1. Field of the Invention
The present invention is directed to pharmaceutical formulations containing 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide, as well as to methods of making such pharmaceutical formulations and a method of treating a subject with such pharmaceutical formulations.
2. Related Background Art
The hepatitis C virus inhibitor 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide is a potent inhibitor of the hepatitis C virus and has shown very favorable toxicological and pharmacological profiles. The structure of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide is as follows:
However, formulating 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide for oral dosage has proven very difficult, as 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide is insoluble in aqueous medium at gastrointestinal pHs. Accordingly, there is a need to develop an oral dosage form containing 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide, which has good bioavailability properties and which can be produced according to a reliable and robust process.
In a first aspect, the present invention is directed to a pharmaceutical formulation comprising a therapeutically effective amount of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide and pharmaceutically acceptable additives, wherein said pharmaceutically acceptable additives comprise at least one surfactant. In a particularly preferred embodiment, the pharmaceutically acceptable additives further comprise at least one solubilizer.
In a second aspect, the present invention is directed to a method of making a pharmaceutical formulation comprising the steps of (a) granulating 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide and pharmaceutically acceptable additives to form a granulate, wherein said pharmaceutically acceptable additives comprise at least one surfactant; and (b) blending the granulate with pharmaceutically acceptable additives to form a final blend. Optionally, the inventive method further comprises the step of (c) encapsulating the final blend to form the pharmaceutical formulation or (c) compressing the final blend to form the pharmaceutical formulation. In a particularly preferred embodiment, the pharmaceutically acceptable additives in step (a) further comprise at least one solubilizer.
In preferred embodiments of the inventive method step (a) comprises the steps of (a1) screening 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide, at least one diluent, at least one solubilizer, at least one disintegrant, and at least one surfactant into a granulator to form a screened material; (a2) blending the screened material to form a screened/blended material; (a3) dissolving at least one surfactant in water to form a surfactant solution; (a4) granulating the screened/blended material with the surfactant solution to form a wet granulation; (a5) drying the wet granulation to form a dried granulation; and (a6) milling the dried granulation to form the granulate. In still other preferred embodiments, step (b) comprises the steps of (b1) blending at least one screened glidant with the granulate from step (a) to form a first blend; (b2) blending the first blend with at least one screened solubilizer and at least one screened disintegrant to form a second blend; (b3) blending a portion of the second blend with an equal amount of at least one screened lubricant to form a third blend; and (b4) blending the third blend with the remaining second blend to form the final blend.
In a third aspect, the present invention is directed to a pharmaceutical formulation made according to the inventive method.
In a fourth aspect, the present invention is directed to a method of inhibiting hepatitis C virus, wherein the method comprises administering a pharmaceutical formulation of the present invention to a subject in need of such treatment.
In certain preferred embodiments of this invention, the therapeutically effective amount of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide ranges from about 1 mg to about 2000 mg, more preferably from about 10 mg to about 400 mg and most preferably from about 25 mg to about 200 mg, and/or the 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide is micronized to a particle size specification of 50% less than or equal to 5 μm and 90% less than or equal to 20 μm. In other preferred embodiments, the at least one surfactant is a blend of surfactants, more preferably a blend of sodium lauryl sulfate and polysorbate 80; in still other preferred embodiments, the at least one solubilizer is povidone. In still other preferred embodiments, the pharmaceutically acceptable additives further comprise ingredients selected from the group consisting of diluents, surfactants, solubilizers, disintegrants, glidants, lubricants, colorants and combinations thereof.
The first embodiment of the invention is directed to a pharmaceutical formulation comprising a therapeutically effective amount of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide and pharmaceutically acceptable additives. The pharmaceutically acceptable additives of the first embodiment necessarily comprise at least one surfactant to effect fast and complete dissolution of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide, given its insolubility in aqueous medium at gastrointestinal pHs.
In fact, according to a preferred embodiment, the at least one surfactant is a blend of surfactants, more preferably a blend of sodium lauryl sulfate and polysorbate 80 (Tween 80). In a particularly preferred embodiment, the pharmaceutically acceptable additives further comprise at least one solubilizer. Preferably the solubilizer is povidone. Hence a pharmaceutical formulation of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide with the combination of sodium lauryl sulfate, polysorbate 80 and povidone is a preferred embodiment of this invention; the present inventors have found that this combination is effective in achieving fast and complete dissolution of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide. Solubility of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide is improved from 0.02 mg/mL to 0.64, 0.16 and 0.14 mg/mL in 2% sodium lauryl sulfate, 10% povidone, and 2% polysorbate 80, respectively.
In more preferred embodiments, the sodium lauryl sulfate is present in an amount ranging from about 1% to about 10%, more preferably from about 4% to about 6%, and most preferably is about 5%, by weight of the pharmaceutical formulation. In more preferred embodiments, the polysorbate 80 is present in an amount ranging from about 1% to about 5%, more preferably from about 2% to about 4%, and most preferably is about 3%, by weight of the pharmaceutical formulation. In more preferred embodiments, the povidone is present in an amount ranging from about 1% to about 20%, more preferably from about 8% to about 12%, and most preferably is about 10%, by weight of the pharmaceutical formulation. In preferred embodiments of this invention, the 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide is present in an amount of at least about 60% by weight of the pharmaceutical formulation.
5-Cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide suitable for use in the present invention can be prepared as previously described in U.S. Patent Application Publication No. 2004-0162318. 5-Cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide may be used for purposes of this invention in any of its amorphous, crystalline, hydrated or solvated forms. Polymorphic forms of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide and methods of making the same are disclosed in co-pending U.S. patent application Ser. No. ______ (based on U.S. Provisional Application No. 60/735,190, which is incorporated by reference herein. In a preferred embodiment of this invention, the 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide is micronized to a particle size specification of 50% less than or equal to 5 μm and 90% less than or equal to 20 μm.
5-Cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide is employed in a therapeutically effective amount. A “therapeutically effective amount” is intended to mean the amount of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide that, when administered to a subject in need thereof, is sufficient to effect treatment for disease conditions alleviated by the inhibition of hepatitis C virus. The amount of a given compound of the invention that will be therapeutically effective will vary depending upon factors such as the disease condition and the severity thereof, the identity of the subject in need thereof, etc., which amount may be routinely determined by artisans of ordinary skill in the art. Typically, the therapeutically effective amount ranges from about 1 mg to about 2000 mg, more preferably from about 10 mg to about 400 mg, and most preferably from about 25 mg to about 200 mg.
Pharmaceutically acceptable additives suitable for use in the present invention include, without limitation, diluents, surfactants, solubilizers, disintegrants, glidants, lubricants, colorants and combinations thereof.
Suitable diluents include, without limitation, microcrystalline cellulose, silicified microcrystalline cellulose, starches, mannitol, lactose, celluloses, calcium phosphates and combinations thereof. When present, a diluent may be employed in an amount ranging from about 10% to about 80%, preferably from about 15% to about 70%, and more preferably is about 16% or about 66% by weight of the pharmaceutical formulation.
Suitable surfactants include, without limitation, polysorbate 80, sodium lauryl sulfate, sugar esters of fatty acids, poloxamer, docusate sodium, polyoxyethylene sorbitan fatty acid esters, and combinations thereof. The surfactant or mixture of surfactants is employed in an amount ranging from about 2% to about 15%, preferably from about 6% to about 10% and more preferably is about 8% by weight of the pharmaceutical formulation.
Suitable solubilizers include, without limitation, povidone, poloxamer, glycerides of fatty acids, polyoxyethylene castor oil derivatives, and combinations thereof. When present, a solubilizer may be employed in an amount ranging from about 1% to about 20%, preferably from about 8% to about 12%, and more preferably is about 10% by weight of the pharmaceutical formulation.
Suitable disintegrants include, without limitation, sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, modified cellulose, pregelatinized starch, ion exchange resins, and combinations thereof. When present, a disintegrant may be employed in an amount ranging from about 1% to about 10%, preferably from about 4% to about 6%, and more preferably is about 5% by weight of the pharmaceutical formulation.
Suitable glidants include, without limitation, colloidal silicon dioxide, talc, metal stearates, magnesium carbonate, calcium silicate, fumed silicon dioxide, and combinations thereof. When present, a glidant may be employed in an amount ranging from about 0.1% to about 1%, preferably from about 0.1% to about 0.3%, and more preferably is about 0.2% by weight of the pharmaceutical formulation.
Suitable lubricants include, without limitation, magnesium stearate, other metal stearates, glyceryl behenate, sodium stearyl fumarate, hydrogenated vegetable oils, fatty acids, and combinations thereof. When present, a lubricant may be employed in an amount ranging from about 0.2% to about 2%, preferably from about 0.4% to about 0.6%, and more preferably is about 0.5% by weight of the pharmaceutical formulation.
Suitable colorants include, without limitation, FD&C approved colorants or combinations thereof. When present, a colorant may be employed in an amount readily determinable by one of ordinary skill in the art.
In one preferred embodiment, the pharmaceutical formulation takes the form of granulated 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide in a capsule. In an additional preferred embodiment, the pharmaceutical formulation takes the form of granulated and compressed 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide, i.e., the form of a tablet. Granulation may be accomplished by the method of the second embodiment of the invention (see below) or by any other suitable means. Any suitable capsule of any suitable size may be used; typically, the capsule is a hydroxypropyl methylcellulose, hypromellose or gelatin capsule, though the capsule is not limited thereto. Compression or tabletting may be accomplished by any convention compression or tabletting means or method; tablets of any suitable size or shape are possible.
The second embodiment of the present invention is directed to a method of making a pharmaceutical formulation comprising the steps of (a) granulating 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide and pharmaceutically acceptable additives to form a granulate, wherein said pharmaceutically acceptable additives comprise at least one surfactant; and (b) blending the granulate with pharmaceutically acceptable additives to form a final blend. In a particularly preferred embodiment of the invention, the pharmaceutically acceptable additives of step (a) further comprise at least one solubilizer. Optionally the inventive method comprises the step of (c) encapsulating the final blend to form the pharmaceutical formulation in the form of a capsule or the step of (c) compressing the final blend to form the pharmaceutical formulation in the form of a tablet. All details regarding ingredient identities, amounts, etc. are the same as noted above with regard to the first embodiment of the invention.
Preferably, step (a) comprises a wet granulation process. More preferably, step (a) comprises the steps of (a1) screening 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide, at least one diluent, at least one solubilizer, at least one disintegrant, and at least one surfactant into a granulator to form a screened material; (a2) blending the screened material to form a screened/blended material; (a3) dissolving at least one surfactant in water to form a surfactant solution; (a4) granulating the screened/blended material with the surfactant solution to form a wet granulation; (a5) drying the wet granulation to form a dried granulation; and (a6) milling the dried granulation to form the granulate. Optionally step (a) further comprises the step of (a4*) adding additional water to facilitate granulation.
In step (a1), the 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide, at least one diluent, at least one solubilizer, at least one disintegrant, and at least one surfactant are screened (sieved, milled, etc.) into a granulator. Screening can be accomplished using any suitable means. Likewise, the granulator can be any suitable equipment. Typically the screened material is sieved through a 20 mesh sieve. It is important to note that, prior to step (a1), it is preferable to micronize the 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide to a particle size specification of 50% less than or equal to 5 μm and 90% less than or equal to 20 μm. In a preferred embodiment, the at least one surfactant in step (a1) is sodium lauryl sulfate and the at least one solubilizer is povidone.
In step (a2), the screened material is blended in a granulator to form a screened/blended material. Blending can be accomplished using any suitable means.
In steps (a3) and (a4), at least one surfactant is dissolved in water to form a surfactant solution, and the screened/blended material is blended with the surfactant solution to form a wet granulation. A surfactant solution is employed in the present inventive method in order to carry out a wet granulation process. A wet granulation process is believed necessary to accommodate the particle size of the ingredients and to improve powder flowability and density. Here again blending may be accomplished using any suitable means. In a preferred embodiment, the at least one surfactant is step (a3) is polysorbate 80. In an optional step (a4*), additional water may be added during blending to facilitate granulation.
In step (a5), the wet granulation is dried. Drying may be accomplished using any suitable means such as a fluid bed dryer at about 50° C. and is carried out until a loss on drying ranging from about 1% to about 4% is achieved.
In step (a6), the dried granulation is milled to form the granulate. Milling can be accomplished using any suitable means. Typically the dry granulation is milled through a screening mill with a screen size of about 0.0394 inches.
Alternatively, step (a), i.e., the provision of a granulate, can be accomplished by any known granulation technique which results in a granulate having the desired properties of density and flowability.
Further preferably, step (b) comprises the steps of (b1) blending at least one screened glidant with the granulate from step (a) to form a first blend; (b2) blending the first blend with at least one screened solubilizer and at least one screened disintegrant to form a second blend; (b3) blending a portion of the second blend with an equal amount of at least one screened lubricant to form a third blend; and (b4) blending the third blend with the remaining second blend to form the final blend.
In step (b1), at least one screened glidant is blended with the granulate from step (a) to form a first blend. First, at least one glidant is screened using any suitable means. Typically a 20 mesh sieve is used. Then the screened glidant is blended with the granulate from step (a). Blending can be accomplished using any suitable means.
In step (b2), the first blend is blended with at least one screened solubilizer and at least one screened disintegrant to form a second blend. First, at least one solubilizer and at least one disintegrant are screened using any suitable means. Typically a 20 mesh sieve is used. Then the screened solubilizer and disintegrant are blended with the first blend from step (b1). Blending can be accomplished using any suitable means.
In step (b3), an equal portion of the second blend and an equal amount of at least one screened lubricant are blended to form a third blend. First, at least one lubricant is screened using any suitable means. Typically a 20 mesh sieve is used. Then the screened lubricant is blended with a portion of the second blend from step (b2) in equal amounts. Blending can be accomplished using any suitable means.
In step (b4), the third blend from step (b3) is blended with the remaining second blend from step (b3) to form the final blend. Blending can be accomplished using any suitable means.
Alternatively, step (b), i.e., the provision of a final blend, can be accomplished by any known blending technique which results in a final blend having the desired properties.
Optional step (c) of the present inventive method may entail encapsulating the final blend of step (b) to form the pharmaceutical formulation. Encapsulation is accomplished by any suitable means, i.e., an encapsulation device. Likewise any suitable capsule may be used; typically, the capsule is of any suitable size and is a hydroxypropyl methylcellulose, hypromellose or gelatin capsule, though the capsule is not limited thereto. In a preferred embodiment of the present invention, a #0E sized capsule is used and filled to a target fill weight ranging from about 50 mg to about 500 mg.
Alternative step (c) of the present inventive method may entail compressing the final blend to form the pharmaceutical formulation in the form of a tablet. Compression or tabletting can be accomplished by any suitable means.
A third embodiment of the present invention is directed to a pharmaceutical formulation made according to the method of the second embodiment.
The fourth embodiment of the present invention is directed to a method of inhibiting hepatitis C virus, wherein the method comprises administering a pharmaceutical formulation as defined by the first or third embodiment of this invention to a subject in need of such treatment. In a preferred embodiment, the pharmaceutical formulation is orally administered to the subject.
Specific embodiments of the invention will now be demonstrated by reference to the following examples. It should be understood that these examples are disclosed solely by way of illustrating the invention and should not be taken in any way to limit the scope of the present invention.
A 2.5 kg batch of a pharmaceutical formulation of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide was made as follows:
A 2.5 kg batch of a pharmaceutical formulation of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide was made as follows:
A 150 mg tablet formulation of 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide was made to contain the following:
The above-listed ingredients were wet granulated using a process similar to that in Examples 1 and 2 above. Then, the final blend was compressed on a Colton 204 Tablet Press equipped with 11/32 in standard concave, round punch. Tablets with target hardness ranging from 2-8 kp were made and tested for tablet weight, thickness, diameter, hardness, friability and dissolution. All tested parameters were within satisfactory limits.
The pharmaceutical formulation of Example 2 was tested in fasted and fed dogs in a cross over fashion. Previously, 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide exhibited a 4.7-fold food effect when dosed in dogs from a 2% Tween 80/0.5% methylcellulose Tox suspension at a high dose of 300 mg/kg. The dose of Tween is also high at 100 mg/kg, which is not practical for human formulation. Another formulation containing 5% sodium lauryl sulfate was also used as a reference for simple dry blend formulation. The simple dry blend formulation exhibited 5.2-fold food effect consistent with the Tween suspension result. The pharmaceutical formulation of the present invention enhanced bioavailability about 3 times at fasted state and as a result reduced the food effect. The fed/fast ratio for the pharmaceutical formulation of the present invention is 1.1. The food effect study results are shown in Table 2 below.
*dry blend also includes ProSolv SMCC 50, sodium starch glycolate and magnesium stearate;
**tox suspension also includes water
In addition, the pharmaceutical formulation of Example 3 was evaluated in four female Beagle dogs (7.0-8.8 kg). A single 150 mg dose (tablet) was administered to each dog following an overnight fast. Blood samples were drawn at 0 (predose), 0.25, 0.5, 1, 2, 3, 4, 8, 12 and 24 hours after dosing, plasma was separated and assayed for 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide content. The pharmacokinetic parameters were determined for each dog and descriptive statistics (AUC0-∞, Cmax, tmax and t1/2) were calculated. The results are summarized in Table 3 below and in
In conclusion, 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide tablet formulation showed similar mean plasma level profiles to wet granulation capsule formulation (secondary peak in capsule profile due to one dog) at the comparable doses on a per kg basis administered (19.6 and 20.1 mg/kg, respectively). There was less variability observed with the tablet formulation relative to the wet granulation capsule, % CV's for AUC, 25% and 50%, respectively, and for Cmax, 43% and 71%, respectively. The dose-normalized AUC from the tablet, 287 ng·hr/mL per mg/kg, was lower than that from the wet granulation capsule, 458 ng·hr/mL per mg/kg. However, the higher AUC from the wet granulation capsule was influenced by secondary peak in one dog. Excluding the dog with secondary peak, the dose-normalized AUC of the wet granulation capsule will be 363 ng·hr/mL per mg/kg.
The accelerated stability of the capsules of Examples 1 and 2 was studied. The capsules were packaged in HDPE bottles and stored at 40° C./75% RH and under ICH option 2 light condition. The samples were assayed by HPLC for potency and impurities and by dissolution apparatus. No apparent decrease in potency or increase in impurities was observed after 2 weeks under ICH option 2 light condition and after 3 months of storage at 40° C./75% RH for both the 25 mg and the 200 mg strengths. No change in dissolution was also observed under all conditions. The stability data for 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide pharmaceutical formulations of Examples 1 and 2 is shown in Table 4 below.
*Dissolution method: 1% SLS in water, peddle 100 RPM followed by HPLC analysis.
While the invention has been described above with reference to specific embodiments thereof, it is apparent that many changes, modifications, and variations can be made without departing from the inventive concept disclosed herein. Accordingly, it is intended to embrace all such changes, modifications, and variations that fall within the spirit and broad scope of the appended claims. All patent applications, patents, and other publications cited herein are incorporated by reference in their entirety.
This application claims the benefit of U.S. Provisional Patent Application No. 60/735,191, filed Nov. 10, 2005, the entire disclosure of which is incorporated by reference herein.
Number | Date | Country | |
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60735191 | Nov 2005 | US |