Compound of Zinc salicylate and Sulphur combination as a novel treatment for asthma and COPD FIELD OF THE INVENTION:

Asthma and COPD (chronic obstructive pulmonary disease, smoker's lung) are the most frequent chronic inflammatory diseases of the lung and both are not curable with any of the existing medication. For both diseases, the available medications only control disease symptoms but have no effect on the structural changes of the lung tissue.

Besides chronic inflammation, both diseases are characterized by significant pathological changes of the airway structure which impairs its function. It is well documented in clinical studies that the existing drugs only reduce airway constriction and inflammation, but have no effect on the pathological changes of the airway structure. Despite decades of investigation, the cause of asthma and most other chronic inflammatory lung diseases is not well understood. Recently, the dogma that asthma and COPD result from chronic inflammation was challenged by the hypothesis that lung structural changes cause chronic inflammation.

The components that build organ tissue are constantly metabolized and any small change in this mechanism will be accumulated over time as structural tissue change, which affects organ function. Most of these metabolic events depend on the availability of metal ions such as iron (Fe), magnesium (Mg), calcium (Ca) or zinc (Zn). Recent studies suggested that specifically zinc deficiency is linked to childhood asthma, asthma severity and also with COPD progression.

Preliminary studies in small cohorts of asthma patients indicate that zinc salicylate supplementation may reduce asthma symptoms and help to reduce the application of conventional asthma drugs which all have unwanted side effects.

BACKGROUND OF THE INVENTION

Chronic inflammatory lung diseases including asthma, chronic obstructive pulmonary disease (COPD), the recently defined Asthma COPD Overlapped Syndrome (ACOS), lung fibrosis, and other dust induced lung diseases are increasing worldwide (http://www.who.int/gard/ publications/ chronic respiratory diseases.pdf). Beside COPD which is clearly related to cigarette smoking, the increase of other chronic inflammatory lung diseases is not well understood.

In recent models which were based on data from the USA, Europe and Asia, it was projected that the exposure to outdoor air pollution causes 3.3 million pre-mature deaths worldwide per year, and this number is expected to double by 2050 (Leiieveld 2015). In addition, further 3.5 million people die per year due to indoor pollution from cooking and heating (Lim 2012). It is important to note that industries and traffic are not the only sources for air pollution. In less industrialized countries, fine dusts (< 10pm) mainly originating from agricultural sources also increase the prevalence of chronic inflammatory lung diseases which leads to premature mortality (Jerrett 2015). These studies imply that there might be even more people worldwide who will develop chronic inflammatory lung diseases as a consequent of air pollution than it was estimated by the corresponding mathematical disease models. However, an individual's sensitivity to developxhronic inflammatory lung diseases depends on his/her genetic pre-disposition. So far, no genetic study (genomics, transcriptomics) has identified these presumed genetic factors leading to chronic inflammatory lung diseases

susceptibility in humans (Postma 2015a). Therefore, the most important task is to identify the condition of each individual, which defines the individual's susceptibility for developing chronic inflammatory lung diseases (Yayan 2016). However, several candidate genes which were linked to the pathogeneses of asthma and COPD encoded for proteins which either control the synthesis of tissue formation, its degradation or the proliferation of connective tissue cells such as fibroblasts (Koppelman 2011, Young 2011). In asthma, remodeling in the lung results from over shooting production of connective tissue and sub-epithelial cell proliferation, while in COPD, increased connection tissue formation is a sign of early disease and complete destruction of the lung structure by enzymes is a sign of end stage (Postma 2015b).

Most pro-inflammatory and remodeling regulating proteins depend on the availability of zinc and some studies correlated zinc deficiency with the pathogeneses of asthma and COPD (Hirota 2013, Khanbabaee 2014, azi 2012). Zinc controls cell proliferation, cell differentiation and the turnover of the extracellular matrix (e.g. collagens). Together, these factors are essential to maintain the tissue structure and therefore organ function.

Lung and airway wall remodeling are two prominent pathologies of chronic lung diseases are they are not affected by available drugs (King 2016, Postma 2014). Tissue remodeling consist of two major events: 1) increased cell number of fibroblasts and/or smooth muscle and 2) of increased deposition of connective tissue, also called extracellular matrix. The deposition of the different components of the connective tissue is regulated on different levels: first the pre-cursors have to be generated via gene activation, second the pre-cursors have to be activated by enzymes and third they have to be deposed by other enzymes (Bienkowski 1995, Roche 2014). Finally, all components of the connective tissue are target for digesting enzymes, matrix metalloproteinases (MMP) which are disease specifically regulated in asthma and COPD, as we have shown earlier (Papakonstantinou 2015).

Minerals such as zinc, copper, selenium, magnesium and others are essential for many proteins to exert their functions as regulator of the homeostasis of organs. Deficiencies of any of these minerals may lead to the development of acute and chronic inflammatory diseases such as asthma and COPD (Bonaventura 2015, Hamon 2014). Zinc is an essential ion necessary for the function of many proteins, which therefore were assigned as either metalloproteinases or zinc finger motive proteins. The latter act as transcription factors for genes relevant for cell proliferation and differentiation.

Zinc is also controlling the activity of matrix metalloproteinases (MMP), which are the major regulators of connective tissues composition and function (Aureli 2008, Loffek 2011). Taking into account that MMPs are responsible for the daily turnover of the lung connective tissue (10% per day), this underlines the eminent role of zinc supplementation. In regard to asthma, two recent reports suggested that most children suffering from childhood asthma have insufficient supply of zinc and selenium (Razi 2012, Khanbabaee 2014). Unfortunately, there is not much information on zinc deficiency in adult asthmatics.

In regard to COPD, a deficiency of zinc has been correlated to the severity of the disease and it was suggested that zinc deficiency was responsible for increase apoptosis of the airway epithelium (Rosioli 2013, Tanrikulu 2011). Supplementation of zinc has reduced oxidative stress in COPD patients; however, it did not improve their lung function (Kirkil 2008). The lack of functional improvement of the lung in this study may be explained by a relative small number of patients (n=30) and a treatment period of only 8 weeks, since the available literature suggests that the improvement of lung repair needs at least 6 months (Pain 2014, Hirota 2013). The regulatory role of MMPs on the repair processes of the damaged lung, as it occurs in asthma and COPD, is essential to understand the beneficial effects of zinc supplementation (Oikonomidi 2009). Furthermore, the malfunction of MMPs due to zinc deficiency is well in line with the hypothesis that COPD results from an imbalance of MMPs and their inhibitors (TIMP).

In addition, asthma and COPD progress with the frequency of exacerbation, which are most often caused by viral and/or bacterial infections inducing the generation of reactive oxygen species (ROS) (Escaffre 2015, He 2013, Kash 2014). ROS lead to increased tissue damage and thus further lung injury. Zinc is a well-known scavenger of ROS and therefore its deficiency may increase lung injury and delay tissue repair (Zhang 2016). Hence, we claim that the adjustment of zinc will help to control and reduce the pathologies which lead to airway wall remodeling in asthma and COPD. In this context, it is of great importance that inorganic zinc as it can be inhaled through industrial dust, welding or traffic pollution causes inflammation of the lung (Wu 2013); in contrast, organic zinc salts such as zinc salicylates, counteract inflammation as we have described in the previous paragraph. However, the mechanism(s) how organic zinc salts achieve the beneficial effects on chronic inflammatory diseases is only partly understood. This patent we claim that zinc salicylates supplementation guarantees the function of zinc dependent proteins which control inflammation and tissue remodeling.

SUMMARY AND DETAILS OF THE INVENTION

The therapeutic compound that has been composed and tested in our pilot research consists of zinc salicylate (20%) and methylsulfonylmethane (80%). The ratio of zinc salicylate:

methylsulfonylmethane can range from 10:90 to 50:50 and may to be adjusted for specific disease conditions.

Zinc Salicylate can be replaced by any nutritional or pharmaceutical Zinc compounds, such as: Zinc Aspirinate, Zinc Picolinate, Zinc Sulfate, Zinc Gluconate, Zinc Methionine, or Zinc Ascorbate.

This salt mixture is branded with methyl cellulose in a 50:50 ratio (weight).

This mixture is branded in gelatin capsules of 400mg.

This salt combination can be applied orally, rectally, trans-dermally, parenterally or in any other acceptable formula to treat and prevent asthma and COPD.

EXAMPLES

Treatment for acute asthma attacks

Example 1:

The following treating methods were employed in treating asthma, in accordance with the present invention.

Participants: 15 patients (16 - 65 years) were treated for acute asthma exacerbation with peak expiratory flow rate (PEFR) < 60% of the predicted value after receiving 2-agonist inhalers. All patients experienced typical severe physical symptoms of asthma attacks.

Intervention: Zinc salicylate (20%) and MSM (80%) was given orally at a dose of 250mg every 8 hours over 30 days.

Findings: Initial significant relief of symptoms and improvement of lung function as determined by peak expiratory flow rate (PEFR), were achieved within 30 minutes after the first application of the treatment. After 3 days, all patients were symptom-free and normal lung function was restored.

All patients reported that they had never experienced such a complete relief and improved well- being before receiving the applied therapy. No adverse effects were reported. No patient showed significant changes of blood pressure or other vital parameters. All patients were able to either completely avoid the usage of 2-agonist or could reduce it by 70%. No patients needed further hospitalization or professional medical care.

Example 2:

Participants: 12 patients (15-69 years) were treated for acute asthma attacks, and all were resistant to 2-agonist therapy and oral theophylline preparation. The peak expiratory flow rate (PEFR) < 60% of the predicted value. Eight patients suffered excessive mucus production, were also resistant to conventional therapies.

Interventions: Zinc salicylate (20%) and MSM (80%) was given at a dose of 200mg orally every 8 hours over 30 days.

Findings: The first significant improvement in both subjective and functional parameters was registered by the patients 30 minutes after the application of zinc salicylate. Within three days of the therapy, most patients were completely symptom-free and their lung function improved to 70-90% of the predicted PEFR.

The eight patients who had experienced high mucus production before the treatment reported a significantly reduction of mucus and subjective well-being. None of the patients reported any side effects.

Example 3:

Participants: 10 asthma (14- 76 years) who suffered from low resistance to exercise, cold weather and exercises-induced asthma. None of the patients exhibited history of allergy, although recurring infections were common. These patients developed long-lasting episodes of active asthma which required treatment by combined drugs comprising of 2-agonist, oral or inhaled steroids, theophylline and anti-biotics.

Intervention: Patients were treated with 200 mg of Zinc salicylate (20%) and MSM (80%) orally every 8 hours over a period of 30 days.

Findings: All patients reported remarkable improvement of lung function and subjective well-being within a period of 60 minutes to 2 hours after the first zinc salicylate application. Symptoms of asthma and infections disappeared within three days after the start of the zinc salicylate therapy. All patients experienced significant subjective improvement in their over-all health and well-being. After 10 days of zinc salicylate (20%) and MSM (80%) application, all patients were able to eliminate their prescribed symptom controlling drugs for both asthma arid infection.

No known side effects had been reported by any patient during the treatment period.

Therapy for chronic asthma in adults and children

Example 1:

Participants: 12 patients diagnosed with chronic asthma (12-78 years) who experienced

unsatisfactory results using conventional therapy were recruited for treatment with zinc salicylate. The conventional drugs for these patients included combination of inhaled long-acting 2-agonist, steroids, chrome salts, theophyllines, and/or leukotriene antagonists. Despite these therapies the patient's health was significantly hampered due to continued asthma symptoms and the

medication's side effects.

Intervention: patients were then treated with 250 mg zinc salicylate (20%) and MSM (80%) 3 times daily over a period of 30 days.

Findings: All patients responded well immediately to zinc salicylate. After 3 days of therapy, asthma symptoms were significantly reduced and the need of broncho-dilating 2-agonists was reduced by 70 -90%. All patients were able to decrease the use of their preventive conventional medication gradually. Within a period of 3 weeks, all patients were able to avoid conventional medications completely. No adverse reaction or intolerance to zinc salicylate occurred during the course of the therapy. Mode of action, biological mechanisms of zinc salicylates (in vitro examples):

Example 1:

Lung fibroblast

Based on our earlier studies in which we showed disease specific cellular pathologies for asthma and COPD cells compared to healthy human lung cells, we have used human lung fibroblasts obtained from healthy controls, asthma patients and COPD patients (6 in each group) and assessed the inhibitory effect of zinc salicylate on serum and platelet derived growth factor-BB (PDGF-BB) induced proliferation over 48 hours.

la PDGF-BB

PDGF-BB was used as a well-defined proliferation stimulus and cell counts were performed manually using an improved Neugebaur chamber slide. As shown in figure 1, PDGF-BB (10 ng/ml) significantly increased cell numbers (proliferation) in all 3 groups (control, asthma, COPD). Increasing concentration of zinc salicylate (1 pM - 100 nM) significantly reduced PDGF-BB stimulated proliferation. Moreover, the inhibitory effect was stronger in COPD cells compared to asthma cells suggesting a disease specific response. The highest concentration of zinc salicylate (100 nM) alone had no effect on proliferation (Figure 1). This finding suggests that zinc salicylate has no lethal effect on human lung fibroblasts. lb Serum stimulation of proliferation

Fetal calf serum served as standard un-specific proliferation stimulator. In vitro fetal calf serum is used instead of human serum for most proliferation studies. It is accepted that 10% fetal calf serum represents the tissue concentration of human serum under inflammatory conditions.

Stimulating fibroblasts with 10% serum increased the cell numbers (proliferation) significantly in all 3 groups (control, asthma, COPD) within 3 days (Figure 2). Incubation with zinc salicylate dose dependently (1 pM - 100 nM) reduced fibroblast proliferation in all 3 groups at concentrations > 100 pM. In cells of controls, a significant reduction was observed already at 10 pM (Figure 2). The highest concentration of zinc salicylate (100 nM) alone had no effect on proliferation (Figure 2). This finding suggests that zinc salicylate has no lethal effect on human lung fibroblasts.

Example 2:

Lung fibroblasts

This inhibitory effect of zinc salicylate on fibroblast proliferation was further investigated by detection of its effect on two regulators of cell proliferation: proliferating cell nuclear antigen (PCNA) and p2i ^Wafl/Cipl . As shown in figure 3, PCNA expression was up-regulated by PDGF-BB (10 ng) within 24 hours and this was dose dependently (1 nM - 100 nM) reduced by zinc salicylate. This finding suggests that zinc salicylate reduces the fibroblast ability to undergo proliferation as one of its essential signaling proteins (PCNA) is down-regulated. To determine the effect of a drug on a specific protein it is essential to determine so called house-keeping protein which is not altered by most drugs or cell culture conditions. In these studies, we have used the well-known g protein named glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

PCNA is negatively correlated to the expression of the cell cycle regulator _p 2i ^{Wafl Cipl} _/ therefore, we analyzed the expression of _p 2i ^Wafl/0pl j _n the same protein samples tested for PCNA and we observed that the expression of _p 2i ^{Wafl Cipl} was up-regulated in the presence of zinc salicylate dose dependently (figure 4). The anti-proliferative effect of p21 ^{Wafl Cipl} depends on its location within the cell; while located in the cytosol, it has no inhibitory effect on proliferation. Only when activated and translocated into the nucleus, it is able to interrupt cell proliferation (Chiappara 2014, Gehen 2007). Our results suggest that zinc salicylate in a mild activator of p21 ^{1 9} but together with other cell proliferation inhibitors such as formoterol, it potentiates their effect (Figure 4).

Example 3:

MMP-2 and MMP-9 are the major enzymes that degrade collagens in any type of tissue and therefore also regulate organ function or pathology. We determined the gelatin digesting activity (zymography) which shows each MMP in form of two separate bands (figure 5), the upper band shows the amount of inactive MMP and the lower band that of activated MMP. Furthermore, zymography provides two types of information: a) the amount of MMP which is the sum of the inactive (large white band) and the active MMP (lower band); and b) the activating effect of a drug or substance on MMPs (ratio of upper to lower band).

As described above, chronic inflammatory lung diseases (asthma, COPD) are characterized by remodeling of the airway wall which reduces the lumen of the airway and therefore limits breathing. None of the existing drugs which are used for the therapy of asthma and COPD have any proven inhibitory effect on airway wall remodeling. MMPs, especially MMP-2 and MMP-9 are the major enzymes that degrade the deposition of collagens in the airway wall (Black 2003, Greenllee 2007, Okamoto 2004). Importantly, all MMPs need a zinc atom within their enzymatic, and without zinc, M MPs cannot exert their degrading activity on collagens and gelatin region (Aureli 2008, Loffek 2011). Based on our experience, we assessed the expression and activity of the major two MMPs (MMP-2, MMP-9) produced by lung fibroblasts using gelatin zymography (Papakonstantinou 2015). As shown in figure 5, zinc salicylate increased both the expression and activity of MMP-2 and MMP-9 in fibroblasts obtained from healthy probands, and asthma patients, while the effect was less pronounced in cells of COPD patients (figure 5). Surprisingly, zinc salicylate seems to immediately activate MMP-2 and MMP-9 as the band s for the inactive pre-cursor MMPs is

This data suggests that zinc salicylate applied over a period of 1 month has the potency to reduce existing airway wall thickening which will lead to improved flexibility, relaxation, and opening of the airways as it was observed indirectly by patients treated with zinc salicylate (see examples for acute and chronic asthma above).

Detailed Methods:

The therapeutic compound consists of:

2 parts of Zinc Salicylate Ci ₄ H ₁₀ O ₆ Zn (MW339.6056 g/mol, PubChem CID 54693468; UNII- 960Q2F972S)

8 parts of Methylsulfonylmethane (MSM) C ₂ H ₆ 0 ₂ S (MW94.1328 g/mol, PubChem CID 6213) 10 parts of Methylcellulose C ₂₉ H ₅₄ 0 ₁₆ (MW958.7295 g/mol, PubChem CID 44263857)

This mixture is blended in capsules or tablets of a total weight of 500 mg.

The daily dosage is 1 to 3 capsules, 3 times daily after meals.

The treatment course can range from 30 to 180 days, or as long as needed to prevent asthma and COPD symptoms; or to compensate for zinc deficiency.

The efficacy of the treatment can be evaluated by symptom recording and standard lung function testing. Concept proof in vitro:

1. We have reported earlier that bronchial mesenchymal cells of asthma patients proliferate faster than that of controls which may explain their increased number and activity in asthma (Johnson 2002, Roth 2004). Therefore, we will investigate in isolated human fibroblasts and airway smooth muscle cells from healthy controls as well as from patients with asthma and COPD (each group = 10 - 20) if zinc salicylate (0.1 pM to 100 nM) reduces proliferation. Cells will be pre-incubated with zinc salicylate for different time periods (0, 5, 10, 15, 30 minutes as well as for 0, 1, 2 and 4 days) in the presence and absence of mitogenic stimuli (PDGF-BB 10 ng/ml, 5% fetal calf serum). Proliferation will be detected by MTT assay and in parallel by manual cell counts (Johnson 2002, Roth 2004).

End-Points: Tissue remodeling: MTT = cell-activity and cell number = proliferation

2. Proliferation is regulated by nuclear proteins that control the duplication of the DNA including PCNA, ERK1/2 mitogen activated protein kinase and AP-1 (Eickelberg 2001, Roth 2006). These proteins are present in cells in a latent form and need to be activated by phosphorylation which can be detected by immune-blotting. For the short term periods, we will determine the activation of cell proliferation inducing proteins including PCNA, AP-1 and Erkl/2 mitogen activation protein kinase. We expect that pre-incubation with zinc salicylate reduces or completely blocks the activation of these mitogenic proteins. Protein activity will be determined by immune-blotting (Eickelberg 2001).

End-Points: Tissue remodeling: activity of proliferation regulating protein activity.

3. In regard to airway wall remodeling, we will assess the effect of zinc salicylate in increasing

concentrations (0.1 pM to 100 nM) on the deposition of collagen type I and fibronectin by disease fibroblasts obtained from patients with COPD and asthma. Cells will be exposed to TGF-β for 24 and 48 hours.

End-Points: Tissue remodeling

4. Reduction of remodeling and degradation of excessive deposed connective tissue (extracellular matrix consisting mainly of collagen type-l and fibronectin) is a major goal for future asthma and COPD therapies (King 2016, Postma 2014). In regard In the cell culture medium of these cells, we will determine the expression and activity of two major MMPs (MMP2, MMP9) by gelatinolitic activity.

End-Points: Tissue remodeling: Connective tissue degrading enzyme activity.

5. We will prove that zinc salicylate has an anti-inflammatory effect on cytokine secretion over a period of 2 days in fibroblasts and airway smooth muscle cells that were stimulated with asthma and COPD relevant pro-inflammatory cytokines (PDGF-BB, TGF-β and TNF-ct). The concentrations of zinc salicylate will increase from 0.1 pM to 100 nM. The pro-inflammatory cytokines that will be tested are: IL-6, IL-8 and eotaxin).

End-Points: Inflammation: Secretion of pro-inflammatory cytokines. References

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