DIETHYL 4-(4-FLUOROBENZYLAMINO)- 1 ,2-PHENYLENEDICARBAMATE,

AND SALTS THEREOF

This application claims the benefit of priority of the United States Provisional Application No. 61/229,138, filed 28 July 2009.

BACKGROUND OF THE INVENTION

Retigabine (compound I) is the international common accepted name for ethyl 2-amino-4-(4-fluorobenzylamino)phenylcarbamate, and has an empirical formula of C16H18FN3O2, and a molecular weight of 303.33 g/mol.

Retigabine is a pharmaceutical substance with anticonvulsive, antipyretic and analgesic activity, and can thus be employed in pharmaceutical preparations. In the United States of America, the non-proprietary name retigabine has been superseded by ezogabine.

The preparation of retigabine dihydrochloride and similar compounds is disclosed in U.S. Patent No. 5,384,330 ("the '330 patent"). Example 1 of the '330 patent describes the preparation of retigabine dihydrochloride by hydrogenating 2- amino-4-(4-fluorobenzylamino)nitrobenzene (compound II), mono- ethoxycarbonylating the obtained 4-(4-fluorobenzylamino)-l,2-phenylenediamine (compound III) by using ethyl chloroformate as an ethoxycarbonylating agent, and treating the obtained mixture with ethanolic hydrochloric acid, and isolating retigabine dihydrochloride (Scheme 1). Ni-Raney

dioxane

(I) -2HCI

Scheme 1

Applicants have found that retigabine, or salts thereof, often contains the impurity diethyl 4-(4-fluorobenzylamino)-l,2-phenylenedicarbamate or salts thereof.

In the view of the foregoing, there is an unmet need for detecting,

isolating and quantifying the impurity diethyl 4-(4-fluorobenzylamino)-l,2- phenylenedicarbamate, or salts thereof, in samples comprising retigabine, or salts thereof.

BRIEF SUMMARY OF THE INVENTION

The invention provides diethyl 4-(4-fluorobenzylamino)-l,2- phenylenedicarbamate, or salts thereof, a process for preparing diethyl 4-(4- fluorobenzylamino)-l,2-phenylenedicarbamate, or salts thereof, and the use of diethyl 4-(4-fluorobenzylamino)-l,2-phenylenedicarbamate or salts thereof, as a reference marker and reference standard for analyzing samples comprising retigabine, or salts thereof. DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that a compound of formula (IV), i.e. diethyl 4-(4- fluorobenzylamino)-l,2-phenylenedicarbamate, or a salt thereof,

can be formed as an impurity in some preparations of retigabine by unexpectedly di-ethoxycarbonylating 4-(4-fluorobenzylamino)- 1 ,2-phenylenediamine (compound III)

(1 )

which is a key intermediate for the preparation of retigabine.

To date, this impurity compound of formula (IV) has not yet been reported, nor suggested, as an impurity of retigabine. Surprisingly, in some preparations of retigabine by mono-ethoxycarbonylating compound (III), the present inventors have found that compound (IV) is formed as an impurity of retigabine, which due to its similar structure and so physical properties, as compared with retigabine, is difficult to detect and quantify. Therefore, in an embodiment, the invention provides diethyl 4-(4- fluorobenzylamino)- 1 ,2-phenylenedicarbamate,

or salts thereof, particularly in isolated form and/or having a purity higher than 80% a/a, preferably having a purity higher than 90% area/area (a/a), and more preferably having a purity higher than 99% a/a, as measured by HPLC.

There is further provided, therefore, use of compound of formula (IV), or a salt thereof

as a reference marker to detect the presence of compound (IV), or a salt thereof, in a sample comprising retigabine, or a salt thereof; and/or as a reference standard to quantify the amount of compound (IV) in a sample comprising retigabine, or a salt thereof; and/or as a reference standard to ensure the purity of a sample comprising retigabine, or a salt thereof, so as to ensure that retigabine, or a salt thereof, preferably retigabine free base, as present in said sample has less than 0.15% a/a, preferably less than 0.08% a/a, as measured by HPLC of a compound of formula (IV), or a salt thereof.

Also, there is provided, a method of use of compound of formula (IV), or a salt thereof as a reference marker to detect the presence of compound (IV), or a salt thereof, in a sample comprising retigabine, or a salt thereof; and/or as a reference standard to quantify the amount of compound (IV) in a sample comprising retigabine, or a salt thereof; and/or as a reference standard to ensure the purity of a sample comprising retigabine, or a salt thereof, so as to ensure that retigabine, or a salt thereof, preferably retigabine free base, as present in said sample has less than 0.15% a/a, preferably less than 0.08% a/a, as measured by HPLC of a compound of formula (IV), or a salt thereof. The term "reference marker", as used herein, refers to a compound that is used in qualitative analysis to identify components of a mixture based on their position, e.g. in a HPLC or a GC chromatogram or on a Thin Layer Chromatography (TLC) plate.

The term "reference standard", as used herein, refers to a compound that is used in quantitative analysis to determine the amount of a compound present in a sample.

Furthermore, there is provided a method for determining the presence of a compound (IV), or a salt thereof

, in a test sample comprising retigabine, or a salt thereof, preferably retigabine free base, the method comprising:

(a) providing a reference sample comprising (i) retigabine, or a salt thereof, preferably retigabine free base, and (ii) a reference marker which is a compound of formula (IV), or a salt thereof;

(b) carrying out chromatographic separation on said reference sample to obtain a reference chromatographic result of said reference marker relative to said retigabine, or a salt thereof;

(c) carrying out chromatographic separation on said test sample to obtain a test chromatographic result;

(d) comparing the chromatographic results obtained in steps (b) and (c); wherein if the test chromatographic result is substantially the same as the reference chromatographic result for said reference marker, then a compound of formula (IV), or a salt thereof, is present in said test sample.

By "substantially the same results", as used herein, is meant to refer that the chromatographic results are considered to be equivalent by the one skilled in the art. Further, by "substantially the same results" it can be meant to refer that the compared chromatographic results share a similarity of more than 80%, preferably more than 90%, even more preferably more than 95%, and yet even more preferably more than 99%.

Preferably, the chromatographic separation comprises HPLC or GC and as such the steps (b) to (d) of the method above comprise: (b) carrying out HPLC or GC on said reference sample to determine the relative retention time of said reference marker compared to said retigabine, or a salt thereof;

(c) carrying out HPLC or GC on said test sample;

(d) comparing relative retention times determined in steps (b) and (c); wherein if there is observed a relative retention time in step (c) substantially the same as the relative retention time of said reference marker compared to said retigabine, or a salt thereof, preferably retigabine free base, in step (b), then a compound of formula (IV), or a salt thereof, is present in said test sample.

Alternatively, the chromatographic separation can comprise TLC and as such the steps (b) to (d) of the method above comprise:

(b) carrying out TLC on said reference sample to determine the relative component position on a chromatographic support of said reference marker compared to said retigabine, or a salt thereof;

(c) carrying out TLC on said test sample;

(d) comparing the relative component positions determined in steps (b) and (c); wherein if there is observed on said chromatographic support a relative component position in step (c) substantially the same as the relative component position of said reference marker compared to said retigabine, or a salt thereof, preferably retigabine free base, in step (b), then a compound of formula (IV), or a salt thereof, is present in said test sample. The present invention still further comprises a method for quantifying the amount of compound (IV), or a salt thereof

in a test sample comprising retigabine, or a salt thereof, the method comprising:

(a) providing a test sample of retigabine, or a salt thereof, preferably retigabine free base, containing an unknown concentration of a compound of formula (IV), or a salt thereof;

(b) subjecting said test sample to chromatographic separation;

(c) obtaining a chromatographic quantitative measurement for a compound of formula (IV), or a salt thereof, in said test sample; and

(d) calculating the amount of a compound of formula (IV), or a salt thereof, in said test sample based on the measurement of step (c) and also chromatographic quantitative measurement for a compound of formula (IV), or a salt thereof, obtained from at least one reference sample having a known concentration of a compound of formula (IV), or a salt thereof.

In a first embodiment of the above quantification method, the following steps are carried out:

(a) providing a test sample of retigabine, or a salt thereof, preferably retigabine free base, containing an unknown concentration of a compound of formula (IV), or a salt thereof;

(b) providing at least one reference sample having a known concentration of a compound of formula (IV), or a salt thereof;

(c) subjecting said test sample and said reference sample to chromatographic separation;

(d) obtaining chromatographic quantitative measurements for a compound of formula (IV), or a salt thereof, in said test sample and said reference sample; and

(e) calculating the amount of a compound of formula (IV), or a salt thereof, in said test sample from the measurements of step (d). More precisely, the following steps are typically carried out in the steps (b) to (e) of the above first embodiment of the quantification method according to the present invention:

(b) providing at least one reference sample having a known concentration of a compound of formula (IV), or a salt thereof;

(c) subjecting said test sample and said reference sample to HPLC or GC;

(d) measuring the area or height of peaks obtained for a compound of formula (IV), or a salt thereof, in said test sample and said reference sample; and

(e) calculating the concentration of a compound of formula (IV), or a salt thereof, in said test sample from the measurements of step (d).

Alternatively, in a second embodiment of the quantification method, the following steps are carried out: (a) providing a test sample of retigabine, or a salt thereof, preferably retigabine free base, containing an unknown concentration of a compound of formula (IV), or a salt thereof;

(b) subjecting said test sample to chromatographic separation;

(c) obtaining chromatographic quantitative measurement for a compound of formula (IV), or a salt thereof, in said test sample; and

(d) calculating the concentration of a compound of formula (IV), or a salt thereof, in said test sample based on the measurement of step (c) and also a calibration curve that is representative of chromatographic quantitative measurement for a compound of formula (IV), or a salt thereof, obtained from more than one reference sample each having a respectively defined concentration of a compound of formula (IV), or a salt thereof.

Typically, the calibration curve referred to above is obtained with at least two reference samples, preferably with at least five or six reference samples, and the concentration of a compound of formula (IV), or a salt thereof, is then calculated by reference to the thus obtained calibration curve. The chromatographic quantitative measurements of the quantitative methods of the invention is preferably carried out by (a) subjecting said test sample and said at least one reference sample to HPLC or GC, and (b) measuring the area or height of peaks obtained for a compound of formula (IV), or a salt thereof, in said test sample and said at least one reference sample. The method is preferably an HPLC method, and more preferably the HPLC method comprises the following features: the column is a Waters Sunfire C 18, 5 μm, 4.6 x 250 mm column at 30 ⁰ C; the mobile phase is a filtered and degassed mixture of buffer solution and methanol (35:65), wherein the buffer solution is prepared by dissolving 0.5 mL of triethylamine in 500 mL of water, and then adjusting the pH to 7.1 with acetic acid; the detector is a UV detector at 254 nm wavelength; the flow rate is 0.8 mL per minute; and the samples are prepared by dissolving the appropriate amount of sample in mobile phase in order to obtain 1.0 mg per mL. There is still further provided by the present invention a method for ensuring the purity of a test sample comprising retigabine, the method carrying out a quantification method substantially as hereinbefore described so as to ensure that retigabine, or a salt thereof, preferably retigabine free base, as present in said test sample has less than 0.15% a/a, preferably less than 0.08% a/a, as measured by HPLC of a compound of formula (IV), or a salt thereof.

There is also provided by the present invention retigabine, or a salt thereof, preferably retigabine free base, having equal to or less than 0.20% (a/a), preferably having equal to or less than 0.15% (a/a), more preferably equal to or less than 0.08% (a/a), even more preferably having equal to or less than 0.10% (a/a), yet even more preferably having equal to or less than 0.05% (a/a), as measured by HPLC of a compound of formula (IV), or a salt thereof

Accordingly, the present invention further provides a pharmaceutical composition comprising an effective amount of retigabine, preferably retigabine free base, substantially as hereinbefore described, together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The term "effective amount" as used herein means an amount of retigabine, preferably retigabine free base, which is capable of providing an anticonvulsive, antipyretic and / or analgesic therapeutic effect. By "pharmaceutically acceptable composition" is meant that the carrier, diluent or excipient must be compatible with retigabine and not be deleterious to a recipient thereof.

The present invention further provides retigabine, preferably retigabine free base, substantially as hereinbefore described, for use in the treatment of a disease state alleviated by administration of retigabine, in particular for the treatment of epilepsy. The present invention also provides a method of treatment of a disease state alleviated by administration of retigabine, in particular epilepsy, which method comprises administering to the patient an effective amount of retigabine, preferably retigabine free base, substantially as hereinbefore described, and one or more pharmaceutically acceptable excipients or carriers, in a patient in need thereof.

Further, the present invention provides the use of retigabine, preferably retigabine free base, substantially as hereinbefore described, for the preparation of a medicament for the treatment of a disease state alleviated by administration of retigabine, in particular for the treatment of epilepsy.

Still further, the present invention provides a process for preparing diethyl 4- (4-fluorobenzylamino)-l,2-phenylenedicarbamate (compound of formula IV)

or a salt thereof, said process comprising:

(i) treating ethyl 2-amino-4-(4-fluorobenzylamino)phenylcarbamate, compound I

(i.e., retigabine),

with a base in an organic solvent,

(ii) adding an ethoxycarbonylating agent, optionally in an organic solvent, to the mixture of step (i),

(iii) isolating compound (IV) from the mixture,

(iv) optionally, purifying (IV),

(v) optionally, converting compound (IV) into a salt thereof, and

(vi) optionally, purifying the salt of compound (IV).

The base of step (i) is preferably an organic base, more preferably is an organic base with a pKa higher than 5.77, even more preferably is an amine organic base with a pKa higher than 5.77, even more preferably is a tertiary amine with a pKa higher than 5.77, even more preferably is a tertiary amine selected from the group of triethylamine and N,N-diisopropylethylamine, and even more preferably is N ₅ N- diisopropy lethy lamine .

The organic solvent of steps (i) and (ii) is preferably an aprotic organic solvent, more preferably is a solvent selected from the group consisting of an ether, an alkane and an aromatic organic solvent, even more preferably is a solvent selected from the group consisting of tert-butylmethylether, dioxane, diethyl ether, tetrahydrofuran and toluene, and yet even more preferably is dioxane.

The step (i) is preferably held above ambient temperature, preferably at 40 ⁰ C to 60 ⁰ C, and more preferably at 50 ⁰ C. The mixture of step (ii) is preferably heated, more preferably is heated to a temperature of from 40 ⁰ C to 60 ⁰ C, and more preferably is heated to a temperature of from 5O ⁰ C t ₀ 55 ⁰ C.

The step (iii) preferably comprises (a) adding water to the mixture to form a suspension comprising compound (IV) and (b) filtering the suspension.

The step (iv) preferably comprises crystallizing compound (IV) from ethyl acetate.

In an embodiment, the invention provides a process for preparing diethyl 4-(4- fluorobenzylamino)-l,2-phenylenedicarbamate (compound IV), or salts thereof, the process comprising (i) forming a mixture of ethyl 2-amino-4-(4- fluorobenzylamino)phenylcarbamate, compound I (i.e., retigabine), with N ₅ N- diisopropylethylamine and dioxane, which mixture is preferably held above ambient temperature, e.g., at 40 ⁰ C to 60 ⁰ C, preferably 50 ⁰ C, (ii) adding a solution of ethyl chloroformate in dioxane to the mixture in (i), which mixture is optionally heated, e.g., to a temperature of from 40 ⁰ C to 60 ⁰ C, preferably 50 ⁰ C to 55 ⁰ C, (iii) adding water to the combined mixture in (ii) to form a suspension comprising compound (IV), (iv) filtering the suspension to isolate compound (IV), (v) optionally, crystallizing compound (IV) from ethyl acetate, (vi) optionally, converting compound (IV) into a salt thereof, and (vii) optionally, purifying the salt of compound (IV). In still another embodiment, the invention provides a process for analyzing the purity of a composition containing retigabine, or salts thereof, comprising monitoring the amount of compound (IV), or salts thereof, present in a sample of the composition.

In yet another embodiment, the invention provides a method for monitoring the presence of compound (IV), or salts thereof, in the product obtained from the reaction o f 4-(4-fluorobenzylamino)-l,2-phenylenediamine (compound III) with an ethoxycarbonylating agent such as, for example, ethyl chloroformate. In addition, the invention provides a composition comprising compound of formula IV (diethyl 4-(4-fluorobenzylamino)-l,2-phenylenedicarbamate),

or a salt thereof, wherein the composition is free of retigabine, or a salt thereof.

Still further, the present invention provides a composition comprising compound of formula IV (diethyl 4-(4-fluorobenzylamino)-l,2- phenylenedicarbamate),

or a salt thereof, in an amount of at least 0.2%, preferably of at least 0.05%, based on the weight of the composition, and a carrier.

The composition of the invention above preferably comprises up to 99%, more preferably up to 99.5%, even more preferably up to 99.8%, and yet even more preferably up to 99.95% by weight of retigabine, or a salt thereof, based on the combined weight of compound of formula (IV), or a salt thereof, and retigabine, or a salt thereof. Also, the invention provides a composition comprising compound of formula

IV (diethyl 4-(4-fluorobenzylamino)-l,2-phenylenedicarbamate),

or a salt thereof, a carrier, and up to 99%, preferably up to 99.95%, by weight of retigabine, or a salt thereof, based on the combined weight of compound of formula (IV), or a salt thereof, and retigabine, or a salt thereof.

Preferably, the compositions of the invention as hereinabove described are pharmaceutical compositions and the carrier is a pharmaceutically acceptable carrier.

Retigabine or its salts can exist in solvated, as well as unsolvated forms, including hydrated forms, i.e. retigabine can contain in its structure stoichiometric amounts of solvent in the case of solvates, or of water in the case of hydrates. It is to be understood that the invention encompasses all such solvated, as well as unsolvated, forms. The preparation of solvates and hydrates depends on the solvent or mixture of solvents used and the crystallization conditions that can be determined by the skilled person.

The following examples further illustrate embodiments of the invention but, of course, should not be construed as in any way limiting its scope.

General Experimental Conditions

HPLC method:

The chromatographic separation was carried out in a Waters Sunfϊre C 18, 5 μm, 4.6 x 250 mm column at 30 ⁰ C.

The mobile phase was a filtered and degassed mixture of buffer solution and methanol (35 :65). The buffer solution was prepared by dissolving 0.5 mL of triethylamine in 500 mL of water, and then adjusting the pH to 7.1 with acetic acid.

The chromato graph was equipped with a 254 nm detector, and the flow rate was 0.8 mL per minute. The test samples (10 μL) were prepared by dissolving the appropriate amount of sample in mobile phase in order to obtain 1.0 mg per mL. The chromatogram was run for at least 45 minutes.

Approximate HPLC Retention Times:

Example 1 . This Example illustrates a preparation of diethyl 4-(4- fluorobenzylamino)-l,2-phenylenedicarbamate (i.e. compound of formula IV) in accordance with an embodiment of the invention.

A mixture of retigabine, compound I (15.2 g, 0.050 mol) [prepared by synthesizing retigabine dihydrochloride following the teachings of Example 1 of the '330 patent, and preparing the corresponding free base by following the same process described in Step C, Example 2, described below] and N,N-diisopropylethylamine (10.5 mL, 0.060 mol) in dioxane (150 mL) was heated to 50 ⁰ C. A solution of ethyl chloroformate (6.75 g, 0.062 mol) in 50 mL of dioxane was added dropwise over 30 minutes. The resulting mixture was stirred at 50-55 ⁰ C for 90 minutes. After cooling the reaction mixture to 30 ⁰ C, 500 mL of water were added to the reaction mixture. The resulting suspension was stirred at room temperature for 1 hour and filtered in a Bϋchner funnel. The filtered solid was dried, thereby obtaining 19.0 g of crude material as a white solid. The obtained white solid was dissolved in 100 mL of ethyl acetate, and the resulting solution was filtered to remove insoluble particles. The solvent was partially evaporated under reduced pressure to a final volume of 40 mL. After overnight cooling in a refrigerator, the resulting suspension was filtered using a Bϋchner funnel. 7.2 g of diethyl 4-(4-fluorobenzylamino)-l,2-phenylenedicarbamate (38.3% yield) was obtained as a white solid.

Analytical data: Purity (HPLC): 94.7%; m.p.: 124.4 - 126.3 ⁰ C; IR (cm ¹ ): 3385, 3290, 2987, 1735, 1677, 1625, 1604, 1546, 1519, 1476, 1433, 1889, 1367,

1322, 1305, 1293, 1258, 1235, 1172, 1158, 1127, 1096, 1073, 1053, 1019, 988, 859, 849, 828, 806, 776, 764; ¹ H NMR (400.1 MHz, CDCl ₃ ): δ (ppm) 7.29 (complex signal, 2 H), 7.21 - 6.78 (broad signal + complex signal, 6 H), 6.40 (broad signal, 1 H), 6.30 (dd, J=8.8 Hz, J=I .6 Hz, 1 H), 4.26 (s, 2 H), 4.19 (q, J=7.2 Hz, 4 H), 1.29 (complex signal, 6 H); ¹³ C NMR (100.6 MHz, CDC13): δ (ppm) 162.3 (d, J _C - _F =245.1 Hz), 156.0, 154.3, 147.1, 134.9, 133.7, 129.3 (d, J _C - _F =8.0 Hz), 127.5, 115.6 (d, Jc- _F =21.4 Hz), 109.1, 106.5, 61.8, 61.6, 47.9, 14.7; MS (ESI+) calculated for Ci ₉ H ₂₂ FN ₃ O ₄ 375.39. Found 376 [M+H]. Elemental Analysis (obtained): C 60.87%, H 5.98%, N 11.07%, F 4.98%; Elemental Analysis (expected): C 60.79%, H 5.91%, N 11.19%, F 5.06%.

Example 2. This example illustrates a preparation of retigabine (compound I) in a highly purified form in accordance with an embodiment of the invention.

Step A: Preparation of 4-(4-fluorobenzylamino)-l,2-phenylenediamine (compound III).

A mixture of 2-amino-4-(4-fluorobenzylamino)nitrobenzene (51.0 g, 0.196 mol) [prepared by following the teachings of the process described in the '330 patent] and 5% palladium over charcoal (15 g) were suspended in 1 L of dioxane at room temperature. The resulting mixture was hydrogenated by reaction with hydrogen gas and Ni-Raney at 50 ⁰ C for 7 days. The catalyst was removed by filtration, and the filtrate solution containing 4-(4-fluorobenzylamino)-l,2-phenylenediamine was used directly in the next step.

Step B: Preparation of retigabine dihydro chloride (compound I-2HC1).

N,N-diisopropylethylamine (31.4 g, 0.245 mol) was added over the dioxane solution containing 4-(4-fluorobenzylamino)-l,2-phenylenediamine of step A. Ethyl chloroformate (22.6 g, 0.208 mol) was added dropwise over the solution, keeping the internal temperature between 10 to 20 ⁰ C. The resulting mixture was stirred at room temperature for 2 hours. Then, a 3.37 M hydrochloric acid solution in isopropanol (170 mL, 0.573 mol) was added dropwise to the reaction mixture, while keeping the internal temperature at 0 ⁰ C. A white solid crystallized out the reaction mixture during the addition. The suspension was stirred at room temperature for 1 hour and was filtered using a Bϋchner funnel. The crude solid was dried at 40 ⁰ C. Step C: Preparation of retigabine (compound I) from retigabine dihydro chloride (compound I-2HC1).

The crude solid of step B was dissolved in 700 mL of water at room temperature. 10% NaOH (130 mL, 0.325 mol) was added dropwise over the solution. A solid precipitated off the reaction mixture during the addition. The solid was filtered off, and then recrystallized in a 1 : 1 mixture of ethyl acetate and petroleum oil (400 mL, equivalent to 13 volumes per gram). 29 g of retigabine (49% global yield) was obtained as a brownish solid.

Analytical data: HPLC purity: 99. 5 % a/a; Content of diethyl 4-(4- fluorobenzylamino)-l,2-phenylenedicarbamate (compound IV): 0.2 %. The purity was determined by employing compound IV as a reference standard.

Example 3. This example illustrates a preparation of retigabine (compound I) in accordance with an embodiment of the invention.

Retigabine dihydro chloride prepared under similar conditions as described in

Example 2 was dissolved in water at room temperature. 10% NaOH was added dropwise to the solution. A solid precipitated from the reaction mixture during the addition. The precipitated solid was filtered, dried, and then recrystallized in a 1 : 1 mixture of ethyl acetate and petroleum oil (21 volumes per gram), thereby obtaining 51.5 g of retigabine (46% global yield) as slightly pink solid.

Analytical data: HPLC purity: 99.8 % a/a; Content of diethyl 4-(4- fluorobenzylamino)-l,2-phenylenedicarbamate (compound IV): not detected. The purity was determined by employing compound IV as a reference standard.

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

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