RU2789128C2 - Compositions and methods for treatment of lung emphysema and other forms of copd - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: group of inventions relates to the field of pharmacotherapy and concerns a combination for the lung emphysema treatment and treatment method. The combination contains an active agent containing a copper compound, where the copper compound is copper sulfate, in an amount of about 0.5 mg to about 1 mg, and glycosaminoglycan or its physiologically acceptable salt, where glycosaminoglycan is heparin, in an amount of about 150,000 IU. A method for treating a subject suffering from lung emphysema involves the administration by inhalation to said subject of a combination containing a copper compound, where the copper compound is copper sulfate, in an amount of about 0.5 mg to about 1 mg, and glycosaminoglycan or its physiologically acceptable salt, where glycosaminoglycan is heparin, in an amount of about 150,000 IU.

EFFECT: beneficial effect on the restoration and development of elastin fibers in the lungs of patients with emphysema while preventing copper-induced stimulation of collagen crosslinking.

13 cl, 17 dwg

Description

Technical field

The present invention relates to the field of pharmacotherapy. In particular, the present invention relates to compositions and methods for the treatment of emphysema with or without airflow limitation and other forms of chronic obstructive pulmonary disease (COPD).

State of the art

COPD is one of the most common non-communicable conditions. COPD is a complex clinical situation that has as a common factor smoking-related fixed airflow limitation that does not change significantly over several months of follow-up. Moreover, airway obstruction shows an abnormally rapid progressive worsening with age. The course of the disease progresses to chronic respiratory symptoms.

The pathogenesis of COPD is characterized by chronic inflammation and accelerated loss of elastic fibers [1] (numbers in square brackets refer to numbers in the list of references at the end of the description.). Chronic airflow limitation in COPD is caused by small airway disease, destruction of the lung parenchyma (ie, emphysema), or a combination of the two [1]. Emphysema is a COPD phenotype characterized by excessive loss of elastin fibers in the lung parenchyma due to protease/anti-protease imbalance and elastin breakdown/repair. Although the role of collagen accumulation in the lung parenchyma in the pathogenesis of emphysema is evaluated to a lesser extent than the role of elastin destruction, it is another important pathogenetic characteristic of emphysema [2].

Elastin, the main component of elastic fibers, is a unique protein that provides elasticity, resilience and deformability to the lungs, and therefore it is the main condition for respiration [3]. Elastin is mainly produced in the womb and in early childhood [4].

The production of elastin fibers begins with the synthesis of tropoelastin, a precursor of elastin, by several cell types [4]. Tropoelastin is subsequently secreted into the extracellular matrix, transported to the fibrillar scaffold, coordinated with many other tropoelastin proteins into polymers, and finally cross-linked with other tropoelastin polymers into mature and strong elastin fibers that are essential throughout life [4]. The crosslinking process is facilitated by LOX enzymes and LOX-like proteins (LOXL) 1 to 4 [4]. Fibulins 4 and 5 also play an important role in the development and maintenance of elastin fibers. While prototype LOX and fibulin-4 are primarily involved in the initial development of elastin fibers, LOXL1 and fibulin-5 are important in elastin repair.

The elastic properties of the lungs are impaired by the destruction of elastin [4], which is enhanced in patients with COPD due to an imbalance between the protective action of antiproteases and the destructive properties of proteases [3]. Another factor in elastin degradation is an imbalance between elastin degradation and elastin repair, given that damaged elastin fibers are more susceptible to further degradation by proteases than native fibers [5]. Moreover, elastin fibers crosslinked by LOX enzymes are relatively resistant to proteases, while non-crosslinked proteins are easily degraded [6-8]. Accelerated degradation of lung elastin is an important pathogenetic mechanism of emphysema leading to loss of lung function [9].

Besides the accelerated loss of elastin, there is another problem in the extracellular matrix of patients with emphysema. Collagen levels in the lungs of patients with emphysema have been shown to be elevated compared to controls [10] and inversely related to forced expiratory volume in one second (FEV ₁ ) [11].

Copper serves as a cofactor in the activation of LOX enzymes (i.e., prototypes of LOX and LOXL1-4) [12]. Induced copper deficiency in chickens disrupts elastin cross-linking due to a decrease in LOX activity and leads to a pronounced decrease in elastin content [12]. The reason for the lower elastin content in copper deficiency appears to be due to increased degradation, as uncrosslinked tropoelastin is much more protease sensitive than properly crosslinked elastin [12]. Replenishing copper deficiency in copper-deficient chickens restores the deposition of protease-resistant elastin fibers to values close to normal [7].

The loss of elastin fibers causes COPD in the lungs, at the same time it causes the formation of wrinkles in the skin [13]. A cream containing copper in healthy people causes an increase in elastin cross-links in the skin [14].

There is evidence of copper deficiency in pulmonary emphysema. Emphysematous changes can be induced in rats and hamsters when fed a copper-deficient diet [15,16]. Copper deficiency caused a decrease in elastin content by 17% and an increase in alveolar spaces by 35% in the lungs of rats [15]. Replenishment of the lack of copper restored the ultrastructure of lung elastin almost to normal [15].

Expression of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) is increased in emphysema [17, 18]. Transgenic mice overexpressing lung-specific TNF-α develop emphysematous lesions [19]. It was concluded that copper deficiency occurs after TNF-α-induced chronic lung inflammation and likely plays an important role in inflammation-induced lung damage.

There is also a human study suggesting local copper deficiency in areas with emphysema. The protein containing the copper metabolism domain 1 (COMMD1) is a key regulator of copper metabolism [20]. It has been demonstrated that the levels of COMMD1, as well as active LOX, LOXL1 and LOXL2, are reduced in pulmonary emphysema [21].

The concentration of copper in the exhaled air condensate in patients with COPD is reduced and is inversely proportional to FEV ₁ [22]. This may indicate the presence of copper deficiency in the lungs in COPD. Accordingly, people with Menkes disease, a genetic disorder of copper transport, can develop severe emphysema [23].

Although the prior art may suggest that copper is a suitable stimulant for elastin repair and development in emphysema lungs, there is one important problem that prevents the use of copper as a therapy in patients with emphysema. LOX enzymes are not only stimulators of elastin cross-linking, but also of collagen cross-linking. Increased collagen cross-linking will lead to improved organization, maturation, and thus accumulation of collagen in emphysema lungs, which is highly undesirable given the fact that collagen levels are already elevated in emphysema patients and will induce transition from emphysema to pulmonary fibrosis, which is another severe lung disease. Thus, copper-induced stimulation of collagen accumulation suggests that copper should not be used as a therapy in patients with emphysema.

The most important complaints of patients with COPD are dyspnea on exertion, and in later stages also at rest, and exercise intolerance. From a mechanistic point of view, it seems more appropriate to consider COPD as a syndrome rather than as a single disease. Airway obstruction in patients with COPD is caused by small airway disease, emphysema, or a combination of the two. Significant emphysema is also often found on computed tomography (CT) in (former) smokers without COPD (i.e., no airflow obstruction).

Clinically, it is difficult to distinguish emphysema from chronic bronchitis because of the similar symptoms of dyspnea, cough, and wheezing. A significant proportion of patients present with combinations of characteristics attributed to chronic bronchitis or emphysema.

The Fleischner Chest Imaging Society has published a report describing the subtypes of COPD that are identified by CT scans. The main pathological categories that can be distinguished are airway wall thickening, bronchiectasis, small airway disease, and emphysema.

It should be understood that these radiological abnormalities can also be identified in people without COPD. Emphysema is characterized by irreversible damage to the lungs. As a result, elasticity of the lung tissue is lost, causing airway collapse and airway obstruction. Chronic bronchitis is an inflammatory disease that starts in the smaller airways in the lungs and gradually progresses to the larger airways. It increases the amount of mucus in the airways and increases the infection of the bronchi with bacteria, which in turn obstructs the passage of air.

Modern pharmacological therapy for COPD can improve respiratory symptoms and exacerbation rates, as well as improve quality of life and physical performance [1]. Inhaled therapy with long-acting bronchodilators and corticosteroids has also been reported to reduce the rate of deterioration in lung function [2–4]. Unfortunately, inhaled bronchodilators and corticosteroids primarily target the airway component of COPD and do not have the same beneficial effects in dominant emphysema as they do in patients with COPD with a dominant airway component. However, the presence of emphysema on CT is an important finding as it is strongly associated with mortality.

No single intervention for COPD, with the exception of lung transplantation, has been proven to be effective in restoring lung function [1]. Therefore, there is an urgent need to develop a specific pharmacological therapy for a large group of individuals with emphysema.

WO 03/068187 A1 discloses the use of glycosaminoglycans, eg heparin, for the treatment of respiratory diseases such as COPD, in particular chronic airflow limitation (CAL).

WO 2012/073025 A1 discloses glycosaminoglycans such as heparin for use in the treatment and/or prevention of COPD, in which, after administration to a subject, heparin reduces inflammation in the subject's lungs.

Brief description of the invention

The present invention is based on the surprising discovery that a combination of copper and specific glycosaminoglycans, in particular heparin, can be used to treat emphysema and other forms of COPD. The combination has a beneficial effect on the repair and development of elastin fibers in the lungs of emphysema patients and at the same time prevents copper-induced stimulation of collagen cross-linking.

Although the prior art discloses the use of heparin as an inhaled monotherapy for patients with COPD [24,25], it does not demonstrate or suggest a synergistic value of adding heparin to inhaled copper therapy to further stimulate recovery/development processes through stimulation of tropoelastin crosslinking. and, more importantly, preventing copper-induced stimulation of collagen cross-linking.

Accordingly, in one aspect, the present invention provides a composition for use in a method of treating pulmonary emphysema and other forms of COPD comprising an active agent comprising a copper compound and a glycosaminoglycan or a physiologically acceptable salt thereof. Particularly preferred is administration by inhalation.

In a preferred embodiment, the composition of the present invention is used as an adjunct to standard pharmacological treatment for COPD, which includes bronchodilators and immunomodulators such as inhaled corticosteroids and oral macrolides.

In another aspect, the present invention provides a method of treating a subject suffering from pulmonary emphysema or another form of COPD, which comprises administering to said subject a therapeutically active amount of an active agent composition comprising a copper compound and glycosaminoglycan or a physiologically acceptable salt thereof.

The significance of the active ingredients of the composition according to the present invention in relation to the repair and development of elastin fibers and the prevention of collagen accumulation will be more fully described in the detailed description that follows.

Brief description of the drawings

Fig. 1: Explanted right lung of a 55-year-old male patient with a combination of fibrosis and emphysema. A. Macro anatomy of the resection specimen showing extensive changes throughout the lung, with advanced emphysema in the upper and middle lobes, blistering in the upper lobe, and micronodular pleural changes in the middle (*) and lower lobes consistent with extensive parenchymal fibrosis. B. Representative microscopy (hematoxylin-eosin stain, 2.5x magnification) of the upper lobe showing extensive emphysematous changes (*) and mild to moderate interstitial fibrosis. C. Representative microscopy (hematoxylin-eosin stain, 10x magnification) of the lower lobe, characterized by severe fibrosis with architectural disturbances and numerous fibroblastic foci (**), consistent with the usual picture of interstitial pneumonia. The median proportion showed a combination of emphysema and severe fibrosis (not shown).

Fig. 2: Relative concentrations of elastin, collagen, (iso)desmosin (DES) and hydroxyproline (Hypro) in the lungs of control subjects, patients with emphysema, idiopathic pulmonary fibrosis and combined pulmonary fibrosis (CPFE; in the basal and apical zones of the lungs). Levels in the control group are set to 100%.

Fig. 3: Relative serum copper concentrations and exhaled breath condensate (EBC) in control subjects (set as 100%) and patients with emphysema and idiopathic pulmonary fibrosis (IPF).

Fig. 4: Relative copper concentrations in lung parenchyma of control subjects (set as 100%) and patients with emphysema, idiopathic pulmonary fibrosis (IPF), and combined pulmonary fibrosis and emphysema (CPFE; apical and basal regions of the lungs).

Fig. 5: Levels of (iso)desmosin (DES) in fibroblast medium ( in vitro cell culture) without supplemental copper (baseline) or (starting copper concentration) + 0.5, 1, 2, 4, 8, 16 and 32 * baseline copper level.

Fig. 6: Relative gene expression of lysyl oxidase (LOX), lysyl oxidase-like enzyme 1 (LOXL1), elastin (ELN), fibulin-5, and levels of tropoelastin, insoluble elastin, (iso)desmosin (DES), and collagen in fibroblasts ( in vitro cell cultures), grown at initial concentration + 8*initial concentration of copper only, copper plus retinoic acid (RA), copper plus minoxidil and copper plus heparin.

Fig. 7: Relative levels of insoluble elastin and (iso)desmosin (DES) in fibroblasts ( in vitro cell cultures) grown at initial concentration + 8*initial concentration of copper only, copper plus vitamin K1, copper plus vitamin K2, and copper plus magnesium sulfate.

Fig. 8: Total lung capacity (TLC) and mean linear length (Lm) in control mice, mice treated with copper, and mice treated with copper/heparin.

Fig. Figure 9: (Iso)desmosine (DES), collagen and hydroxyproline (Hypro) in control mice, mice treated with copper, and mice treated with copper/heparin.

Fig. 10: Microscopy (10x magnification) of the lung of a mouse from the placebo group shows extensive emphysematous changes.

Fig. 11: Microscopy (10x magnification) of a lung from a copper/heparin mouse group shows normal alveoli without emphysematous changes.

Fig. 12: First measurement of particle size distribution in 5 ml of 0.9% sodium chloride solution with 5000 IU of heparin and 0.5 mg of copper using laser diffraction analysis.

Fig. 13: Remeasurement of particle size distribution in 5 ml of 0.9% sodium chloride solution with 5000 IU of heparin and 0.5 mg of copper using laser diffraction analysis.

Fig. 14: First measurement of particle size distribution in 5 ml of 0.9% sodium chloride solution with 100,000 IU of heparin and 1.0 mg of copper using laser diffraction analysis.

Fig. 15: Remeasurement of particle size distribution in 5 ml of 0.9% sodium chloride solution with 100,000 IU of heparin and 1.0 mg of copper using laser diffraction analysis.

Detailed description of the invention

Reactivation of the production of lung elastin fibers and repair of damaged elastin fibers are necessary conditions for the restoration of lung function. Three steps are critical to the production of new and repair of damaged elastin fibers in adults: (a) activation of tropoelastin synthesis, (b) activation of assembly of tropoelastin proteins into polymer chains, and (c) activation of lysyl oxidase-mediated crosslinking.

Compositions and methods for the treatment of pulmonary emphysema and other forms of COPD are proposed. These compositions include a copper-containing active agent and a glycosaminoglycan or a physiologically acceptable salt thereof.

Compositions of the present invention should be used to treat subjects suffering from or at risk of developing pulmonary emphysema, with or without airflow limitation, and other forms of COPD. Typically, the subject is a mammal, in particular a human, but may be a vertebrate. The airflow limitation is usually progressive and is associated with a decrease in the elasticity of the elastin fibers in the lungs.

Proposed methods for the treatment of emphysema and other forms of COPD. Such methods include diagnosing one or more lung diseases in a subject and administering a therapeutically effective amount of a composition comprising a copper-containing active agent and a glycosaminoglycan or a physiologically acceptable salt thereof.

The term "treat" or "treatment" refers to the implementation of a protocol, which may include the administration of one or more compositions or active ingredients to a patient (human or other) with the aim of repairing damaged lungs and/or preventing the progression of a disease or disorder. "Treat" or "treatment" does not imply complete cessation of disease progression, does not imply complete repair of all lung damage, and in particular includes protocols that have little effect on the patient.

The term "therapeutically effective amount" means the amount of the instant composition which, when administered to a patient, is sufficient to result in an improvement in the patient's condition. Improvement does not mean a cure, and may involve only a minor change in the patient's condition. It also includes the amount of active agents that prevent the condition or stop or delay its progression.

For purposes of the present invention, "other forms of COPD" may be defined as a condition of airway wall thickening, bronchiectasis, chronic bronchitis, and/or small airway disease.

The subject is usually an adult. For example, the subject may be 21 to 85, preferably 25 to 70, more preferably 30 to 60, and even more preferably 40 to 50 years old. The onset of any of the symptoms mentioned herein or specific symptoms usually occurs in adulthood. For example, the subject could be at least 25, more preferably at least 30, even more preferably at least 35, and even more preferably at least 40 years old prior to the onset of a particular symptom. In particular, symptoms associated with later stages of emphysema, such as any of those mentioned herein, may appear at such later stages of life. Subjects with a genetic predisposition to develop emphysema, such as those deficient in alpha ₁ antitrypsin, may develop the disease earlier. For example, they may exhibit one or more or a particular symptom between the ages of 20 and 31, preferably between 22 and 28, or more preferably between 24 and 26 years of age. Alternatively, they may first experience a symptom in any of the age ranges mentioned herein. A subject may be diagnosed at any age or within any of the age ranges specified herein.

In cases where the subject is not a human, it may be a domestic animal or an animal important to agriculture. The animal may be, for example, a sheep, pig, cow, bull, poultry, or other commercially raised animal. In particular, the animal may be a cow or a bull, and preferably a dairy cow. The animal may be a pet, such as a dog, cat, bird, or rodent. In a preferred embodiment, the animal may be a cat or other feline animal. The animal may be a monkey, such as a non-human primate. For example, the primate may be a chimpanzee, a gorilla, or an orangutan. In a preferred embodiment of the present invention, the animal may be a horse and, for example, may be a racehorse.

The main therapeutically active ingredients of the compositions according to the present invention are copper and glycosaminoglycan. These ingredients will be discussed in more detail below.

Copper

In the compositions according to the present invention, an active agent containing a copper compound is used. As used herein, the term "active agent" refers to a chemical element of a compound that has a stimulating effect on the recovery and development of lung elastin. The active agent includes a copper compound, in particular a copper salt. Various copper salts can serve as a source of copper compounds. Suitable copper salts include, but are not limited to, copper sulfate, copper chloride, copper gluconate, copper acetate, copper heptanoate, copper oxide, copper methionate, copper (I) oxide, copper chlorophyllin, and copper calcium edetate. Of these, copper sulfate is preferred.

Glycosaminoglycan

Glycosaminoglycans and, in particular, heparin are used in the compositions of the present invention. Glycosaminoglycans are linear heteropolysaccharides having characteristic repetitive disaccharide sequences that are typically highly N- and O-sulfated at D-glucosamine, galactosamine, and uronic acid residues.

Any suitable glycosaminoglycan may be used in the present invention. Glycosaminoglycans and salts of glycosaminoglycans suitable for use in the present invention will have an average molecular weight of 12 to 18 kDa. Can be offered glycosaminoglycan or salt with different molecular weight in this range. For further details, reference may be made to the prior art, in particular WO 03/068187 and its counterpart EP 1511466, the contents of which are incorporated herein by reference.

The glycosaminoglycan may be any suitable commercially available glycosaminoglycan and may, for example, be unfractionated glycosaminoglycan. Glycosaminoglycan is usually isolated from natural sources such as animal. In some cases, the glycosaminoglycan may be synthesized rather than being a naturally occurring molecule.

Any suitable physiologically acceptable glycosaminoglycan salt may be used in the present invention, in particular a metal salt such as sodium salt, alkali metal salt or alkaline earth metal salt. Other salts include calcium, lithium and zinc salts. Ammonium salts can also be used. The salt may be sodium glycosaminoglycanate or glycosaminoglycan sulfate. Salts of derivatives of specific glycosaminoglycans mentioned herein can also be used in the present invention. In the present application, where glycosaminoglycan is mentioned, such mention also includes its physiologically acceptable salts.

In a particularly preferred embodiment of the present invention, the glycosaminoglycan used is any of chondroitin sulfates A to E, heparin, heparin sulfate, heparan, heparan sulfate, hyaluronic acid, keratan sulfate, a derivative of any of these, or a physiologically acceptable salt thereof, or a mixture thereof, or any two of them.

Heparin is a naturally occurring mucopolysaccharide present in various organs and tissues, especially in the liver, lungs and large arteries. Heparin is a polymer consisting of alternating a-D-glucosamine and hexuronate residues connected by (1,4)-glycosidic bonds. When glycosaminoglycans are synthesized in nature, they are usually conjugated to the central core of the protein. Preferably, however, the glycosaminoglycans used in the invention do not have such a central core. Commercially available preparations of glycosaminoglycans usually do not have a core, and they can be used.

Preferably, unfractionated heparin is used in the formulation. Instead of unfractionated heparin, low molecular weight heparins, including dalteparin and enoxaparin, and other members of the glycosaminoglycan family, including heparan sulfate, with a copper compound in the inhalation formulation can be used to promote tropoelastin polymerization and/or prevent copper-induced collagen cross-linking.

Heparin is clinically used as an anticoagulant and is thought to exert its effects through interactions with antithrombin III (AT-III), heparin cofactor II, and other coagulation factors. Typically, heparin retains some anticoagulant activity, ie it can increase the clotting time of the individual. Thus, preferably, heparin will be able to bind antithrombin III (AT-III) and/or heparin cofactor II (HCII) and therefore inhibit blood clotting. Preferably it will be capable of complexing with AT-III, thrombin and a blood coagulation factor. However, in some embodiments, heparin that does not have anticoagulant activity or that has reduced anticoagulant activity can also be used. Therefore, heparin can be modified such that it has 0 to 80%, preferably 5 to 60%, more preferably 10 to 40%, and even more preferably 10 to 30% of the activity of the unmodified form or compared to unmodified heparin. . Other glycosaminoglycans, in particular dermatan sulfate, also have anticoagulant activity. Therefore, it is preferred that the glycosaminoglycans and their derivatives used retain some anticoagulant activity, as discussed above for heparin and its derivatives.

Other components

To reactivate lung fiber elastin production, repair damaged elastin fibers, slow down the rate of elastin degradation, and inhibit advanced glycation end product (AGE) formation, combining a composition of the present invention containing a copper compound and a glycosaminoglycan with other health or pharmaceutically active ingredients in a single composition or in the form of a kit for simultaneous, sequential or separate administration may be preferred. For example, it is contemplated that the composition of the present invention may be provided in combination with drugs or substances that affect the metabolism of elastin in the vasculature, selected from the polyphenols epigallocatechin-(3-)gallate (EGCG) and pentagalloylglucose (PGG), ATP-dependent potassium channel openers, e.g. minoxidil, nicorandil, diazoxide, pinacidil and cromacalin, magnesium, vitamin K1, vitamin K2, arterial AGE degrading agents, e.g. aminoguanidine, pyridoxamine, N-phenacylthiazolium bromide, alagebrium, and flavonoids kaempferol, genistein, quercitrin, quercetin, and epicatechin), compounds with potential effects on lung elastin metabolism, selected from vitamin A, vitamin D, and pentagalloylglucose.

Subject score

The present invention relates to compositions containing an active agent containing a copper compound and a glycosaminoglycan or a salt thereof, for use in facilitating the repair and development of elastin fibers in the lungs of emphysema patients and preventing copper-induced stimulation of collagen cross-linking. The copper compound and glycosaminoglycan or salt used, the route of delivery, and any other parameters of the composition and the subject being treated may be the same as described herein for any of the other embodiments of the present invention.

Compositions according to the present invention preferably cause an improvement in the condition of the subject and/or prevention/slowing the progression of the disease. Thus, the compositions can be used to treat a patient suffering from or prone to emphysema and/or other forms of COPD as defined herein. They can prevent, alleviate, improve, or cure a condition. They can slow or stop the progressive deterioration that is characteristic of emphysema and other forms of COPD or, in some cases, even reverse some of the deterioration. They may prevent, reduce, or reverse one or more of the symptoms associated with emphysema and other forms of COPD. They also preferably improve the subject's well-being and quality of life.

The composition according to the present invention preferably reduces, eliminates or at least prevents a further increase in one or more of the following:

- accelerated decline in lung function parameters, including but not limited to FEV ₁ and diffusive capacity;

- damage to the structure of the lung.

Treatment with the compositions of the present invention may also mean that the FEV1/FVC ratio is no longer reduced or improved. For example, this ratio may be closer to that expected in a healthy subject.

The compositions can reduce the breakdown of lung elastin and help restore lung elastin. They may also have a preventive effect on collagen accumulation in emphysema lungs.

Compositions according to the present invention can reduce the destruction of the structure of the lungs, such as the destruction of elastin in the airways and alveoli, and therefore the loss of elasticity of the lungs. They can reduce or prevent the collapse of parts of the lung and/or the development of enlarged air spaces into which air can enter. The compositions can prevent or reduce any of the pathological changes associated with emphysema and other forms of COPD described herein. In particular, they can prevent the progression of the pathological change. They can also prevent or delay the occurrence of a specific pathological change.

The compositions of the present invention can generally reduce the decline in lung function parameters such as diffusing capacity and FEV ₁ by 10 to 100%, preferably 20 to 80%, more preferably 30 to 60%, and even more preferably 40 to 50%. . They can reduce the annual decrease in FEV ₁ by 10 to 100 ml, preferably 20 to 60 ml and even more preferably 30 to 40 ml per year. In some cases, with treatment, the subject will show improvement in lung function parameters such that FEV ₁ and diffusive capacity are between 25% and 100%, preferably between 40% and 100%, more preferably between 60% and 100%, and even more preferably between 80% and 100%. % of predicted value.

Measurement of lung density using computed tomography is a convenient way to quantify the severity of emphysema. Compositions of the present invention can slow or halt the progressive decline in CT lung density in patients with emphysema, or even increase lung density.

Compositions of the present invention can reduce lung tissue destruction and facilitate repair of damaged lung tissue.

The compositions of the present invention can eliminate, delay the onset or reduce the severity of any of the symptoms and signs of emphysema and other forms of COPD mentioned herein.

Introduction and composition

Pharmaceutical compositions according to the present invention can be prepared by preparing at least one copper compound, preferably copper sulfate, and a glycosaminoglycan, preferably heparin, with a standard physiologically and in particular pharmaceutically acceptable carrier and/or excipient, as is usually done in this the field of technology.

The exact nature of the composition will depend on several factors, including the particular copper compound and glycosaminoglycan used and the desired route of administration. Suitable formulation types are fully described in Remington's Pharmaceutical Sciences, 22nd Edition, Mack Publishing Company, Eastern Pennsylvania, USA, the disclosure of which is incorporated herein by reference in its entirety.

In a particularly preferred embodiment, the compositions containing the copper compound and the glycosaminoglycan are administered in the form of an inhalation therapy, including but not limited to inhalation of the formulation using a nebulizer, metered dose inhalers, or in a form suitable for a dry powder inhaler. The composition may be in a blister pack or in a split capsule. Thus, the introduction can usually be done through the mouth.

Since the compositions of the present invention are usually administered by inhalation or insertion, it is preferred that they be in a form suitable for administration by this route. In particular, the compositions may be in a form suitable for inhalation and/or insertion.

Suitable methods for the preparation and preparation of compositions for administration by inhalation are well known in the art and can be used in the present invention. The composition provided by copper sulfate and heparin as a nebulizer therapy can be used with saline containing excipients. The composition in the form of a dry powder can be used with excipients containing lactose. The metered dose inhaler formulation can be used with excipients including hydrofluoroalkane (HFA) containing propellants, ethanol containing cosolvents and oleic acid containing stabilizers.

The necessary dose for administration will usually be determined by the physician, but will depend on a number of factors such as the condition being treated and the condition of the patient. Examples of doses and dose ranges will be given below. The preferred duration of administrations, the preferred frequency of administrations, and the preferred dosage of administrations depend on a variety of factors including, but not limited to, age, body weight, and severity of emphysematous lesions as quantified by CT lung densitometric measurements and lung function tests. The duration of treatment can usually be from two weeks, a month, six months a year or more. In many cases, the subject will use the compositions according to the present invention constantly or for extended periods of time. In patients with more severe forms of emphysema, the preferred duration of use of the present invention is for life, and the preferred frequency of administration is once a day. In milder forms of emphysema, a temporary period of administration may be sufficient, and administration less frequently than once a day.

The severity of lung copper deficiency is another factor that determines the intensity of treatment. Measurement of copper in exhaled breath condensate is a convenient way to calculate lung copper deficiency in order to determine the intensity and duration of inhaled copper therapy.

Pharmaceutical compositions of the present invention, exemplified by copper sulfate and heparin, are preferably and effectively administered at the following dosages depending on, among other things, factors such as age, sex, body weight, and condition of the patient. Preferred doses of both copper sulfate and heparin are derived from the fibroblast cell culture studies described below (see Experimental section), in which different doses and combinations are evaluated for their effect on elastin repair and development.

(a) For the copper salt, 1 µg to 10 mg per day, preferably 50 µg to 2 mg per day, more preferably 100 µg to 1 mg per day, and most preferably 200 to 500 µg per day. These doses are usually administered one, two or three times a day, preferably once a day.

(b) For heparin, 100 to 1,500,000 IU per day, preferably 5,000 to 1,000,000 IU per day, more preferably 25,000 to 500,000 IU per day, and most preferably 50,000 to 250,000 IU per day. A unit of heparin activity (USP) is defined as the amount of heparin that prevents clotting of 1 ml of citrate sheep plasma within one hour after the addition of 0.2 ml of 1% CaCl ₂ . These doses are usually administered one, two or three times a day, preferably once a day.

A preferred composition for inhalation contains about 0.5-1 mg of copper sulfate and about 150,000 IU of heparin.

The therapeutically active components that make up the composition according to the present invention are preferably administered simultaneously, but if desired, they can also be administered sequentially or separately.

In some preferred embodiments, the compositions of the present invention may be formulated into aerosols. The preparation of pharmaceutical aerosols is common to those skilled in the art, see, for example, Sciarra, J. in Remington (supra). The agents may be formulated as aerosol solutions, dispersions, or suspension aerosols from dry powders, emulsions, or semi-solid drugs. The aerosol can be delivered using any propellant system known to those skilled in the art. Aerosols can be applied to the lower respiratory tract. Compositions containing a copper compound and heparin can be delivered using liposome and nanoparticle delivery methods known to those skilled in the art. Liposomes, especially cationic liposomes, can be used in carrier formulations.

Compositions for use in accordance with the present invention may include, in addition to the active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. In particular, they may include a pharmaceutically acceptable excipient. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration. Suitable pharmaceutical carriers are described in Remington (see above).

Compositions of the present invention may be delivered by any device adapted to deliver one or more therapeutic compositions to the lower respiratory tract. In some preferred embodiments, the devices of the present invention may be metered dose inhalers. The devices can be adapted to deliver the therapeutic compositions of the present invention in the form of a fine aerosol liquid, foam or powder. The device may use a piezoelectric effect or ultrasonic vibration to expel a powder attached to a surface, such as a tape, to create an inhalable aerosol. The devices may use any propellant system known to those skilled in the art, including, but not limited to, pumps, liquefied gas, compressed gas, and the like.

In cases where the copper compound and heparin are administered in the form of particles or droplets, the size of the particle/droplet and/or other properties of the particle/droplet may be selected to ensure delivery of the particles to a specific area of the respiratory tract. For example, they may be designed to reach only the lower respiratory tract. In cases where the copper compound and heparin are delivered in aqueous form, preferably the solution is isotonic to facilitate efficient delivery to the subject. In particular, 10 µm particles are believed to be effective in reaching the lower parts of the respiratory tract and therefore can be used where such a site is a necessary target for compositions. In embodiments where the composition is to be delivered to the lower respiratory tract, such as, for example, the alveoli, the particle diameter administered may be less than 10 µm, preferably less than 8 µm, more preferably less than 6 µm, and even more preferably less than 4 µm. In a preferred embodiment, the particles may have a diameter of 3 μm or less, and more preferably may have a diameter of 2 μm or less. In a particularly preferred embodiment, the particles have a diameter of 3 to 5 microns. In some cases, the introduced particles may have a diameter of less than 1000 nm, preferably less than 500 nm, more preferably less than 250 nm, and even more preferably less than 100 nm. The sizes may refer to solid particles or droplets of solutions and suspensions.

The particle size required to penetrate a particular part of the respiratory tract is known in the art, and therefore the particle size can be selected according to the given size. Techniques such as milling can be used to obtain the desired very fine particles. In some cases, the upper respiratory tract may be a necessary part of the respiratory tract and therefore larger particles can be used. The density of the particles and their shape can also be selected to facilitate their delivery to the desired location.

Compositions according to the present invention may take various forms. They may be in the form of powders, powder microspheres, solutions, suspensions, gels, nanoparticle suspensions, liposomes, emulsions or microemulsions. The liquids present may be water or other suitable solvents such as CFC or HFA. In the case of solutions and suspensions, they may be aqueous or include solutions other than water.

The devices of the present invention are generally understood to be a container with one or more valves through which the therapeutic composition flows and an activator to control the flow. Suitable devices for use in the present invention can be found, for example, in Remington (see above). Suitable devices for administering the compositions of the present invention include inhalers and nebulizers, such as those commonly used to deliver steroids to asthmatics. In some cases, a spacer may be used in conjunction with an inhaler to ensure efficient delivery.

Various designs of inhalers are commercially available that can be used to deliver the compositions of the present invention. These include Accuhaler, Aerohaler, Aerolizer, Airmax, Autohaler, Breezhaler, Clickhaler, Diskhaler, Easi-breathe inhaler, Easyhaler, Evohaler, Ellipta, Fisonair, Handihaler, Integra, Jet inhaler, Miat-haler, Nexthaler, Novolizer inhaler, Pulvinal devices inhaler, Respimat, Rotahaler, Spacehaler, Spinhaler, Syncroner inhaler and Turbohaler. A number of techniques for preparing particularly desirable particles are known in the art and can be used. For example, nanocrystal, pulmosol and pulmosphere technologies can be applied.

In some cases, compositions can be entered through the installation. In such cases, typically the composition is in liquid form and will be administered via an artificial airway such as, for example, an endotracheal tube. Fluid is typically aspirated with a syringe and then passed through an artificial airway into the subject's airway. The installation is often used in emergency cases. In many cases, it can be used when the subject has a relatively severe form of CAL and has been hospitalized.

The compositions may include various components to optimize their suitability for the particular delivery route chosen. The viscosity of the compositions can be maintained at the desired level using a pharmaceutically acceptable thickener. Thickeners that can be used include methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, polyvinyl alcohol, alginates, gum arabic, chitosans, and combinations thereof. The thickener concentration will depend on the selected agent and the desired viscosity.

In some embodiments, the compositions may include a humectant. It can help reduce or prevent drying of the mucous membrane and prevent irritation of the membrane. Suitable humectants include sorbitol, mineral oil, vegetable oil and glycerin; soothing agents; shell conditioning agents; sweeteners; and their combinations.

The compositions may include a surfactant. Suitable surfactants include non-ionic, anionic and cationic surfactants. Examples of surfactants that can be used include, for example, polyoxyethylene derivatives of partial fatty acid esters of sorbitol anhydrides, such as, for example, tween 80, polyoxyl 40 stearate, polyoxyethylene 50 stearate, fusieates, bile salts and octoxynol. .

The synergistic effects of compositions according to the present invention containing an active agent comprising a copper compound and a glycosaminoglycan in inhalation therapy, using copper sulfate and heparin, respectively, will be further demonstrated in the Experimental section.

experimental part

The studies on which the present invention is based are part of a research project based on a systematic approach to develop specific therapy for patients with pulmonary emphysema.

The main focus of this project is the pulmonary extracellular matrix macroproteins elastin and collagen, as well as other proteins that play a decisive role in the development and repair of elastin and collagen fibers: that is, tropoelastin, fibulin-4, fibulin-5 "prototype" LOX and LOXL1.

The level of elastin crosslinking was quantified by measuring the elastin-specific crosslinking amino acids desmosine and isodesmosine (collectively referred to as DES) [3], and the level of collagen crosslinking was quantified by measuring the collagen-specific crosslinking amino acid hydroxyproline.

In the research project of the applicants, the experiments were carried out in the following sequential order:

1. Histological examination of lung biopsies from patients with emphysema, idiopathic pulmonary fibrosis (IPF), combined pulmonary fibrosis and emphysema (CPFE) and controls without lung parenchyma disease.

2. Staining of lung biopsy specimens with antibodies against active LOXL1 and LOXL2.

3. Studies of gene expression in lung tissues of patients with emphysema, IPF, CPFE and control subjects without lung parenchyma disease.

4. Measurements of copper levels in exhaled breath condensate in patients with emphysema and IPF and in the control group without lung disease.

5. Measurements of copper levels in lung tissue in patients with emphysema, IPF, CPFE and controls without lung parenchyma disease.

6. Cell cultures with rat lung fibroblasts.

7. Mechanisms of recovery in a mouse model of emphysema induced by porcine pancreatic protease.

8. Nebulization analysis of sodium heparin and copper sulfate solutions by laser diffraction.

1. Histological examination of lung biopsies

Scientific Intention: The authors began this project by studying the extracellular matrix of patients with emphysema, IPF, CPFE and control subjects. The reason for studying the lungs of patients with fibrosis was the authors' expectation that elucidation of the so-called "divergence factor" in the pathogenesis of IPF and emphysema could help identify the defect responsible for the failed elastin repair process in emphysema lungs and facilitate the identification of specific therapy for patients with emphysema.

Methods: Lung tissue was obtained from surgical lung resection specimens from patients with emphysema (n=10) and healthy controls without COPD/emphysema (n=10); Tumor-free lung tissue was taken in the subpleural region at an appropriate distance from the tumor. Lung tissue was obtained from diagnostic surgical lung biopsy from patients with IPF (n = 10). Lung tissues from apical (emphysematous) and basal (fibrous) lung sections of CPFE patients were obtained from lung explants (n = 4; Fig. 1). First, the authors examined the extracellular matrix of the lungs using histological analysis: Masson trichrome stain for collagen and Verhoff-Van Gieson stain for elastin.

Results: The authors found that the content of elastin in the lung parenchyma decreased in patients with pulmonary emphysema and increased in patients with IPF (Fig. 2). Collagen content was increased in both emphysema and IPF patients compared to lungs of control patients; however, the authors observed a more pronounced increase in collagen fibers with IPF. In patients with CPFE, the elastin content increased in the parenchyma with fibrosis of the basal part of the lung and decreased in the parenchyma with emphysema of the apical part of the lung. The content of collagen in patients with CPFE increased both in the parenchyma with apical emphysema and in the parenchyma with fibrosis of the basal part of the lung, but was more pronounced in the basal regions of the lung with fibrosis. DES levels were reduced in emphysema lungs and increased in IPF lungs. Hydroxyproline levels were elevated in both emphysema and IPF lungs, but were much higher in the latter. Notably, the relative difference in collagen levels between emphysema and IPF lungs was much lower than the relative difference in hydroxyproline levels between emphysema and IPF lungs, indicating that collagen is less cross-linked in emphysema compared to IPF lungs.

Conclusions: From these analyzes on lungs with fibrosis and emphysema, the authors concluded that their specific therapy for patients with emphysema should not only stimulate the repair and development of elastin fibers, but should also interfere with the maturation, organization and accumulation of collagen, since collagen in large quantities present in lungs with emphysema.

2. Staining lung biopsies to detect active LOXL1 and active LOXL2

The Scientific Concept: LOX enzymes are not only responsible for crosslinking tropoelastin precursors into strong elastin fibers, but they also crosslink procollagen precursors into strong collagen fibers. While elastin fibers provide elasticity, resilience and deformability, collagen fibers give the lungs tensile strength. Excessive collagen deposition is a sign of pulmonary fibrosis. Stimulation of pulmonary fibrosis formation would be an undesirable side effect of LOX stimulation. The authors hypothesized that LOX enzymes would decrease in emphysema and increase in fibrosis.

Methods: We stained the same lung biopsies used for histological analysis with antibodies to active LOXL1 (Novus Biologicals; NBP1-82827) and active LOXL2 (Novus Biologicals; NBP1-32954).

Results: Compared to controls, both active LOXL1 and active LOXL2 staining intensity was increased in IPF patients and decreased in emphysema patients. In CPFE patients, active LOXL1 and active LOXL2 staining was increased in the basal lung parenchyma with fibrosis and decreased in the apical lung parenchyma with emphysema.

3. Analyzes of gene expression in lung tissues

Scientific Intention: The authors continued their systematic research project by analyzing gene expression (quantitative real-time polymerase chain reaction; qRT-PCR) in the lungs of patients with emphysema, IPF and CPFE compared to lungs of the control group to identify those elastin repair genes/proteins , which are not sufficiently activated in emphysema and should be stimulated to achieve effective elastin recovery.

Methods: The authors analyzed the expression of the following genes in previously mentioned lung biopsies: tropoelastin (ELN), LOX, LOXL1, LOXL2, fibulin-4 and fibulin-5.

Results: To the authors' surprise, they found that ELN and fibulin-5 activity was strongly elevated in both emphysema and IPF patients, suggesting that these proteins are not a "divergence factor" between emphysema and fibrosis. LOXL1 was upregulated in IPF patients compared to controls. No significant differences were found in LOXL1 gene expression between patients with emphysema and controls.

Conclusions: Based on the study of gene expression, the authors concluded that stimulation of ELN and fibulin-5 synthesis cannot be the main target of lung elastin restoration therapy, since these proteins are already activated in the lungs of patients with emphysema.

Interim Analysis

The authors faced the paradox that activated LOXL1 levels were reduced in emphysema lungs, yet LOXL1 expression was not reduced in qRT-PCR. Based on an interim analysis of the results of the inventors' systematic research project, they concluded that patients do not develop emphysema due to a decrease in the LOXL1 protein, and suggested that patients may develop emphysema due to a decrease in the level of the critical cofactor LOXL1, i.e. copper. The authors conducted several studies to test this hypothesis.

4. Copper in exhaled air condensate and serum

Scientific Design: Because copper is a critical cofactor for the activation of LOX enzymes, the authors hypothesized that copper concentrations would be reduced in patients with emphysema.

Methods: First, the authors measured the level of copper in the blood serum of patients with emphysema (n = 10) and controls (n = 10). Subsequently, the authors collected exhaled breath condensate (EBC) using RTube ^TM (Respiratory Research; www.repiratoryresearch.com) and measured copper levels.

Results: Contrary to the authors' hypothesis, serum copper levels did not decrease, but increased in patients with emphysema (Fig. 3). However, EBC copper concentrations were reduced in patients with emphysema compared to controls.

Conclusions: In emphysema, there is a local pulmonary deficiency and there is no systemic copper deficiency.

5. Copper concentration in lung biopsies

Scientific Design: Since copper is a critical cofactor for the activation of LOX enzymes, the authors hypothesized that copper concentrations would be reduced in patients with emphysema and increased in patients with IPF.

Methods: The authors measured copper concentrations in the lung parenchyma of patients with emphysema, IPF and CPFE.

Results: The authors found that the concentration of copper did decrease in emphysema and increase in fibrosis compared with control lungs (Fig. 4). The authors also found a strong copper concentration gradient between the parenchyma of the apical lung with emphysema (low copper levels) and the basal lung with fibrosis (high copper levels) in the lungs of patients with CPFE. The authors' explanation for these surprising differences in CPFE lung copper concentrations is that copper delivery to the upper lung zones is much lower than to the lower lung zones, which seems logical given that the apical lung zones are very poorly irrigated.

Conclusions: Inhaled copper therapy is preferred over the systemic route of administration (a) because the upper lungs are much better ventilated than irrigated, and (b) because there is local and systemic copper deficiency. There is also a third important reason for preferring inhaled copper therapy over oral administration. Serum copper levels are positively associated with the risk of developing Alzheimer's disease [38]. To achieve the same concentrations of copper in the lungs (especially in the apical zones of the lungs), much lower doses of copper are needed with inhalation therapy than with oral therapy. The authors intratracheally administered copper to mice and indeed found no effect of this intervention on the concentration of copper in the brain.

High concentrations of copper in the lung parenchyma of IPF patients and in the basal areas of the lungs with fibrosis in CPFE patients formed the basis of the authors' concerns that stimulation of LOX enzyme activation by inhaled copper therapy would stimulate collagen cross-linking and thus maturation. organization of collagen, since LOX enzymes are cross-linking agents not only for elastin, but also for collagen [39]. Copper-induced collagen accumulation in the emphysema lung would be detrimental because (a) collagen levels are already elevated in emphysema lungs and (b) it can cause the transition from emphysema to fibrosis (i.e., the transition from one severe lung disease to another).

Thus, the authors concluded that copper should be combined with one or more other ingredients in a given inhalation formulation to prevent copper-induced collagen accumulation.

6a. Fibroblast Cell Culture with Copper Supplement

Scientific Design: Based on the fact that copper deficiency is the most likely cause of an ineffective elastin repair process in patients with emphysema, the authors hypothesized that copper supplementation would stimulate the elastin development/repair process by activating more LOX enzymes.

Methods: Fibroblasts were grown for 21 days, after which they were lysed and mRNA was extracted. The medium was replenished twice a week. qPCR was performed to measure the expression of the genes LOX, LOXL1 and elastin (ELN encoding tropoelastin). LOX activity was measured using the Amplite Fluorimetrix LOX Assay Kit (AAT Bioquest, Sunnyvale, CA, USA). Total insoluble elastin deposited in cell layers and soluble tropoelastin were measured using the Fastin™ Elastin assay kit (Biocolor, UK). DES levels were measured using a liquid chromatography-tandem mass spectrometry method at the Canisius-Wilhelmina Hospital (Nijmegen, The Netherlands) as previously described [9]. Collagen in the medium and matrix was quantified using Sircol™ INSOLUBLE Collagen Assays (Biocolor, UK). First, the authors measured the level of copper in the environment of fibroblasts. We then added additional copper sulfate in increasing concentrations, i.e. + 0.5 * initial concentration of copper in the fibroblast medium, + 1 * initial concentration of copper, + 2 * initial concentration of copper, + 4 * initial concentration of copper, + 8 * initial concentration of copper, + 16 * initial concentration of copper and + 32 * initial copper concentration to effect a dose-response relationship between copper concentration and other variables.

Results: Copper sulfate increased LOX and LOXL1 gene expression, LOX activity, DES levels (all favorable; FIG. 5), and insoluble collagen levels (unfavorable) in a dose-dependent manner. Copper sulfate did not affect the expression of the ELN gene.

Conclusions: The addition of additional copper sulfate to the cell culture medium had a favorable stimulating effect on the accumulation of cross-linked elastin fibers; however, it also had an adverse stimulating effect on the accumulation of insoluble collagen. The dose-response curve for DES levels peaks at about +8* the initial copper concentration in the fibroblast medium (FIG. 5).

6b. Fibroblast cell cultures to test potential synergistic effects of retinoic acid, minoxidil and heparin in addition to copper sulfate

Scientific Design: In the second part of the cell culture studies, the authors assessed whether the addition of other substances to copper sulfate additionally stimulates the development/repair of elastin.

Methods: We added retinoic acid, minoxidil, and heparin to copper-enriched fibroblast medium (copper concentration + 8*initial copper concentration in fibroblast medium).

Results (Fig. 6) : In contrast to copper sulfate monotherapy, the addition of retinoic acid to copper sulfate had a stimulatory effect on ELN gene expression and tropoelastin levels. The addition of retinoic acid to copper sulfate also had an additional stimulating effect on insoluble elastin levels; however, retinoic acid had no additional effect on DES levels. Retinoic acid had no additional effect to copper sulfate monotherapy on LOX and LOXL1 gene expression. The addition of minoxidil to copper sulfate had a stimulating effect on the expression of the LOX, LOXL1, ELN and fibulin-5 genes. The addition of minoxidil to copper sulfate had an additional stimulatory effect on tropoelastin, insoluble elastin, and DES levels compared to copper sulfate alone. The addition of minoxidil to copper sulfate had no additional stimulatory effect on collagen accumulation compared to copper sulfate alone; however, minoxidil supplementation did not have an inhibitory effect on collagen accumulation. The addition of heparin to copper sulfate had no additional effect compared to copper sulfate alone on LOX, LOXL1, ELN, and fibulin-5 gene expression; and had no effect on tropoelastin levels. The addition of heparin to copper sulfate had a small stimulatory effect on the total level of insoluble elastin compared with copper sulfate alone; however, this had no additional effect on DES levels. More importantly and surprisingly, the addition of heparin to copper sulfate had a strong inhibitory effect on collagen accumulation compared to copper sulfate alone.

Conclusions: The addition of retinoic acid, minoxidil, and heparin had some additional effects on elastin development and repair compared to copper sulfate alone. Surprisingly, the addition of heparin to copper sulfate had a strong inhibitory effect on collagen levels. From this study, the authors concluded that heparin appears to be an ideal adjunct to copper for the treatment of patients with emphysema to prevent copper-induced collagen buildup.

6c. Fibroblast cell cultures to test potential synergistic effects of vitamin K and magnesium sulfate in addition to copper sulfate

Scientific Design: In the third part of the cell culture studies, the authors assessed whether the addition of other substances to copper sulfate would inhibit the rate of elastin degradation.

Methods: We added vitamin K1, vitamin K2, and magnesium sulfate to copper-enriched fibroblast medium (copper concentration +8 *initial copper concentration in fibroblast medium).

Results: The addition of vitamin K1, K2 and magnesium sulfate to copper sulfate had no stimulatory effect on ELN gene expression, tropoelastin levels, or LOX and LOXL1 gene expression. However, the addition of vitamins K1, K2 and magnesium sulfate to copper sulfate had an additional stimulatory effect on insoluble elastin levels and DES accumulation (FIG. 7). The addition of vitamin K1, vitamin K2, and magnesium sulfate to copper sulfate had no additional stimulatory effect on collagen accumulation compared to copper sulfate alone; however, the addition of vitamin K1, vitamin K2, and magnesium sulfate did not have an inhibitory effect on collagen accumulation.

Conclusions: Supplementation with vitamin K1, vitamin K2, and magnesium sulfate had an additional effect on elastin and DES accumulation compared to copper sulfate alone. The most likely mechanical reason for this effect is the inhibitory effect of vitamin K1, vitamin K2, and magnesium sulfate on elastin degradation, since vitamin K1, vitamin K2, and magnesium sulfate had no effect on elastin development. Based on this study, the authors concluded that vitamin K1, vitamin K2, and magnesium sulfate appear to be appropriate copper supplements for the treatment of patients with emphysema to slow the rate of elastin degradation.

7. Emphysema induced by intratracheal injection of porcine pancreatic elastase

Scientific Design: Based on the very promising effects of adding heparin to copper sulfate on elastin and collagen metabolism in cell culture studies, the authors further evaluated these effects in an animal model of emphysema.

Methods: To evaluate the effect of copper sulfate and heparin on elastin and collagen metabolism in vivo , we used a model of porcine pancreatic emphysema induced by pancreatic elastase (PPE). The study was carried out on male BALB/c mice at 7 weeks of age with an initial body weight of approximately 25 g. During the study period, all mice were housed in conventional animal vivariums with a 12/12 hour light-dark cycle in cages with overhead filters, and they received pelleted food and water ad libitum. 1.5 units porcine pancreatic elastase in 25 μl saline was administered intratracheally on day 1 under light anesthesia. 25 μl copper sulfate monotherapy (12.5 μg in 25 μl saline; n = 4), copper sulfate (12.5 μg in 12.5 μl saline)/heparin combination (1000 IU in 12.5 μl saline; n = 4) or placebo (25 µl saline; n = 4) was administered intratracheally under light anesthesia on days 1, 8, 15, 22 and 29. On day 35, mice were intraperitoneally anesthetized with a mixture of xylazine (8.5 mg/kg) and ketamine (130 mg/kg), tracheotomy was performed, and the whole body was placed on a plethysmograph to assess lung function. After measuring lung function, mice were sacrificed by intracardiac administration of pentobarbital. The left lung was flash frozen in liquid nitrogen and stored at -80°C for subsequent gene expression studies in which ELN, LOX and LOXL1 were measured. The right lung was fixed in 6% paraformaldehyde at a constant hydrostatic pressure of 25 cm of fluid for 24 hours. After dehydration and paraffin embedding, sagittal sections were stained with various stains and used for histological analysis to measure airspace expansion (midline line segment); and subsequently this lung was used to measure the concentrations of both DES and insoluble collagen. The brain was removed to measure the concentration of copper.

Results: In tests of lung function of mice in the placebo group, more overstretch was present than in the copper sulfate and copper sulfate/heparin groups (FIG. 8). DES levels in lung tissue were higher in copper sulfate and copper sulfate/heparin treated mice than in placebo treated mice (FIG. 9). The mean linear length was lower in mice treated with copper sulfate and copper sulfate/heparin than in mice treated with placebo (FIGS. 8, 10 and 11). The levels of insoluble collagen and hydroxyproline were increased in mice treated with copper sulfate monotherapy compared with placebo (Fig. 9). The levels of insoluble collagen and hydroxyproline were significantly lower in mice treated with copper sulfate/heparin compared with mice treated with copper sulfate alone, and slightly lower compared with mice treated with placebo. Heparin is well known as an anticoagulant; however, intratracheal heparin had no effect on systemic coagulation in mice.

Conclusions: The authors found that copper sulfate was very effective in stimulating the elastin repair process, but also inducing the accumulation and maturation of collagen fibers in the lungs. The combination of copper sulfate and heparin was very effective in repairing damaged elastin fibers (even better than copper sulfate alone), and the authors found that, unlike copper sulfate alone, copper sulfate and heparin did not lead to collagen accumulation. Collagen and hydroxyproline levels after copper sulfate/heparin treatment were even lower than after placebo treatment. Thus, inhaled heparin is an ideal adjuvant to a copper inhalation formulation to prevent copper-induced collagen accumulation and stimulate the elastin repair process.

8. Nebulization analysis of sodium heparin and copper sulfate solutions by laser diffraction

Scientific Design: It is important to know whether it is possible to nebulize a solution of both heparin and copper with a conventional nebulization system and whether this results in an appropriate percentage of particles < 5 µm.

Methods 1: The authors started their nebulization experiments with relatively low concentrations of copper and heparin. 26 mg of sodium heparin (191 IU/mg) was dissolved in 1 ml of 0.9% sodium chloride, and 12.5 mg of copper sulfate (5 mg of copper) was dissolved in 10 ml of 0.9% sodium chloride, of which 1 ml was used. 3 ml of 0.9% sodium chloride was added to 1 ml of sodium heparin solution (5000 IU) and 1 ml of copper sulfate solution (0.5 mg of copper). 5 ml of the nebulization solution was loaded into a reusable nebulizer (PARI LC® Plus) and nebulized with a compressor (PARI BOY® SX). The aerosol was analyzed every 30 seconds using laser diffraction analysis (LDA) when the nebulizer began to spray.

Results 1: Spray time was about 3 minutes. X ₁₀ was 0.81 µm, X ₅₀ was 2.34 µm, and X ₉₀ was 6.58 µm. The percentage of particles <5 μm was 82.44% (FIG. 12). Experiment 1 was remeasured: the spray time was about 3 minutes, X ₁₀ was 0.80 µm, X ₅₀ was 2.29 µm, X ₉₀ was 6.34 µm, and the percentage of <5 µm particles was 83.58% ( Fig. 13).

Methods 2: 100,000 IU of heparin sulfate and 1 mg of copper were combined in solution and 0.9% sodium chloride was added to a total volume of 5 ml.

Results 2: Spray time was approximately 4 minutes. X ₁₀ was 0.80 µm, X ₅₀ was 2.32 µm, and X ₉₀ was 6.86 µm (FIG. 14). The percentage of particles <5 μm was 82.13%. Experiment 2 was remeasured: the spray time was about 5 minutes, X ₁₀ was 0.80 µm, X ₅₀ was 2.33 µm, X ₉₀ was 7.05 µm, and the percentage of particles <5 µm was 81.19% ( Fig. 15).

Conclusions: It is possible to combine heparin and copper in a nebulizer formulation and using the conventional nebulization system results in a high percentage of <5 µm particles that can effectively reach the target alveolar regions in the human lung.

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While the present invention has been described in connection with the illustrated embodiments, it should be understood that they are not intended to be limiting to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents which may be included in the present invention as defined by the appended claims.

Claims (17)

1. Combination for the treatment of pulmonary emphysema, containing an active agent containing a copper compound, where the copper compound is copper sulfate, in an amount of from about 0.5 mg to about 1 mg, and glycosaminoglycan or a physiologically acceptable salt thereof, where the glycosaminoglycan is heparin , in an amount of approximately 150,000 IU. 2. Combination according to claim 1, characterized in that the treatment of pulmonary emphysema includes the treatment of a condition of airway wall thickening, bronchiectasis, chronic bronchitis and/or small airway disease. 3. A combination according to claim 1 or 2, characterized in that the combination is used to treat an adult. 4. A combination according to any one of the preceding claims, characterized in that the glycosaminoglycan or salt has an average molecular weight of 12 to 18 kDa. 5. Combination according to any one of the preceding claims, characterized in that heparin is heparin sodium or heparin sulfate. 6. A combination according to any one of the preceding claims, characterized in that the combination is administered once, twice or three times a day. 7. Combination according to any one of the preceding claims, characterized in that the therapeutically active components that make up the combination are administered simultaneously. 8. Combination according to any one of the preceding claims, characterized in that the therapeutically active components that make up the combination are administered sequentially or separately. 9. A combination according to any of the preceding claims, further comprising at least one drug or substance that affects the metabolism of elastin in the vasculature or lungs, agents that degrade advanced glycation end products (AGEs) in the arteries. 10. A combination according to any one of the preceding claims, characterized in that the combination is administered by inhalation and/or dropwise. 11. Combination according to any one of the preceding paragraphs, said combination being intended to: (a) reactivating lung fiber elastin production in the subject; (b) repairing damaged elastin fibers; (c) slowing down the rate of degradation of elastin; and/or (d) inhibition of advanced glycation end product (AGE). 12. Combination according to any one of the preceding claims, characterized in that the combination is used with bronchodilators, inhaled corticosteroids or oral macrolides. 13. A method of treating a subject suffering from pulmonary emphysema, comprising administering by inhalation to said subject a combination containing a copper compound, where the copper compound is copper sulfate, in an amount of from about 0.5 mg to about 1 mg, and glycosaminoglycan or a physiologically acceptable salt thereof , where glycosaminoglycan is heparin, in an amount of about 150,000 IU.

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