BOWEL DISEASES (IBDS)

FIELD OF THE INVENTION

The present invention relates to compositions and methods that can treat, delay progression, or cure gastrointestinal (GI) disorders such as Inflammatory Bowel Disease (IBD), including, for example, Crohn's Disease (CD) and ulcerative colitis (UC).

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract suffered by approximately one million patients in the United States. The two most common forms of IBD are Crohn's disease (CD) and ulcerative colitis (UC). Although CD can affect the entire gastrointestinal tract, it primarily affects the ileum (the distal or lower portion of the small intestine) and the large intestine. UC primarily affects the colon and the rectum. Current treatments for both CD and UC include aminosalicylates (e.g., 5-aminosalicylic acid, sulfasalazine, and mesalamine), antibiotics (e.g., ciprofloxacin and metronidazole), corticosteroids (e.g., budesonide or prednisone), immunosuppressants (e.g., azathioprine or methotrexate), and tumor necrosis factor (TNF) antagonists (e.g., infliximab (Remicade)). Patient response to these therapies varies with disease severity, and may vary over cycles of active inflammation and remission. Many patients do not respond to the initial treatment or lose response over time. Moreover, many of the current therapies for IBD are associated with undesirable side effects. There is an unmet need for more effective treatments for IBD that are easier to administer, cause less side effects and that can cure or treat this debilitating condition.

Alpha- 1 -antitrypsin (AAT) is a heavily glycosylated plasma protein of 52 kDa. AAT is produced by the liver and secreted into the circulation, and is also produced locally by lung epithelial cells. Circulating levels of AAT increase during an acute phase response. This increase is due to the presence of IL-l and IL-6 responsive elements inside the promoter region of the AAT encoding gene. AAT functions as a serine protease inhibitor that primarily targets elastase, trypsin, and proteinase-3, three inflammatory and immune cell-derived enzymes that are involved in protease-activated receptor (PAR) activation and the onset and progression of inflammation (Vergnolle N. 2009. Pharmacol Ther l23(3):292-309). AAT induces the production and release of anti-inflammatory mediators such as IL-10 and IL-l -receptor antagonist (IL-lRa) (Lewis E C et al. 2008. Proc Natl Acad Sci USA. 105(42): 16236-41). In individuals with AAT deficiency, (serum level below the normal range 80 mg/dl), there is an excess of neutrophil elastase that increases the breakdown of elastin leading to airway destruction. This manifests clinically as chronic obstructive pulmonary disease (COPD) with emphysema and/or chronic bronchitis.

Several clinical trials have addressed the potential benefit of AAT therapy for individuals with normal AAT production (i.e. not defined as AAT deficient subjects) who are suffering from inflammatory conditions. Such indications include islet and lung transplantation, Type 1 diabetes mellitus (T1DM), graft- versus-host disease, acute myocardial infarction, and cystic fibrosis.

SUMMARY OF THE INVENTION

The present invention provides compositions, methods, and uses for Alpha- 1 antitrypsin (AAT), derivatives or analogs thereof for the treatment or prevention of bowel disease. In certain embodiments, treatment or prevention of bowel disease includes, but is not limited to, treatment or prevention of inflammatory bowel disease (IBS or IBD). In other embodiments, IBD can be ulcerative colitis and/or Crohn's disease (CD). According to some embodiments, AAT or a peptide derivative thereof may be used to treat or prevent IBD in a subject. According to some embodiments, AAT or a peptide derivative thereof may be used to attenuate, prevent or delay disease progression of IBD. Yet other embodiments herein concern reducing side effects of these conditions in a subject by employing a multiple-dose regimen of AAT administration.

According to one aspect, the present invention provides a method for treating inflammatory bowel disease in a subject in need thereof, said method comprising administering to a subject in need thereof Alpha- 1 antitrypsin (AAT) in a multiple dosage regimen.

According to certain embodiments, the present invention provides AAT in a multiple variable dosage regimen for use in treating inflammatory bowel disease in a subject in need thereof. According to certain embodiments, the present invention provides a use of the composition comprising AAT or functional variant thereof in the manufacture of a medicament for the treatment of inflammatory bowel disease in a subject in need thereof.

According to certain embodiments, the inflammatory bowel disease comprises Crohn's disease (CD), ulcerative colitis (UC), collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis, or a combination thereof.

According to other embodiments, the AAT treatment comprises multiple administrations of multiple portion doses. According to certain embodiments, each portion dose comprises from about 40 mg AAT/KgBW to about 240 mg AAT/KgBW. According to other embodiments, each portion dose comprises 40, 60, 80, 120, 180 or 240 mg AAT/KgBW. Each possibility represents a separate embodiment of the present invention. According to other embodiments, each portion dose comprises 40-240 mg.

According to certain embodiments, the treatment comprises administering AAT in multiple portion doses at intervals of from 2-4 days to two weeks. According to certain embodiments, the interval between AAT portion dose administrations is constant. According to other embodiments, the interval between AAT portion dose administrations is variable. According to certain embodiments, the portion doses contain the same AAT amount. According to other embodiments, the portion doses contain variable AAT amounts.

According to certain exemplary embodiments, the AAT portion dose is administered once a week during the entire treatment. According to certain exemplary embodiments, the AAT amount in each of the portion doses is constant during the entire treatment.

The total cumulative dose of AAT to be administered during the treatment is variable and depends on the age, gender and the IBD condition to be treated.

According to certain exemplary embodiments, the amount of AAT in the treatment dose portion decreases from the first portion dose administered to the last portion dose administered.

According to some embodiments, the duration of the treatment is in the range from 1 day, 2 weeks, to 74 weeks. According to other embodiments, the duration of the treatment is in the range from 8 weeks to 44 weeks. According to yet additional embodiments, the duration of the treatment is in the range from 6 weeks to 16 weeks. According to certain exemplary embodiments, the duration of the treatment is selected from the group consisting of 2, 4, 6, 8, 16, 44, and 74 weeks. According to some embodiments, the administration is carried out for a plurality of months. According to certain embodiments, the present invention employs a long-term multiple-dose regimen of AAT administration. Each possibility represents a separate embodiment of the present invention.

According to another aspect, the present invention provides a method for treating an exacerbation of inflammatory bowel disease, which comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising Alpha- 1 antitrypsin (AAT).

According to certain embodiments, the AAT alleviates imbalanced proteinase/antiproteinase ratios which develop during the exacerbation period.

According to certain embodiments, the pharmaceutical composition is administered at the time of an exacerbation episode. According to certain embodiments, the subject is human.

Any route of administration as is known in the art to be suitable for AAT administration can be used according to the teachings of the present invention. According to certain embodiments, the AAT is administered parenterally. According to certain exemplary embodiments, the AAT is administered intravenously (i.v.). According to other embodiments, the AAT is administered by oral administration. According to other embodiments, the AAT is administered by rectal administration. The AAT is typically administered within a pharmaceutical composition formulated to complement the route of administration.

According to some embodiments, the AAT is administered in a dry powder form. According to some embodiments, the AAT is administered in a liquid form. According to some embodiments, the AAT is administered by oral administration to a subject with mild to moderate Crohn's disease as measured by the Crohn's Disease Activity Index (CDAI) where 150 < CDAI < 450.

According to some embodiments, the AAT is administered intravenously to a subject with severe Crohn's disease (CDAI > 450). According to some embodiments, the AAT is administered intravenously and/or by oral administration to a subject with acute Crohn's disease. According to some embodiments, the AAT is administered intravenously and/or by oral administration for the maintenance of a subject with Crohn's disease.

According to yet another aspect, the present invention provides a kit for the treatment of IBD comprising: (a) at least one portion of a pharmaceutical composition comprising an induction dose of AAT; (b) a plurality of portions of a pharmaceutical composition each comprising a treatment dose of AAT; and (c) instructions for administration of the induction dose of the AAT within the treatment phase.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates the dose response of stimulated macrophages to AAT treatment. RAW 264.7 cells (l0 ⁵ /well) were incubated with the indicated concentrations of AAT (none (-) to 4 mg/ml) overnight, followed by stimulation with LPS (5 ng/ml). Supernatants were collected 24 hours after stimulation and assayed for the indicated cytokines by magnetic multiplex. All samples were tested in triplicate. Graphs show Mean ± SEM. CT, unstimulated control, Dex, Dexamethasone (10 pg/ml).

FIG. 2 demonstrates the dose response of stimulated THP-l cells to AAT treatment. THP-l cells (2xl0 ⁴ /well) were induced to become macrophages by incubation with 80 nM PMA for 48 h. Cells were then treated with 3 or 5 mg/ml of AAT for 4 h. followed by stimulation with LPS (100 ng/ml). Supernatants were collected 24 hours after stimulation and assayed for the indicated cytokines by magnetic multiplex. All samples were tested in triplicate. Graphs show Mean ± SEM. Dex, Dexamethasone (10 ng/ml).

FIG. 3 demonstrates the dose response of stimulated macrophages to AAT treatment. RAW 264.7 cells (l0 ⁵ /well) were incubated with the indicated concentrations of AAT overnight, followed by stimulation with LPS, as described above. Supernatants were collected 24 hours after stimulation and assayed for G-CSF levels by magnetic multiplex. All samples were tested in triplicate. Graphs show Mean ± SEM. CT, unstimulated control, Dex, Dexamethasone (10 mg/ml).

FIG. 4 demonstrates dose response of stimulated THP-l cells to AAT treatment. THP-l cells (2xl0 ⁴ /well) were induced to become macrophages followed by AAT treatment and LPS induction as described above. Supernatants were collected 24 hours after stimulation and assayed for the indicated cytokines by magnetic multiplex. All samples were tested in triplicates. Graphs show Mean ± SEM. Dex, Dexamethasone (10 ng/ml).

FIG. 5 demonstrates the dose response of stimulated macrophages to AAT treatment. RAW 264.7 cells (l0 ⁵ /well) were incubated with the indicated concentrations of AAT (mg/ml) overnight, followed by stimulation with LPS as described above. Supernatants were collected 24 hours after stimulation and assayed for the indicated cytokines by magnetic multiplex (TNFa and IL-6) or by ELISA (IL-lRa). All samples were tested in triplicate. Graphs show Mean ± SEM. CT unstimulated control, Dex, Dexamethasone (10 pg/ml).

FIG. 6 demonstrates the dose response of stimulated THP-l cells to AAT treatment. THP-l cells (2xl0 ⁴ /well) were induced to become macrophages followed by AAT treatment and LPS induction as described above. Supernatants were collected 24 hours after stimulation and assayed for the indicated cytokines by magnetic multiplex. All samples were tested in triplicate. Graphs show Mean ± SEM. Dex, Dexamethasone (10 ng/ml).

FIG. 7 demonstrates the dose response of stimulated PBMCs to AAT treatment. PBMC cells (250,000 cells/well) were treated with 1 or 3 mg/ml of AAT or Dexamethasone (10 ng/ml) for 2 h, followed by stimulation with LPS (10 ng/ml). Supernatants were collected 24 hours after stimulation and assayed for the indicated cytokine by ELISA. All samples were tested in triplicate. Graphs show Mean ± SEM. Dex, Dexamethasone (10 ng/ml).

FIGS. 8A-B demonstrate the length of the small and large intestines in DSS- induced IBD rats with and without AAT. * P value<0.0l

FIG. 9 demonstrates that AAT attenuates weight loss in DSS-induced IBD rats. * P value<0.05 FIG. 10 demonstrates that AAT-treated animals had less diarrhea, starting from day four onwards.

FIG. 11 shows that AAT-treated animals demonstrated a lower disease score compared to animals not treated with AAT.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses multiple-variable dosage method for treating IBD, particularly in human subjects with Crohn's disease or ulcerative colitis.

Thl and Thl7 cells are both important mediators of inflammation in Crohn's disease. According to the present invention, AAT treatment reduces inflammation, inhibits Thl and Thl7, protects epithelial cells, and induces tolerance in autoimmune diseases such as IBD.

Lymphocyte infiltration and transmural (full-thickness) inflammation in the intestinal wall are characteristics of Crohn’s disease (CD). AAT attenuates inflammation via reduced secretion of pro -inflammatory cytokines, reduced inflammatory cell infiltration and reduced tissue injury.

Definitions

"A patient with IBD" as used herein, refers to a patient suffering from any of the symptoms or manifestations of IBD, a patient who may suffer from any of the symptoms or manifestations of IBD, or any patient who might benefit from a method of the invention for treating or evaluating treatment for IBD. A patient in need may include a patient who is diagnosed with a risk of developing IBD, a patient who has suffered from IBD in the past, or a patient who has previously been treated for IBD. In some embodiments, the patient with IBD is a Crohn's disease (CD) patient. In some embodiments, the patient with IBD is an ulcerative colitis (UC) patient.

Crohn's disease

Crohn's disease (CD) is identified as one of the types of IBD. Usually, the symptoms of inflammation, congestion, or swollen lymph nodes may occur in the colon, small intestine, or stomach of the patient. The main difference between CD and UC lies in in the location and nature of the inflammation. Crohn's disease may affect any part of the digestive system, e.g. small intestine, colon, anus, stomach, appendix, esophagus, and mouth although it is most common in the terminal ileum and adjacent colon segment, and the right-half colon. In contrast, inflammation in UC is usually restricted to the colon, most often the sigmoid colon, and rectum, although it too may affect the entire large intestine (pancolitis). Very rarely, UC can also affect the distal region of the small intestine (Backwash Ileitis).

The inflammation is accompanied usually by diarrhea, which may be profuse and bloody. Micro-ulcers form in places where inflammation has destroyed the cells lining the bowel; these areas bleed and produce pus and mucus. Ulcerative colitis, especially when mild, can be difficult to diagnose because the symptoms are similar to those of other intestinal disorders, most notably the other type of IBD - Crohn's disease and also irritable bowel syndrome (IBS). Crohn's disease differs from ulcerative colitis because it causes inflammation throughout the whole thickness of the intestinal wall and produces deep ulcers and fistulae, whereas ulcerative colitis is usually restricted to the surface layers. Crohn's and ulcerative colitis may co-exist in the same patient.

Microscopically, Crohn's disease may affect all the inner wall of the bowel, while UC is restricted to the mucosa.

Crohn's disease is a chronic and recurrent disease with an unknown etiology for which there are currently no known curative drugs. Treatment for Crohn's disease typically involves glucocorticoids, salicylic acid formulations, immunosuppressive agents, antibiotics, methotrexate, and biological agents (e.g. infliximab). Although these drugs have been proven to be able to change the natural process of the disease, they cannot completely alleviate the pathology and prevent complications. Many patients do not respond to the initial drug treatment or response may be lost over time. Moreover, the known western medicines such as glucocorticoids and immunosuppressive agents are associated with significant adverse reactions, and long-term administration is likely to cause long-term damage to the body. Hence, there is a need to develop a new medicine and its formulation thereof for the treatment of Crohn's disease.

As used herein, "Crohn's Disease Activity Index" or "CDAI" refers to a measurement or index used to assess the progress of patients suffering from CD as described by Best et ah, Gastroenterology, 70:439-44 (1976). CDAI scores of 150 or below are generally associated with inactive disease and are indicative of better prognosis than higher scores. Values above 150 are generally associated with active disease and values above 450 are associated with extremely severe disease. CDAI scores may be used to determine how well a patient is responding to therapy and may be used to identify patients in remission. In certain embodiments, a benchmark clinical response means that the CDAI score of a subject decreases by at least 100 points. In a clinical trial, a CDAI score of 150 or below is generally associated with remission.

CD inflammation Etiology

CD is associated with defective defence mechanisms in the intestinal mucosa and barrier function, and by altered numbers and fewer species of the bacteria normally found in the gut (dysbiosis).

The CD inflammatory response is believed to be driven by the activation of T- helper (Th) 1 cells, as evidenced by the increased levels of IL-2 and interferon gamma (IFN-g) in the intestinal mucosa of CD patients.

CD is also mediated by Thl7 cells, a subset of T cells associated with autoimmune inflammation. Specifically, Thl7-induced IL-17 overexpression in the lamina propria of CD patients has been linked to disease pathogenesis (in particular, related to the IL-23R signalling-pathway).

Environmental factors such as smoking, diet, hygiene, and geography are also thought to play important roles in CD pathogenesis.

As CD can occur anywhere in the gastrointestinal tract between the mouth and the anus, and since the disease can occur throughout the layers of the bowel walls, it seems to be a suitable condition to respond to a systemic treatment by IV administration. AAT could be administered IV to patients with severe disease (acute disease or for maintenance).

Treatment by AAT in a powder or liquid form may be useful for a specific patient population with inflammation located in the small intestine. AAT could be administered by oral administration for mild disease in the small intestine (acute or maintenance).

Ulcerative colitis

Ulcerative colitis occurs most often in peaks between the ages of 15 to 30 and then again, between 50-70 years old, although the disease may strike at any age. It affects men and women equally and appears to run in some families.

There are a number of types of ulcerative colitis. As used herein, "ulcerative proctitis" refers to a form of disease where the bowel inflammation is limited to the rectum. Because of the limited penetration (usually less than the six inches from the rectum into the colon), ulcerative proctitis tends to be a milder form of ulcerative colitis. It is associated with fewer complications and offers a better outlook than more widespread disease. For approximately 30% of patients with ulcerative colitis, the illness begins as ulcerative proctitis.

As used herein, "proctosigmoiditis" refers to a form of colitis affecting the rectum and the sigmoid colon, the lower segment of colon located immediately above the rectum. Symptoms include bloody diarrhea, cramps, and a constant feeling of the need to pass stool, known as tenesmus. Moderate pain on the lower left side of the abdomen may occur in active disease.

As used herein, "left-sided colitis" refers to continuous inflammation that begins at the rectum and extends as far as a bend in the colon near the spleen called the splenic flexure. Symptoms include loss of appetite, weight loss, diarrhea, severe pain on the left side of the abdomen, and bleeding.

As used herein, "pan-ulcerative (total) colitis" affects the entire colon. Symptoms include diarrhea, severe abdominal pain, cramps, and extensive weight loss. Potentially serious complications include massive bleeding and acute dilation of the colon (toxic megacolon), which may lead to an opening (perforation) in the bowel wall. Serious complications may require surgery.

Several theories have been proposed regarding the cause of ulcerative colitis. There is some evidence to suggest that the body's immune system reacts to an environmental, dietary or infectious agent in genetically susceptible individuals causing inflammation in the intestinal wall. Although ulcerative colitis is not actually caused by emotional distress or sensitivity to certain foods or food products, these factors may trigger symptoms in some people.

The most common symptoms of ulcerative colitis are bloody diarrhea and abdominal pain. Patients also may experience fever, rectal bleeding, fatigue, anemia, loss of appetite, weight loss and loss of body fluids and nutrients resulting in nutritional deficiencies. These symptoms occur as intermittent attacks (flare-ups) in between periods when the symptoms go away (remissions). These disease-free periods can last for months or even years. Usually a flare-up begins with increased urgency to defecate, mild lower abdominal cramps, and blood and mucus in the stools.

Ulcerative colitis may cause long-term problems such as arthritis, inflammation of the eye, liver disease (fatty liver, hepatitis, cirrhosis, and primary sclerosing cholangitis), osteoporosis, skin rashes, anaemia, and kidney stones. These complications may occur when the immune system triggers inflammation in other parts of the body and may disappear when the colitis is treated effectively.

Treatment for ulcerative colitis depends on the seriousness of the disease. Most people are treated with medication. Some people whose symptoms are triggered by certain foods are able to control the symptoms by avoiding the types of foods that upset their intestines (like highly seasoned foods or dairy products). Each person experiences ulcerative colitis differently, so treatment is adjusted for each individual.

Many patients with mild or moderate disease are first treated with 5-ASA agents, including a combination of the drugs 5-aminosalicylic acids and sulfasalazine to help control inflammation. Sulfasalazine is the most commonly used of these drugs. Sulfasalazine can be used for as long as needed and can be given in combination with other drugs. Patients who do not do well on sulfasalazine may respond to newer 5-ASA agents. Possible side effects of 5-ASA preparations include nausea, vomiting, heartburn, diarrhea, and headache.

People with severe disease and those who do not respond to 5-ASA preparations may be treated with added corticosteroids. Prednisone, budesonide, and hydrocortisone are corticosteroids used to reduce inflammation. They can be given orally, intravenously, through an enema, or in a suppository, depending on the location of the inflammation. Corticosteroids can cause side effects such as weight gain, acne, facial hair, hypertension, diabetes, mood swings, and increased risk of infection, so doctors carefully monitor patients taking these medications.

Immunosuppressants such as azathioprine, 6-mercaptopurine (6-MP), and methotrexate are often used and can make a marked improvement at a low dose with few side effects. Other drugs may be given to relax the patient or to relieve pain, diarrhoea, or infection. Occasionally, symptoms are severe and the person must be hospitalized. For example, a person may have severe bleeding or severe diarrhoea that causes dehydration. In such cases the doctor will act to try to stop the diarrhoea and loss of blood, fluids, and minerals. The patient may need a special diet, parenteral feeding, medications, or sometimes surgery.

In severe cases, a patient may need surgery to remove the diseased part of the colon. Sometimes the doctor will recommend removing the colon if medical treatment fails or if the side effects of treatment medications threaten the patient's health.

As used herein, "Ulcerative Colitis Disease Activity Index" or "UCDAI" refers to a measurement or index used to assess the progress of patients suffering from UC as described by Sutherland et al., Gastroenterology, 92:1894-98 (1987). The UCDAI is a series of qualifiers about the symptoms of UC including stool frequency, rectal bleeding, the appearance of the colon lining, and a physician's rating of disease activity. Each of these qualifiers is given a score from 0 to 3, with 3 being indicative of the most serious disease. The UCDAI may be used in clinical trials to determine how well a patient is responding to therapy and may be used to identify patients in remission. In a clinical trial, remission is often defined as a UCDAI score of 1 or less, and improvement is a reduction of 3 or more points from the baseline score at the beginning of the trial. Other commonly used indices for measuring disease severity in UC patients include the Truelove and Witts Index, the St. Mark's Index, the Simple Clinical Colitis Activity Index (SCCAI), the Lichtiger Index, the Ulcerative Colitis Symptom Score (UCSS), and the Mayo Clinic Score.

As used herein, "remission, cure, or resolution rate" refers to the percentage of patients that are cured or obtain remission or complete resolution of a condition in response to a given treatment. Remission, cure, or resolution of ulcerative colitis refers to complete cessation of rectal bleeding, urgency, and increased stool frequency. Quantitatively, remission, cure, or resolution is achieved when a patient's UCDAI score, assessed after 8 weeks of treatment, is below or equal to 2. Remission, cure, or resolution can be further confirmed by endoscopic specimen examination and observation of mucosal healing.

As used herein, "response rate" refers to the percentage of patients who respond positively (e.g., reduced severity or frequency of one or more symptoms) to a given treatment. Quantitatively, a positive response to treatment may be defined as a decrease of at least 2 UCDAI points from baseline to week 8.

As used herein, the term“Alpha- 1 Antitrypsin” (AAT) refers to a glycoprotein that in nature is produced by the liver and lung or intestinal epithelial cells and secreted into the circulatory system. AAT belongs to the Serine Proteinase Inhibitor (Serpin) family of proteolytic inhibitors. This glycoprotein consists of a single polypeptide chain containing one cysteine residue and 12-13% of the total molecular weight of carbohydrates. AAT has three N-glycosylation sites at asparagine residues 46, 83 and 247, which are occupied by mixtures of complex bi- and triantennary glycans. This gives rise to multiple AAT isoforms, having isoelectric points in the range of 4.0 to 5.0. The glycan monosaccharides include N-acetylglucosamine, mannose, galactose, fucose, and sialic acid. AAT serves as a pseudo-substrate for elastase; elastase attacks the reactive center loop of the AAT molecule by cleaving the bond between the methionine358 - serine359 residues to form an AAT-elastase complex. This complex is rapidly removed from the blood circulation. AAT is also referred to as “alpha- 1 Proteinase Inhibitor” (API). The term“glycoprotein” as used herein refers to a protein or peptide covalently linked to a carbohydrate. The carbohydrate may be monomeric or composed of oligosaccharides. It is to be explicitly understood that any AAT as is or will be known in the art, including plasma-derived AAT, and recombinant AAT, derivatives or analogs thereof can be used according to the teachings of the present invention.

The terms "treat" and "treating" include alleviating, ameliorating, halting, restraining, slowing or reversing the progression, or reducing the severity of the pathological conditions described above.

The term "dosage" as used herein refers to the amount, frequency, and duration of AAT given to a subject during a therapeutic period.

The term "dose" as used herein, refers to the amount of AAT given to a subject in a single administration.

The terms "multiple-variable dosage" and“multiple dosage” are used herein interchangeably and include different doses of AAT administration to a subject and/or variable frequency of administration of the AAT for therapeutic treatment. "Multiple dose regimen" or "multiple-variable dose regimen" describe a therapy schedule, which is based on administering different amounts of AAT at various time points throughout the course of therapy. According to some embodiments, the AAT is administered by the intravenous route followed by transition to the oral route.

The term "treatment phase" or "maintenance phase", as used herein, refers to a period of therapy comprising administration of AAT to a subject in order to maintain a desired therapeutic effect.

The terms“treatment dose” or "treatment dose portion" as used herein refer to a dose of AAT administered to a subject to maintain or continue a desired therapeutic effect during the treatment phase.

As used herein, the terms "exacerbation" "exacerbation period" and "exacerbation episode" are used interchangeably to describe an increase in the severity of symptoms during the course of a disease, which is mostly associated with a worsening of quality of life. By definition, exacerbations are worsening and/or increase in severity and/or magnitude of the disease symptoms.

As used herein the term "about" refers to the designated value ± 10%.

The term "simultaneous administration," as used herein, means that the AAT and additional treatments are administered with a time separation of no more than about 15 minute(s), such as no more than about any of 10, 5, or 1 minutes.

The term "sequential administration" as used herein means that the AAT and the additional treatment are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either the AAT or the additional IBD treatment may be administered first.

The term "dry powder" refers to a powder composition that contains finely dispersed dry particles that are capable of being dispersed.

The particles of the dry powder composition have a particle size distribution that enables the particles to target the mucosal region of the intestine when delivered via tablets. The particle-size distribution (PSD) of a powder is a list of values or a mathematical function that defines the relative amount of particles present according to size. The powders of the invention are generally polydispersed (i.e., consist of a range of particle sizes). In particular embodiments, the term“particle size distribution” refers to the size distribution of particle system and represents the number of solid particles that fall into each of the various size ranges, given as a percentage of the total solids of all sizes in the sample of interest.

As used herein, the term "particle size distribution D ₉₀ value" is defined as the numerical value, expressed in microns, at which 90 percent of the particles have particle sizes which are less than or equal to that value. As used herein, the term "particle size distribution D ₅₀ value" is defined as the numerical value, expressed in microns, at which 50% of the particles have particle sizes that are less than or equal to a given value.

According to some embodiments, the average particle size is below 10 pm. In other embodiments, the average particle size is below 9, 8, or 7 pm. Each possibility represents a separate embodiment of the invention. In additional embodiments, the average particle size is between 1 to 10 pm, 2 to 9 pm, 3 to 8 pm, 4 to 8 pm, or 4-8 pm. Each possibility represents a separate embodiment of the invention. The average particle size of the powder may be measured as mass mean diameter (MMD) by conventional techniques.

The term "dry" means that the particles of the powder have moisture content such that the powder is physically and chemically stable when stored at room temperature. According to some embodiments, the moisture content of the particles is below 10%, 8%, 6%, 4%, 2%, or 1% by weight. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, the dry powder composition consists of AAT and at least one excipient selected from the group consisting of Trehalose, Glycine, Dipalmitoylphosphatidylcholine (DPPC), and Ectoin, wherein the AAT is at least 60% (w/w) of the composition and wherein at least 90% of the AAT is in a monomeric form.

Pharmaceutical Compositions

According to certain embodiments, AAT is administered in the form of a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to a preparation of AAT with other chemical components such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to an organism and enhance its stability and turnover.

Any available AAT as is known in the art, including plasma-derived AAT, and recombinant AAT, derivatives or analogs thereof, can be used according to the teachings of the present invention. According to certain exemplary embodiments, the AAT is produced by the method described in U.S. Patent No. 7,879,800 to the Applicant of the present invention.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, and more particularly in humans.

The term "carrier" refers to a diluent or vehicle that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included in this category. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, isotonic buffers and physiological pH and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

The pharmaceutical compositions of the invention can further comprise an excipient. Herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, lipids, phospholipids, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.

The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, spray drying, or lyophilizing processes.

According to certain exemplary embodiments, pharmaceutical compositions, which contain AAT as an active ingredient, are prepared as injectable, either as liquid solutions or suspensions, however, solid forms, which can be suspended or solubilized prior to injection, can also be prepared. According to yet additional embodiments, the AAT-containing pharmaceutical composition is formulated in a form suitable for subcutaneous administration. Subcutaneous administration may be a preferred mode of administration, because administration of AAT at multiple low doses was shown to have a positive effect on islet protection. From the patient point of view, multiple injections are not a favorable treatment, and thus this mode of treatment may be replaced by slow and/or controlled release subcutaneous administration. Any other forms of slow and/or controlled release are also explicitly encompassed within the scope of the present invention.

The compositions can also take the form of emulsions, tablets, capsules, gels, syrups, slurries, powders, creams, depots, sustained-release formulations, and the like.

Methods of introduction of a pharmaceutical composition comprising AAT include, but are not limited to, intravenous, subcutaneous, intramuscular, intraperitoneal, oral, topical, intradermal, transdermal, intranasal, epidural, ophthalmic, vaginal, and rectal routes. The pharmaceutical compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and may be administered together with other therapeutically active agents. The administration may be localized, or may be systemic.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, typically in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be transversed are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores. Suitable excipients may be fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in a conventional manner.

Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.

According to certain exemplary embodiments, the A AT -containing pharmaceutical composition used according to the teachings of the present invention is a ready-to-use solution. According to further exemplary embodiments the AAT- containing pharmaceutical composition is marketed under the trade name Glassia ^® .

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1: Expanding the characterization of immunological pathways affected by Alpha- 1 Antitrypsin

The aim of this study was to further characterize the immunological pathways affected by AAT in order to better understand the mechanism of action and assess the therapeutic potential in IBD. The effect of AAT (“Glassia ^® ”) on the profile of a number of cytokines was examined using in-vitro systems that include human monocytes and mouse macrophages cell-lines as well as human-derived peripheral blood mononuclear cells. To better elucidate the immunological mechanism of action, the study was focused on the effects of AAT on the Thl immune response, the systemic effect on immune cell maturation and activation, and the anti-inflammatory properties of the protein.

Materials & Methods

Materials

Cells systems

Three cell lines were used in these assays:

1) A human monocyte cell line, THP-l that was induced by PM A to become macrophages before exposure to AAT and activation. 2) The mouse macrophage cell line RAW 264.7 that was treated similarly with AAT before activation. 3) Human peripheral-blood mononuclear cells (PBMC) derived from a whole blood sample.

Alpha- 1 Antitrypsin (AAT)

The preparation of AAT that was used in these studies was“Glassia ^® ”, a human plasma-derived AAT that is produced by KAMADA Ltd. Glassia ^® was supplied in vials containing 2% (20 mg/ml) of AAT as liquid, ready for use.

Dexamethasone Dexamethasone (SIGMA Cat# 4902) was used at the indicated concentrations as a positive control, of inhibition of the cellular immune responses.

Lipopolysaccharide (LPS)

LPS (SIGMA Cat# L2880) at the indicated concentrations was used to stimulate the cells.

Methods

AAT effect in RAW 264.7 cells

RAW 264.7 cells (l0 ⁵ /well) were seeded in 96-well plates. Cells were incubated with the indicated concentrations of AAT overnight and subsequently stimulated with LPS (5 ng/ml). Supernatants were collected 24 hours post stimulation and kept frozen at - 80°C until testing. Levels of mouse IL-lRa and TGFP were determined by ELISA (Mouse IL-lra/IL-lF3 DuoSet, Cat# DY480, and Mouse TGF-Bl Quantikine, Cat# MB100B, both from R&D SYSTEMS). The cytokines: KC, G-CSF, GM-CSF, IL-10, IL-l2p70, CXCL-10, IL-lb, IL-6, IL-17A, IFNy and TNFa were evaluated by magnetic Luminex assay (R&D Systems, LXSAMSM-l l). All samples were tested in triplicate. AAT effect in THP-1 cells

THP-l cells (2xl0 ⁴ /well) were differentiated to macrophages by incubation with 80 nM phorbol l2-myristate l3-acetate (PMA) for 48 h (Park et ah, 2007). The cells were then incubated with the indicated concentrations of AAT or dexamethasone for 4 h, followed by stimulation with LPS at 100 ng/ml. Supernatants were collected 24 hours post stimulation and kept frozen at -80 °C until testing. Assays for the cytokines: G-CSF, GM-CSF, IL-10, IL-l2p70, IL-lb, IL-6, IL-8, and TNFa were conducted by magnetic multiplex (MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay, Cat # HCYTOMAG-60K-08). All samples were tested in triplicate.

AAT effect in Peripheral Blood Mononuclear Cells

Peripheral Blood Mononuclear Cells (PBMCs) were prepared from a sample of human whole blood using the SepMate™-50 tubes (STC Inc. Cat# 15450). The cells were plated at 250,000 cells/well and incubated for 2 h with AAT or dexamethasone, at the indicated concentrations, followed by stimulation with LPS (10 ng/ml). Supernatants were collected 24 hours post stimulation and kept frozen at -80 °C until use. TNFa levels were determined by ELISA (Human TNF alpha Elisa, Cat# ELH-TNFA-l, RayBiotech). All samples were tested in triplicate.

Results

The effects of AAT on the Thl immunological arm

The effect of AAT treatment on secretion of the Thl -related cytokines CXCL10 and IL-12 as well as on the Thl-inhibitor cytokine IL-10, was tested using two leukocytic cell lines, the mouse macrophage cell line, RAW 264.7 and the human monocyte cell line THP-l.

RAW 264.7 cells were incubated with various concentrations of AAT (Glassia ^® ), and subsequently stimulated with LPS (5 ng/ml). Supernatants were collected 24 hours post stimulation and the effect of AAT treatment on various Thl -related cytokines was studied. As shown in figure 1 (left panel), AAT treatment inhibited the secretion of the Thl chemokine C-X-C motif chemokine 10 (CXCL10), also known as Interferon gamma- induced protein 10 (IP- 10). There was a clear dose response that led to over 60% reduction of IP-10 (from 15848 to 6050 pg/ml in samples without added AAT and with 4 mg/ml AAT, respectively). Levels of the Thl inducer cytokine IL-12 were also tested but were below the limit of detection, at least under the current conditions.

In contrast to the effect on IP- 10, treating the cells with AAT produced a dose dependent increase in IL-10 secretion. An approximately threefold increase (30.9 to 85.9 pg/ml) in IL-10 was obtained with an optimal AAT concentration of 2 mg/ml (Fig 1, right panel).

The effect of AAT on the Thl immunological arm was validated with the human monocyte cell line THP-l. THP-l cells were first differentiated to macrophages by incubation for 48 hours with 80 nM Phorbol-l2-myristate- 13 -acetate (PM A). The cells were then incubated for 4 hours with AAT at the indicated concentrations or with dexamethasone as control, followed by stimulation with LPS (100 ng/ml). Supernatants were collected 24 hours following stimulation and cytokines levels were detected by magnetic multiplex.

THP-l cells seem to be less sensitive to AAT treatment than RAW 264.7 cells, at least under these tested conditions. The results shown are for 3 and 5 mg/ml, AAT, since there was no effect seen with concentrations of 1 mg/ml or less. As shown in figure 2, treatment with AAT at 5 mg/ml reduced the secreted IL-l2p70 (the heterodimer whole IL-12 molecule), an initiator of the Thl response, by more than 80% (1.79 to 0.33 pg/ml). The inhibition was dose dependent and the results were similar to those seen with the powerful positive steroid control dexamethasone (0.38 pg/ml). The levels of CXCL-10 levels were below the level of detection under these assay conditions.

As shown above for the mouse RAW 264.7 cells, treatment of THP-l cells with 5 mg/ml AAT (Fig. 2) more than doubled the levels of secreted IL-10 (16.37 to 36.98 pg/ml).

Systemic effects of AAT on the immune system

The systemic effects of AAT on immune cell maturation and activation were studied by monitoring the secretion levels of the growth factors G-CSF and GM-CSF. These factors affect the maturation of several different types of immune cells in the bone marrow, and influence their proliferation and recruitment to sites of inflammation.

The results demonstrate a strong inhibitory effect of AAT on G-CSF secretion levels from the mouse macrophage cell line, reaching over 90% inhibition (39,995 to 3611 pg/ml) at an optimal concentration of 2 mg/ml (Figure 3). GM-CSF was not detected in the culture medium.

Further support of the systemic effect of AAT on inhibition of proliferation and maturation of precursor immune cells is shown in figure 4. AAT treatment at 5 mg/ml reduced the levels of G-CSF and GM-CSF secreted by THP-l human monocytes induced by LPS by about 60%-70% (552 to 229 and 252 to 84 pg/ml, respectively).

Anti-inflammatory effects of AAT

In addition to inhibition of the Thl arm and systemic maturation and activation of immune cells by specific growth factors, the data also support an anti-inflammatory effect of AAT, on the secretion of the major inflammatory cytokines TNFa, IL-6 and IL-Ib, as well as on secretion of the anti-inflammatory cytokine IL-lRa.

Treatment of RAW 264.7 cells with AAT inhibited the secretion of key pro- inflammatory cytokines, such as TNFa (3494 to 1954 pg/ml) and IL-6 (120 to 31.4 pg/ml) (Figure 5, upper panel). In addition, there was a more than 3 fold (53102.2 to 185241.2 pg/ml), enhanced secretion of the anti-inflammatory cytokine IL-lRa (Figure 5 lower panel). IL-lRa is an inhibitor of the IL-1 receptor that blocks the pro- inflammatory effect of IL- 1 b and IL-la and modulates a variety of interleukin- 1 related immune and inflammatory responses.

AAT treatment also induced an anti-inflammatory effect in THP-l cells mediated by the inhibition of the pro-inflammatory cytokines TNFa, IL-6, and IL- 1 b (Figure 6). Although there was no inhibition with AAT at 3 mg/ml and there was actually a slight increase in cytokines, at 5 mg/ml AAT, there was a significant reduction (about 60% and 35%, respectively) in the levels of IL-6 (2460 to 1034 pg/ml) and TNFa (21 to l3.8pg/ml). AAT treatment also gave rise to a smaller reduction (1211 to 1009 pg/ml) in the levels of the pro-inflammatory cytokine IL-1. The results demonstrate that a high concentration of AAT (5 mg/ml) inhibits the secretion of pro-inflammatory cytokines by THP-l cells.

The effect of AAT on Peripheral-Blood Mononuclear Cells

In addition to the use of cell lines to study the effect of AAT, peripheral blood mononuclear cells (PBMCs) were isolated from a sample of whole human blood and were then incubated with AAT or dexamethasone as control for 2 hours, followed by stimulation with LPS (10 ng/ml). Supernatants were collected 24 h. following stimulation, and cytokine levels were detected by ELISA.

PBMC were tested for IL-8 and TNFa. Whereas treatment with AAT at 3 mg/ml resulted in a clear reduction in the secreted levels of TNFa (Fig. 7, 67% reduction, 4223.2 to 1404 pg/ml), no effect was found on IL-8 secretion levels (data not shown). These results shed light on the importance of the role of AAT in the inhibition of neutrophil maturation, proliferation, differentiation, and function, in addition to its known direct protease-inhibitory effect on the neutrophil elastase, secreted by neutrophils during inflammation.

AAT treatment significantly inhibited GM-CSF secretion, (67%) from THP-l human monocytes induced by LPS. GM-CSF is involved in the maturation, proliferation, and activation of granulocytes, and the maturation of monocytes to macrophages in the bone marrow.

All together these results suggest that AAT has a strong effect on maturation proliferation and activation of immune cells, and the resulting cell recruitment from the bone marrow to the circulation. Example 2: Effect of local treatment of DSS-induced IBD Model in rats using AAT in a powder formulation

This study used the Dextran Sulfate Sodium (DSS)-induced IBD model in rats. AAT was administered directly to the intestine in a powder formulation, by a surgical procedure. Control animals underwent the same procedure without AAT administration.

Methods

Two groups of five rats were each given 3% Dextran Sulphate Sodium (DSS) in their drinking water ad libitum for five days, followed by a recovery period of five days with no DSS in the drinking water. One of these groups receive powdered AAT directly to the intestine, while the second group underwent a sham procedure with no drug. Two untreated rats were used as controls.

Dosing Regimen: 2 mg of AAT in a powder formulation were administered once every other day for 9 days (days 0, 2, 4, 6, 8).

Route of administration: directly to the duodenum by a surgical procedure, or rectally to the colon using gelatine capsules.

Table 1: Groups treatments and details

* detailed in Table 2.

Animal testing: Animals were observed daily and were graded for the severity of the IBD as indicated in Table 2.

Table 2: IBD tests Results

AAT prevents shortening of the large and small intestines

At the end of the study (day 10), animals were sacrificed and the length of the small and large intestines were measured. Representative small and large intestines from an animal of each treatment group and control are shown in figure 8A. The average length of the small and the large intestines for each group are depicted in figure 8B.

AAT attenuates weight loss in rats with DSS-induced IBD

Animals were weighed on a daily basis. As shown in Figure 9, animals in both DSS- induced groups lost weight in a similar manner, however upon removal of DSS from the drinking water at the end of day 5, the AAT-treated group maintained their weight, while the rats in the untreated group continued to lose weight.

AAT treated animals demonstrated a lower disease score

The stool form as shown in Figure 10, was recorded during the study. Starting from day four and onward, AAT-treated animals had fewer events of diarrhoea than the group that did not receive AAT. Due to the surgical procedure that the animals underwent every other day and the resulting bleeding, blood in the stool could not be used in the determination of the disease score. Hence, the total disease score was calculated by weight loss and diarrhoea. As shown in figure 11, the AAT-treated group demonstrated a lower disease score starting from day 4 onwards, until the end of the study.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.