Patent Application
20100216739
The present invention relates to methods and pharmaceutical compositions for promoting tissue repair, and more particularly, to methods which utilize in-tissue cross-linkable compositions for promoting repair of damaged muscle tissue such as infracted myocardial tissue.
Cross-linked polymer gel materials are widely utilized in the biomedical industry. For example, polysaccharide gels have been applied in contact lenses, blood contact materials, controlled release formulations, wound dressings, bioadhesives, membranes, superabsorbents, cell encapsulation and immunoisolation materials, and tissue engineering scaffolds (Suggs et al., J. Biomater. Sci. Polym. 9: 653-666, 1998; Aebischer et al., Transplantation 58: 1275-1277, 1994; and Atala et al. (J. Urol. 150: 745-747, 1993).
The potential use of polysaccharide gel materials for treating damaged heart tissue has been intensively researched during the past decade.
The main focus of research has been on utilizing polysaccharide gels for treating the heart tissue following acute myocardial infarction (AMI). AMI typically causes an acute loss of myocardial tissue and an abrupt increase in loading conditions which induces left ventricular (LV) remodeling. The early phase of LV remodeling involves expansion of the infarct zone, which often results in early ventricular rupture or aneurysm formation. Late remodeling encompasses the entire LV and involves time-dependent dilatation, recruitment of border zone myocardium into the scar, distortion of ventricular shape and mural hypertrophy. Consequently, it may lead to progressive deterioration in contractile function, heart failure and eventually death (Sutton and Sharpe, Circulation 101:2981-2988, 2000; Mann, D. L., Circulation 100:999-1008, 1999; and Jugdutt, B. I., Circulation 108:1395-1403, 2003).
Accordingly, cessation or reversal of progressive chamber remodeling is an important aim of heart failure therapy. Clinical attempts to minimize the devastating effects of AMI have thus far failed to effectively repair the irreversible damage inflicted to the heart tissue (Khand et al., Eur. Heart J. 22:153-164, 2001; Jessup and Brozena, S. N. Engl. J. Med. 348:2007-2018, 2003; and Redfield, M. M., N. Engl. J. Med. 347:1442-1444, 2000).
Recently, attempts to implant living cells in damaged myocardium have given hope for repairing the damaged tissue via promoting tissue regeneration (Etzion et al., J. Mol. Cell. Cardiol. 33:1321-1330, 2000; Leor et al., Expert Opin. Biol. Ther. 3:1023-39, 2003; and Beltrami et al., Cell; 114:763-776, 2003). This approach has advanced considerably with the development of 3-D biomaterial scaffolds aimed at supporting implantation of donor cells (e.g., cardiac cells or stem cells) in the myocardium. Lately, 3-D biomaterial scaffolds made of polysaccharide gel were successfully implanted onto damaged myocardium with promising results (Leor et al., Circulation 102:56-61, 2000). However, clinical use of such cell seeded 3-D biomaterial scaffolds is limited due to scarcity of suitable donor cells and the high risk involved in major surgery.
While reducing the present invention to practice the present inventor has surprisingly and unexpectedly uncovered that an injectable non cross-linked polymer solution can effectively promote the repair of a damaged heart muscle tissue in rats.
According to one aspect of the present invention there is provided a method of treating a muscle tissue. The method is effected by providing the muscle tissue with an effective amount of a sterile polymer solution being essentially devoid of free multivalent cations and capable of self-gelling following deposition in or around the muscle tissue.
According to another aspect of the present invention there is provided a method of treating a heart infarction. The method is effected by providing a subject in need thereof with an effective amount of a sterile polymer solution being essentially devoid of free multivalent cations and capable of self-gelling following deposition in or around infarcted myocardium tissue.
According to yet another aspect of the present invention there is provided a method of treating a heart condition. The method is effected by providing a subject in need thereof with an effective amount of a sterile polymer solution being essentially devoid of free multivalent cations and capable of self-gelling following deposition in or around a damaged myocardium tissue, thereby treating the heart condition.
According to still another aspect of the present invention there is provided a method of treating an ischemic muscle tissue. The method is effected by providing a subject in need thereof with an effective amount of a sterile polymer solution being essentially devoid of free multivalent cations and capable of self-gelling following deposition in or around the ischemic muscle tissue, thereby treating the ischemic muscle tissue.
According to yet an additional aspect of the present invention there is provided an article of manufacturing which includes a sterile polymer solution being essentially devoid of free multivalent cations and capable of self-gelling following deposition in or around an ischemic muscle tissue and a packaging material identifying the polymer solution for use in treatment of ischemic muscle tissue.
According to further features in preferred embodiments of the invention described below, the muscle tissue is a myocardium tissue.
According to still further features in the described preferred embodiments, the muscle tissue is selected having a concentration of the multivalent cations sufficient to cause the self-gelling.
According to still further features in the described preferred embodiments, the polymer solution is a polysaccharide solution.
According to still further features in the described preferred embodiments, the polysaccharide solution is an alginate solution.
According to still further features in the described preferred embodiments, the alginate solution is a sodium alginate solution.
According to still further features in the described preferred embodiments, the alginate having an average molecular weight ranging from 10 to 300 kDa.
According to still further features in the described preferred embodiments, the alginate concentration ranges from 0.1 to 10% (w/v).
According to still further features in the described preferred embodiments, the alginate monomer ratio between α-L-guluronic acid and β-D-mannuronic acid ranges between 1:1 and 3:1.
According to still further features in the described preferred embodiments, the step of providing the polymer solution to the muscle tissue is effected via injection or catheterization.
According to still further features in the described preferred embodiments, the catheterization is intra-arterial catheterization.
According to still further features in the described preferred embodiments the effective amount ranges between about 0.5 and 5 ml.
According to still further features in the described preferred embodiments, the polymer solution further comprises at least one therapeutic agent.
According to still further features in the described preferred embodiments, the at least one therapeutic agent is selected from the group consisting of a growth factor, a hormone, an anti ischemic drug, an anti-inflammatory drug, an anti-apoptotic drug and an antibiotic drug.
According to still further features in the described preferred embodiments, the heart condition is congestive heart failure.
According to still further features in the described preferred embodiments, the ischemic muscle tissue is an ischemic myocardium tissue.
According to still further features in the described preferred embodiments, the ischemic muscle tissue is an ischemic striated muscle tissue.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a novel method of treating a damaged muscle tissue by safely administering to the muscle tissue a therapeutically beneficial polymer solution capable of forming a flexible gel following deposition in or around the tissue, thereby providing substantial therapeutic benefits to the damaged muscle tissue.
The present invention utilizes novel pharmaceutical compositions to promote repair of damaged tissue such as infracted myocardial tissue by delivering to the damaged tissue a polymer solution which is sterile and essentially devoid of free multivalent cations yet capable of self-gelling following deposition in or around the tissue.
The principles and operation of the present invention may be better understood with reference to the accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
U.S. patent application Ser. No. 11/229,119 teaches a cross-linked polymer solution capable of maintaining a liquid state in storage and assuming a gel state following deposition within a body tissue. The cross-linked polymer solution can be administered into a body tissue via injection or catheterization. This application provides a partially cross-linked alginate solution and thus requires careful mixing for preparation.
The present inventors unexpectedly uncovered that an alginate solution devoid of exogenous cations is capable of forming a gel scaffold following administration into muscle tissue. As is illustrated in the Examples section hereinbelow administration of a sterile sodium alginate solution which is essentially devoid of free multivalent cations into myocardium tissue following AMI, promoted cellular repair of such tissue and thus effectively reversed the damage caused by the infarction.
Thus, according to one aspect of the present invention, there is provided a method of treating a muscle tissue. The method is effected by providing the muscle tissue with an effective amount of a sterile polymer solution which is essentially devoid of free multivalent cations and yet is capable of self-gelling without addition of exogenous multivalent cations following deposition in or around the muscle tissue. The in situ transition of the polymer solution into gel may be effected via cross-linking with extracellular multivalent cations being present in the muscle tissue.
It should be pointed out that in contrast to the cross-linked polymer solution taught by U.S. patent application Ser. No. 11/229,119, the polymer solution of the present intention is not partially or wholly cross-linked prior to being deposited in the target tissue
The term “treating” used herein encompasses the complete range of therapeutically positive effects of administrating the polymer solution of the present invention to a muscle tissue, including improving the tissue function, providing mechanical support and promoting tissue healing and repair processes. The term treating further includes reduction of, alleviation of, and relief of diseases or disorders associated with an ischemic muscle tissue. In addition, the term treating includes prevention or postponement of development of diseases or disorders associated with a damaged tissue.
The phrase “muscle tissue” used herein encompasses any mammalian muscle tissue, preferably a human muscle tissue. The phrase further encompasses a smooth muscle tissue, a striated muscle tissue and a cardiac muscle tissue. Striated (skeletal) muscle tissue is under voluntary control. The muscle fibers are syncytial and contain myofibrils, tandem arrays of sarcomeres. Smooth muscle tissue is made up from long tapering cells, generally involuntary and differs from striated muscle in the much higher actin/myosin ratio, the absence of conspicuous sarcomeres and the ability to contract to a much smaller fraction of its resting length. Smooth muscle cells are found particularly in blood vessel walls, surrounding the intestine (especially the gizzard in birds) and in the uterus. Cardiac muscle tissue is a striated but involuntary tissue responsible for the pumping activity of the vertebrate heart. The individual cardiac muscle cells are not fused together into multinucleate structures as they are in striated muscle tissue. As used herein, the phrases “cardiac muscle tissue” and “myocardium tissue” are interchangeable.
Without being bound to theory, gelling of the polymer solution may result from cross linking of the polymer via multivalent cations which are present in or around the muscle tissue. Accordingly, the muscle tissue of the present invention can be any muscle tissue having a concentration of multivalent cations which is sufficient to cause gellation of the polymer solution.
The phrase “polymer solution” used herein refers to a liquid in which a polymer is uniformly dispersed in a solvent such as water.
A suitable polymer, according to the teaching of the present invention, can be any biocompatible, non-immunogenic and, preferably, bio-erodible polymer which can be cross-linked via multivalent cations to form a hydrogel. The polymer can be in a form of a free-base, an acid, a basic, an anion salt, or a monovalent cation salt.
The term “sterile” used herein refers to a solution substantially free of living cells. Sterility of the polymer solution of the present invention can be ascertained using, for example, the following Microbiological Tests in USP 24: Microbial Limit Tests <61>, Sterility Tests <71> and Sterilization and Sterility Assurance of Compendial Articles <12211>.
The phrase “essentially devoid of free multivalent cations” used herein refers to a polymer solution having a concentration of free multivalent ions being less than 0.001% (w/v). It will be appreciated that in the case of sodium alginate trace amounts of calcium and other multivalent cations may be present in commercial preparations (often up to 1.5% on dry weight basis), since such preparations are derived from algae extracts which contain ions. As such, although trace amounts of ions may exist in the polymer compositions utilized by the present invention, such amounts are so low that the present compositions can be considered essentially free of multivalent ions.
The term “gel” used herein refers to a semisolid colloidal suspension of a solid in a liquid. The term “hydrogel” used herein refers to a gel which contains water as the liquid.
The polymer of the present invention can be, but not limited to, a polysaccharide such as alginate or chitosan, gellan gum, collagen, carageenan, hyaluronic acid, poly lactic acid, a polyphosphazine and a polyacrylate. Preferably, the polymer of the present invention is an alginate.
The term “alginate” used herein refers to a polyanionic polysaccharide copolymer derived from sea algae (e.g., Laminaria hyperborea, L. digitata, Eclonia maxima, Macrocystis pyrifera, Lessonia nigrescens, Ascophyllum codosum, L. japonica, Durvillaea antarctica, and D. potatorum) and which includes β-D-mannuronic (M) and α-L-guluronic acid (G) residues in varying proportions.
Preferably, the alginate of the present invention has a monomer ratio between α-L-guluronic acid and β-D-mannuronic ranging between 1:1 to 3:1 and a molecular weight ranging between 10 to 300 kDa, more preferably between 25 to 50 kDa.
The alginate of the present invention is preferably a soluble alginate salt such as, but not limited to, sodium alginate, potassium alginate, lithium alginate, rubidium alginate and cesium salts of alginic acid, as well as the ammonium salt, and the soluble alginates of an organic base such as mono-, di-, or tri-ethanolamine alginates, aniline alginates, and the like. More preferably, the polymer of the present invention is sodium alginate. Pharmaceutical grade sodium alginate which comply with all the quality and safety requirements of the European and United States of America (USA) pharmacological regulatory authorities, are readily available from several commercial manufacturers such as, for example, Novamatrix FMC Biopolymers (Drammen, Norway). The final concentration of sodium alginate in the polymer solution of the present invention preferably ranges between 0.1 to 10% (w/v).
Once the polymer of the invention is completely dissolved the solution is sterilized using standard sterilization methods well known in the art such as, but not limited to, autoclaving (e.g., at 120° C. for 30 min), gamma-irradiation or filter sterilization. Preferably the sterilization is effected via filter sterilization using a 0.2 μm membrane.
Optionally, the polymer solution of the invention further includes at least one therapeutic agent. Suitable therapeutic agents which may be included in the polymer solution of the present invention can be, for example, growth factors (e.g., basic fibroblast growth factor; bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), members of the TGF-family, bone morphogenic proteins (BMP), platelet-derived growth factors, angiopoietins, and other factors such as myogenic factors, transcription factors, cytokines, and homeobox gene products, polynucleotides, polypeptides, hormones, anti-inflammatory drugs, anti ischemic drugs, anti-apoptotic drugs or antibiotic drugs.
Advantageously, the therapeutic agent or agents can be chemically linked to the polymer of the invention. Such linkage can be effected via any known chemical bonding approach, preferably a covalent bond. A suitable covalent bond can be, for example, an ester bond (e.g., a carboxylic ester bond, an oxyalkyl carboxylic ester bond, an amide bond, or a thioester bond), a glycosidic bond, a carbonate bond, a carbamate bond, a thiocarbamate bond, a urea bond or a thiourea bond.
Preferably the polymer is alginate and the bond to the therapeutic agent is effected on a mannuronate monomer location such the alginate polymer retains its ability to cross-link via a glucoronate monomer location.
The polymer solution may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the therapeutic compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for medical devices or drugs, or of an approved product insert. Therapeutic compositions may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.
Administration of the polymer solution of the present invention to a subject in need thereof can be effected via injection directly into the target muscle tissue or via parenteral administration. For treating AMI, the polymer solution can be administered intra-arterially, preferably intra-coronarily, via a suitable catheter such as a balloon catheter (see, for example, in Knight et al., Circulation 95:2075-2081, 1997).
The polymer solution can be administered on the same day of the AMI during or after initiating reperfusion, preferably about one day after the AMI, preferably about one day after the AMI, preferably about two days after the AMI, preferably about three days after the AMI, preferably about four days after the AMI, preferably about five days after the AMI, preferably about six days after the AMI, preferably about seven days after the AMI.
The phrase “effective amount” used herein refers to an amount effective to provide a significant therapeutic benefit. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. An effective amount ranges between 0.1 and 10 ml (dwt between 1 and 1000 mg), preferably between 1 and 5 ml (dwt between 10 and 500 mg) of a concentration ranging between 0.1 and 10% (w/v), preferably between 0.2 and 5% (w/v), preferably between 0.3 and 2% (w/v), preferably between 0.5 and 1.5% (w/v), preferably 1% (w/v).
The polymer solution of the present invention can be used to treat a heart infarction by providing a subject in need thereof with an effective amount of the polymer solution in or around the infarcted myocardium.
In addition, the polymer solution of the present invention can be used to treat a heart condition such as congestive heart failure or mitral valve rigurgitation, by providing a subject in need thereof with an effective amount of the polymer solution in or around a damaged myocardium tissue, thereby treating the heart condition.
In addition, the polymer solution of the present invention can be used to treat diseases and conditions associated with an ischemic muscle tissue, such as an ischemic striated muscle tissue, by providing the ischemic muscle tissue with an effective amount of the polymer solution.
Hence, the present invention provides a novel method of treating a muscle tissue by providing the muscle tissue with a sterile polymer solution being essentially devoid of free multivalent cations and capable of self-gelling in situ. The treatment provides substantial therapeutic benefits to the muscle tissue safely and effectively.
As used herein, the term “about” denotes ±10%.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions; illustrate the invention in a non limiting fashion.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
Materials and Methods
Test Solutions:
An aqueous solution of 1% (w/v) sodium alginate (UP-VLVG, Mw=41 kDa; NovaMatrix FMC Biopolymers, Drammen, Norway) was filter-sterilized using a 0.2 μm membrane (Millipore) prior to use. A cross-linked alginate solution (BL-1040) was prepared by thoroughly mixing equal parts of filter-sterilized aqueous solutions of 2% (w/v) sodium alginate and of 0.6% (w/v) calcium D-gluconate (USP/NF Grade Dr. Paul Lauman lot. 623784).
Induction of Acute Myocardial Infarction (AMI)
The rat model of AMI was essentially as described by Leor et al. (Circulation 102:III56-61, 2000), Battler et al. (J Mol Cell Cardiol. 33: 1321-133, 2001) and Leor et al. (Circulation 114: I9.4-100, 2006). Briefly, male Sprague-Dawley rats (250-350 g) were anesthetized with a combination of 40 mg/kg ketamine and 10 mg/kg xylazine, intubated and mechanically ventilated. To induce AMI, animal chests were opened by left thoracotomy, their pericardia were removed and the proximal left coronary arteries were permanently occluded by intramural stitches.
Administration of Test Solutions
Seven days following AMI, animals were randomized, anesthetized and their chests were opened under sterile conditions. The infarcted myocardial area was visually recognized based on the appearance of surface scar and wall motion akinesis. A single injection (100-150 μL volume) of BL-1040 (positive control), saline (negative control) or 1% sodium alginate was administered into the infarcted myocardial area using a 29-gauge needle. Following injection the surgical incision was sutured closed.
Ecocardiography
Transthoracic echocardiography was performed within 24 hours (baseline echocardiogram) and 30 days following AMI. Echocardiograms were performed using a commercially available echocardiography system (Sonos 5500, Philips, Andover, Mass.) equipped with 12-MHz phased-array transducer, as described by Leor et al. (Circulation 114: 194-100, 2006). Diastolic function was evaluated using pulse-wave Doppler interrogation of mitral inflow performed in a modified four-chamber view. All measurements were averaged for three consecutive cardiac cycles and were performed blinded to the treatment groups.
Statistical Analysis
Echocardiographic measurements were analyzed using paired t-tests. All tests applied were two-tailed, and p value of 5% or less was considered statistically significant.
Results
As shown in Table 1 hereinbelow, administering 1% sodium alginate solution to rat infracted myocardium resulted in:
4. A significant reduction in the LV diastolic diameter changes as compared with the saline control (26.0% difference between the means, p≦0.05).
5. A significant reduction in the LV mass changes as compared with the saline control (51.5% difference between the means, p≦0.01).
1 % change = [((echo day 30) − (echo day 1))/(echo day 1)] × 100
2 P value = the estimated probability, using paired t-test, that the difference between the mean values (treatment vs. saline control) can be ascribed to chance alone.
3 LVEDD = LV end diastolic dimension
4 LVESD = LV end systolic dimension
5 LV EDA = LV end diastolic area
6 LV ESA = LV end systolic area
7 LV FS = LV fractional shortening − [(LVIDd − LVIDs)/LVIDd] × 100
8 FAC = Fractional area change
Hence, the results clearly illustrate that sodium alginate solution administered to animal myocardium tissue following AMI can effectively and substantially reduce damage to the infarcted tissue and effectively improve the heart function.
Material and Methods
Test Solutions:
Test solutions are: saline (negative control), a cross-linked alginate solution (BL-1040; positive control) and sodium alginate solution (1%). The sodium alginate and BL-1040 solutions are prepared as described in Example 1 hereinabove.
Induction of Acute Myocardial Infarction (AMI)
Two years old (20-25 kg) dogs are anesthetized and intubated and respired with room air. Animals are sedated with intravenous oxymorphone hydrochloride (0.22 mg/kg) and diazepam (0.17 mg/kg) and a plane of anesthesia is maintained with 1-2% isofluorane. The procedures are performed with the chest closed and under sterile conditions. To produce the coronary occlusion, a 2.7 French PTCA balloon catheter is advanced introduced over a 0.014 inch wire through a 4JL guiding catheter through a femoral arteriotomy. A 4.0 mm diameter, 15 mm balloon is used. The guiding catheter and positioned in the Left Main coronary artery and the wire, advanced under fluoroscopic guidance, is positioned in the distal Circumflex coronary artery. The balloon catheter is advanced into the coronary artery over the wire and positioned proximal to the first marginal branch. In all instances, the animals are anti-coagulated with 750-1000 USP units of intravenous sodium heparin.
Intra-Coronary Administration
Once in place, the PTCA balloon is inflated to occlude the coronary artery. Complete occlusion of the coronary artery is confirmed by ST-segment elevation on the electrocardiogram and near absence of LV posterior wall motion assessed by transthoracic 2-dimensional echocardiography. In all instances, the coronary occlusion is maintained for 3 to 5 hours. At the end of the occlusion, the balloon is deflated to allow for coronary reperfusion.
The test solution (2 ml volume) is injected into the animals' Circumflex coronary artery. To simulate the likely clinical setting for use of the test solution, the coronary injection of the test solution is made either on the same day of the AMI (about one hour following initiating reperfusion), or 2-5 days following the AMI. Dogs are randomly assigned to receive any of the test solution. All animals are followed for 4 months following the AMI. Angiographic and echocardiographic measurements are made at baseline, prior to inducing the AMI, just prior to administering the test solutions and at two and four months thereafter.
End Points
1. Prevention or attenuation of progressive LV dilation based on ventriculographic measurements of LV end-systolic and end-diastolic volume.
2. Prevention of progressive deterioration of LV systolic function based on ventriculographic measurement of LV ejection fraction (EF) and echocardiographic measurement of LV fractional area of shortening (FAS).
3. Prevention or attenuation of infarct expansion based on echocardiographic measurements of systolic thickening of the infarcted LV wall.
Angiographic and Echocardiographic Measurements
All angiographic and echocardiographic measurements are made during cardiac catheterization under general anesthesia and sterile conditions. Left ventriculograms are obtained with the dog placed on its right side and digitally recorded during the injection of 20 ml of contrast material (RENO-M-60 Squibb, Princeton, N.J.). Correction for image magnification is made with a radiopaque calibrated grid placed at the level of the LV. LV end-systolic (ESV) and end-diastolic (EDV) volumes are calculated from LV silhouettes using the area-length method (Dodge et al., Am J. Cardiol. 1966; 18:10-24, 1966). Left ventricular EF is calculated as the ratio of the difference of EDV and ESV to EDV times 100 (Sabbah et al., Circulation 89:2852-2859, 1994).
All 2-dimensional echocardiographic measurements are made with the dog placed in the right lateral decubitus position. Echocardiographic studies are performed using a General Electric VIVID 7 Dimension ultrasound system with a 3.5 MHz transducer and recorded on a Mitsubishi MD3000 VHS recorder for off-line analysis. A LV short-axis view at mid-papillary muscle level is recorded and used to calculate the percent fractional shortening (FAS; Kono et al. J Am Coll Cardiol 1992; 19:1101-1105, 1992), defined as the difference between the end-diastolic area and the end-systolic area divided by the end-diastolic area times 100. Percent systolic wall thickening of the LV anterior wall, posterior wall and inter-ventricular septum are also measured using the short axis view and each calculated as the ratio of wall thickness at end-diastole to wall thickness at end-systole divided by wall thickness at end-diastole times 100. Extrasystolic and post-extrasystolic beats are excluded from all analysis.
Statistical Analysis
Within group angiographic and echocardiographic data are analyzed using repeated measures analysis of variance (ANOVA) with alpha set at 0.05. If the overall ANOVA is significant, then pairwise comparisons between pre-treatment (PRE) and 2 and 4 months are performed using the Student-Newman-Keuls test. For this test a p-value lower than 0.05 is considered significant. To assess treatment effect, the change (Δ) in each measure from PRE to 2 months and PRE to 4 months post-treatment is calculated for each of the two study arms. To determine whether significant differences in Δ were present between the control groups and the sodium alginate treated group, a t-statistic for two means is used with p<0.05 considered significant.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IL2008/000348 | 3/13/2008 | WO | 00 | 3/5/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60906521 | Mar 2007 | US |
