REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/826,590, filed on May 23, 2013, which is hereby incorporated by reference for all purposes as if fully set forth herein.

STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant no. 1 UH2 TR 000966-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Duchenne muscular dystrophy is a profoundly devastating and fatal X-linked disorder that begins to manifest in the toddler years with difficulty with running and progresses to loss of ambulation in the early teens followed by loss of arm, trunk, respiratory and cardiac function. Death is common in early adulthood from respiratory or cardiac failure. DMD, and the milder allelic disorder, Becker muscular dystrophy (BMD), are due to mutations in the gene for dystrophin. The incidence of DMD is estimated to be

approximately 1 :3500 male births worldwide or approximately 20,000 new cases of DMD worldwide per year. Furthermore, while DMD and each of the other 30-plus muscular dystrophies are each rare and considered "orphan diseases" (<200,000 cases in the U.S.), cumulatively, their numbers are significant.

[0004] There is no FDA approved treatment for this disorder. Current care guidelines recommend that boys with DMD be treated chronically with corticosteroids. Although prednisone has been proven in clinical trials to mildly reduce the severity of the disease, it is generally recognized as a "bad drug for a bad disease" with a host of serious side effects. A novel therapy that could be used in conjunction with, or ultimately as replacement for, prednisone would have a tremendous positive impact on this disorder.

[0005] As such, in DMD, BMD, and other muscular dystrophies, there is an urgent, critical need for efficacious and safe treatments that slow disease progression in both skeletal and cardiac muscle. Such therapies will reduce disease burden, improve quality of life and buy time until the establishment of treatments that address the primary gene defect.

Treatments that attenuate muscle degeneration and fibrosis and enhance muscle strength will increase longevity and improve patient quality of life.

SUMMARY OF THE INVENTION

[0006] In accordance with an embodiment, the present invention provides a method for ameliorating the symptoms associated with dystrophic disorder in a subject comprising administering to a subject a pharmaceutical composition comprising an effective amount of a nitric oxide (NO) independent activator of soluble guanylate cyclase (sGC).

[0007] In accordance with another embodiment, the present invention provides a method for ameliorating the symptoms associated with dystrophic disorder in a subject comprising administering to a subject a pharmaceutical composition comprising a NO independent activator of sGC selected from the group consisting of: 2-(4-chloro-phenylsulfonylamino)- 4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benza mide; 2-(4-chloro- phenylsulfonylamino)-N-(4-(cis-2,6-dimethyl-morpholine-4-sul fonyl)-phenyl)-4,5- dimethoxy-benzamide; 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino)-N-(4-(morpho line- 4-sulfonyl)-phenyl)-benzamide; and 5-chloro-2-(3,5-dimethyl-isoxazole-4-sulfonylamino)-N- (4-(cis-2,6-dimethyl-morpholine-4-sulfonyl)-phenyl)-benzamid e, and pharmaceutically acceptable salts solvates or stereoisomers of any of the foregoing.

[0008] In accordance with a further embodiment, the present invention provides a method for ameliorating the symptoms associated with dystrophic disorder in a subject comprising administering to a subject a pharmaceutical composition comprising 5-chloro-2-(5-chloro- thiophene-2-sulfonylamino)-N-(4-(morpholine-4-sulfonyl)-phen yl)-benzamide or a pharmaceutically acceptable salt solvate or stereoisomer thereof.

[0009] In accordance with yet another embodiment, the present invention provides a method for ameliorating the symptoms associated with Duchenne muscular dystrophy (DMD) or a related dystrophic disorder in a subject comprising administering to a subject a pharmaceutical composition comprising a NO independent activator of sGC and at least one additional biologically active agent.

[0010] In accordance with another embodiment, the present invention provides a method of assessing the effect of a pharmaceutical composition comprising a NO independent activator of sGC on the diaphragm function of a subject or population of subjects comprising: a) measuring the diaphragm function of the subject to give a control measurement; b) administering the pharmaceutical composition to the subject or population of subjects; c) measuring the diaphragm function of the subject or population of subjects to give a treatment measurement; and d) comparing the measurements of a) and c) to determine the effect of pharmaceutical composition on the diaphragm function of the subject or population of subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a sample image from an 8 month-old male C57B110 mouse using the diaphragm measuring methods of the present invention. Upper panel shows a Brightness (B) mode image of the diaphragm. Lower panel is the M-mode image corresponding to three diaphragm contractions. The distance the diaphragm moves is measured from a baseline to the peak of the contraction, as indicated. In this example, the diaphragm movement is 0.95 mm.

[0012] Figure 2 is a graph showing the comparison of diaphragm function by in vivo ultrasonography and ex vivo contractile measurement.

[0013] Figure 3 shows data for IVS (d mm), FS%, EF% and IVRT (ms) assessed in conscious mice using transthoracic echocardiogram. From these data, fractional shortening (maximal diameter - minimal diameter)/maximal diameter * 100; chamber cross sectional diameters at end-diastole and end-systole, heart rate, wall thickness in the intraventricular septum, posterior wall, wall mass (estimated from a spherical model), isovolumic contraction time (rVCT), ejection time (ET), isovolumic relaxation time (IVRT), and myocardial performance index (MPI = (IVCT+IVRT)/ET) were determined.

[0014] Figure 4 is a group of pressure -volume curves for treated and control animals. LV function was assessed by pressure-volume (PV) analysis in anesthetized mice, (n=5 from each group of either vehicle treated or HMR 1766 treated). Pressure volume data were recorded at steady state, and during transient preload reduction produced by obstruction venous return from the inferior vena cava. Pressure-volume data were analyzed by custom software (WinPVAN). Steady state data were signal averaged over 5-10 sequential cardiac cycles. Sequential cycles measured during preload reduction were used to determine load- independent measures of cardiac contractility. [0015] Figures 5A and 5B are average data of treated (red) and control (black) animals assessing activity by open field testing. The experimental animals are dystrophic (mdx) mice greater than a year old (n=9 per group) and are considered a good preclinical model for Duchenne muscular dystrophy. Animals from both groups are initially more active when placed in an open field as measured by laser beam breaks and this activity declines over time. Animals treated with HMR 1766 are consistently more active than untreated controls on two different days (Figure 5A and Figure 5B).

[0016] Figure 6 is similar open field data on old mdx either treated (red) or untreated (black). In this experiment, animals were run on a treadmill until exhaustion and then placed in the open field. Activity is increased in treated animals compared to untreated animals.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Dystrophin is a large protein underlying the muscle cell membrane, the sarcolemma, and associates with multiple proteins making up the dystrophin protein complex (DPC). The DPC serves as a scaffold that targets signaling proteins to the sarcolemma. Disruption of this scaffold alters key signaling pathways necessary for muscle health and function and is a common theme in most muscular dystrophies. In recent years, the realization that these proteins may be new therapeutic targets has stimulated interest in drugs that augment dystrophin-related signaling pathways. One especially important signaling protein in the DPC is neuronal nitric oxide synthase (nNOS). ηΝΟδμ (the muscle specific form of this enzyme) is linked to syntrophin and to dystrophin at the muscle sarcolemma. One of the functions of sarcolemmal ηΝΟδμ is local regulation of vascular flow during exercise; nitric oxide (NO) promotes smooth muscle relaxation and vasodilation. NO also regulates cardiac, skeletal and smooth muscle contractile function and modulates the immune response in muscle. The major signaling pathway for NO is stimulation of cGMP production by soluble guanylyl cyclase (sGC). NO at concentrations less than 1 nM activates sGC, producing cGMP at μΜ levels, effectively amplifying the NO signal 1000 fold. In turn, cGMP directly activates protein kinase G (PKG) and certain ion channels

[0018] It was known that ηΝΟδμ is lost from the sarcolemma and down regulated when dystrophin is absent in DMD, but is retained in some, more mild, BMD cases. The variation in severity in some BMD cases may be explained by the recent observation that certain spectrin-like repeats of dystrophin are required for sarcolemmal targeting of ηΝΟδμ. [0019] Loss of sarcolemmal ηΝ08μ is not limited to DMD and BMD, however. Certain human limb girdle muscular dystrophies lack sarcolemmal nNOS, especially those caused by deficiency of the sarcoglycans including LGMD2C (gamma sarcoglycan), LGMD2D (alpha- sarcoglycan), and LGMD2E (beta-sarcoglycan). Muscle atrophy caused by amyotrophic lateral sclerosis and myasthenia gravis is also accompanied by loss of ηΝ08μ from the sarcolemma. Thus, the present inventors conceptualized that the nNOS signaling pathway is a potential therapeutic target not only for DMD, but for other devastating neuromuscular diseases, both acquired and inherited.

[0020] Phosphodiesterases (PDEs) hydro lyze cGMP and turn down the gain of NO- cGMP signaling. The present inventors understood that the NO-cGMP-PKG pathway is disrupted in muscular dystrophy beginning with reduction in nNOS activity, and that transgenic expression of nNOS in mdx mouse skeletal and cardiac muscle reduces the severity of the dystrophic phenotype. Thus, the present inventors hypothesized that a NO- independent activator of sGC, would be expected to bypass a major deficiency in NO-cGMP- PKG signaling in muscular dystrophy.

[0021] Thus, in accordance with an embodiment, the present invention provides a method for ameliorating the symptoms associated with Duchenne muscular dystrophy (DMD) in a subject comprising administering to a subject a pharmaceutical composition comprising an effective amount of a nitric oxide (NO) independent activator of soluble guanylate cyclase

(sGC).

[0022] One such sGC activator is HMR1766, also known as ataciguat, (5-chloro-2-((5- chloro-2-thienyl)sulfonylamino)-N-(4-(morpholin-4-ylsulfonyl )phenyl)benzamide) and has the following chemical structure of Formula I:

(I). [0023] Ataciguat is a NO independent activator of sGC. It preferentially activates the oxidized form of sGC leading to an increase in cyclic GMP, a key component of several intracellular signal transduction pathways. Toxicology studies (6/12 months),

embryotoxicology and fertility studies showed an appropriate safety profile. Multiple clinical trials have been conducted with ataciguat including DBPCTs in chronic angina pectoris, peripheral artery disease and neuropathic pain. In these Phase II trials, as well as those with healthy subjects, ataciguat was shown to be well tolerated with a safety profile

indistinguishable from placebo. Over 600 subjects have received ataciguat without a serious adverse event. This excellent safety and tolerability profile as well as the fact that ataciguat is orally delivered as a once daily drug, make it desirable for repurposing for a pediatric indication.

[0024] Ataciguat and its derivatives are derived from a novel class of sulfur substituted sulfonylaminocarboxylic acid N-arylamide compounds. See, U.S. Patent No. 6,335,334, and incorporated by reference herein. These compounds are capable of modulating the body's production of cyclic guanosine monophosphate (cGMP) and are generally suitable for the therapy and prophylaxis of diseases which are associated with a disturbed cGMP balance.

[0025] Thus, in accordance with an embodiment, the present invention provides a method for ameliorating the symptoms associated with a dystrophic disorder in a subject comprising administering to a subject a pharmaceutical composition comprising a NO independent activator of sGC selected from the group consisting of:. 2-(4-chloro-phenylsulfonylamino)- 4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benza mide; 2-(4-chloro- phenylsulfonylamino)-N-(4-(cis-2,6-dimethyl-morpholine-4-sul fonyl)-phenyl)-4,5- dimethoxy-benzamide; 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino)-N-(4-(morpho line- 4-sulfonyl)-phenyl)-benzamide; and 5-chloro-2-(3,5-dimethyl-isoxazole-4-sulfonylamino)-N- (4-(cis-2,6-dimethyl-morpholine-4-sulfonyl)-phenyl)-benzamid e, and pharmaceutically acceptable salts solvates or stereoisomers of any of the foregoing.

[0026] In the compound of formula I, various substitutions may be made and are exemplified by a straight-chained or cyclic hydrocarbon group (e.g., an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an aralkyl group, alkylol group, hydroxyalkyl group, alkoxy group, alkoxyalkyl group, etc.) or the like. Among these, straight-chained or cyclic hydrocarbon groups having 1 to 16 carbon atoms are preferred.

[0027] The term "substituted" is also contemplated to include all permissible substituents of organic compounds such as the sGC activators of interest. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

[0028] Examples of the "alkyl group" preferably include a Ci_ ₆ alkyl group (e.g., a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec -butyl group, a tert-butyl group, a pentyl group, a hexyl group, etc.) and the like.

[0029] Examples of the "alkenyl group" preferably include a C2-6 alkenyl group (e.g., a vinyl group, an allyl group, an isopropenyl group, a 1-butenyl group, a 2-butenyl group, a 3- butenyl group, a 2-methyl-2-propenyl group, a 1 -methyl-2-propenyl group, a 2-methyl-l- propenyl group, etc.) and the like.

[0030] Examples of the "alkynyl group" preferably include a C 2-6 alkynyl group (e.g., an ethynyl group, a propargyl group, a 1-butynyl group, a 2-butynyl group, a 3-butynyl group, a 1-hexynyl group, etc.) and the like.

[0031] Examples of the "cycloalkyl group" preferably include a C 3-6 cycloalkyl group (e.g., a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc.) and the like. The "cycloalkyl group" may be condensed with one or two C 6-14 aromatic carbon rings (e.g., a benzene ring, etc.) (e.g., a 1-indanyl group, a 1,2,3,4-tetrahydro-l- naphthyl group, etc.).

[0032] Examples of the "aryl group" preferably include a C 6-14 aryl group (e.g., a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a 2-biphenylyl group, a 3-biphenylyl group, a 4-biphenylyl group, a 2-anthryl group, etc.) and the like.

[0033] Examples of the "aralkyl group" preferably include a C 7-16 aralkyl group (e.g., a benzyl group, a phenethyl group, a diphenylmethyl group, a 1-naphthylmethyl group, a 2- naphthylmethyl group, a 2,2-diphenylethyl group, a 3-phenylpropyl group, a 4-phenylbutyl group, a 5-phenylpentyl group, etc.) and the like.

[0034] The terms "alkylol" and "hydroxyalkyl" embrace linear or branched alkyl groups having one to about ten carbon atoms any one of which may be substituted with one or more hydroxyl groups. The term "alkenyl" embraces linear or branched radicals having two to about twenty carbon atoms, preferably three to about ten carbon atoms, and containing at least one carbon-carbon double bond. The term "alkynyl" embraces linear or branched radicals having two to about twenty carbon atoms, preferably two to about ten carbon atoms, and containing at least one carbon-carbon triple bond. The terms "cycloalkenyl" and

"cycloalkynyl" embrace cyclic radicals having three to about ten ring carbon atoms including, respectively, one or more double or triple bonds involving adjacent ring carbons. The terms "alkoxy" and "alkoxyalkyl" embrace linear or branched oxy-containing radicals having alkyl portions of one to about ten carbon atoms, such as methoxy group. The "alkoxy" or

"alkoxyalkyl" radicals may be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide haloalkoxy or haloalkoxyalkyl groups.

[0035] Specific examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec -butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, methylbutyl, dimethylbutyl and neopentyl. Typical alkenyl and alkynyl groups may have one unsaturated bond, such as an allyl group, or may have a plurality or unsaturated bonds, with such plurality of bonds either adjacent, such as allene-type structures, or in conjugation, or separated by several saturated carbons.

[0036] Included within the family of compounds of Formula I are the tautomeric forms of the described compounds, isomeric forms including diastereoisomers, and the

pharmaceutically-acceptable salts thereof. The term "pharmaceutically-acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. Since the NO independent activator of sGC compounds of Formula I contain basic nitrogen atoms, such salts are typically acid addition salts or quaternary salts. The nature of the salt is not critical, provided that it is pharmaceutically acceptable, and acids which may be employed to form such salts are, of course, well known to those skilled in this art. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulphuric acid and phosphoric acid, and such organic acids as maleic acid, succinic acid and citric acid. Other pharmaceutically acceptable salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, or with organic bases, such as dicyclohexylamine. All of these salts may be prepared by conventional means by reacting, for example, the appropriate acid or base with the corresponding NPSA compound of General Formula I. [0037] In accordance with an embodiment, the present invention provides a method for ameliorating the symptoms associated with dystrophic disorder in a subject comprising administering to a subject a pharmaceutical composition comprising 5-chloro-2-(5-chloro- thiophene-2-sulfonylamino)-N-(4-(morpholine-4-sulfonyl)-phen yl)-benzamide or a pharmaceutically acceptable salt solvate or stereoisomer thereof.

[0038] In accordance with a further embodiment, the present invention further includes derivatives of ataciguat. In one embodiment, the term "derivative" includes, but is not limited to, ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like.

Methods of preparing these derivatives are known to a person skilled in the art. For example, ether derivatives are prepared by the coupling of the corresponding alcohols. Amide and ester derivatives are prepared from the corresponding carboxylic acid by a reaction with amines and alcohols, respectively.

[0039] In addition, this invention further includes hydrates of ataciguat. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like. Hydrates of ataciguat may be prepared by contacting the NO independent activator of sGC with water under suitable conditions to produce the hydrate of choice.

[0040] In an embodiment, the pharmaceutical compositions of the present invention comprise ataciguat and derivatives thereof together with a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carriers include soluble carriers such as known buffers which can be physiologically acceptable (e.g., phosphate buffer) as well as solid compositions such as solid-state carriers or latex beads.

[0041] In accordance with an embodiment, the present invention provides a

pharmaceutical composition comprising a NO independent activator of sGC and a pharmaceutically acceptable carrier, for use in the treatment of the symptoms associated with a dystrophic disorder in a subject. In some embodiments, the NO independent activator of sGC is a compound of Formula I, or a salt, solvate or stereoisomers thereof.

[0042] In accordance with another embodiment, the NO independent activators of sGC for use in the treatment of the symptoms associated with a dystrophic disorder comprise 2-(4- chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholi ne-4-sulfonyl)-phenyl)- benzamide; 2-(4-chloro-phenylsulfonylamino)-N-(4-(cis-2,6-dimethyl-morp holine-4- sulfonyl)-phenyl)-4,5-dimethoxy-benzamide; 5-chloro-2-(5-chloro-thiophene-2- sulfonylamino)-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamid e; and 5-chloro-2-(3,5- dimethyl-isoxazole-4-sulfonylamino)-N-(4-(cis-2,6-dimethyl-m orpholine-4-sulfonyl)- phenyl)-benzamide, and pharmaceutically acceptable salts solvates or stereoisomers of any of the foregoing

[0043] This invention further includes a process for preparing pharmaceutical products comprising ataciguat and derivatives thereof. The term "pharmaceutical product" means a composition suitable for pharmaceutical use (pharmaceutical composition), as defined herein. Pharmaceutical compositions formulated for particular applications comprising ataciguat and derivatives thereof are also part of this invention, and are to be considered an embodiment thereof.

[0044] As used herein, the term "dystrophic disorder" includes any skeletal muscle disease in which treatment with an NO independent sGC activator improves muscle function. Included in the term are DMD and related dystrophic disorders, including, for example, BMD, Limb Girdle Muscular Dystrophy (LGMD) 2C, LGMD2D, LGMD2E, LGMD2F, LGMD2I, Muscular dystrophy congenital type 1A (MCDIA), MDCDIC, Bethlem myopathy (BM), and Ulrich congenital muscular dystrophy (UCMD).

[0045] According to another embodiment of the present invention, a method is provided for treating DMD and related dystrophic disorders in a subject, comprising administering to the subject, at least one compound of the present invention and/or its analog, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, hydrate, or N- oxide, or any combination thereof, in an amount effective to treat the DMD and related dystrophic disorders in the subject.

[0046] "Treating" or "treatment" is an art-recognized term which includes curing as well as ameliorating at least one symptom of any condition or disease. Treating includes reducing the likelihood of a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing any level of regression of the disease; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, even if the underlying pathophysiology is not affected or other symptoms remain at the same level.

[0047] "Prophylactic" or "therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0048] As used herein, the term "effective amount" is an equivalent phrase refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and/or duration of a disease, ameliorate one or more symptoms thereof, prevent the advancement of a disease or cause regression of a disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating a disease. For example, a treatment of interest can increase the use of skeletal muscle in a subject, based on baseline of the diseased muscle, by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In another embodiment, an effective amount of a therapeutic or a prophylactic agent of interest reduces the symptoms of a disease, such as a symptom of DMD by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Also used herein as an equivalent is the term, "therapeutically effective amount."

[0049] In accordance with one or more embodiments, the symptoms associate with a dystrophic disorder include impaired cardiac function, including, but not limited to, cardiac output, ejection fraction, end-systolic elastance, PRSW, dp/dt max end-diastolic volume. As such, in accordance with one or more embodiments, administration of an effective amount of a NO independent activators of sGC results in increased systolic function and/or increased contractility in the heart of the subject. One of ordinary skill would understand that this effect can also be characterized as increasing cardiac function in the subject.

[0050] It is also understood by those of ordinary skill in the art that additional symptoms of dystrophic disorder include weakness of limb and trunk muscle and fatigue of these muscles. Therefore, in accordance with an embodiment, the administration of an effective amount of a NO independent activator of sGC would result in increased strength of muscles, decreased fatigue of muscles and increased function in activities of daily living such as walking, climbing, lifting etc.

[0051] As used herein, the term "subject" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0052] In another embodiment, the term "contacting" means that the at least one compound of the present invention is introduced into a subject receiving treatment, and the compound is allowed to come in contact with the sGC or the sGC complex in vivo.

[0053] In one embodiment, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used, and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The

pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use.

[0054] The carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.

[0055] Solid carriers or diluents include, but are not limited to, gums, starches (e.g. corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0056] For liquid formulations, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media. [0057] Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0058] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain

anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0059] Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[0060] In addition, in an embodiment, the compounds of the present invention may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., cremophor, glycerol, polyethylene glycerol, benzlkonium chloride, benzyl benzoate, cyclodextrins, sorbitan esters, stearic acids), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweetners (e.g., aspartame, citric acid), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates), and/or adjuvants.

[0061] The choice of carrier will be determined, in part, by the particular compound, as well as by the particular method used to administer the compounds. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, and interperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the compounds, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0062] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-P-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0063] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the compounds of the present invention in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants, for example, having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

[0064] The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0065] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ^SHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).

[0066] For purposes of the invention, the amount or dose of the sGC activator compound administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular compound and the condition of a human, as well as the body weight of a human to be treated.

[0067] The dose of the sGC activator compound also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular compound. Typically, an attending physician will decide the dosage of the sGC activator compound with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, the compound to be administered, route of administration, and the severity of the condition being treated. By way of example, and not intending to limit the invention, the dose of the sGC activator compound can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1 mg/kg body weight/day. In a preferred embodiment, the dose of the sGC activator compound is from about 0.5 mg/kg body weight/day to about 3 mg/kg body weight/day.

[0068] Alternatively, the compounds of the present invention can be modified into a depot form, such that the manner in which the compound is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450, 150). Depot forms of the compounds of the present invention can be, for example, an implantable composition comprising the compounds of the present invention and a porous or non-porous material, such as a polymer, wherein the compound is encapsulated by or diffused throughout the material and/or degradation of the non-porous material. The depot is then implanted into the desired location within the body and the compounds of the present invention are released from the implant at a predetermined rate. [0069] In one embodiment, the pharmaceutical compositions provided herein are controlled release compositions, i.e., compositions in which the one or more compounds are released over a period of time after administration. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). In another embodiment the composition is an immediate release composition, i.e., a composition in which the entire compound is released immediately after administration.

[0070] In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, or other modes of

administration. In an embodiment, a pump may be used (see Langer, Science 249: 1527-1533 (1990); Sefton, CRC Crit. Rev. Biomed. Eng. 14:201-401 (1987); Buchwald et al, Surgery 88:507-516 (1980); Saudek et al, N. Engl. J. Med. 321 :574-576 (1989). In one embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer, supra.

[0071] The sGC activator compositions of the present invention may also include incorporation of the active ingredients into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts). Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

[0072] An active agent and a biologically active agent are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "active agent,"

"pharmacologically active agent" and "drug" are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc. The active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.

[0073] In accordance with an embodiment, the present invention provides a method for ameliorating the symptoms associated with a dystrophic disorder in a subject comprising administering to a subject a pharmaceutical composition comprising an effective amount of a NO independent activator of sGC, wherein the NO independent activator of sGC is administered with at least one additional biologically active agent.

[0074] In some embodiments, measurement of the effect of the NO independent activator of sGC is done by measuring contractile force of diaphragm or tibialis muscle strips ex vivo. In other embodiments, measurement of the effect of the NO independent activator of sGC is done by measuring voluntary exercise of subjects. In one embodiment, the exercise can be voluntary wheel running of mice. In other embodiments, measurement of the effect of the NO independent activator of sGC is done by measuring cardiac function via

echocardiaography.

[0075] It will be understood by those of ordinary skill in the art that measurements of the diaphragm function of a subject can be accomplished by a number of known means. In an embodiment, the compounds and methods of the present invention can utilize a non-invasive method for measuring the diaphragm function of a subject. This non-invasive method comprises: a) one or more individual transducers including at least a first individual ultrasound transducer, the transducer having an array comprising a plurality of transducer elements and a contact face, wherein the arrays are arranged to transmit ultrasonic signals through an abdominal surface and to the diaphragm of a subject being measured; b) locating the diaphragm in B mode by arranging the one or more transducers in a spaced relation on the abdominal surface perpendicular to the subject, dorsal side down at the ventral wall to allow a view of the diaphragm movement during respiration; c) recording the ultrasonic images in M mode on a device which has which has electronic circuits, at least one microprocessor, memory storage and means for controlling the ultrasound signal transmission and reception of the at least- first transducer arrays, processing the ultrasound signals received by the at least-first transducer arrays, and registering the monitoring measurements; d) establishing a baseline along the diaphragm image during relaxation; and e) measuring the amplitude of the diaphragm contraction from the baseline d) to the peak of contraction.

[0076] In some embodiments the subject being measured is placed on a stage. [0077] In some embodiments the method further comprises a means for electrically monitoring respiration of the subject.

[0078] In some embodiments the subject on the stage is oriented at a specified angle which deviates from horizontal from 1 to 60 degrees. In a preferred embodiment, the stage is oriented at a specified angle which deviates from horizontal from about 15 to 45 degrees, in a more preferred embodiment, the stage is oriented at a specified angle which deviates from horizontal by about 30 degrees, with the subject's head downward.

[0079] In accordance with another embodiment, the present invention provides a method of assessing the effect of a pharmaceutical composition on the diaphragm function of a subject or population of subjects comprising: a) measuring the diaphragm function of the subject using any known methods to give a control measurement; b) administering the pharmaceutical composition to the subject or population of subjects; c) measuring the diaphragm function of the subject or population of subjects using any known methods to give a treatment measurement; and d) comparing the measurements of a) and c) to determine the effect of pharmaceutical composition on the diaphragm function of the subject or population of subjects.

[0080] It will be understood by those of ordinary skill that the effect of any

pharmaceutical composition, compound, drug, antibody, protein, peptide on the skeletal muscle or cardiac muscle function of a subject or population of subjects can be assessed using the methods of the present invention, including, for example, NO independent activators of sGC.

EXAMPLES

[0081] The methods of the present invention are useful to assess the efficacy of ataciguat in improving skeletal muscle function and preventing fibrosis, a reliable indicator of prognosis and severity of the disease in humans. The inventors have previously published studies of sildenafil demonstrating that amplification of the NO-cGMP pathway suppresses profibrotic pathways in the mdx mouse. Other histological parameters are also to be evaluated. Two different skeletal muscles will be studied for functional improvement:

diaphragm and tibialis anterior. In addition to analyses of skeletal muscle, subjects are tested for improvements in cardiac function by echocardiography and cardiac physiology (pressure volume loops). Finally, voluntary exercise performance (open field testing) is tested which is indicative of cardiac, diaphragmatic and skeletal muscle function. Without being limited to any particular theory, the present inventors believe that by activating directly the oxidized form of sGC, ataciguat and its derivatives will bypass the deficit in NO bioavailability and boost cGMP levels. Based on the inventor's published results demonstrating the efficacy of sildenafil, it is thought that ataciguat will improve the functional deficits exhibited by dystrophic skeletal muscle and heart.

EXAMPLE 1

[0082] Non-invasive assessment of diaphragm muscle function in vivo.

[0083] The compounds and methods of the present invention, in some embodiments, will utilize a non-invasive measurement of diaphragm function. Using ultrasound M-mode imaging, the decline in diaphragm function, and the impact of drug treatment, can be measured over time in the same animal. This technique dramatically improves the ability to assess the time course of the development of the dystrophic phenotype in the diaphragm and the effect of drug treatment.

[0084] A comparison of diaphragm contraction amplitude was conducted by M-mode in vivo imaging and ex vivo specific force generation of mdx diaphragm muscle at 8 and 18 months of age. The linear correlation coefficient of these methods is very high (R ² = 0.70; P < 0.01), validating this ultrasonography method as a reliable non-invasive, in vivo technique for measuring diaphragm function.

[0085] The experiments of the present invention are conducted on male mdx:utr het mice on the C57BL/10 genetic background (Circulation 124: 582-588 (2011); J Neurol Sci 264: 106-1 11(2008)). The mdx:utr het mouse, lacking dystrophin and having half the quantity of the homologous and compensatory utrophin protein, exhibits a more severe dystrophic phenotype in skeletal muscle than the mdx model used for in earlier studies. This increased severity has the advantages of more accurately recapitulating the skeletal muscle defects in DMD boys as well as developing the phenotype more rapidly in the mdx:utr het mouse. Wild type controls are age-matched males on the same background. Proper genetic background controls are essential in the evaluation of experimental outcomes. Animal experiments are performed according to AALAC requirements and approved by the University of Washington IACUC prior to starting the experiments. Animals are randomized to treatment group and investigators conducting analysis are blinded to treatment group in accordance with recent recommendations by NIH on the conduct of preclinical trials. In addition, when appropriate and to the extent possible, the protocols described below follow the standard operating procedures recommended by TREAT -NMD: (treat-nmd.eu/research/preclinical/dmd-sops/).

[0086] For the in vivo experiments, 20 mdx/C57B16 mice born March to November of 2012 were used. The experiment was initiated about December of 2013, with the mice aged 12 to 20 months (Group A mean 13.7 months, Group B mean 14 months).

[0087] Drug Administration. In a set of experiments, the aged mdx mice were then randomized to different diets, one being a placebo diet and the other diet containing about 0.5% ataciguat by weight. The animals were on the respective diets for 4 months.

[0088] In some other embodiments, the sGC activator of interest or placebo is delivered orally as a suspension by gavage. Concentrations of 3, 10 and 30 mg/kg/day (plus placebo) of the test compounds of the present invention will be administered to male mdx:utr het mice, beginning at 3 weeks of age. In a mouse model of neuropathic pain, ataciguat or a derivative thereof, administered orally for 14 days at 3 and 10 mg/kg/day reduced tactile allodynia by 64% and 94%, respectively. Similarly, a dose of 30 mg/kg/day significantly improved several aspects of ischemia-induced muscle weakness in the ZDF rat (data not shown). The NOAEL for mice treated for 13 weeks with ataciguat is 500mg/kg/day. Untreated C57BL10 male mice are subjected to the same analyses. The drug is not administered to control C67BL10 mice but they will serve as a baseline/goal of function.

[0089] Outcome measures. Although sGC activators of interest are expected to have both cardiac and skeletal muscle effects, skeletal muscle outcome measures may be selected to be the primary outcome measures in these preclinical trials which are specifically focused on providing evidence of efficacy to justify clinical trials in pediatric, ambulatory DMD. In this stage of DMD disease, skeletal muscle dysfunction predominates and skeletal muscle functional outcome measures have been validated while cardiac dysfunction is nascent and cardiac function outcome measures such as cardiac strain at this stage are exploratory for clinical trials. As described below, about a 5-15% improvement in one of three skeletal muscle functions will be considered an outcome measure indicating preclinical efficacy for the proposed trial. Skeletal muscle function is assessed by diaphragm and tibialis ex vivo specific force and by voluntary wheel running. Improvement of about 5-15% in skeletal muscle contractile function as indicative of preclinical efficacy is based on the inventors' studies of sildenafil on diaphragm function (Am J Physiol Lung Cell Mol Physiol 297 (2008)). Data on the effect of drug treatment targeting the NO-cGMP on voluntary running are not available. However, genetic correction by AAV-delivered dystrophin comparing a construct that restores sarcolemmal nNOS versus one that does not revealed up to 40% improvement in distance run. Since we would not expect drug treatment to be as efficacious as genetic correction, about a 5-15% increase is a valid target.

[0090] Blinded assessments of the mdx mice with the placebo diet and ataciguat diet included treadmill running, open field test spontaneous activity (with and without pre- running), echocardiograms, cardiac physiology, hydroxyproline content of diaphragms as described below.

[0091] Preclinical cardiac outcome measures are also determined which will inform the current trial and potentially future clinical trials in other populations with more severe cardiomyopathy than pediatric DMD.

Skeletal muscle studies. Skeletal muscle studies are focused on the diaphragm and the tibialis anterior. The mdx:utr het diaphragm more accurately reflects the severity of the dystrophic phenotype in DMD than limb muscle or standard mdx diaphragm, which displays a comparatively mild phenotype. Since failure of respiratory muscles is a major cause of death in DMD boys, any treatment that improves diaphragm function has a strong potential for therapeutic value. Restoration of dystrophin expression in diaphragm alone is sufficient to reduce cardiomyopathy in the mdx mouse. Thus, improvement of respiratory DMD muscle function could have profound effects on longevity and quality of life. Also in contrast to the standard mdx mouse, leg muscles in the mdx:utr het mouse show earlier fibrosis and fatty replacement typical of DMD.

EXAMPLE 2

[0092] Functional analyses of skeletal muscle. In an embodiment, this is performed using non-invasive measurement of diaphragm function, using ultrasound imaging. This technique dramatically improves the ability to assess the time course of the development of the dystrophic phenotype in the diaphragm and the effect of drug treatment on the severity of the disease.

[0093] Since the heart can introduce unwanted movement, the right side of the diaphragm is used for imaging. The diaphragm is located and observed in B-mode, then images are recorded in M-mode. From the M-mode image, the amplitude of the diaphragm contraction is measured. A baseline is established along the diaphragm image during relaxation (end of exhalation) and the amplitude of the contraction is measured from this baseline to the peak of contraction (inhalation) (Figure 1). From each mouse, at least five measurements are acquired and used for statistical analysis of the diaphragm contraction. An 8 month-old C57B110 control mouse normally has a peak contraction amplitude of about 1.2 mm, while an mdx mouse has an average peak contraction amplitude of around 0.8 mm. Old mdx mice (18 months of age) show a more severe dystrophic phenotype: average peak contraction amplitude of 0.45 mm. We expect that the mdx:utr het mouse will exhibit the more robust signal difference at 3-4 months of age.

[0094] A comparison of diaphragm contraction amplitude was conducted by M-mode in vivo imaging and ex vivo specific force generation of mdx diaphragm muscle at 8 and 18 months of age. As shown in Figure 2, the linear correlation coefficient of these methods is very high (R2 = 0.70; P < 0.01), validating this novel ultrasonography method as a reliable non-invasive, in vivo technique for measuring diaphragm function (Bible and Whitehead, unpublished results).

[0095] Diaphragm imaging is performed with the Visual Sonics High Frequency Ultrasound Vevo 2100 with the MS-400 (40 MHz) probe, a system specifically designed for mouse ultrasound imaging. After hair removal, the mouse is anesthetized using isoflurane (induced with 3%, maintained at 1%) and placed on a heated imaging stage. The stage is heated to 37 °C and has contact terminals imbedded to which the feet of the mouse are taped with electrode gel to record the respiration rate throughout the procedure. For consistent diaphragm imaging the respiration rate is maintained between 140 to 200 breaths per minute. Images are recorded with the transducer placed perpendicular to the mouse mounted on the stage, dorsal side down at the ventral wall. For optimal diaphragm imaging, the stage and mouse are tilted at a 30-degree angle, head downward. This positioning provides a clear consistent view of the diaphragm movement during respiration.

EXAMPLE 3

[0096] Physiological studies of muscle contractile function are performed to confirm changes in ex vivo studies of diaphragm strips. These studies are conducted using methods described by Whitehead et al. for wild type and mdx mouse muscles (PLoS ONE 5: el 5354). Diaphragm muscle strips, 2 to 3 mm wide, are dissected in physiological buffer, bubbled with 95% O ₂ and 5% CO ₂ (pH 7.4). The diaphragm strip is then placed in the experimental chamber, which is continuously perfused with solution. The central tendon of the strip is attached, via a metal clip, to the lever of a dual-mode force transducer/length controller (Aurora Scientific). At the other end of the chamber, the strip is attached to a stainless steel hook, via a small hole in the rib bone. Muscle stimulation is provided by two platinum electrodes, attached to the inside walls of the chamber, which are connected to a stimulator (Aurora Scientific). Supramaximal stimulus voltage is set at 20% greater than the voltage required for maximum twitch force. A length-force curve is then measured by tetanic contractions (120 Hz, 300 ms duration), spaced 1 minute apart, over a range of muscle lengths (from short to long). The optimum length (L ₀ ) is the length at which maximum tetanic force is generated. Muscle fiber length (Lf) at L ₀ is then measured using calipers, for later calculation of specific force (force normalized to muscle cross-sectional area). At this stage, the muscle is subjected to either a fatigue or eccentric contraction protocol. For fatigue, the muscle is stimulated at 1 Hz for 45 seconds. This typically reduces diaphragm force by approximately 50%. Recovery of force following fatigue is measured at 1 minute intervals up to 10 minutes.

[0097] Functional analysis of the tibialis anterior is conducted by the in situ method described in several of our recent publications (J. Clin. Invest. 120: 816-826 (2010)). This preparation has the advantage that muscle contractile function is tested under conditions in which innervation and vascular function are preserved.

[0098] Although eccentric contraction-induced "injury" is a widely used method for assessment of improvement in the dystrophic phenotype, this functional parameter is probably not relevant to in vivo diaphragm function. To our knowledge, diaphragm muscle does not undergo eccentric contractions under normal physiological conditions. In contrast, eccentric contraction injury is relevant for the tibialis anterior. For eccentric contractions, the muscle is stretched by 20% of Lf during tetanic stimulation, at a velocity of 2 Lf sec ^"1 , with the stretch starting 150 ms after the onset of stimulation. A series of 10 eccentric

contractions are carried out, 30 seconds apart. At 15 minutes after the eccentric contractions, the tetanic length- force curve is measured again to determine the force drop. Re-measuring the length-force curve after the eccentric contractions is important, since Lo is known to shift to a longer length, due to increased series compliance in the muscle.

EXAMPLE 4

[0099] Histological analyses. Treated and untreated mice are compared by standard histological assays that are well established in our laboratory. Cryosections are stained by the H&E method to assess the general pathology of the muscle. Central nuclei (CN) are counted to determine the percentage of muscle fibers that have undergone damage and regeneration. Fibronectin immunostaining and immunoblotting are reliable markers for fibrosis, and are conducted as described in our recent publication, as well as immunoanalysis of collagen deposition, and measurements of hydroxyproline levels.

[00100] A hallmark of dystrophic muscle is heterogeneity of fiber cross-sectional area. Fibers in mdx and mdx:utr het muscle display a broader range of sizes and a higher proportion of small diameter fibers, as compared to wild type. To assess the effect of ataciguat on this parameter, the minimal 'Feret's diameter' is measured and yields a reliable measure of muscle cross sectional size, and provides numbers that are independent of the orientation of the plane of section.

EXAMPLE 5

[0100] Cardiac studies. Cardiac function is examined in treated and untreated mice mdx:utr het for comparison with control C57BL/10 by echocardiography. Thus, data on both diaphragm and cardiac function are obtained in a single experimental session. This has proven to be extremely useful in analyzing drug efficacy on both respiratory muscle and cardiac systems.

[0101] Temporal echocardiography analysis of treated and untreated groups of wild type and mdx mice largely follows the methods used in our previous publication (Neuromuscular Disorders, 2007; 17(4): 290-296). Initially, anesthetized mice at 1, 2, and 3 months of age are analyzed. Echocardiography is performed using a Visualsonics Vevo 2100. This machine is designed for murine cardiac analysis and provides much higher resolution images and superior analysis capabilities similar to those in human echocardiography. The mice are treated with dobutamine to stress the heart, in order to observe more consistent measurements and a greater revelation of cardiac defects.

[0102] In vivo cardiac morphology was assessed in conscious mice using transthoracic echocardiography (Acuson Sequoia C256, 13-MHz transducer; Siemens Medical Solutions USA). M-mode LV end-systolic and end-diastolic dimensions were averaged from 3-5 beats. From these data, fractional shortening (maximal diameter - minimal

diameter)/maximal diameter * 100; chamber cross sectional diameters at end-diastole and end-systole, heart rate, wall thickness in the intraventricular septum, posterior wall, wall mass (estimated from a spherical model), isovolumic contraction time (IVCT), ejection time (ET), isovolumic relaxation time (IVRT), and myocardial performance index (MPI =

(rVCT+IVRT)/ET) were determined.

[0103] Heart wall dimensions and fractional shortening. Transthoracic M-mode echocardiography obtained at the level of the papillary muscles on anesthetized mice is used to determine cardiac wall dimensions and the percentage of fractional shortening (FS, calculated as (LVIDd-LVIDs) /LVIDd x 100%, where LVIDd is the left ventricular internal dimension at end diastole and LVIDs is the left ventricular internal dimension at end-systole. LVM (left ventricle mass) is calculated as 1.04 * (IVSd + LVIDd + PWd)3 - (LVIDd3), where IVSd is the interventricular septal thickness at end-diastole, PWd is the posterior wall thickness at end-diastole, and LVIDs is the left ventricular internal dimension at end-systole. LVMI (left ventricle mass index) will be calculated by normalizing LVM to body weight.

[0104] Diastolic and systolic function. Doppler tissue imaging (DTI) is used to measure E' (early passive diastolic myocardium velocity) and A' (peak myocardium diastolic velocity upon atrial contraction) values. E' and A' velocities is used to calculate the Ea/Aa ratio. An Ea/Aa ratio of < 1 represents diastolic (left ventricle myocardium relaxation) dysfunction. The MPI is obtained using pulse-wave Doppler imaging of mitral valve inflow from the apical four chamber view as (IVCT + IVRT) / LVET, where IVCT is the isovolemic contraction time, IVRT is the isovolemic relaxation time, and LVET is left ventricular ejection time. An increase in MPI indicates that a greater fraction of diastole is spent to cope with the pressure changes during isovolemic phases, which are a sign of left ventricular systolic and diastolic dysfunction. Data from the aged mdx mice in placebo and ataciguat groups are shown in Table 1 and Figure 3.

[0105] TABLE 1. Echocardiogram Results

Group A is the control group and Group B is the ataciguat group

[0106] In vivo hemodynamics. In vivo LV function was assessed by pressure-volume (PV) analysis in anesthetized mice, (n=5 from each group of either vehicle treated or HMR 1766 treated) (Am. J. Physiol. Heart Circ. Physiol, 201 l;301 :H2198-2206). Mice were anesthetized with urethane (800-1200 mg/kg), etomidate (5-10 mg/kg) and morphine (2 mg/kg), and then intubated, and mechanically ventilated (tidal volume 250-300 \L, rate 120/min) as previously described (Nat. Med., 2005; 1 1 :214-222). In addition, pancuronium (2 mg/kg) was administered i.p. once anesthesia was confirmed. The LV apex was exposed through an incision between the seventh and eighth ribs, and a 1.4-Fr PV catheter (SPR-839, Millar Inc.) was advanced through the apex so its distal tip lay in the aortic root. The conductance signal and micromanometer pressure signal were recorded using the Millar pressure-volume catheter system (Model MPCU-200, Millar Inc.), and digitized at 2 kHz using custom designed software (XLC, Hopkins). Pressure volume data were recorded at steady state, and during transient preload reduction produced by obstruction venous return from the inferior vena cava. The volume signal was calibrated using a directly measured mean aortic flow by ultrasound probe (Transonics, Inc), and the hypertonic saline calibration method.

[0107] Pressure-volume data were analyzed by custom software (WinPVAN). Steady state data were signal averaged over 5-10 sequential cardiac cycles. These data Sequential cycles measured during preload reduction were used to determine load-independent measures of cardiac contractility. These were end-systolic elastance (Ees), the slope of the end-systolic pressure volume relationship, preload recruitable stroke work (PRSW), the slope of the stroke-work end-diastolic volume (EDV) relationship, and the slope of the dP/dt _mx - EDV relationship. For each beat in the preload-varying data set, loop area (stroke work), maximal rate of pressure rise (dP/dt _mx ), and end-systolic pressure and volume points were determined using well established algorithms, and used to determine these relations.

[0108] Pressure volume curves from the aged mdx mice in placebo and ataciguat groups are shown in Figure 4. A significant increase in pressure/volume was found in the hearts of ataciguat treated mdx mice.

[0109] Histology is performed on cryosections of hearts from treated and untreated mdx:utr animals as described above for skeletal muscle. These studies are conducted to support the potential efficacy of ataciguat in an animal model of DMD. However, there are no specific histological criteria for efficacy.

[0110] Imaging and PV and image analysis were performed by investigators blinded to treatment group.

EXAMPLE 6

[0111] Voluntary exercise performance. Mice are run voluntarily in a wheel placed in their cages and the total running distance can be influenced by cardiac, respiratory and/or skeletal muscle function. Voluntary wheel running distance is one of the outcome measures assessing overall function in mdx:utr het mice treated with ataciguat or derivatives thereof. Adult mdx mice ran 48, 38, 40 and 31% of the distance that adult C57 mice ran after 1, 2, 3 and 4 weeks, respectively, of voluntary running. At 2-3 months of age (exact time to be determined in preparatory studies of mdx:utr het mice), treated and untreated mdx and control mice (6 animals in each group) are caged individually with free access to a running wheel and the distance run is measured daily. Wheel revolutions and the calculated distance run are monitored simultaneously.

[0112] In experiments with aged mdx mice being fed placebo or ataciguat diets, open field movement was measured. Subjects are placed in an box and after a period of acclimation, their vertical and horizontal movements are recorded by the number of lasere beam breaks. In Figs 5A and 5B, the total movement is compared between the two groups. There is an improved trend in movement in the ataciguat treated group but not significant at the p< 0.05 level. In addition, open filed movement of the aged mdx mice being fed placebo or ataciguat diets was measured after placing the mice on a treadmill until they are exhausted. Subjects were run on a treadmill at 10 meters per minute until exhaustion. Exhaustion was determined by the subject sitting on an electric grid. After 1 minute of sitting on an electric grid without returning to the treadmill, animals were placed in the open field for activity testing. Again, there is a marked improvement of the ataciguat treated group but not significant at the p< 0.05 level (Fig. 6).

[0113] Statistical Analyses. For cardiac and skeletal muscle studies, univariate analysis of variance (ANOVA) is used to determine the statistical significance of the effects of treatment on experimental measures using SPSS software (SPSS Inc.). For all other comparisons, values are compared by unpaired Student's ?-tests using Graphpad Prism software. P values less than 0.05 will be considered significant.

EXAMPLE 7

Platelet VASP phosphorylation assay. As a surrogate for cGMP levels, the level of cGMP- stimulated phosphorylation of VASP in platelets is measured. This is a well-established assay for efficacy of drugs that alter cGMP levels (phosphodiesterase inhibitors, sGC activators). The assay is based on the preferential phosphorylation of VASP Ser239 by protein kinase G (J Biol Chem, 1998. 273(32): p. 20029-20035). VASP phospho-specific monoclonal antibodies that recognize the S239 phosphorylated site in both human and mouse are commercially available.

EXAMPLE 8

[0114] Single and multiple ascending dose Phase lb studies to evaluate the safety, tolerability, pharmacokinetic and pharmcodynamic effects of ataciguat and/or derivatives thereof, in boys with Duchenne muscular dystrophy.

[0115] The Phase lb studies are designed to provide initial safety, tolerability, pharmacokinetic (PK) and pharmacodynamics (PD) data of ataciguat in this pediatric population. A Single Ascending Dose (SAD) study will be conducted in a limited number of subjects at two clinical sites (Kennedy Krieger and Nationwide Childrens'). Following review of safety and tolerability in the SAD study, the highest tolerated doses will be compared to placebo in a 14 day Multiple Ascending Dose (MAD) study. The Phase lb studies are designed to provide a complete PK plasma profile following administration of single and multiple doses of ataciguat, administered in dose-escalating fashion [0116] Phase lb Study Design. This is an open-labeled single (SAD) followed by a placebo-controlled multiple (MAD) ascending dose study to evaluate the safety, tolerability, PK, and PD effects of ataciguat, an orally-delivered, soluble guanylate cyclase activator, in participants with DMD. Dose escalation and initiation of the MAD phase will occur only with approval by the Data and Safety Monitoring Board (DSMB). After successful completion of the SAD phase of the study and minimum 1-week wash-out period, study participants will be given the option to continue in the MAD phase. Participants remaining in the MAD phase will receive ataciguat or placebo (2: 1) for 14 consecutive days. Blood sampling for PK analysis and routine safety evaluations will be performed.

[0117] The SAD and MAD studies are designed to determine the highest safe and tolerated dose of ataciguat in a pediatric disease population. To date, across all studies performed in adult volunteers, oral dosages of 25 to 200 mg ataciguat have been given as single doses and oral dosages of 5 to 200 mg have been given as repeated doses, the highest dosage regimen being 200 mg once daily for 26 weeks in the ACCELA trial in adults with peripheral arterial disease. It is known that the correlation between dose and exposure to ataciguat is fairly linear, and that steady state conditions at 5, 25, and 100 mg per day are reached after 5-7 days of dosing. Consistent with the elimination half-life of ataciguat, a doubling of exposure was observed between multiple and single doses (i.e. accumulation ratio of approximately 2). These pharmacokinetic properties of ataciguat support the 14-day duration of the MAD trial.

[0118] A clinically significant number of subjects will be enrolled in 4-5 dose cohorts in the SAD and another group of subjects (2: 1 ratio of drug to placebo) will be enrolled in 3-4 cohorts in the subsequent MAD.

[0119] Inclusion Criteria: Participants must meet the following criteria to be included:

1) Male gender

2) years of age to be determined at time of dosing

3) Diagnosis of DMD confirmed by at least one of the following:

3 a) Positive gene deletion test (missing one or more exons) of dystrophin, where reading frame can be predicted as "out-of-frame", and clinical presentation consistent with typical DMD, or

3b) Complete dystrophin gene sequencing showing an alteration (point mutation, duplication, or other mutation resulting in a stop codon mutation) definitively associated with DMD, and clinical presentation consistent with typical DMD

4) Corticosteroid therapy for at least 12 calendar months prior to dosing

5) Participant, Parent/Guardian must provide written informed consent and assent for participating in the study

6) Participant must possess the ability, per the Principal Investigator (PI), to understand and comply with protocol instruction for the entire duration of the study.

[0120] Exclusion Criteria: Participants meeting any of the following criteria are to be excluded:

1) Previous treatment with another investigational product within 12 weeks prior to dosing

2) Use of nitrates, sildenafil or other phosphodiesterase inhibitor within 1 month of dosing

3) Use of Coumadin or other drugs metabolized by CYP2C9 within 1 month of dosing

4) Use of midazolam or other drugs metabolized by CYP3A within 1 month of dosing

5) Any clinically significant cardiac, endocrine, hematologic, hepatic, immunologic, metabolic, urologic, pulmonary, neurologic, dermatologic, psychiatric, renal, and/or other major disease as determined by the PI that is not known to be related to DMD.

6) History of severe allergic or anaphylactic reactions as defined as one or more severe allergic or anaphylactic reactions requiring medical intervention.

7) Unwillingness or inability to comply with the requirements of this protocol (in the opinion of the PI) including, but not limited to, the presence of any condition (physical, mental, or social) that is likely to affect the participant's ability to return for study visits or adhere to the visit schedule.

[0121] Rationale for inclusion and exclusion criteria:

1) Age: The age range for the Phase lb study is similar to that expected to be enrolled in the Phase lib trial.

2) Ambulatory status: The ability of the subject to ambulate is not expected to affect the safety, tolerability, PK, or PD as assessed in this Phase lb study and therefore is not a criteria for inclusion in this study. 3) Concomitant medications: All subjects must be receiving corticosteroid treatment for a minimum of 1 year in efforts to enroll a homogenous population. Nitrates and PDEi are known to have effects within the NO-cGMP pathway and may potentiate activity of ataciguat and are therefore excluded. Ataciguat is a moderate inhibitor of CYP2C9. Co-administration of ataciguat with Coumadin let to a 3 fold increase in Coumadin. Ataciguat is a weak CYP3A inhibitor. Co-administration of ataciguat with midazolam led to a 1.4 fold increase in midazolam. Drugs which are metabolized by these enzymes are excluded.

[0122] Duration of treatment. Study duration for each subject participating in both SAD and MAD is for a clinically significant time period. In an embodiment, this time periods is 2 months, including a minimum 1 -week wash-out period between SAD phase and MAD phase of the trial. SAD Phase: 14 days (1 day of screening, 1 day dosing, and 6 days of follow-up, and minimum 1 week wash-out period). MAD Phase: 45 days (1 day re-confirmation of eligibility, 14 days of dosing, and 30 days of follow-up).

[0123] Scaling for size suggests that an exposure in a pediatric population corresponding to the top adult exposure would be approximately 3 mg/kg. Dosing cohorts will be adjusted following the juvenile toxicology studies in dogs and following recommendations from the FDA. However, possible SAD dosing cohorts are: Single oral delivery of ataciguat at 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg and 3 mg/kg. MAD Phase dosing will be based on safety and PK data from SAD and should include the top 3-4 safe and tolerated doses of the SAD study. Simulations will be performed by outside contractors prior to the start of the SAD study to ensure that pediatric subjects will not be exposed to ataciguat concentrations greater than those previously observed in healthy adults.

[0124] Standard safety assessments will include: Adverse event (AE) collection, Clinical laboratory testing (hematology, chemistry, coagulation, urinalysis), Vital signs, ECG and Physical examinations.

[0125] Pharmacokinetic (PK) Plasma studies. Venous blood samples will be obtained at the below time points to determine plasma concentrations of ataciguat. Subjects will be admitted during the first 24 hours to the Johns Hopkins or Nationwide Children's Clinical Research Units (NIH CTSA funded units) to facilitate blood sampling through a PICC line (Peripherally Inserted Central Catheter) and for safety monitoring. [0126] SAD phase: Post dosing at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours. A follow-up visit (with blood sampling) will occur on Day 6 (144 hours) post dosing.

[0127] MAD phase: Day 0 (first day of treatment, post-dosing): 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 12 hours. On Day 13 (2 weeks of continuous dosing), samples will be obtained pre-dosing (baseline), then post-dosing at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours.

[0128] Regulatory-complaint PK-PD analysis, biostatistics and reporting will be performed.

[0129] Pharmacodynamics (PD). The specific target of guanylate cyclase in skeletal muscle is unknown. Further, muscle biopsy will substantially add to the discomfort, complexity and cost of the Phase lb and Phase lib studies. In an embodiment, the study will follow a surrogate biomarker of ataciguat function, vasodilator-stimulated phosphoprotein (VASP) phosphorylation for assessment of ataciguat activity. Determination of cyclic nucleotide (cGMP) levels is possible but the very rapid cellular turnover of cGMP severely complicates the interpretation of results. Instead, detection of cyclic nucleotide-dependent protein phosphorylation, which is much more stable, has been used, including in clinical trials of ataciguat, as an assay to monitor intracellular cyclic nucleotide levels. A common substrate of cyclic nucleotide-dependent protein kinases is the platelet phosphoprotein VASP. VASP phosphorylation is sensitive to elevated cyclic nucleotide levels and stable over time. Treatment of platelets with ataciguat results in activation of oxidized sGC with dose dependent VASP-phosphorylation (Hoechst Internal Document F19999PHM0273, 1999).

[0130] Statistical methods. Determination of sample size: The determination of sample size for this safety study was based on clinical considerations. A clinically significant number of eligible participants are to be enrolled at each of 4-5 doses of the SAD phase for a total of 8-10 subjects. A clinically significant number of participants per will be enrolled in each of 3-4 cohorts in the MAD phase at 2: 1 drug: placebo ratio, including 1) participants who completed the SAD phase and are eligible for enrollment into the MAD phase and 2) new participants replacing initial SAD participants ineligible for the MAD.

[0131] Statistical analysis plan: In general, descriptive statistics for all endpoints captured will be performed. In the MAD phase of the trial, within-group estimates of the change from baseline will be summarized along with 95% confidence intervals for all PD effects. [0132] Analysis of safety. Exposure to study drug and reasons for discontinuation of study will be tabulated, and demographics will be presented using descriptive statistics (i.e., mean, standard deviation, median, and range). Safety variables will be tabulated and presented for all participants who receive the assigned dose of ataciguat. AEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA). Incidence of treatment- emergent AEs will be presented by system organ class (SOC) and preferred term. AEs will also be presented by severity (as evaluated by the Common Terminology Criteria for Adverse Events) and relationship to study drug. Change from baseline in clinical laboratory parameters and vital signs will be summarized across time. Shift tables will be presented for selected laboratory parameters, ECGs, and vital signs. Physical examination results will be presented in listings.

[0133] Pharmacokinetic analysis. Plasma concentrations will be assayed using a validated LC/MS/MS method. The PK population is defined as all participants enrolled in the study who received at least 1 dose of ataciguat and have at least 1 post dose PK measurement. Listing of individual participant ataciguat plasma concentrations, actual blood sampling times, and PK parameters as well as graphs of concentration versus time will be prepared by actual dose regimen. Summary statistics will be presented for plasma concentrations by nominal sampling time, and for the PK parameters of interest. Those treatment periods with at least 2 time points with measurable concentrations will be included in the analysis of PK parameters. The following PK parameters will be calculated: The following PK parameters will be calculated: Area under the curve (AUC), maximum concentration (Cmax), time to maximum concentration (Tmax), elimination half-life (Tl/2), apparent clearance (CL/F) and apparent volume of distribution (Vd/F).

[0134] Upon successful completion of Phase I studies, a Phase lib study will be conducted using the two highest tolerated doses administered in the MAD trial in the multicenter Phase lib study, in which patients will undergo daily dosing of oral ataciguat, and/or a derivative thereof, or placebo in a 2:2: 1 ratio over a 24 week time period. Efficacy can be evaluated primarily by motor function in the 6 Minute Walk Test (6MWT) and/or left ventricular end systolic volume as determined by cardiac MRI, for example. Multiple secondary endpoints of skeletal, cardiac and pulmonary muscle function will be assessed to more comprehensively determine the effect of ataciguat on the function of DMD boys.

During this period of repeat daily dosing, population PK parameters will be obtained, thereby expanding the ataciguat set in DMD. The Phase lib trial is powered to be a pivotal study for seeking provisional FDA approval for ataciguat given the life-threatening and disabling nature of DMD.

[0135] Test product, dose and mode of administration. In one embodiment, the product will be supplied for oral administration with food, as soft capsules containing 5 mg, 25 mg and 50 mg of ataciguat or a derivative thereof (calculated with reference to the active moiety) and the following excipients: macrogols, macrogolglycerol hydroxystearate, medium-chain partial glycerides, and glycerol. The soft capsule shell contains the following excipients: gelatin, glycerol, and the commercially available Anidrisorb 85/70 (contains sorbitol, sorbitan, mannitol, and higher polyols). The soft capsules are packaged in blister packs. Drug and placebo will be shipped directly to each clinical site from the FDA approved

manufacturer.

[0136] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0137] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0138] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.