Delay of peak effect and / or extension of reaction duration

Abstract

The present disclosure describes certain techniques for administering large agents (e.g., bioactive large agents, such as biotherapeutic agents, including, for example, botulinum toxins) to subjects, including techniques for obtaining unexpected results, such as delayed peak effects and / or extended response durations.

Description

[0001] Cross-references to related applications

[0002] This application is a divisional application of Chinese Patent Application No. 202080036448.2, filed on May 12, 2020, entitled "Delayed Peak Effect and / or Extended Response Duration". The original application was the national phase application with international application No. PCT / US2020 / 032458.

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 847,901, filed May 14, 2019, the entire contents of which are incorporated herein by reference. Background Technology

[0004] Significant resources have been invested in developing application techniques for large-scale pharmaceuticals (such as bioactive large-scale pharmaceuticals). Summary of the Invention

[0005] This disclosure describes certain techniques for administering large doses (e.g., bioactive large doses, such as biotherapeutic agents, including, for example, botulinum toxin) to subjects and further describes unintended results achieved by such techniques, including, for example, delayed peak effects and / or prolonged duration of response. In some embodiments, such unintended results are achieved relative to administration of the same large dose (e.g., botulinum toxin) according to a reference regimen (e.g., via a formulation, route, and / or administration regimen understood in the art). Those skilled in the art will appreciate relevant (e.g., appropriately comparable) reference regimens, including reference regimens for approved products described herein. Furthermore, those skilled in the art will understand that in some embodiments, an appropriate reference regimen is one that achieves a peak effect comparable in magnitude to the provided regimen, and will also be familiar with comparative regimens (and / or the effects of regimens) that may involve administration of the relevant large dose (e.g., botulinum toxin) via different formulations, different doses, different number of doses, different dose intervals, different routes of administration, etc. To cite just one example, those skilled in the art will know that the individual dose of parenteral botulinum toxin is typically much lower than that of topical botulinum toxin (i.e., contains much fewer botulinum toxin units) [because topical application of any active ingredient typically delivers only a small fraction of the active ingredient to the skin, while parenteral application delivers 100% to the skin]; nevertheless, those skilled in the art can still evaluate comparable parenteral and topical regimens and compare parameters relevant to this disclosure (e.g., one or more outcome parameters), such as the time to peak effect and duration of effect, which can be evaluated, for example, based on the biological effects of the agent (e.g., the reduction of facial wrinkles at maximum contraction as measured by a physician using a wrinkle assessment scale, etc.).

[0006] Among other things, this disclosure identifies certain sources of problems with conventional techniques for administering large doses, particularly for administering botulinum toxin, including, for example, that many such techniques are designed to achieve rapid onset and / or rapid peak effect. This disclosure teaches, in particular, that in certain circumstances (including, for example, for many subjects receiving wrinkle treatment), delayed onset and / or delayed peak effect may be desirable and / or beneficial. This disclosure further records that the techniques provided can achieve surprisingly and unexpectedly delayed onset and / or delayed peak effect.

[0007] Furthermore, this disclosure demonstrates that the provided technology can surprisingly achieve a surprisingly and unexpectedly prolonged duration of effect. Among other things, this disclosure believes that such a prolonged duration of effect may have various beneficial effects, including, for example, allowing for a reduction in the frequency of dose administration, which in turn may provide greater convenience (e.g., fewer visits to a doctor's office or clinic) and / or cost savings.

[0008] The techniques provided herein specifically include transdermal delivery techniques. Those skilled in the art are well aware of the challenges associated with achieving effective transdermal delivery, particularly for large pharmaceutical formulations. It is generally accepted that transdermal permeability decreases with increasing molecular size, reaching negligible or even non-existent levels. This disclosure provides specific techniques in which the topical application of large pharmaceutical formulations (e.g., nanoemulsion formulations) combined with microneedle skin conditioning (e.g., preconditioning) achieves surprising and unexpected results, in some embodiments of which may include one or both of delayed peak effects and / or prolonged response duration.

[0009] Attached Figure Description

[0010] Figure 1 : Figure 1 A bar graph comparing the observed response rates using the provided technique with approved injectable botulinum toxin therapies is presented, illustrating the extended duration of effect achieved, for example, by the provided technique described herein.

[0011] Figure 2 : Figure 2 Line graphs comparing the peak effect times of two different approved injectable botulinum toxin therapies with the provided local + MSC therapy are presented, and delayed peak effects, such as those achieved by the techniques provided herein, are recorded.

[0012] definition

[0013] In this application, unless clearly indicated from the context, (i) the term “a” is to be understood as “at least one”; (ii) the term “or” is to be understood as “and / or”; (iii) the terms “comprising” and “including” are to be understood as covering the listed components or steps, whether they are presented on their own or together with one or more other components or steps; and (iv) the terms “about” and “approximately” are to be understood as allowing standard variations as understood by one of ordinary skill in the art; and (v) if a scope is provided, endpoints are included.

[0014] Abrasion: As used herein, the term “abrasion” refers to any means of altering, disrupting, removing, or destroying the top layer of the skin. In some embodiments, abrasion refers to mechanical means of altering, disrupting, removing, or destroying the top layer of the skin. In some embodiments, abrasion refers to chemical means of altering, disrupting, removing, or destroying the top layer of the skin. For example, agents such as exfoliants, fine particles (e.g., magnesium or aluminum particles), acids (e.g., alpha-hydroxy acids or beta-hydroxy acids), and alcohols can cause abrasion. Generally, penetration enhancers, such as those described, for example, by Donovan (e.g., U.S. Publications 2004 / 009180 and 2005 / 175636, and PCT Publication WO 04 / 06954) and Graham (e.g., U.S. Patent 6,939,852 and U.S. Publication 2006 / 093624), are expected to cause abrasion. Of course, those skilled in the art will understand that a particular agent may cause abrasion when present at a certain concentration or in combination with one or more other agents, but may not cause abrasion under different circumstances. Therefore, whether a particular material is an "abrasive" depends on the context. Abrasion can be readily assessed by a person skilled in the art, for example, by observing redness or irritation of the skin and / or by histological examination of the skin, which shows changes, damage, removal, or erosion of the stratum corneum.

[0015] Administration: As used herein, the term "administration" generally refers to administering the composition to a subject or system. Those skilled in the art will recognize the various routes of administration that may be used to administer the composition to a subject (e.g., a person) where appropriate. For example, in some embodiments, administration may be parenteral; in some embodiments, administration may be topical. In some embodiments, administration may include intermittent dosing (e.g., multiple doses spaced apart at time) and / or periodic dosing (e.g., individual doses spaced apart by a common period of time). In some embodiments, administration may include continuous dosing for at least a selected period of time.

[0016] Pharmaceutical Agent: Generally, as used herein, the term "pharmaceutical agent" can be used to refer to any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. Where appropriate, as will be clearly apparent from the context to a person skilled in the art, the term can be used to refer to an entity that is or comprises a cell or organism or a part thereof, extract or component thereof. Alternatively or additionally, as will be clearly apparent from the context, the term can be used to refer to a natural product because it is present in and / or obtained from nature. In some cases, also as will be clearly apparent from the context, the term can be used to refer to one or more man-made entities because they are designed, engineered and / or produced by human hand and / or do not exist in nature. In some embodiments, the pharmaceutical agent may be utilized in isolated or pure form; in some embodiments, the pharmaceutical agent may be utilized in crude form. In some embodiments, the potential pharmaceutical agent may be a collection or library used, for example, for screening to identify or characterize the active agents therein. In some cases, the term "pharmaceutical 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 comprising one or more polymer moieties. In some embodiments, the term "pharmaceutical" may refer to a compound or entity that is not a polymer and / or substantially free of any polymer and / or one or more specific polymeric moieties. In some embodiments, the term may refer to a compound or entity lacking or substantially free of any polymeric moieties. In some embodiments, the term may refer to a molecular complex. In many embodiments as described herein, a pharmaceutical agent may be a macropharmaceutical (e.g., a bioactive macropharmaceutical, such as a biotherapeutic agent, including, for example, botulinum toxin).

[0017] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising classical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As known in the art, naturally occurring complete antibodies are tetrameric agents of approximately 150 kDa, consisting of two identical heavy-chain polypeptides (each approximately 50 kDa) and two identical light-chain polypeptides (each approximately 25 kDa), linked together in a structure commonly 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-shape), followed by three constant domains: CH1, CH2, and a carboxyl-terminal CH3 (located at the base of the Y-stem). A short region, referred to as a "switch," connects the heavy-chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region link the two heavy-chain polypeptides together in a complete antibody. Each light chain consists of two domains – a variable (VL) domain at the amino terminus followed by a constant (CL) domain at the carboxyl terminus, separated from each other by another “switch.” A complete antibody tetramer consists of two heavy-chain-light-chain dimers, where the heavy and light chains are linked together by a single disulfide bond; two additional disulfide bonds link the heavy-chain hinge regions together, causing the dimers to link together and form a tetramer. Naturally occurring antibodies are also glycosylated, typically at the CH2 domain. Each domain in a natural antibody is characterized by an “immunoglobulin fold” structure formed by two β-sheets (e.g., 3-, 4-, or 5-chain sheets) packaged opposite 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 a natural antibody folds, the FR region forms a β-sheet that provides a structural framework for the domain, and the CDR loop regions from both the heavy and light chains aggregate in three-dimensional space, creating a single hypervariable antigen-binding site at the apex of the Y structure. The Fc region of a naturally occurring antibody binds elements of the complement system and also binds to receptors on effector cells, including, for example, effector cells mediating cytotoxicity. As is known in the art, the affinity and / or other binding properties of the Fc region to Fc receptors can be modulated by glycosylation or other modifications. In some embodiments, antibodies generated and / or utilized according to the invention comprise glycosylated Fc domains, including modified or engineered glycosylated Fc domains. For the purposes of this invention, in some embodiments, any polypeptide or polypeptide complex comprising a sufficient sequence of immunoglobulin domains as found in natural antibodies may be referred to as and / or used as an "antibody," whether such polypeptide is naturally occurring (e.g., produced by an organism reacting with an antigen) or generated by recombinant engineering, chemical synthesis, or other artificial systems or methods.In some embodiments, the antibody is polyclonal; in other embodiments, the antibody is monoclonal. In some embodiments, the antibody has a constant region sequence specific to mouse, rabbit, primate, or human antibodies. In some embodiments, as known in the art, the antibody sequence element is humanized, primate-based, chimeric, etc. Furthermore, the term "antibody" as used herein may, in appropriate embodiments (unless otherwise stated or clear from the context), refer to any construct or form known or developed in the art for utilizing antibody structural and functional characteristics in alternative presentations. For example, in embodiments, the antibody utilized according to the invention is in a form selected from, but not limited to, intact IgG, IgE, and IgM, bispecific or multispecific antibodies (e.g., [missing information]). (etc.), single-chain Fv, peptide-Fc fusion, Fab, colloidal antibodies, masking antibodies (e.g., Small modular immunotherapy (SMIP) TM () Single-chain or tandem double antibodies VHH, Microantibodies Ankylosing spondylogenetics or DART, TCR-like antibodies, Microproteins and In some embodiments, the antibody may lack the covalent modifications (e.g., glycan attachment) that it would have if naturally produced (e.g., in mammals). In some embodiments, the antibody may contain covalent modifications (e.g., glycan, payload [e.g., detectable portion, therapeutic portion, catalytic portion, etc.] attachments, or other side groups (e.g., polyethylene glycol, etc.).

[0018] Antibody agents: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes sufficient to confer specific binding immunoglobulin structural elements. Exemplary antibody agents include, but are not limited to, human antibodies, primate-derived antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused with other proteins, radiolabels, or cytotoxins), and small modular immunopharmaceuticals ("SMIPs"). TMSingle-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 from at least two intact antibodies, and antibody fragments, provided they exhibit the desired biological activity. In some embodiments, the term covers stapled peptides. In some embodiments, the term covers one or more antibody-like binding peptide mimics. In some embodiments, the term covers one or more antibody-like binding scaffold proteins. In some embodiments, the term covers single-chain antibodies, colloidal antibodies, and antibody fragments. Cloning antibodies or adnectin proteins. 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 complementarity-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) substantially identical to the CDRs present in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs, wherein the included CDRs are sequence-identical or contain 1-5 amino acid substitutions compared to the reference CDRs. In some embodiments... The included CDRs are substantially identical to the reference CDR, 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 with the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDR, wherein the included CDRs exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDR, wherein at least one of the included CDRs exhibits sequence identity compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, wherein 1-5 amino acids in 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 included CDR is substantially identical to the reference CDR, wherein at least one amino acid in the included CDR is 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 included CDR is substantially identical to a 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 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 homologous or substantially homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is or comprises an antibody-drug conjugate.

[0019] Antibody component: As used herein, refers to a polypeptide element (which may be a full polypeptide or a portion of a larger polypeptide, such as a fusion polypeptide as described herein) that specifically binds to an epitope or antigen and includes one or more immunoglobulin structural features. Typically, an antibody component is any polypeptide whose amino acid sequence includes elements specific to an antibody-binding region (e.g., an antibody light chain or variable region or one or more of its complementarity-determining regions (“CDRs”), or an antibody heavy chain or variable region or one or more of its CDRs, optionally containing one or more frame regions). In some embodiments, the antibody component is or comprises a full-length antibody. In some embodiments, the antibody component is less than full-length but includes at least one binding site (containing at least one, and preferably at least two, sequences having a known antibody “variable region” structure). In some embodiments, the term “antibody component” covers any protein having a binding domain homologous or substantially homologous to an immunoglobulin-binding domain. In a particular embodiment, the included “antibody component” covers a polypeptide having a binding domain showing at least 99% identity with an immunoglobulin-binding domain. In some embodiments, the included "antibody component" is any polypeptide having a binding domain that shows at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity with an immunoglobulin binding domain (e.g., a reference immunoglobulin binding domain). The included "antibody component" may have the same amino acid sequence as antibodies (or portions thereof, such as their antigen-binding portions) found in naturally derived sources. The antibody component may be monospecific, bispecific, or multispecific. The antibody component may include structural elements specific to any immunoglobulin class, including any human class: IgG, IgM, IgA, IgD, and IgE. Antigen-binding function of antibodies has been demonstrated to be achieved via fragments of full-length antibodies. Such antibody embodiments may also be bispecific, dual-specific, or multispecific forms that specifically bind two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of antibody include: (i) Fab fragments, which are formed by V... H V L C H 1 and CL (ii) a monovalent segment composed of structural domains; (iii) a divalent segment consisting of two Fab segments connected by disulfide bridges in the hinge region; and (iv) a segment composed of V. H and C H (iv) The Fd fragment composed of a single-arm antibody; H and V L The Fv segment consists of a domain; (v) a dAb segment containing a single variable domain (Ward et al. (1989) Nature 341:544-546); and (vi) a separated complementarity-determining region (CDR). Furthermore, although the two domains of the Fv segment (V... H and V L These genes are encoded by independent genes, but they can be joined by synthetic linkers using recombination methods, which enable them to be made into genes containing V. H and V L Regions pair to form a single protein chain of a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, 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, the “antibody component” is or comprises such a single-chain antibody. In some embodiments, the “antibody component” is or comprises a biantibody. A biantibody is a bivalent bispecific antibody, wherein V H and V L The domain is expressed on a single polypeptide chain, but a linker that is too short to allow pairing between two domains on the same chain is used, thereby forcing the domain to pair with a complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger, P., et al., (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ., (1994) Structure 2(12): 1121-1123). Such antibody-binding moieties 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” containing a pair of tandem Fv fragments (V) that together with the complementary light chain polypeptide form a pair of antigen-binding regions. H -C H 1-V H -C H1)(Zapata et al., (1995) Protein Eng. 8(10):1057-1062; and U.S. Patent No. 5,641,870). In some embodiments, the antibody component may have structural elements specific to chimeric or humanized antibodies. Typically, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the complementarity-determining region (CDR) of the recipient are replaced by residues from the CDR of a non-human species (donor antibody) (such as mouse, rat, or rabbit) having the desired specificity, affinity, and capability. In some embodiments, the antibody component may have structural elements specific to human antibodies.

[0020] Antibody fragment: As used herein, an "antibody fragment" includes a portion of a complete antibody, such as an antigen-binding region or variable region like an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triantibodies; tetraantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, an antibody fragment includes a separated fragment, an "Fv" fragment consisting of variable regions of a heavy chain and a light chain; a recombinant single-chain polypeptide molecule wherein the light chain variable region and the heavy chain variable region are linked by a peptide linker ("ScFv protein"); and a minimal recognition unit consisting of amino acid residues mimicking a hypervariable region. In many embodiments, the antibody fragment contains a sufficient parent antibody sequence, wherein it is a fragment that binds the same antigen to the parent antibody; in some embodiments, the fragment binds the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for antigen binding. 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 biantibodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs). Antigen-binding fragments of antibodies can be generated by any means. For example, antigen-binding fragments of antibodies can be enzymatically or chemically generated by fragmentation of the complete antibody and / or can be generated by recombination of genes encoding partial antibody sequences. Alternatively or additionally, antigen-binding fragments of antibodies can be synthesized wholly or partially. Antigen-binding fragments of antibodies may optionally comprise single-chain antibody fragments. Alternatively or additionally, antigen-binding fragments of antibodies may comprise multiple chains, said multiple chains being linked together, for example, by disulfide bonds. Antigen-binding fragments of antibodies may optionally comprise multi-molecular complexes. Functional antibody fragments typically contain at least about 50 amino acids, and more typically contain at least about 200 amino acids.

[0021] 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 ​​(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 any 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).

[0022] Related: When used herein, the term means that two events or entities are “related” to each other if the presence, level, and / or form of one is related to the presence, level, and / or form of another. For example, an entity is considered related to a particular disease, condition, or symptom if the presence, level, and / or form of a particular entity (e.g., polypeptide, genetic trait, metabolite, microorganism, etc.) is related to the incidence and / or susceptibility to a disease, condition, or symptom (e.g., across relevant populations). In some embodiments, two or more entities are physically “related” to each other if they interact directly or indirectly such that they are physically close to each other and / or remain physically close. In some embodiments, two or more physically related entities are covalently connected to each other; in some embodiments, two or more physically related entities are not covalently connected but non-covalently related, for example, through hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0023] Biocompatibility: As used herein, the term "biocompatibility" refers to a material that does not cause significant damage to living tissue when it comes into contact with such tissue, for example, in vivo. In some embodiments, materials are "biocompatible" if they are non-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 application does not cause significant inflammation or other such adverse reactions.

[0024] Biodegradable: As used herein, the term "biodegradable" refers to a material that, when introduced into cells, is broken down (e.g., by cellular machinery, such as by enzymatic degradation, by hydrolysis, and / or by combinations thereof) into components that are reusable or treatable by the cells without significant toxicity. In some embodiments, the components resulting from the breakdown of a biodegradable material are biocompatible and therefore do not cause significant inflammation and / or other adverse effects in vivo. In some embodiments, a biodegradable polymeric material breaks down into its component monomers. In some embodiments, the breakdown of a biodegradable material (including, for example, a biodegradable polymeric material) involves the hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, the breakdown of a biodegradable material (including, for example, a biodegradable polymeric material) involves the breaking of urethane bonds. 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, blends, and copolymers of PEG, polyanhydrides, poly(ortho-esters), polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), and their blends and copolymers. Many naturally occurring polymers are also biodegradable, including, for example, proteins (such as albumin, collagen, gelatin, and prolyl gluten, such as zein) and polysaccharides (such as alginates, cellulose derivatives, and polyhydroxyalkanoates, such as polyhydroxybutyrate), their blends, and copolymers. Those skilled in the art will understand or be able to determine when such polymers are biocompatible and / or biodegradable derivatives of their kind (e.g., associated with a parent polymer by substantially the same structure, differing only in terms of substitutions or additions of specific chemical groups known in the art).

[0025] Bioactive Agent: As used herein, the term "bioactive agent" refers to a pharmaceutical agent that has a specific biological effect when administered to a subject (e.g., a human). In some embodiments, the bioactive agent may be a therapeutic, cosmetic, and / or diagnostic agent. In some embodiments, the bioactive agent may be or comprises an entity or portion classified as an "active pharmaceutical ingredient" by the U.S. Food and Drug Administration. In some embodiments, the bioactive agent is a large pharmaceutical agent. In some embodiments, the bioactive agent may be or comprises an agent whose presence is associated with a desired pharmacological and / or therapeutic, cosmetic, and / or diagnostic effect. In some embodiments, the bioactive agent is characterized in that its biological effect is dose-dependent (e.g., optionally increases linearly with increasing dose over at least a first concentration range).

[0026] Botulinum toxin crude emulsion composition: As used herein, the term "botulinum toxin crude emulsion composition" refers to a crude emulsion composition in which at least one crude emulsion includes botulinum toxin. Botulinum toxin may be present within the crude emulsion, on the surface of the crude emulsion, and / or within the micelle membrane defining the crude emulsion.

[0027] Botulinum toxin nanoemulsion compositions: As used herein, the term "botulinum toxin nanoemulsion composition" refers to a nanoemulsion composition in which at least one nanoemulsion includes botulinum toxin. Botulinum toxin may be present within the nanoemulsion, on the surface of the nanoemulsion, and / or within the micelle membrane defining the nanoemulsion.

[0028] Botulinum toxin: As used herein, the term "botulinum toxin" refers to a neurotoxin produced by Clostridium botulinum. Unless otherwise stated, the term encompasses fragments or portions (e.g., its light and / or heavy chains) of such neurotoxin that retain appropriate activity (e.g., muscle relaxant activity). As used herein, the phrase "botulinum toxin" may refer to botulinum toxin or serotypes A, B, C, D, E, F, or G. As those skilled in the art will understand, in some embodiments, as used herein, the term botulinum toxin may encompass botulinum toxin complexes (i.e., complexes of, for example, 300, 600, and 900 kDa) or purified (i.e., isolated) botulinum toxin (i.e., for example, about 150 kDa). "Purified botulinum toxin" is understood to be botulinum toxin isolated or substantially isolated from other proteins, such as proteins that may be associated with botulinum toxin in nature, including those involved in botulinum toxin complexes. The purity of purified toxin may be greater than 95%, and in some embodiments greater than 99%. Those skilled in the art will understand that this invention is not limited to any specific source of botulinum toxin. For example, the botulinum toxin used according to this invention can be isolated from Clostridium botulinum, can be chemically synthesized, can be recombinantly produced (i.e., in host cells or organisms other than Clostridium botulinum), etc. The botulinum toxin can be genetically engineered or chemically modified to make its duration of action longer or shorter than that of botulinum toxin serotype A.

[0029] Carrier: As used herein, this refers to a diluent, adjuvant, excipient, or medium applied with the composition (e.g., it is an integral part of the formulation of the composition). In some exemplary embodiments, the carrier may include sterile liquids, such as water and oils, including petroleum, animal, plant, and synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid components.

[0030] 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, one therapeutic agent, and one mode of treatment, 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 dose of the second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy may include administering one or more agents and / or modes of treatment to a subject receiving other agents or modes of treatment in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even simultaneously), although in some embodiments, two or more agents or their active portions may be administered together in the combination composition, or even in the combination compound (e.g., as part of a single chemical complex or covalent entity).

[0031] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, condition groups, etc., that may be different from each other but are similar enough to allow for comparison between them, such that a person skilled in the art will understand that reasonable conclusions can be drawn based on observed differences or similarities. In some embodiments, comparable conditions, environments, individuals, or groups are characterized by a number of substantially identical features and one or a few different features. In the context, a person skilled in the art will understand the degree of similarity required for two or more such agents, entities, situations, or condition groups, etc., to be considered comparable in any given context. For example, a person skilled in the art will understand that environments, individuals, or groups are comparable to each other when they possess a sufficient number and type of substantially identical features to ensure that reasonable conclusions can be drawn that differences in results or observed phenomena under different environments, individuals, or groups are caused by or indicate changes in those different features.

[0032] Composition: Those skilled in the art will understand that, as used herein, the term "composition" can be used to refer to a discrete physical entity comprising one or more specified components. Generally, unless otherwise stated, a composition can be in any form—e.g., gas, gel, liquid, solid, etc.

[0033] Composition: The description herein of a composition or method “comprising” one or more named elements or steps is open-ended, meaning that the named element or step is necessary, but other elements or steps may be added within the scope of the composition or method. To avoid verbosity, it should also be understood that any composition or method described as “comprising” (or “containing”) one or more named elements or steps also describes a corresponding, more limited composition or method that “consists substantially of” (or “consists substantially of”): the same named element or step, meaning that the composition or method includes the necessary named element or step, and may also include additional elements or steps that do not substantially affect the essential and novel characteristics of the composition or method. It should also be understood that any composition or method described herein as “comprising” or “consists substantially of”: one or more named elements or steps also describes a corresponding, more limited and closed composition or method that “consists of” (or “consists of”): the named element or step, excluding any other unnamed element or step. In any composition or method disclosed herein, any known or disclosed equivalent of any named basic element or step may replace said element or step.

[0034] Dosage Form or Unit Dosage Form: Those skilled in the art will understand that the term "dosage form" can be used to refer to physically discrete units of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such an amount is a unit dose (or a whole portion thereof) suitable for administration according to a dosing regimen that has been determined to be associated with desired or beneficial outcomes when administered to the relevant population (i.e., with a therapeutic dosing regimen). Those skilled in the art will understand that the total amount of therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may include the administration of multiple dosage forms.

[0035] Dosing regimen: Those skilled in the art will understand that the term "dosing regimen" can be used to refer to a set of unit doses (usually more than one) administered individually to a subject, typically separated by time periods. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, a dosing regimen includes multiple doses, each time separate from the other doses. In some embodiments, multiple doses are separated from each other by time periods of equal length; in some embodiments, a dosing regimen includes multiple doses and at least two distinct time periods separating the individual doses. In some embodiments, all doses within a dosing regimen have the same unit dosage. In some embodiments, the different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen includes a first dose administered at a first dosage, followed by one or more additional doses administered at a second dosage different from the first dosage. In some embodiments, a dosing regimen includes a first dose administered at a first dosage, followed by one or more additional doses at a second dosage identical to 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).

[0036] Emulsion: The term “emulsion” is used herein in accordance with the understanding in the field of “a system consisting of liquids, with or without emulsifiers, generally dispersed in an immiscible liquid in droplets larger than colloidal size.” See, for example, the definition in the Medline PlusOnline Medical Dictionary, Merriam Webster (2005).

[0037] Excipients: As used herein, these are non-therapeutic agents that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc.

[0038] Human: In some implementations, a human is an embryo, fetus, infant, child, adolescent, adult, or elderly person.

[0039] Hydrophilic: As used herein, the terms “hydrophilic” and / or “polar” refer to the tendency to mix with or be readily soluble in water.

[0040] Hydrophobic: As used herein, the terms “hydrophobic” and / or “nonpolar” refer to a tendency to repel, not bind with water, or be poorly soluble in water.

[0041] Improvement, increase, or reduction: As used herein or its grammatical equivalents, the terms “improvement,” “increase,” or “reduction” refer to a value relative to a baseline measurement (such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein). In some embodiments, a “control individual” is an individual suffering from the same form of disease or injury as the treated individual.

[0042] Macromolecules: The term "macromolecule" is generally used herein to describe molecules with a size greater than about 100 kilodaltons (kDa). In some embodiments, macromolecules are larger than about 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, or 500 kDa. In some embodiments, macromolecules are polymers or contain polymer portions or entities. In some embodiments, macromolecules are or contain peptides. In some embodiments, macromolecules are or contain nucleic acids.

[0043] Large-molecule pharmaceuticals: As used herein, the term "large-molecule pharmaceuticals" generally refers to pharmaceuticals with a molecular weight greater than about 100 kilodaltons (kDa) in terms of size. In some embodiments, the large molecules are greater than about 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, or 500 kDa. In some embodiments, the large-molecule pharmaceuticals are bioactive agents. In some embodiments, the large-molecule pharmaceuticals are or comprise one or more large molecules. In some embodiments, the large-molecule pharmaceuticals are or comprise one or more molecular complexes. In some embodiments, the large-molecule pharmaceuticals are or comprise peptides. In some embodiments, the large-molecule pharmaceuticals are or comprise peptide complexes. In some embodiments, the large-molecule pharmaceuticals are or comprise bacterial toxins (e.g., botulinum toxin). In some embodiments, the large-molecule pharmaceuticals are or comprise antibody agents.

[0044] Crude Emulsion: As used herein, the term "crude emulsion" refers to an emulsion in which at least some droplets have diameters ranging from hundreds of nanometers to micrometers. As will be understood by those skilled in the art, a crude emulsion is characterized by droplets with diameters greater than 300 nm. In some embodiments, the crude emulsion composition utilized according to this disclosure comprises one or more macropharmaceutical agents or one or more bioactive agents. In some embodiments, the macropharmaceutical agent included in the crude emulsion composition may be a bioactive agent. Those skilled in the art will understand that the crude emulsion composition used according to this disclosure can be prepared by any available means, including, for example, chemical or mechanical means. In some embodiments, the droplets in the crude emulsion have a size ranging from about 301 nm to about 1000 μm. In some embodiments, the crude emulsion has droplets with a size distribution between about 301 nm and about 1000 μm. In some embodiments, the droplets in the crude emulsion have a size ranging from about 500 nm to about 5000 μm. In some embodiments, the crude emulsion has droplets with a size distribution between about 500 nm and about 5000 μm.

[0045] Microneedles: As used herein, the term "microneedle" generally refers to an elongated structure having a suitable length, diameter, and shape for penetrating the skin. In some embodiments, microneedles are arranged and constructed (either on their own or within a device) to minimize contact with nerves upon insertion into the skin while still creating an efficient pathway for drug delivery. In some embodiments, the microneedle has a diameter consistent along its length. In some embodiments, the microneedle has a diameter that varies along its length. In some embodiments, the microneedle has a diameter that tapers gradually along its length. In some embodiments, the diameter of the microneedle is narrowest at the tip where it 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 an array. 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 and 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.

[0046] Microneedle array imprinting: As used herein, the term "microneedle array imprinting" refers to a microneedle imprint obtained by imprinting microneedles and / or microneedle arrays onto the skin and then removing them from the skin. In some embodiments, the microneedle array may be stamped onto the skin (e.g., using a microneedle array impression). In some embodiments, the microneedle array may be rolled onto the skin (e.g., using a microneedle array roller).

[0047] 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; and 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 that the microneedles in the array may have. However, those skilled in the art will understand that microneedle density can be expressed regardless of whether the relevant area is flat (e.g., a microneedle array impression), curved (e.g., a microneedle array roller), or irregular. Those skilled in the art will understand that assessing microneedle density as microneedle punctures per unit area of ​​the microneedle puncture site can be particularly useful, for example, if the array has needles of different lengths and / or the microneedle puncture sites have topological diversity, such that when the array is applied to the site, not every needle actually punctures the skin.

[0048] Microneedle puncture size: As used herein, the term "microneedle puncture size" or "microneedle aperture size" refers to the calculated puncture area produced by each microneedle of the microneedle array after microneedles and / or microneedle arrays have been imprinted 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.

[0049] Nanoemulsion: As used herein, the term "nanoemulsion" refers to an emulsion in which at least some droplets have diameters in the nanoscale range. As will be understood by those skilled in the art, a nanoemulsion is characterized by droplets with a diameter of 300 nm or less. In some embodiments, the nanoemulsion composition utilized according to this disclosure comprises one or more macropharmaceutical agents or one or more bioactive agents. In some embodiments, the macropharmaceutical agent included in the nanoemulsion composition may be a bioactive agent. Those skilled in the art will understand that the nanoemulsion composition used according to this disclosure can be prepared by 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.

[0050] Nanoparticles: As used herein, the term "nanoparticle" refers to solid particles with a diameter of less than 300 nm, as defined by the National Science Foundation. In some implementations, the nanoparticles have a diameter of less than 100 nm, as defined by the National Institutes of Health.

[0051] Patient: As used herein, the term "patient" means any organism to which, or to which the provided composition may be administered, for example, experimental, diagnostic, preventative, 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 a human being. In some embodiments, the patient suffers from or is susceptible to one or more diseases or conditions. In some embodiments, the patient exhibits one or more symptoms of a disease or condition. In some embodiments, the patient is diagnosed with one or more diseases or conditions. In some embodiments, the disease 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 a disease, disease, or condition.

[0052] Permeation enhancer: As used herein, the term "permeation enhancer" refers to an agent whose presence or level is associated with an increase in the permeation of the agent of interest through the skin compared to its observed state in the absence of the agent. In some embodiments, the permeation enhancer is characterized by its degradation and / or disruption of skin structure. In some embodiments, the permeation enhancer is or comprises a chemical agent (e.g., a chemical or enzyme, for example) that can damage, disrupt, and / or degrade one or more components of the stratum corneum, including, 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, etc. Derivatives, pyrrolidones, or pyrrolidone derivatives; terpenes and terpene derivatives; fatty acids and their esters; macrocyclic compounds; surfactants; or sulfoxides (e.g., dimethyl sulfoxide (DMSO), decylmethyl 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 inflammatory and / or anaphylactic reactions occur when said 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 contain a chemical agent that does not damage, destroy, or degrade skin structure, but its presence or level is still associated with increased penetration of the agent of interest across the skin compared to its absence. In some embodiments, coneptides, carrier molecules, and carrier peptides may be penetration enhancers that do not damage, destroy, and / or degrade skin structure. In some embodiments, coneptides, carrier molecules, and carrier peptides may be non-irritating penetration enhancers. The term "penetration enhancer" does not include mechanical devices (e.g., needles, scalpels, etc.) or their equivalents (e.g., other invasive procedures). Furthermore, those skilled in the art will understand that the structure of nanoparticles or emulsions is not a chemical agent, and therefore not a chemical penetration enhancer, even if its presence is related to skin penetration enhancement of agents of interest that may be structure-related.

[0053] 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 dose suitable for administration in a treatment regimen that demonstrates a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those suitable for topical application, such as sterile solutions or suspensions, or sustained-release formulations, as gels, creams, ointments, or controlled-release patches or sprays applicable to the skin, lungs, or mouth; intravaginally or rectally, for example, as a pessary, cream, or foam; sublingually; via the eyes; transdermally; or on the nasal, pulmonary, and other mucosal surfaces.

[0054] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to carriers, diluents, or excipients used to formulate the compositions disclosed herein means that the carrier, diluent, or excipient must be compatible with the other components of the composition and harmless to its recipient.

[0055] Pharmaceutically acceptable carriers: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, used to carry or transport the subject compound from one organ or part of the body to another. Each carrier must be acceptable in the sense that it is compatible with other components of the formulation and does not harm 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; 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 non-toxic and compatible substances used in pharmaceutical formulations.

[0056] Premix: As used herein, the term "premix" refers to a combination of components subsequently used to generate an emulsion composition (e.g., a nanoemulsion composition). For example, in some embodiments, the premix is ​​a collection of components that, when subjected to high shear forces, produce a nanoemulsion useful according to this disclosure. In some embodiments, the premix is ​​a collection of components that, when subjected to high shear forces, produce a homogeneous nanoemulsion. Premixes often contain a liquid dispersion medium and other components sufficient to generate a nanoemulsion in the dispersion medium. According to some embodiments of this disclosure, one or more large pharmaceutical agents may be included in the premix. According to some embodiments of this disclosure, one or more biological agents may be included in the premix. According to the invention, botulinum toxin may be included in the premix. According to the 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 in which the premix contains botulinum toxin, an antibody, another bioactive agent, and / or a penetration enhancer, the botulinum toxin, antibody, another bioactive agent, and / or penetration enhancer are in solution before a high shear force is applied to the premix.

[0057] Prevention or prevention: As used herein, when used in connection with the occurrence of a disease, condition, and / or symptom, refers to reducing the risk of developing a disease, condition, and / or symptom and / or delaying the onset of one or more features or symptoms of a disease, condition, and / or symptom. Prevention is considered complete when the onset of a disease, condition, or symptom is delayed by a predetermined period of time.

[0058] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). Proteins may contain portions other than amino acids (e.g., they may be glycoproteins, proteoglycans, etc.), and / or may be otherwise processed or modified. Those skilled in the art will understand that a "protein" can be a complete polypeptide chain produced by a cell (with or without a signal sequence), or it can be its characteristic portion. Those skilled in the art will understand that proteins may sometimes comprise more than one polypeptide chain, for example, linked by one or more disulfide bonds or otherwise. 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, proteins 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 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 length. In some embodiments, proteins are antibodies, antibody fragments, their biologically active portions, and / or their characteristic portions.

[0059] Polypeptide: As used herein, the term "polypeptide" generally has the technically accepted meaning of a polymer having at least three amino acids. Those skilled in the art will understand that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having the complete sequence listed herein, but also polypeptides representing functional fragments (i.e., fragments retaining at least one active component) of such complete polypeptides. Furthermore, those skilled in the art will understand that protein sequences generally allow for some substitutions without destroying activity. Therefore, any polypeptide that retains activity and shares 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 includes at least one region with higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%, typically encompassing at least 3-4 and often up to 20 or more amino acids, is covered 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, and methylation. 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 with 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, its biologically active portion, and / or its characteristic portion.

[0060] References: As used herein, standards or controls are described relative to which comparisons are made. For example, in some embodiments, a drug, animal, individual, population, sample, protocol, sequence, or value of interest is compared with a reference or control drug, animal, individual, population, sample, protocol, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as understood by those skilled in the art, the reference or control is determined or characterized under conditions or settings comparable to those under evaluation. Those skilled in the art will understand when sufficient similarity exists to demonstrate dependence on and / or comparison with a particular possible reference or control.

[0061] Self-application: As used herein, the term "self-application" refers to a situation where the subject has the ability to apply the composition to himself or her without medical supervision. In some embodiments of the invention, self-application may be performed outside of a clinical setting. As an example, in some embodiments of the invention, a facial beauty cream may be applied by the subject at his or her own home.

[0062] Small molecules: Generally, "small molecules" are understood in the art to be organic molecules with a size less than about 5 kilodaltons (Kd). In some embodiments, small molecules are less than about 3 Kd, 2 Kd, or 1 Kd. In some embodiments, small molecules are less than about 800 Daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, small molecules are non-polymerized. In some embodiments, small molecules are not proteins, peptides, or amino acids. In some embodiments, small molecules are not nucleic acids or nucleotides. In some embodiments, small molecules are not sugars or polysaccharides.

[0063] 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, condition, or symptom. In some embodiments, the subject is susceptible to a disease, condition, or symptom. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, condition, or symptom. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, condition, or symptom. In some embodiments, the subject is a person who has one or more characteristics of susceptibility to a disease, condition, or symptom or who is at risk of such susceptibility. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who has received and / or has received a diagnostic and / or treatment.

[0064] Essentially: As used herein, the term “essentially” refers to a qualitative situation that exhibits a general or near-general extent or degree of the characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely (if they have ever occurred) reach completeness and / or proceed to completeness or achieve or avoid absolute results. Therefore, the term “essentially” is used herein to capture the inherent lack of completeness in many biological and chemical phenomena.

[0065] Therapeutic agent: As used herein, the phrase "therapeutic agent" generally refers to any agent that, when administered to an organism, induces a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect in an appropriate population. In some embodiments, an appropriate population may be a model organism population. In some embodiments, an appropriate population may be defined by various criteria, such as a specific age group, sex, genetic background, pre-existing clinical symptom, etc. In some embodiments, a therapeutic agent is a substance that can be used to reduce, improve, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, condition, and / or symptom. In some embodiments, a "therapeutic agent" is an agent that has been or requires approval by a government agency before it can be sold to humans for administration. In some embodiments, a "therapeutic agent" is an agent for human administration requiring a medical prescription. In some embodiments, an agent is not considered a "therapeutic agent" if it merely enhances the delivery of a different agent that actually achieves the desired effect.

[0066] Therapeutic effective amount: As used herein, this means the amount that produces the desired effect achieved by administering this amount. In some embodiments, the term refers to an amount sufficient to treat a disease, condition, and / or symptom when administered to a population suffering from or susceptible to such a disease, condition, and / or symptom according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity and / or delays the onset of one or more symptoms of a disease, condition, and / or symptom. Those skilled in the art will understand that the term "therapeutic effective amount" does not actually require successful treatment in a particular individual. Rather, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a considerable number of subjects when administered to a patient requiring such treatment. In some embodiments, reference to a therapeutically effective amount can refer to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, condition, or symptom) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, a specific agent or therapy of a therapeutically effective amount may be formulated and / or administered in a single dose. In some implementations, the therapeutically effective agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0067] Treatment regimen: When used herein, “treatment regimen” means a dosing regimen that, when administered in the relevant population, may be associated with a desired or beneficial treatment outcome.

[0068] Treatment: As used herein, the term "treatment" (also "treat" or "treating") means any application of a therapy that partially or completely reduces, improves, alleviates, suppresses, delays the onset of a particular disease, condition, and / or symptom, reduces its severity, and / or reduces the incidence of one or more of its symptoms, features, and / or causes. In some embodiments, such treatment may be used on subjects who do not exhibit signs of the relevant disease, condition, and / or symptom, and / or subjects who exhibit only early signs of the disease, condition, and / or symptom. Alternatively or additionally, such treatment may be used on subjects who exhibit one or more established signs of the relevant disease, condition, and / or symptom. In some embodiments, treatment may be directed at subjects who have been diagnosed with the relevant disease, condition, and / or ailment. In some embodiments, treatment may be directed at subjects who are known to have one or more susceptibility factors statistically associated with an increased risk of developing the relevant disease, condition, and / or symptom.

[0069] Uniformity: When used herein to refer to a nanoemulsion composition, the term "uniform" means a nanoemulsion composition in which each droplet has a specific range of droplet diameter dimensions. For example, in some embodiments, a uniform nanoemulsion composition is a nanoemulsion composition in which the difference between the minimum and 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 macropharmaceutical nanoemulsion composition of the present invention (e.g., droplets containing macropharmaceuticals) have diameters smaller than about 300, 250, 200, 150, 130, 120, 115, 110, 100, 90, 80 nm or smaller. In some embodiments, the droplets in the uniform macropharmaceutical nanoemulsion composition of the present invention (e.g., droplets containing macropharmaceuticals) have diameters in the range of about 10 to about 300 nm. In some embodiments, the droplets in the uniform macropharmaceutical nanoemulsion composition of the present invention have a diameter in 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 macropharmaceutical nanoemulsion composition of the present invention (e.g., droplets containing macropharmaceuticals) have an average droplet size lower than about 300, 250, 200, 150, 130, 120, 115, 110, 100, or 90 nm. In some embodiments, the average droplet size is in the range of about 10-300, 50-250, 60-200, 65-150, or 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, most droplets in the uniform nanoemulsion composition of the present invention (e.g., droplets containing large pharmaceutical agents) have a diameter below a specified size or within a specified range. In some embodiments, most are 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.

[0070] Variant: As used herein, the term "variant" refers to an entity that exhibits significant structural identity with a reference entity compared to the reference entity, but differs structurally from the reference entity in the presence or level of one or more chemical components. In many embodiments, the variant is also functionally different from its reference entity. Generally, whether a particular entity is properly considered a "variant" of a reference entity is based on the degree of its structural identity with the reference entity. As those skilled in the art will understand, any biological or chemical reference entity possesses certain characteristic structural elements. By definition, a variant is a unique chemical entity that shares one or more of these characteristic structural elements. To cite just a few examples, small molecules may have characteristic core structural elements (e.g., a macrocyclic core) and / or one or more characteristic overhangs. Therefore, variants of small molecules are molecules that share the core structural elements and characteristic overhangs, but differ in the types of bonds (single vs. double, E vs. Z, etc.) present in other overhangs and / or within the core. Peptides may have characteristic sequence elements consisting of multiple amino acids at designated positions relative to each other in linear or three-dimensional space and / or contributing to a specific biological function. Nucleic acids may have characteristic sequence elements consisting of multiple nucleotide residues at designated positions relative to each other in linear or three-dimensional space. For example, variant peptides may differ from reference peptides due to one or more differences in the amino acid sequence and / or one or more differences in the chemical parts (e.g., carbohydrates, lipids, etc.) covalently linked to the peptide backbone. In some embodiments, the variant peptide exhibits an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% with the reference peptide, optionally excluding conserved amino acid substitutions. Alternatively or additionally, in some embodiments, the variant peptide does not share at least one characteristic sequence element with the reference peptide. In some embodiments, the reference peptide has one or more biological activities. In some embodiments, the variant peptide shares one or more biological activities of the reference peptide. In some embodiments, the variant peptide lacks one or more biological activities of the reference peptide. In some embodiments, the variant peptide exhibits a reduced level of one or more biological activities compared to the reference peptide. In many embodiments, if the peptide of interest has an amino acid sequence identical to that of the parent but with a small number of sequence changes at specific positions, the peptide of interest is considered a “variant” of the parent or reference peptide. Typically, compared to the parent, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of residues are substituted in the variant. In some embodiments, the variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue 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 specific biological activity). Furthermore, variants typically have no more than 5, 4, 3, 2, or 1 additions or deletions compared to the parent, and often have none. Additionally, 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, or about 6 residues, 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.

[0071] Detailed description of certain implementation schemes

[0072] Transdermal drug delivery

[0073] In some embodiments, this disclosure provides techniques for improving the transdermal delivery and / or bioavailability of large pharmaceutical agents (e.g., botulinum toxin, antibodies). In some embodiments, this disclosure teaches achieving particularly advantageous results when microneedle treatment techniques are combined with emulsion compositions. In some embodiments, the microneedle treatment techniques are combined with lotions, creams, or liquid compositions; in some embodiments, such compositions may be or include emulsion compositions (e.g., crude emulsion compositions, microemulsion compositions, and / or nanoemulsion compositions). Alternatively, in some embodiments, the provided techniques combine microneedle treatment techniques with transdermal delivery that does not utilize nanoemulsions, microemulsions, crude emulsions, or even any emulsion. In some embodiments, the provided techniques do not utilize penetration enhancers. In some embodiments, the provided techniques do not utilize chemical penetration enhancers that damage, disrupt, and / or degrade the skin. In some embodiments, the provided techniques do not utilize chemical penetration enhancers.

[0074] Human skin consists of the dermis and the epidermis. The epidermis has several layers, namely the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale (identified in order from the outer surface of the skin inward).

[0075] The stratum corneum is typically the most important barrier in transdermal delivery (and is presumed to be particularly important for large-volume drugs). The stratum corneum is usually about 10–15 μm thick and consists of several layers of flattened keratinocytes (keratinocytes). The intercellular spaces between the keratinocytes are filled with lipid structures and play an important role in the permeation of substances through the skin (Bauerova et al., 2001, European Journal of Drug Metabolism and Pharmacokinetics, 26:85).

[0076] The remaining epidermis beneath the stratum corneum is approximately 150 μm thick. The dermis is about 1-2 mm thick and lies beneath the epidermis. The dermis is innervated by various capillary and neuronal processes.

[0077] Transdermal administration has long been the subject of research seeking alternative routes of administration without the undesirable consequences associated with injection and oral delivery. For example, needles often cause local pain and can expose patients receiving injections to bloodborne diseases. Oral administration often has poor drug bioavailability due to the extremely acidic environment of the patient's stomach.

[0078] Efforts have been made to develop transdermal delivery technologies for certain drugs in an attempt to overcome these drawbacks by providing non-invasive administration. Transdermal delivery is generally desired to minimize damage to the patient's skin. Therefore, transdermal delivery can reduce or eliminate injection-related pain, reduce the likelihood of blood contamination, and improve drug bioavailability once they are systemically incorporated.

[0079] Traditionally, attempts at transdermal application have focused on disrupting and / or degrading the stratum corneum. Some attempts have involved the use of chemical penetration enhancers. Penetration enhancers can act to degrade and / or disrupt skin structure. In some embodiments, penetration enhancers are or contain chemical agents (e.g., chemical agents or enzymes, which can disrupt and / or degrade one or more components of the stratum corneum). In some embodiments, penetration enhancers can be irritants because inflammatory and / or allergic reactions occur when said agents are applied to the skin.

[0080] "However, a major limitation of penetration enhancers is that their efficacy is often closely associated with the occurrence of skin irritation." Alkilani, AZ, et al., "Transdermal drug delivery: Innovative pharmaceutical developments based on disruption of the barrier properties of the stratumcorneum." 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 delivery across the skin is low and variable." Ibid.

[0081] Some attempts involve using mechanical devices to bypass or ablate portions of the stratum corneum. Other attempts include using ultrasound or iontophoresis to facilitate drug penetration through the skin. In most cases, the goal is to deliver the drug, typically a small molecule, to the capillary bed 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.

[0082] Microneedle technology has been shown to enhance the transdermal delivery of various small drugs, such as calcein (approximately 623 Da), desmopressin (approximately 1070 Da), diclofenac (approximately 270 Da), nicotinic acid methyl ester (approximately 40 Da), diclofenac sodium (approximately 214 Da), insulin (approximately 5.8 kDa), bovine serum albumin (approximately 66.5 kDa), and ovalbumin (approximately 45 kDa). However, prior to this disclosure, the delivery and / or improvement of bioavailability of large drugs, particularly those of 100 kDa or greater, remained problematic.

[0083] Transdermal delivery of large doses of medicine

[0084] Transdermal delivery of large pharmaceutical agents (e.g., macromolecules) is considered a significant challenge. The applicant has demonstrated (see, for example, U.S. Patent Application Nos. 62 / 774,677, 62 / 789,407, and 62 / 808,274) that microneedling treatments, particularly microneedling skin preconditioning using relatively low microneedle densities and / or relatively small microneedle puncture sizes (e.g., puncture size per microneedle), have a surprising effect and / or efficacy on the transdermal administration of large pharmaceutical agents (e.g., botulinum toxin). Given the current level of technology, this demonstration is surprising, including studies, for example, on the delivery of four low molecular weight hydrophilic peptides using solid microneedles: tetrapeptide-3 (456.6 Da); hexapeptide (498.6 Da); acetyl hexapeptide-3 (889 Da); and oxytocin (1007.2 Da) as well as L-carnitine (161.2 Da). This study shows that while microneedle pretreatment significantly enhances the permeability of each tested peptide, the skin permeability of a peptide depends on its 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).

[0085] Furthermore, when comparing sandpaper abrasion, tape peeling, and single-puncture subcutaneous needle models of MSCs in studies on the effect of molecular size of larger FITC (fluorescein isothiocyanate) conjugate molecules on transdermal delivery, it was found that transdermal drug delivery decreased with increasing molecular size (4.3, 9.6, and 42.0 kDa FITC conjugates) for all methods, as well as when tested on untreated skin. Tape peeling 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).)

[0086] 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, skin punctures were performed using microneedles of different lengths (300, 550, 700, or 900 μm) to assess the diffusion of each of the aforementioned compounds. While transport of each compound was observed in all but 300 μm microneedle arrays, degradation of DCB and FITC-Dex was observed.

[0087] Prior to the applicant's work, it was understood in the art that transdermal permeability using MSCs (“microneedle dermatology”) decreases with increasing molecular size, to the point of being negligible or even nonexistent. Even where negligible permeability is observed, larger molecules are observed to degrade and lack biological activity. The applicant's work (including, for example, described in International Patent Application No. PCT / US17 / 53333; published as WO2018 / 093465) has demonstrated various advantages for the transdermal delivery of large agents of interest by combining microneedle treatment techniques with emulsion techniques; in some embodiments, these techniques have shown a particularly surprising enhancement in the transdermal delivery of large molecular structures without the use of mechanical or chemical permeation enhancers. For example, in some embodiments, these techniques have enabled the transdermal delivery of botulinum toxin, which is approximately 150 kDa, more than twice the size of FITC-Dex.

[0088] This disclosure provides certain techniques that have surprising properties and / or achieve unexpected benefits for the administration of macromolecular pharmaceutical agents, particularly botulinum toxin (e.g., via transdermal delivery).

[0089] Those skilled in the art will recognize a variety of protein agents that have been approved by relevant regulatory agencies for therapeutic use. For example, the U.S. Food and Drug Administration maintains a list of approved biological therapeutics arranged by year of approval, which can be found at www.fda.gov / biologicsbloodvaccines / developmentapprovalprocess / biologicalapprovalsbyyear / ucm547553.htm. Those skilled in the art who read this disclosure will understand that its teachings are applicable to any of a variety of such agents; those intended and / or formulated for topical application are of particular interest, including, for example, those that may be undergoing or have undergone clinical trials (e.g., those that may be approved or are undergoing approval by the U.S. Food and Drug Administration or its equivalent in another jurisdiction, or may be included in clinical trials at one or more clinical sites, such as those listed in the records of www.clinicaltrials.gov and / or the Institutional Review Board or its equivalent).

[0090] Those skilled in the art will understand upon reading this disclosure that, in some embodiments, the macropharmaceutical associated with its teaching may be or include antibody agents.

[0091] In some embodiments, the antibody agent may be suitable for 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 part. In some embodiments, the antibody agent may be conjugated to polyethylene glycol. In some embodiments, the antibody may be multispecific (e.g., bispecific) and capable of linking to two or more different target antigens or epitopes.

[0092] In some embodiments, the antibody agent targets TNFα (e.g., epitope-binding elements found in anti-TNFα antibodies such as infliximab, adalimumab, golimumab, etanercept, etanercept-szzs, and / or pegylated cetrusumab). In some embodiments, the antibody agent targets CD2 (e.g., epitope-binding elements found in anti-CD2 antibodies such as ciplelizumab). In some embodiments, the antibody agent targets CD4 (e.g., epitope-binding elements found in anti-CD4 antibodies such as zamumab).

[0093] In some embodiments, the antibody agent targets IL-12 (e.g., including epitope-binding elements found in anti-IL-12 antibodies such as brenumab). In some embodiments, the antibody agent targets IL-17 (e.g., including epitope-binding elements found in anti-IL-17 antibodies such as secukinumab and / or brodatumab). In some embodiments, the antibody agent targets IL-22 (e.g., including epitope-binding elements found in anti-IL-22 antibodies such as fezanumbab). In some embodiments, the antibody agent targets IL-23 (e.g., including epitope-binding elements found in uteroclosporine and / or gusecurumab).

[0094] Those skilled in the art will understand upon reading this disclosure that, in some embodiments, the large pharmaceutical agent associated with its teaching may be or include a prophylactic agent, such as a vaccine. In some embodiments, a vaccine may comprise 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 may be combined with interleukins, interferons, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc. In some implementations, 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, Tula Francisella, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira, Borrelia burgdorferi, Campylobacter jejuni, etc.; and antigens of viruses such as: smallpox, influenza A and B viruses, respiratory syncytial virus, parainfluenza virus, measles, etc. HIV, varicella-zoster virus, herpes simplex virus 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, human papillomavirus, poliovirus, mumps, rabies, rubella, Coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, etc.; antigens of fungi, protozoa, and parasites such as: Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsia, Rickettsia typhus, 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 intact, killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

[0095] Those skilled in the art will recognize that the preceding paragraphs provide an exemplary, rather than comprehensive, list of pharmaceutical agents that can be delivered using the techniques according to the present invention; and will understand the applicability of the teachings provided to the administration of other pharmaceutical agents.

[0096] Botulinum toxin therapy

[0097] Those skilled in the art will know that botulinum toxin is a potent and effective inhibitor of acetylcholine release. Acetylcholine is a neurotransmitter that is active, for example, at neuromuscular junctions and certain other synapses (e.g., within the central nervous system and ganglia, particularly in the visceral motor system). Botulinum toxin can be used to treat and / or prevent a variety of diseases, conditions, and symptoms, particularly those related to acetylcholine release and / or activity.

[0098] In nature, botulinum toxin is produced by Clostridium botulinum, a Gram-positive anaerobic bacterium. Botulinum toxin is classified into one of seven antigenically distinct but structurally similar categories: A, B, C (C1 or C2), D, E, F, and G. All seven categories are understood to be proteins required for the docking and fusion processes involved in the release of acetylcholine (through enzymatic cleavage).

[0099] Botulinum toxin is naturally produced as a single polypeptide (approximately 150 kDa), forming intrapeptide disulfide bonds and being cleaved to produce dipeptide toxins whose chains are interconnected by disulfide bonds. The light chain (approximately 50 kDa) has endopeptidase activity; the heavy chain binds to presynaptic receptors and also promotes the translocation of the light chain across the endosome membrane.

[0100] Botulinum toxin is released by Clostridium bacteria as a complex containing a 150 kDa botulinum toxin protein molecule along with associated non-toxin proteins. Therefore, the BTX-A complex can be produced by Clostridium bacteria in forms of 900 kDa, 500 kDa, and 360 kDa. Type B and C1 botulinum toxins apparently only produce a 500 kDa complex. Type D botulinum toxin produces complexes of 300 kDa and 500 kDa. Finally, type E and F botulinum toxins only produce a complex of approximately 300 kDa.

[0101] The U.S. Food and Drug Administration has approved several botulinum toxin treatments, including, for example:

[0102] a) AbobotulinumtoxinA (as...) Sold and approved for use in the treatment of achalasia, blepharospasm associated with dystonia, cervical dystonia (spastic torticollis), chronic anal fissure, detrusor hyperactivity (detrusor hyperreflexia) or detrusor-sphincter incoordination caused by spinal cord injury or disease, hand dystonia, hand tremor, hemifacial spasm, hyperhidrosis including gustatory sweating (Frederick syndrome), moderate to severe glabellar lines in adults, oromandibular dystonia, drooling, spasmodic dysarthria (laryngeal dystonia), spasms associated with cerebral palsy or multiple sclerosis or neuromyelitis optica or stroke or other brain or spinal cord injury or tumor, strabismus, tongue dystonia, torsional dystonia and upper and lower limb spasticity, including lower limb spasticity in children 2 years of age or older, and vocal tremor.

[0103] b) IncobotulinumtoxinA (as...) Sold and approved for use in blepharospasm, cervical dystonia, chronic drooling, moderate to severe glabellar lines, spasms associated with cerebral palsy or multiple sclerosis or neuromyelitis optica or stroke or other brain or spinal cord injury or tumors, etc.

[0104] c) OnabotulinumtoxinA (as...) Sold and approved for use in the treatment of achalasia, blepharospasm, cervical dystonia, chronic anal fissure, chronic migraine, neurogenic detrusor overactivity, hand dystonia, hand tremor, hemifacial spasm, hyperhidrosis including gustatory sweating (Frederick syndrome), oromandibular dystonia, overactive bladder, drooling, spasmodic dysarthria (laryngeal dystonia), severe primary axillary hyperhidrosis, spasms associated with cerebral palsy or multiple sclerosis or neuromyelitis optica or stroke or other brain or spinal cord injury or tumor, strabismus, tongue dystonia, torsional dystonia, voice tremor, etc.; and Botox. Approved for the treatment of moderate to severe lateral crow's feet (also known as crow's feet and moderate to severe frown lines);

[0105] d) Prabotulinumtoxin A-xcfs (marketed as Jeuveau™, approved for the temporary improvement of the appearance of moderate to severe frown lines associated with activity of the corrugator supercilii and / or depressor supercilii muscles in adults); and

[0106] e) RimabotulinumtoxinB (as...) It is marketed and approved for use in cases of cervical dystonia, detrusor hyperactivity (detrusor hyperreflexia), drooling, and spasms associated with cerebral palsy, multiple sclerosis, neuromyelitis optica, stroke, or other brain or spinal cord injury or tumor.

[0107] Those skilled in the art are familiar with the standard and / or approved administration protocols for such commercially available botulinum toxin compositions, and upon reading this disclosure, will understand how and to what extent such compositions and / or protocols can be utilized with microneedle treatment techniques (e.g., particularly with MSCs), as described herein. Alternatively or additionally, those skilled in the art, upon reading this disclosure, will understand to what extent such approved products and / or protocols can be considered appropriate references to assess the time to peak effect and / or duration of response relative to said references, as described herein.

[0108] The use of botulinum toxin is considered cosmetic when it relates to improving appearance or when there is no physiological impairment that is expected to be improved by the application of the toxin; (in the presence of such physiological impairment) it is considered therapeutic; and if it is applied prior to the appearance of significant symptoms or features of physiological impairment related to the cosmetic feature, it is considered preventative.

[0109] Specifically regarding the treatment of wrinkles, the U.S. Food and Drug Administration (FDA) has provided a draft industry guidance recommending that "measurements at maximum contraction should be used to evaluate the efficacy of botulinum toxin drug products to demonstrate their paralyzing effect," and further suggesting that "success should be defined as...a simultaneous improvement of two grades from baseline on both the [Investigator's Assessment] and [Subject's Self-Assessment] scales to ensure clinical significance." See www.fda.gov / downloads / Drugs / GuidanceComplianceRegulatoryinformation / Guidances / UCM407983.pdf.

[0110] Botulinum toxin is a complex protein that requires three regions, or functional parts, to be intact in order to be biologically active. Therefore, damage to any of these three regions renders the protein biologically inactive. Per Johnson, E., et al., “Botulinum toxin is very susceptible to todenaturation due to surface denaturation, heat, and alkaline conditions.” U.S. Patent Publication No. 5,512,547. Many conventional microneedling treatment conditions (such as those described in Wu) are expected to carry a significant risk of botulinum toxin degradation and inactivation. Given this known tendency of botulinum toxin, the findings documented in this disclosure—for example, the application of nanoemulsion compositions in conjunction with microneedling dermatological conditioning—can delay the peak effect and / or prolong the duration of the response, are particularly surprising.

[0111] Those skilled in the art will understand upon reading this disclosure that its teachings are applicable to the administration of botulinum toxin peptides and / or botulinum toxin complexes, and / or any portion or fragment or variant of botulinum toxin protein or complex that retains the associated activity.

[0112] In some embodiments, the botulinum toxins utilized according to this disclosure may be selected from the group consisting of: types A, Ab, Af, B, Bf, C1, C2, D, E, F, and 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 biological activity lifetime than a suitable, comparable reference form (e.g., wild-type form). In some embodiments, the botulinum toxin exists in any of the subtypes 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.

[0113] In some embodiments, the botulinum toxin provided and / or utilized according to the present invention may be or include one or more approved or developing botulinum toxin products, such as Abobotulinumtoxin A, Daxibotulinumtoxin A, Hengli, Incobotulinumtoxin A, Medy-Tox, Neuronox, NT-201, Onabotulinumtoxin A, Prabotulinumtoxin A-xcfs, PurTox, Rimabotulinumtoxin B, etc.

[0114] Some embodiments of the present invention contemplate pharmaceutical compositions comprising stable botulinum toxin for transdermal delivery to human patients. The botulinum toxin may be selected from the group consisting of: botulinum toxins of types A, B, C1, D, E, F, and G; isolated and / or purified (i.e., about 150 kDa) botulinum toxin; and naturally or recombinantly prepared botulinum toxin. In some embodiments, the composition may comprise and / or deliver a unit amount of botulinum toxin, which may be, for example, between about 1 unit and about 100,000 units, and / or may comprise and / or deliver an amount of botulinum toxin sufficient to achieve a therapeutic effect lasting about 1 month to about 5 years.

[0115] Reaction duration and peak effect

[0116] As noted in a recent review, "The time to onset and duration of the reaction are key measures of botulinum toxin efficacy and have a significant impact on patient satisfaction." See Nestor et al., Aesthetic Surg J.37:S20, 2017. This review also reports that: "Generally, some patients notice improvement in wrinkles within one day of treatment, while the recovery of muscle function generally appears to occur 3 to 6 months after treatment. Patients who have undergone multiple treatments may find that the duration of effect is longer, thus extending the interval between injections," citing Chauhan et al. J Maxillofac Oral Surg. 12(2):173, 2013; Hexsel et al. J Drugs Dermatol. 2013; 12(12):1356-1362; Jaspers et al. Int JOral Maxillofac Surg. 2011; 40(2):127-133; Michaels et al. Aesthet Surg J. 2012; 32(1):96-102; Nestor & Ablon J Drugs Dermatol. 2011; 10(10):1148-1157; Nestor & Ablon J Clin Aesthet Dermatol. 2011; 4(9):43-49; Rzany et al. J Drugs Dermatol. 2013; 12(1):80-84; Schlessinger et al. Dermatol Surg. 2011; 37(10):1434-1442; Yu et al. Arch Facial Plast Surg. 2012; 14(3):198-204; Small Am Fam Physician. 2014; 90(3):168-175.

[0117] Multiple treatments may be necessary to achieve a longer duration of reported effect, as many studies have concluded that the associated clinical effect declines significantly earlier. For example, leading botulinum toxin researcher Carruthers described that "in most people, the clinical effect of botulinum toxin A begins to appear in 1–2 days, peaks in 1–4 weeks, and gradually declines after 3–4 months" (Carruthers J and Carruthers A; Using Botulinum Toxic Cosmetically, A Practical Guide 2011 Informa Healthcare, London, UK). Furthermore, a controlled clinical trial using injectable botulinum toxin to treat crow's feet found that the median duration of effect in responders at day 30 was approximately 4.5 months (Carruthers A et al., "Efficacy and Safety of Onabotulinumtoxin A for the Treatment of Crows Feet Lines: A Multicenter, Randomized, Controlled Trial" Dermatol Surg 2014; 40:1181-1190). In addition, Botox... TM The product's packaging instructions state that the effects of injectable botulinum toxin treatment for frown lines last for approximately 3 to 4 months (Botox Cosmetic packaging instructions).

[0118] While many in the community have reported that expecting a rapid response is desirable, for example, reports that “patients expect the effects of treatment to be visible as soon as possible after surgery” (see Nestor et al. Aesthetic Surg J.37:S20,2017), this disclosure understands that an excessively rapid response may have disadvantages, including, for example, an increased risk of a “frozen face” effect and / or feelings of negative social commentary or embarrassment if immediate and significant changes in facial features can be observed by others.

[0119] Therefore, among other things, this disclosure identifies a problem with certain available botulinum toxin treatments that are designed to achieve rapid onset of action (typically within one day of application, e.g., about 1-3 days) and / or rapid peak effect (typically within less than one month of application). This disclosure recognizes that, particularly for treatments with visible effects (including cosmetic treatments), designing and / or applying botulinum toxin therapies with delayed onset of action and / or peak effect may be desirable. In some embodiments, such onset of action or peak effect is evaluated against (e.g., each) a single dose; in other embodiments, such onset of action or peak effect is evaluated against the last of a plurality of doses in a regimen.

[0120] Those skilled in the art will understand that, in the event of multiple administrations, the time to peak effect for a later dose administered to a particular individual may be shorter than the time to peak effect for an earlier dose administered to the same individual, due to the presence of weakened muscle. Those skilled in the art are familiar with the expected variation in the time to peak effect for a given subject and are therefore able to assess and understand the relevant effects described herein relative to comparable doses (e.g., doses administered to subjects who have received equivalent previous doses).

[0121] In some implementations, the provided botulinum toxin treatment regimens reach peak effect more than one month after administration, and in some implementations more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months or even more than one year later (e.g., for one or more doses in the regimen).

[0122] Among other things, this disclosure provides an insight that the delayed peak effect, as described herein, can have certain advantages, particularly in certain patient populations. For example, many subjects, especially those receiving cosmetic or other visually noticeable botulinum toxin treatments (e.g., wrinkle reduction), may prefer a relatively slow onset and / or rate of effect development, either or both of which can manifest as a delayed peak effect so that the fact that they have received treatment may not be immediately apparent or surprising to onlookers. In some embodiments, delayed peak effect protocols, as described herein, allow for a sufficiently gradual onset so that onlookers may not be aware that the subject has received botulinum toxin therapy.

[0123] Alternatively or additionally, this disclosure provides the insight that the delayed peak effect, as described herein, may present a reduced risk of developing another undesirable potential side effect, such as a "frozen face," or certain other botulinum toxin treatments.

[0124] Furthermore, this disclosure documents the remarkable effects of certain botulinum toxin therapies (e.g., delayed peak effect therapies) as described herein, as they exhibit a longer median duration of response than reported by various approved botulinum toxin therapies.

[0125] Without wishing to be bound by any particular theory, this disclosure observes that the delayed peak effect documented herein reveals pharmacokinetic and / or pharmacodynamic alterations achieved by the therapy compared to certain other botulinum toxin treatments, including, for example, injection therapy. Those skilled in the art will understand upon reading this disclosure that such alterations in pharmacokinetic and / or pharmacodynamics may also be the basis for the prolonged duration of effect observed herein.

[0126] As noted in this article, the median duration of response for currently available botulinum toxin products is approximately 3-4 months (e.g., reported for treatment of wrinkles under approved protocols), and the maximum duration of response is typically less than 6 months.

[0127] This disclosure discloses botulinum toxin treatments, such as those involving the application of botulinum toxin in conjunction with microneedling dermatological conditioning, characterized by a median duration of effect (e.g., for a single dose of botulinum toxin) exceeding 6 months. In some embodiments, such treatments are characterized by a median duration of response exceeding 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or longer.

[0128] Therefore, in some embodiments, the provided botulinum toxin therapy (e.g., involving the application of botulinum toxin in conjunction with microneedling dermatological conditioning) involves the administration of two or more separate doses, which are spaced apart from each other for a period of more than about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or a year or longer. In some embodiments, the different dose pairs in the provided regimen are spaced apart from each other at different such time intervals; in some embodiments, some or all dose pairs may be spaced apart from each other at the same such time intervals.

[0129] Those skilled in the art will recognize that increasing the dosage can sometimes prolong the duration of effect of certain therapies. However, the prolonged duration of effect presented herein cannot be attributed solely to changes in dosage. Those skilled in the art know that increasing the dosage prolongs the duration of effect without affecting the timing of the peak effect. For example, studies have shown that increasing the dosage of botulinum toxin by a factor of three increases the median duration of effect; however, this dosage increase has no effect on the timing of the peak effect. That is, the peak effect of all dosages is achieved within 4 weeks of injection, consistent with other studies on botulinum toxin injections (Ascher B. et al., “Efficacy and Safety of Botulinum Toxin Type A in the Treatment of Lateral Crow's Feet: Double-Blind, Placebo-Controlled, Dose-Ranging Study” Dematol Surg, 2009; 35:1478-1486).

[0130] In stark contrast, as described herein, the therapies offered (e.g., combining certain local botulinum toxin treatments with MSP) both prolong the duration of effect and result in a delayed peak effect (i.e., a peak effect much later than 4 weeks). Without wishing to be bound by any particular theory, it is believed, as stated above, that these combined effects reflect pharmacokinetic and / or pharmacodynamic alterations achieved through the techniques presented herein.

[0131] Therefore, this disclosure provides techniques for both delaying the peak effect and prolonging the duration of response of applied large agents (e.g., botulinum toxin), for example by combining topical application (e.g., emulsion formulations, such as nanoemulsion formulations) with MSCs, as described and / or illustrated herein.

[0132] Microneedle treatment

[0133] In some embodiments, microneedling treatments (e.g., microneedling skin conditioning) according to this disclosure are performed using an array of microneedles (MNs) that is a minimally invasive system or shares features with a minimally invasive system. In some embodiments, the microneedling treatment techniques useful according to this disclosure avoid and / or overcome one or more disadvantages commonly associated with the use of subcutaneous injection needles and / or subcutaneous needles, and improve patient comfort and compliance. Such disadvantages include, for example, the possibility of needle tip misalignment with subcutaneous injection needles, as the healthcare professional cannot see the exact location of the needle; such misalignment is particularly problematic for the administration of botulinum toxin, as it can cause adverse reactions such as ptosis (“drooping eyelid”) if, for example, botulinum toxin is mistakenly injected into the face. Microneedling treatment techniques are less prone to this problem. Other advantages of microneedling treatment techniques include that they are unlikely to cause bleeding, minimize the entry of pathogens into the body through the pores created by the microneedles, and / or may eliminate the variability of transdermal drug delivery. Other advantages include the possibility of self-administration, reduced risk of accidental needlestick injury, reduced risk of spreading infection, and ease of disposal. In some implementations, microneedles are multiple micro-protrusions (e.g., impressions, rollers, arrays, applicators, pens) assembled on one side of a support, such as a patch or device.

[0134] In some embodiments, the microneedles used according to this disclosure can be designed and / or configured in an array, which can, for example, improve skin contact and / or facilitate skin penetration. In some embodiments, the microneedles used have appropriate length, width, and shape to minimize contact with nerves when inserted into the skin, while still creating an efficient 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).

[0135] In some implementations, suitable microneedles can be solid, coated, porous, soluble, hollow, or hydrogel microneedles. Solid microneedles create micropores in the skin, thereby increasing the delivery of drug formulations (e.g., "puncture and patch" methods). Coated microneedles allow the coated drug to dissolve rapidly into the skin (e.g., "coating and puncture" methods). Soluble microneedles allow for rapid and / or controlled release of the drug incorporated into the microneedle. Hollow microneedles can be used to puncture the skin and are capable of releasing the composition after active infusion or diffusion of the formulation through the microneedle pores (e.g., "puncture and flow" methods).

[0136] In the case of soluble microneedles, the microneedles can act as drug reservoirs, holding the drug composition until it is released by dissolution in the case of soluble microneedles or by swelling in the case of hydrogel microneedles (e.g., the "insertion and release" method). However, as described herein, in many embodiments, the bulk drug is not delivered by injection via one or more microneedles. That is, in many embodiments, any microneedles utilized according to such embodiments are not coated, loaded, or manufactured in any way with a bulk drug to achieve bulk drug delivery.

[0137] Alternatively or additionally, in some embodiments, as described herein, if the macropharmaceutical is formulated into a crude emulsion or nanoemulsion composition as described herein, the microneedles utilized according to this disclosure (whether in MSCs or otherwise) may contain and / or deliver the macropharmaceutical. Therefore, as will be understood by those skilled in the art upon reading this specification, treating the skin with microneedles that deliver macropharmaceuticals (e.g., by microneedle injection, by release through a microneedle coating, or by release from dissolved microneedles) is not microneedle dermatology.

[0138] In some embodiments, the microneedles have a uniform diameter along their entire length. In some embodiments, the diameter of the microneedles is largest at their base. In some embodiments, the microneedles taper to a point distal to their base. In some embodiments, the microneedles may be solid. In some embodiments, the microneedles may be hollow. In some embodiments, the microneedles may be tubular. In some embodiments, the microneedles may be sealed at one end. In some embodiments, the microneedles are part of a microneedle array.

[0139] In some embodiments, the length of the microneedle can be between about 1 μm and about 4,000 μm. In some embodiments, the length of the microneedle can be between about 1 μm and about 2,000 μm. In some embodiments, the length of the microneedle can be between about 50 μm and about 400 μm. In some embodiments, the length of the microneedle can be between about 50 μm and about 500 μm. In some embodiments, the length of the microneedle can be between about 50 μm and about 600 μm. In some embodiments, the length of the microneedle can be between about 50 μm and about 700 μm. In some embodiments, the length of the microneedle can be between about 50 μm and about 800 μm. In some embodiments, the length of the microneedle can be between about 800 μm and about 1500 μm. In some embodiments, the length of the microneedle can be less than about 1400 μm. In some embodiments, the length of the microneedle can be less than about 1100 μm. In some embodiments, the length of the microneedle can be less than about 1000 μm. In some embodiments, the length of the microneedle can be less than about 800 μm. In some implementations, the length of the microneedles can be between approximately 100 μm and approximately 800 μm.

[0140] In some embodiments, microneedling treatment as described herein includes applying a plurality of microneedles of common length (e.g., a microneedle array) to the skin; in some embodiments, microneedling treatment as described herein includes applying a plurality of microneedles of different lengths (e.g., a microneedle array) to the skin.

[0141] Microneedles of various lengths can be used in the microneedling techniques described herein. In some implementations, such as the MSC described herein, the length of the microneedles is adjusted according to the skin thickness of the treatment site.

[0142] In some implementations, the microneedles or microneedle arrays include microneedles with lengths of about 25, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, or about 1500 μm.

[0143] In some implementations, microneedles or microneedle arrays include multiple needles. In some implementations, the microneedles or microneedle arrays include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or more microneedles per cm².

[0144] Microneedles of any shape can be used in the microneedle processing techniques 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 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.

[0145] Microneedles of various cross-sectional areas can be used in the microneedling techniques described herein. As described herein, the cross-sectional area of ​​each microneedle in an MN array for MSC (“microneedle dermatology”) can, in turn, define the microneedle puncture size (e.g., the puncture size per microneedle) of the MN array for MSC. In some embodiments, the microneedle puncture size can be from about 100 to about 60,000 μm. 2 Within the range of microneedles. In some implementations, the microneedle puncture size can be from approximately 100 to approximately 30,000 μm. 2 / Within the range of microneedles.

[0146] In some embodiments, the microneedle or microneedle array includes needles with multiple microneedle puncture sizes. In some embodiments, the microneedle or microneedle array includes needles with at least two different microneedle puncture sizes. In some embodiments, the microneedle or microneedle array includes needles with at least three different microneedle puncture sizes. In some embodiments, the microneedle or microneedle array includes needles with at least four different microneedle puncture sizes. In some embodiments, the microneedle or microneedle array includes needles with at least five different microneedle puncture sizes. In some embodiments, the microneedle or microneedle array includes needles with up to ten different microneedle puncture sizes. In some embodiments, the microneedle or microneedle array includes needles with at least eleven different microneedle puncture sizes. In some embodiments, the microneedle or microneedle array includes needles with at least twelve different microneedle puncture sizes. In some embodiments, the microneedle or microneedle array includes needles with at most one different microneedle puncture size.

[0147] In some embodiments, microneedles or microneedle arrays comprising microneedles having various microneedle puncture sizes can be used in the microneedle processing techniques described herein. In some embodiments, the microneedles or microneedle arrays include microneedle puncture sizes of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, and about... 2000, approximately 2500, approximately 3000, approximately 3500, approximately 4000, approximately 4500, approximately 5000, approximately 5500, approximately 6000, approximately 6500, approximately 7000, approximately 7500, approximately 8000, approximately 8500, approximately 9000, approximately 9500, approximately 10000, approximately 10500, approximately 11000, approximately 11500, or approximately 12000 μm 2Microneedles of microneedles. In some embodiments, microneedles or microneedle arrays include microneedle puncture sizes less than about 13,000, less than about 14,000, less than about 15,000, less than about 20,000, less than about 25,000, less than about 30,000, less than about 35,000, less than about 40,000, less than about 45,000, less than about 50,000, less than about 55,000, or less than about 60,000 μm. 2 / Microneedles of microneedles.

[0148] In some embodiments, the microneedles used may be solid; in some embodiments, the microneedles used may be hollow; in some embodiments, the microneedles used may be uncoated; in some embodiments, the microneedles used may be coated (e.g., with a composition that may be or includes the large pharmaceutical agents described herein).

[0149] In some embodiments, the MN used according to this disclosure may be manufactured from different materials using techniques including, but not limited to, micromolding processes or lasers. In some embodiments, the MN may be manufactured from various types of biocompatible materials, including polymers, metals, ceramics, semiconductors, organics, composites, or silicon. Unless they are designed to rupture into the skin and dissolve, in some embodiments, the microneedles have mechanical strength to remain intact and deliver drugs or collect biofluids when inserted into the skin and / or when removed from the skin after insertion. In some embodiments, the MN is capable of remaining in situ for up to several days before complete removal. In some embodiments, the microneedles may be sterilized using standard techniques. In some embodiments, the MN is biodegradable. In some embodiments, the MN comprises polymeric materials. In some embodiments, the polymeric materials comprise poly-L-lactic acid, polyglycolic acid, polycarbonate, polylactic-co-glycolic acid (PLGA), polydimethylsiloxane, polyvinylpyrrolidone (PVP), copolymers of methyl vinyl ether and maleic anhydride, sodium hyaluronate, carboxymethyl cellulose, maltose, dextrin, galactose, starch, gelatin, or combinations thereof.

[0150] In some embodiments, the MSC as described herein includes one imprinting of an MN or MN array. In some embodiments, the MSC includes two imprintings of an MN or MN array. In some embodiments, the MSC includes three imprintings of an MN or MN array. In some embodiments, the MSC includes four imprintings of an MN or MN array. In some embodiments, the MSC includes five imprintings of an MN or MN array. In some embodiments, the MSC includes six imprintings of an MN or MN array. In some embodiments, the MSC includes seven imprintings of an MN or MN array. In some embodiments, the MSC includes eight imprintings of an MN or MN array. In some embodiments, the MSC includes nine imprintings of an MN or MN array. In some embodiments, the MSC includes ten imprintings of an MN or MN array. In some embodiments, the MSC includes eleven imprintings of an MN or MN array. In some embodiments, the MSC includes twelve imprintings of an MN or MN array. In some embodiments, the MSC includes thirteen imprintings of an MN or MN array. In some embodiments, the MSC includes fourteen imprintings of an MN or MN array. In some embodiments, the MSC includes fifteen imprintings of an MN or MN array. In some embodiments, the MSC includes sixteen imprints of a microneedle or microneedle array. In some embodiments, the MSC includes seventeen imprints of a microneedle or microneedle array. In some embodiments, the MSC includes eighteen imprints of a microneedle or microneedle array. In some embodiments, the MSC includes nineteen imprints of a microneedle or microneedle array. In some embodiments, the MSC includes twenty imprints of a microneedle or microneedle array. In some embodiments, the MSC involves rolling a microneedle or microneedle array onto the skin one or more times. In some embodiments, the microneedle array rotates between imprints. In some embodiments, the microneedle array does not rotate between imprints. In some embodiments, imprinting is performed on the same site. In some embodiments, imprinting is performed on overlapping sites. In some embodiments, imprinting is performed on different sites. In some embodiments, imprinting is performed by punching the microneedle array. In some embodiments, imprinting is performed by rolling a microneedle roller onto the site one or more times. According to established MN practices, in some implementations, the skin imprint of the MN array lasts for less than one second, or in some implementations, they last for more than one second, for example, they can last for 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 on.

[0151] In some embodiments, this disclosure understands that as the total surface area of ​​the skin punctured by microneedles decreases, the bioavailability of the macropharmaceutical in the emulsion applied to the skin increases. See, for example, U.S. Patent Application No. 62 / 789,407. Therefore, in some embodiments, relatively fewer imprints may be preferred. In some embodiments, fewer microneedle array imprints may be preferred when the macropharmaceutical applied with microneedles is in a topical formulation that is not (or does not contain) an emulsion (e.g., an emulsion containing the pharmaceutical agent). In some embodiments, shorter microneedle lengths may be preferred. In some embodiments, relatively shorter microneedle lengths may be preferred when the macropharmaceutical applied with microneedles is in a topical formulation that is not (or does not contain) an emulsion (e.g., an emulsion containing the pharmaceutical agent).

[0152] Furthermore, in some embodiments, this disclosure has shown that applying a relatively reduced amount (e.g., volume and / or dose) of a product containing a bioactive agent (e.g., a large dose) in conjunction with MSCs can achieve greater bioefficacy. See, for example, U.S. Patent Application No. 62 / 808274. Therefore, in some embodiments, a relatively reduced volume of the product containing the active agent (e.g., a large dose) may be preferred. In some embodiments, a relatively small product volume may be preferred when the large dose, applied in conjunction with microneedle dermatological treatment, is in a topical formulation that is or contains an emulsion (e.g., a nanoemulsion). In some embodiments, a relatively small product volume may be preferred when the large dose, applied in conjunction with microneedle dermatological treatment, is in a topical formulation that is not (or does not contain) an emulsion (e.g., an emulsion containing the agent). Suitable MN arrays and MSC devices for use in combination with compositions containing large doses to deliver large doses transdermally include those described, for example, in U.S. Patents 6,334,856; 6,503,231; 6,908,453; 8,257,324; and 9,144,671.

[0153] In some embodiments, the site is subjected to MSCs prior to the application (e.g., before a specific application and / or before each application) of a formulation containing and / or delivering a large agent to the site (e.g., a formulation containing an emulsion composition, such as a nanoemulsion composition). In some embodiments, the site is subjected to MSCs after the application of such a formulation to the site. In some embodiments, the MSCs of the site and the application of such a formulation to the site occur substantially simultaneously.

[0154] In some embodiments, the formulation is not injected via one or more microneedles. In some embodiments, the microneedles are part of a microneedle array. In some embodiments, the microneedles may have a length between about 1 μm and about 4,000 μm. In some embodiments, the microneedles may have a length between about 1 μm and 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 1,500 μm.

[0155] emulsion composition

[0156] In some embodiments, this disclosure provides and / or utilizes emulsion compositions (e.g., comprising or otherwise utilizing with large pharmaceutical agents such as botulinum toxin). In some embodiments, the formulations described herein that comprise and / or deliver large pharmaceutical agents may be or comprise emulsions (e.g., emulsions comprising large pharmaceutical agents).

[0157] This disclosure covers the understanding that emulsion technology can provide stable benefits for pharmaceuticals of interest, including large pharmaceuticals as described herein, and particularly including botulinum toxin and / or antibody pharmaceuticals.

[0158] Furthermore, this 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 Publications 2012 / 0328701, 2012 / 0328702, 8,318,181, and 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.

[0159] This disclosure provides certain techniques in which the application of an emulsion composition, together with microneedle treatment, achieves surprising results, such as delayed peak effects and / or prolonged reaction duration.

[0160] The specific emulsion composition of interest may be or include a water-in-oil or oil-in-water emulsion (e.g., a liquid emulsion comprising an oil phase in which water droplets are dispersed, or a liquid emulsion comprising an aqueous phase in which oil droplets are dispersed).

[0161] In some embodiments, the emulsion may be or include a coarse emulsion, for example, characterized by a droplet size in the range of about 300 nm to about 5,000 μm in diameter. In some embodiments, the emulsion may be or include a nanoemulsion, for example, characterized by a droplet size in the range of about 1 nm to about 300 nm in diameter.

[0162] In some embodiments, the use of nanoemulsions can achieve broader and / or deeper transdermal penetration, which can be attributed, for example, at least in part, to the nanoemulsions themselves (e.g., compared to penetration achieved using alternative compositions, including, in some embodiments, the use of macroemulsions).

[0163] In some embodiments, a useful emulsion may be characterized by one or more of the following: the ratio of the aqueous dispersion medium to the oil is between about 0.01:1 and about 20:1; the ratio of the oil to the surfactant is between about 0.1 and about 40; and / or the zeta potential is between about -80 mV and about +80 mV.

[0164] In some embodiments, the provided emulsion (e.g., nanoemulsion) composition comprises oil and surfactant in a ratio between about 0.1:1 and about 2:1. In some embodiments, the provided emulsion composition comprises oil and surfactant in a ratio between about 0.1:1 and about 1:1. In some embodiments, the provided emulsion composition comprises oil and surfactant in a ratio between about 0.5:1 and about 1:1. In some embodiments, the provided emulsion composition comprises oil and surfactant in a ratio between about 0.5:1 and about 1:1.5. In some embodiments, the provided emulsion composition comprises an oil and a 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 0.55:1, 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, the provided emulsion composition comprises an oil and a surfactant in a ratio of about 0.67:1.

[0165] In some embodiments, an aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) and a surfactant are used in a ratio ranging from 0.01 to 20. In some embodiments, an aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) and a surfactant are used in a ratio ranging from 0.1 to 20. In some embodiments, an aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) and a surfactant are used in a ratio ranging from 0.5 to 10. In some embodiments, an aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) and a surfactant are used in a ratio ranging from 0.5 to 1. In some embodiments, the ratio of the aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) to the surfactant is approximately 0.01:1, approximately 0.02:1, approximately 0.03:1, approximately 0.04:1, approximately 0.05:1, approximately 0.06:1, approximately 0.07:1, approximately 0.08:1, approximately 0.0:1, approximately 0.1:1, approximately 0.2:1, approximately 0.3:1, approximately 0.4:1, approximately 0.5:1, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1. In some embodiments, the ratio of surfactant to water is approximately 0.5:1, approximately 1:1, approximately 2:1, approximately 3:1, approximately 4:1, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, approximately 10:1, approximately 11:1, approximately 12:1, approximately 13:1, approximately 14:1, approximately 15:1, approximately 16:1, approximately 17:1, approximately 18:1, approximately 19:1, or approximately 20:1. In some embodiments, the aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) and surfactant are used in a ratio ranging from 0.5 to 2. In some embodiments, the ratio of aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) to surfactant is approximately 0.5:1, approximately 1:1, or approximately 2:1. In some embodiments, the ratio of surfactant to aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) is approximately 0.5:1, approximately 1:1, or approximately 2:1. In some embodiments, the ratio of the aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) to the surfactant is approximately 1:1. In some embodiments, compositions utilizing such a ratio of aqueous dispersion medium (e.g., water, buffer solution, salt solution, etc.) to surfactant comprise water-in-oil emulsions.

[0166] In some embodiments, the droplets in the nanoemulsion composition have diameters (e.g., average diameter and / or median diameter) in the ranges 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 diameters (e.g., average diameter and / or median diameter) in the ranges 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 diameters 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 (e.g., average diameter and / or median diameter).

[0167] In some embodiments, the total droplet distribution is within a specified droplet diameter range. In some embodiments, less than 50%, 25%, 10%, 5%, or 1% of the total droplet distribution exceeds the specified droplet diameter range. In some embodiments, less than 1% of the total droplet distribution exceeds the specified droplet diameter range.

[0168] In some embodiments, the nanoemulsion composition is substantially free of droplets with diameters greater than 300 nm, 250 nm, 200 nm, 150 nm, 120 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, less than 50%, 25%, 10%, 5%, or 1% of the total droplet distribution has a diameter greater than 300 nm, 250 nm, 200 nm, 150 nm, 120 nm, 100 nm, 75 nm, 50 nm, or 25 nm.

[0169] In some embodiments, the droplets in the nanoemulsion composition have an average droplet size below about 300 nm, about 250 nm, about 200 nm, about 150 nm, about 130 nm, about 120 nm, about 115 nm, about 110 nm, about 100 nm, about 90 nm, or about 50 nm. In some embodiments, the average droplet size is in the range of about 10 nm to about 300 nm, about 50 nm to about 250 nm, about 60 nm to about 200 nm, about 65 nm to about 150 nm, or about 70 nm to about 130 nm. In some embodiments, the average droplet size is about 80 nm to about 110 nm. In some embodiments, the average droplet size is about 90 nm to about 100 nm.

[0170] In some embodiments, the zeta potential of the emulsion (e.g., nanoemulsion) droplets is between -80 mV and +80 mV. In some embodiments, the zeta potential of the emulsion droplets is between -50 mV and +50 mV. In some embodiments, the zeta potential of the emulsion droplets is between -25 mV and +25 mV. In some embodiments, the zeta potential of the emulsion droplets is between -10 mV and +10 mV. In some embodiments, the zeta potential of the emulsion droplets is approximately -80 mV, approximately -70 mV, approximately -60 mV, approximately 50 mV, approximately -40 mV, approximately -30 mV, approximately -25 mV, approximately -20 mV, approximately -15 mV, approximately -10 mV, or approximately -5 mV. In some embodiments, the zeta potential of the emulsion droplets is 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 zeta potential of the emulsion droplets is about 0 mV.

[0171] Among other things, this disclosure recognizes that the use of emulsion compositions can provide stability to large pharmaceutical agents, such that, for example, the relevant pharmaceutical agent can remain intact to a greater extent and / or remain active to a greater extent and / or for a longer period of time compared to its maintenance under comparable conditions in other comparable compositions.

[0172] This disclosure understands that, although some reports in the art state that microneedling dermatology helps facilitate the transdermal delivery of small compounds, the transdermal delivery of large agents (e.g., botulinum toxin) can be significantly facilitated by using emulsion compositions in conjunction with microneedling dermatology as described herein, including achieving unintended results such as delayed peak effects and / or prolonged response duration. This disclosure provides the insight that such techniques are particularly useful in certain situations (e.g., for treating certain subjects and / or their sites, especially those with specific disease conditions or symptoms and / or their reaction sites, for whom delayed peak effects and / or prolonged response duration may be particularly desirable).

[0173] This disclosure particularly recognizes that the combination of emulsion compositions with microneedle treatment can achieve transdermal delivery with surprising results, as it has been reported that the combination of microneedle conditioning with encapsulation of even small molecule agents in solid nanoparticles (e.g., 105 ± 2.92 nm) provides only minimal permeation up to 6 hours after application, and material permeation is not observed until 24 hours after application. 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 permeation was evaluated. See Gomaa, Y. et al., “Effect of microneedle treatment on the skin permeation of a nanoencapsulated dye.” JPharm Pharmacol. 2012 Nov; 64(11):1592-1602. The data showed that very small amounts of dye began to permeate the skin after 6 hours of continuous application; a significant increase in permeation was not observed until 24 hours of continuous skin treatment. The researchers explain that "a new consensus exists that NP [nanoparticles] generally cannot penetrate the stratum corneum, although they can deposit well in hair follicles." Therefore, prior to this disclosure, those skilled in the art anticipated that microneedle treatment techniques using media significantly larger than 105 nm would not be able to effectively deliver even small molecule drugs (such as rhodamine dye) transdermally; naturally, delivery of large drugs was considered impossible. However, this disclosure teaches that microneedle treatment can significantly enhance the transdermal delivery of large drugs, particularly when used in conjunction with emulsion techniques, even including coarse emulsion techniques, which involve particles or droplets much larger than those used by Gomaa, and are therefore unlikely to achieve transdermal delivery.

[0174] Among other things, this disclosure recognizes that the provided techniques can enhance transdermal delivery (e.g., large agents, particularly from crude emulsion compositions) without the use of other disruptive agents (i.e., without chemical penetration enhancers and without other techniques that disrupt or puncture skin structures). Therefore, in some embodiments, the provided techniques can thus achieve efficient delivery without the inflammation, irritation, and / or allergic reactions that are typically associated with the use of skin disruptors.

[0175] Previous studies using microneedles for transdermal delivery of agents as large as botulinum toxin (i.e., approximately 150 kDa) have reported that delivery is unsuccessful unless additional treatment is 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 applied to disrupt the skin structure at the microneedle site.

[0176] This disclosure particularly recognizes that the provided techniques can enable transdermal delivery (e.g., large agents, particularly from crude emulsion and nanoemulsion compositions) when no coating or loading of microneedles is utilized and / or the microneedles are not designed to remain in the skin. Among other things, as already noted, this disclosure recognizes that such a coating or loading of microneedles may be commercially unfeasible, at least due to the instability of the coating or the material loaded therein (e.g., large agents such as botulinum toxin). For example, according to Johnson, E., et al., “Botulinum toxin is very prone to denaturation due to surface denaturation, heat, and alkaline conditions. Lyophilization or freeze-drying of botulinum toxin is the most economical, rational, and practical method for dispensing the product in a stable and easily usable form by clinicians.” U.S. Patent No. 5,512,547.

[0177] Additionally, as those skilled in the art will understand upon reading this specification, the techniques described herein have certain advantages, including the elimination of the need to leave microneedles in or associated with tissue. For example, those skilled in the art will understand that leaving microneedles in the skin can carry risks of skin irritation, inflammation, allergic reactions, and / or cosmetically undesirable scarring. In contrast to the present invention, the techniques described in U.S. Patent Publication No. 2017 / 0209553 utilize microneedle arrays loaded with botulinum toxin inserted into the needles and designed to cause the microneedles to break off into the skin (according to U.S. Patent Nos. 2017 / 0209553 and 2016 / 0263362; see also International Patent Publication No. WO / 2018 / 151832).

[0178] This disclosure provides a surprisingly efficient technique for the transdermal delivery of large pharmaceutical agents, such as botulinum toxin, antibodies, etc. In particular, this disclosure teaches that the transdermal delivery of such agents can be significantly enhanced by using microneedle processing techniques without requiring any other destructive strategies. Therefore, the provided technique enables efficient delivery without inflammation, irritation, and / or allergic reactions, which are often associated with the use of skin disruptors. As will be understood by those skilled in the art upon reading this specification, this disclosure teaches that the transdermal delivery of such large pharmaceutical agents can be significantly enhanced even if they are not loaded into, coated onto, and / or manufactured as part of microneedles, by using the provided technique. Similarly, as will be understood by those skilled in the art upon reading this specification, this disclosure teaches that the delivery of large pharmaceutical agents as described herein can be significantly enhanced by using the provided technique (particularly by using MSCs) without leaving microneedles in the skin (e.g., by breaking them and / or otherwise retaining them in situ and / or degrading them). For example, those skilled in the art will understand that the provided technique avoids the long-term stability issues of large dosage forms required for commercially viable products and enables efficient 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, this disclosure explicitly teaches MSC-promoting transdermal delivery of botulinum toxin from topical (e.g., creams, ointments) compositions and particularly from compositions comprising coarse or nanoemulsions using microneedles free of botulinum toxin.

[0179] In some embodiments, this disclosure teaches achieving particularly advantageous results when microneedling techniques are combined with emulsion compositions. In some embodiments, the microneedling techniques are combined with lotions, creams, or liquid compositions, which may in turn be or include emulsion compositions. In some embodiments, the provided techniques do not utilize skin-damaging techniques, such as chemical penetration enhancers.

[0180] In some embodiments, the present invention utilizes emulsion compositions comprising large pharmaceutical agents that are particularly effective and / or useful in a medical context, such as for therapeutic purposes. In some embodiments, specific emulsion compositions are particularly effective and / or useful for topical application of the agent to a subject in need. In some embodiments, the emulsion composition may comprise one or more large pharmaceutical agents.

[0181] In some embodiments, the emulsion may be formulated as a composition suitable for topical application to the skin. In some embodiments, the composition suitable for topical application may be a lotion, cream, powder, ointment, liniment, gel, or drops.

[0182] In some embodiments, the 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.

[0183] preparation

[0184] In some embodiments, a large agent (e.g., botulinum toxin) may be provided and / or utilized in a composition formulated for appropriate application (e.g., for topical application, such as local administration to the skin surface, such as achieving transdermal delivery, or, in some embodiments, for parenteral administration) in accordance with this disclosure.

[0185] In some embodiments, the large pharmaceutical composition (e.g., a botulinum toxin composition) may be or include an emulsion composition, such as a nanoemulsion composition.

[0186] In some embodiments, the large pharmaceutical composition (e.g., a botulinum toxin composition) may be formulated as a cream, drops, foam, gel, liniment, liquid, lotion, ointment, powder, spray, etc. (e.g., in some embodiments, it may be or include an emulsion composition, such as a nanoemulsion composition).

[0187] Those skilled in the art will understand that compositions for topical application can be formulated as, for example, skin softeners, nourishing lotion-type emulsions, cleansing lotions, cleansing creams, skin lotions, moisturizing lotions, massage creams, moisturizing creams, cosmetic primers, masks or facial gels, cleansing formulations such as shampoos, mouthwashes, body cleansers, hair conditioners or soaps, or dermatological components such as lotions, ointments, gels, creams, patches or sprays. In some embodiments, compositions for topical application are not formulated for application to mucous membranes (e.g., not suitable for application to mucous membranes and / or not formulated to deliver a suitable amount of large dose to or across mucous membranes).

[0188] In some embodiments, the formulation used according to this disclosure (e.g., for topical application) may comprise one or more of purified water, methylparaben, mineral oil, isopropyl myristate, white petrolatum, emulsified wax, and propylparaben; in some embodiments, the formulation used according to this disclosure (e.g., for topical application) may comprise one or more of purified water, mineral oil, isopropyl myristate, white petrolatum, and emulsified wax.

[0189] Generally, formulations used according to this disclosure can be prepared by any suitable method (e.g., methods known or later developed in the field of pharmacology). Typically, such preparation methods include the steps of combining the provided composition with one or more excipients, and then, if desired and / or wished, shaping and / or packaging it into a suitable form for administration, for example, as a single-dose or multi-dose unit.

[0190] In some embodiments, the useful substance may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or in multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition comprising a predetermined amount of the provided composition. In some embodiments, the formulation comprises and / or delivers a single dose of the associated active agent (e.g., a large dose, such as botulinum toxin), or a suitable fraction thereof.

[0191] In some embodiments, excipients suitable for 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 media, dispersing or suspending agents, surfactants and / or emulsifiers, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, disintegrants, adhesives, 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 aroma agents may be utilized. Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2005; incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation.

[0192] In some embodiments, the appropriate excipient (e.g., a pharmaceutically and / or cosmetically acceptable excipient) is 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 Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other international pharmacopoeias.

[0193] Specific exemplary formulations can be prepared as, for example, cosmetic formulations such as skin softeners, nourishing lotion-type emulsions, cleansing lotions, cleansing creams, skin lotions, moisturizing lotions, massage creams, moisturizing creams, makeup bases, masks, or facial gels; cleansing formulations such as shampoos, mouthwashes, body cleansers, hair conditioners, or soaps; or dermatological components such as lotions, ointments, gels, creams, liniments, patches, deodorants, or sprays. In some embodiments, the composition for topical application is not formulated for application to mucous membranes (e.g., not suitable for application to mucous membranes and / or not formulated to deliver a suitable amount of large dose to or across the mucous membrane).

[0194] In some embodiments, the present invention provides specific cream and / or lotion formulations as described herein. In some embodiments, the provided cream and / or lotion formulations comprise water. In some embodiments, the provided cream and / or lotion formulations comprise methylparaben. In some embodiments, the provided cream and / or lotion formulations comprise mineral oil. In some embodiments, the provided cream and / or lotion formulations comprise isopropyl myristate. In some embodiments, the provided cream and / or lotion formulations comprise white petrolatum. In some embodiments, the provided cream and / or lotion formulations comprise emulsified wax. In some embodiments, the provided cream and / or lotion formulations comprise propylparaben. In some embodiments, the provided cream and / or lotion formulations do not contain any parabens. In some embodiments, the provided cream and / or lotion formulations do not contain methylparaben. In some embodiments, the provided cream and / or lotion formulations do not contain propylparaben.

[0195] Exemplary lotion formulations are provided in Table 1.

[0196] Table 1. Exemplary Cream and / or Lotion Formulations

[0197] %w / w Element 72.00 Purified water 0.200 Methylparaben 5.00 mineral oil 5.00 Isopropyl myristate 2.000 Vaseline 15.00 Emulsified wax 0.800 propylparaben 100 total

[0198] In some embodiments, cream and / or lotion formulations are available for topical and / or transdermal application. This disclosure covers the understanding that the provided cream and / or lotion formulations are particularly suitable for delivering pharmaceutical agents to the dermis layer of the skin. In some embodiments, the provided cream and / or lotion formulations are formulated for topical and / or transdermal delivery to a subject in need. In some embodiments, the provided cream and / or lotion formulations are administered to a subject in need via topical and / or transdermal delivery.

[0199] In some embodiments, the provided composition is formulated with cosmetically acceptable components. For example, in some embodiments, the provided composition is formulated with water and any cosmetically acceptable solvent, particularly monohydric alcohols, such as alkanols having 1 to 8 carbon atoms (like ethanol, isopropanol, benzyl alcohol, and phenethyl alcohol), polyhydric alcohols, 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 proportions up to 60%, 70%, 80%, or 90% by weight relative to the total composition.

[0200] In some embodiments, the provided composition for topical application includes one or more cosmetically acceptable components that impart desired or appropriate appearance properties to the subject to whom the composition is to be applied (e.g., a matte appearance, which may be particularly desired or appropriate for subjects with oily skin).

[0201] In some embodiments, the provided composition is formulated with at least one cosmetically acceptable filler, for example, to obtain a matte product, which may be particularly desirable for individuals with oily skin.

[0202] In some embodiments, the botulinum toxin composition formulated for application (e.g., as a cream or lotion) comprises about 1 to about 200,000 units of botulinum toxin per milliliter, or about 1 to about 100,000 units of botulinum toxin per milliliter, or about 1 to about 50,000 units of botulinum toxin per milliliter, or about 500 to about 20,000 units of botulinum toxin per milliliter, or about 100 to about 2,000 units of botulinum toxin per milliliter, or about 50 to about 500 units of botulinum toxin per milliliter, or about 25 to about 400 units of botulinum toxin per milliliter. In some embodiments, the botulinum toxin composition formulated for application (e.g., as a cream or lotion) contains about 2 to about 40,000 units of botulinum toxin per milliliter, or about 12,000 units of botulinum toxin per milliliter, or about 100 to about 2,000 units of botulinum toxin per milliliter, or about 50 to about 1,000 units of botulinum toxin per milliliter.

[0203] Those skilled in the art will understand that the units herein refer to units that are biologically equivalent or bioactively equivalent to units as defined by the commercial manufacturers of botulinum toxin.

[0204] In some embodiments, the present invention provides a topical formulation of botulinum toxin that avoids potential complications, including but not limited to systemic toxicity or botulism. In some embodiments, the dosage range of botulinum toxin (including types A, B, C, D, E, F, or G, or 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. Specific dosages may vary depending on the condition being treated and the treatment regimen used. For example, treating subcutaneous, overactive muscles may require high transdermal doses of botulinum toxin (e.g., 1,000 to 20,000 units). In contrast, treating neurogenic inflammation or overactive sweat glands may require relatively small transdermal doses of botulinum toxin (e.g., about 1 unit to about 1,000 units).

[0205] In some embodiments, the large-dose composition may be formulated and delivered in combination with MSCs as described herein for systemic delivery; in other embodiments, the provided composition may be formulated and / or delivered for local rather than systemic delivery.

[0206] In some embodiments, the large-dose composition may be formulated and delivered in combination with MSCs as described herein for systemic delivery; in other embodiments, the provided composition may be formulated and / or delivered for local rather than systemic delivery.

[0207] In some embodiments, the composition suitable for topical formulations comprises a penetration enhancer. In some embodiments, the penetration enhancer degrades, disrupts, and / or damages skin structure and / or skin. In some embodiments, the penetration enhancer does not degrade, disrupt, 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.

[0208] This disclosure specifically demonstrates the effective and efficient delivery of large pharmaceutical agents (and particularly large biological agents, such as botulinum toxin and / or antibody agents and / or prophylactic agents such as vaccines) to the dermis using the provided compositions in combination with MSCs as described herein. For example, in some embodiments, the invention provides a method comprising administering the compositions as described herein without clinically significant side effects. As an example, when considering local delivery, clinically significant side effects include, but are not limited to, undesirable systemic side effects, damage to nerve tissue beneath the dermis (e.g., nerve paralysis), undesirable effects on muscles (e.g., muscle paralysis), and / or undesirable blood levels of large pharmaceutical agents.

[0209] Those skilled in the art who have read this disclosure will understand that, in some embodiments, the provided composition can be incorporated into a device (e.g., a patch). Various transdermal patch structures are known in the art; those skilled in the art will understand that the provided composition can be readily incorporated into any of these various structures. In some embodiments, the transdermal patch may include a plurality of needles extending from the 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 nerves in the dermis of the skin.

[0210] In some embodiments, the transdermal patch includes an adhesive. Examples of adhesive patches are well known (e.g., see U.S. Design Patent 296,006; and U.S. Patents 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). Adhesive patches are generally characterized by having an adhesive layer to be applied to a patient's skin, a reservoir or reservoir for holding the provided composition, and an outer surface to prevent leakage of the provided composition from the reservoir. The outer surface of the patch may be non-adhesive.

[0211] According to some embodiments, the macropharmaceutical composition may be a patch; in some embodiments, the incorporated macropharmaceutical remains stable over a long period. In some embodiments, the provided composition may be incorporated into a polymer matrix stabilizing the macropharmaceutical, allowing the drug to diffuse from the matrix and the patch. In some embodiments, the macropharmaceutical composition may be incorporated into an adhesive layer of the patch, such that the provided composition diffuses through the skin once the patch is applied. In some embodiments, the adhesive layer may be thermally activated, wherein a temperature of approximately 37°C causes the adhesive to liquefy slowly, allowing the drug to diffuse through the skin. When stored below 37°C, the adhesive may remain tacky, and upon application to the skin, the adhesive loses its tacky nature upon liquefaction.

[0212] In some embodiments, a large pharmaceutical composition may be provided in a reservoir within a patch, such that pressure applied to the patch causes the provided composition to be guided out of the patch and through the stratum corneum via microneedles. Exemplary embodiments of microneedles have been described above. Suitable devices for intradermal application of the provided composition include those described in U.S. Patent 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 may be applied 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 their functional equivalents.

[0213] In some embodiments, such as to prolong the effect of a large pharmaceutical composition, it may be desirable to slow the absorption of the provided composition into the skin. In some embodiments, this can be accomplished by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. The absorption rate of the provided composition depends on its dissolution rate, which in turn depends on the crystal size and crystal form. In some embodiments, the release rate of the provided composition can be controlled depending on the ratio of the provided composition to the polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides).

[0214] In some embodiments, the provided formulation comprises a mixture of the provided emulsion composition (e.g., a nanoemulsion composition) and one or more pharmaceutically acceptable excipients. In some embodiments, cream and / or lotion formulations comprise a mixture of the provided nanoemulsion composition and / or a saline solution.

[0215] In some embodiments, the provided composition comprises the provided nanoemulsion composition. In some embodiments, the provided composition is a cream and / or lotion formulation. In some embodiments, the provided cream and / or lotion formulation comprises a nanoemulsion composition. In some embodiments, the composition comprises the provided nanoemulsion composition but is not a cream and / or lotion formulation. In some embodiments, a suitable composition is formulated as a cream and / or lotion but does not contain a nanoemulsion composition.

[0216] In some embodiments, the provided composition comprises a mixture of the provided nanoemulsion composition and one or more pharmaceutically acceptable excipients, for example, for topical and / or transdermal application (e.g., via lotion, cream, powder, ointment, liniment, gel, drops, etc.).

[0217] In some embodiments, for nanoemulsion compositions comprising known therapeutic agents and / or independently active bioactive agents, such nanoemulsion compositions are arranged and constructed for administration in combination with MSCs, such that a sufficient amount of therapeutic agent to treat a symptom or condition is delivered to the desired target site (e.g., to the epidermis and / or dermis). In some embodiments, the provided nanoemulsion compositions are arranged and constructed (e.g., by selecting and / or combining agents, component structures, etc.) such that they achieve the desired therapeutic effect upon application to the skin. In some embodiments, the provided nanoemulsion compositions are arranged and constructed such that they do not cause undesirable clinical effects within and / or outside the desired site of action (e.g., the skin surface, dermis, etc.). In some embodiments, the provided nanoemulsion compositions are arranged and constructed and administered in combination with MSCs such that they have a systemic effect.

[0218] In some embodiments, the provided composition may be formulated and delivered in combination with MSCs as described herein for whole-body delivery; in other embodiments, the provided composition may be formulated and / or delivered for local rather than whole-body delivery.

[0219] This disclosure specifically demonstrates the effective and efficient delivery of therapeutic agents (and particularly large biological agents such as botulinum toxin or antibody agents) to the dermis using the provided compositions in combination with MSCs. For example, in some embodiments, the invention provides a method comprising administering the compositions as described herein without clinically significant side effects. As an example, when considering local delivery, clinically significant side effects include, but are not limited to, undesirable systemic side effects, damage to nerve tissue beneath the dermis (e.g., nerve paralysis), undesirable effects on muscles (e.g., muscle paralysis), and / or undesirable blood levels of the therapeutic agent.

[0220] In some embodiments, the present invention provides a topical formulation of a large dose (e.g., botulinum toxin or an antibody agent) that allows the agent to penetrate through the skin of a subject without significantly penetrating through the blood vessels. For example, in some embodiments of the invention, when applied according to this disclosure, 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 agent present in the formulation penetrates into the blood vessels.

[0221] Those skilled in the art will understand that the compositions of the present invention for transdermal administration of botulinum toxin or antibody agents can be incorporated into devices (e.g., patches, rollers, pens, impressions, etc.).

[0222] use

[0223] In some embodiments, this disclosure provides a macropharmaceutical therapy (e.g., botulinum toxin therapy) that involves administering one or more doses of a related macropharmaceutical composition (e.g., botulinum toxin composition) in conjunction with a microneedling treatment (e.g., microneedling dermatology) for one or more doses within the regimen, according to a protocol that has been shown to achieve delayed onset and / or delayed peak effect as described herein (e.g., a detectable onset of effect later than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or more days after administration, and / or a peak effect later than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 months or even more than one year).

[0224] This disclosure provides the insight that such techniques are particularly useful in certain situations (e.g., for treating certain subjects and / or their sites, especially those with specific disease conditions or symptoms and / or their reaction sites, for whom a delayed peak effect and / or prolonged duration of response may be particularly desirable).

[0225] This disclosure demonstrates that certain dosing regimens (e.g., a prolonged time interval between two administrations, given the prolonged duration of effect documented herein) can be surprisingly effective and even particularly useful in certain circumstances (e.g., when administered to a subject and / or site suffering from a disease, condition, or symptom, for which a delayed peak effect and / or prolonged duration of response may be particularly desirable, including for the reasons described herein).

[0226] This invention particularly provides techniques for administering large agents, such as botulinum toxin or antibody preparations, 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 conjunction with MSCs as described herein. These techniques surprisingly provide delayed peak effects and / or prolonged durations of action compared to certain alternative techniques for delivering the relevant large agents. The inventors have surprisingly discovered that when used in conjunction with MSCs employing microneedles or arrays of microneedles with relatively low microneedle density or relatively small microneedle puncture sizes (e.g., puncture size per microneedle, cross-sectional area per microneedle), not only are the transdermal permeability and bioavailability of botulinum toxin or antibody preparations incorporated into the nanoemulsion composition significantly improved, but the benefit of this invention is the ability to administer such large agents intradermally while minimizing skin irritation or damage. Furthermore, certain unexpected results can be achieved. Therefore, this disclosure teaches that, in certain situations (e.g., when administered to specific subjects and / or sites), the local application of emulsion compositions containing and / or delivering large agents in conjunction with MSCs is particularly useful. Furthermore, this disclosure identifies certain dosing regimens (e.g., formulations containing and / or delivering large doses—e.g., emulsion compositions such as nanoemulsions—with prolonged intervals between individual administrations, for example, taking into account the prolonged duration of effect).

[0227] In some embodiments, the bulk agent is botulinum toxin. In some embodiments, a botulinum toxin emulsion composition (e.g., in a formulation as described herein) is applied directly to the skin and absorbed through the epidermis before MSCs. In some embodiments, such a botulinum toxin emulsion composition is applied directly to the skin and absorbed through the epidermis after MSCs. In some embodiments, a botulinum toxin emulsion composition is applied directly to the skin and absorbed through the epidermis in substantially the same time as MSCs.

[0228] In some embodiments, botulinum toxin emulsion (e.g., nanoemulsion) compositions combined with MSCs can penetrate the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, without the use of penetration enhancers. In some embodiments, botulinum toxin emulsion compositions combined with MSCs can penetrate the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, without the use of degrading agents, irritants, and / or abrasives.

[0229] In some embodiments, the antibody emulsion composition in combination with MSCs can penetrate the top stratum corneum, including the stratum corneum, dermal pores, and / or dermal glands, without the use of penetration enhancers. In some embodiments, the bulk agent is an antibody agent. In some embodiments, the antibody emulsion composition is applied directly to the skin and is intended for absorption through the epidermis prior to MSCs. In some embodiments, the antibody emulsion composition is applied directly to the skin and is intended for absorption through the epidermis after MSCs. In some embodiments, the antibody emulsion composition is applied directly to the skin and is intended for absorption through the epidermis at substantially the same time as MSCs. In some embodiments, the antibody emulsion composition is applied directly to the skin and is intended for systemic absorption.

[0230] In some embodiments, antibody emulsion compositions combined with MSCs can penetrate the top layer of the skin, including the stratum corneum, dermal pores, and / or dermal glands, without the use of degrading agents, irritants, and / or abrasives.

[0231] Diseases, symptoms and conditions

[0232] The technology disclosed herein can be used to treat and / or prevent any of a variety of diseases, conditions and / or symptoms, particularly including certain systemic or skin diseases, conditions or illnesses.

[0233] In some embodiments, the present invention provides techniques for treating and / or preventing diseases, conditions, or symptoms related to sweat and / or sebaceous gland activity. In some embodiments, the present invention provides techniques for treating and / or preventing infection-related diseases, conditions, or symptoms. In some embodiments, the present invention provides techniques for treating and / or preventing inflammation-related diseases, conditions, or symptoms. In some embodiments, the present invention provides techniques for treating and / or preventing cancer-related diseases, conditions, or symptoms. In some embodiments, the present invention provides techniques for treating and / or preventing systemic diseases, conditions, or symptoms. In some embodiments, the present invention provides techniques for treating and / or preventing autoimmune diseases, conditions, or symptoms. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, conditions, or symptoms related to the epidermal and / or dermal levels of the skin. In some embodiments, the present invention provides techniques for treating and / or preventing eye diseases, conditions, or symptoms.

[0234] In some embodiments, the present invention provides techniques for treating and / or preventing one or more of the following: acne, unwanted sweating, body odor, hyperhidrosis, bromhidrosis, chromhidrosis, rosacea, hair loss, psoriasis, actinic keratosis, eczematous dermatitis (e.g., atopic dermatitis), conditions of excessive sebum secretion (e.g., seborrheic dermatitis), burns, Raynaud's phenomenon, lupus erythematosus, conditions of hyperpigmentation (e.g., melasma), conditions of hypopigmentation (e.g., vitiligo), skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma), dermal infections (e.g., bacterial infections, viral infections, fungal infections), facial wrinkles (e.g., Wrinkles involving the forehead, glabella, and / or periorbital area; headaches; unsightly facial expressions (e.g., due to underlying overactivity of facial muscle tissue); neck wrinkles; hyperfunctional facial lines; hyperactive facial lines; platysma band; neck and shoulder wrinkles; neuromuscular disorders and conditions involving muscle spasms and / or contractures (including various forms of facial nerve palsy, cerebral palsy, blepharospasm, facial contractures); dystonia; benign prostatic hyperplasia; headaches; strabismus; hemifacial spasm; tremor; spasms such as those caused by multiple sclerosis, posterior orbital muscle, various ophthalmic and urological conditions (e.g., penile and / or bladder conditions and penile and scrotal wrinkles) and / or combinations thereof.

[0235] In some implementations, the provided techniques may be particularly useful for treating wrinkles, including, for example, wrinkles involving the forehead, glabellar area, wrinkles and / or periorbital area (including crow's feet), unsightly facial expressions (e.g., due to underlying overactivity of facial muscle tissue), neck wrinkles, hyperfunctional facial lines, hyperactive facial lines, platysma band, neck and shoulder wrinkles, hand wrinkles, foot wrinkles, breast wrinkles, penile wrinkles, and scrotal wrinkles.

[0236] In some embodiments, the present invention provides techniques for treating and / or preventing rheumatoid arthritis. In some embodiments, the present invention provides techniques for treating and / or preventing psoriatic arthritis. In some embodiments, the present invention provides techniques for treating and / or preventing osteoarthritis.

[0237] 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. In some embodiments, the present invention provides techniques for treating and / or preventing Crohn's disease. In some embodiments, the present invention provides techniques for treating and / or preventing inflammatory bowel disease. In some embodiments, the present invention provides techniques for treating and / or preventing ulcerative colitis.

[0238] In some embodiments, the present invention provides techniques for treating and / or preventing lung diseases. In some embodiments, the lung disease may be asthma or chronic obstructive pulmonary disease.

[0239] In some embodiments, the present invention provides techniques for treating and / or preventing amyloidosis. In some embodiments, amyloidosis is systemic or cutaneous.

[0240] In some embodiments, the present invention provides techniques for treating and / or preventing cancer. In some embodiments, the cancer is of the skin, blood, breast, colon, or lung.

[0241] In some embodiments, the present invention provides techniques for treating and / or preventing dyslipidemia. In some embodiments, dyslipidemia is hypercholesterolemia.

[0242] In some embodiments, the present invention provides techniques for treating and / or preventing infections. In some embodiments, the infection is caused by Clostridium difficile or Staphylococcus aureus.

[0243] In some embodiments, the present 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.

[0244] In some embodiments, the present invention provides techniques for treating and / or preventing neurological symptoms. In some embodiments, the neurological symptoms are Alzheimer's disease, Parkinson's disease, or stroke.

[0245] In some implementations, the provided technology can be used to treat and / or prevent one or more diseases, conditions, and symptoms, such as certain skin conditions (e.g., acne, rosacea), certain eye conditions (e.g., blepharospasm, strabismus, etc.), various muscle and / or movement disorders (e.g., cervical dystonia, muscle contractures, muscle spasms, muscle stiffness, torticollis, etc.), certain bladder and / or bowel conditions (e.g., urinary incontinence, overactive bladder, including subjects who cannot tolerate side effects associated with other therapies, urinary urgency, etc.), migraines, hyperhidrosis (e.g., bromhidrosis, chromhidrosis, hyperhidrosis, etc.), wrinkles, etc.

[0246] In some embodiments, the technology provided in this disclosure relates to administration according to a dosing regimen sufficient to achieve a reduction of at least about 20% in the severity and / or prevalence of the relevant skin condition; in some embodiments, according to a dosing regimen sufficient to achieve a reduction of at least about 25%; in some embodiments, according to a dosing regimen sufficient to achieve a reduction of at least about 30%; and in some embodiments, according to a dosing regimen sufficient to achieve a reduction of at least about 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%, or about 48%. Dosing regimens of approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, or more.

[0247] In some embodiments, the present invention relates to at least one provided composition administered in combination with MSCs according to a dosing regimen sufficient to achieve a reduction of at least about 20% in the degree and / or prevalence of the associated skin condition in a specific percentage of a patient population in which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve a reduction of at least about 25% in a specific percentage of a patient population in which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve a reduction of at least about 30% in a specific percentage of a patient population in which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve a reduction of at least about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% in a specific percentage of a patient population in which the composition is administered. Approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately Dosing regimens of 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, or more. In some embodiments, a specific percentage of the patient population to which the composition is applied 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, for the purpose of providing illustrative examples, the present invention relates to applying at least one provided composition according to a dosing regimen sufficient to achieve a reduction of at least about 20% in the degree and / or prevalence of the associated skin condition in at least about 50% of the patient population to which the composition is applied. In some embodiments, the present invention relates to applying at least one provided composition according to a dosing regimen sufficient to achieve a reduction of at least about 30% in the degree and / or prevalence of the associated skin condition in at least about 50% of the patient population to which the composition is applied.

[0248] This invention provides techniques for treating and / or preventing skin conditions, including applying the provided composition in combination with MSCs to a subject who has, is susceptible to, and / or exhibits symptoms of a skin condition. In some embodiments, compositions for treating skin conditions as described herein are provided for use via any of the routes of administration described herein. In some embodiments, the provided compositions are formulated for topical application. In some embodiments, the provided compositions are formulated as creams, lotions, lotions, gels, shampoos, conditioners, sunscreens, deodorants, and / or antiperspirants (e.g., as rollers, sticks, gels, creams, aerosols, etc.), depending on the condition to be treated.

[0249] In some embodiments, such provided compositions are applied topically in combination with MSCs to the affected area (e.g., armpits, 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 application is achieved through topical application in combination with MSCs.

[0250] combination

[0251] In some implementations, the techniques described herein can be used to administer a large dose (e.g., botulinum toxin) in combination with another dose and / or another treatment.

[0252] In some implementations, the provided technology can be used to administer multiple large agents (e.g., a combination of botulinum toxin with one or more other large agents such as one or more antibodies).

[0253] In some embodiments, the provided technology may be used in conjunction with one or more penetration-enhancing technologies (e.g., one or more penetration enhancers); in some embodiments, no penetration enhancer is used. In some embodiments, the provided technology may be used with one or more non-irritating and / or non-degrading, non-destructive, and / or non-damaging skin structure and / or skin penetration enhancers. In some embodiments, the non-irritating penetration enhancer may be selected from, for example, coneptides, carrier molecules, and carrier peptides. In some embodiments, the carrier molecule is positively charged. In some embodiments, the carrier molecule may be a coneptide. In some embodiments, the carrier molecule may be a long-chain positively charged polypeptide or a positively charged non-peptide polymer, such as polyalkylimide. In some embodiments, the carrier peptide may be a cationic peptide. In some embodiments, the carrier peptide has the sequence RKKRRQRRRG-(K). 15 -GRKKRRQRRR is a positively charged carrier. In some embodiments, the carrier molecule may be the carrier molecule disclosed in U.S. Patent Publication 2010 / 0168023 or U.S. Patent Publication 2009 / 0247464, the contents of which are incorporated herein by reference in their entirety.

[0254] In some embodiments, the provided technology can be used in combination with one or more therapies that act on or in the skin and / or impart therapeutic and / or cosmetic effects. In some embodiments, the bioactive agent used in combination with antibody agents as described herein can be an agent that acts on or in the skin and / or imparts therapeutic and / or cosmetic effects. For example, the provided technology can be used in combination with one or more anesthetics (e.g., lidocaine), steroids (e.g., hydrocortisone) and / or retinoids (e.g., visual pigment A), cosmetic agents such as dermal fillers (e.g., hyaluronic acid or other elastin materials), collagen, and / or silicones.

[0255] Subjects

[0256] The techniques disclosed herein are applicable to human and animal use. Subjects who may benefit from treatment with the techniques described herein include, in particular, subjects suffering from diseases, conditions, or ailments as described herein. In certain embodiments, the disease, condition, or ailment suffered by the subject is cosmetic and / or includes visually noticeable features, effects, or characteristics. Another contribution of this disclosure is the insight that a delayed peak effect and / or prolonged duration of effect may be particularly desirable for certain such subjects.

[0257] In some embodiments, the application site is the skin covering the muscle or muscle group of the subject. In some embodiments, the site is hairless. In some embodiments, the site is on the torso. 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 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 mucous membrane.

[0258] In some embodiments, the site is affected by a skin condition. In some embodiments, the site is the skin covering a muscle or muscle group affected by a neuromuscular condition. In some embodiments, the length of the microneedles used in MSCs is adjusted based on the skin thickness at the treatment site.

[0259] In many implementations, the subject has wrinkles and / or the techniques described herein are applied to wrinkled areas (e.g., to areas of wrinkled skin).

[0260] In some embodiments, the provided technology enables the controlled and / or improved delivery of active agents to biologically relevant target sites (e.g., specific tissues, intradermal locations, cells, etc.) in a controlled manner. In some embodiments, the invention demonstrates that controlled delivery and / or the achievement of therapeutic effects in certain biologically relevant target sites are achieved without significant side effects associated with delivery to other areas.

[0261] In some embodiments, the provided technology can effectively improve the delivery and / or bioavailability of active agents, specifically for delivery to the dermis, and / or for cosmetic and / or therapeutic effects upon application to the skin of a subject. In some embodiments, the invention demonstrates improved delivery and / or bioavailability and / or therapeutic effects through dermal delivery without significant side effects associated with delivery to other areas (e.g., subcutaneous or subcutaneous structures and / or tissues outside the dermis). In some embodiments, the provided technology can improve the transdermal delivery and / or bioavailability of active agents, such as therapeutic agents (e.g., botulinum toxin, antibody agents, etc.).

[0262] In some embodiments, the large dose applied according to this disclosure penetrates the skin within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes after application. In some embodiments, the large dose penetrates the skin within about 5 to about 60 minutes after application. In some embodiments, the large dose penetrates the skin within about 5 to about 12 minutes after application. In some embodiments, the large dose penetrates the skin within about 5 to about 15 minutes after application. In some embodiments, the large dose penetrates the skin within about 15 to about 30 minutes after application. In some embodiments, the large dose penetrates the skin within about 1 hour after application. In some embodiments, the large dose penetrates the skin within about 2 hours after application. In some embodiments, the large dose penetrates the skin within about 3 hours after application. In some embodiments, the large dose penetrates the skin within about 4 hours after application. In some embodiments, the large dose penetrates the skin within about 5 hours after application. In some embodiments, the large dose penetrates the skin within about 6 hours after application.

[0263] In some embodiments, the large agent penetrates the skin layer within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes after application. In some embodiments, the large agent penetrates the skin layer within about 5 to about 60 minutes after application. In some embodiments, the large agent penetrates the skin layer within about 5 to about 12 minutes after application. In some embodiments, the large agent penetrates the skin layer within about 5 to about 15 minutes after application. In some embodiments, the large agent penetrates the skin layer within about 15 to about 30 minutes after application. In some embodiments, the large agent penetrates the skin layer within about 1 hour after application. In some embodiments, the large agent penetrates the skin layer within about 2 hours after application. In some embodiments, the large agent penetrates the skin layer within about 3 hours after application. In some embodiments, the large agent penetrates the skin layer within about 4 hours after application. In some embodiments, the large agent penetrates the skin layer within about 5 hours after application. In some embodiments, the large agent penetrates the skin layer within about 6 hours after application.

[0264] In some embodiments, the large agent penetrates the dermis within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes after application. In some embodiments, the large agent penetrates the dermis within approximately 5 to approximately 60 minutes after application. In some embodiments, the large agent penetrates the dermis within approximately 5 to approximately 12 minutes after application. In some embodiments, the large agent penetrates the dermis within approximately 5 to approximately 15 minutes after application. In some embodiments, the large agent penetrates the dermis within approximately 15 to approximately 30 minutes after application. In some embodiments, the large agent penetrates the dermis within approximately 1 hour after application. In some embodiments, the large agent penetrates the dermis within approximately 2 hours after application. In some embodiments, the large agent penetrates the dermis within approximately 3 hours after application. In some embodiments, the large agent penetrates the dermis within approximately 4 hours after application. In some embodiments, the large agent penetrates the dermis within approximately 5 hours after application. In some embodiments, the large agent penetrates the dermis within approximately 6 hours after application.

[0265] In some embodiments, the large agent penetrates the top layer of 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 after application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 5 to about 60 minutes after application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 5 to about 12 minutes after application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 5 to about 15 minutes after application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 15 to about 30 minutes after application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within about 1 hour after application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within approximately 2 hours of application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within approximately 3 hours of application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within approximately 4 hours of application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within approximately 5 hours of application. In some embodiments, the large agent penetrates the top layer of skin, including the stratum corneum, dermal pores, and / or dermal glands, within approximately 6 hours of application.

[0266] Reagent test kit

[0267] In some embodiments, the present invention provides a pharmaceutical package or kit comprising one or more emulsion compositions and one or more microneedle devices used according to the invention. In some embodiments, the pharmaceutical package or kit comprises a formulation or pharmaceutical composition containing the provided composition in one or more containers, said containers optionally filled with one or more additional components 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 accompanying such containers may be a tabular report issued by a government agency that regulates the manufacture, use, or sale of the pharmaceutical product, reflecting approval for human administration by the agency that manufactures, uses, or sells it.

[0268] In some embodiments, a kit containing a therapeutic agent is provided. By way of non-limiting example only, the provided composition may be provided as a topical formulation and, in combination with a microneedle device, administered as a therapy. Instructions for drug dosage or self-administration may be provided in the kit for administration to individuals who have or are at risk of a condition or ailment (e.g., those related to the dermal level of the skin).

[0269] In some embodiments, the kit may include (i) the provided composition; and (ii) at least one pharmaceutically acceptable excipient; and (iii) at least one device for microneedling skin treatment; and (iv) instructions for use. In some embodiments, at least one device may include a relatively low microneedle density (e.g., about 2 microneedles / cm²). 2 Approximately 50 microneedles / cm 2 Microneedles (within the range of...). In some embodiments, for example, at least one device may include microneedles with a relatively small microneedle puncture size (e.g., the puncture size of each microneedle is about 100 μm). 2 / Microneedles approximately 30,000 μm 2 Within the microneedle range, the puncture size of each microneedle is approximately 100 μm. 2 / Microneedles up to 60,000 μm 2 Microneedles within the range of microneedles.

[0270] The present invention also includes the following embodiments:

[0271] 1. A method for treating a subject in need of treatment, the method comprising the following steps:

[0272] According to the dosing regimen, multiple doses of a composition are administered to the subject's skin site in conjunction with microneedle skin conditioning (MSC), the composition delivering a large dose with a molecular weight of 100,000 Da or greater, the dosing regimen comprising at least two consecutive doses spaced at least one month apart.

[0273] 2. The method according to implementation plan 1, wherein the time period is at least two months.

[0274] 3. The method according to implementation plan 1, wherein the time period is at least four months.

[0275] 4. The method according to implementation plan 1, wherein the time period is at least six months.

[0276] 5. The method according to implementation plan 1, wherein the time period is at least eight months.

[0277] 6. The method according to implementation scheme 1, wherein the time period is at least ten months.

[0278] 7. The method according to implementation scheme 1, wherein the time period is at least twelve months.

[0279] 8. The method according to embodiment 1, wherein the dosing regimen comprises administering at least three doses, separated by at least first and second time periods, wherein the average time period is at least one month.

[0280] 9. The method according to implementation scheme 8, wherein the average time period is at least two months.

[0281] 10. The method according to embodiment 8, wherein the average time period is at least four months.

[0282] 11. The method according to embodiment 8, wherein the average time period is at least six months.

[0283] 12. The method according to embodiment 8, wherein the average time period is at least eight months.

[0284] 13. The method according to implementation scheme 8, wherein the average time period is at least ten months.

[0285] 14. The method according to embodiment 8, wherein the average time period is at least twelve months.

[0286] 15. The method according to implementation scheme 8, wherein each of the time periods is the same.

[0287] 16. The method according to embodiment 15, wherein each of the time periods is at least one month.

[0288] 17. The method according to implementation scheme 15, wherein each of the time periods is at least two months.

[0289] 18. The method according to implementation scheme 15, wherein each of the time periods is at least four months.

[0290] 19. The method according to embodiment 15, wherein each of the time periods is at least six months.

[0291] 20. The method according to embodiment 15, wherein each of the time periods is at least eight months.

[0292] 21. The method according to embodiment 15, wherein each of the time periods is at least ten months.

[0293] 22. The method according to embodiment 15, wherein each of the time periods is at least twelve months.

[0294] 23. The method according to any one of the foregoing embodiments, wherein the application comprises applying the composition locally to the site.

[0295] 24. The method according to any one of the foregoing embodiments, wherein the composition comprises a nanoemulsion.

[0296] 25. The method according to any one of the foregoing embodiments, wherein the composition comprises a crude emulsion.

[0297] 26. The method according to any one of the foregoing embodiments further includes the step of applying a non-irritating penetration enhancer.

[0298] 27. The method according to embodiment 26, wherein the non-irritating permeability enhancer is selected from carrier peptides and co-peptides.

[0299] 28. The method according to any one of embodiments 26 to 27, wherein the non-irritating penetration enhancer is selected from cationic peptides and peptides having the sequence RKKRRQRRRG-(K). 15 -GRKKRRQRRR is a positively charged carrier.

[0300] 29. The method according to any one of the foregoing embodiments, wherein the application step includes performing the MSC prior to applying the composition to the site.

[0301] 30. The method according to any one of the foregoing embodiments, wherein the application step includes performing the MSC after applying the composition to the site.

[0302] 31. The method according to any one of the foregoing embodiments, wherein the application step includes performing the MSC while applying the composition to the site.

[0303] 32. The method according to any one of the foregoing embodiments, wherein the large agent is botulinum toxin.

[0304] 33. The method according to embodiment 32, wherein the application step is further combined with a cosmetic or therapeutic agent.

[0305] 34. The method according to embodiment 33, wherein the cosmetic agent or therapeutic agent is selected from the group consisting of anesthetics, collagen, fillers, retinoids, silicones, steroids, and combinations thereof.

[0306] 35. The method according to embodiment 34, wherein the cosmetic agent or therapeutic agent is selected from the group consisting of hydrocortisone, visual pigment A, lidocaine, and combinations thereof.

[0307] 36. The method according to any one of the foregoing embodiments, wherein the large agent is an antibody agent.

[0308] 37. The method according to embodiment 36, wherein the antibody agent is selected from the group consisting of anti-TNFα antibody, anti-CD2 antibody, anti-CD4 antibody, anti-IL-12 antibody, anti-IL-17 antibody, anti-IL-22 antibody and anti-IL-23 antibody.

[0309] 38. The method according to any one of embodiments 36 to 37, wherein the antibody agent is selected from the group consisting of antibodies having an epitope binding element found in one or more of the following: infliximab, adalimumab, golimumab, etanercept, etanercept-szzs, pegylated cetrus, ciplelizumab, zamumab, brenumab, secukinumab, brodatumab, non-zanumab, uteroclosab, and / or gusekumab.

[0310] 39. The method according to any one of embodiments 36 to 38, wherein the application step is further combined with a cosmetic or therapeutic agent.

[0311] 40. The method according to any one of embodiments 36 to 39 further includes the step of applying a non-irritating penetration enhancer.

[0312] 41. The method according to embodiment 40, wherein the non-irritating penetration enhancer is selected from conopeptides and carrier peptides.

[0313] 42. The method according to any one of the foregoing embodiments, wherein the MSC is performed using a device comprising at least one microneedle (MN).

[0314] 43. The method according to embodiment 42, wherein the device comprises a plurality of MNs.

[0315] 44. The method according to embodiment 42 or 43, wherein the device is a patch, roller, mold, or pen.

[0316] 45. The method according to any one of the foregoing embodiments, wherein the site is the skin surface covering the muscle or muscle group.

[0317] 46. ​​The method according to any one of the foregoing embodiments, wherein the site is the skin surface containing sweat glands.

[0318] 47. The method according to any one of the foregoing embodiments, wherein the site is the skin surface containing sebaceous glands.

[0319] 48. The method according to any one of the foregoing embodiments, wherein the site is the skin surface containing hair follicles.

[0320] 49. The method according to any one of embodiments 42 to 47, wherein the MN has a length sufficient to penetrate the stratum corneum of the skin.

[0321] 50. The method according to any one of embodiments 42 to 49, wherein the MN has a length insufficient to reach the nerves in the dermis of the skin.

[0322] 51. The method according to any one of embodiments 42 to 50, wherein the MN is composed of a biocompatible material.

[0323] 52. The method according to any one of embodiments 42 to 51, wherein the MN is made of metal.

[0324] 53. The method according to any one of embodiments 42 to 51, wherein the MN is composed of at least one soluble polymer.

[0325] 54. The method according to any one of the foregoing embodiments, wherein the MSC comprises applying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 microneedle (MN) imprints, wherein each imprint is performed during a period of continuous contact between the site and a device comprising one or more MNs.

[0326] 55. The method according to embodiment 54, wherein the device is or includes an impression, the impression including the one or more MNs.

[0327] 56. The method according to embodiment 54, wherein the device is or includes a roller, the roller comprising the one or more MNs.

[0328] 57. The method according to embodiment 54, wherein the device is or includes a patch, the patch including the one or more MNs.

[0329] 58. The method according to any one of embodiments 54 to 57, wherein the device comprises a plurality of MNs, and the plurality of MNs are arranged in a geometric pattern.

[0330] 59. The method according to embodiment 54, wherein the MSC includes applying multiple imprints.

[0331] 60. The method according to embodiment 59, wherein the two or more imprints are performed on substantially the same location.

[0332] 61. The method according to any one of embodiments 59, wherein the two or more imprintings are performed on overlapping portions.

[0333] 62. The method according to any one of embodiments 54 to 59, wherein individual imprints are performed on different locations.

[0334] 63. The method according to any one of embodiments 54 to 62, wherein the embossing is performed by stamping.

[0335] 64. The method according to any one of embodiments 54 to 62, wherein the imprinting is performed by roller pressing.

[0336] 65. The method according to any one of the foregoing embodiments, wherein the application step delivers the bulk agent to the skin within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

[0337] 66. The method according to any one of the foregoing embodiments, wherein the application step delivers the bulk agent to the skin in about 5 to about 60 minutes, about 5 to about 12 minutes, about 5 to about 15 minutes, or about 15 to about 30 minutes.

[0338] 67. The method according to any one of the foregoing embodiments, wherein the application step delivers the large dose to the skin within approximately 1, 2, 3, 4, 5, or 6 hours.

[0339] 68. A method for treating skin diseases, conditions, or symptoms, comprising the method according to any one of the foregoing embodiments.

[0340] 69. The method according to any one of the foregoing embodiments, wherein the subject suffers from or is susceptible to a skin disease, condition, or symptom, and the application achieves improvement of one or more features or symptoms of the skin disease, condition, or symptom.

[0341] 70. The method according to embodiment 68 or 69, wherein the skin disease, condition or symptom is selected from acne, actinic keratosis, body odor, bromhidrosis, burns, chromhidrosis, skin infections, eczematous dermatitis, conditions of excessive sebum secretion, facial wrinkles, hair loss, hyperactive facial lines, hyperhidrosis, hyperactive facial lines, hyperpigmentation, hypopigmentation, keloids, linear scleroderma, lupus erythematosus, neck wrinkles, platysma band, psoriasis, Raynaud's syndrome, rosacea, scleroderma, skin cancer, unsightly facial expressions, unwanted sweating and / or combinations thereof.

[0342] 71. A method for treating or preventing diseases, conditions, or symptoms selected from: acne (e.g., which may be associated with excessive sebum secretion, infection, etc.), amyloidosis (e.g., cutaneous amyloidosis), asthma, body odor, burns, cancers (e.g., leukemia, breast cancer, colon cancer, lung cancer, skin cancer), sun-related diseases (e.g., actinic keratosis, sunburn, skin cancers such as melanoma, etc.), inflammatory conditions (e.g., chronic obstructive pulmonary disease, Crohn's disease), sweating conditions (e.g., bromhidrosis, chromhidrosis, skin infections, discoid lupus, drug-induced lupus, eczematous dermatitis, conditions of excessive sebum secretion, facial wrinkles, hair loss, hyperactive facial lines, hyperhidrosis, hyperactive facial lines, hyperpigmentation, hyperpigmentation, hyperplasia (e.g., benign prostatic hyperplasia), hypopigmentation, inflammatory bowel disease, keloids, linear scleroderma, lupus erythematosus, neck wrinkles, neonatal lupus, osteoarthritis). Arthritis, platysma, psoriasis, psoriatic arthritis, lung disease, Raynaud's syndrome, rheumatoid arthritis, rosacea, scleroderma, skin cancer, systemic amyloidosis, systemic lupus erythematosus, ulcerative colitis, unsightly facial expressions, unwanted sweating, dyslipidemia, hypercholesterolemia, infection, Clostridium difficile infection, Staphylococcus aureus infection, dystonia, headache, pain, arthritis-related pain, rheumatoid arthritis-related pain, psoriatic arthritis-related pain, osteoarthritis-related pain, certain ophthalmic conditions, certain urinary tract conditions, neuromuscular disorders, conditions involving muscle spasms and / or contractures, strabismus, hemifacial spasm, tremor, spasms such as those caused by multiple sclerosis, retro-orbital muscle, neuropathies, migraines or other headaches, Alzheimer's disease, Parkinson's disease, or stroke, and / or combinations thereof, the method comprising the method according to any one of the foregoing embodiments.

[0343] 72. The method according to any one of the foregoing embodiments, wherein the composition is formulated as a lotion, cream, powder, ointment, liniment, gel or drops.

[0344] 73. A method for treating a subject in need of treatment, the method comprising the following steps:

[0345] According to the dosing regimen, the composition is administered to the skin site of the subject in conjunction with microneedle skin conditioning (MSC), the composition delivering a large dose with a molecular weight of 100,000 Da or greater, the dosing regimen such that the peak effect of the large dose is reached no earlier than about 1 month after administration. Example

[0346] Example 1 The impact of MSC preconditioning on the peak treatment time and duration of treatment effect in reducing crow's feet wrinkles.

[0347] This embodiment describes a study that evaluated the time to peak therapeutic effect and the duration of therapeutic effect of topical botulinum toxin combined with microneedle skin preconditioning (in the form of a nanoemulsion formulation) in reducing crow's feet.

[0348] The study included two test groups, Group A and Group B, in which participants had moderate to severe crow's feet. Each participant's crow's feet area was treated once topically with a botulinum toxin emulsion (e.g., nanoemulsion) formulation (Group A) or its placebo control (Group B). Participants in both Group A (N=9) and Group B (N=9) received skin preconditioning using the same matched microneedle array. The application of the topical formulation to the skin took approximately 5 minutes, during which time it was fully absorbed. Prior to the application of the botulinum toxin formulation, all participants underwent preconditioning with eight microneedle imprints from the microneedle array.

[0349] The expected effect of botulinum toxin treatment is to reduce crow's feet at the treated area. The severity of wrinkles was measured by each researcher and subject using a five-point wrinkle scale (Wrinkle Scale): 0 = none, 1 = very few, 2 = mild, 3 = moderate, 4 = severe. As is generally accepted in the art, subjects were considered "responders" for peak effect purposes when both researchers and subjects assessed a reduction in wrinkle severity at contraction relative to baseline of two or more points; subjects were considered "responders" for effect duration purposes when researchers assessed a reduction in wrinkle severity at contraction relative to baseline of one or more points.

[0350] The study described in this embodiment found that, at baseline, the average severity of crow's feet measured by the wrinkle scale was approximately the same in groups A and B.

[0351] Regarding peak effect, at one month after treatment, the response rate in group A was 11% and the response rate in group B was 0%; at two months after treatment, the response rate in group A was 11% and the response rate in group B was 0%; and at three months after treatment, the response rate in group A was 33% and the response rate in group B was 0%.

[0352] Regarding the duration of treatment effect, the response rate in group A was 56% at approximately 6 months post-treatment. In contrast, literature reports on the use of commercially available botulinum toxin injections for crow's feet describe a response rate of 15% at approximately six months using the same responder metrics; other studies have found a median duration of treatment effect for wrinkles using commercially available botulinum toxin to 3.5 to 4 months.

[0353] Figure 1This study presents a comparison of results (particularly the observed response rates) achieved in Group A at specific time points (particularly at weeks 18 and 26) with those of approved botulinum toxin injection products administered according to their approved treatment regimens for crow's feet, compared to literature reports. It can be seen that at week 18, the response rate for the injections was less than 50%, while the response rate for the technique described herein was greater than 80%. Furthermore, the response rate achieved by the presented technique at week 26 was higher than that of the injections at week 18 (i.e., greater than 70%).

[0354] The study described in this embodiment documents an unexpected result that the topical application of botulinum toxin (e.g., in the form of a nanoemulsion formulation) combined with microneedling skin pretreatment can increase the time to peak therapeutic effect and further increase the duration of botulinum toxin therapy (e.g., for wrinkles).

[0355] Example 2 The impact of MSC preconditioning on the time to peak therapeutic effect compared to commercially available injectable botulinum toxin.

[0356] This embodiment describes a study that compares the time to peak treatment effect of topical application (in nanoemulsion formulation) of botulinum toxin with microneedling skin preconditioning as described herein to a report describing the time to peak treatment effect of injectable botulinum toxin on reducing crow's feet.

[0357] As described above, Group A (N=9) of subjects with moderate to severe crow's feet received a single topical treatment with botulinum toxin emulsion (combined with skin preconditioning with a microneedling array, specifically, eight microneedling impressions of the microneedling array prior to the application of the botulinum toxin preparation). The application of the topical preparation to the skin takes approximately 5 minutes, during which time the preparation is fully absorbed into the skin.

[0358] The results of this study were compared with literature reports describing treatments received by subjects who had previously received approved injectable botulinum toxin therapies. Specifically, group B (N=48) was treated with a first commercially available injectable botulinum toxin, and group C (N=222) was treated with a different second commercially available injectable botulinum toxin.

[0359] Figure 2The results of these studies were compared. The expected effect of botulinum toxin treatment is to reduce crow's feet at the treatment site. For group A, the severity of wrinkles was measured by each researcher using a five-point wrinkle scale (Wrinkle Scale): 0 = none, 1 = very few, 2 = mild, 3 = moderate, 4 = severe. For groups B and C, the severity of wrinkles was measured by each researcher using a four-point wrinkle scale (Wrinkle Scale): 0 = none, 1 = mild, 2 = moderate, 3 = severe. When researchers assessed wrinkle scores as mild or a more favorable score (e.g., none), subjects were considered "responders" for peak effect purposes.

[0360] Reference Figure 2 It can be seen that all three therapies ultimately achieved comparable peak effects; therefore, those skilled in the art will understand that although they may involve different absolute “dosages” included in a single dose administered (whether considered in units of botulinum toxin and / or amounts of botulinum toxin [e.g. ng]), their relative regimens can be considered comparable.

[0361] In terms of peak effect, Figure 2 The response rates were recorded as follows: one month after treatment, Group A had a response rate of 44%, Group B had a response rate of 60%, and Group C had a response rate of 67%; two months after treatment, the response rates were 44% for Group A, 54% for Group B, and 56% for Group C; and three months after treatment, the response rates were 56% for Group A, 30% for Group B, and 39% for Group C. See also Figure 1 Peak effects were observed in Group A at time points within three months following the administration of botulinum toxin; peak effects were observed in Groups B and C one month after administration.

[0362] The study described in this embodiment documents an unexpected result: topical application of botulinum toxin (e.g., in the form of a nanoemulsion) combined with microneedling skin pretreatment can increase the time to peak therapeutic effect. As will be understood by those skilled in the art, this demonstration of a delayed peak effect also indicates an extension of the duration of the response.

[0363] Example 3 The effect of MSC preconditioning on the time to peak therapeutic effect compared to local treatment without MSC preconditioning.

[0364] This embodiment describes a study that evaluated the time to peak treatment effect of topical botulinum toxin combined with microneedling skin preconditioning (in nanoemulsion form) on reducing crow's feet compared to a group receiving topical botulinum toxin treatment without microneedling skin preconditioning (in nanoemulsion form).

[0365] The study included two test groups, Group A and Group B, in which participants had moderate to severe crow's feet. Each participant's crow's feet area was treated once topically with a comparable dose of botulinum toxin emulsion (e.g., nanoemulsion). Participants in Group A (N=26) did not receive microneedling skin pre-treatment, while participants in Group B (N=9) received skin pre-treatment using a microneedling array. The application of the topical formulation to the skin took approximately 5 minutes, during which time it was fully absorbed.

[0366] The expected outcome of botulinum toxin treatment is to reduce crow's feet at the treated area. The severity of wrinkles was measured by each researcher and subject using a five-point wrinkle scale (Wrinkle Scale): 0 = none, 1 = very few, 2 = mild, 3 = moderate, 4 = severe. As is generally accepted in the art, subjects were considered "responders" for peak effect purposes when researchers assessed a reduction in wrinkle severity at contraction of two or more points relative to baseline.

[0367] The study found that Group A reached its peak response rate one month after treatment, while Group B reached its peak response rate three months after treatment.

[0368] The study described in this embodiment documents an unexpected result: that topical application of botulinum toxin in the form of a nanoemulsion formulation, in conjunction with microneedling skin preconditioning, can increase the time to peak therapeutic effect compared to topical application of botulinum toxin in the form of a nanoemulsion formulation without microneedling skin preconditioning.

[0369] Example 4 The effect of needle length on peak effect time

[0370] U.S. Patent Application No. 62 / 808,274 describes a single-dose topical study of the bioavailability of botulinum toxin in the human body following a topical application of a botulinum toxin nanoemulsion formulation in conjunction with microneedling dermatology. The study tested the effect of varying microneedle length on enhancing botulinum toxin bioavailability in the human body by measuring the reduction in wrinkles after topical treatment with this formulation following microneedling dermatology.

[0371] The study included three test groups: Group A, Group B, and Group C, which included subjects with moderate to severe crow's feet. Each subject's crow's feet area was treated once with a topical botulinum toxin formulation, specifically a nanoemulsion. Subjects in Group A (N=9) received a 500μm needle pretreatment, those in Group B (N=9) received an 800μm needle pretreatment, and those in Group C (N=9) received a 1400μm needle pretreatment.

[0372] In this specific study, the topical formulation was applied to the skin over approximately 5 minutes, during which time it was fully absorbed. Prior to application of the botulinum toxin formulation, all subjects underwent pretreatment with the same number of microneedle imprints from a microneedle array. The botulinum toxin dosage used in groups A, B, and C was perfectly matched among the subjects.

[0373] The expected effect of botulinum toxin nanoemulsion treatment is to reduce crow's feet at the treatment site; this reduction was measured against the different treatment methods used in this example. The severity of wrinkles was measured by each researcher and subject using a five-point wrinkle scale (Wrinkle Scale): 0 = none, 1 = very few, 2 = mild, 3 = moderate, 4 = severe. Respondents in this study were those assessed by researchers and subjects as having a reduction in wrinkle severity of two or more levels compared to baseline.

[0374] This study found that at baseline, the average severity of crow's feet, as measured by a wrinkle scale, was roughly equal in groups A, B, and C. At twelve weeks post-treatment, the response rate was 36% in group A, 14% in group B, and 13% in group C. This study determined that using shorter microneedles during microneedling skin pretreatment unexpectedly increased the bioavailability of topical macropharmaceutical nanoemulsions, especially those containing botulinum toxin.

[0375] This embodiment further establishes that using such shorter microneedles surprisingly prolongs the peak effect compared to observations using longer microneedles. Without this disclosure, it would be reasonable to expect that microneedle length would have no effect on the duration of the peak effect, or if it did, that effect would be to accelerate (rather than delay) the peak effect.

[0376] The above studies were conducted using shorter (e.g., 500 μm long) microneedles and longer (e.g., 1500 μm long) microneedles. The peak effect was observed to be significantly delayed (e.g., approximately 4–8 weeks later) using the shorter microneedles.

[0377] Example 5 Exemplary emulsion compositions

[0378] In some implementations, an exemplary crude emulsion may be:

[0379] Table 1: Exemplary crude emulsion formulations

[0380] 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 Sodium hydrogen phosphate 0.04 0.04 gelatin 0.02 0.02 Large-volume drugs (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

[0381] *In view of this specification, those skilled in the art can make reasonable adjustments to this and other components based on the volume, weight and / or dosage of the large pharmaceutical preparation to be used.

[0382] In some embodiments, the nanoemulsion may be prepared from a premixed composition (e.g., by applying shear force to it, such as through microfluidization, which in some embodiments may be single-pass microfluidization). In some embodiments, exemplary premixed compositions may include:

[0383] Table 2. Exemplary Premixes

[0384] %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* ** Large doses 100 total

[0385] *The buffer solution contains (w / w) 0.199% gelatin, 0.398% disodium hydrogen phosphate, and 99.4% purified water, with the pH adjusted to 6.0 ± 0.2 using hydrochloric acid.

[0386] Table 3 provides exemplary nanoemulsion formulations, which are not intended to be limiting.

[0387] Table 3: Exemplary Nanoemulsion Formulations

[0388]

[0389]

[0390] *In view of this specification, those skilled in the art can make reasonable adjustments to this and other components based on the volume, weight and / or dosage of the large pharmaceutical preparation to be used.

[0391] Table 4 provides exemplary formulations of botulinum toxin nanoemulsion premixes, which are not intended to limit the scope of the formulation.

[0392] Table 4. Exemplary Botulinum Toxin Nanoemulsion Formulations (Premixes)

[0393]

[0394] *The buffer solution contains (w / w) 0.199% gelatin, 0.398% disodium hydrogen phosphate, and 99.4% purified water, with the pH adjusted to 6.0 ± 0.2 using hydrochloric acid.

[0395] Equivalence

[0396] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. The scope of the invention is not intended to be limited to the foregoing description, but rather is set forth in the claims.

Claims

1. A method for treating a subject in need of treatment, the method comprising the following steps: According to the dosing regimen, multiple doses of a composition are administered to the subject's skin site in conjunction with microneedle skin conditioning (MSC), the composition delivering a large dose with a molecular weight of 100,000 Da or greater, the dosing regimen comprising at least two consecutive doses spaced at least one month apart.

2. The method according to claim 1, wherein the time period is at least two months.

3. The method according to claim 1, wherein the time period is at least four months.

4. The method according to claim 1, wherein the time period is at least six months.

5. The method according to claim 1, wherein the time period is at least eight months.

6. The method of claim 1, wherein the time period is at least ten months.

7. The method of claim 1, wherein the time period is at least twelve months.

8. The method of claim 1, wherein the dosing regimen comprises administering at least three doses, separated by at least first and second time periods, wherein the average time period is at least one month.

9. The method of claim 8, wherein the average time period is at least two months.

10. The method of claim 8, wherein the average time period is at least four months.

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