Improved delivery of large agents

Abstract

Methods, compositions, and devices for enhancing transdermal delivery and / or bioavailability of large pharmaceutical agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese invention patent application with application number 201980091096.8, application date November 26, 2019, and invention name “Improved Delivery of Large Drugs”. The original application is the Chinese national phase application of the PCT patent application with international application number PCT / US2019 / 063351, which claims priority to U.S. provisional patent applications No. 62 / 774,677 filed on December 3, 2018, No. 62 / 789,407 filed on January 7, 2019, and No. 62 / 808,274 filed on February 20, 2019. The entire contents of each of these applications are hereby incorporated by reference in their entirety. Background Art

[0003] Considerable resources have been invested in developing effective transdermal delivery technologies. Those skilled in the art are well aware of the challenges associated with achieving effective transdermal delivery, particularly for large pharmaceutical agents. As the size of the molecule increases, transdermal permeability decreases, reaching a point where it is negligible or even nonexistent. Summary of the invention

[0004] Transdermal administration is often the subject of research in an attempt to provide an alternative route of administration of pharmaceutical agents without the adverse consequences associated with injection and oral delivery. For example, needles tend to cause local pain, bleeding, and bruising, and may expose the patient to transmissible diseases; oral administration may suffer from poor bioavailability of the drug due to the extremely acidic environment of the patient's stomach. In some embodiments, transdermal delivery has a more uniform, regular, and / or consistent pharmacokinetic profile compared to other routes of administration.

[0005] Although there are many advantages, transdermal drug delivery brings many logistical problems. Only a limited number of drugs have been proven to be administered by this approach. Transdermal delivery of active agents is difficult, including but not limited to hydrophilic molecules, macromolecular structures (e.g., greater than several hundred Daltons), genetic therapeutic agents, vaccines, etc. Prausnitz, MR & Langer, R., "Transdermal drug delivery", Nat Biotechnol. 26 (11): 1261-1268 (2008).

[0006] The present disclosure provides an improved technology for transdermal delivery of an agent of interest. In some embodiments, the present disclosure teaches that the combination of some microneedle technology and the topical application of a composition comprising a large agent can promote and / or otherwise improve the delivery of a topical agent. In some embodiments, such a composition can be or include an emulsion (e.g., nanoemulsion), emulsions, for example, comprising large agents. In some embodiments, such a composition may include or may not include an emulsion and / or may be formulated into, for example, liquids, gels, creams, or other preparations suitable for topical application.

[0007] Recently developed technologies (see, e.g., International Publication No. PCT / US17 / 53333, incorporated herein by reference) achieve various advantages by combining microneedle technology with emulsion technology for the transdermal delivery of agents of interest; in some embodiments, these technologies have been shown to achieve particularly surprising enhancements in the transdermal delivery of large molecular structures.

[0008] The present disclosure demonstrates that even greater advantages can be achieved when utilizing microneedle methods having a particular microneedle density and / or a particular microneedle puncture size. Surprisingly, the present disclosure teaches that particularly desirable results are achieved with microneedle methods having a relatively low density and / or a relatively small puncture size.

[0009] A variety of microneedle technologies have been developed that can be used to administer certain agents of interest. Microneedles can avoid certain disadvantages (e.g., pain and / or amount of bleeding) that are often associated with the use of larger needles (e.g., using standard injection techniques). Microneedle technology can utilize one or more (e.g., a series of) hollow or solid microneedles. The agent of interest can be placed in the microneedle (e.g., if the microneedle is hollow and / or if the agent is incorporated into the microneedle material) or on the microneedle (i.e., on its surface), and / or can be applied to the site before, during, or after microneedle treatment of the skin site. The agent in or on the microneedle can be released, for example, by diffusion or expulsion from the microneedle, or by rupture and / or disintegration of the microneedle material after application to the site.

[0010] In some embodiments, the present disclosure provides strategies in which microneedle treatment is used to "condition" the skin to which a transdermal product has been, is being, or will be applied (and in particular to pre-condition the skin before applying a large agent), such as microneedle skin conditioning (MSC). The present disclosure provides the following insights: such microneedle conditioning as described herein (e.g., microneedle conditioning of relatively low density and / or relatively small puncture size), even compared to microneedle conditioning methods of higher density and / or larger puncture size, can also provide significant benefits in enhancing the transdermal delivery of large agents (e.g., molecular weight exceeding about 100KDa or more). In some embodiments, the present disclosure teaches that microneedle treatment can provide surprising improvements in the delivery of large agents. In some embodiments, the present disclosure establishes that microneedle treatment may be particularly beneficial for the delivery of large agents in emulsion compositions (e.g., nanoemulsions).

[0011] Previous reports have found that microneedle conditioning strategies may only be useful for small molecular weight agents, as studies analyzing the transdermal delivery of small molecules (specifically, short hydrophilic peptides with a molecular weight in the range of 400-1000 Da) found that "skin permeability of peptides depends on their molecular weight and decreases as the molecular weight increases." Zhang, S. et al., "Enhanced delivery of hydrophilic peptides in vitro by transdermal microneedle pretreatment", Acta Pharmaceutica Sinica B. 4(1): 100-104 (2014). In addition, studies evaluating the effects of microneedle density, microneedle length, and / or microneedle puncture size on such small molecule delivery have determined that microneedle density has no effect on small molecule delivery and / or bioavailability, while relatively longer microneedle lengths and larger microneedle puncture sizes improve small molecule delivery and / or bioavailability. Although Yan's early report (Yan, G. et al., "Evaluation needle length and density of microneedle arrays in the pretreatment of skin for transdermal drug delivery", International Journal of Pharmaceutics, 391:7-12, 2010) pointed out that "if microneedles with sufficiently long needle length (>600 μm) are used and the needle density is low (<2000 needles / cm 2) are more effective in increasing drug flux”. Later work found that the method used by Yan had shortcomings, including that the type of assay used by Yan “may be prone to artifacts” and “may lead to biomechanical changes in skin tension and / or hydration levels, which may adversely affect the size of the microchannels” (see Donnelly, RF et al., Optical coherence tomography is a valuable tool in the study of the effects of microneedle arrays on skin penetration characteristics and in-skin dissolution, Journal of Controlled Release, 147:333-341, 2010). In addition, even the lowest microneedle density studied by Yan (400 needles / cm 2 ) is also relatively high. Furthermore, the data presented by Yan itself show that for needles less than 1100 μm in length, needle density has no significant effect on small molecule delivery (e.g., “drug flux” through the skin).

[0012] The Donnelly study cited above reported that microneedle (MN) penetration depth (rather than density or other factors) was the most important factor in determining effective drug permeation. Donnelly also determined that “varying MN spacing had no effect on the penetration depth achieved.” Ibid.

[0013] Thus, prior to the present disclosure, the art demonstrated that, at least at the density levels studied in the literature, different microneedle densities were not expected to affect the delivery of a drug through the skin. Of course, based on first principles, one might expect that if microneedle treatment improves delivery, then a relatively high microneedle density (and / or a relatively large microneedle puncture size) might be more effective. As each factor of microneedle density and microneedle diameter increases, the total surface area of ​​the skin that has been punctured also increases, which would be expected to allow more active ingredients to percutaneously penetrate the skin. However, the present disclosure records a surprising discovery that, at least for the delivery of large agents and / or agents in emulsions (e.g., nanoemulsions), relatively low microneedle densities and / or relatively small microneedle puncture sizes achieve better results. That is, it was surprisingly found that as the total surface area of ​​the skin punctured by the microneedles decreases, the bioavailability of large agents in the emulsion applied to the skin increases. In some embodiments, at about 2 to about 50 microneedles / cm 2Microneedle densities in the range of about 100 to about 60,000 μm can significantly improve transdermal delivery and / or bioavailability, even when compared to microneedle treatments using relatively higher microneedle densities. 2 Microneedle conditioning of the skin with a microneedle puncture size within a range of about 100 to about 30,000 μm can achieve significant transdermal delivery and / or bioavailability. In addition, the present disclosure further demonstrates that the use of smaller microneedle puncture sizes (e.g., between about 100 and about 30,000 μm) can achieve significant transdermal delivery and / or bioavailability. 2 Microneedle conditioning of the skin (within the range of 100 to 30,000 μm) can significantly improve transdermal delivery and / or bioavailability even compared to microneedle treatment using relatively larger microneedle puncture sizes. In some embodiments, the microneedle puncture size can be between about 100 and about 30,000 μm. 2 / within the scope of microneedles.

[0014] Prior to the present disclosure, one skilled in the art would understand from the literature that microneedle conditioning of the skin using any particular microneedle density would not be expected to enhance transdermal delivery of even small molecules, let alone large pharmaceutical agents. The present disclosure unexpectedly demonstrates that microneedle conditioning of the skin using from about 2 to about 50 microneedles / cm 2 Microneedle conditioning of the skin with microneedle densities ranging from about 100 to about 30,000 μm can significantly enhance the transdermal delivery of agents such as botulinum toxin, which has a molecular weight of about 150,000 Da. Standard antibodies also have a similar molecular weight. In addition, the present disclosure unexpectedly demonstrates that the use of microneedle densities ranging from about 100 to about 30,000 μm can significantly enhance the transdermal delivery of agents such as botulinum toxin, which has a molecular weight of about 150,000 Da. Standard antibodies also have a similar molecular weight. 2 Microneedle conditioning of the skin with microneedle puncture sizes within the microneedle range can significantly enhance the transdermal delivery of such large pharmaceutical agents.

[0015] Those skilled in the art who read this disclosure will understand that, logically, in addition to reducing the total surface puncture by reducing microneedle density and / or reducing microneedle puncture size, a comparable effect can be achieved by minimizing (e.g., reducing) the number of impressions made by the microneedle array on the skin of the treatment area. Consistent with this understanding, given the surprising observations above that a reduction in the total punctured surface area (e.g., can be achieved at least in part by a (smaller) microneedle puncture size, which may have additional advantages), the present disclosure further documents that a relatively small number of microneedle array impressions can result in greater bioavailability than a relatively large number of impressions. In some embodiments, at about 1 impression / cm 2 Up to about 5 impressions / cm 2 Microneedle imprinting within a range can achieve significant transdermal delivery and / or bioavailability.

[0016] This disclosure proves that with about 1 impression / cm 2 Up to about 4 impressions / cm 2In some embodiments, microneedle conditioning of the skin with microneedle impressions within a range of about 1 to about 20 impressions to the treatment site can achieve significant transdermal delivery and / or bioavailability. In addition, the present disclosure demonstrates that microneedle conditioning of the skin with microneedle impressions within a range of about 1 to about 13 impressions to the treatment site can significantly improve transdermal delivery and / or bioavailability even compared to microneedle treatments using relatively more microneedle impressions.

[0017] In addition, those skilled in the art who read this disclosure will understand that reducing the total surface puncture of the applied large agent by reducing the microneedle density, reducing the microneedle puncture size and / or reducing the microneedle array impression results in a reduction in the total product volume (i.e., the volume of the product formulation containing the large agent) applied to the skin area punctured by the microneedles compared to relatively high surface punctures. Therefore, the present disclosure teaches those of ordinary skill in the art that improved transdermal delivery and / or greater bioavailability of large agents can be achieved by reducing the total product volume (e.g., formulation containing the large agent) applied.

[0018] Without the teachings embodied herein, one of ordinary skill in the art would typically expect that applying more product would result in increased biological effect. Thus, prior to the present disclosure, it has been expected that applying increasing amounts of a product containing a bioactive agent (e.g., a large agent), including in combination with MSCs and / or by including an emulsion (e.g., a nanoemulsion) product composition, should achieve increasingly greater biological effect. However, the present disclosure surprisingly demonstrates that, beyond a certain "critical" or "threshold" product volume, further increasing the volume of the product composition applied to a given skin treatment area in combination with MSCs becomes less effective (rather than more effective). Thus, once certain critical product volumes are exceeded, despite an increase in the dose of the bioactive agent applied, applying (e.g., in combination with MSCs) an increasing volume of a product composition containing a bioactive agent (e.g., a large agent, and particularly including in the case where the product composition comprises an emulsion) reduces the biological effect.

[0019] In some embodiments, about 1 / 100 drop / cm 2 Up to about 5 drops / cm 2 In some embodiments, a product volume within a range of about 1 / 100 drops / cm2 is applied to the skin, combined with conditioning the skin with one or more impressions of the microneedle array, to achieve significant transdermal delivery and / or bioavailability. 2 To about 4 drops / cm 2In some embodiments, a product volume within a range of about 1 / 100 drops / cm2 is applied to the skin, combined with conditioning the skin with one or more impressions of the microneedle array, to achieve significant transdermal delivery and / or bioavailability. 2 To about 3 drops / cm 2 In some embodiments, a product volume within a range of about 1 / 100 drops / cm2 is applied to the skin, combined with conditioning the skin with one or more impressions of the microneedle array, to achieve significant transdermal delivery and / or bioavailability. 2 To about 2.5 drops / cm 2 The application of a product volume within a range of about 1 / 100 drops / cm to the skin, combined with conditioning the skin with one or more impressions of the microneedle array, can achieve significant transdermal delivery and / or bioavailability. 2 To about 2 drops / cm 2 Application of a product volume within a range (e.g., a composition comprising a large pharmaceutical agent) to the skin, combined with conditioning the skin with one or more impressions of a microneedle array, can significantly improve transdermal delivery and / or bioavailability, even when compared to the use of relatively higher product volumes (and / or doses) (e.g., a large pharmaceutical agent).

[0020] In some embodiments, about 0.0001 mls / cm 2 To about 0.04mls / cm 2 In some embodiments, a product volume within a range of about 0.0001 mls / cm2 (e.g., a composition containing a large pharmaceutical agent) is applied to the skin, combined with conditioning the skin with one or more impressions of the microneedle array, to achieve significant transdermal delivery and / or bioavailability. 2 To about 0.05mls / cm 2 In some embodiments, a product volume within a range of about 0.0001 mls / cm2 (e.g., a composition containing a large pharmaceutical agent) is applied to the skin, combined with conditioning the skin with one or more impressions of the microneedle array, to achieve significant transdermal delivery and / or bioavailability. 2 To about 0.06mls / cm 2 In some embodiments, a product volume within a range of about 0.0001 mls / cm2 (e.g., a composition containing a large pharmaceutical agent) is applied to the skin, combined with conditioning the skin with one or more impressions of the microneedle array, to achieve significant transdermal delivery and / or bioavailability. 2 To about 0.065mls / cm 2In some embodiments, the application of a product volume within a range of about 0.0025 mls / cm to the skin, combined with conditioning the skin with one or more impressions of the microneedle array, can achieve significant transdermal delivery and / or bioavailability. 2 To about 0.07mls / cm 2 Application of a range of product volumes (e.g., a composition comprising a large pharmaceutical agent) to the skin, combined with conditioning the skin with one or more impressions of a microneedle array, can significantly improve transdermal delivery and / or bioavailability, even when compared to the use of relatively higher product volumes (and / or doses) of large pharmaceutical agents.

[0021] One of the advantages provided by certain embodiments of the present disclosure (e.g., those that reduce the volume of applied product compositions) is a reduction in the time it takes to apply (e.g., rub in) a topical formulation. As already noted, the present disclosure documents that, surprisingly, reducing the volume of a topically applied agent (and / or reducing the dose in some embodiments) can actually achieve greater delivery (e.g., bioavailability) of the agent across the skin, particularly (but not limited to), for example, when the agent is an applied emulsion (e.g., nanoemulsion) formulation.

[0022] As previously mentioned, studies on the effect of microneedle length on small molecule delivery have determined that relatively long microneedle lengths improve small molecule delivery and / or bioavailability. See Yan's report (Yan, G. et al., "Evaluation needle length and density of microneedle arrays in the pretreatment of skin for transdermal drug delivery", International Journal of Pharmaceutics, 391: 7-12, 2010). However, those of ordinary skill in the art who read this disclosure will understand that reducing total surface puncture improves transdermal delivery and / or active agent (e.g., large agent bioavailability) compared to relatively large total surface puncture. Therefore, logically, those of ordinary skill in the art who read this disclosure will understand that by reducing the microneedle length of a microneedle skin conditioning array, improved transdermal delivery and / or greater bioavailability of large agents can be achieved. Those of ordinary skill in the art will understand that the longer the microneedle, the larger the base of the microneedle must be in order to structurally support the increased length, and the larger the base of the needle, the larger the puncture area produced by the needle. Those of ordinary skill in the art will appreciate that the longer the microneedle, the greater the surface area of ​​the punctured tissue due to the increased surface area of ​​the microneedle itself. The present disclosure surprisingly demonstrates that microneedle skin conditioning using relatively short microneedle lengths can result in greater bioavailability than when using relatively long microneedle lengths. In some embodiments, microneedle lengths in the range of about 1 μm to about 900 μm can achieve significant transdermal delivery and / or bioavailability. In some embodiments, microneedle lengths below 1400 μm, and in some embodiments below about 1100 μm or even 1000 μm are desirable. In fact, the present disclosure specifically records the surprising effectiveness of microneedle lengths of about 800 μm or less. In some embodiments, microneedle lengths in the range of about 15 μm to about 800 μm can achieve significant transdermal delivery and / or bioavailability. In some embodiments, the present disclosure records the surprising effectiveness of microneedle lengths of about 500 μm or less. In some embodiments, microneedle lengths ranging from about 15 μm to about 500 μm can achieve significant transdermal delivery and / or bioavailability.

[0023] In some embodiments, the microneedle length may be in the range of about 50 μm to about 900 μm. In some embodiments, the present disclosure demonstrates that microneedle conditioning of the skin at a treatment site with a microneedle length in the range of about 50 μm to about 900 μm or about 100 μm to about 700 μm can significantly improve transdermal delivery and / or bioavailability, even compared to microneedle treatments using relatively longer microneedle lengths. In some embodiments, microneedle conditioning of the skin at a treatment site with a microneedle length in the range of about 100 μm to about 800 μm can significantly improve transdermal delivery and / or bioavailability, even compared to microneedle treatments using relatively longer microneedle lengths. In some embodiments, microneedle conditioning of the skin at a treatment site with a microneedle length in the range of about 15 μm to about 500 μm can significantly improve transdermal delivery and / or bioavailability, even compared to microneedle treatments using relatively longer microneedle lengths. In some embodiments, microneedle conditioning of the skin at a treatment site using microneedle lengths less than about 800 μm can significantly increase transdermal delivery and / or bioavailability, even compared to microneedle treatments using relatively longer microneedle lengths.

[0024] The unexpected discovery documented herein that shorter needle lengths can achieve more effective drug delivery (i.e., described as "increasing bioavailability") provides one advantage in reducing pain in subjects receiving microneedle skin conditioning (e.g., associated with and / or as part of a topical treatment) to administer large pharmaceutical agents. Microneedles of varying lengths (including, for example, in the range of about 500 to about 1400 μm) are readily available and have been described as being particularly useful because bleeding can often be minimized or even avoided using these lengths. However, the present disclosure recognizes that even in the absence of bleeding, significant pain can be experienced, particularly for lengths up to 1400 μm, and sometimes even shorter. The present disclosure provides significant advantages in avoiding or reducing pain for topical delivery, even for large pharmaceutical agents, particularly (but not limited to) those administered in the context of emulsion (or even nanoemulsion) formulations, by documenting the effectiveness of microneedle lengths significantly less than 1400 μm, and in some embodiments even less than 1100 μm, 1000 μm, 900 μm, or less.

[0025] The present disclosure demonstrates, inter alia, that microneedle technology (e.g., microneedle conditioning of the skin using relatively low needle density, relatively small microneedle puncture size (e.g., the puncture size of each microneedle), relatively few microneedle impressions, relatively small product volume (and / or dose), and relatively short needle length) can significantly enhance the transdermal delivery of large pharmaceutical agents, particularly (but not limited to) in emulsion compositions (e.g., macroemulsion compositions and / or nanoemulsion compositions). As exemplified, for example, prior to administration of a large pharmaceutical agent of interest (botulinum toxin), microneedle density of less than about 31 microneedles / cm 2 The microneedle array of the present invention uses microneedles to precondition the skin, which surprisingly enhances the delivery of large pharmaceutical agents across the skin. The specific examples included herein record this enhanced delivery under various conditions and / or environments (e.g., different skin sites, application quantities, etc.). Those skilled in the art will recognize other changes that fall within the scope of the present disclosure (e.g., application sites, dosage quantities, etc.).

[0026] Nanoemulsion compositions of particular interest include water-in-oil and oil-in-water nanoemulsions characterized by droplet sizes ranging from about 10 nm to about 300 nm in diameter, ratios of aqueous dispersion medium to oil ranging between about 0.01:1 and about 20:1; oil to surfactant ratios spanning from about 0.1 to about 40, and / or zeta potentials spanning from about -80 mV to about +80 mV (see, e.g., descriptions of nanoemulsion compositions in one or more of PCT / US2006 / 26918; PCT US06 / 46236; PCT / US2012 / 22276; and PCT / US2012 / 22279, the disclosures of each of which are incorporated herein by reference in their entireties).

[0027] International Publication No. PCT / US17 / 53333 has described certain surprising features achieved by the combination of microneedle treatment and emulsion (e.g., nanoemulsion) technology, considering that there are reports that transdermal delivery of solid nanoparticles of comparable droplet size (e.g., 105±2.92 nm) to the nanoemulsion composition utilized herein does not effectively transdermally deliver (or enhance delivery) even small molecule agents. For example, Gomaa et al. describe a study in which a solution of rhodamine dye (molecular weight 479 Da) encapsulated in PLGA nanoparticles was applied to skin that had been preconditioned by microneedle treatment and skin penetration was evaluated. See Gomaa, Y. et al., "Effect of microneedle treatment on the skin permeation of a nanoencapsulated dye", J Pharm Pharmacol. 2012 November; 64(11): 1592-1602. The data showed that after 6 hours of continuous application, a very small amount of dye began to penetrate the skin; no significant increase in penetration was observed until the skin was treated for 24 hours. The researchers explain that "there is an emerging consensus that NPs [nanoparticles] generally do not penetrate the stratum corneum, although they may be well deposited in hair follicles." Therefore, prior to the present disclosure, those skilled in the art expected that even small molecule agents (e.g., rhodamine dyes) would not be effectively delivered transdermally using microneedle technology with nanosized vehicles; certainly, delivery and / or enhanced bioavailability of large agents was considered impossible. International Publication No. PCT / US17 / 53333 demonstrates that microneedle treatment can significantly enhance the transdermal delivery of large agents, particularly when used in conjunction with an emulsion (e.g., nanoemulsion) system. The present disclosure further demonstrates that the use of microneedle densities of about 2 to about 50 microneedles / cm2 can significantly enhance the transdermal delivery of large agents, particularly when used in conjunction with an emulsion system (e.g., nanoemulsion system), when compared to utilizing relatively higher microneedle densities. 2 Microneedle treatment within a range can significantly enhance the transdermal delivery of large pharmaceutical agents and / or improve bioavailability. In some embodiments, when compared to using relatively higher microneedle densities, especially when used in conjunction with an emulsion system (e.g., a nanoemulsion system), using a microneedle density of less than about 40 microneedles / cm 2 (For example, between about 2 and about 40 microneedles / cm 2 or more preferably using a microneedle density of less than about 35 microneedles / cm 2 (For example, between about 2 and about 35 microneedles / cm 2 microneedle treatment within the range of about 32 microneedles / cm 2 (For example, between about 2 and about 32 microneedles / cm 2microneedle treatment within the range of about 31 microneedles / cm 2 (For example, between about 2 and about 31 microneedles / cm 2 microneedle treatment in the range of about 30 microneedles / cm 2 (For example, between about 2 and about 30 microneedles / cm 2 microneedle treatment in the range of about 29 microneedles / cm 2 (For example, between about 2 and about 29 microneedles / cm 2 microneedle treatment within the range of about 28 microneedles / cm 2 (For example, between about 2 and about 28 microneedles / cm 2 Microneedle treatment within the range of can significantly enhance the transdermal delivery of large pharmaceutical agents and / or improve bioavailability.

[0028] Furthermore, the present disclosure unexpectedly demonstrates that the use of a molten-liquid crystal structure in the range of about 100 to about 60,000 μm 2 Microneedle conditioning of the skin with microneedle puncture sizes in the range of about 100 to about 30,000 μm can achieve significant transdermal delivery and / or bioavailability. In addition, the present disclosure demonstrates that the use of smaller microneedle puncture sizes, such as between about 100 and about 30,000 μm, can achieve significant transdermal delivery and / or bioavailability when compared to the use of relatively larger microneedle puncture sizes, particularly when used in conjunction with an emulsion system (e.g., a nanoemulsion system). 2 Microneedle treatment within the range of about 50,000 μm / microneedle can significantly enhance the transdermal delivery of large pharmaceutical agents and / or improve bioavailability. In some embodiments, when compared to using relatively larger microneedle puncture sizes, especially when used in conjunction with emulsion systems (e.g., nanoemulsion systems), the use of microneedle puncture sizes below about 50,000 μm is preferred. 2 / microneedles (e.g., between about 100 and about 50,000 μm 2 Microneedle treatment with a microneedle having a puncture size of less than about 45,000 μm 2 / microneedles (e.g., between about 100 and about 45,000 μm 2 Microneedle treatment with a microneedle having a puncture size of less than about 40,000 μm 2 / microneedles (e.g., between about 100 and about 40,000 μm 2 / microneedle range) or, more preferably, using microneedle puncture size less than about 35,000 μm 2 / microneedles (e.g., between about 100 and about 35,000 μm 2 Microneedle treatment with a microneedle having a puncture size of less than about 30,000 μm 2 / microneedles (e.g., between about 100 and about 30,000 μm 2 Microneedle treatment with a microneedle having a puncture size of less than about 25,000 μm 2 / microneedles (e.g., between about 100 and about 25,000 μm 2 Microneedle treatment (within the scope of microneedles) can significantly enhance the transdermal delivery of large pharmaceutical agents and / or improve bioavailability.

[0029] Among other things, the present disclosure demonstrates that microneedle technology as described herein can enhance transdermal delivery (e.g., of large agents, particularly large agents from macroemulsion or nanoemulsion compositions) when no other disrupting agents are utilized (i.e., no chemical permeation enhancers, and no other techniques for disrupting or piercing skin structure). Previous studies using microneedles to transdermally deliver agents as large as botulinum toxin (i.e., about 150 kDa) have reported that delivery was unsuccessful unless additional treatment was performed to disrupt the skin. For example, U.S. Patent Publication No. 2010 / 0196445 reports that botulinum toxin cannot be effectively delivered from pre-coated microneedles unless skin digesting enzymes are also administered to disrupt the skin structure at the site of the microneedles.

[0030] In some embodiments, the present disclosure provides a technique for achieving enhanced transdermal delivery and / or improved bioavailability of large agents (e.g., botulinum toxin, antibodies, etc.) by utilizing microneedle technology as described herein, without the need for additional use of a penetration enhancer. Alternatively or additionally, in some embodiments, the present disclosure provides a technique for achieving enhanced transdermal delivery and / or improved bioavailability of large agents (e.g., botulinum toxin, antibodies, etc.) by utilizing microneedle technology, without the need for any other disruptive strategies. Thus, the provided techniques can achieve effective delivery and / or improved bioavailability without the inflammation, irritation, and / or allergic reactions that often accompany the use of skin disruptive agents.

[0031] Alternatively or additionally, the present disclosure identifies the source of the problem with certain existing methods that incorporate large agents, and particularly large protein agents (e.g., botulinum toxin, antibodies, etc.) into or onto microneedle structures. Typically, such conventional incorporation strategies utilize liquid solutions of the relevant agents, which are applied to the microneedles and allowed to air dry. This strategy was utilized in the aforementioned U.S. Patent Publication No. 2010 / 0228225 to coat microneedles with botulinum toxin. U.S. Patent Publication No. 2017 / 0209553 describes a microneedle array with botulinum toxin in the needles. The present disclosure recognizes that the resulting botulinum toxin coating or loading material is unstable and therefore not commercially viable when used to prepare a product. In fact, even if such a liquid is prepared from a powdered material, the present disclosure recognizes that for many large agents (e.g., botulinum toxin), powders and other solid materials that are not formed through a freeze-drying process may be highly unstable. For example, per Johnson, E., et al., "Botulinum toxin is very susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. Lyophilization or freeze-drying of botulinum toxin is the most economically sound and practical method of distributing the product in a form that is stable and readily used by the clinician," U.S. Pat. No. 5,512,547. Similarly, this approach does not work for the administration of therapeutic antibodies, which have their own stability and storage challenges. The present disclosure provides the following insights: the use of emulsion compositions (e.g., in some embodiments, nanoemulsion compositions, and / or in some embodiments, macroemulsion compositions) as described herein can protect or otherwise improve the stability of large agents, particularly large protein agents, and specifically including botulinum toxin and / or antibody agents, for use in conjunction with microneedles.

[0032] The present disclosure provides surprisingly effective technologies for transdermal delivery of large medicaments and / or improving bioavailability. Specifically, the present disclosure teaches that the transdermal delivery of such medicaments can be significantly enhanced by using certain microneedle technologies. Those skilled in the art who read the present disclosure will understand that its teachings may be applicable to any topical preparations of large medicaments. In some embodiments, the present disclosure particularly teaches that particularly favorable results are achieved when microneedle technology is combined with an emulsion composition (e.g., in some embodiments, a nanoemulsion composition, and / or in some embodiments, a coarse emulsion composition). In some embodiments, microneedle technology is combined with a lotion, an ointment, or a liquid composition, which in turn can be or include an emulsion composition (e.g., in some embodiments, a nanoemulsion embodiment and / or in some embodiments, a coarse emulsion composition). In some embodiments, the technology provided does not utilize skin disruption technology, such as a chemical penetration enhancer.

[0033] Description of the drawings

[0034] Figure 1 Depicted are the effects of varying microneedle array densities on the bioavailability of botulinum nanoemulsion formulations following MSC ("microneedle skin conditioning"), as determined by survival in a rat study.

[0035] Figure 2 The effects of different microneedle puncture sizes on the bioavailability of botulinum toxin nanoemulsion formulations after MSC, as determined by survival in a rat study, are depicted.

[0036] definition

[0037] In this application, unless the context clearly indicates otherwise, (i) the term "a" or "an" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising" and "including" may be understood to cover the listed components or steps, whether presented by themselves or together with one or more additional components or steps; and (iv) the terms "about" and "approximately" may be understood to allow for standard variations as understood by those of ordinary skill in the art; and (v) if a range is provided, the endpoints are included.

[0038] Abrasion: As used herein, the term "abrasion" refers to any means of altering, damaging, removing or destroying the top layer of the skin. In some embodiments, abrasion refers to mechanical means of altering, damaging, removing or destroying the top layer of the skin. In some embodiments, abrasion refers to chemical means of altering, damaging, removing or destroying the top layer of the skin. To name a few examples, agents such as exfoliants, fine particles (e.g., magnesium or aluminum particles), acids (e.g., alpha-hydroxy acids or beta-hydroxy acids), alcohols can cause abrasion. In general, penetration enhancers such as those described by Donovan (e.g., U.S. Publications 2004 / 009180 and 2005 / 175636, and PCT Publication WO 04 / 06954) and Graham (e.g., U.S. Pat. No. 6,939,852 and U.S. Publication 2006 / 093624), etc., are expected to cause abrasion. Of course, one of ordinary skill in the art will understand that a particular agent may cause abrasion when present in one concentration, or in combination with one or more other agents, but may not cause abrasion under different circumstances. Thus, whether a particular material is an "abrasive" depends on the context.Abrasion can be readily assessed by one of ordinary skill in the art, for example by observation of redness or irritation of the skin and / or histological examination of the skin, which shows alteration, disruption, removal or erosion of the stratum corneum.

[0039] Administration: As used herein, the term "administering" generally refers to administering a composition to a subject or system. One of ordinary skill in the art will appreciate that various routes of administration to a subject (e.g., a human) may be used where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some embodiments, administration may be bronchial (e.g., by bronchial instillation), oral, dermal (which may be or include, for example, topical to the dermis, intradermal, interdermal, transdermal, etc., one or more), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, in a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may include intermittent administration (e.g., multiple doses separated in time) and / or periodic administration (e.g., individual doses separated by a common period of time). In some embodiments, administering can include continuous administration (eg, perfusion) for at least a selected period of time.

[0040] Agent: In general, as used herein, the term "agent" may be used to refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. Where appropriate, as will be clear from the context to one skilled in the art, the term may be used to refer to an entity that is or comprises a cell or organism, or a part, extract, or component thereof. Alternatively or additionally, as will be clear from the context, the term may be used to refer to a natural product, because it occurs in nature and / or is obtained from nature. In some cases, as will also be clear from the context, the term may be used to refer to one or more artificial entities, because it is designed, engineered, and / or produced by the action of the human hand and / or does not occur in nature. In some embodiments, an agent may be utilized in an isolated or pure form; in some embodiments, an agent may be utilized in a crude form. In some embodiments, a potential agent may be a collection or library that is used, for example, to screen to identify or characterize an active agent therein. In some cases, the term "agent" may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymer moieties. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymer moieties. In some embodiments, the term may refer to a molecular complex.

[0041] Antibody: As used herein, the term "antibody" refers to a polypeptide that includes classical immunoglobulin sequence elements sufficient to confer specific binding to a specific target antigen. As known in the art, a naturally occurring complete antibody is a tetrameric agent of approximately 150 kDa, which is composed of two identical heavy chain polypeptides (each approximately 50 kDa) and two identical light chain polypeptides (each approximately 25 kDa), which are bound to each other in a structure generally referred to as a "Y shape". Each heavy chain consists of at least four domains (each approximately 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CH1, CH2, and carboxyl-terminal CH3 (located at the base of the Y stem). A short region called a "switch" connects the heavy chain variable region and the constant region. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in a complete antibody. Each light chain consists of two domains - an amino-terminal variable (VL) domain, followed by a carboxyl-terminal constant (CL) domain, separated from each other by another "switch". A complete antibody tetramer consists of two heavy chain-light chain dimers, in which the heavy chain and the light chain are connected to each other by a single disulfide bond; two additional disulfide bonds connect the heavy chain hinge regions to each other, so that the dimers are connected to each other and form a tetramer. Naturally produced antibodies are also glycosylated, usually on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed by two β folds (e.g., 3-, 4- or 5-chain sheets) packed relative to each other in a compressed antiparallel β barrel. Each variable domain contains three hypervariable loops called "complementarity determining regions" (CDR1, CDR2 and CDR3) and four slightly invariant "framework" regions (FR1, FR2, FR3 and FR4). When the natural antibody is folded, the FR region forms a beta fold that provides a structural framework for the domain, and the CDR loop regions from both the heavy chain and the light chain are gathered together in three-dimensional space so that they produce a single hypervariable antigen binding site located at the top of the Y structure. The Fc region of the naturally occurring antibody binds to elements of the complement system and also binds to receptors on effector cells (including, for example, effector cells that mediate cytotoxicity). As known in the art, the affinity and / or other binding properties of the Fc region to Fc receptors can be regulated by glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized according to the present invention include glycosylated Fc domains, including such glycosylated Fc domains with modifications or engineering. For the purposes of the present invention, in some embodiments, any polypeptide or polypeptide complex including enough immunoglobulin domain sequences as found in natural antibodies can be referred to and / or used as "antibodies", whether such polypeptides are naturally produced (e.g., produced by an organism reacting with an antigen), or produced by recombinant engineering, chemical synthesis or other artificial systems or methods.In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has a constant region sequence specific to a mouse, rabbit, primate or human antibody. In some embodiments, as known in the art, antibody sequence elements are humanized, primatized, chimeric, etc. In addition, the term "antibody" as used herein may refer to any construct or form known in the art or developed in appropriate embodiments (unless otherwise specified or clear from the context) for utilizing antibody structure and functional characteristics in alternative presentations. For example, embodiments, antibodies utilized according to the present invention are in a form selected from, but not limited to, complete IgG, IgE and IgM, bi- or multi-specific antibodies (e.g.,. etc.), single-chain Fv, polypeptide-Fc fusion, Fab, colloidal antibody, masked antibody (e.g., ), small modular immunopharmaceuticals (“SMIPs TM ”), single-chain or tandem diabodies VHH, Microantibodies, Ankyrin repeat protein or DART, TCR-like antibodies, Microprotein, and In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally (e.g., in a mammal). In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other side groups [e.g., polyethylene glycol, etc.].

[0042] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a specific antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), small modular immunopharmaceuticals ("SMIPs"), TM"), single-chain antibodies, colloidal antibodies, and antibody fragments. As used herein, the term "antibody agent" also includes intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies (e.g., bispecific antibodies) formed by at least two intact antibodies, and antibody fragments, as long as they exhibit the desired biological activity. In some embodiments, the term encompasses stapled peptides. In some embodiments, the term encompasses one or more antibody-like binding peptide mimetics. In some embodiments, the term encompasses one or more antibody-like binding scaffold proteins. In some embodiments, the term encompasses monoclonal antibodies. Clonal antibody or adnectin protein (adnectin). In many embodiments, the antibody agent is or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementary determining regions (CDRs); in some embodiments, the antibody agent is or includes a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to the CDR present in the reference antibody. In some embodiments, the included CDR is substantially identical to the reference CDR, wherein the included CDR is identical in sequence or contains between 1-5 amino acid substitutions compared to the reference CDR. In some embodiments , the included CDRs are substantially identical to the reference CDRs, wherein the included CDRs exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs, wherein the included CDRs exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs, wherein at least one of the included CDRs is identical to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs, wherein 1-5 amino acids within the included CDRs are deleted, added or substituted compared to the reference CDRs, but the included CDRs have otherwise identical amino acid sequences to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs, wherein at least one amino acid within the included CDRs is substituted compared to the reference CDRs, but the included CDRs have otherwise identical amino acid sequences to the reference CDRs.In some embodiments, the included CDR is substantially identical to the reference CDR, wherein 1-5 amino acids within the included CDR are deleted, added or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements that are recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or mostly homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is or comprises an antibody-drug conjugate.

[0043] Antibody component: As used herein, refers to a polypeptide element that specifically binds to an epitope or antigen and includes one or more immunoglobulin structural features (which may be a complete polypeptide, or a part of a larger polypeptide (e.g., such as a fusion polypeptide as described herein)). Generally, an antibody component is any polypeptide whose amino acid sequence includes elements unique to an antibody binding region (e.g., an antibody light chain or variable region or one or more complementary determining regions ("CDR") thereof, or an antibody heavy chain or variable region or one or more CDRs thereof, optionally with one or more framework regions). In some embodiments, an antibody component is or comprises a full-length antibody. In some embodiments, an antibody component is less than the full length but includes at least one binding site (comprising at least one, and preferably at least two sequences having a known antibody "variable region" structure). In some embodiments, the term "antibody component" encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In a specific embodiment, the included "antibody component" encompasses a polypeptide having a binding domain that shows at least 99% identity to an immunoglobulin binding domain. In some embodiments, the "antibody component" included is any polypeptide having a binding domain that shows at least 70%, 75%, 80%, 85%, 90%, 95% or 98% identity to an immunoglobulin binding domain (e.g., a reference immunoglobulin binding domain). The "antibody component" included may have an amino acid sequence identical to an antibody (or a portion thereof, such as an antigen binding portion thereof) found in a natural source. The antibody component may be monospecific, bispecific or multispecific. The antibody component may include structural elements unique to any immunoglobulin class, including any human class: IgG, IgM, IgA, IgD and IgE. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual-specific or multispecific forms that specifically bind to two or more different antigens. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include: (i) a Fab fragment, which is a fragment of a V H 、V L , C H 1 and CL (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a fragment consisting of V H and C H (iv) a single-arm V fragment of the antibody; H and V L (v) a dAb fragment comprising a single variable domain (Ward et al. (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR). In addition, although the two domains (V H and V L ) are encoded by separate genes, but they can be joined using recombinant methods by synthetic linkers that enable them to be made into H and V L The single protein chain that pairs with the V domain to form a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). In some embodiments, as described herein, an "antibody component" is or comprises such a single-chain antibody. In some embodiments, an "antibody component" is or comprises a diabody. A diabody is a bivalent, bispecific antibody in which V H and V L The domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen binding sites (see, e.g., Holliger, P., et al., (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, (1994) Structure 2 (12): 1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel, eds., Antibody Engineering (2001) Springer-Verlag. New York. p. 790 (ISBN 3-540-41354-5). In some embodiments, the antibody component is or comprises a single-chain "linear antibody" comprising a pair of tandem Fv fragments (VFs) that form a pair of antigen binding regions together with complementary light chain polypeptides. H -C H 1-V H -C H1) (Zapata et al., (1995) Protein Eng. 8(10): 1057-1062; and U.S. Pat. No. 5,641,870). In some embodiments, the antibody component may have structural elements that are characteristic of chimeric or humanized antibodies. Typically, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit with the desired specificity, affinity and capacity. In some embodiments, the antibody component may have structural elements that are characteristic of human antibodies.

[0044] Antibody fragment: As used herein, "antibody fragment" includes a portion of an intact antibody, such as, for example, an antigen binding region or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed by antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments consisting of the variable regions of the heavy and light chains; recombinant single-chain polypeptide molecules in which the light chain variable region and the heavy chain variable region are connected by a peptide linker ("ScFv protein"); and a minimum recognition unit consisting of amino acid residues that mimic a hypervariable region. In many embodiments, an antibody fragment contains enough parent antibody sequence, wherein it is a fragment that binds to the same antigen as the parent antibody; in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementary determining regions (CDR) regions. Antigen-binding fragments of antibodies can be produced by any means. For example, antigen-binding fragments of antibodies can be produced enzymatically or chemically by fragmentation of intact antibodies and / or they can be produced recombinantly from genes encoding partial antibody sequences. Alternatively or additionally, antigen-binding fragments of antibodies can be produced synthetically in whole or in part. Antigen-binding fragments of antibodies can optionally include single-chain antibody fragments. Alternatively or additionally, antigen-binding fragments of antibodies can include multiple chains, which are, for example, linked together by disulfide bonds. Antigen-binding fragments of antibodies can optionally include multimolecular complexes. Functional antibody fragments typically include at least about 50 amino acids, and more typically include at least about 200 amino acids.

[0045] Approximately: As used herein, when applied to one or more values ​​of interest, the term "approximately" or "about" refers to a value similar to the reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​within a range of (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the reference value, unless otherwise stated or otherwise apparent from the context (e.g., when one or more values ​​of interest define a sufficiently narrow range, applying such a percentage variance will eliminate the range).

[0046] Related: When the term is used herein, two events or entities are "associated" with each other if the presence, level and / or form of one is related to the presence, level and / or form of the other. For example, if the presence, level and / or form of a particular entity (e.g., a polypeptide, a genetic feature, a metabolite, a microorganism, etc.) is associated with the incidence and / or susceptibility of a disease, disorder or condition (e.g., across a related population), it is considered to be associated with a particular disease, disorder or condition. In some embodiments, if two or more entities interact directly or indirectly so that they are in physical proximity and / or remain in physical proximity to each other, they are physically "associated" with each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0047] Biocompatibility: As used herein, the term "biocompatible" refers to materials that do not cause significant damage to living tissue when in contact with such tissue, such as in vivo. In some embodiments, materials are "biocompatible" if they are not toxic to cells. In some embodiments, a material is "biocompatible" if its addition to cells in vitro results in less than or equal to 20% cell death and / or its in vivo administration does not cause significant inflammation or other such adverse reactions.

[0048] Biodegradable: As used herein, the term "biodegradable" refers to a material that, when introduced into a cell, is broken down (e.g., by cellular machinery, such as by enzymatic degradation, by hydrolysis, and / or by a combination thereof) into components that can be reused or disposed of by the cell without significant toxic effects on the cell. In some embodiments, the components produced by the decomposition of the biodegradable material are biocompatible and therefore do not cause significant inflammation and / or other adverse effects in the body. In some embodiments, the biodegradable polymeric material decomposes into its component monomers. In some embodiments, the decomposition of a biodegradable material (including, for example, a biodegradable polymeric material) involves the hydrolysis of an ester bond. Alternatively or additionally, in some embodiments, the decomposition of a biodegradable material (including, for example, a biodegradable polymeric material) involves the cleavage of a carbamate bond. Exemplary biodegradable polymers include, for example, hydroxy acids, such as lactic acid and glycolic acid, including but not limited to polymers of poly(hydroxy acids), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), and copolymers with PEG, polyanhydrides, poly(o-)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates, poly(lactide-co-caprolactone), blends and copolymers thereof. Many naturally occurring polymers are also biodegradable, including, for example, proteins (such as albumin, collagen, gelatin, and prolamins, such as zein) and polysaccharides (such as alginates, cellulose derivatives, and polyhydroxyalkanoates, such as polyhydroxybutyrate), blends and copolymers thereof. One of ordinary skill in the art will understand or be able to determine when such polymers are biocompatible and / or biodegradable derivatives thereof (e.g., related to the parent polymer by substantially the same structure, the structure differing only in substitution or addition of specific chemical groups as known in the art).

[0049] Bioactive agent: As used herein, the term "bioactive agent" refers to an agent that has a specific biological effect when administered to a subject (e.g., a human). In some embodiments, a bioactive agent may be a therapeutic agent, a cosmetic agent, and / or a diagnostic agent. In some embodiments, a bioactive agent may be or include an entity or portion classified as an "active pharmaceutical ingredient" by the U.S. Food and Drug Administration. In some embodiments, a bioactive agent is a large agent. In some embodiments, a bioactive agent may be or include an agent whose presence is associated with a desired pharmacological and / or therapeutic, cosmetic, and / or diagnostic effect. In some embodiments, a bioactive agent is characterized in that its biological effect is dose-dependent (e.g., optionally increasing with increasing dose in a linear manner over at least a first concentration range). In some embodiments, an agent is not considered a "bioactive agent" if it merely enhances the delivery of a different agent that actually achieves the desired effect.

[0050] Botulinum macroemulsion composition: As used herein, the term "botulinum macroemulsion composition" refers to any macroemulsion composition wherein at least one macroemulsion includes a botulinum toxin. The botulinum toxin may be present within the macroemulsion, on the surface of the macroemulsion, and / or within the micellar membrane defining the macroemulsion.

[0051] Botulinum nanoemulsion composition: As used herein, the term "botulinum nanoemulsion composition" refers to any nanoemulsion composition wherein at least one nanoemulsion includes a botulinum toxin. The botulinum toxin may be present within the nanoemulsion, on the surface of the nanoemulsion, and / or within the micellar membrane defining the nanoemulsion.

[0052] Botulinum toxin: As used herein, the term "botulinum toxin" refers to any neurotoxin produced by Clostridium botulinum. Unless otherwise indicated, the term encompasses fragments or portions (e.g., light and / or heavy chains thereof) of such toxins that retain the appropriate activity (e.g., muscle relaxant activity). As used herein, the phrase "botulinum toxin" encompasses botulinum toxin serotypes A, B, C, D, E, F, and G. As used herein, botulinum toxin also encompasses both botulinum toxin complexes (i.e., e.g., 300, 600, and 900 kDa complexes) and purified (i.e., e.g., isolated) botulinum toxins (i.e., e.g., about 150 kDa). "Purified botulinum toxin" is defined as a botulinum toxin that is separated or substantially separated from other proteins, including proteins used in the botulinum toxin complex. The purified toxin may be greater than 95% pure, and preferably greater than 99% pure. One of ordinary skill in the art will appreciate that the present invention is not limited to any particular source of botulinum toxin. For example, the botulinum toxin used in accordance with the present invention can be isolated from Clostridium botulinum, can be chemically synthesized, can be recombinantly produced (i.e., in a host cell or organism other than Clostridium botulinum), etc. Clostridium botulinum can be genetically engineered or chemically modified to have a longer or shorter duration of action than botulinum toxin serotype A.

[0053] Carrier: As used herein, refers to a diluent, adjuvant, excipient or vehicle with which a composition is administered. In some exemplary embodiments, carriers may include sterile liquids such as, for example, water and oils, including oils of petroleum, animal, vegetable and synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, a carrier is or includes one or more solid components.

[0054] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents, a therapeutic agent and a treatment modality, etc.). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any doses of the second regimen are administered); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may include administering one or more agents and / or modalities to a subject receiving other agents or modalities in the combination. For clarity, combination therapy does not require that the separate agents be administered together in a single composition (or even must be administered simultaneously), although in some embodiments, two or more agents or their active portions may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0055] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, condition groups, etc., which may be different from each other but similar enough to allow comparison between them, so that those skilled in the art will understand that conclusions can be reasonably drawn based on the observed differences or similarities. In some embodiments, comparable conditions, environments, individuals or groups of people are characterized by a variety of substantially identical features and one or a few different features. In the context, those of ordinary skill in the art will understand that for two or more such agents, entities, situations or condition groups, etc., under any given environment, what degree of identity is required to be considered comparable. For example, those of ordinary skill in the art will understand that environments, individuals or groups of people are comparable to each other when characterized by: a sufficient number and type of substantially identical features to ensure that the following reasonable conclusions are drawn: the differences in the results or observed phenomena obtained under or with different environments, individuals or groups of people are caused by changes in those different features or indicate changes in those different features.

[0056] Composition: Those skilled in the art will appreciate that, as used herein, the term "composition" may be used to refer to a discrete physical entity comprising one or more specified components. Generally, unless otherwise specified, a composition may be in any form - e.g., gas, gel, liquid, solid, etc.

[0057] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it is also understood that any composition or method described as "comprising" (or "including") one or more named elements or steps also describes a corresponding, more limited composition or method, which "consists essentially of" (or "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps, and may also include additional elements or steps that do not substantially affect the basic and novel features of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of": one or more named elements or steps also describes a corresponding, more limited and closed composition or method, which "consists of" (or "consisting of") the named elements or steps, to the exclusion of any other unnamed elements or steps. In any composition or method disclosed herein, a known or disclosed equivalent of any named basic element or step may replace the element or step.

[0058] Dosage form or unit dosage form: It will be understood by those skilled in the art that the term "dosage form" can be used to refer to physically discrete units of an active agent (e.g., a therapeutic agent or a diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of an active agent. In some embodiments, such an amount is a unit dose (or its entire portion) suitable for administration according to a dosing regimen that has been determined to be associated with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). It will be understood by those of ordinary skill in the art that the total amount of a therapeutic composition or medicament administered to a particular subject is determined by one or more attending physicians and may include the administration of a variety of dosage forms.

[0059] Dosing regimen: Those skilled in the art will understand that the term "dosing regimen" can be used to refer to a group of unit doses (usually more than one) that are administered separately to a subject, usually separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen includes multiple doses, each dose separated in time from the other doses. In some embodiments, multiple doses are separated from each other by a period of time of the same length; in some embodiments, the dosing regimen includes multiple doses and at least two different time periods that separate the individual doses. In some embodiments, all doses within the dosing regimen have the same unit dosage. In some embodiments, different doses within the dosing regimen have different amounts. In some embodiments, the dosing regimen includes a first dose administered in a first dosage, followed by one or more additional doses administered in a second dosage different from the first dosage. In some embodiments, the dosing regimen includes a first dose administered in a first dosage, followed by one or more additional doses of the same second dosage as the first dosage. In some embodiments, when administered in a relevant population, the dosing regimen is associated with a desired or beneficial outcome (i.e., a therapeutic dosing regimen).

[0060] Emulsion: The term "emulsion" is used herein consistent with the art's understanding of "a system consisting of a liquid dispersed in an immiscible liquid, usually in droplets larger than a colloid size, with or without an emulsifier." See, e.g., the definition in Medline Plus Online Medical Dictionary, Merriam Webster (2005).

[0061] Excipient: As used herein, refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilization effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc.

[0062] Human: In some embodiments, the human is an embryo, fetus, infant, child, adolescent, adult, or elderly.

[0063] Hydrophilic: As used herein, the terms "hydrophilic" and / or "polar" refer to the tendency to mix with or dissolve readily in water.

[0064] Hydrophobic: As used herein, the terms "hydrophobic" and / or "non-polar" refer to the tendency to repel, not bind to, or not readily dissolve in water.

[0065] Improvement, increase or decrease: As used herein or their grammatical equivalents, the terms "improvement," "increase," or "decrease" indicate a value relative to a baseline measurement (such as a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein). In some embodiments, a "control individual" is an individual with the same form of disease or injury as the individual being treated.

[0066] Macromolecule: The term "macromolecule" is generally used herein to describe a molecule having a size greater than about 100 kilodaltons (KDa). In some embodiments, the macromolecule is greater than about 110KDa, 120KDa, 130KDa, 140KDa, 150KDa, 160KDa, 170KDa, 180KDa, 190KDa, 200KDa, 250KDa, 300KDa, 400KDa, or 500KDa. In some embodiments, the macromolecule is a polymer or comprises a polymer portion or entity. In some embodiments, the macromolecule is or comprises a polypeptide. In some embodiments, the macromolecule is or comprises a nucleic acid.

[0067] Large agent: As used herein, the term "large agent" generally refers to an agent having a molecular weight greater than about 100 kilodaltons (KDa) in size. In some embodiments, the macromolecule is greater than about 110KDa, 120KDa, 130KDa, 140KDa, 150KDa, 160KDa, 170KDa, 180KDa, 190KDa, 200KDa, 250KDa, 300KDa, 400KDa, or 500KDa. In some embodiments, the large agent is a bioactive agent. In some embodiments, the large agent is or comprises one or more macromolecules. In some embodiments, the large agent is or comprises one or more molecular complexes. In some embodiments, the large agent is or comprises a polypeptide. In some embodiments, the large agent is or comprises a polypeptide complex. In some embodiments, the large agent is or comprises a bacterial toxin (e.g., botulinum toxin). In some embodiments, the large agent is or comprises an antibody agent.

[0068] Macroemulsion: As used herein, the term "macroemulsion" refers to an emulsion in which at least some of the droplets have a diameter in the range of hundreds of nanometers to micrometers. As will be understood by one of ordinary skill in the art, a macroemulsion is characterized by droplets having a diameter greater than 300 nm. In some embodiments, the macroemulsion composition utilized in accordance with the present disclosure includes one or more macropharmaceuticals or one or more bioactive agents. In some embodiments, the macropharmaceutical contained in the macroemulsion composition can be a bioactive agent. One of ordinary skill in the art will understand that the macroemulsion composition used in accordance with the present disclosure can be prepared according to any available means, including, for example, chemical or mechanical means. In some embodiments, the droplets in the macroemulsion have a size in the range of about 301 nm to about 1000 μm. In some embodiments, the macroemulsion has droplets with a size distribution between about 301 nm and about 1000 μm. In some embodiments, the droplets in the macroemulsion have a size in the range of about 500 nm to about 5000 μm. In some embodiments, the macroemulsion has droplets with a size distribution between about 500 nm and about 5000 μm.

[0069] Microneedle: The term "microneedle" as used herein generally refers to an elongated structure having a suitable length, diameter, and shape to penetrate the skin. In some embodiments, the microneedle is arranged and constructed (by itself or within the device) to minimize contact with nerves when inserted into the skin, while still creating an effective pathway for drug delivery. In some embodiments, the microneedle has a consistent diameter along the length of the microneedle. In some embodiments, the microneedle has a diameter that varies along the length of the microneedle. In some embodiments, the microneedle has a diameter that tapers along the length of the microneedle. In some embodiments, the diameter of the microneedle is narrowest at the tip that penetrates the skin. In some embodiments, the microneedle may be solid. In some embodiments, the microneedle may be hollow. In some embodiments, the microneedle may be tubular. In some embodiments, the microneedle may be sealed at one end. In some embodiments, multiple microneedles are utilized. In some embodiments, multiple microneedles are utilized in array form. In some embodiments, the microneedle may have a length ranging from about 1 μm to about 4,000 μm. In some embodiments, the microneedle may have a length between about 1 μm to about 2,000 μm. In some embodiments, the microneedles may have a length between about 50 μm and about 400 μm. In some embodiments, the microneedles may have a length between about 800 μm and about 1500 μm.

[0070] Microneedle array impression: As used herein, the term "microneedle array impression" refers to a microneedle impression obtained by imprinting microneedles and / or microneedle arrays onto the skin and then removing them from the skin. In some embodiments, the microneedle array can be stamped onto the skin (e.g., with a microneedle array stamp). In some embodiments, the microneedle array can be rolled onto the skin (e.g., with a microneedle array roller).

[0071] Microneedle density: As used herein, the term "microneedle density" refers to the number of microneedles per unit area (e.g., square centimeters). In some embodiments, microneedle density is assessed as the number of microneedles per unit area of ​​the microneedle array; in some embodiments, microneedle density is assessed as the number of microneedle punctures per unit area of ​​the microneedle puncture site; in some embodiments, microneedle density is assessed as the number of microneedles per unit area that simultaneously achieves the maximum or near-maximum skin penetration possible for the microneedles in the array. In any case, one of ordinary skill in the art will understand that microneedle density can be expressed regardless of whether the area of ​​interest is flat (e.g., a microneedle array stamp), curved (e.g., a microneedle array roller), or irregular. One of ordinary skill in the art will understand that assessing microneedle density as microneedle punctures per unit area of ​​the microneedle puncture site may be particularly useful, for example, if the array has needles of different lengths and / or the microneedle puncture site has topological diversity, such that when the array is applied to the site, not every needle can actually puncture the skin.

[0072] Microneedle puncture size: As used herein, the term "microneedle puncture size" or "microneedle hole puncture size" refers to the calculated puncture area produced by each microneedle of a microneedle array after the microneedle and / or microneedle array is impressed onto the skin and then removed from the skin. In most embodiments, the microneedle puncture size is calculated as the area of ​​the microneedle base.

[0073] Nanoemulsion: As used herein, the term "nanoemulsion" refers to an emulsion in which at least some of the droplets have a diameter in the nanometer size range. As will be understood by one of ordinary skill in the art, a nanoemulsion is characterized by droplets having a diameter of 300 nm or less. In some embodiments, the nanoemulsion compositions utilized in accordance with the present disclosure include one or more macro-agents or one or more bioactive agents. In some embodiments, the macro-agents contained in the nanoemulsion compositions can be bioactive agents. One of ordinary skill in the art will understand that the nanoemulsion compositions used in accordance with the present disclosure can be prepared according to any available means, including, for example, chemical or mechanical means. In some embodiments, the droplets in the nanoemulsion have a size in the range of about 1 nm to about 300 nm. In some embodiments, the nanoemulsion has droplets with a size distribution between about 1 nm and about 300 nm.

[0074] Nanoparticle: As used herein, the term "nanoparticle" refers to a solid particle having a diameter less than 300 nm, as defined by the National Science Foundation of the United States. In some embodiments, the nanoparticle has a diameter less than 100 nm, as defined by the National Institutes of Health.

[0075] Patient: As used herein, the term "patient" refers to any organism to which a provided composition may be administered, for example, for experimental, diagnostic, preventive, cosmetic and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates and / or humans). In some embodiments, the patient is human. In some embodiments, the patient suffers from or is susceptible to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient is diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer or the presence of one or more tumors. In some embodiments, the patient is receiving or has received certain therapies to diagnose and / or treat the disease, disorder or condition.

[0076] Permeation enhancer: As used herein, the term "permeation enhancer" refers to an agent whose presence or level is associated with increased permeation of an agent of interest through the skin compared to that observed in its absence. In some embodiments, a permeation enhancer is characterized in that it degrades and / or disrupts skin structure. In some embodiments, a permeation enhancer is or comprises a chemical agent (e.g., a chemical or an enzyme, for example). For example, a chemical agent that can damage, disrupt and / or degrade one or more components of the stratum corneum can include, for example, alcohols, such as short chain alcohols, long chain alcohols, or polyols; amines and amides, such as urea, amino acids, or their esters, amides, Derivatives, pyrrolidones or pyrrolidone derivatives; terpenes and terpene derivatives; fatty acids and their esters; macrocyclic compounds; surfactants; or sulfoxides (e.g., dimethyl sulfoxide (DMSO), decyl methyl sulfoxide, etc.); surfactants, such as anionic, cationic and nonionic surfactants; polyols; essential oils; and / or hyaluronidase. In some embodiments, the penetration enhancer may be an irritant because an inflammatory and / or allergic reaction occurs when the agent is applied to the skin. In some embodiments, the penetration enhancer is not an irritant. In some embodiments, the penetration enhancer may be or include a chemical agent that does not damage, destroy or degrade the skin structure, but its presence or level is still associated with an increased penetration of the agent of interest through the skin compared to that observed in its absence. In some embodiments, co-peptides, carrier molecules and carrier peptides may be penetration enhancers that do not damage, destroy and / or degrade the skin structure. In some embodiments, co-peptides, carrier molecules and carrier peptides may be penetration enhancers that do not irritate the skin. The term "penetration enhancer" does not include mechanical devices (e.g., needles, scalpels, etc.) or their equivalents (e.g., other damaging treatments). Additionally, one skilled in the art will appreciate that a structure such as a nanoparticle or emulsion is not a chemical agent, and therefore is not a chemical penetration enhancer, even though its presence is associated with enhanced skin penetration of an agent of interest that may be related to the structure.

[0077] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage amount suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those suitable for topical administration, for example, sterile solutions or suspensions, or sustained release formulations, as gels, creams, ointments, or controlled release patches or sprays suitable for use on the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, lungs, and other mucosal surfaces.

[0078] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" as applied to a carrier, diluent or excipient used to formulate the compositions disclosed herein means the carrier, diluent or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0079] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject or patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; maltose; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, medium chain triglycerides, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances used in pharmaceutical formulations.

[0080] Premix: As used herein, the term "premix" refers to any combination of components that are subsequently used to produce a nanoemulsion composition or according to the present invention. For example, a premix is ​​a collection of any ingredients that, when subjected to high shear forces, produces a nanoemulsion according to the present invention. In some embodiments, a premix is ​​a collection of ingredients that, when subjected to high shear forces, produces a nanoemulsion composition, such as a uniform nanoemulsion composition. A premix often contains a liquid dispersion medium and other components sufficient to produce a nanoemulsion in the dispersion medium. According to some embodiments of the present disclosure, one or more large pharmaceutical agents may be included in the premix. According to some embodiments of the present disclosure, one or more biological pharmaceutical agents may be included in the premix. According to the present invention, botulinum toxin may be included in the premix. According to the present invention, one or more antibodies may be included in the premix. In some embodiments, the premix may contain one or more surfactants, penetration enhancers and / or other agents. In some embodiments, the premix comprises a solution. In some embodiments where the premix comprises a botulinum toxin, an antibody, another bioactive agent, and / or a permeation enhancer, the botulinum toxin, the antibody, another bioactive agent, and / or a permeation enhancer are in solution prior to applying high shear forces to the premix.

[0081] Prevent or prevention: As used herein, when used in conjunction with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing a disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of a disease, disorder, and / or condition. Prevention is considered complete when the onset of a disease, disorder, or condition is delayed for a predetermined period of time.

[0082] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids connected to each other by peptide bonds). Proteins may contain parts other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.), and / or may be processed or modified in other ways. One of ordinary skill in the art will understand that a "protein" may be a complete polypeptide chain produced by a cell (with or without a signal sequence), or may be a characteristic part thereof. One of ordinary skill in the art will understand that a protein may sometimes include more than one polypeptide chain, for example, linked by one or more disulfide bonds or linked by other means. A polypeptide may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, a protein may contain natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, a protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0083] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will understand that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having the complete sequences listed herein, but also polypeptides representing functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). In addition, those of ordinary skill in the art understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, retaining activity and sharing at least about 30%-40% total sequence identity with another polypeptide of the same class, often greater than about 50%, 60%, 70% or 80%, and further generally including at least one region with higher identity, often greater than 90% or even 95%, 96%, 97%, 98% or 99% in one or more highly conserved regions, generally covering at least 3-4 and often up to 20 or more amino acids Any polypeptide is included within the relevant term "polypeptide" as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, the protein may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0084] Reference: As used herein, describes a standard or control relative to which a comparison is made. For example, in some embodiments, an agent, animal, individual, colony, sample, regimen, sequence, or value of interest is compared to a reference or control agent, animal, individual, colony, sample, regimen, sequence, or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as understood by those skilled in the art, a reference or control is determined or characterized under conditions or environments comparable to those under evaluation. Those skilled in the art will understand when there is sufficient similarity to justify reliance on and / or comparison with a particular possible reference or control.

[0085] Self-administration: As used herein, the term "self-administration" refers to a situation where a subject has the ability to administer a composition to him or herself without medical supervision. In some embodiments of the invention, self-administration can be performed outside of a clinical setting. As an example, in some embodiments of the invention, a facial cosmetic cream can be applied by a subject in his or her own home.

[0086] Small molecule: Generally, "small molecule" is understood in the art as an organic molecule having a size of less than about 5 kilodaltons (Kd). In some embodiments, a small molecule is less than about 3Kd, 2Kd, or 1Kd. In some embodiments, a small molecule is less than about 800 Daltons (D), 600D, 500D, 400D, 300D, 200D, or 100D. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not a protein, peptide, or amino acid. In some embodiments, a small molecule is not a nucleic acid or nucleotide. In some embodiments, a small molecule is not a carbohydrate or polysaccharide.

[0087] Subject: As used herein, "subject" means an organism, typically a mammal (e.g., a human, including prenatal human forms in some embodiments). In some embodiments, the subject suffers from a relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics of susceptibility to a disease, disorder, or condition or having a risk thereof. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom diagnosis and / or treatment is administered and / or has been administered.

[0088] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting a characteristic or property of interest in total or near total extent or degree. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, reach perfection and / or proceed to completeness or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0089] Therapeutic agent: As used herein, the phrase "therapeutic agent" generally refers to any agent that triggers a desired pharmacological effect when administered to an organism. In some embodiments, if the agent shows a statistically significant effect in an appropriate population, the agent is considered to be a therapeutic agent. In some embodiments, the appropriate population can be a model organism population. In some embodiments, the appropriate population can be defined by various criteria, such as a specific age group, gender, genetic background, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, alleviate, inhibit, prevent, delay the onset, reduce its severity and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder and / or condition. In some embodiments, a "therapeutic agent" is an agent that has been or needs to be approved by a government agency before it can be sold to a person for administration. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription for administration to a person. In some embodiments, if the agent only enhances the delivery of a different agent that actually achieves the desired effect, the agent is not considered to be a "therapeutic agent."

[0090] Therapeutically effective amount: As used herein, it means the amount that produces the desired effect achieved by applying this amount. In some embodiments, the term refers to an amount sufficient to treat the disease, disorder and / or condition when applied to a population suffering from or susceptible to a disease, disorder and / or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder and / or condition and / or delays its onset. One of ordinary skill in the art will understand that the term "therapeutically effective amount" does not actually need to achieve successful treatment in a particular individual. On the contrary, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a considerable number of subjects when applied to a patient who needs such treatment. In some embodiments, a therapeutically effective amount can refer to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of ordinary skill in the art will understand that in some embodiments, a therapeutically effective amount of a specific agent or therapy can be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0091] Therapeutic regimen: As the term is used herein, a "therapeutic regimen" refers to a regimen of dosing, the administration of which in the relevant population is associated with desired or beneficial therapeutic outcomes.

[0092] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be used for subjects who do not show signs of the relevant disease, disorder, and / or condition, and / or subjects who only show early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be used for subjects who show one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0093] Uniform: When used herein to refer to a nanoemulsion composition, the term "uniform" refers to a nanoemulsion composition in which each droplet has a droplet diameter size within a specific range. For example, in some embodiments, a uniform nanoemulsion composition is a nanoemulsion composition in which the difference between the minimum diameter and the maximum diameter does not exceed about 300, 250, 200, 150, 100, 90, 80, 70, 60, 50 or less nm. In some embodiments, the droplets in the uniform large-dose nanoemulsion composition of the present invention (e.g., droplets containing a large drug) have a diameter of less than about 300, 250, 200, 150, 130, 120, 115, 110, 100, 90, 80nm or less. In some embodiments, the droplets in the uniform large-dose nanoemulsion composition of the present invention (e.g., droplets containing a large drug) have a diameter within the range of about 10 and about 300 nanometers. In some embodiments, the droplets in the uniform large-drug nanoemulsion composition of the present invention have a diameter within the range of about 10-300, 10-200, 10-150, 10-130, 10-120, 10-115, 10-110, 10-100, or 10-90 nm. In some embodiments, the droplets in the large-drug nanoemulsion composition of the present invention (e.g., droplets containing large-drug) have an average droplet size of less than about 300, 250, 200, 150, 130, 120, or 115, 110, 100, or 90 nm. In some embodiments, the average droplet size is within the range of about 10-300, 50-250, 60-200, 65-150, 70-130 nm. In some embodiments, the average droplet size is about 80-110 nm. In some embodiments, the average droplet size is about 90-100 nm. In some embodiments, the majority of droplets in the uniform nanoemulsion composition of the present invention (e.g., droplets containing large agents) have a diameter below a specified size or within a specified range. In some embodiments, the majority is more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more of the droplets in the composition. In some embodiments of the present invention, the uniform nanoemulsion composition is achieved by microfluidization of the sample.

[0094] Variant: As used herein, the term "variant" refers to an entity that, compared to a reference entity, exhibits significant structural identity to a reference entity, but differs in structure from the reference entity in the presence or level of one or more chemical moieties. In many embodiments, a variant is also functionally different from its reference entity. In general, whether a particular entity is properly considered a "variant" of a reference entity is based on the degree of its structural identity to the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a variant is a unique chemical entity that shares one or more such characteristic structural elements. To name just a few examples, a small molecule may have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic pendant moieties, and thus a variant of a small molecule is a molecule that shares the core structural element and the characteristic pendant moieties, but differs in the type of bonds (single vs. double, E vs. Z, etc.) present in the other pendant moieties and / or in the core, a polypeptide may have a characteristic sequence element consisting of a plurality of amino acids that have a specified position relative to one another in linear or three-dimensional space and / or contribute to a specific biological function, and a nucleic acid may have a characteristic sequence element consisting of a plurality of nucleotide residues that have a specified position relative to one another in linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide due to one or more differences in the amino acid sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the variant polypeptide shows an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% with a reference polypeptide, optionally except for conservative amino acid substitutions. Alternatively or additionally, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more biological activities of the reference polypeptide. In some embodiments, the variant polypeptide lacks one or more biological activities of the reference polypeptide. In some embodiments, compared with the reference polypeptide, the variant polypeptide shows a reduced level of one or more biological activities. In many embodiments, if the polypeptide of interest has the same amino acid sequence as the parent, but has a small amount of sequence changes in a specific position, the polypeptide of interest is considered to be a "variant" of the parent or reference polypeptide. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted compared to the parent. In some embodiments, the variant has 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substituted residues compared to the parent.Typically, variants have a very small number (e.g., less than 5, 4, 3, 2, or 1) of substituted functional residues (i.e., residues involved in a particular biological activity). In addition, variants typically have no more than 5, 4, 3, 2, or 1 additions or deletions, and often no additions or deletions, compared to the parent. In addition, any additions or deletions are typically less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is a polypeptide found in nature.

[0095] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0096] Transdermal Drug Delivery

[0097] In some embodiments, the present invention provides technology for improving transdermal delivery and / or bioavailability of large pharmaceutical agents (e.g., botulinum toxin, antibodies). In some embodiments, the disclosure teaches that particularly advantageous results are achieved when microneedle technology is combined with an emulsion composition. In some embodiments, microneedle technology is combined with a lotion, cream, or liquid composition, which in turn may be or include an emulsion composition (e.g., a coarse emulsion composition and / or a nanoemulsion composition). In some embodiments, the technology provided does not utilize a penetration enhancer. In some embodiments, the technology provided does not utilize a chemical penetration enhancer that damages, destroys, and / or degrades the skin. In some embodiments, the technology provided does not utilize a chemical penetration enhancer.

[0098] Human skin includes the dermis and the epidermis. The epidermis has several layers of tissue, namely the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale (identified in order from the outer surface of the skin inward).

[0099] The stratum corneum is generally the most important barrier in transdermal delivery (and presumably especially for large pharmaceutical agents). The stratum corneum is generally about 10-15 μm thick and is composed of flat cornified cells (keratinocytes) arranged in several layers. The intercellular spaces between the keratinocytes are filled with lipid structures and may play an important role in the penetration of substances through the skin (Bauerova et al., 2001, European Journal of Drug Metabolism and Pharmacokinetics, 26:85).

[0100] The remaining epidermis below the stratum corneum is approximately 150 μm thick. The dermis is approximately 1-2 mm thick and lies beneath the epidermis. The dermis is innervated by various capillaries as well as neuronal processes.

[0101] Transdermal administration has generally been the subject of research attempting to provide alternative routes of administration without the undesirable consequences associated with injection and oral delivery. For example, needles often cause local pain and may expose the patient receiving the injection to blood-borne diseases. Oral administration often has poor drug bioavailability due to the extremely acidic environment of the patient's stomach.

[0102] Efforts have been made to develop transdermal administration techniques for certain drugs in an attempt to overcome these disadvantages by providing non-invasive administration. Transdermal administration is generally desired to minimize damage to the patient's skin. Thus, transdermal administration can reduce or eliminate the pain associated with injections, reduce the possibility of blood contamination, and improve the bioavailability of drugs once they are systemically incorporated.

[0103] Traditionally, attempts at transdermal administration have focused on the destruction and / or degradation of the stratum corneum. Some attempts include the use of chemical penetration enhancers. A penetration enhancer can act to degrade and / or disrupt the structure of the skin. In some embodiments, a penetration enhancer is or comprises a chemical agent (e.g., a chemical agent or an enzyme, e.g., that can destroy and / or degrade one or more stratum corneum components). In some embodiments, a penetration enhancer can be an irritant, because an inflammatory and / or allergic reaction occurs when the agent is applied to the skin.

[0104] "However, the main limitation of penetration enhancers is that their efficacy is often closely related to the occurrence of skin irritation." Alkilani, AZ, et al., "Transdermal drug delivery: Innovative pharmaceutical developments based on disruption of the barrier properties of the stratum corneum." Pharmaceutics. 7: 438-470 (2015). Penetration enhancers often have poor efficacy and safety profiles. "They fail to achieve the desired skin disruption, and their ability to increase transport across the skin is low and variable." Ibid.

[0105] Some attempts include using mechanical devices to bypass or ablate portions of the stratum corneum. Additionally, attempts include using ultrasound or iontophoresis to facilitate drug penetration through the skin. In most cases, the goal is to deliver an agent, typically a small molecule, so that it can be delivered to the capillary beds in the dermis where it can be systemically incorporated into the subject to achieve a therapeutic effect. These methods are limited by the amount of energy that can be applied to the skin without causing discomfort and / or skin damage.

[0106] Transdermal delivery of macromolecules

[0107] Microneedle technology has been demonstrated to enhance the transdermal delivery of various small agents, such as calcein (about 623 Da), desmopressin (about 1070 Da), diclofenac (about 270 Da), methyl nicotinate (about 40 Da), bischloroethylnitrosourea (about 214 Da), insulin (about 5.8 KDa), bovine serum albumin (about 66.5 KDa) and ovalbumin (about 45 KDa), however, prior to the present disclosure, the delivery and / or improved bioavailability of large agents, particularly large agents of 100 KDa or greater, remained problematic.

[0108] Transdermal delivery of macromolecules is considered a major challenge. Prior to the present disclosure, microneedle treatment, particularly microneedle skin preconditioning using relatively low microneedle density and / or relatively small microneedle puncture size (e.g., puncture size per microneedle), has not been considered to affect transdermal administration of large agents. For example, studies using solid microneedles to deliver four low molecular weight hydrophilic peptides tetrapeptide-3 (456.6 Da); hexapeptide (498.6 Da); acetyl hexapeptide-3 (889 Da); and oxytocin (1007.2 Da) and L-carnitine (161.2 Da) showed that while microneedle pretreatment significantly enhanced the penetration of each of these peptides, the skin penetration of the peptides depended on their molecular weight and decreased with increasing molecular weight. Zhang, S., et al., "Enhanced delivery of hydrophilic peptides in vitro by transdermal microneedle pretreatment." Acta Pharmaceutica Sinica B. 4(1): 100-104 (2014).

[0109] When sandpaper abrasion, tape stripping, and a single puncture hypodermic needle model of MSCs were compared in a study of the effect of molecular size of larger FITC (fluorescein isothiocyanate) conjugated molecules on transdermal delivery, it was found that for all methods, as well as when tested on untreated skin, transdermal drug delivery was again shown to decrease with increasing size of the tested molecule (4.3, 9.6, and 42.0 KDa FITC conjugates). Tape stripping was the most effective technique, while sandpaper abrasion was found to be the most skin damaging. Wu, X., et al., "Effects of pretreatment of needle puncture and sandpaperabrasion on the in vitro skin permeation of fluorescein isothiocyanate (FITC)-dextran." International Journal of Pharmaceutics. 316: 102-108 (2006).

[0110] Other studies have attempted to deliver even larger molecules: Cascade Blue (CB, Mw 538), Dextran-Cascade Blue (DCB, Mw 10 kDa), and FITC-conjugated dextran (FITC-Dex, Mw 72 kDa). In these studies, microneedles of varying lengths (300, 550, 700, or 900 μm) were used to puncture the skin of human patients, and the diffusion of each of the above compounds was evaluated. Although transport of each compound was observed with all but the 300 μm microneedle array, degradation of DCB and FITC-Dex was observed.

[0111] As shown in the prior art, as the size of the molecule increases, transdermal penetration using MSC ("microneedle skin conditioning") decreases, to the point where it is minimal and even non-existent. Even in cases where some trace penetration is observed, larger molecules are observed to become degraded and biologically inactive. Recently developed technologies (see, e.g., International Publication No. PCT / US17 / 53333) achieve various advantages by combining microneedle technology with emulsion technology for transdermal delivery of large agents of interest; in some embodiments, these technologies show that transdermal delivery of large molecular structures can be achieved without the use of mechanical or chemical permeation enhancers, which is a particularly surprising enhancement. For example, in some embodiments, these technologies have achieved transdermal delivery of botulinum toxin, which is about 150 KDa, more than twice the size of FITC-Dex. The present disclosure demonstrates that microneedle technology as described herein can unexpectedly further enhance transdermal delivery and / or improve bioavailability of large agents (e.g., molecular weight greater than 100 kDa (e.g., botulinum toxin)). Botulinum toxin is a complex protein that requires three regions or functional parts to be intact in order for the protein to be biologically active. Thus, damage to any of the three regions of the protein renders the protein biologically inactive. Per Johnson, E., et al., “Botulinum toxin is very susceptible to denaturation due to surface denaturation, heat, and alkaline conditions.” U.S. Patent Publication No. 5512547. Thus, under the microneedle conditions described by Wu, one would expect a significant level of degradation and inactivation of botulinum toxin.

[0112] Among other things, the present disclosure demonstrates that microneedle technology as described herein can enhance transdermal delivery and / or improve bioavailability (e.g., large agents, particularly large agents from macroemulsion or nanoemulsion compositions) when no other penetration enhancers, particularly disruptive agents, are utilized (e.g., no chemical penetration enhancers, and no other techniques that disrupt or pierce the skin structure).

[0113] Microneedling

[0114] The present disclosure provides surprising findings that MSC as described herein can surprisingly improve the transdermal delivery of large medicaments. In some embodiments, large medicaments can be formulated into creams and / or lotions. In some embodiments, large medicaments can be combined with one or more bioactive agents. In some embodiments, large medicaments can be formulated into emulsions (e.g., as coarse emulsions or nanoemulsions) compositions or in emulsion compositions. In some embodiments, emulsions comprising one or more large medicaments can be formulated into creams and / or lotions.

[0115] In some embodiments, the microneedle (MN) array used in accordance with the present disclosure is a minimally invasive system or shares features with a minimally invasive system, developed to overcome some of the disadvantages commonly associated with subcutaneous injections and the use of subcutaneous needles, as well as to improve patient comfort and compliance. Such disadvantages include, for example, the possibility of misalignment of the needle with the subcutaneous needle because the health care professional cannot see the exact location where the needle is going; when botulinum toxin is incorrectly injected into the face, this needle misalignment may lead to adverse reactions such as drooping eyelids ("ptosis"). MNs are less prone to such problems. Other advantages of MNs are that they may not cause bleeding, minimize the introduction of pathogens through the holes created by MNs, and eliminate the variability of transdermal administration. Other advantages are the possibility of self-administration, reduced risk of accidental needle sticks, reduced risk of spreading infection, and easy disposal. In some embodiments, MNs are multiple microscopic protrusions assembled on one side of a support, such as a patch or device (e.g., a stamp, a roller, an array, an applicator, a pen).

[0116] In some embodiments, the MNs used in accordance with the present disclosure can be designed and / or constructed in an array format to improve skin contact and facilitate penetration into the skin. In some embodiments, the MNs utilized have a suitable length, width, and shape to minimize contact with nerves when inserted into the skin while still creating an effective pathway for drug delivery. Alkilani, AZ, et al., "Transdermal drug delivery: Innovative pharmaceutical developments based on disruption of the barrier properties of the stratum corneum." Pharmaceutics. 7: 438-470 (2015).

[0117] In some embodiments, suitable MNs may be solid, coated, porous, soluble, hollow or hydrogel MNs. Solid MNs create micropores in the skin, thereby increasing the transport of drug formulations (e.g., "pierce and patch" methods). Coated MNs allow the coated drug to dissolve rapidly into the skin (e.g., "coat and pierce" methods). Soluble MNs allow for rapid and / or controlled release of drugs incorporated into the microneedles. Hollow MNs can be used to pierce the skin and can release the composition after active infusion or diffusion of the formulation through the microneedle holes (e.g., "pierce and flow" methods). In the case of soluble MNs, MNs can act as drug reservoirs, holding the drug composition until it is released by dissolution in the case of soluble MNs or swollen in the case of hydrogel MNs (e.g., "pierce and release" methods). However, as already described herein, in many embodiments, large medicaments are not delivered by injection via one or more microneedles. That is, in many embodiments, any microneedle utilized according to such embodiments is not coated, loaded or manufactured with a large medicament in any manner that will achieve large medicament delivery. Alternatively, in some embodiments, as described herein, if the large agent is formulated as a coarse or nanoemulsion composition as described herein, the MNs utilized in accordance with the present disclosure (whether in MSCs or otherwise) can contain and / or deliver a large agent. Thus, as will be understood by one of skill in the art reading the descriptions described herein, treating the skin with microneedles that deliver a large agent (e.g., by microneedle injection, by release from a microneedle coating, or by release from a dissolved microneedle) is not microneedle skin conditioning.

[0118] In some embodiments, the microneedle has a diameter that is consistent throughout the length of the microneedle. In some embodiments, the diameter of the microneedle is the largest at the base end of the microneedle. In some embodiments, the microneedle tapers to a point at the distal end of the microneedle base. In some embodiments, the microneedle may be solid. In some embodiments, the microneedle may be hollow. In some embodiments, the microneedle may be tubular. In some embodiments, the microneedle may be sealed on one end. In some embodiments, the microneedle is part of a microneedle array. In some embodiments, the length of the microneedle may be between about 1 μm and about 4,000 μm. In some embodiments, the length of the microneedle may be between about 1 μm and about 2,000 μm. In some embodiments, the length of the microneedle may be between about 50 μm and about 400 μm. In some embodiments, the length of the microneedle may be between about 50 μm and about 500 μm. In some embodiments, the length of the microneedle may be between about 50 μm and about 600 μm. In some embodiments, the length of the microneedle may be between about 50 μm and about 700 μm. In some embodiments, the length of the microneedle may be between about 50 μm and about 800 μm. In some embodiments, the length of the microneedle may be between about 800 μm and about 1500 μm. In some embodiments, the length of the microneedle may be less than about 1400 μm. In some embodiments, the length of the microneedle may be less than about 1100 μm. In some embodiments, the length of the microneedle may be less than about 1000 μm. In some embodiments, the length of the microneedle may be less than about 800 μm. In some embodiments, the length of the microneedle may be between about 100 μm and about 800 μm.

[0119] In some embodiments, microneedle treatment as described herein comprises applying a plurality of microneedles of a common length (e.g., a microneedle array) to the skin; in some embodiments, microneedle treatment as described herein comprises applying a plurality of microneedles of different lengths (e.g., a microneedle array) to the skin.

[0120] Microneedles of various lengths can be used for the microneedle technology described herein. In some embodiments, the length of the microneedles used in the MSC as described herein is adjusted according to the skin thickness of the treatment site. In some embodiments, MN or MN arrays include microneedles with a length of about 100 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 150 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 200 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 250 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 300 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 350 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 400 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 450 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 500 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 550 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 600 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 650 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 700 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 750 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 800 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 850 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 900 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 950 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 1000 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 1100 μm. In some embodiments, MN or MN arrays include microneedles with a length of about 1200 μm. In some embodiments, the MN or MN array comprises microneedles having a length of about 1300 μm. In some embodiments, the MN or MN array comprises microneedles having a length of about 1400 μm. In some embodiments, the MN or MN array comprises microneedles having a length of about 1500 μm.

[0121] In some embodiments, the MN or MN array comprises a plurality of needles. In some embodiments, the MN or MN array comprises 2 microneedles / cm 2 In some embodiments, the MN or MN array comprises 3 microneedles / cm 2 In some embodiments, the MN or MN array comprises 4 microneedles / cm 2In some embodiments, the MN or MN array comprises 5 microneedles / cm 2 In some embodiments, the MN or MN array comprises 6 microneedles / cm 2 In some embodiments, the MN or MN array comprises 7 microneedles / cm 2 In some embodiments, the MN or MN array comprises 8 microneedles / cm 2 In some embodiments, the MN or MN array comprises 9 microneedles / cm 2 In some embodiments, the MN or MN array comprises 10 microneedles / cm 2 In some embodiments, the MN or MN array comprises 11 microneedles / cm 2 In some embodiments, the MN or MN array comprises 12 microneedles / cm 2 In some embodiments, the MN or MN array comprises 13 microneedles / cm 2 In some embodiments, the MN or MN array comprises 14 microneedles / cm 2 In some embodiments, the MN or MN array comprises 15 microneedles / cm 2 In some embodiments, the MN or MN array comprises 16 microneedles / cm 2 In some embodiments, the MN or MN array comprises 17 microneedles / cm 2 In some embodiments, the MN or MN array comprises 18 microneedles / cm 2 In some embodiments, the MN or MN array comprises 19 microneedles / cm 2 In some embodiments, the MN or MN array comprises 20 microneedles / cm 2 In some embodiments, the MN or MN array comprises 21 microneedles / cm 2 In some embodiments, the MN or MN array comprises 22 microneedles / cm 2 In some embodiments, the MN or MN array comprises 23 microneedles / cm 2 In some embodiments, the MN or MN array comprises 24 microneedles / cm 2 In some embodiments, the MN or MN array comprises 25 microneedles / cm 2 In some embodiments, the MN or MN array comprises 26 microneedles / cm 2 In some embodiments, the MN or MN array comprises 27 microneedles / cm 2 In some embodiments, the MN or MN array comprises 28 microneedles / cm 2 In some embodiments, the MN or MN array comprises 29 microneedles / cm 2In some embodiments, the MN or MN array comprises 30 microneedles / cm 2 In some embodiments, the MN or MN array comprises 31 microneedles / cm 2 In some embodiments, the MN or MN array comprises 35 microneedles / cm 2 In some embodiments, the MN or MN array comprises 40 microneedles / cm 2 In some embodiments, the MN or MN array comprises 45 microneedles / cm 2 In some embodiments, the MN or MN array comprises 50 microneedles / cm 2 In some embodiments, the MN or MN array comprises 55 microneedles / cm 2 In some embodiments, the MN or MN array comprises 60 microneedles / cm 2 In some embodiments, the MN or MN array comprises 65 microneedles / cm 2 In some embodiments, the MN or MN array comprises 70 microneedles / cm 2 In some embodiments, the MN or MN array comprises 75 microneedles / cm 2 In some embodiments, the MN or MN array comprises 80 microneedles / cm 2 In some embodiments, the MN or MN array comprises 85 microneedles / cm 2 In some embodiments, the MN or MN array comprises 90 microneedles / cm 2 In some embodiments, the MN or MN array comprises 95 microneedles / cm 2 In some embodiments, the MN or MN array comprises 100 microneedles / cm 2 In some embodiments, the MN or MN array comprises 200 microneedles / cm 2 In some embodiments, the MN or MN array comprises 300 microneedles / cm 2 In some embodiments, the MN or MN array comprises 400 microneedles / cm 2 In some embodiments, the MN or MN array comprises 500 microneedles / cm 2 In some embodiments, the MN or MN array comprises less than 1000 microneedles / cm 2 In some embodiments, the MN or MN array comprises less than 2000 microneedles / cm 2 .

[0122] Microneedles of any shape may be used in the microneedle technology described herein. In some embodiments, the microneedle may have a circular cross-section. In some embodiments, the microneedle may have a triangular cross-section. In some embodiments, the microneedle may have a rectangular cross-section. In some embodiments, the microneedle may have a square cross-section. In some embodiments, the microneedle may have a quadrilateral cross-section. In some embodiments, the microneedle may have a pentagonal cross-section. In some embodiments, the microneedle may have a hexagonal cross-section. In some embodiments, the microneedle may have a heptagonal cross-section. In some embodiments, the microneedle may have an octagonal cross-section. In some embodiments, the microneedle may have a nonagonal cross-section. In some embodiments, the microneedle may have a decagonal cross-section.

[0123] Microneedles of various cross-sectional areas can be used in the microneedle technology described herein. As described herein, the cross-sectional area of ​​each microneedle in the MN array for MSC ("microneedle skin conditioning") can in turn define the microneedle puncture size (e.g., the puncture size of each microneedle) of the MN array for MSC. In some embodiments, the microneedle puncture size can be between about 100 and about 60,000 μm. 2 In some embodiments, the microneedle puncture size may be between about 100 and about 30,000 μm. 2 / within the scope of microneedles.

[0124] In some embodiments, MN or MN arrays include needles having multiple microneedle puncture sizes. In some embodiments, MN or MN arrays include needles having at least 2 different microneedle puncture sizes. In some embodiments, MN or MN arrays include needles having at least 3 different microneedle puncture sizes. In some embodiments, MN or MN arrays include needles having at least 4 different microneedle puncture sizes. In some embodiments, MN or MN arrays include needles having at least 5 different microneedle puncture sizes. In some embodiments, MN or MN arrays include needles having up to 10 different microneedle puncture sizes. In some embodiments, MN or MN arrays include needles having at least 11 different microneedle puncture sizes. In some embodiments, MN or MN arrays include needles having at least 12 different microneedle puncture sizes. In some embodiments, MN or MN arrays include needles having up to 1 different microneedle puncture size.

[0125] MNs or MN arrays comprising microneedles of various microneedle puncture sizes can be used in the microneedle techniques described herein. In some embodiments, MNs or MN arrays comprise microneedles with a puncture size of 100 μm. 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 200 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 300 μm 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 400 μm. 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 500 μm. 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 600 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 700 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 800 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 900 μm 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 1000 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 1100 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 1200 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 1300 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 1400 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 1500 μm 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 1600 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 1700 μm 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 1800 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 1900 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 2000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 2500 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 3000 μm2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 3500 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 4000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 4500 μm 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 5000 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 5500 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 6000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 6500 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 7000 μm 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of 7500 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 8000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 8500 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 9000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 9500 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 10000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 10500 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 11000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 11500 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of 12000 μm 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 13000 μm. 2In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 14000 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 15000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 20000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 25000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 30000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 35000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 40000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 45000 μm 2 In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 50000 μm. 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 55000 μm 2 / microneedle. In some embodiments, the MN or MN array comprises a microneedle puncture size of less than 60000 μm 2 / Microneedling of microneedles.

[0126] In some embodiments, the MN used in accordance with the present disclosure can be made of different materials using techniques including, but not limited to, micromolding processes or lasers. In some embodiments, the MN can be made using various types of biocompatible materials, including polymers, metals, ceramics, semiconductors, organics, composite materials, or silicon. Unless they are designed to break into the skin and dissolve, in some embodiments, the microneedles have mechanical strength to remain intact and deliver drugs or collect biological fluids when inserted into the skin and / or removed from the skin after insertion. In some embodiments, the MN can remain in place for up to several days before complete removal. In some embodiments, the microneedles can be sterilized using standard techniques. In some embodiments, the MN is biodegradable. In some embodiments, the MN comprises a polymer material. In some embodiments, the polymer material comprises poly-L-lactic acid, polyglycolic acid, polycarbonate, polylactic acid-co-glycolic acid (PLGA), polydimethylsiloxane, polyvinyl pyrrolidone (PVP), copolymers of methyl vinyl ether and maleic anhydride, sodium hyaluronate, carboxymethyl cellulose, maltose, dextrin, galactose, starch, gelatin, or a combination thereof.

[0127] In some embodiments, a MSC as described herein comprises one impression of a MN or MN array. In some embodiments, a MSC comprises two impressions of a MN or MN array. In some embodiments, a MSC comprises three impressions of a MN or MN array. In some embodiments, a MSC comprises four impressions of a MN or MN array. In some embodiments, a MSC comprises five impressions of a MN or MN array. In some embodiments, a MSC comprises six impressions of a MN or MN array. In some embodiments, a MSC comprises seven impressions of a MN or MN array. In some embodiments, a MSC comprises eight impressions of a MN or MN array. In some embodiments, a MSC comprises nine impressions of a MN or MN array. In some embodiments, a MSC comprises ten impressions of a MN or MN array. In some embodiments, a MSC comprises eleven impressions of a MN or MN array. In some embodiments, a MSC comprises twelve impressions of a MN or MN array. In some embodiments, a MSC comprises thirteen impressions of a MN or MN array. In some embodiments, a MSC comprises fourteen impressions of a MN or MN array. In some embodiments, a MSC comprises fifteen impressions of a MN or MN array. In some embodiments, the MSC comprises sixteen impressions of the MN or MN array. In some embodiments, the MSC comprises seventeen impressions of the MN or MN array. In some embodiments, the MSC comprises eighteen impressions of the MN or MN array. In some embodiments, the MSC comprises nineteen impressions of the MN or MN array. In some embodiments, the MSC comprises twenty impressions of the MN or MN array. In some embodiments, the MSC comprises rolling the MN or MN array on the skin one or more times. In some embodiments, the MN array is rotated between impressions. In some embodiments, the MN array is not rotated between impressions. In some embodiments, the impressions are performed on the same site. In some embodiments, the impressions are performed on overlapping sites. In some embodiments, the impressions are performed on different sites. In some embodiments, the impressions are performed by stamping the MN array. In some embodiments, the impressions are performed by rolling a microneedle roller on the site one or more times. In accordance with established MN practice, in some embodiments, skin impressions of the MN array last less than one second, or, in some embodiments, they last longer than one second, for example, they may last 30 seconds or more, 60 seconds or more, two minutes or more, five minutes or more, ten minutes or more, thirty minutes or more, and so forth.

[0128] As described above, the present disclosure teaches that as the total surface area of ​​the skin punctured by the microneedles decreases, the bioavailability of a large pharmaceutical agent in an emulsion applied to the skin increases. Therefore, in some embodiments, relatively fewer impressions may be preferred. In some embodiments, when the large pharmaceutical agent administered in conjunction with microneedle skin conditioning is in a topical formulation that is not (or does not include) an emulsion (e.g., an emulsion containing the pharmaceutical agent), fewer microneedle array impressions may be preferred. In some embodiments, shorter microneedle lengths may be preferred. In some embodiments, when the large pharmaceutical agent administered in conjunction with microneedle skin conditioning is in a topical formulation that is not (or does not include) an emulsion (e.g., an emulsion containing the pharmaceutical agent), relatively shorter microneedle lengths may be preferred.

[0129] In addition, as described above, a person of ordinary skill in the art who reads this disclosure will understand that, in some embodiments, a product containing a biologically active agent (e.g., a large agent) in a relatively reduced amount (e.g., volume and / or dose) in conjunction with MSCs can achieve a greater biological effect. Therefore, in some embodiments, a relatively reduced product volume containing an active agent (such as a large agent) may be preferred. In some embodiments, when a large agent applied in conjunction with microneedle skin conditioning is in a topical formulation that is or contains an emulsion (e.g., a nanoemulsion), a relatively small product volume may be preferred. In some embodiments, when a large agent applied in conjunction with microneedle skin conditioning is in a topical formulation that is not (or does not contain) an emulsion (e.g., an emulsion containing an agent), a relatively small product volume may be preferred. Suitable MN arrays and MSC devices for use in combination with compositions containing large agents for transdermal delivery of large agents include devices such as those described in, for example, U.S. Patents 6,334,856; 6,503,231; 6,908,453; 8,257,324; and 9,144,671.

[0130] Big Pharmacy

[0131] In some embodiments, the compositions provided and / or utilized as described herein comprise one or more large agents. In some embodiments, the large agent utilized is a bioactive agent (e.g., a therapeutically active agent). In addition, the present disclosure provides strategies and surprising improvements for topical administration and transdermal delivery of compositions comprising large agents in combination with MSCs as described herein. In addition, the present disclosure establishes that microneedle therapy may be particularly advantageous for the delivery of large agents in emulsion compositions (e.g., nanoemulsions).

[0132] 1. Protein pharmaceuticals

[0133] Any of the various protein agents can be incorporated into the provided compositions and administered in combination with MSCs. In some embodiments, the protein agent can be a peptide agent. In some embodiments, the peptide has a molecular weight greater than 100 KDa. In some embodiments, the peptide agent has a molecular weight of at least 150 KDa. In some embodiments, the peptide agent is composed only of naturally occurring amino acids. In some embodiments, the peptide agent comprises one or more non-naturally occurring amino acids.

[0134] Those skilled in the art will be aware of various protein agents that have been approved for therapeutic use by relevant regulatory agencies. For example, the U.S. Food and Drug Administration maintains a list of approved biotherapeutics arranged by approval year, which can be found at the following website: www.fda.gov / biologicsbloodvaccines / developmentapprovalprocess / biologicalapprovalsbyyear / ucm547553.htm. Those skilled in the art who read this disclosure will understand that its teachings can be applied to any of a variety of such agents; of particular interest are those intended and / or formulated for topical administration, including, for example, those that may be undergoing or may have been undergoing clinical trials (e.g., may be approved or undergoing approval by the U.S. Food and Drug Administration or its equivalent in another jurisdiction, or may be included in a clinical trial at one or more clinical sites, such as may be listed in the records of www.clinicaltrials.gov and / or an institutional review board or its equivalent).

[0135] (i) Botulinum toxin

[0136] In some embodiments, the large agent can be botulinum toxin. Botulinum toxin (BTX) BTX is produced in nature by the anaerobic Gram-positive bacillus Clostridium botulinum and is a potent polypeptide neurotoxin. Most notably, BTX causes a neuroparalytic disease in humans and animals, known as botulism. BTX can apparently pass through the intestinal lining and attack peripheral motor neurons. Symptoms of botulinum toxin poisoning can develop from difficulty walking, swallowing, and speaking to respiratory muscle paralysis and death.

[0137] For all seven known botulinum toxin serotypes, the molecular weight of the botulinum toxin protein molecule is about 150kDa. Botulinum toxin is released by Clostridium bacteria as a complex comprising a 150kDa botulinum toxin protein molecule together with related non-toxin proteins. Therefore, the BTX-A complex can be produced by Clostridium bacteria in the form of 900kDa, 500kDa and 360kDa. Type B and C1 botulinum toxins apparently only produce a complex of 500kDa. Type D botulinum toxins produce a complex of 300kDa and 500kDa. Finally, Type E and F botulinum toxins only produce a complex of about 300kDa.

[0138] It is believed that BTX complexes (i.e., those compositions having a molecular weight greater than about 150 kDa) contain non-toxin hemagglutinin proteins and non-toxin non-hemagglutinin proteins. These two non-toxin proteins (which together with the botulinum toxin molecule constitute the relevant neurotoxin complex) can be used to provide stability to prevent denaturation of the botulinum toxin molecule and protection against digestive acids when the toxin is ingested.

[0139] According to the present invention, BTX protein or BTX complex can be used as known therapeutic agents and / or independently active biologically active agents. In fact, those of ordinary skill in the art will understand that any portion or fragment of the BTX protein or complex that retains appropriate activity can be utilized as described herein.

[0140] In some embodiments, the botulinum toxin may be selected from the group consisting of: type A, type Ab, type Af, type B, type Bf, type C1, type C2, type D, type E, type F, and type G; mutants thereof; variants thereof; fragments thereof; characteristic portions thereof; and / or fusions thereof. In some embodiments, the botulinum toxin may be a variant toxin, for example, having one or more structural changes relative to a reference (e.g., wild-type) toxin (or a related fragment thereof). In some specific embodiments, the variant toxin may have a longer or shorter biologically active life than an appropriate, comparable reference form (e.g., wild-type form). In some embodiments, the botulinum toxin exists in any subtype described in Sakaguchi, 1982, Pharmacol. Ther., 19: 165; and / or Smith et al., 2005, Infect. Immun., 73: 5450; both of which are incorporated herein by reference.

[0141] In some embodiments, the present invention provides botulinum toxin compositions. In some embodiments, the present invention provides nanoemulsion botulinum toxin compositions. Commercially available botulinum toxin sources that can be utilized in accordance with the present invention include, but are not limited to (clostridium botulinum toxin type A hemagglutinin complexed with human serum albumin and lactose; Ispen Limited, Berkshire UK), Medy-Tox, NT-201 (Merz Pharmaceuticals) and / or (an injection consisting of botulinum toxin type B, human serum albumin, sodium succinate and sodium chloride, pH 5.6, Elan Pharmaceuticals, Dublin, Ireland), NEURONOX (Medytox), HENGLI (Lanzhou Institute), etc. Those skilled in the art are aware of the standard and / or approved administration regimens for such commercially available botulinum toxin compositions, and will understand that any relevant such compositions and / or regimens can be used with microneedle technology (e.g., particularly with MSCs), as described herein.

[0142] In some embodiments, the compositions provided include botulinum toxin compositions and are formulated into creams and / or lotions and include about 1 to about 200,000 units of botulinum toxin per mL. In some embodiments, the compositions provided include botulinum toxin compositions and are formulated into creams and / or lotions and include about 1 to about 100,000 units of botulinum toxin per mL. In some embodiments, the compositions provided include botulinum toxin compositions and are formulated into creams and / or lotions and include about 1 to about 50,000 units of botulinum toxin per mL. In some embodiments, the compositions provided include botulinum toxin compositions and are formulated into creams and / or lotions and include about 500 to about 20,000 units of botulinum toxin per mL. In some embodiments, the compositions provided include botulinum toxin compositions and are formulated into creams and / or lotions and include about 100 to about 2,000 units of botulinum toxin per mL. In some embodiments, provided compositions comprising a botulinum toxin composition and formulated as a cream and / or lotion comprise from about 50 to about 500 units of botulinum toxin per mL. In some embodiments, provided compositions comprising a botulinum toxin composition and formulated as a cream and / or lotion comprise from about 25 to about 400 units of botulinum toxin per mL.

[0143] In some embodiments, the botulinum toxin composition comprises about 2 to about 40,000 units of botulinum toxin per mL. In some embodiments, the botulinum toxin composition comprises about 2 to about 12,000 units of botulinum toxin per mL. In some embodiments, the botulinum toxin composition comprises about 100 to about 2,000 units of botulinum toxin per mL. In some embodiments, the botulinum toxin composition comprises about 50 to about 1,000 units of botulinum toxin per mL.

[0144] In some embodiments, the botulinum toxin composition comprises at least one bioactive agent other than botulinum toxin. Alternatively or additionally, in some embodiments, the botulinum toxin composition is administered in combination with at least one other composition comprising such a bioactive agent. In some embodiments, the botulinum toxin composition is administered in combination with a penetration enhancer. In some embodiments, the botulinum toxin composition is administered in combination with another bioactive agent. In some embodiments, the botulinum toxin composition is administered in combination with another bioactive agent and a penetration enhancer.

[0145] In some embodiments, the bioactive agent used in combination with a botulinum toxin as described herein can be an agent that acts on or in the skin and / or imparts a therapeutic and / or cosmetic effect. For example, in some embodiments, such a bioactive agent can be selected from therapeutic agents such as anesthetics (e.g., lidocaine), steroids (e.g., hydrocortisone) and / or retinoids (e.g., retinoid A), cosmetic agents such as dermal fillers (e.g., hyaluronic acid or other elastic materials), collagen, and / or silicone. In some embodiments, the botulinum composition is administered in combination with a delivery modifier (e.g., a penetration enhancer) (which, in some embodiments, is not an irritant and / or does not degrade, disrupt, and / or damage skin structure and / or skin).

[0146] In some embodiments, the non-irritating penetration enhancer can be selected from, for example, co-peptides, carrier molecules, and carrier peptides. In some embodiments, the carrier molecule is positively charged. In some embodiments, the carrier molecule can be a co-peptide. In some embodiments, the carrier molecule can be a long chain positively charged polypeptide or a positively charged non-peptidyl polymer, such as a polyalkylene imine. In some embodiments, the carrier peptide can be a cationic peptide. In some embodiments, the carrier peptide is a peptide having the sequence RKKRRQRRRG-(K) 15 -GRKKRRQRRR's positively charged carrier. In some embodiments, the carrier molecule may be a carrier molecule disclosed in US Patent Publication No. 2010 / 0168023 or US Patent Publication No. 2009 / 0247464, the contents of which are incorporated herein by reference in their entirety.

[0147] In some embodiments, the composition provided comprising both a botulinum toxin nanoemulsion composition and a cream and / or lotion formulation comprises about 1 to about 100,000 units of botulinum toxin per mL. In some embodiments, the composition provided comprising both a botulinum toxin nanoemulsion composition and a cream and / or lotion formulation comprises about 1 to about 100,000 units of botulinum toxin per mL. In some embodiments, the composition provided comprising both a botulinum toxin nanoemulsion composition and a cream and / or lotion formulation comprises about 1 to about 50,000 units of botulinum toxin per mL. In some embodiments, the composition provided comprising both a nanoemulsion composition and a cream and / or lotion formulation comprises about 500 to about 20,000 units of botulinum toxin per mL. In some embodiments, the composition provided comprising both a nanoemulsion composition and a cream and / or lotion formulation comprises about 100 to about 2,000 units of botulinum toxin per mL. In some embodiments, provided compositions comprising both a botulinum toxin nanoemulsion composition and a cream and / or lotion formulation comprise from about 50 to about 500 units of botulinum toxin per mL. In some embodiments, provided compositions comprising both a botulinum toxin nanoemulsion composition and a cream and / or lotion formulation comprise from about 25 to about 400 units of botulinum toxin per mL.

[0148] In some embodiments, the botulinum toxin nanoemulsion composition comprises about 2 to about 40,000 units of botulinum toxin per mL. In some embodiments, the botulinum toxin nanoemulsion composition comprises about 2 to about 12,000 units of botulinum toxin per mL. In some embodiments, the botulinum toxin nanoemulsion composition comprises about 100 to about 2,000 units of botulinum toxin per mL. In some embodiments, the botulinum toxin nanoemulsion composition comprises about 50 to about 1,000 units of botulinum toxin per mL.

[0149] (ii) Antibody Agents

[0150] In some embodiments, the disclosure relates to the delivery of antibody agents. In some embodiments, the large agent can be an antibody or a fragment or derivative thereof. In addition, the disclosure provides certain compositions comprising antibody agents, and also provides administration techniques for compositions comprising antibody agents, which are combined with MSCs as described herein.

[0151] In some embodiments, the antibody agent may be applicable to treating skin conditions. In some embodiments, the antibody agent may be a fusion protein. In some embodiments, the antibody agent may be conjugated to another portion. In some embodiments, the antibody agent may be conjugated to polyethylene glycol. In some embodiments, the antibody may be multispecific (e.g., bispecific) and may be connected to two or more different target antigens or epitopes.

[0152] In some embodiments, the antibody agent targets TNFα (e.g., including epitope binding elements found in anti-TNFα antibodies (such as infliximab, adalimumab, golimumab, etanercept, etanercept-szzs, and / or certolizumab pegol). In some embodiments, the antibody agent targets CD2 (e.g., including epitope binding elements found in anti-CD2 antibodies (such as siplizumab). In some embodiments, the antibody agent targets CD4 (e.g., including epitope binding elements found in anti-CD4 antibodies (such as zalimumab)).

[0153] In some embodiments, the antibody agent targets IL-12 (e.g., including an epitope binding element found in an anti-IL-12 antibody such as Briakinumab). In some embodiments, the antibody agent targets IL-17 (e.g., including an epitope binding element found in an anti-IL-17 antibody such as secukinumab and / or brodalumab). In some embodiments, the antibody agent targets IL-22 (e.g., including an epitope binding element found in an anti-IL-22 antibody such as fezakinumab). In some embodiments, the antibody agent targets IL-23 (e.g., including an epitope binding element found in ustekinumab and / or guselkumab).

[0154] In some embodiments, the antibody pharmaceutical composition comprises at least one bioactive agent other than the antibody pharmaceutical. Alternatively or additionally, in some embodiments, the antibody pharmaceutical composition is administered in combination with at least one other composition comprising such a bioactive agent. In some embodiments, the antibody pharmaceutical composition is administered in combination with a penetration enhancer. In some embodiments, the antibody pharmaceutical composition is administered in combination with another bioactive agent. In some embodiments, the antibody pharmaceutical composition is administered in combination with another bioactive agent and a penetration enhancer. In some embodiments, the antibody pharmaceutical composition is a nanoemulsion. In some embodiments, the antibody pharmaceutical composition is a cream and / or lotion formulation.

[0155] In some embodiments, the bioactive agent used in combination with an antibody agent as described herein can be an agent that acts on or in the skin and / or imparts a therapeutic and / or cosmetic effect. For example, in some embodiments, such a bioactive agent can be selected from a therapeutic agent, such as an anesthetic (e.g., lidocaine), a steroid (e.g., hydrocortisone) and / or a retinoid (e.g., visual pigment A), a cosmetic agent, such as a dermal filler (e.g., hyaluronic acid or other elastic material), collagen, and / or silicone. In some embodiments, the antibody agent composition is administered in combination with a delivery modifier (e.g., a penetration enhancer) (in some embodiments, it is not an irritant and / or does not degrade, destroy, and / or damage the skin structure and / or skin).

[0156] In some embodiments, the non-irritating penetration enhancer can be selected from, for example, co-peptides, carrier molecules, and carrier peptides. In some embodiments, the carrier molecule is positively charged. In some embodiments, the carrier molecule can be a co-peptide. In some embodiments, the carrier molecule can be a long chain positively charged polypeptide or a positively charged non-peptidyl polymer, such as a polyalkylene imine. In some embodiments, the carrier peptide can be a cationic peptide. In some embodiments, the carrier peptide is a peptide having the sequence RKKRRQRRRG-(K) 15 -GRKKRRQRRR's positively charged carrier. In some embodiments, the carrier molecule may be a carrier molecule disclosed in US Patent Publication No. 2010 / 0168023 or US Patent Publication No. 2009 / 0247464, the contents of which are incorporated herein by reference in their entirety.

[0157] 2. Preventive agents

[0158] Any of the various prophylactic agents can be incorporated into the provided compositions and administered in combination with the MSCs of the present invention. In some embodiments, the prophylactic agent includes, but is not limited to, a vaccine. In some embodiments, the vaccine can include isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts. In some embodiments, the prophylactic agent can be combined with interleukins, interferons, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, and the like. In some embodiments, the prophylactic agent may include antigens of bacterial organisms such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira, Borrelia burgdorferi, Campylobacter jejuni, etc.; antigens of viruses such as smallpox, influenza A and B viruses, respiratory syncytial virus, parainfluenza virus, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D and E, etc.; antigens of fungi, protozoa and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsiae, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, etc. In some embodiments, these antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates or a combination thereof.

[0159] Those skilled in the art will recognize that the preceding paragraphs provide an exemplary, but not comprehensive, list of pharmaceutical agents that can be delivered using techniques according to the present invention.Any pharmaceutical agent may be associated with the compositions provided according to the present invention.

[0160] Topical preparations

[0161] Compositions as described herein are particularly useful because they can be used to deliver large pharmaceutical agents to subjects in need thereof via topical and / or transdermal (e.g., by lotion, cream, powder, ointment, liniment, gel, drops, etc.) administration. In some embodiments, provided cream and / or lotion formulations containing large pharmaceutical agents are administered to subjects in need thereof via topical and / or transdermal (e.g., by lotion, cream, powder, ointment, liniment, gel, drops, etc.) administration.

[0162] In some embodiments, the cream and / or lotion formulation comprises purified water, methylparaben, mineral oil, isopropyl myristate, white petrolatum, emulsifying wax, and propylparaben. In some embodiments, the cream and / or lotion formulation comprises purified water, mineral oil, isopropyl myristate, white petrolatum, and emulsifying wax.

[0163] In some embodiments, the present invention provides specific cream and / or lotion formulations as described herein. In some embodiments, the cream and / or lotion formulation provided comprises water. In some embodiments, the cream and / or lotion formulation provided comprises methylparaben. In some embodiments, the cream and / or lotion formulation provided comprises mineral oil. In some embodiments, the cream and / or lotion formulation provided comprises isopropyl myristate. In some embodiments, the cream and / or lotion formulation provided comprises white vaseline. In some embodiments, the cream and / or lotion formulation provided comprises emulsifying wax. In some embodiments, the cream and / or lotion formulation provided comprises propylparaben. In some embodiments, the cream and / or lotion formulation provided does not comprise any paraben. In some embodiments, the cream and / or lotion formulation provided does not comprise methylparaben. In some embodiments, the cream and / or lotion formulation provided does not comprise propylparaben. Exemplary lotion formulations are provided in Table 1.

[0164] Table 1. Exemplary cream and / or lotion formulations

[0165]

[0166]

[0167] In some embodiments, cream and / or lotion formulations can be used for topical and / or transdermal administration. The present invention encompasses the recognition that provided cream and / or lotion formulations can be particularly useful for delivering agents to the dermis of the skin. In some embodiments, provided cream and / or lotion formulations are formulated for topical and / or transdermal delivery to a subject in need. In some embodiments, provided cream and / or lotion formulations are administered to a subject in need via topical and / or transdermal delivery.

[0168] In some embodiments, the provided compositions are formulated with cosmetically acceptable components. For example, in some embodiments, the provided compositions are formulated with water and any cosmetically acceptable solvent, particularly monohydric alcohols, such as alkanols (like ethanol, isopropanol, benzyl alcohol and phenylethyl alcohol) used alone or in combination, such as alkylene glycols (like glycerol, ethylene glycol and propylene glycol), and glycol ethers, such as mono-, di- and tri-ethylene glycol monoalkyl ethers, for example, ethylene glycol monomethyl ether and diethylene glycol monomethyl ether. Such components may be present, for example, in a proportion of up to 60 wt %, 70 wt %, 80 wt % or 90 wt % relative to the weight of the total composition.

[0169] In some embodiments, provided compositions for topical administration include one or more cosmetically acceptable components that impart desirable or appropriate cosmetic attributes to the subject to which the composition is to be applied (e.g., a matte appearance, which may be particularly desirable or appropriate for application to subjects with greasy skin).

[0170] In some embodiments, provided compositions are formulated with at least one cosmetically acceptable filler material, for example, to achieve a matte product, which may be particularly desirable for individuals with greasy skin.

[0171] In some embodiments, the macro-agents are formulated into compositions suitable for topical administration. Exemplary macro-agents include those described herein. In some embodiments, provided compositions can be formulated and delivered in combination with MSCs as described herein to achieve systemic delivery; in some embodiments, provided compositions can be formulated and / or delivered to achieve local but non-systemic delivery.

[0172] In some embodiments, compositions suitable for topical formulations include a penetration enhancer. In some embodiments, the penetration enhancer degrades, destroys, and / or damages skin structure and / or skin. In some embodiments, the penetration enhancer does not degrade, destroy, and / or damage skin structure and / or skin. In some embodiments, the penetration enhancer is an irritant. In some embodiments, the penetration enhancer is not an irritant.

[0173] The present disclosure specifically demonstrates the effective and efficient delivery of therapeutic agents (and particularly large biological agents, such as botulinum toxin and / or antibody agents) to the dermis using provided compositions in combination with MSCs as described herein. For example, in some embodiments, the present invention provides methods comprising administering a composition as described herein without clinically significant side effects. As an example, when local delivery is considered, clinically significant side effects include, but are not limited to, undesirable systemic side effects, damage to neural tissue underlying the dermis (e.g., neuropathic paralysis), undesirable effects on muscle (e.g., muscle paralysis), and / or undesirable blood levels of the therapeutic agent, etc.

[0174] Those of ordinary skill in the art will appreciate that the compositions provided can be incorporated into a device (e.g., a patch). Various transdermal patch structures are known in the art; those of ordinary skill in the art will appreciate that the compositions provided can be easily incorporated into any of the various such structures. In some embodiments, a transdermal patch may include a plurality of needles extending from one side of the patch applied to the skin, wherein the needles extend from the patch to protrude through the stratum corneum of the skin. In some embodiments, the needles do not rupture blood vessels. In some embodiments, the needles do not penetrate deeply enough to reach the nerves of the dermis of the skin.

[0175] In some embodiments, transdermal patch includes adhesive. Some examples of adhesive patch are well known (for example, referring to U.S. Design Patent 296,006; and U.S. Patent 6,010,715; 5,591,767; 5,008,110; 5,683,712; 5,948,433; and 5,965,154; all of which are incorporated herein by reference). The feature of adhesive patch is generally to have an adhesive layer to be applied to the patient's skin, a reservoir or a reservoir for keeping the composition provided, and an outer surface preventing the composition provided from leaking from the reservoir. The outer surface of the patch can be non-adhesive.

[0176] According to the present invention, the composition provided can be incorporated into the patch so that it remains stable for a long time. For example, in some embodiments, the composition provided can be incorporated into a polymer matrix that stabilizes a large agent, and allows the agent to diffuse from the matrix and the patch. The composition provided can also be incorporated into the adhesive layer of the patch so that once the patch is applied to the skin, the composition provided can diffuse through the skin. In some embodiments, the adhesive layer can be heat-activated, wherein a temperature of about 37°C causes the adhesive to slowly liquefy, allowing the agent to diffuse through the skin. When stored below 37°C, the adhesive can remain viscous, and once applied to the skin, the adhesive loses its viscosity when it liquefies.

[0177] In some embodiments, the provided compositions can be provided in a reservoir in the patch so that pressure applied to the patch directs the provided compositions out of the patch and through the stratum corneum through the microneedles. Exemplary embodiments of microneedles are described above. Suitable devices for intradermal administration of the provided compositions include those described in: U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions can be administered by devices that limit the effective penetration length of the needle into the skin, such as those described in PCT Publication WO 99 / 34850 and functional equivalents thereof.

[0178] In some embodiments, for example, to prolong the effect of the provided composition, it may be desirable to slow down the absorption of the provided composition into the skin. In some embodiments, this can be accomplished by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the provided composition depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. In some embodiments, depending on the ratio of the provided composition to the polymer and the properties of the specific polymer employed, the rate at which the provided composition releases can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).

[0179] Lotion

[0180] The present disclosure encompasses the recognition that emulsion technology can provide stabilization benefits to pharmaceutical agents of interest, including large agents as described herein, and particularly including botulinum toxin and / or antibody agents.

[0181] In addition, the present disclosure recognizes that certain liquid nanoemulsion technologies have been shown to provide significant transdermal delivery properties, even for very large molecules, such as botulinum toxin and / or antibody agents. See, for example, U.S. Patent Publication Nos. 2012 / 0328701, 2012 / 0328702, 8,318,181, and U.S. Patent No. 8,658,391, the disclosures of which are incorporated herein by reference in their entirety. These liquid nanoemulsions are far superior to solid nanoparticle drug delivery, particularly transdermal drug delivery, where, as Gomaa points out, solid nanoparticles cannot penetrate the skin but only accumulate in hair follicles. These liquid nanoemulsions are also stable for at least 34 months, making them commercially viable from this perspective.

[0182] 1. Coarse emulsion

[0183] In some embodiments, the present disclosure provides a strategy for "conditioning" the skin with microneedles, where a transdermal product has been, is being, or will be applied to the skin. The present disclosure provides an insight that such microneedle conditioning can surprisingly provide significant benefits in enhancing the transdermal delivery of large agents (e.g., having a molecular weight greater than about 100 KDa or more), although it has been previously reported that such strategies may only be useful for small molecular weight agents, because studies analyzing the transdermal delivery of small molecules (specifically, short hydrophilic peptides with a molecular weight in the range of 400-1000 Da) found that "skin penetration of peptides depends on their molecular weight and decreases with increasing molecular weight." Zhang, S. et al., "Enhanced delivery of hydrophilic peptides invitro by transdermal microneedle pretreatment." Acta Pharmaceutica Sinica B.4(1):100-104(2014).

[0184] The present disclosure provides insight that by combining macroemulsion application with microneedle skin conditioning (MSC) as described herein (e.g., using relatively low microneedle density and / or relatively small microneedle puncture size), effective and rapid (i.e., applied within minutes) transdermal delivery of macromolecules via such liquid macroemulsion compositions can be surprisingly improved.

[0185] The present disclosure specifically demonstrates that microneedle technology (e.g., microneedle conditioning of the skin) can significantly enhance the transdermal delivery of large agents (e.g., botulinum toxin and / or antibody agents), particularly when used in conjunction with macroemulsion technology. Macroemulsion compositions of particular interest include water-in-oil and oil-in-water macroemulsions characterized by droplet sizes ranging from greater than about 300 nm to about 5,000 μm in diameter, an aqueous dispersion medium to oil ratio ranging from about 0.01:1 to about 20:1; an oil to surfactant ratio in the range of about 0.1 to about 40 and / or a zeta potential in the range of about -80 mV to about +80 mV. The surfactant portion of the composition may contain one or more surfactants.

[0186] Crude emulsion preparation

[0187] Components Weight(g) Percentage (by weight) 1349 Oil 22.0 22 Tween-80 1.0 1 Span-65 3.0 3 Propylparaben 0.2 0.2 Sodium chloride (a) 0.63 0.63 Disodium hydrogen phosphate 0.04 0.04 gelatin 0.02 0.02 Large agents (e.g., botulinum toxin and / or antibodies) * * Isopropyl myristate 0.62 0.62 Purified water (c) 72.49 72.49 total 100.22 100.00

[0188] * In view of this specification, a skilled artisan can make reasonable adjustments to this and other ingredients based on the volume, weight and / or dosage of the bulk drug to be utilized.

[0189] Coarse emulsion formulations can be used to stabilize large pharmaceutical agents (e.g., botulinum toxin and / or antibody agents). Although coarse emulsion formulations by themselves cannot necessarily be expected to achieve transdermal delivery of large pharmaceutical agents, the present disclosure encompasses the insight that combining the improved stability that can be provided by incorporation into coarse emulsion compositions with microneedle technology as described herein can achieve a synergistic enhancement of transdermal delivery.

[0190] The present disclosure teaches that, although MSCs are only expected to help facilitate transdermal delivery of small compounds, transdermal delivery of large agents from a macroemulsion composition can be achieved by combining with microneedle technology. Furthermore, the present disclosure is particularly surprising given that, as described by Gomaa, microneedle conditioning combined with encapsulation of even small molecule agents in solid nanoparticles provides only a small amount of penetration after 6 hours of application, with no penetration of the substance observed until 24 hours after application.

[0191] In some embodiments, a coarse emulsion formulation containing a large agent is applied together with microneedle conditioning with a solid microneedle. In some embodiments, before the coarse emulsion formulation containing a large agent is applied to the site (e.g., before a specific application and / or before each application), the MSC of the site is performed. In some embodiments, after the coarse emulsion formulation containing a large agent is applied to the site, the MSC of the site is performed. In some embodiments, the MSC of the site and the application of the coarse emulsion formulation containing a large agent to the site occur substantially simultaneously. In some embodiments, the coarse emulsion formulation containing a large agent is not injected via one or more microneedles. In some embodiments, the microneedle is part of a microneedle array. In some embodiments, the microneedle may have a length between about 1 μm and about 4,000 μm. In some embodiments, the microneedle may have a length between about 1 μm and about 2,000 μm. In some embodiments, the microneedle may have a length between about 50 μm and about 400 μm. In some embodiments, the microneedle may have a length between about 800 μm and about 1500 μm.

[0192] The findings provided herein are particularly surprising in view of reports that solid nanoparticles with a transdermal delivery size (e.g., 105 ± 2.92 nm) much smaller than the droplets in the coarse emulsion composition used herein cannot even effectively deliver (or enhance the delivery of) small molecule agents through the skin. For example, Gomaa et al. describe a study in which a solution of rhodamine dye (molecular weight 479 Da) encapsulated in PLGA nanoparticles was applied to skin that had been pretreated with microneedles and skin penetration was evaluated. See Gomaa, Y. et al., "Effect of microneedle treatment on the skin permeation of a nanoencapsulated dye." J Pharm Pharmacol. 2012 November; 64 (11): 1592-1602. The data showed that after 6 hours of continuous application, a very small amount of dye began to penetrate the skin; it was not until the skin was treated for 24 hours that a significant increase in penetration was observed. The researchers explain that "there is an emerging consensus that NPs [nanoparticles] generally cannot penetrate the stratum corneum, although they may be well deposited in hair follicles." Therefore, prior to this disclosure, those skilled in the art would have expected that even small molecule agents (e.g., rhodamine dyes) would not be effectively delivered transdermally using microneedle technology utilizing vehicles significantly larger than 105 nm; certainly, delivery of large agents was thought to be impossible. However, this disclosure demonstrates that microneedles can significantly enhance transdermal delivery of large agents, particularly when used in conjunction with macroemulsion technology.

[0193] Among other things, the present disclosure demonstrates that microneedle technology can enhance transdermal delivery (e.g., of large agents, particularly large agents from coarse emulsion compositions) when no other disrupting agents are utilized (i.e., no chemical permeation enhancers, and no other techniques for disrupting or piercing skin structure). Previous studies using microneedles to transdermally deliver agents as large as botulinum toxin (i.e., about 150 kDa) have reported that delivery was unsuccessful unless additional treatment was performed to disrupt the skin. For example, U.S. Patent Publication No. 2010 / 0196445 reports that botulinum toxin cannot be effectively delivered from pre-coated microneedles unless skin-digesting enzymes are also administered to disrupt skin structure at the site of the microneedles.

[0194] In addition, the present disclosure demonstrates that microneedle technology can achieve transdermal delivery (e.g., of large agents, particularly from macroemulsion and nanoemulsion compositions) when coating or loading of the microneedles is not utilized and / or when the microneedles are not designed to remain in the skin. In addition, as already noted, the present disclosure recognizes that such coating or loading of microneedles may not be commercially viable due, at least to the instability of the botulinum coating or loading materials. For example, according to Johnson, E., et al., "Botulinum toxin is very susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. Lyophilization or freeze drying of botulinum toxin is the most economically reasonable and practical method for dispensing the product in a form that is stable and easy for clinicians to use. U.S. Patent No. 5,512,547. In addition, as will be appreciated by those skilled in the art reading this specification, the technology described herein has certain advantages, including that it is not necessary to leave microneedles in or associated with tissue. For example, it will be appreciated by those skilled in the art that leaving microneedles in the skin may have the risk of skin irritation, inflammation, allergic reaction, and / or cosmetically undesirable scarring. In contrast to the present invention, the technology as described in U.S. Patent Publication No. 2017 / 0209553 utilizes a microneedle array that is loaded with botulinum toxin into the needle and is designed to break the microneedle into the skin (according to U.S. Patent Nos. 2017 / 0209553 and 2016 / 0263362.

[0195] The present disclosure provides a surprisingly effective technology for transdermal delivery of large agents (e.g., botulinum toxin, antibodies, etc.). Specifically, the present disclosure teaches that by using microneedle technology without any other destruction strategy, the transdermal delivery of such agents can be significantly enhanced. Therefore, the technology provided can achieve effective delivery without the inflammation, irritation and / or allergic reactions that often accompany the use of skin destroyers. As will be understood by those skilled in the art reading this specification, the present disclosure teaches that by using microneedle technology, the transdermal delivery of such large agents can be significantly enhanced, even when the large agent is not loaded into the microneedle, coated on the microneedle and / or manufactured as a part of the microneedle. Similarly, as will be understood by those skilled in the art reading this specification, the present disclosure teaches that by using microneedle technology (and in particular by using MSCs), the delivery of large agents as described herein can be significantly enhanced without leaving the microneedles in the skin (e.g., by causing them to fall off and / or otherwise retain and / or degrade in situ). For example, one skilled in the art will appreciate that the provided technology can avoid the problem of long-term stability of large doses required for commercially viable products, and can achieve effective delivery without inflammation, irritation, and / or allergic reactions caused by skin disrupting agents and / or microneedles remaining in the skin. Indeed, in the Examples and elsewhere, the present disclosure explicitly teaches MSC-facilitated transdermal delivery of botulinum toxin from topical (e.g., creams, ointments) compositions, and particularly from compositions comprising macro- or nanoemulsions, using microneedles that do not contain botulinum toxin.

[0196] In some embodiments, the present disclosure teaches that particularly advantageous results are achieved when microneedle technology is combined with a coarse emulsion composition. In some embodiments, microneedle technology is combined with a lotion, cream, or liquid composition, which in turn can be or contain a coarse emulsion composition. In some embodiments, the provided technology does not utilize skin-disrupting technology, such as chemical penetration enhancers.

[0197] In some embodiments, the present invention utilizes macroemulsion compositions containing macropharmaceuticals that are particularly effective and / or useful in a medical setting, e.g., for therapeutic purposes. In some embodiments, a particular macroemulsion composition is particularly effective and / or useful for topically administering a pharmaceutical agent to a subject in need thereof. In some embodiments, a macroemulsion composition may contain one or more macropharmaceuticals.

[0198] In some embodiments, the crude emulsion can be formulated into a composition suitable for topical application on the skin. In some embodiments, the composition suitable for topical application can be a lotion, cream, powder, ointment, liniment, gel or drops.

[0199] In some embodiments, the crude emulsion formulation comprises water, medium chain triglycerides, span 65, polysorbate 80, methylparaben, and propylparaben. In some embodiments, the crude emulsion formulation comprises water, medium chain triglycerides, span 65, and polysorbate 80.

[0200] In some embodiments, provided compositions include a mixture of provided crude emulsion compositions and one or more pharmaceutically acceptable excipients. In some embodiments, cream and / or lotion formulations include a mixture of provided crude emulsion compositions and / or saline solutions.

[0201] In some embodiments, the provided compositions include a coarse emulsion composition containing one or more large pharmaceutical agents. In some embodiments, the provided compositions are cream and / or lotion formulations. In some embodiments, the provided cream and / or lotion formulations include a coarse emulsion composition. In some embodiments, the composition includes a coarse emulsion composition provided but is not a cream and / or lotion formulation. In some embodiments, suitable compositions are formulated as creams and / or lotions, but do not include a coarse emulsion composition.

[0202] In some embodiments, provided compositions include a mixture of provided crude emulsion compositions and one or more pharmaceutically acceptable excipients, e.g., for topical and / or transdermal (e.g., by lotion, cream, powder, ointment, liniment, gel, drops, etc.) administration.

[0203] 2. Nanoemulsion

[0204] In some embodiments, the present disclosure provides strategies in which microneedles are used to "condition" the skin to which a transdermal product has been, is being, or will be applied. The present disclosure provides an insight that such microneedle conditioning can surprisingly provide significant benefits in enhancing the transdermal delivery of large agents (e.g., having a molecular weight greater than about 100KDa or more), although it has been previously reported that such strategies may only be useful for small molecular weight agents, because studies analyzing the transdermal delivery of small molecules (specifically, short hydrophilic peptides with a molecular weight in the range of 400-1000Da) found that "skin penetration of peptides depends on their molecular weight and decreases with increasing molecular weight." Zhang, S. et al., "Enhanced delivery of hydrophilic peptides invitro by transdermal microneedle pretreatment." Acta Pharmaceutica Sinica B.4(1):100-104(2014).

[0205] The present disclosure provides insight that by combining nanoemulsion application with microneedle skin conditioning (MSC) as described herein, efficient and rapid (ie, applied within minutes) transdermal delivery of macromolecules via such liquid nanoemulsion compositions can be surprisingly improved.

[0206] The present disclosure specifically demonstrates that microneedle technology (e.g., microneedle conditioning of the skin) can significantly enhance the transdermal delivery of large agents (e.g., botulinum toxin and / or antibody agents), particularly when used in conjunction with nanoemulsion technology. Nanoemulsion compositions of particular interest include water-in-oil and oil-in-water nanoemulsions characterized by droplet size diameters ranging from about 1 nm to about 300 nm, an aqueous dispersion medium to oil ratio ranging from about 0.01:1 to about 20:1; an oil to surfactant ratio in the range of about 0.1 to about 40 and / or a zeta potential in the range of about -80 mV to about +80 mV.

[0207] In some embodiments, provided nanoemulsion compositions comprise oil and surfactant in a ratio ranging from about 0.1:1 to about 2:1. In some embodiments, provided nanoemulsion compositions comprise oil and surfactant in a ratio of about 0.1:1 to about 1:1. In some embodiments, provided nanoemulsion compositions comprise oil and surfactant in a ratio of about 0.5:1 to about 1:1. In some embodiments, provided nanoemulsion compositions comprise oil and surfactant in a ratio of about 0.5:1 to about 1:1.5. In some embodiments, provided nanoemulsion compositions comprise oil and surfactant in a ratio of about 0.1: 1, about 0.15: 1, about 0.2: 1, about 0.25: 1, about 0.3: 1, about 0.35: 1, about 0.4: 1, about 0.45: 1, about 0.5: 1, about 0.5: 1, about 0.55: 1, about 0.6: 1, about 0.65: 1, about 0.7: 1, about 0.75: 1, about 0.8: 1, about 0.85: 1, about 0.9: 1, about 0.95: 1, or about 1: 1. In some embodiments, provided nanoemulsion compositions comprise oil and surfactant in a ratio of about 0.67: 1.

[0208] In some embodiments, an aqueous dispersion medium (e.g., water, buffer, saline, etc.) and a surfactant are utilized in a ratio ranging between 0.01 and 20. In some embodiments, an aqueous dispersion medium (e.g., water, buffer, saline, etc.) and a surfactant are utilized in a ratio ranging between 0.1 and 20. In some embodiments, an aqueous dispersion medium (e.g., water, buffer, saline, etc.) and a surfactant are utilized in a ratio ranging between 0.5 and 10. In some embodiments, an aqueous dispersion medium (e.g., water, buffer, saline, etc.) and a surfactant are utilized in a ratio ranging between 0.5 and 1. In some embodiments, the ratio of aqueous dispersion medium (e.g., water, buffer, saline, etc.) to surfactant is about 0.01:1, about 0.02:1, about 0.03:1, about 0.04:1, about 0.05:1, about 0.06:1, about 0.07:1, about 0.08:1, about 0.0:1, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments, the ratio of surfactant to water is about 0.5: 1, about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, or about 20: 1. In some embodiments, the aqueous dispersion medium (e.g., water, buffer, saline, etc.) and the surfactant are utilized in a ratio ranging between 0.5 and 2. In some embodiments, the ratio of aqueous dispersion medium (e.g., water, buffer, saline, etc.) to surfactant is about 0.5: 1, about 1: 1, or about 2: 1. In some embodiments, the ratio of surfactant to aqueous dispersion medium (e.g., water, buffer, saline, etc.) is about 0.5: 1, about 1: 1, or about 2: 1. In some embodiments, the ratio of aqueous dispersion medium (e.g., water, buffer, saline, etc.) to surfactant is about 1: 1. In some embodiments, compositions utilizing such ratios of aqueous dispersion medium (e.g., water, buffer, saline, etc.) to surfactant comprise water-in-oil emulsions.

[0209] In some embodiments, the droplets in the nanoemulsion composition have a diameter (e.g., average diameter and / or median diameter) in the range of about 10 nm to about 300 nm, about 10 nm to about 200 nm, about 10 nm to about 150 nm, about 10 nm to about 130 nm, about 10 nm to about 120 nm, about 10 nm to about 115 nm, about 10 nm to about 110 nm, about 10 nm to about 100 nm, or about 10 nm to about 90 nm. In some embodiments, the droplets in the nanoemulsion composition have a diameter (e.g., average diameter and / or median diameter) in the range of 1 nm to 300 nm, 1 nm to 200 nm, 1 nm to 150 nm, 1 nm to 120 nm, 1 nm to 100 nm, 1 nm to 75 nm, 1 nm to 50 nm, or 1 nm to 25 nm. In some embodiments, the droplets in the nanoemulsion composition have a diameter (eg, mean diameter and / or median diameter) of 1 nm to 15 nm, 15 nm to 200 nm, 25 nm to 200 nm, 50 nm to 200 nm, or 75 nm to 200 nm.

[0210] In some embodiments, the total droplet distribution is contained within a specified droplet diameter size range. In some embodiments, less than 50%, 25%, 10%, 5% or 1% of the total droplet distribution exceeds the specified droplet diameter size range. In some embodiments, less than 1% of the total droplet distribution exceeds the specified droplet diameter size range. In some embodiments, the nanoemulsion composition is substantially free of droplets having a diameter greater than 300nm, 250nm, 200nm, 150nm, 120nm, 100nm, 75nm, 50nm or 25nm. In some embodiments, less than 50%, 25%, 10%, 5% or 1% of the total droplet distribution has a diameter greater than 300nm, 250nm, 200nm, 150nm, 120nm, 100nm, 75nm, 50nm or 25nm.

[0211] In some embodiments, the droplets in the nanoemulsion composition have an average droplet size of less than about 300nm, about 250nm, about 200nm, about 150nm, about 130nm, about 120nm, about 115nm, about 110nm, about 100nm, about 90nm, or about 50nm. In some embodiments, the average droplet size is in the range of about 10nm to about 300nm, about 50nm to about 250, about 60nm to about 200nm, about 65nm to about 150nm, or about 70nm to about 130nm. In some embodiments, the average droplet size is about 80nm to about 110nm. In some embodiments, the average droplet size is about 90nm to about 100nm.

[0212] In some embodiments, the nanoemulsion droplets have a zeta potential ranging between -80mV and +80mV. In some embodiments, the nanoemulsion droplets have a zeta potential ranging between -50mV and +50mV. In some embodiments, the nanoemulsion droplets have a zeta potential ranging between -25mV and +25mV. In some embodiments, the nanoemulsion droplets have a zeta potential ranging between n-10mV and +10mV. In some embodiments, the nanoemulsion droplets have a zeta potential of about -80mV, about -70mV, about -60mV, about 50mV, about -40mV, about -30mV, about -25mV, about -20mV, about -15mV, about -10mV, or about -5mV. In some embodiments, the nanoemulsion droplets have a zeta potential of about +50 mV, about +40 mV, about +30 mV, about +25 mV, about +20 mV, about +15 mV, about +10 mV, or about +5 mV. In some embodiments, the nanoemulsion droplets have a zeta potential of about 0 mV.

[0213] The present disclosure provides a surprisingly effective technology for transdermal delivery of large agents (e.g., botulinum toxin, antibodies, etc.). Specifically, the present disclosure teaches that by using microneedle technology without any other destruction strategy, the transdermal delivery of such agents can be significantly enhanced. Therefore, the technology provided can achieve effective delivery without the inflammation, irritation and / or allergic reactions that often accompany the use of skin destroyers. As will be understood by those skilled in the art reading this specification, the present disclosure teaches that by using microneedle technology, the transdermal delivery of such large agents can be significantly enhanced, even when the large agent is not loaded into the microneedle, coated on the microneedle and / or manufactured as a part of the microneedle. Similarly, as will be understood by those skilled in the art reading this specification, the present disclosure teaches that by using microneedle technology (and in particular by using MSCs), the delivery of large agents as described herein can be significantly enhanced without leaving the microneedles in the skin (e.g., by causing them to fall off and / or otherwise retain and / or degrade in situ). For example, one skilled in the art will appreciate that the provided technology can avoid the problem of long-term stability of large doses required for commercially viable products, and can achieve effective delivery without inflammation, irritation, and / or allergic reactions caused by skin disrupting agents and / or microneedles remaining in the skin. Indeed, in the Examples and elsewhere, the present disclosure explicitly teaches MSC-facilitated transdermal delivery of botulinum toxin from topical (e.g., creams, ointments) compositions, and particularly from compositions comprising macro- and nanoemulsions, using microneedles that do not contain botulinum toxin.

[0214] In some embodiments, the present disclosure teaches that particularly advantageous results are achieved when microneedle technology is combined with a nanoemulsion composition. In some embodiments, microneedle technology is combined with a lotion, cream, or liquid composition, which in turn may be or contain a nanoemulsion composition. In some embodiments, the provided technology does not utilize skin-disrupting technology, such as chemical penetration enhancers.

[0215] The findings provided herein are particularly surprising given that solid nanoparticles with a reported transdermal delivery size (e.g., 105 ± 2.92 nm) comparable to the droplets in the nanoemulsion composition used herein are not able to effectively deliver (or enhance their delivery) even small molecule agents through the skin. For example, Gomaa et al. describe a study in which a solution of rhodamine dye (molecular weight 479 Da) encapsulated in PLGA nanoparticles was applied to skin that had been pretreated by microneedles and skin penetration was evaluated. See Gomaa, Y. et al., "Effect of microneedle treatment on the skin permeation of ananoencapsulated dye." J Pharm Pharmacol. 2012 November; 64(11): 1592-1602. The data showed that after 6 hours of continuous application, very small amounts of dye began to penetrate the skin; a significant increase in penetration was not observed until the skin was treated for 24 hours. The researchers explain that "there is an emerging consensus that NPs [nanoparticles] generally cannot penetrate the stratum corneum, although they may be well deposited in hair follicles." Therefore, prior to this disclosure, those skilled in the art would have expected that even small molecule agents (e.g., rhodamine dyes) would not be effectively delivered transdermally using microneedle technology utilizing nanosized vehicles; certainly, delivery of large agents was thought to be impossible. However, this disclosure demonstrates that microneedles can significantly enhance transdermal delivery of large agents, particularly when used in conjunction with a nanoemulsion system.

[0216] Among other things, the present disclosure demonstrates that microneedle technology can enhance transdermal delivery (e.g., of large agents, particularly large agents from nanoemulsion compositions) when no other disrupting agents are utilized (i.e., no chemical permeation enhancers, and no other techniques for disrupting or piercing skin structure). Previous studies using microneedles to transdermally deliver agents as large as botulinum toxin (i.e., about 150 kDa) have reported that delivery was unsuccessful unless additional treatment was performed to disrupt the skin. For example, U.S. Patent Publication No. 2010 / 0196445 reports that botulinum toxin could not be effectively delivered from pre-coated microneedles unless skin-digesting enzymes were also administered to disrupt the skin structure at the site of the microneedles.

[0217] In addition, the present disclosure demonstrates that microneedle technology can achieve transdermal delivery (e.g., large agents, particularly large agents from coarse emulsions and nanoemulsion compositions) when microneedle coating or loading is not utilized and / or when the microneedles are not designed to remain in the skin. In addition, as already noted, the present disclosure recognizes that such microneedle coating or loading may not be commercially viable, at least due to the instability of the botulinum coating or loading materials. For example, according to Johnson, E., et al., "Botulinum toxin is very susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. Lyophilization or freeze drying of botulinum toxin is the most economically reasonable and practical method of distributing the product in a stable and easy-to-use form for clinicians." U.S. Patent No. 5,512,547. In addition, as will be understood by those skilled in the art who read this specification, the technology described herein has certain advantages, including that it is not necessary to leave the microneedles in the tissue or associated with the tissue. For example, it will be understood by those skilled in the art that leaving the microneedles in the skin may have the risk of skin irritation, inflammation, allergic reaction, and / or cosmetically undesirable scarring. In contrast to the present invention, the technology as described in U.S. Patent No. 2017 / 0209553 utilizes a microneedle array that is loaded with botulinum toxin into the needles and is designed to break the microneedles into the skin (according to U.S. Patent Nos. 2017 / 0209553 and 2016 / 0263362).

[0218] The present disclosure teaches that, although MSCs are only expected to help facilitate transdermal delivery of small compounds, the already efficient transdermal delivery of large agents via nanoemulsion compositions can be significantly enhanced by combination with microneedle technology. Furthermore, the present disclosure is particularly surprising because, as described by Gomaa, microneedle conditioning combined with encapsulation of even small molecule agents in solid nanoparticles provides only a small amount of penetration after 6 hours of application, and no penetration of the substance was observed until 24 hours after application.

[0219] In some embodiments, the nanoemulsion formulation containing a large agent is applied together with microneedle conditioning with a solid microneedle. In some embodiments, before the nanoemulsion formulation containing a large agent is applied to the site (for example, before a specific application and / or before each application), the MSC of the site is performed. In some embodiments, after the nanoemulsion formulation containing a large agent is applied to the site, the MSC of the site is performed. In some embodiments, the MSC of the site and the nanoemulsion formulation containing a large agent are applied to the site substantially simultaneously. In some embodiments, the coarse emulsion formulation containing a large agent is not injected via one or more microneedles. In some embodiments, the microneedle is part of a microneedle array. In some embodiments, the microneedle may have a length between about 1 μm and about 4,000 μm. In some embodiments, the microneedle may have a length between about 1 μm and about 2,000 μm. In some embodiments, the microneedle may have a length between about 50 μm and about 400 μm. In some embodiments, the microneedle may have a length between about 800 μm and about 1500 μm.

[0220] In some embodiments, the present invention utilizes nanoemulsion compositions containing large pharmaceutical agents that are particularly effective and / or useful in a medical setting, e.g., for therapeutic purposes. In some embodiments, a particular nanoemulsion composition is particularly effective and / or can be used to locally administer a pharmaceutical agent to a subject in need thereof. In some embodiments, a nanoemulsion composition may contain one or more large pharmaceutical agents. Exemplary nanoemulsion compositions and methods of preparation are described, e.g., in WO2012 / 103035, the disclosure of which is incorporated by reference in its entirety.

[0221] In some embodiments, the nanoemulsion can be formulated into a composition suitable for topical administration. In some embodiments, the composition suitable for topical administration can be a lotion, cream, powder, ointment, liniment, gel or drops.

[0222] In some embodiments, the nanoemulsion formulation comprises water, medium chain triglycerides, polysorbate 80, methylparaben, and propylparaben. In some embodiments, the nanoemulsion formulation comprises water, medium chain triglycerides, and polysorbate 80. Exemplary premixes are provided in Table 2 and are not meant to be limiting.

[0223] Table 2. Exemplary premixes

[0224] %w / w Element 6.375 1349 Oil 9.562 Polysorbate 80 0.199 Propylparaben 63.75 Isotonic sodium chloride solution 0.199 Methylparaben 19.92 Buffer solution* ** Big Pharmacy 100 total

[0225] * The buffer solution contained (w / w) 0.199% gelatin, 0.398% sodium hydrogen phosphate, 99.4% purified water, and the pH was adjusted to 6.0 ± 0.2 with hydrochloric acid.

[0226] **Given this specification, one of ordinary skill can make reasonable adjustments to this and other ingredients based on the volume, weight and / or dosage of the bulk drug to be utilized.

[0227] These compositions are particularly useful because they can be used to deliver the agent to a subject in need via topical and / or transdermal (e.g., by lotion, cream, powder, ointment, liniment, gel, drops, etc.) administration. In some embodiments, the cream and / or lotion formulations provided can be administered to a subject in need via topical and / or transdermal (e.g., by lotion, cream, powder, ointment, liniment, gel, drops, etc.) administration. In some embodiments, the nanoemulsion compositions provided can be formulated into cream and / or lotion formulations. In some embodiments, the cream and / or lotion formulations provided containing the nanoemulsion compositions can be used for and / or are effectively topically administered to a subject. In some embodiments, the nanoemulsion compositions provided can be mixed with one or more cream components in a cream formulation (e.g., a cream formulation provided) and / or a saline solution for preparing a pharmaceutical composition.

[0228] The present invention encompasses the recognition that emulsion compositions (e.g., macroemulsion compositions and nanoemulsion compositions) can be formulated into cream and / or lotion formulations for administration to a subject. The present invention encompasses the recognition that provided cream and / or lotion formulations can be particularly useful for formulating emulsions (such as those described herein) for administration to a subject. Exemplary nanoemulsion formulations are provided in Table 3 and are not meant to be limiting.

[0229] Table 3: Nanoemulsion formulations

[0230] Components Weight(g) Percentage (by weight) 1349 Oil 3.2 3.19 Tween-80 4.8 4.79 Methylparaben 0.2 0.2 Propylparaben 0.2 0.2 Sodium chloride (a) 0.63 0.63 Disodium hydrogen phosphate 0.04 0.04 gelatin 0.02 0.02 Large agents (e.g., botulinum toxin and / or antibodies) * * Mineral oil 0.63 0.63 Isopropyl myristate 0.62 0.62 White Vaseline 0.25 0.25 Emulsified wax 1.87 1.87 Purified water (c) 87.76 87.57 total 100.22 100.00

[0231] * In view of this specification, a skilled artisan can make reasonable adjustments to this and other ingredients based on the volume, weight and / or dosage of the bulk drug to be utilized.

[0232] In some embodiments, provided compositions comprise a mixture of provided nanoemulsion compositions and one or more pharmaceutically acceptable excipients. In some embodiments, cream and / or lotion formulations comprise a mixture of provided nanolotion compositions and / or saline solutions.

[0233] In some embodiments, provided compositions include provided nanoemulsion compositions. In some embodiments, provided compositions are cream and / or lotion formulations. In some embodiments, provided cream and / or lotion formulations include nanoemulsion compositions. In some embodiments, compositions include provided nanoemulsion compositions but are not cream and / or lotion formulations. In some embodiments, suitable compositions are formulated as creams and / or lotions but do not include nanoemulsion compositions.

[0234] In some embodiments, provided compositions include a mixture of provided nanoemulsion compositions and one or more pharmaceutically acceptable excipients, e.g., for topical and / or transdermal (e.g., by lotion, cream, powder, ointment, liniment, gel, drops, etc.) administration.

[0235] In some embodiments, for nanoemulsion compositions containing known therapeutic agents and / or independently active bioactive agents, such nanoemulsion compositions are arranged and constructed and administered in combination with MSCs so that a certain amount of therapeutic agent sufficient to treat a condition or disorder is delivered to a desired target site (e.g., to the epidermis and / or dermis structure). In some embodiments, the provided nanoemulsion compositions are arranged and constructed (e.g., by selecting and / or combining agents, component structures, etc.) so that they achieve the desired therapeutic effect after being applied to the skin. In some embodiments, the provided nanoemulsion compositions are arranged and constructed so that they do not cause undesirable clinical effects within and / or outside the desired site of action (e.g., skin surface, dermis, etc.). In some embodiments, the provided nanoemulsion compositions are arranged and constructed and administered in combination with MSCs so that they have a systemic effect.

[0236] In some embodiments, provided compositions can be formulated and delivered in combination with MSCs to achieve systemic delivery; in some embodiments, provided compositions can be formulated and / or delivered to achieve localized but non-systemic delivery.

[0237] The present disclosure specifically demonstrates that therapeutic agents (and particularly large biological agents, such as botulinum toxin or antibody agents) are effectively and efficiently delivered to the dermis using provided compositions in combination with MSCs. For example, in some embodiments, the present invention provides methods comprising administering a composition as described herein without clinically significant side effects. As an example, when local delivery is considered, clinically significant side effects include, but are not limited to, undesirable systemic side effects, damage to neural tissue beneath the dermis (e.g., neurotic paralysis), undesirable effects on muscle (e.g., muscle paralysis), and / or undesirable blood levels of therapeutic agents, etc. Exemplary formulations of botulinum nanoemulsion premixes are provided in Table 4, and are not meant to be limiting.

[0238] Table 4. Botulinum toxin nanoemulsion formulation (premix)

[0239]

[0240] * The buffer solution contained (w / w) 0.199% gelatin, 0.398% sodium hydrogen phosphate, 99.4% purified water, and the pH was adjusted to 6.0 ± 0.2 with hydrochloric acid.

[0241] Diseases, illnesses and conditions

[0242] The present invention provides techniques for treating and / or preventing any of a variety of systemic or dermatological diseases, disorders and / or conditions. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders or conditions associated with sweat and / or sebaceous gland activity. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders or conditions associated with infection. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders or conditions associated with inflammation. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders or conditions associated with inflammation. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders or conditions associated with cancer. In some embodiments, the present invention provides techniques for treating and / or preventing systemic diseases, disorders or conditions. In some embodiments, the present invention provides techniques for treating and / or preventing autologous diseases, disorders or conditions. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders or conditions associated with the epidermis and / or dermis level of the skin.

[0243] In some embodiments, the present invention provides a technology for treating and / or preventing one or more of the following: acne, unwanted sweating, body odor, excessive sweating, bromhidrosis, chromhidrosis, rosacea, hair loss, psoriasis, actinic keratosis, eczematous dermatitis (e.g., atopic dermatitis, etc.), conditions of excessive sebum secretion (e.g., seborrhea, seborrheic dermatitis, etc.), burns, Raynaud's phenomenon, lupus erythematosus, hyperpigmentation conditions (e.g., melasma, etc.), hypopigmentation conditions (e.g., vitiligo, etc.), skin cancer (e.g., squamous cell skin cancer, basal cell skin cancer, etc.), dermal infections (e.g., bacterial infections, viral infections, fungal infections, etc.), Facial wrinkles (e.g., involving the forehead, glabellar lines, wrinkles, and / or periorbital areas), headaches, unsightly facial expressions (e.g., due to overactivity of the underlying facial musculature), neck lines, hyperfunctional facial lines, hyperkinetic facial lines, platysma bands, neuromuscular disorders and conditions involving muscle spasms and / or contractures (including various forms of facial palsy, cerebral palsy, blepharospasm, facial contractures), dystonia, prostatic hyperplasia, headaches, strabismus, hemifacial spasm, tremors, spasms, such as those caused by multiple sclerosis, retroorbital muscles, various ophthalmic and urological conditions (e.g., penile and / or bladder disorders), and / or combinations thereof.

[0244] In some embodiments, the present invention provides technology for treating and / or preventing rheumatoid arthritis. In some embodiments, the present invention provides technology for treating and / or preventing psoriatic arthritis. In some embodiments, the present invention provides technology for treating and / or preventing osteoarthritis.

[0245] In some embodiments, the present invention provides techniques for treating and / or preventing lupus erythematosus. In some embodiments, the lupus erythematosus is systemic, discoid, drug-induced, or neonatal.

[0246] In some embodiments, the present invention provides technologies for treating and / or preventing Crohn's disease. In some embodiments, the present invention provides technologies for treating and / or preventing inflammatory bowel disease. In some embodiments, the present invention provides technologies for treating and / or preventing ulcerative colitis.

[0247] In some embodiments, the present invention provides techniques for treating and / or preventing a lung disease. In some embodiments, the lung disease may be asthma or chronic obstructive pulmonary disease.

[0248] In some embodiments, the present invention provides techniques for treating and / or preventing amyloidosis.In some embodiments, the amyloidosis is systemic or cutaneous.

[0249] In some embodiments, the invention provides techniques for treating and / or preventing cancer. In some embodiments, the cancer is skin, blood, breast, colon, or lung.

[0250] In some embodiments, the present invention provides techniques for treating and / or preventing dyslipidemia. In some embodiments, dyslipidemia is hypercholesterolemia.

[0251] In some embodiments, the invention provides techniques for treating and / or preventing infection. In some embodiments, the infection is or is caused by Clostridium difficile or Staphylococcus.

[0252] In some embodiments, the invention provides techniques for treating and / or preventing pain. In some embodiments, the pain is associated with arthritis. In some embodiments, the arthritis is rheumatoid arthritis, psoriatic arthritis, or osteoarthritis.

[0253] In some embodiments, the present invention provides techniques for treating and / or preventing neurological conditions. In some embodiments, the neurological condition is Alzheimer's disease, Parkinson's disease, or stroke.

[0254] In some embodiments, the invention relates to administering at least one of the provided compositions administered in combination with MSCs according to a dosing regimen sufficient to achieve at least about a 20% reduction in the extent and / or prevalence of a skin condition of interest; in some embodiments, according to a dosing regimen sufficient to achieve at least about a 25% reduction; in some embodiments, according to a dosing regimen sufficient to achieve at least about a 30% reduction; in some embodiments, according to a dosing regimen sufficient to achieve at least about a 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about %, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90% or more dosage regimens.

[0255] In some embodiments, the invention relates to administering at least one provided composition administered in combination with MSCs according to a dosing regimen sufficient to achieve at least about a 20% reduction in the extent and / or prevalence of a relevant skin condition in a specific percentage of a patient population to which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve at least about a 25% reduction in a specific percentage of a patient population to which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve at least about a 30% reduction in a specific percentage of a patient population to which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve at least about a 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 1 about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90% or more dosage regimens. In some embodiments, the specific percentage of the patient population to which the composition is administered is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, to give some illustrative examples, the invention relates to administering at least one provided composition according to a dosing regimen sufficient to achieve at least about a 20% reduction in the extent and / or prevalence of a relevant skin condition in at least about 50% of the patient population to which the composition is administered. In some embodiments, the invention relates to administering at least one provided composition according to a dosing regimen sufficient to achieve at least about a 30% reduction in the extent and / or prevalence of a relevant skin condition in at least about 50% of the patient population to which the composition is administered.

[0256] The present invention provides a technology for treating and / or preventing skin conditions, including applying the provided composition in combination with MSC to a subject suffering from, susceptible to and / or showing symptoms of skin conditions. In some embodiments, a composition for treating skin conditions as described herein is provided, for any administration route described herein. In some embodiments, the provided composition is formulated for topical application. In some embodiments, the provided composition is formulated into a cream, liniment, lotion, gel, shampoo, conditioner, sunscreen, deodorant and / or antiperspirant (e.g., as a roller, solid stick, gel, cream, aerosol, etc.), etc., depending on the condition to be treated.

[0257] In some embodiments, such provided compositions are topically administered in combination with MSCs to the affected area (e.g., underarms, hands, feet, scalp, hair follicles, face, neck, back, arms, chest, legs, groin, crotch, etc., depending on the specific condition to be treated). In some embodiments, topical administration is achieved by topical administration in combination with MSCs.

[0258] Compositions and preparations

[0259] As noted herein, the present invention provides and / or utilizes compositions comprising one or more macro-agents for combined administration with MSCs. In some embodiments, the compositions provided can be formulated for topical and / or transdermal delivery (e.g., as lotions, creams, liniments, ointments, powders, gels, drops, etc.). In some embodiments, the compositions provided can be or include nanoemulsions. In some embodiments, the compositions provided can be or include macroemulsions.

[0260] The formulations of the provided compositions can be prepared by any suitable method (e.g., methods known or later developed in the art of pharmacology). Typically, such preparation methods include the steps of combining the provided compositions with one or more excipients, and then, if necessary and / or desired, shaping and / or packaging them into a suitable form for administration, e.g., as or in a single or multiple dose unit.

[0261] In some embodiments, the composition can be prepared, packaged, and / or sold in bulk as a single unit dose and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of a provided composition. The amount of the provided composition is generally equal to the dose of the provided composition to be administered to a subject and / or a convenient fraction of such a dose, such as, for example, half or one-third of such a dose.

[0262] In some embodiments, excipients suitable for use in the composition (e.g., pharmaceutically and / or cosmetically acceptable compositions) may, for example, include one or more excipients such as solvents, dispersion media, granulation media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants and / or emulsifiers, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, disintegrants, binders, preservatives, buffers, etc., suitable for the desired specific dosage form. In some embodiments, excipients such as cocoa butter and / or suppository waxes, colorants, coating agents, sweeteners, flavoring agents, and / or aromatics may be utilized. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2005; which is incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation.

[0263] In some embodiments, suitable excipients (e.g., pharmaceutically and / or cosmetically acceptable excipients) are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other international pharmacopoeias.

[0264] In some embodiments, the compositions provided are formulated into creams, liniments, ointments, oils, foams, sprays, lotions, liquids, powders, thickening lotions or gels (e.g., formulated for transdermal delivery as described herein). Specific exemplary such preparations can be, for example, prepared into cosmetic preparation products, such as skin softeners, nutrient lotion-type emulsions, cleansing lotions, cleansing creams, skin milks, moisturizing lotions, massage creams, moisturizing creams, cosmetic bases, facial masks or facial gels, detergent preparations, such as shampoos, mouthwashes, body cleansers, hair nourishers or soaps, or dermatological components, such as lotions, ointments, gels, creams, liniments, patches, deodorants or sprays. In some embodiments, the compositions for topical application are not formulated for application to mucosa (e.g., not suitable for application to mucosa and / or not formulated to deliver an appropriate amount of large medicament to or through the mucosa).

[0265] Treatment Area

[0266] The technology of the present invention is applicable to both human and veterinary use.A subject suffering from any condition that would benefit from the topical application of an active agent can be treated with the disclosed transdermal drug delivery technology.

[0267] Any site suitable for MSC is a suitable site of administration. In some embodiments, the site of administration is the skin covering a muscle or muscle group of the subject. In some embodiments, the site is hairless. In some embodiments, the site is on the trunk. In some embodiments, the site is on the back. In some embodiments, the site is on the chest. In some embodiments, the site is on the buttocks. In some embodiments, the site is on the crotch. In some embodiments, the site is on the groin. In some embodiments, the site is on the head. In some embodiments, the site is on the scalp. In some embodiments, the site is on the face. In some embodiments, the site is on the neck. In some embodiments, the site is on the shoulder. In some embodiments, the site is in the armpit. In some embodiments, the site is on the armpit. In some embodiments, the site is on the hand. In some embodiments, the site is on the foot. In some embodiments, the site is on the arm. In some embodiments, the site is on the leg. In some embodiments, the site is not a mucosa.

[0268] In some embodiments, the site is affected by a skin condition. In some embodiments, the site is the skin overlying a muscle or group of muscles affected by a neuromuscular condition. In some embodiments, the length of the microneedles used in the MSC is adjusted based on the thickness of the skin at the treatment site.

[0269] Application

[0270] The present invention provides techniques for delivering emulsion compositions (e.g., botulinum toxin emulsion compositions or antibody agent emulsion compositions) for various applications, including, for example, cosmetics, nutritional products, and medical applications. Such emulsion compositions may include one or more bioactive agents. In some embodiments, the emulsion composition includes botulinum toxin. In some embodiments, the emulsion composition includes an antibody agent. In some embodiments, the emulsion composition is a nanoemulsion composition and / or a macroemulsion composition.

[0271] The present invention provides techniques for treating a condition or disorder using any of the provided compositions (e.g., provided emulsion compositions; cream and / or lotion formulations; a combination of provided emulsion compositions and cream and / or lotion formulations; etc.) as described herein in combination with MSCs.

[0272] In some embodiments, such methods involve applying the provided compositions in combination with MSCs to patients suffering from and / or susceptible to diseases, conditions or illnesses. In some embodiments, such methods involve applying the provided nanoemulsion compositions in combination with MSCs as described herein (e.g., using microneedles with relatively low microneedle density and / or relatively small microneedle puncture size) to patients suffering from and / or susceptible to diseases, conditions or illnesses associated with the dermis of the skin. In some embodiments, such methods include applying the emulsion composition in combination with MSCs to patients suffering from and / or susceptible to diseases, conditions or illnesses, the emulsion composition comprising at least one known therapeutic agent and / or independently active bioactive agent. In some embodiments, such methods include applying the emulsion composition and / or at least one known therapeutic agent and / or independently active bioactive agent formulated with the provided cream and / or lotion formulations in combination with MSCs to patients suffering from and / or susceptible to diseases, conditions or illnesses. In some embodiments, such methods involve administering the composition via topical and / or transdermal (e.g., by lotion, cream, powder, ointment, liniment, gel, drops, etc.) administration in combination with MSCs. Some embodiments further comprise administering a penetration enhancer. Some embodiments further comprise administering a non-irritating penetration enhancer.

[0273] In some embodiments, the present invention provides techniques for treating any condition or disorder. In some embodiments, the present invention demonstrates that certain compositions as described herein in combination with MSCs as described herein can achieve controlled and / or improved delivery of active agents to biologically relevant target sites (e.g., specific tissues, locations within the skin, cells, etc.). In some embodiments, the present invention demonstrates controlled delivery and / or achievement of therapeutic effects in certain biologically relevant target sites without significant side effects associated with delivery to other areas.

[0274] In some embodiments, the present invention provides an improved technology for treating conditions or disorders associated with epidermal and / or dermal structures (e.g., sweat glands, sebaceous glands, hair follicles, etc.). In some embodiments, the present invention demonstrates that the provided compositions (e.g., provided nanoemulsion compositions; cream and / or lotion formulations; provided nanoemulsion compositions and cream and / or lotion formulations in combination; etc.) as described herein and MSCs (e.g., using microneedles with relatively low microneedle density and / or relatively small microneedle puncture size) can effectively improve the delivery and / or bioavailability of active agents and specifically deliver to the dermis, and provides that the compositions as described herein can have a therapeutic effect when applied to the skin of a subject. In some embodiments, the present invention demonstrates improved delivery and / or bioavailability and / or therapeutic effects achieved by dermal delivery without significant side effects associated with delivery to other areas (e.g., subcutaneous or subcutaneous structures and / or tissues other than the dermis). In some embodiments, provided compositions as described herein (e.g., provided emulsion compositions; cream and / or lotion formulations; a combination of provided emulsion compositions and cream and / or lotion formulations; etc.) in combination with MSCs as described herein can improve the transdermal delivery and / or bioavailability of active agents, such as therapeutic agents (e.g., botulinum toxin, antibody agents, etc.).

[0275] The present invention provides techniques for treating a condition or disorder by administering to a patient a provided composition as described herein (e.g., a provided emulsion composition; a cream and / or lotion formulation; a combination of a provided emulsion composition and a cream and / or lotion formulation; etc.) in combination with MSCs as described herein (e.g., using microneedles having a relatively low microneedle density and / or a relatively small microneedle puncture size). In some embodiments, the present invention provides techniques for treating a condition or disorder by topically administering to a patient a composition containing a provided emulsion composition as described herein in combination with MSCs.

[0276] In some embodiments, the large medicament penetrates the skin within about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes of application. In some embodiments, the large medicament penetrates the skin within about 5 to about 60 minutes of application. In some embodiments, the large medicament penetrates the skin within about 5 to about 12 minutes of application. In some embodiments, the large medicament penetrates the skin within about 5 to about 15 minutes of application. In some embodiments, the large medicament penetrates the skin within about 15 to about 30 minutes of application. In some embodiments, the large medicament penetrates the skin within about 1 hour of application. In some embodiments, the large medicament penetrates the skin within about 2 hours of application. In some embodiments, the large medicament penetrates the skin within about 3 hours of application. In some embodiments, the large medicament penetrates the skin within about 4 hours of application. In some embodiments, the large medicament penetrates the skin within about 5 hours of application. In some embodiments, the large medicament penetrates the skin within about 6 hours of application.

[0277] In some embodiments, the large medicament penetrates the skin layer within about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes of application. In some embodiments, the large medicament penetrates the skin layer within about 5 to about 60 minutes of application. In some embodiments, the large medicament penetrates the skin layer within about 5 to about 12 minutes of application. In some embodiments, the large medicament penetrates the skin layer within about 5 to about 15 minutes of application. In some embodiments, the large medicament penetrates the skin layer within about 15 to about 30 minutes of application. In some embodiments, the large medicament penetrates the skin layer within about 1 hour of application. In some embodiments, the large medicament penetrates the skin layer within about 2 hours of application. In some embodiments, the large medicament penetrates the skin layer within about 3 hours of application. In some embodiments, the large medicament penetrates the skin layer within about 4 hours of application. In some embodiments, the large medicament penetrates the skin layer within about 5 hours of application. In some embodiments, the large medicament penetrates the skin layer within about 6 hours of application.

[0278] In some embodiments, the large medicament penetrates the top layer of the skin within about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 5 to about 60 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 5 to about 12 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 5 to about 15 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 15 to about 30 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 1 hour of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 2 hours of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 3 hours of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 4 hours of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 5 hours of application. In some embodiments, the large medicament penetrates the top layer of the skin within about 6 hours of application.

[0279] In some embodiments, the large medicament penetrates the top layer of the skin, including the stratum corneum, dermal pores and / or dermal glands, within about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin, including the stratum corneum, dermal pores and / or dermal glands, within about 5 to about 60 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin, including the stratum corneum, dermal pores and / or dermal glands, within about 5 to about 12 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin, including the stratum corneum, dermal pores and / or dermal glands, within about 5 to about 15 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin, including the stratum corneum, dermal pores and / or dermal glands, within about 15 to about 30 minutes of application. In some embodiments, the large medicament penetrates the top layer of the skin, including the stratum corneum, dermal pores and / or dermal glands, within about 1 hour of application. In some embodiments, the large agent penetrates the top layer of the skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 2 hours of administration. In some embodiments, the large agent penetrates the top layer of the skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 3 hours of administration. In some embodiments, the large agent penetrates the top layer of the skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 4 hours of administration. In some embodiments, the large agent penetrates the top layer of the skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 5 hours of administration. In some embodiments, the large agent penetrates the top layer of the skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 6 hours of administration.

[0280] Reagent test kit

[0281] In some embodiments, the present invention provides a pharmaceutical package or kit, which includes one or more emulsion compositions according to the present invention and one or more microneedle devices. In some embodiments, the pharmaceutical package or kit includes a preparation or pharmaceutical composition containing a provided composition in one or more containers, and the container is optionally filled with one or more additional ingredients of the pharmaceutical composition. In some embodiments, the pharmaceutical package or kit includes additional approved therapeutic agents (e.g., benzoyl peroxide for treating acne; aluminum compounds for treating hyperhidrosis; etc.) for combination therapy. In some embodiments, optionally accompanied by such containers may be a report in a form issued by a government agency that regulates the manufacture, use or sale of pharmaceutical products, which reflects approval by the agency for manufacture, use or sale for human use.

[0282] In some embodiments, kits are provided that include therapeutic agents. As a non-limiting example only, provided compositions can be provided as topical preparations and administered as therapy in combination with the use of a microneedle device. Instructions for the dosage of the drug or its self-administration can be provided in the kit for administration to individuals suffering from or at risk of a condition or disorder, such as those associated with the dermal level of the skin.

[0283] In some embodiments, the kit may include (i) a provided composition; and (ii) at least one pharmaceutically acceptable excipient; and (iii) at least one device for microneedle treatment of skin; and (iv) instructions for use. In some embodiments, at least one device may include a microneedle having a relatively low density (e.g., about 2 microneedles / cm 2 Up to about 50 microneedles / cm 2 In some embodiments, for example, at least one device may include microneedles having a relatively small puncture size (e.g., each microneedle has a puncture size of about 100 μm). 2 / Microneedle to about 30,000μm 2 / microneedle range, each microneedle has a puncture size of about 100 μm 2 / Microneedle to about 60,000μm 2 / microneedle range).

[0284] In addition, the present invention provides a technique for administering large agents (e.g., botulinum toxin or antibody agents), improving transdermal delivery, and / or improving the bioavailability of such large agents by incorporating one or more large agents into one or more emulsion compositions and then administering them in combination with MSCs as described herein. The inventors surprisingly found that when used in combination with MSCs using microneedles or microneedle arrays having a relatively low microneedle density or a relatively small microneedle puncture size (e.g., the puncture size of each microneedle, the cross-sectional area of ​​each microneedle), the transdermal penetration and bioavailability of botulinum toxin or antibody agents incorporated into nanoemulsion compositions are significantly improved. The benefit of the present invention is the ability to administer such large agents intradermally while minimizing irritation or damage to the skin. The use of other agents or steps in the emulsion composition and MSCs is not necessarily excluded in all embodiments of the present invention, but is also not required.

[0285] Thus, the present invention provides a technique for administering a large agent by topical administration of a superior emulsion composition (e.g., a macroemulsion composition and / or a nanoemulsion composition) in combination with MSCs. In some embodiments, the large agent is botulinum toxin. In some embodiments, the botulinum emulsion composition is applied directly to the skin and is used for absorption through the epidermis before MSCs. In some embodiments, the botulinum emulsion composition is applied directly to the skin and is used for absorption through the epidermis after MSCs. In some embodiments, the botulinum emulsion composition is applied directly to the skin and is used for absorption through the epidermis at substantially the same time as MSCs.

[0286] In some embodiments, a botulinum toxin emulsion composition combined with MSCs can penetrate the top layers of the skin, including the stratum corneum, dermal pores, and / or dermal glands, without the use of a penetration enhancer. In some embodiments, a botulinum toxin emulsion composition combined with MSCs can penetrate the top layers of the skin, including the stratum corneum, dermal pores, and / or dermal glands, without the use of a degrading agent, an irritant, and / or an abrasive.

[0287] In some embodiments, the antibody agent emulsion composition combined with MSC can penetrate the top layer of the skin, including the stratum corneum, dermal pores and / or dermal glands, without the use of a penetration enhancer. In some embodiments, the large agent is an antibody agent. In some embodiments, the antibody agent emulsion composition is directly applied to the skin and used for absorption through the epidermis before MSC. In some embodiments, the antibody agent emulsion composition is directly applied to the skin and used for absorption through the epidermis after MSC. In some embodiments, the antibody agent emulsion composition is directly applied to the skin and used for absorption through the epidermis at substantially the same time as MSC. In some embodiments, the antibody agent emulsion composition is directly applied to the skin and used for systemic absorption.

[0288] In some embodiments, the antibody agent emulsion composition combined with MSCs can penetrate the top layers of the skin, including the stratum corneum, dermal pores, and / or dermal glands, without the use of degrading agents, irritants, and / or abrasives.

[0289] It will be appreciated by one of ordinary skill in the art that the compositions of the present invention for topical administration may have a cosmetic formulation such as a skin softener, a nourishing lotion-type emulsion, a cleansing lotion, a cleansing cream, a skin milk, a moisturizing lotion, a massage cream, a moisturizing cream, a makeup base, a mask or a facial gel, a cleanser formulation such as a shampoo, a mouthwash, a body cleanser, a hair nourisher or a soap, or a dermatological component such as a lotion, an ointment, a gel, a cream, a patch or a spray. In some embodiments, the composition for topical administration is not formulated for application to a mucosa (e.g., not suitable for application to a mucosa and / or not formulated to deliver an appropriate amount of a large agent to or across a mucosa).

[0290] One of ordinary skill in the art will understand that the units herein refer to units that are biologically equivalent or biologically equivalent to the units defined by the commercial manufacturers of botulinum toxin.

[0291] In some embodiments, the therapeutic effect of the botulinum toxin administered according to the present invention can last as long as the effect of the injection solution. In some embodiments, the effect of such an injection solution can last up to about 6 to 7 months. In some embodiments, the therapeutic effect of the botulinum toxin administered according to the present invention can last up to 6 to 7 months. In some embodiments, the use of a synthetic polymer carrier that can retain the botulinum toxin for slow release can extend the effect by up to about five years (U.S. Pat. No. 6,312,708).

[0292] In some embodiments, the present invention provides topical formulations of botulinum toxin that avoid potential complications, including but not limited to systemic toxicity or botulism. In some embodiments, the dosage range of botulinum toxin (including A, B, C, D, E, F or G types or botulinum toxin genetically engineered or chemically modified to have a longer or shorter duration of action than botulinum toxin serotype A) can be from as low as about 1 unit to as high as about 50,000 units, with minimal risk of adverse side effects. The specific dosage can vary depending on the condition being treated and the treatment regimen being utilized. For example, a high transdermal dose (e.g., 1000 units to 20,000 units) of botulinum toxin may be required to treat subcutaneous, overactive muscles. In contrast, a relatively small transdermal dose (e.g., from about 1 unit to about 1,000 units) of botulinum toxin may be required to treat neurogenic inflammation or overactive sweat glands.

[0293] Some embodiments of the present invention contemplate pharmaceutical compositions comprising a stable botulinum toxin for transdermal delivery to a human patient. The botulinum toxin may be selected from the group consisting of botulinum toxin types A, B, C1, D, E, F, and G, isolated and / or purified (i.e., about 150 kDa) botulinum toxin, and natural or recombinant botulinum toxin. In some embodiments, the composition may comprise from about 1 unit to about 50,000 units of botulinum toxin, and the composition may comprise an amount of botulinum toxin sufficient to achieve a therapeutic effect lasting from 1 month to 5 years.

[0294] In some embodiments, the present invention provides topical formulations of botulinum toxin (e.g., botulinum emulsion compositions) that allow the botulinum toxin to penetrate through the skin of a subject without penetrating substantially through blood vessels. For example, in some embodiments of the present invention, upon administration of the topical and / or transdermal formulations of the present invention, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the botulinum toxin present in the pharmaceutical composition penetrates into blood vessels.

[0295] In some embodiments, the present invention provides topical formulations of antibody agents (e.g., antibody agent emulsion compositions) that allow the antibody agent to penetrate through the subject's skin without penetrating substantially through blood vessels. For example, in some embodiments of the present invention, after application of the topical and / or transdermal formulations of the present invention, less than about 25% or even less than about 5% of the antibody agent present in the pharmaceutical composition penetrates into the blood vessels.

[0296] In some embodiments, the present invention provides topical preparations of antibody agents (e.g., antibody agent emulsion compositions), which allow the antibody agent to penetrate through the skin of the subject and penetrate in large quantities through the blood vessels. In some embodiments, the present invention provides topical preparations of antibody agents (e.g., antibody agent emulsion compositions), which allow the antibody agent to penetrate through the skin of the subject and penetrate through the blood vessels in a therapeutically effective amount. For example, in some embodiments of the present invention, after applying the topical and / or transdermal preparations of the present invention, greater than about 25%, 50%, 75%, 90% or 95% of the antibody agent present in the pharmaceutical composition penetrates into the blood vessels. In some embodiments, the present invention provides topical preparations of antibody agents (e.g., antibody agent emulsion compositions), which allow the antibody agent to have a systemic effect on the subject.

[0297] One of ordinary skill in the art will appreciate that the compositions of the invention that effect transdermal administration of a botulinum toxin or antibody agent may be incorporated into a device (eg, a patch, roller, pen, stamp, etc.). Example

[0298] Example 1: Effect of Microneedle Skin Conditioning (MSC) Pretreatment on the Bioavailability of Botulinum Toxin

[0299] A single-dose topical study of botulinum toxin bioavailability following topical application of a botulinum nanoemulsion was conducted. The skin was conditioned with a microneedle array by stamping the array onto the skin prior to application of the topical treatment. The array was then removed prior to application of the botulinum treatment. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities and microneedle puncture sizes (e.g., puncture area per microneedle) on botulinum toxin bioavailability.

[0300] The study included four test groups of eight rats each. Each group had microneedle skin conditioning as described above. Except for the control group, each group received a topical treatment once with a fixed volume and concentration of botulinum nanoemulsion on the skin overlying the biceps femoris, gastrocnemius, and tibialis anterior muscles of the right hind limb. The nanoemulsion was applied with a gloved finger. It took about 10 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin. The effect of this treatment was measured by mortality, as it is also known that doses of botulinum toxin applied in sufficient concentrations may induce death in animals. Therefore, the mortality rates of the four treatment groups were compared. Specifically, the mortality rate on day 3 after treatment was used to measure the relative bioavailability of botulinum toxin, where an increase in mortality represents a higher bioavailability of the toxin.

[0301] Table 5 describes the properties of each type of microneedle used in each treatment group, including needle density, needle length, and the size of the puncture each needle made in the skin before treatment (such as the microneedle puncture size described herein). Table 5 also details the mortality rate of each group.

[0302] It can be seen that each microneedle approach (i.e., each of Groups A, B, and C) achieved meaningful bioavailability.

[0303] In addition, by comparing Group C with any of Groups A and B, it was unexpectedly observed that fewer and smaller puncture holes provided higher bioavailability than more and larger puncture holes. That is, reducing the microneedle density improves bioavailability, and reducing the microneedle puncture size also improves bioavailability. By comparing Groups A and B with each other, it was also unexpectedly observed that increasing the microneedle length did not improve bioavailability.

[0304] That is, the results of the study determined that when such botulinum toxin is contained in a nanoemulsion and applied to the skin after microneedle skin conditioning as described herein, the microneedle density and microneedle puncture size can significantly improve the bioavailability of botulinum toxin. Specifically, when the microneedle array is used to condition the skin before the macromolecule in the emulsion composition is topically applied, reducing the needle density of the microneedle array to less than 31 needles per square centimeter can improve the bioavailability of the macromolecule. Moreover, when the microneedle array is used to condition the skin before the macromolecule in the emulsion composition is topically applied, reducing the needle puncture size of the microneedle array (each microneedle) to less than 36,000 square microns per microneedle can improve the bioavailability of the macromolecule.

[0305] Table 5. Research results of Example 1

[0306]

[0307] Example 2: Effect of MSC preconditioning on botulinum toxin bioavailability in humans: Effect on sweat reduction

[0308] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities on enhancing botulinum toxin bioavailability in humans by measuring the reduction in sweat from the skin following topical treatment with a botulinum nanoemulsion formulation.

[0309] The study included one subject. Three points, each approximately 2 cm2 in area and 5 cm apart from each other, were selected on the abdomen and marked with a marker. Each point was topically treated once with a fixed volume of a botulinum nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin. The first point was not preconditioned with the microneedle array and was a control site. The second point was preconditioned with a microneedle array at a density of 9 microneedles / cm prior to application of the botulinum formulation. 2 The third site was preconditioned with three impressions of a microneedle array of 85 microneedles / cm before the application of the botulinum toxin formulation. 2 Three impressions of the microneedle array were preconditioned and were intervention site No. 2.

[0310] The intended effect of this treatment is to reduce sweating at the site of botulinum nanoemulsion treatment. The amount of sweating at the treatment site is measured by two methods: 1) an evaporometer test, in which an instrument used to measure the rate of evaporation of water from the skin is used to detect the sweating rate (so that when the amount of sweating increases, a greater amount of evaporation can be detected); or 2) a starch-iodine test, in which the subject applies povidine to the treatment site; allows it to dry; corn starch is sprinkled on the treatment site; when the subject sweats into the white corn starch, it turns purple; if the subject is not sweating, it remains white; this is called a starch-iodine test. For either method of sweat detection, in order to induce sweating, the subject is placed under a heat lamp and then the sweat detection method is used.

[0311] The sweat detection method was used at baseline before botulinum nanoemulsion treatment; two weeks after treatment and four weeks after treatment. The study found that at baseline, the average sweat volume detected by the evaporator test or starch-iodine test was approximately equal in the control and intervention sites. At two weeks and four weeks after treatment, the average sweat volume detected by the evaporator test or starch-iodine test at the control site was greater than the average sweat volume detected at the intervention site in these post-treatment weeks. The study also found that at two weeks and four weeks after treatment, the average sweat volume detected by the evaporator test or starch-iodine test at intervention site No. 2 was greater than the average sweat volume detected at intervention site No. 1 in these post-treatment weeks.

[0312] This study determined that microneedle preconditioning with a relatively low microneedle density unexpectedly increased the bioavailability of a topical large-dose nanoemulsion containing botulinum toxin.

[0313] Example 3: Effect of MSC preconditioning with different microneedle densities on the bioavailability of botulinum toxin in humans Effect: Effect on sweat reduction

[0314] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities on enhancing botulinum toxin bioavailability in humans by measuring the reduction in sweat from the skin following topical treatment with a botulinum nanoemulsion formulation.

[0315] Twelve subjects were included in the study. Three points, each approximately 2 cm2 in area and 5 cm apart from each other, were selected on the back and marked with a marker. Each point was topically treated once with a fixed volume of a botulinum nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin. The first point was not preconditioned with the microneedle array and was a control site. The second point was preconditioned with a microneedle array at a density of 9 microneedles / cm prior to application of the botulinum formulation. 2 The third site was preconditioned with five impressions of a microneedle array of 85 microneedles / cm before the application of the botulinum toxin preparation. 2 Five impressions of the microneedle array were preconditioned and were intervention site No. 2.

[0316] The intended effect of this treatment is to reduce sweating at the site of treatment with the botulinum nanoemulsion. The amount of sweating at the treatment site is measured by two methods: 1) an evaporometer test, in which an instrument used to measure the rate of evaporation of water from the skin is used to detect the sweating rate (so that as the amount of sweating increases, greater evaporation can be detected); or 2) a starch-iodine test, in which the subject applies povidone to the treatment site; allows it to dry; corn starch is sprinkled on the treatment site; when the subject sweats into the white corn starch, it turns purple; if the subject is not sweating, it remains white; this is called a starch-iodine test. For either method of sweat detection, the subject is placed in a sauna to induce sweating, and then the sweat detection method is used.

[0317] The sweat detection method was used at baseline before botulinum nanoemulsion treatment; two weeks after treatment and four weeks after treatment. The study found that at baseline, the average sweat volume detected by the evaporator test or starch-iodine test was approximately equal in the control and intervention sites. At two weeks and four weeks after treatment, the average sweat volume detected by the evaporator test or starch-iodine test at the control site was greater than the average sweat volume detected at the intervention site in these post-treatment weeks. The study also found that at two weeks and four weeks after treatment, the average sweat volume detected by the evaporator test or starch-iodine test at intervention site No. 2 was greater than the average sweat volume detected at intervention site No. 1 in these post-treatment weeks.

[0318] This study determined that microneedle preconditioning with a relatively low microneedle density unexpectedly increased the bioavailability of a topical large-dose nanoemulsion containing botulinum toxin.

[0319] Example 4: Effect of MSC preconditioning with different microneedle puncture sizes on the bioavailability of botulinum toxin in humans Effect: Effect on sweat reduction

[0320] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percentage of bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle puncture sizes (e.g., puncture area per microneedle) on significantly enhancing botulinum toxin bioavailability in humans by measuring the reduction in sweat in the skin following topical treatment with a botulinum nanoemulsion formulation.

[0321] Twelve subjects were included in the study. Three points, each approximately 2 cm2 in area and 5 cm apart from each other, were selected on the back and marked with a marker. Each point was topically treated once with a fixed volume of a botulinum nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin. The first point was not preconditioned with the microneedle array and was a control site. The second point was punctured with a microneedle array having a size of approximately 11,000 μm prior to application of the botulinum formulation. 2 The third site was preconditioned with five impressions of a microneedle array of 100 μm / microneedles and was intervention site No. 1. The third site was punctured with a microneedle with a size of approximately 60,000 μm before the application of the botulinum toxin formulation. 2 / Five impressions of a microneedle array of microneedles were preconditioned and were intervention site No. 2.

[0322] The intended effect of this treatment is to reduce sweating at the site of treatment with the botulinum nanoemulsion. The amount of sweating at the treatment site is measured by two methods: 1) an evaporometer test, in which an instrument used to measure the rate of evaporation of water from the skin is used to detect the sweating rate (so that as the amount of sweating increases, greater evaporation can be detected); or 2) a starch-iodine test, in which the subject applies povidone to the treatment site; allows it to dry; corn starch is sprinkled on the treatment site; when the subject sweats into the white corn starch, it turns purple; if the subject is not sweating, it remains white; this is called a starch-iodine test. For either method of sweat detection, the subject is placed in a sauna to induce sweating, and then the sweat detection method is used.

[0323] The sweat detection method was used at baseline before botulinum nanoemulsion treatment; two weeks after treatment and four weeks after treatment. The study found that at baseline, the average sweat volume detected by the evaporator test or starch-iodine test was approximately equal in the control and intervention sites. At two weeks and four weeks after treatment, the average sweat volume detected by the evaporator test or starch-iodine test at the control site was greater than the average sweat volume detected at the intervention site in these post-treatment weeks. The study also found that at two weeks and four weeks after treatment, the average sweat volume detected by the evaporator test or starch-iodine test at intervention site No. 2 was greater than the average sweat volume detected at intervention site No. 1 in these post-treatment weeks.

[0324] This study determined that microneedle preconditioning with a relatively small microneedle puncture size unexpectedly increased the bioavailability of a topical large-dose nanoemulsion containing botulinum toxin.

[0325] Example 5: Effect of MSC on the bioavailability of botulinum toxin in humans: Effect on sweat and wrinkle reduction

[0326] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities on enhancing botulinum toxin bioavailability in humans by measuring sweat reduction and wrinkle reduction in the skin following topical treatment with a botulinum nanoemulsion formulation.

[0327] The study included a subject with severe frontalis (or horizontal) wrinkles on his forehead. Three points, each approximately 2 square centimeters in area and 5 cm apart from each other, were selected on the subject's forehead and marked with a marker. Each point was topically treated once with a fixed volume of a botulinum nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin. The first point was not preconditioned with the microneedle array and was a control site. The second point was preconditioned with a microneedle array at a density of 9 microneedles / cm prior to application of the botulinum formulation. 2 The third site was preconditioned with five impressions of a microneedle array of 85 microneedles / cm before the application of the botulinum toxin preparation. 2 Five impressions of the microneedle array were preconditioned and were intervention site No. 2.

[0328] The intended effect of this treatment is to reduce sweating at the site of treatment with the botulinum nanoemulsion. The amount of sweating at the treatment site is measured by two methods: 1) an evaporometer test, in which an instrument used to measure the rate of evaporation of water from the skin is used to detect the sweating rate (so that when the amount of sweating increases, a greater amount of evaporation can be detected); or 2) a starch-iodine test, in which the subject applies povidone to the treatment site; allows it to dry; corn starch is sprinkled on the treatment site; when the subject sweats into the white corn starch, it turns purple; if the subject is not sweating, it remains white; this is called the starch-iodine test. For either method of sweat detection, the subject is placed in a sauna to induce sweating and then the method is used.

[0329] The expected effect of botulinum nanoemulsion treatment is to reduce frontalis wrinkles at the botulinum nanoemulsion treatment site.The severity of wrinkles was measured using a four-level wrinkle rating scale (Wrinkle Scale): 0 = none, 1 = mild, 2 = moderate, 3 = severe.

[0330] The sweat detection method was used at baseline before botulinum nanoemulsion treatment; two weeks after treatment and four weeks after treatment. The study found that at baseline, the average sweat volume detected by the evaporator test or starch-iodine test was approximately equal in the control and intervention sites. At two weeks and four weeks after treatment, the average sweat volume detected by the evaporator test or starch-iodine test at the control site was greater than the average sweat volume detected at the intervention site in these weeks after treatment. The study also found that at two weeks and four weeks after treatment, the average sweat volume detected by the evaporator test or starch-iodine test at intervention site No. 2 was greater than the average sweat volume detected at intervention site No. 1 in these weeks after treatment. The study found that at baseline, the average severity of frontal muscle wrinkles measured by the wrinkle scale was approximately equal in the control and intervention sites. At two weeks and four weeks after treatment, the average severity of frontal muscle wrinkles measured by the wrinkle scale at the control site in these weeks after treatment was greater than the average severity detected at the intervention site. The study also found that at two and four weeks post-treatment, the mean severity of frontalis wrinkles measured by the wrinkle rating scale at intervention site No. 2 was greater than the mean severity detected at intervention site No. 1 in the weeks following these treatments.

[0331] This study determined that microneedle preconditioning with a relatively low microneedle density unexpectedly increased the bioavailability of a topical large-dose nanoemulsion containing botulinum toxin.

[0332] Example 6: Effect of MSCs with different microneedle densities on the bioavailability of botulinum toxin in humans: Effect of sweat reduction on sweat subjects

[0333] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities on enhancing botulinum toxin bioavailability in humans by measuring the reduction in sweat from the skin following topical treatment with a botulinum nanoemulsion formulation.

[0334] The study included three treatment groups of twenty subjects each with axillary hyperhidrosis characterized by excessive underarm sweating: Group 1 was a control group and a botulinum nanoemulsion was applied to the underarms of each subject; Group 2 was intervention group 1 and a microneedle density of 9 microneedles / cm was applied to the underarms of each subject before the application of the botulinum nanoemulsion formulation. 2 Each section of the underarm skin was preconditioned with five impressions of a microneedle array of ; Group 3 was intervention group 2 and was primed with a microneedle array at a density of 85 microneedles / cm before the application of the botulinum toxin nanoemulsion formulation. 2 Each section of the underarm skin was preconditioned with five impressions of a microneedle array of . Each subject in Groups 1, 2, and 3 was topically treated once with a fixed volume of a botulinum toxin nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin.

[0335] The intended effect of this treatment is to reduce sweating at the site of botulinum nanoemulsion treatment, i.e., the underarm. The amount of sweating at the treatment site is measured by gravimetric sweat measurement (GS test): the subject's underarm is wiped dry with a paper towel; filter paper is weighed; the filter paper is applied to the underarm for 5 minutes and then reweighed; the excess weight after reweighing the paper is the weight of sweat produced by the subject in five minutes. The severity of the subject's hyperhidrosis condition is measured by the subject using the Hyperhidrosis Sweat Severity Scale (HDSS), which is a four-level scale rated by the subject: 0 = none, 1 = mild, 2 = moderate, 3 = severe.

[0336] The GS test and HDSS were used at baseline before botulinum nanoemulsion treatment; at two weeks after treatment and at four weeks after treatment. The study found that at baseline, the average sweat volume detected by the GS test or the severity of the disease measured by the HDSS was approximately equal in Groups 1-3. At two weeks and four weeks after treatment, the average sweat volume or disease severity detected in Group 1 was greater than the average sweat volume or disease severity detected in Groups 2 or 3 in these post-treatment weeks. The study also found that at two weeks and four weeks after treatment, the average sweat volume or disease severity detected in Group 3 in these post-treatment weeks was greater than the average sweat volume or disease severity detected in Group 2.

[0337] This study determined that microneedle preconditioning using relatively low microneedle density unexpectedly increased the bioavailability of topical large-dose nanoemulsions containing botulinum toxin.

[0338] Example 7: Effect of MSCs with different microneedle puncture sizes on the bioavailability of botulinum toxin in humans: Effects on sweat reduction in hyperhidrotic subjects

[0339] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle puncture sizes on enhancing botulinum toxin bioavailability in humans by measuring the reduction in sweat in the skin following topical treatment with a botulinum nanoemulsion formulation.

[0340] The study included three treatment groups of twenty subjects each with axillary hyperhidrosis characterized by excessive underarm sweating: Group 1 was a control group and a botulinum nanoemulsion was applied to the underarms of each subject; Group 2 was intervention group 1 and a microneedle with a size of approximately 11,000 μm was used to puncture the underarms of each subject before the application of the botulinum nanoemulsion formulation. 2 Each section of the underarm skin was preconditioned with five impressions of a microneedle array of microneedles / microneedles; Group 3 was intervention group No. 2 and was punctured with microneedles with a size of approximately 60,000 μm before the application of the botulinum toxin nanoemulsion formulation. 2 Each section of underarm skin was preconditioned with five impressions of a microneedle array of 100 μl / microneedles. Each subject in Groups 1, 2, and 3 was topically treated once with a fixed volume of a botulinum toxin nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin.

[0341] The intended effect of this treatment is to reduce sweating at the site of botulinum nanoemulsion treatment, i.e., the underarm. The amount of sweating at the treatment site is measured by gravimetric sweat measurement (GS test): the subject's underarm is wiped dry with a paper towel; filter paper is weighed; the filter paper is applied to the underarm for 5 minutes and then reweighed; the excess weight after reweighing the paper is the weight of sweat produced by the subject in five minutes. The severity of the subject's hyperhidrosis condition is measured by the subject using the Hyperhidrosis Sweat Severity Scale (HDSS), which is a four-level scale rated by the subject: 0 = none, 1 = mild, 2 = moderate, 3 = severe.

[0342] The GS test and HDSS were used at baseline before botulinum nanoemulsion treatment; at two weeks after treatment and at four weeks after treatment. The study found that at baseline, the average sweat volume detected by the GS test or the disease severity measured by the HDSS was approximately equal in Groups 1-3. At two weeks and four weeks after treatment, the average sweat volume or disease severity detected in Group 1 was greater than the average sweat volume or disease severity detected in Groups 2 or 3 in these post-treatment weeks. The study also found that at two weeks and four weeks after treatment, the average sweat volume or disease severity detected in Group 3 in these post-treatment weeks was greater than the average sweat volume or disease severity detected in Group 2.

[0343] This study determined that microneedle preconditioning using a relatively small microneedle puncture size unexpectedly increased the bioavailability of a topical large-dose nanoemulsion containing botulinum toxin.

[0344] Example 8: Effect of MSC on the bioavailability of botulinum toxin in humans: Effect on the reduction of crow's feet

[0345] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities on enhancing botulinum toxin bioavailability in humans by measuring wrinkle reduction in the skin following topical treatment with a botulinum nanoemulsion formulation.

[0346] The study included a subject who had severe crow's feet on the sides of her eyes. A botulinum nanoemulsion was applied to the subject's crow's feet. When the botulinum nanoemulsion was applied without microneedle skin preconditioning, the dose of botulinum toxin applied to the skin was approximately 15% of the amount of the effective dose. The effective dose is defined as the dose that will result in at least a two-level improvement in the appearance of the wrinkles when the subject contracts the muscles that cause crow's feet, as measured by a five-level wrinkle assessment scale. The subject was preconditioned: before the botulinum nanoemulsion formulation was applied to side 1 of the face, microneedles were applied with a density of 9 microneedles / cm 2 Each part of the skin where the crow's feet were located was preconditioned by five impressions of a microneedle array and the other side of the face (side 2) was irradiated with a microneedle density of 85 microneedles / cm 2 Five impressions of the microneedle array precondition each portion of the skin where crow's feet are located. Application of the topical formulation to the skin takes about 5 minutes, at which time the topical formulation is completely absorbed into the skin.

[0347] The expected effect of botulinum nanoemulsion treatment is to reduce crow's feet at the botulinum nanoemulsion treatment site. Wrinkle severity was measured using a five-level wrinkle rating scale (Wrinkle Scale): 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.

[0348] Baseline before botulinum nanoemulsion treatment; two weeks after treatment and four weeks after treatment, a wrinkle scale was used. The study found that at baseline, the subjects had severe wrinkles, as assessed by the wrinkle scale, with a score of 4 on the 5-level wrinkle scale. Two weeks after treatment, the average severity of wrinkles on side 2 of the face decreased by one level on the wrinkle scale, scoring 3 (moderate). Four weeks after treatment, the average severity of wrinkles on side 2 of the face decreased by two levels on the wrinkle scale, scoring 2 (mild). The study also found that two weeks after treatment, the average severity of wrinkles on side 1 of the face decreased by two levels on the wrinkle scale, scoring 2 (mild). Four weeks after treatment, the average severity of wrinkles on side 1 of the face decreased by three levels on the wrinkle scale, scoring 1 (very little).

[0349] This study determined that microneedle preconditioning using relatively low microneedle density unexpectedly increased the bioavailability of topical large-dose nanoemulsions containing botulinum toxin.

[0350] Example 9: Effect of MSCs with different microneedle densities on the bioavailability of botulinum toxin in humans: fish Effect of reducing tail wrinkles

[0351] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities on enhancing botulinum toxin bioavailability in humans by measuring wrinkle reduction in the skin following topical treatment with a botulinum nanoemulsion formulation.

[0352] The study included three treatment groups, each with twenty subjects who had severe crow's feet on the sides of their eyes: Group 1 was a control group, and botulinum toxin nanoemulsion was applied to the crow's feet of each subject; Group 2 was intervention group 1, and before the application of the botulinum toxin nanoemulsion formulation, the crow's feet of each subject were treated with microneedles at a density of 9 microneedles / cm 2 The third group was the intervention group 2, which was preconditioned with a microneedle array with a density of 85 microneedles / cm before applying the botulinum toxin nanoemulsion formulation. 2Each part of the skin where the crow's feet are located was preconditioned with five impressions of a microneedle array. Each subject in Groups 1, 2, and 3 was topically treated once with a fixed volume of a botulinum toxin nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin.

[0353] The expected effect of botulinum nanoemulsion treatment is to reduce crow's feet at the botulinum nanoemulsion treatment site.The severity of wrinkles was measured using a four-level wrinkle rating scale (Wrinkle Scale): 0 = none, 1 = mild, 2 = moderate, 3 = severe.

[0354] The wrinkle scale was used at baseline before botulinum nanoemulsion treatment; at two weeks after treatment and at four weeks after treatment. The study found that at baseline, the average severity of wrinkles detected by the wrinkle scale was approximately equal in Groups 1-3. At two weeks and four weeks after treatment, the average severity of wrinkles in Group 1 was greater than the average severity of wrinkles detected in Groups 2 and 3 in these post-treatment weeks. The study also found that at two weeks and four weeks after treatment, the average severity of wrinkles in Group 3 was greater than the average severity of wrinkles detected in Group 2 in these post-treatment weeks.

[0355] This study determined that microneedle preconditioning with a relatively low microneedle density unexpectedly increased the bioavailability of a topical large-dose nanoemulsion containing botulinum toxin.

[0356] Example 10: Effect of MSCs with different microneedle puncture sizes on the bioavailability of botulinum toxin in humans: Effect on reducing crow’s feet

[0357] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle puncture sizes on enhancing botulinum toxin bioavailability in humans by measuring wrinkle reduction in the skin following topical treatment with a botulinum nanoemulsion formulation.

[0358] The study included three treatment groups, each with twenty subjects who had severe crow's feet wrinkles on the sides of their eyes: Group 1 was a control group, and a botulinum nanoemulsion was applied to the crow's feet of each subject; Group 2 was an intervention group 1, in which microneedles with a size of about 11,000 μm were punctured on the crow's feet of each subject before the application of the botulinum nanoemulsion formulation. 2 / microneedle array was used to precondition each part of the skin where the crow's feet were located; Group 3 was intervention group 2, which was punctured with microneedles with a size of about 60,000 μm before applying the botulinum toxin nanoemulsion formulation. 2Five impressions of a microneedle array of 100 microneedles / microneedles preconditioned each portion of the skin where the crow's feet were located. Each subject in Groups 1, 2, and 3 was topically treated once with a fixed volume of a botulinum toxin nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin.

[0359] The expected effect of botulinum nanoemulsion treatment is to reduce crow's feet at the botulinum nanoemulsion treatment site.The severity of wrinkles was measured using a four-level wrinkle rating scale (Wrinkle Scale): 0 = none, 1 = mild, 2 = moderate, 3 = severe.

[0360] The wrinkle scale was used at baseline before botulinum nanoemulsion treatment; at two weeks after treatment and at four weeks after treatment. The study found that at baseline, the average severity of wrinkles detected by the wrinkle scale was approximately equal in Groups 1-3. At two weeks and four weeks after treatment, the average severity of wrinkles in Group 1 was greater than the average severity of wrinkles detected in Groups 2 and 3 in these post-treatment weeks. The study also found that at two weeks and four weeks after treatment, the average severity of wrinkles in Group 3 was greater than the average severity of wrinkles detected in Group 2 in these post-treatment weeks.

[0361] This study determined that microneedle preconditioning with a relatively small microneedle puncture size unexpectedly increased the bioavailability of a topical large-dose nanoemulsion containing botulinum toxin.

[0362] Example 11: Effect of MSC on the bioavailability of botulinum toxin in humans: Effect of dose variation on the reduction of crow's feet wrinkles Influence

[0363] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical botulinum nanoemulsion formulation in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities on enhancing botulinum toxin bioavailability in humans by measuring wrinkle reduction in the skin following topical treatment with a botulinum nanoemulsion formulation.

[0364] The study included three treatment groups, each with twenty subjects who had severe crow's feet: Group 1 was a control group, and botulinum toxin nanoemulsion was applied to the crow's feet of each subject; Group 2 was intervention group 1, and before the application of botulinum toxin nanoemulsion formulation, the crow's feet of each subject were treated with microneedles at a density of 9 microneedles / cm 2 The third group was the intervention group 2, which was preconditioned with a microneedle array with a density of 85 microneedles / cm before applying the botulinum toxin nanoemulsion formulation. 2Each section of the skin where the crow's feet were located was preconditioned with five impressions of a microneedle array. Each subject was topically treated once with a fixed volume of a botulinum toxin nanoemulsion formulation having a fixed concentration of botulinum toxin, except that the Group 1 treatment had twice the concentration of botulinum toxin in Groups 2 and 3. Application of the topical formulation to the skin took approximately 5 minutes, at which time the topical formulation was completely absorbed into the skin.

[0365] The expected effect of botulinum nanoemulsion treatment is to reduce crow's feet at the botulinum nanoemulsion treatment site.The severity of wrinkles was measured using a four-level wrinkle rating scale (Wrinkle Scale): 0 = none, 1 = mild, 2 = moderate, 3 = severe.

[0366] Baseline before botulinum nanoemulsion treatment; two weeks after treatment and four weeks after treatment, a wrinkle scale was used. The study found that at baseline, the average severity of wrinkles detected by the wrinkle scale was approximately equal in Groups 1-3. At two weeks and four weeks after treatment, the average severity of wrinkles in Groups 1 and 3 was reduced by approximately the same amount compared to baseline, even though Group 1 was treated with twice the concentration of Group 3. The study also found that at two weeks and four weeks after treatment, the average severity of wrinkles in Group 2 was reduced significantly more than the average severity of wrinkles in Group 3 compared to baseline, even though Groups 2 and 3 were treated with the same concentration of botulinum toxin.

[0367] This study determined that microneedle preconditioning with a relatively low microneedle density unexpectedly increased the bioavailability of a topical large-dose nanoemulsion containing botulinum toxin, allowing lower doses of botulinum toxin to achieve equivalent therapeutic effects when compared to patients who did not receive microneedle skin preconditioning or patients who received MSCs with relatively higher microneedle density.

[0368] Example 12: Effect of MSC on the bioavailability of botulinum toxin in humans: Effect of dose variation on the reduction of crow's feet wrinkles Influence

[0369] A single-dose topical study of botulinum toxin bioavailability following topical administration of a topical macroemulsion formulation of botulinum toxin in humans was conducted. The study was designed not only to evaluate the percent bioavailability achieved with different microneedle systems, but also to allow for comparisons to test the effects of different microneedle array needle densities on enhancing botulinum toxin bioavailability in humans by measuring wrinkle reduction in skin following topical treatment with a macroemulsion formulation of botulinum toxin.

[0370] The study included three treatment groups, each with twenty subjects who had severe crow's feet: Group 1 was a control group, and a botulinum toxin crude emulsion was applied to the crow's feet of each subject; Group 2 was an intervention group 1, and a microneedle density of 9 microneedles / cm was used before the application of the botulinum toxin crude emulsion formulation. 2The third group was the intervention group 2, which was preconditioned with a microneedle array with a density of 85 microneedles / cm before applying the botulinum toxin crude emulsion formulation. 2 Each part of the skin where the crow's feet are located was preconditioned with five impressions of a microneedle array. Each subject was topically treated once with a fixed volume of a botulinum toxin nanoemulsion formulation having a fixed concentration of botulinum toxin. It took about 5 minutes to apply the topical formulation to the skin, at which time the topical formulation was completely absorbed into the skin.

[0371] The expected effect of botulinum nanoemulsion treatment is to reduce crow's feet at the botulinum nanoemulsion treatment site.The severity of wrinkles was measured using a four-level wrinkle rating scale (Wrinkle Scale): 0 = none, 1 = mild, 2 = moderate, 3 = severe.

[0372] The wrinkle scale was used at baseline before botulinum nanoemulsion treatment; at two weeks after treatment and at four weeks after treatment. The study found that at baseline, the average severity of wrinkles detected by the wrinkle scale was approximately equal in Groups 1-3. At two weeks and four weeks after treatment, the average severity of wrinkles in Group 1 was greater than the average severity of wrinkles in Groups 2 and 3. The study also found that at two weeks and four weeks after treatm...

Claims

1. A method comprising the following steps: Applying an emulsion composition comprising a macropharmaceutical having a molecular weight of 100,000 Da or greater to a site, in combination with microneedle skin conditioning (MSC) of the site with a microneedle array having a microneedle density of about 2 to about 50 microneedles / cm 2 within the range.

2. The method of claim 1, wherein the microneedle density is from about 2 to about 10 microneedles / cm 2 within the range.

3. The method of claim 1, wherein the microneedle density is from about 2 to about 35 microneedles / cm 2 within the range.

4. The method of claim 1, wherein the composition comprising the large pharmaceutical agent comprises a nanoemulsion comprising the large pharmaceutical agent.

5. The method of claim 1, wherein the composition comprising the large pharmaceutical agent comprises a macroemulsion comprising the large pharmaceutical agent.

6. The method of any one of claims 1 to 5, further comprising administering a non-irritating penetration enhancer.

7. The method of claim 6, wherein the non-irritating penetration enhancer is selected from the group consisting of a carrier peptide and a co-peptide.

8. The method according to any one of claims 6 to 7, wherein the non-irritating penetration enhancer is selected from cationic peptides and peptides having the sequence RKKRRQRRRG-(K) 15 -GRKKRRQRRR's positively charged carrier.

9. The method of any one of claims 1 to 8, wherein the MSCs of the site are treated prior to applying the composition comprising the large agent to the site.

10. The method of any one of claims 1 to 8, wherein the MSCs of the site are treated after applying the composition comprising the large agent to the site.

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