has an inhibitory role on vascular function. Other in vitro studies demonstrate that MSM has the ability to dampen the expression of vasodilating agents such as nitric oxide (NO) and prostanoids. A reduction of NO protects macrophages against NO stimulated apoptosis.
Furthermore, MSM may provide other immune modulatory effects linked to cell cycle and cell death.
In vitro studies suggest that MSM does not chemically neutralise ROS in stimulated neutrophils but it instead suppresses mitochondrial generation of superoxide, hydrogen peroxide, and hypochlorous acid. Furthermore, MSM is able to restore the reduced glutathione (GSH)Zoxidised glutathione (GSSG) ratio to normal levels, decrease the production of NO, and reduce the production of neuronal ROS following exposure to HIV-1 Tat. Animal studies using MSM as primary treatment for experimentally induced lesions show reductions in malondialdehyde (MDA), GSSG, myeloperoxidase (MPO), NO, carbon monoxide (CO) and increase in GSH, CAT, SOD and GPx.
A clinical trial was conducted to test anti-inflammatory activity in patients with allergic rhinitis. The cohort of patients was treated with capsules of 650 mg of MSM 4 times a day, 2 in the morning and two in the afternoon, for 30 days. The group of patients non-responsive to the therapy continued for another 14 days, doubling the treatment dose.
The outcome of the trial showed improvement in the clinical picture in patients with allergic rhinitis, MSM activity focused on the upper respiratory tract, with minimal side effects only.
MSM seems to be well tolerated and safe. A series of toxicity studies was conducted on various animal species, including rats, mice and dogs. In a preliminary toxicity study report, there was reported a single mortality in a female rat to which there was administered an oral aqueous dose of 15.4 g/kg after two days; however, the post-mortem necroscopic examination did not reveal any serious disease. Other technical reports show that mild skin and eye irritation were observed when MSM is applied topically. The FDA recognises MSM as GRAS, considering it safe at doses below 4845.6 mg/day.
The combination of substances described in the present invention allows to carry out the following actions simultaneously:
- Direct antimicrobial action carried out by chlorhexidine;
- Antioxidant and anti-inflammatory action carried out by methylsulfonylmethane;
-Antioxidant, immunomodulant and mucolytic action carried out by N-acetylcysteine (NAG).
The composition subject of the present invention consists of an association or combination of N- acetylcysteine (NAG), chlorhexidine or a salt thereof and methylsulfonylmethane (MSM) for use in a method for the prevention and/or treatment of diseases of the respiratory system both in humans and in animals.
Such diseases in particular are selected from the group comprising or, alternatively, consisting of; rhinitis, allergic rhinitis, nasopharyngitis or cold, cough, nasal congestion, accumulation of nasal mucus, flu, rhinosinusitis, sinusitis, pharyngitis, epiglottitis, laryngitis, tracheitis, bronchitis, bronchiolitis and bronchiectasis.
In a preferred embodiment, N-acetylcysteine (NAG) is present in an amount comprised between 0.01% and 30%, more preferably in an amount comprised between 0.05% and 20%, even more preferably in an amount comprised between 0.1% and 10%, on the total weight or volume of the composition; chlorhexidine or a salt thereof is present in an amount comprised between 0.01% and 40%, more preferably in an amount comprised between 0.05% and 30%, even more preferably in an amount comprised between 0.1% and 20%, on the total weight or volume of the composition; methylsulfonylmethane (MSM) is present in an amount comprised between 0.05% and 35%, more preferably in an amount comprised between 0.1% and 25%, even more preferably between 0.5% and 5%, on the total weight or volume of the composition.
The dosage form may be a pharmaceutical composition or a medical device or a cosmetic or a dietary supplement, a food for special medical purposes or a complementary feed including the aforementioned active ingredients mixed to each other. The preferred route of administration is nasal, topical, inhalation, oral.
The pharmaceutical forms which can be used according to the present invention are selected from nasal sprays, vials for nasal instillation, vials for inhalation through aerosol, nasal drops, nasal creams, nasal ointments or other pharmaceutical forms suitable for nasal, topical, inhalation, oral administration.
The following examples are given purely by way of illustration and they do not limit the scope of protection of the invention as defined in the attached claims. Changes or variations to the embodiments exemplified herein, obvious to the person skilled in the art, are encompassed by the attached claims.
Besides said association or combination, the composition of the present invention further optionally comprises pharmaceutical or food or cosmetic grade excipients and additives.
Suitable excipients may be selected from those usually known in the state of the art and they include, though not limited thereto: diluents (for example dibasic calcium phosphate, lactose, microcrystalline cellulose and cellulose derivatives), thickeners (for example gums, hydroxypropyl methyl cellulose and other cellulose derivatives), sweeteners (for example sorbitols, mannitol and other polyols, acesulfame K, aspartame, cyclamates, saccharin, sucralose), lubricants (for example magnesium stearate, stearic acid, waxes), dispersants, surfactants (for example sodium lauryl sulfate and polysorbate), flavour-enhancement agents, adsorbents (for example silica gel, talc, starch, bentonite, kaolin), glidants and anti-adherents (for example talc, colloidal silica, maize starch, silicon dioxide), dyes (for example iron oxides), opacifiers (for example titanium oxide), antioxidants, binders (for example gums, starch, gelatine, cellulose derivatives, sucrose, sodium alginate), disaggregating agents (starch, microcrystalline cellulose, alginic acid, crospovidone), plasticisers (for example ethyl cellulose and other cellulose derivatives, acrylates and methacrylates, glycerol and sorbitol), preservatives (for example parabens, sulfur dioxide), viscosifiers, emulsifiers, humectants, wetting agents, chelating agents and mixtures thereof.
Example 1 : spray for nasal application
Example 2: spray for nasal application
Example 3: drops for nasal application
Example 4: vials for nasal instillation
Example 5: solution for inhalation application through aerosol
EXPERIMENTAL PART
1 . Various experimental models may be used to test the efficacy of the combination or association of the functional substances subject of the present invention N-acetylcysteine (NAC), chlorhexidine or a salt thereof and methylsulfonylmethane (MSM)) and to evaluate the synergistic action thereof.
The experimental groups differ in the group treated with single substance, group treated with combination of substances and control group.
Any test for the evaluation of antiviral, antimicrobial, anti-inflammatory, antioxidant and immunomodulating activity known in literature can be used.
By way of example, the antiviral activity is for example evaluated by means of various types of tests which allow to evaluate the effect of the tested compounds on the plaques of the virus, on a particular effect thereof (cytotoxicity), on some proteins fundamental for the viruses, or on particular phases of the reproduction cycle such as attachment, inlet, uncoating, replication, assembly, release etc.
2. Antimicrobial activity is evaluated on the main bacterial strains belonging to the categories of Gram positive and/or Gram negative and/or other microbial species, such as for example S.aureus (respiratory pathogen model).
3. In vitro assays such as for example broth-dilution (the sensitivity of the microorganism is evaluated based on the growth or non-growth thereof in a culture medium at different concentrations of the sample) and diffusion into agar (where a standardised concentration of the sample is applied in a bacterial broth-culture and the diffusivity of the sample in the medium is calculated) are suitable to demonstrate the antibacterial efficacy of the composition according to the present invention.
4. In vitro assays which evaluate the ability to inhibit the release of inflammatory cytokines such as for example IL-1, IL-6 and TNF-o and to inhibit the expression of enzymes such as COX-2 and IL-1 p- induced metalloprotease-13 in primary cultures of cells (for example macrophages, chondrocytes, fibroblasts or others), also following an irritation stimulation with LPS, are suitable to demonstrate the anti-inflammatory efficacy of the composition according to the present invention. 5. As regards immunomodulating activity, it is preferably evaluated through scientific tests capable of detecting the stimulation/inhibition capacity of cells, cytokines or other factors involved in the immune response.
6. In vitro assays, such as for example DPPH tests, radical scavenging activity on nitric oxide or peroxynitrite radical, TEAC (total radical-trapping antioxidant parameter), FRAP (ferric reducing- antioxidant power), HORAC (hydroxyl radical averting capacity), ORAC (oxygen radical absorbance capacity) tests and the like, are suitable to demonstrate the antioxidant efficacy of the composition according to the present invention. The antioxidant activity can also be evaluated by means of cell tests known to the person skilled in the art.
Embodiments FRns of the present invention are reported below:
FR1. A composition comprising a combination of N-acetylcysteine (NAC), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM).
FR2. The composition according to FR1, wherein N-acetylcysteine is present in an amount comprised between 0.01% and 30%, more preferably in an amount comprised between 0.05% and 20%, even more preferably in an amount comprised between 0.1% and 10%, on the total weight or volume of the composition.
FR3. The composition according to any one of FR1-FR2, wherein chlorhexidine or a salt thereof is present in an amount comprised between 0.01% and 40%, more preferably in an amount comprised between 0.05% and 30%, even more preferably in an amount comprised between 0.1% and 20%, on the total weight or volume of the composition.
FR4. The composition according to any one of FR1-FR3, wherein the methylsulfonylmethane (MSM) is present in an amount comprised between 0.05% to 35%, more preferably in an amount comprised between 0.1% and 25%, even more preferably in an amount comprised between 0.5% and 15%, on the total weight or volume of the composition.
FR5. The composition according to any one of FR1-FR4, wherein the composition is a composition for medical device according to regulation EU 2017/745, a cosmetic product, a dietary supplement, a nutraceutical, dietetic and nutritional composition, a food product, a beverage, a nutraceutical product, a medicament, a medicated food, a pharmaceutical composition, a food for special medical purposes, a complementary feed.
FR6. The composition according to any one of FR1-FR5, wherein said composition is for use in a method for the prevention and/or treatment of diseases of the respiratory system and of the related symptoms both in humans and in animals.
FR7. The composition for use according to FR6, wherein the diseases associated with the respiratory system are selected from the group comprising or, alternatively, consisting of: rhinitis, allergic rhinitis, nasopharyngitis or cold, cough, nasal congestion, accumulation of nasal mucus, flu, rhinosinusitis, sinusitis, pharyngitis, epiglottitis, laryngitis, tracheitis, bronchitis, bronchiolitis and bronchiectasis.

Claims

1 . A composition comprising a combination of N-acetylcysteine (NAC), chlorhexidine or a salt thereof, and methylsulfonylmethane (MSM).
2. The composition according to the preceding claim, wherein N-acetylcysteine is present in an amount comprised between 0.01% and 30%, more preferably in an amount comprised between 0.05% and 20%, even more preferably in an amount comprised between 0.1% and 10%, on the total weight or volume of the composition.
3. The composition according to any one of the preceding claims, wherein chlorhexidine or a salt thereof is present in an amount comprised between 0.01% and 40%, more preferably in an amount comprised between 0.05% and 30%, even more preferably in an amount comprised between 0.1% and 20%, on the total weight or volume of the composition.
4. The composition according to any one of the preceding claims, wherein the methylsulfonylmethane (MSM) is present in an amount comprised between 0.05% to 35%, more preferably in an amount comprised between 0.1% and 25%, even more preferably in an amount comprised between 0.5% and 15%, on the total weight or volume of the composition.
5. The composition according to any one of the preceding claims, wherein the composition is a composition for medical device according to regulation EU 2017/745, a cosmetic product, a dietary supplement, a nutraceutical, dietetic and nutritional composition, a food product, a beverage, a nutraceutical product, a medicament, a medicated food, a pharmaceutical composition, a food for special medical purposes, a complementary feed.
6. The composition according to any one of the preceding claims, wherein said composition is for use in a method for the prevention and/or treatment of diseases of the respiratory system and of the related symptoms both in humans and in animals.
7. The composition for use according to claim 6, wherein the diseases associated with the respiratory system are selected from the group comprising or, alternatively, consisting of: rhinitis, allergic rhinitis, nasopharyngitis or cold, cough, nasal congestion, accumulation of nasal mucus, flu, rhinosinusitis, sinusitis, pharyngitis, epiglottitis, laryngitis, tracheitis, bronchitis, bronchiolitis and bronchiectasis.
EP21830494.7A 2020-11-19 2021-11-19 Composition for use in a method for the prevention and treatment of diseases of the respiratory system Pending EP4247350A1 (en)

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