AMPHIPHILIC ENTITY NANOPARTICLES

Abstract

The present invention provides nanoparticle compositions comprising AE nanoparticles. The present invention provides AE nanoparticles comprising one or more amphiphilic entities and pharmaceutical compositions comprising AE nanoparticles. The present invention provides methods of manufacturing AE nanoparticles. The present invention provides methods of delivering a biologically active agent to a subject by administering AE nanoparticles containing a biologically active agent to a subject.

Claims

1-186. (canceled)

187. A method for treating a subject having a dermatological condition, the method comprising transdermally administering to the subject a nanoemulsion comprising a population of particles in a dispersion medium and a botulinum toxin, wherein the nanoemulsion comprises oily particles dispersed within an aqueous dispersion medium, or wherein the nanoemulsion comprises aqueous particles dispersed within an oily dispersion medium; wherein the majority of particles have diameters between 10 and 300 nanometers; wherein the particles comprise one or more amphiphilic entities; wherein said one or more amphiphilic entities is hyaluronic acid, wherein the nanoemulsion penetrates the skin of the subject without changing or altering the structure of the skin; thereby treating the subject having the dermatological condition.

188. The method of claim 187, wherein the one or more amphiphilic entities comprise a pullulan component and a polycaprolactone component.

189. The method of claim 187, wherein fewer than 5% of the particles have a diameter in excess of 300 nm.

190. The method of claim 187, wherein the difference between the minimum particle diameter and the maximum particle diameter does not exceed approximately 600 nm.

191. The method of claim 187, wherein the difference between the minimum particle diameter and the maximum particle diameter does not exceed approximately 300 nm.

192. The method of claim 187, wherein the difference between the minimum particle diameter and the maximum particle diameter does not exceed approximately 100 nm.

193. The method of claim 187, wherein the majority of particles have diameters below a specified size and within a specified range.

194. The method of claim 187, wherein more than 70% of the majority of particles have diameters below a specified size and within a specified range.

195. The method of claim 187, wherein more than 90% of the majority of particles have diameters below a specified size and within a specified range.

196. The method of claim 187, wherein more than 99.5% of the majority of particles have diameters below a specified size and within a specified range.

197. The method of claim 187, wherein more than 99.9% of the majority of particles have diameters below a specified size and within a specified range.

198. The method of claim 187, wherein the particles have an average diameter ranging between 50-250 nm.

199. The method of claim 187, wherein the nanoemulsion comprises an oil and a surfactant.

200. The method of claim 199, wherein the oil and surfactant are present in a ratio ranging from 0.5-2.0.

201. The method of claim 200, wherein the percent of oil in the nanoemulsion ranges from 1%-30%.

202. The method of claim 187, wherein the botulinum toxin (a) is encapsulated within the particles; (b) is nestled within the particle membrane; (c) is associated with the particle surface; or a combination thereof.

203. The method of claim 187, wherein the nanoparticle composition nanoemulsion has a zeta potential ranging between 25 mV to +25 mV.

204. The method of claim 187, wherein the nanoemulsion penetrates the top layer of the skin of the subject without the use of skin permeation enhancers or abrasives.

205. The method of claim 187, wherein the nanoemulsion was generated by microfluidization.

206. The method of claim 205, wherein the microfluidization is a single-pass microfluidization.

Description

EXEMPLIFICATION

[0235] The following examples are only intended to provide illustrations of specific embodiments contemplated by the present invention. The examples are not intended in any way to be limiting.

Example 1

Formulation of a Self-Assembling Pullulan and Polycaprolactone Nanosphere

[0236] A mixture of 2.5 g of soybean oil and 2.5 g polysorbate 80 (Tween-80) was prepared. The mixture was stirred and heated at 40 C. for 5 minutes. 50 ml deionized water was added, and the resulting mixture was stirred and heated at 40 C. for 10 minutes. 5 ml DMSO containing 0.905 g pullulan and polycaprolactone was added, and the resulting mixture was stirred and heated at 45 C. for 10 minutes. 5 ml was taken for a pre-process sample. The remaining mixture was microfluidized in a single pass at 24,000 psi. The particle size of the pre-process sample was >4000 nm. The particle size after microfluidization was 155 nm.

Example 2

Sample Preparation for Microfluidized Sample (Per Sample)

[0237] A mixture of 100 l of microfluidized sample and 900 l of reagent (0.1 M sodium phosphate buffer, 1 mM EDTA, 0.25% Triton X-100, 160 IU/mL of triglyceride hydrolase, and 1 IU/ml of cholesterol esterase) was prepared in a 8 ml glass vial.

[0238] The resulting mixture was incubated at ambient temperature in the dark for 1 hour. 100 l of 5% sodium dodecyl sulfate was added, and the resulting mixture was vortexed for 30 seconds. 1 ml of ethanol was added, and the resulting mixture was vortexed for 30 seconds. 100 l of an internal standard was added, and the resulting mixture was vortexed for 30 seconds. 4 ml of a 1:1 mixture of hexane:ether with 1% ethanol and 0.1% BHT was added. Ethanol and BHT stabilize the ether to prevent peroxide formation. The resulting mixture was vortexed for 60 seconds then centrifuged for 2 minutes on medium speed. The supernatant was extracted with a glass pipet and was stored at 80 C. for up to 30 days. The supernatant was evaporated and redissolved in 40 l of methanol. 30 l was injected into an high-pressure liquid chromatography (HPLC) apparatus.

Example 3

Botulinum Toxin a Formulation with Pullulan and Polycaprolactone

[0239] A mixture of 1.6 g of soybean oil and 1.6 g of polysorbate 80 (Tween-80) is prepared and stirred for five minutes. In a separate container, a mixture of 100 ng of botulinum toxin A and 20 ml 0.9% saline is prepared and stirred for five minutes. The mixture of saline and botulinum toxin A is added to the mixture of oil and Tween-80 and stirred for 10 minutes. 5 ml of DMSO containing 0.905 grams pullulan and polycaprolactone are added, and the resulting mixture is stirred for 10 minutes. A 5 ml pre-process sample is taken. The remaining mixture is microfluidized in a single pass at 24,000 psi. The particle size before and after microfluidization is measured.

Example 4

Vitamin E Formulation with Pullulan and Polycaprolactone

[0240] A mixture of 2.5 g soybean oil and 1 g vitamin E is prepared. 2.5 g polysorbate 80 (Tween-80) is added. The resulting mixture is stirred and heated at 40 C. for 5 minutes. 50 ml water is added to the mixture, and the resulting mixture is stirred and heated at 40 C. for 10 minutes. 5 ml DMSO containing 0.905 g pullulan and polycaprolactone is added. The resulting mixture is stirred and heated at 45 C. for 10 minutes. A 5 ml pre-process sample is taken. The remaining mixture is microfluidized in a single pass at 24,000 psi. The particle size before and after microfluidization is measured.

Example 5

Tocopherol (Vitamin E) Analysis

[0241] Delta tocopherol concentrations can be measured for a patient blood plasma or for nanoparticle compositions. Delta tocopherol concentrations are determined by adding 200 L of plasma or nanoparticle composition with 10 L of retinyl acetate (internal standard; 10 g/mL) and 200 L of ethanol containing butylated hydroxytoluene (BHT) (10 mg/L) and 1.0 mL hexane followed by vortex mixing. The samples are centrifuged at 500 g for 5 minutes and the organic layer transferred to fresh tube. The sample residues are re-extracted with 1.0 mL of hexane and the organic layers are combined. The organic layers are evaporated under N.sub.2 and reconstituted with 200 L of ethanol containing BHT (10 mg/dL) and injected into an HPLC. The HPLC system is a Model 5600 CoulArray 8-channel system with two Model 580 pumps, a high-pressure gradient mixer, a peek pulse damper, a Model 540 autoinjector, a CoulArray Thermostatic Chamber and a serial array of eight coulometric electrodes (ESA Laboratories, Inc., Chelmsford, Mass., USA). The column is a 3.0150 mm, 3M, Supelcosil LC-18 (Supelco, Bellefonte, Pa., USA). The mobile phase consists of methanol/1Propanol/1 M ammonium acetate (78:20:2 v:v:v) at a flow rate of 0.8 mL/min The concentrations of delta tocopherol are determined by external standardization using purified solutions of delta tocopherol standards (Sigma Chemicals, St. Louis, Mo., USA).

Equivalents and Scope

[0242] The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

[0243] In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. For example, it is to be understood that any of the compositions of the invention can be used for inhibiting the formation, progression, and/or recurrence of adhesions at any of the locations, and/or due to any of the causes discussed herein or known in the art. It is also to be understood that any of the compositions made according to the methods for preparing compositions disclosed herein can be used for inhibiting the formation, progression, and/or recurrence of adhesions at any of the locations, and/or due to any of the causes discussed herein or known in the art. In addition, the invention encompasses compositions made according to any of the methods for preparing compositions disclosed herein.

[0244] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term comprising is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

[0245] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0246] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention (e.g., any amphiphilic entity, any component of an amphiphilic entity, any polymer, any biologically active agent, any surfactant, any dispersion medium, any release-retarding ingredient, any AE nanoparticle or composition comprising any AE nanoparticle, any route or location of administration, any purpose for which a composition is administered, etc.), can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects are excluded are not set forth explicitly herein.