Prior to setting forth the invention, it may be helpful to provide an understanding of certain terms that will be used hereinafter.
Analogue(s): As used herein “analogue(s)” include compounds having structural similarity to another compound. For example, the anti-viral compound acyclovir is a nucleoside analogue and is structurally similar to the nucleoside guanosine which is derived from the base guanine. Thus acyclovir mimics guanosine (is “analogous with” biologically) and interferes with DNA synthesis by replacing (competing with) guanosine residues in the viral nucleic acid and prevents translation/transcription. Thus compounds having structural similarity to another (a parent compound) that mimic the biological or chemical activity of the parent compound are analogues. There are no minimum or maximum numbers of elemental or functional group substitutions required to qualify as an analogue as used herein providing the analogue is capable of mimicking, in some relevant fashion, either identically, complementary or competitively, with the biological or chemical properties of the parent compound. Analogues can be, and often are, derivatives of the parent compound (see “derivative” infra). Analogues of the compounds disclosed herein may have equal, less or greater activity than their parent compounds.
Derivative: As used herein a “derivative” is a compound made from (derived from), either naturally or synthetically, a parent compound. A derivative may be an analogue (see “analogue” supra) and thus may possess similar chemical or biological activity. However, as used herein, a derivative does not necessarily have to mimic the activity of the parent compound. There are no minimum or maximum numbers of elemental or functional group substitutions required to qualify as a derivative. As an example, the antiviral compound ganclovir is a derivative of acyclovir. Ganclovir has a different spectrum of anti-viral activity from that of acyclovir as well as different toxicological properties. Derivatives of the compounds disclosed herein may have equal, less, greater or no similar activity to their parent compounds.
Elsamitrucin: As used herein, the term “elsamitrucin” refers to an anti-neoplastic composition having a molecular weight of approximately 825.83 Da and is known chemically as benzo(h)(1)benzopyrano(5,4,3-cde)(1)ebnzopyran-5,12-dione,10((2-O-(2-amino-2,6-dideoxy-3-O-methyl-alpha-D-galactopyranosyl)-6-deoxy-3-C-methyl-beta-D-galactopyranosyl)oxy)-6-hydroxy-1-methyl, and has the structure generally depicted in Formula I. Elsamitrucin is also known as 10-O-elsaminosylelsarosylchartarin, BBM 2478A, BMY-28090, SPI-28090, BRN 5214813, elsamicin A, elsamitrucina, and elsamitrucine. See U.S. Pat. Nos. 4,518,589 and 4,572,895 for methods of isolating and characterizing elsamitrucin from natural sources. See also Konishi M, Sugawara K, Kofu F, Nishiyama Y, Tomita K, Miyaki T, Kawaguchi H. 1986. Elsamicins, new antitumor antibiotics related to chartreusin 1. Production, isolation, characterization and antitumor activity. J. Antibiot. (Tokyo) June;39(6):784-91.
Pharmaceutical Formulation: As used herein the term pharmaceutical formulation refers to a pharmaceutically acceptable preparation comprising one or more of the elsamitrucin salts of the present invention and at least one pharmaceutically acceptable carrier such as, but not limited to water for injection, saline or phosphate buffered saline. Moreover, the pharmaceutical formulations of the present invention may also include stabilizers, preservatives, buffers or additional therapeutic agents. The pharmaceutical formulations of the present invention may be administered by any means known to those skilled in the art and are ideally suited for intravenous administration or infection into the skin, muscle or other tissues of the body. The pharmaceutical formulation may be intended for oral administration.
Salt: As used herein a “salt” or “salts” include any compounds that result from replacement of part or all of the acid hydrogen of an acid by a metal or a group acting like a metal: an ionic crystalline compound. In this case, the salt is a product of a free base and an organic acid that can exist as a stable solid and does not include pseudo salts, or salts made in situ, which only exist in the solution.
Suitable salt form(s): As used herein, the term “suitable salt form(s)” means an elsamitrucin salt prepared in stable solid state either as amorphous or crystalline form.
Solid or solid salt: As used herein the term solid or solid salt refers to an elsamitrucin salt existing in a solid state and having less than 30% residual moisture, preferably less than 10% residual moisture and more preferably less than 5% residual moisture. As used herein “moisture” refers to water or an organic solvent. The term “solid” is also used herein to differentiate the elsamitrucin salts of the present invention from salts formed in situ and exist primarily in the aqueous phase.
Stable: As used herein “stable” refers to an elsamitrucin salt or a parenteral elsamitrucin salt-containing formulation (made by method other than in situ salt formation) wherein the elsamitrucin salt retains NMR data showing a near perfect 1:1 salt ratio (thus indicating no decomposition in the solid state) during drying at elevated temperatures at 75° C. for nine hours or more preferably 98° C. overnight. Moreover, stable as used herein refers to elsamitrucin salt-contained in a parenteral formulation that retains at least 90% of its anti-neoplastic activity as determined by in vitro growth inhibition testing (see Example 4) for at least 24 months in the solid form and for 18 months in the liquid form at a suitable storage temperature.
The present invention provided water soluble, solid elsamitrucin salts useful for preparing stable pharmaceutical formulations intended for use in anti-neoplastic therapeutic regimens. Additionally, methods for preparing the stable, elsamitrucin salts have also been provided.
In one embodiment of the present invention the stable, elsamitrucin salts of the present invention are selected form the group consisting of, but not limited to, lactate, fumarate, maleate, succinate, tartrate, tosylate, methanesulfonate, benzoate, salicylate, hydrochloride, sulfate, phosphate, and others. The elsamitrucin salts of the present invention are stable solids and may be either crystalline or amorphous.
Elsamitrucin and structurally related antibiotics bind to GC-rich tracts in DNA, with a clear preference for B-DNA over Z-DNA. They inhibit RNA synthesis and cause single-strand scission of DNA via the formation of free radicals. Elsamitrucin can also be regarded as the most potent inhibitor of topoisomerase II reported so far and can inhibit the formation of several DNA-protein complexes. Elsamitrucin binds to the P1 and P2 promoter regions of the c-myc oncogene inhibits the binding of the Sp1 transcription factor, thus inhibiting transcription.
Elsamitrucin has shown activity in patients with relapsed or refractory non-Hodgkin's lymphoma and in vivo activity against a wide range of murine neoplasmas including leukemia P388, leukemia L1210, and melanoma B16 and M5076, as well as against MX1 and HCT116 xenografts (see for example Raber M N, Newman R A, Newman B M, Gaver R C, Schacter LP1992 Phase I trial and clinical pharmacology of elsamitrucin. Cancer Res. Mar 15;52(6):1406-10).
Additionally, experimental treatment of refractory/relapsed non-Hodgkin's lymphoma has demonstrated that elsamtrucin-associated toxicity is relatively mild and consisted mainly of asthenia, nausea and vomiting and did not include myelosuppression. The activity of elsamitrucin and its lack of myelosuppression suggest utility in this disease especially when combined with other proven agents. (see Allen SL, Schacter L P, Lichtman S M, Bukowski R, Fusco D, Hensley M, O'Dwyer P, Mittelman A, Rosenbloom B, Huybensz S. 1996. Phase II study of elsamitrucin (BMY-28090) for the treatment of patients with refractory/relapsed non-Hodgkin's lymphoma. Invest. New Drugs. 14(2):213-7.
The in vitro activity of elsamitrucin was also investigated as compared with that of doxorubicin (DX) on two sensitive breast cancer cell lines: one estrogen receptor-positive (ER+, MCF7) and one estrogen receptor-negative (ER−, MDA-MB-231) line, and on a DX-resistant subline (MCF7DX). The activity of the two drugs was also investigated on 19 clinical breast cancer specimens from untreated patients. The drugs were tested at pharamcologically relevant concentrations, as calculated from the area under the curve for a 3 h exposure to the lethal dose producing 10% mortality (LD10) in mice, and at 10- and 100-fold concentrations. In DX-sensitive lines, a greater inhibition of RNA and DNA precursor incorporation, as well as of cell proliferation, was caused by elsamitrucin than by DX. Moreover, the antiproliferative effect was 10-fold higher in the ER+ MCF7 than in the ER− MDA-MB-231 cell line (IC50: 0.25 versus 0.21 micrograms/ml). Elsamitrucin was cross-resistant to DX in the MCF7DX subline. In clinical specimens, effects on DNA precursor incorporation were more often observed for elsamitrucin than for DX at the same drug concentrations. The in vitro sensitivity to elsamitrucin was more pronounced for ER+ than for ER− tumors: minimal inhibiting concentrations of the drug were 0.1 and 3.5 micrograms/ml, respectively, in the two groups. These in vitro results would indicate a promising role for elsamitrucin in clinical treatment, mainly of ER+ breast cancer patients (see Silvestrini R, Sanfilippo O, Zaffaroni N, De Marco C, Catania S. 1992. Activity of a chartreusin analog, elsamitrucin, on breast cancer cells. Anticancer Drugs. December; 3(6):677-81).
U.S. Pat. No. 5,508,268 issued Apr. 16, 1996 to Nassar et al. assigned to Bristol-Myers Squibb (hereinafter the '268 patent) discloses parenteral formulations comprising elsamitrucin base, an organic acid, a stabilizer and a buffer. The elsamitrucin compositions disclosed therein were prepared using various organic acids including hydrochloric, L(+)-lactic, L-tartaric, D-glucuronic, methane-sulfonic, adipic and succinic with the succinic acid being preferred. The elsamitrucin compositions are prepared according to the teachings of an Example occurring at column 4 lines 5-30. In this example only the succinate salt is described. Specifically, the '268 patent, in accordance with the disclosure therein, the elsamitrucin salt is formed in situ using an organic acid in combination with at least one reducing agent (preservative) and the pH adjusted to approximately 4. The resulting solution was filtered and retained in the liquid state for stability testing. In other embodiments disclosed in the '268 patent the organic acid, elsamitrucin base, reducing agent and other suitable pharmaceutical excipients such as, but not limited to sugars, are admixed in solution and the resulting composition is lyophilized.
However, the '268 patent does not disclose, discuss or teach stable solid elsamitrucin salts. In sharp contrast to the teachings of the '268 patent the present inventors have discovered methods that provide stable solid elsamitrucin salts made using elsamitrucin base and selected organic acids. The resulting compositions made in accordance with the teachings of the present invention are solid, dry or partially dried elsamitrucin salt powders, as opposed to lyophilizates described in the '268 patent. Thus, the elsamitrucin salt compositions of the present invention are true salts in solid state, not in situ solutions containing a solubilized base and organic acid admixture.
The present invention offers numerous advantages over in situ formed admixtures as described in the '268 patent. First, the elsamitrucin salts made in accordance with the teachings of the present invention can be carefully analyzed for impurities and refined as needed to meet exceedingly high governmental regulations. Moreover, the true salts of the present invention can be precisely weighed and dissolved in suitable pharmaceutical carriers such as Water for Injection. The selected salts themselves are extremely stable when stored in the solid state and have extended shelf lives as do their corresponding solubilized solutions. Thus, parenteral solutions can be prepared using the elsamitrucin salts of the present invention and stored for extended periods of time.
The following Examples are provided as illustrative embodiments of the present invention. It should be understood that the stable dried, or nearly dried elsamitrucin salts of the present invention are not limited by the following examples. The teachings of the Examples that follow can be used by pharmaceutical chemists of ordinary skill as guidance in making other, variations that result in the same compositions as disclosed here.
Small batches of elsamitrucin salts were prepared prior to optimization and scale up. Eight counter ions based on organic acids were selected, these included lactic acid, maleic acid, succinic acid, L-tartaric acid, p-toluenesulfonic (also referred to herein as p-TSA or tosylate), benzoic acid, salicylic acid, and sulfuric acids. Three solvents were selected based on previous screen methods known to those skilled in the art of pharmaceutical chemistry, the selected solvents included dioxane, dimethylformamide (DMF), and acetic acid (AcOH). An additional combination of p-TSA/MeOH was included for a total of twenty-five variations reactions.
To each reaction vial 3.0×10−5 mol of elsamitrucin base was added. The elsamitrucin base was dissolved in 0.25 mL of DMF or AcOH at 55° C., 1.5 mL of dioxane at 80° C., or 12 mL of MeOH at 70° C. and stirred for five minutes to ensure dissolution. Each vial was then charged with 245-270 μL of a 0.126 M dioxane solution of one of the organic acids listed above (see Table 1) corresponding to 1.05 equivalent of each of the eight acids (tartaric acid was dispensed in a 1:1 mixture of methanol/water due to its insolubility in dioxane).
The initial temperature was held for ten minutes and then ramped down to room temperature at a rate of 20° C./hour for DMF and AcOH, 30° C./hour for dioxane and 25° C./hour for MEOH. Solids were formed in the vials with dioxane/L-tartaric acid, dioxane/p-TSA, dioxane/sulfuric acid, and AcOH/sulfuric acid. Solids were collected by filtration and dried in vacuo at 50° C. and 30 in. Hg. Vials were solids did not form were concentrated to dryness with a stream of nitrogen and dried in vacuo at 50° C. and 30 in. Hg. Methanol was removed under vacuum and the resultant residue dried under high vacuum at room temperature. All samples were analyzed by x-ray diffraction (XRPD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) to determine the crystallinity of the salts. Crystalline solids were obtained from dioxane/sulfuric acid and from AcOH/sulfuric acid; semi-crystalline solids were obtained from dioxane/L-tartaric acid, dioxane/p-TSA, DMF/lactic acid, DMF/maleic acid, DMF/L-tartaric acid, DMF/benzoic acid, DMF/sulfuric acid, AcOH/lactic acid, AcOH/p-TSA, and AcOH/benzoic acid. All other solids were found to be amorphous by XRPD.
Next three elsamitrucin salts made in accordance with the teaching of Example 1 were selected for scale-up development. The salts selected were elsamitrucin tartrate, elsamitrucin sulfate and elsamitrucin tosylate. These were selected because each provided crystalline or semi-crystalline solids that precipitated during the cooling process, which can allow better isolation and purification (if necessary) of the salt and thus lends them more suitable for larger scale manufacturing techniques. However, their selection for purposed of Example 2 should not be considered a limitation.
L-tartaric acid, sulfuric acid and p-TSA were dissolved in dioxane. Suitable reaction containers were each charged with 1.7×10−4 mol of elsamitrucin base which was dissolved in 7.5 mL of dioxane at 80° C., and stirred for five minutes to ensure dissolution. Each vial was then charged with 350-380 μL of a 0.5 M solution of the organic acid in dioxane corresponding to approximately 1.05 equivalent each of the three acids (Table 2).
The initial temperature was held for ten minutes and then ramped down to room temperature at a rate of 30° C./hour for dioxane. Solids were formed upon addition of acid to the vials with dioxane/L-tartaric acid and dioxane/sulfuric acid and for dioxane/p-TSA precipitation occurred during the cooling process. After filtration the solids were dried in vacuo at 50° C. and 30 inch Hg. Samples were analyzed by XRPD, DSC, and TGA to determine the crystallinity (Table 3), and other physical properties.
As Table 3 shows, all solids in Example 2 were semi-crystalline, contained up to about 5% of the residual solvent and were pasty in constancy due to the high amount of solvent that was retained in the solids due to a rapid precipitation.
In another embodiment the elsamitrucin salts of the present invention were prepared using a slower precipitation method than described above. Reaction containers were charged with 7.6×10−5 mol of elsamitrucin base and 5 mL of dioxane at 80° C. After the mixture was stirred for five minutes to ensure dissolution of the base, 400 μL of a 0.2 M aqueous solution of tartaric acid corresponding to 1.05 equivalents was added to the dissolved elsamitrucin base. The temperature was held at 80° C. for ten minutes and then the vials were cooled to room temperature at a rate of 30° C./hour. During the cooling phase precipitation occurred. The solids were collected by filtration and dried in vacuo at 50° C. and 30 inches Hg. Samples were analyzed by XRPD, DSC, and TGA to determine physical properties [Table 3, OVL-A-55(1) and OVL-A-55(2)]. The first sample [dioxane/sulfuric acid, OVL-A-55(1)] was crystalline by XRPD, but it contained 3.6% residual solvent according to TGA analysis and three endothermic peaks on the DSC curve. The second sample [dioxane/L-tartaric acid, OVL-A-55(2)] was semi-crystalline.
Next elsamitrucin salts were prepared in an aqueous environment as follows. Reaction vials were charged with 100 mg of elsamitrucin base, 1.05 equivalents of corresponding acid (p-TSA, succinic, and L-tartaric acid were added as solids; sulfuric acid was dissolved in 0.5 mL of water) and water (10 mL for p-TSA, succinic, and L-tartaric acid, 9.5 mL for sulfuric acid). The suspensions were heated to 80° C. with stirring for ten minutes to form a clear solution and then ramped down to room temperature at a rate of 30° C./hour. After stirring overnight at room temperature precipitates were not formed in any of the experiments. The water was removed under a gentle flow of nitrogen at 35° C. Precipitation was observed in the p-TSA experiment after the removal of one-third of the water, this solid was filtered and dried in vacuo at 50° C. and 30 inches Hg. The filtrate was also analyzed. The other three vials were evaporated to dryness and dried in vacuo at 50° C. and 30 inches Hg. The results showed that the solids produced were semi-crystalline with high amorphous content [Table 3, OVL-A-47(1), OVL-A-47(2-1), OVL-A-47(3), and OVL-A-65].
On a microscope slide, 1-2 mg of amorphous elsamitrucin tosylate was sprinkled with the help of a spatula and a cover-slip was placed. Drops of solvent were placed on the side of the cover-slip so as to allow the solvent to sip under the cover-slip and dissolve the drug. The drug in contact with the solvent was stored at room temperature and examined under microscope with 100× or 400× magnification. The solvents used were isopropyl alcohol, methanol, ethanol, acetonitrile, acetone, propylene glycol, tetrahydrofuran, dichloromethane and 1:1 mixtures of isopropyl alcohol, methanol, ethanol, acetonitrile, acetone with water. Needle or rod-shaped crystals were observed under microscope in solvents—ethanol, methanol, propylene glycol, isopropyl alcohol, acetone, and all the water:solvent mixtures. Microscopic examination of the samples indicated that the Elsamitrucin tosylate salt became crystalline in at least one solvent.
A small amount of the salt (1-2 mg) was placed on a microscope slide and covered with a cover-slip. Several drops of solvent were added at the edge of the cover-slip so that capillary action would draw the solvent between the slide and the cover-stip. If the material was partially dissolved, the slide was slowly heated on a hot-plate until most of the solid had dissolved. Each slide was cooled at room temperature to allow slow crystallization. 1:1 mixtures of methanol, ethanol, isopropyl alcohol and acetonitrile in water were used for the crystallization. Each salt in all the four different solvent systems produced crystals of elsamitrucin. Elsamitrucin tosylate salt crystals showed birefringence under plane polarized light as depicted in
The recrystallization of p-TSA salt was carried out with slow evaporation in the Craig tube. The p-TSA salt was dissolved in 1:1 mixture of acetonitrile and water at elevated temperatures. After hot filtration in the Craig tube, the solvent from the reaction was evaporated slowly, which yielded precipitate. Under microscope, the crystal habit was observed to be needle-shaped. The crystals were isolated by filtration and dried under vacuum. The material was observed to be transparent to XRD, which was confirmed to be crystalline by microscopy. In the differential scanning calorimetry analysis, the material showed a melt followed by degradation or crystallization at 183° C. and 186° C., respectively. 1 H NMR analysis showed the sample to be 1:1 ratio of APl to the counter ion (p-TSA).
The solubility of the p-TSA salt in water was checked by HPLC after stirring the slurry at room temperature for six hours. It was found that the solubility of the p-TSA salt in water is 15.6 mg/mL (Table 5, lot # OVL-A-137). The solubility of the p-TSA salt at pH 4 (benzoate buffer) is 14.7 mg/mL (Table 5, OVL-A-143).
Elsamitrucin salts made in accordance with the teachings of the present invention were tested for stability. Two samples of the isolated p-TSA salt (40 mg each) were placed in a vacuum oven at 75° C. for nine hours. After this exposure, sample #1 was taken out, the temperature was increased to 98° C. and the second sample was dried overnight. The NMR data showed a perfect 1:1 salt ratio, so there was no decomposition in the solid state during drying at elevated temperatures. The weight loss by TGA was approximately 2.5% for both samples. Karl Fischer analysis indicated the two lots still had water present: sample #1 had 4.0% water content and #2 had 4.6%. The p-TSA salt (16 mg) of elsamitrucin was dissolved in 1.6 mL of benzoate buffer (pH 4) and stirred at 50° C. for ten days. Samples for HPLC and MS were taken in 3, 5, and 10 days. No evidence of the decomposition product was found by either MS (peak at 282) or HPLC.
Elsamitrucin (200 mg) was slurried in 1 mL of acetonitrile/water (1:1) and heated up to 75° C. resulting in a very thick slurry. A 1 M HCl solution in water (0.321 mL, 1.05 eq.) was added to the slurry to form a clear solution. The mixture was then slowly cooled to room temperature at a rate of 25° C./h with very gentle stirring. After stirring at room temperature for approximately 6 hours, the solids obtained were isolated by filtration and dried in vacuo at 50° C. and 30 inches Hg to yield 187.5 mg (88.86% yield) of the HCl salt. DSC and XRD analyses confirmed the crystalline nature of the salt.
The following experiment confirms that the elsamitrucin salts made in accordance with the teachings of the present invention retain their in vitro anti-neoplastic activity when compared to elsamitrucin base. Elsamitrucin and Elsamitrucin tosylate were tested in vitro using: B16F10 (murine lung), HCT 116 (human colon), HT29 (human colon) and SK-MES-1 (human non-small cell lung carcinoma ). Cell growth inhibition was evaluated in 96-well micro-culture plates with a semi-automated MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
SK-MES-1 human non-small cell lung carcinoma, B16F10 murine melanoma cells, HCT 116 and HT29 human colon carcinomas (collectively “test cell cultures”) were maintained in buffered RPMI 1640 supplemented with fetal calf serum, antibiotics and other appropriate growth factors such as glutamine. Test cells (1,500-2,000 cells/well) were seeded in a 96-well micro culture plate with a total volume of 100 μL/well. After overnight incubation in a humidified incubator at 37° C. with 5% CO2 and 95% air, elsamitrucin solutions were diluted to various concentrations with RPMI 1640, were added to each well in a 100 μL volume. The elsamitrucin base and the elsamitrucin tosylate solutions (elsamitrucin solutions) were prepared and stored in a −20° C. freezer. The solutions were thawed not more than 10 times for the entire experiment.
Cell culture plates seeded with test cells and various concentrations of elsamitrucin solutions were placed in a humidified incubator at 37° C., with CO2 and 95% air for 5-10 days. The plates were then centrifuged briefly, and 100 μL of the growth medium was removed. The cell cultures were incubated with 50 μL of MTT reagent (1 mg/ml in Dulbecco™ phosphate-buffered saline) for 4 hours at 37° C. The resultant purple formazan precipitate was solubilized with 200 μL of 0.04 N HCl in isopropanol. Absorbance was monitored at a wavelength of 595 nm, and at a reference wavelength of 650 nm, using a TECAN® GENios mocroplate reader. In all experiments, absorbance data was acquired for each agent at two overlapping concentration ranges. In most cases, the study was repeated using broader concentration ranges.
The results of each test were stored and imported into PRISM® 3.03 for graphical analysis and determination of IC50 values. All results were graphed as a percentage of controlled absorbance versus the drug concentration. The IC50 values were estimated with PRISM® 3.03 using nonlinear regression analysis to fit the data to the sigmoidal dose-response curve described by the following four-logistic equation:
Top is the maximal percentage of control absorbance, bottom is the minimal percentage of control absorbance at the highest agent concentration, Y is the observed absorbance, X is the agent concentration, IC50 is the concentration of agent that inhibits cell growth by 50% compared to the control cells, and n is the slope of the curve. Table 4 demonstrates that elsamitrucin and elsamitrucin tosylate salt possess essentially the same anti-proliferative effect on the cell lines tested. Thus as demonstrated in Table 4, the elsamitrucin salts made in accordance with the teachings of the present invention can be expected to have equivalent, or superior in vivo anti-neoplastic activity as therapeutic compositions made using elsamitrucin base alone. The elsamitrucin tosylate comprises an IC50 that is within preferably about 20%, more preferably about 15% and most preferably about 10% of a similar amount of an elsamitrucin base.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements The terms “a” and “an” and “the” and similar references used 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. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual 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 otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of any and all Markush groups used in the appended claims.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those preferred embodiments will 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 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.
Furthermore, references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.
In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.