Delaying peak effect and / or extending duration of response

Abstract

To provide a method for treating a subject in need of treatment, the method comprising administering a large agent (for example, a biologically active large agent, such as a biologic therapeutic, for example, botulinum toxin) to the subject.SOLUTION: A method of treating a subject in need of treatment is provided, the method comprising a step of administering, in combination with microneedle skin conditioning (MSC), a plurality of doses of a composition that delivers a large agent having a molecular weight of 100,000 Da or greater to a site on skin of the subject according to a dosing regimen in which at least two successive doses are separated from one another by a time period of at least one month.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 62 / 847,901, filed May 14, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Significant resources are being invested in developing technologies for the administration of large drugs (eg, large bioactive drugs). Summary of the Invention

[0003] The present disclosure describes techniques for administering large drugs (e.g., biological therapeutics, including bioactive large drugs, e.g., botulinum toxin) to subjects, and describes unexpected results achieved by such techniques, including, for example, delayed peak effect and / or prolonged duration of response. In some embodiments, such unexpected results are achieved compared to administering the same large drug (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 be aware of relevant (e.g., appropriately comparable) reference regimens, including regimens for approved products described herein. Those skilled in the art will also understand that, in some embodiments, an appropriate reference regimen is one that achieves a peak effect of comparable magnitude to that of the provided regimen, and will be familiar with comparing regimens (and / or the effects of the regimens), which may include administering the relevant large drug (e.g., botulinum toxin) via different formulations, different doses, different administration frequencies, different administration intervals, different administration routes, etc. By way of example, one skilled in the art will know that individual doses of parenterally administered botulinum toxin are typically much smaller (i.e., contain much fewer units of botulinum toxin) than individual doses of topically administered botulinum toxin (this is because topical administration of any active ingredient typically delivers only a small portion of the active ingredient to the skin, while parenteral administration delivers 100% to the skin); nevertheless, one skilled in the art will be able to evaluate comparable parenteral versus topical regimens and compare parameters (e.g., one or more outcome parameters) relevant to the present disclosure, such as timing of peak effect and duration of effect, based, for example, on the biological effect of the drug (e.g., reduction in facial wrinkles at maximum contraction as measured by a physician using a wrinkle rating scale).

[0004] In particular, the present disclosure identifies the causes of certain prior art problems related to the administration of large drugs, particularly botulinum toxin, including the fact that many such techniques are designed to achieve rapid onset and / or rapid peak effect. In particular, the present disclosure teaches that in certain situations (including, for example, those involving many subjects undergoing wrinkle treatment), delayed onset and / or delayed peak effect may be desirable and / or beneficial. The present disclosure also teaches that the provided techniques can achieve surprising and unexpected delayed onset and / or delayed peak effect.

[0005] Furthermore, the present disclosure demonstrates that the provided technology can achieve surprising and unexpected extended duration of effect. In particular, the present disclosure recognizes that such extended duration of effect can have a variety of beneficial effects, including, for example, allowing for less frequent administration, which may provide greater convenience (e.g., fewer visits to a doctor's office or clinic) and / or cost savings.

[0006] The technology provided herein includes transdermal delivery technology. Those skilled in the art are well aware of the challenges involved in achieving effective transdermal delivery, especially of large drugs. It is generally understood that as molecular size increases, transdermal penetration decreases until it becomes negligible or non-existent. The present disclosure provides specific technology in which topical application of large drug formulations (e.g., nanoemulsion formulations) in combination with microneedle skin conditioning (e.g., preconditioning) achieves surprising and unexpected results, which in some embodiments may include one or both of delayed peak effect and / or extended duration of response. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows a bar graph comparing the responder rates observed with the provided techniques compared to approved botulinum toxin injection treatments, illustrating, for example, the extended duration of response achieved with the provided techniques described herein.

[0008] [Figure 2] FIG. 2 shows a line graph comparing the timing of peak effect for two different approved botulinum toxin injection treatments provided with topical + MSC treatment, e.g., demonstrating the delayed peak effect achieved by the provided technology described herein.

[0009] (definition) In this application, unless otherwise clear from the context, (i) as used herein, the term "a" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising" and "including" may be understood to encompass the listed elements or steps, whether presented by themselves or with one or more additional elements or steps; and (iv) the terms "about" and "approximately" may be understood to allow for standard variations understood by those of ordinary skill in the art; and (v) when ranges are provided, the endpoints are included.

[0010] Abrasion: As used herein, the term "abrasion" refers to any means that alters, destroys, removes, or disrupts the top layer of skin. In some embodiments, abrasion refers to mechanical means that alters, destroys, removes, or disrupts the top layer of skin. In some embodiments, abrasion refers to chemical means that alters, destroys, removes, or disrupts the top layer of skin. To name a few, agents such as scrubs, microparticles (e.g., magnesium or aluminum particles), acids (e.g., alpha hydroxy acids or beta hydroxy acids), and alcohol can cause abrasion. In general, penetration enhancers, such as those described by Donovan (e.g., U.S. Patent Application Publication Nos. 2004 / 009180 and 2005 / 175636, and WO 04 / 06954), and Graham (e.g., U.S. Patent Application Publication No. 6,939,852 and U.S. Patent Application Publication No. 2006 / 093624), are expected to cause abrasion. Of course, those skilled in the art will understand that a certain drug may cause abrasion when present at one concentration or in combination with one or more other drugs, but may not cause abrasion under different conditions.Therefore, whether a certain substance is an "abrasive" depends on the situation.Abrasion can be easily evaluated by those skilled in the art, for example, by observing redness or irritation of the skin and / or histological examination of the skin, which indicates the change, destruction, removal or erosion of the stratum corneum.

[0011] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system. Those skilled in the art will be aware of various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be parenteral; in some embodiments, administration may be topical. In some embodiments, administration may include intermittent (e.g., multiple administrations separated by time) and / or periodic (e.g., individual administrations separated by a common period of time) administration. In some embodiments, administration may include continuous administration for at least a selected period of time.

[0012] Drug: Generally, as used herein, the term "drug" can be used to refer to any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations or complexes thereof. In appropriate circumstances, as will be clear to those skilled in the art from the context, the term can be used to refer to an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as the context will dictate, the term can be used to refer to a natural product, in that it is found in nature and / or obtained from nature. In some cases, again as will be clear from the context, the term can be used to refer to one or more entities that are artificial, in that they are designed, engineered, and / or produced by the action of man and / or are not found in nature. In some embodiments, the drug can be used in an isolated or pure form; in some embodiments, the drug can be used in a crude form. In some embodiments, potential drugs can be provided, for example, as a collection or library that can be screened to identify or characterize active substances therein. In some cases, the term may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymer molecules. In some embodiments, the term "drug" may refer to a compound or entity that is not a polymer and / or does not substantially comprise any polymer and / or one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or does not substantially comprise any polymer moieties. In some embodiments, the term may refer to a molecular complex. In many embodiments described herein, the drug may be a large drug (e.g., a large biologically active drug, a biological therapeutic agent, such as botulinum toxin).

[0013] Antibody: As used herein, the term "antibody" refers to a polypeptide containing sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, naturally occurring intact antibodies are approximately 150 kDa tetrameric entities composed of two identical heavy chain polypeptides (approximately 50 kDa each) and two identical light chain polypeptides (approximately 25 kDa each) that bind to one another in what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains (each approximately 110 amino acids long): an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains, CH1, CH2, and carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the variable and constant regions of the heavy chain. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Disulfide bonds in two of the hinge regions connect the two heavy chain polypeptides to one another in intact antibodies. Each light chain consists of two domains—an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch." An intact antibody tetramer consists of two heavy-light chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions, thereby connecting the dimers to each other and forming a tetramer. Naturally produced antibodies are also typically glycosylated in the CH2 domain. Each domain of a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., a three-, four-, or five-stranded sheet) packed together within a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complement-determining regions" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a native antibody folds, the FR regions form beta sheets that provide the structural framework for the domain, and the CDR loop regions of both the heavy and light chains are brought together in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure.The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding properties of the Fc region for an Fc receptor can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present invention comprise a glycosylated Fc domain, including Fc domains with modified or engineered glycosylation. For purposes of the present invention, in some embodiments, any polypeptide or polypeptide complex comprising a sufficient immunoglobulin domain sequence as found in a natural antibody can be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., by an organism responding to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody," as used herein, can refer, in appropriate embodiments (unless otherwise specified or clear from the context), to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in surrogate presentation. For example, in embodiments, antibodies utilized in accordance with the present invention include intact IgG, IgE, and IgM, bi- or multispecific antibodies (e.g., Zybodies®), single-chain Fv, Fc fusions of polypeptides, Fab, camelid antibodies, masked antibodies (e.g., Probodies®), etc. S mall M odd I mmuno P harmaceutical ("SMIP TM"), single chain or tandem diabody antibodies (TandAb®), VHH, Anticalin®, Nanobody®, minibody, BiTE®, ankyrin repeat proteins or DARPIN®, Avimer®, DaRT, TCR-like antibodies, Adnectin®, Affilin®, Trans-body®, Affibody®, TrimerX®, MicroProtein, Fynomer®, Centyrin®, and KALBITOR®. In some embodiments, the antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally (e.g., in a mammalian organism). In some embodiments, the antibody may include a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.)).

[0014] Antibody drug: As used herein, the term "antibody drug" refers to a drug that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody drugs include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused with other proteins, radiolabels, or cytotoxins), S mall M odd I mmuno P harmaceutical ("SMIP TM"), single chain antibodies, camelid antibodies, and antibody fragments. As used herein, the term "antibody drug" 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, so long as they exhibit the desired biological activity. In some embodiments, the term encompasses stapled peptides. In some embodiments, the term encompasses one or more antibody-like binding peptidomimetics. In some embodiments, the term encompasses one or more antibody-like binding scaffold proteins. In some embodiments, the term encompasses monobodies or adnectins. In many embodiments, an antibody drug is or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, an antibody drug is or comprises a polypeptide whose amino acid sequence comprises at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are identical in sequence or contain one to five amino acid substitutions compared to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they 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 CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid in the included CDR has been deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR.In some embodiments, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids within the included CDR have been deleted, added, or substituted relative to the reference CDR, but the amino acid sequence is otherwise identical to that of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid has been substituted relative to the reference CDR, but the amino acid sequence is otherwise identical to that of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids within the included CDR have been deleted, added, or substituted relative to the reference CDR, but the amino acid sequence is otherwise identical to that of the reference CDR. In some embodiments, the antibody drug is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody drug is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody drug is or comprises an antibody-drug conjugate.

[0015] Antibody component: As used herein, an antibody component refers to a polypeptide component (which may be a complete polypeptide or a portion of a larger polypeptide, such as, for example, a fusion polypeptide described herein) that specifically binds to an epitope or antigen and comprises one or more immunoglobulin structural features. Generally, an antibody component is any polypeptide whose amino acid sequence comprises elements characteristic of an antibody binding region (e.g., an antibody light chain or variable region or one or more complementarity-determining regions ("CDRs") thereof, or an antibody heavy chain or variable region or one or more CDRs thereof, optionally in the presence of one or more framework regions). In some embodiments, an antibody component is or comprises a full-length antibody. In some embodiments, an antibody component is short but full-length and comprises at least one binding site (comprising at least one, preferably at least two, sequences with the structure of a known antibody "variable region"). In some embodiments, the term "antibody component" encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In certain embodiments, the included "antibody component" encompasses a polypeptide having a binding domain that exhibits at least 99% identity to an immunoglobulin binding domain. In some embodiments, an "antibody component" includes any polypeptide having an immunoglobulin-binding domain, e.g., a binding domain that exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity to a reference immunoglobulin-binding domain. An "antibody component" included can have an amino acid sequence identical to that of an antibody (or a portion thereof, e.g., an antigen-binding portion thereof) found in a natural source. An antibody component can be monospecific, bispecific, or multispecific. An antibody component can include structural elements characteristic of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.Such antibody embodiments may also be bispecific, dual specific, or multispecific formats that specifically bind to two or more different antigens. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) V. H , V L , C H 1 and C L (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H (iv) a single-arm V of an antibody; H and V L (v) a Dab fragment containing a single variable domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR). In addition, the V of the two domains of the Fv fragment may be H and V L are encoded by separate genes, but V H and V Lの The domains can be recombinantly linked by synthetic linkers that allow them to be produced as a single protein chain, pairing to form monovalent molecules (known as single-chain Fvs (scFvs); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). In some embodiments, the "antibody component" described herein is or comprises such a single-chain antibody. In some embodiments, the "antibody component" is or comprises a diabody. Diabodies are molecules that contain a V H and V LBivalent, bispecific antibodies are antibodies in which the domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, and thus the domains pair with complementary domains on the other chain, creating two antigen-binding sites (see, e.g., Holliger, P., et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, (1994) Structure 2(12):1121-1123). Such antibody binding moieties are known in the art (Kontermann and Dubel, eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5)). In some embodiments, the antibody component comprises a pair of tandem Fv segments (V) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. H -C H 1-V H -C H 1) (Zapata et al., (1995) Protein Eng. 8(10): 1057-1062; and U.S. Pat. No. 5,641,870). In some embodiments, the antibody component may possess the characteristic components of a chimeric or humanized antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some embodiments, the antibody component may possess the characteristic components of a human antibody.

[0016] Antibody fragment: As used herein, "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains are connected by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of amino acid residues mimicking the hypervariable regions. In many embodiments, an antibody fragment contains sufficient sequence of the parent antibody that it is a fragment that binds to the same antigen as the parent antibody; in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining region (CDR) regions. Antigen-binding fragments of antibodies can be produced by any means. For example, they can be enzymatically or chemically produced by fragmentation of an intact antibody and / or recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, they can be wholly or partially synthetically produced. Antigen-binding fragments of antibodies can optionally include single-chain antibody fragments. Alternatively or additionally, they can include multiple chains linked together, for example, by disulfide bonds. Antigen-binding fragments of antibodies can optionally include multimolecular complexes. Functional antibody fragments typically contain at least about 50 amino acids, more typically at least about 200 amino acids.

[0017] About: As used herein, the term "approximately" or "about," when applied to one or more target values, refers to a value similar to the stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise specified or otherwise clear from the context (e.g., when one or more target values define a sufficiently narrow range, the application of such percentage variations is unnecessary).

[0018] Associated: As used herein, two events or entities are "associated" with each other when the presence, level and / or form of one correlates with that of the other. For example, a particular entity (e.g., polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder or condition when its presence, level and / or form correlates with the incidence of and / or susceptibility to the disease, disorder or condition (e.g., a related population). In some embodiments, two or more entities are physically "associated" with each other when they interact directly or indirectly, and thus are physically close to each other and / or remain physically close to each other. In some embodiments, two or more entities that are physically associated with each other are covalently bound to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently bound to each other, but are non-covalently bound, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0019] Biocompatible: As used herein, the term "biocompatible" refers to a material that does not cause significant harm to living tissue when placed in contact with such tissue, for example, in vivo. In some embodiments, a material is "biocompatible" if it is not toxic to cells. In some embodiments, a material is "biocompatible" if its addition to cells in vitro results in 20% or less cell death and / or if its administration in vivo does not induce significant inflammation or other such adverse effects.

[0020] Biodegradable: As used herein, the term "biodegradable" refers to a material that, when introduced into cells, is broken down (e.g., by cellular mechanisms such as enzymatic degradation, hydrolysis, and / or a combination thereof) into components that the cells can reuse or discard without significant toxic effects to the cells. In some embodiments, the components produced by the degradation of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, biodegradable polymeric materials decompose into their component monomers. In some embodiments, the degradation of biodegradable materials (e.g., including biodegradable polymeric materials) involves hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, the degradation of biodegradable materials (e.g., including biodegradable polymeric materials) involves cleavage of urethane bonds. Exemplary biodegradable polymers are polymers of hydroxy acids, such as lactic acid and glycolic acid, including, but not limited to, poly(hydroxyl acids), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), lactide-caprolactone copolymers, and mixtures and copolymers thereof. Many naturally occurring polymers are also biodegradable, including, for example, proteins, such as albumin, collagen, gelatin, and prolamines, such as zein, and polysaccharides, such as alginic acid, cellulose derivatives, and polyhydroxyalkanoates, such as polyhydroxybutyrate, and mixtures and copolymers thereof. Those skilled in the art will understand or be able to determine when such polymers (e.g., related to the parent polymer by substantially identical structure differing only by the substitution or addition of certain chemical groups, as known in the art) are biocompatible and / or biodegradable derivatives thereof.

[0021] Bioactive substance: As used herein, the term "bioactive substance" refers to a drug that has a specific biological effect when administered to a subject, e.g., a human. In some embodiments, the bioactive substance can be a therapeutic active substance, a cosmetic active substance, and / or a diagnostic active substance. In some embodiments, the bioactive substance can be or include an entity or moiety that would be classified as an "active pharmaceutical ingredient" by the U.S. Food and Drug Administration. In some embodiments, the bioactive substance is a large drug. In some embodiments, the bioactive substance can be or include a drug whose presence correlates with a desired pharmacological and / or therapeutic, cosmetic, and / or diagnostic effect. In some embodiments, the bioactive substance is characterized in that its biological effect is dose-dependent (e.g., increases linearly with increasing dose, optionally over at least a first concentration range).

[0022] Botulinum macroemulsion composition: As used herein, the term "botulinum macroemulsion composition" refers to a macroemulsion composition in which at least one macroemulsion comprises a botulinum toxin. The botulinum toxin may be present within the macroemulsion, on the surface of the macroemulsion, and / or within the micellar membrane that defines the macroemulsion.

[0023] Botulinum nanoemulsion composition: As used herein, the term "botulinum nanoemulsion composition" refers to a nanoemulsion composition in which at least one nanoemulsion comprises a botulinum toxin. The botulinum toxin may be present within the nanoemulsion, on the surface of the nanoemulsion, and / or within the micellar membrane that defines the nanoemulsion.

[0024] Botulinum toxin: As used herein, the term "botulinum toxin" refers to any neurotoxin produced by Clostridium botulinum. Unless otherwise specified, the term encompasses fragments or portions (e.g., light chains and / or heavy chains) of such neurotoxins that retain the appropriate activity (e.g., muscle relaxant activity). As used herein, the term "botulinum toxin" can refer to botulinum toxin or serotypes A, B, C, D, E, F, and G. As will be understood by those skilled in the art, in some embodiments, the term botulinum toxin as used herein can encompass botulinum toxin complexes (i.e., for example, 300, 600, and 900 kDa complexes) or purified (i.e., for example, isolated) botulinum toxin (i.e., for example, about 150 kDa). A "purified botulinum toxin" is understood to be a botulinum toxin that has been isolated or substantially isolated from other proteins to which it may effectively bind, including those involved in the botulinum toxin complex. A purified toxin can be greater than 95% pure, and in some embodiments, greater than 99% pure. Those skilled in the art will understand that the present invention is not limited to a particular source of botulinum toxin. For example, botulinum toxin for use in accordance with the present invention can be isolated from Clostridium botulinum, chemically synthesized, or produced recombinantly (i.e., in a host cell or organism other than Clostridium botulinum). The botulinum toxin may be genetically engineered or chemically modified to act with a longer or shorter duration than botulinum toxin serotype A.

[0025] Carrier: As used herein, carrier refers to the diluent, adjuvant, additive or vehicle (for example, a component of the formulation of the composition) that the composition is administered together with.In some exemplary embodiments, carrier comprises, for example, sterile liquid, such as water and oil, for example, oil of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil etc.In some embodiments, carrier is or comprises one or more solid components.

[0026] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a patient is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents, therapeutic agents and therapeutic modalities, etc.). In some embodiments, two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all "doses" of a first regimen are administered before any dose of a second regimen is administered). In some embodiments, such drugs are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy can include administration of one or more drugs and / or modalities to a subject that is administered in combination with other drugs or modalities. For clarity, in some embodiments, two or more drugs or active portions thereof can be administered in a combination composition or in a combination compound (e.g., as part of a single chemical complex or covalent entity), but combination therapy does not require that the individual drugs be administered together (or even necessarily simultaneously) in a single composition.

[0027] Equivalent: As used herein, the term "equivalent" refers to two or more substances, entities, circumstances, sets of conditions, etc., which may not be identical to one another, but are sufficiently similar to allow comparisons between them, so that one skilled in the art will understand that one can reasonably draw conclusions based on the observed differences or similarities. In some embodiments, an equivalent set of conditions, environments, individuals, or populations is characterized by multiple substantially identical characteristics and one or a few different characteristics. One skilled in the art will understand what level of identity is required in a given environment for two or more such substances, entities, circumstances, sets of conditions, etc. to be considered equivalent in context. For example, one skilled in the art will understand that sets of circumstances, individuals, or populations are equivalent to one another when they are characterized by a sufficient number and variety of substantially identical characteristics to warrant a reasonable conclusion that differences between results obtained or phenomena observed under or with different sets of environments, individuals, or populations are caused by or indicate alterations in these changed characteristics.

[0028] Composition: Those skilled in the art will understand that the term "composition," as used herein, can be used to refer to a separate physical entity that includes one or more specific components. Generally, unless otherwise specified, a composition can be in any form, such as a gas, gel, liquid, solid, etc.

[0029] Comprising: Compositions or methods described herein as "comprising" one or more specified components or steps are open-ended, meaning that the specified components or steps are essential, but that other components or steps may be added within the composition or method. To avoid redundancy, it is understood that any composition or method described as "comprising" (or "comprises") one or more specified components or steps also describes a corresponding, more limited composition or method "consisting essentially of" (or "consist essentially of") the same specified components or steps, meaning that the composition or method includes the specified essential components or steps, and may also include additional components or steps that do not materially affect the basic and novel characteristics of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more specified components or steps also describes a composition or method "consisting of" (or "consist of") the corresponding, more limited, close-ended specified components or steps, excluding other unspecified components or steps. In any composition or method disclosed herein, known or disclosed equivalents of any specified essential component or step may be substituted for that component or step.

[0030] Dosage form or unit dosage form: Those skilled in the art will understand that the term "dosage form" can be used to refer to a physically discrete unit 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 amount is a unit dosage (or a whole fraction thereof) appropriate for administration according to a dosing regimen (i.e., a therapeutic dosing regimen) determined to correlate with a desired or beneficial outcome when administered to a relevant population. Those skilled in the art will understand that the total amount of a therapeutic composition or drug to be administered to a particular subject is determined by one or more physicians and may include the administration of multiple dosage forms.

[0031] Dosage regimen: Those skilled in the art will understand that the term "dosage regimen" can be used to refer to a series of unit doses (typically multiple) that are administered individually to a subject and are typically separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which can include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, the individual doses are separated from each other by periods of the same length; in some embodiments, a dosing regimen includes multiple doses and at least two different periods separating the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dose. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first administration of a first dose, followed by one or more additional administrations of a second dose that is different from the first dose. In some embodiments, a dosing regimen includes a first administration of a first dose, followed by one or more additional administrations of a second dose that is the same as the first dose. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered to an entire relevant population (ie, is a therapeutic dosing regimen).

[0032] Emulsion: The term "emulsion" is used herein consistent with its understanding in the art as "a system consisting of a liquid dispersed, with or without an emulsifier, in an immiscible liquid, usually in droplets greater than colloidal size." See, e.g., Medline Plus Online Medical Dictionary, Merriam-Webster (2005).

[0033] Additive: As used herein, additive refers to a non-therapeutic substance that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical additives include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.

[0034] Human: In some embodiments, the human is an embryo, fetus, infant, child, teenager, adult, or elderly.

[0035] Hydrophilic: As used herein, the terms "hydrophilic" and / or "polar" refer to the tendency to mix with or dissolve readily in water.

[0036] Hydrophobic: As used herein, the terms "hydrophobic" and / or "non-polar" refer to a tendency to repel, immiscible with, or not readily dissolve in water.

[0037] Improved, increased, or decreased: As used herein or its grammatical equivalents, the terms "improved," "increased," or "decreased" refer to a value relative to a baseline measurement, e.g., a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control individual (or control individuals) in the absence of a treatment described herein. In some embodiments, a "control individual" is an individual suffering from the same form of disease or disorder as the individual being treated.

[0038] Macromolecule: The term "macromolecule" is generally used herein to describe a molecule greater than about 100 kilodaltons (KDa) in size. In some embodiments, the macromolecule is greater than about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 250 kDa, about 300 kDa, about 400 kDa, or about 500 kDa. In some embodiments, the macromolecule is a polymer or comprises a polymer moiety or entity. In some embodiments, the macromolecule is or comprises a polypeptide. In some embodiments, the macromolecule is or comprises a nucleic acid.

[0039] Large drug: As used herein, the term "large drug" generally refers to a drug having a molecular weight greater than about 100 kilodaltons (KDa). In some embodiments, the large molecule is greater than about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 250 kDa, about 300 kDa, about 400 kDa, or about 500 kDa. In some embodiments, the large drug is a biologically active substance. In some embodiments, the large drug is or comprises one or more large molecules. In some embodiments, the large drug is or comprises one or more molecular complexes. In some embodiments, the large drug is or comprises a polypeptide. In some embodiments, the large drug is or comprises a polypeptide complex. In some embodiments, the large drug is or comprises a bacterial toxin (e.g., botulinum toxin). In some embodiments, the large drug is or comprises an antibody drug.

[0040] Macroemulsion: As used herein, the term "macroemulsion" refers to an emulsion in which at least a portion of the droplets have diameters ranging from several hundred nanometers to several micrometers. As will be understood by those skilled in the art, macroemulsions are characterized by droplets greater than 300 nm in diameter. In some embodiments, the macroemulsion compositions utilized in accordance with the present disclosure include one or more larger drugs or one or more bioactive substances. In some embodiments, the large drug included in the macroemulsion composition can be a bioactive substance. It will be understood by those skilled in the art that macroemulsion compositions for use in accordance with the present disclosure can be prepared by any available means, including, for example, chemical or mechanical means. In some embodiments, the droplets in the macroemulsion have a size ranging from about 301 nm to about 1000 μm. In some embodiments, the macroemulsion has a droplet size distribution ranging from about 301 nm to about 1000 μm. In some embodiments, the droplets in the macroemulsion have a size ranging from about 500 nm to about 5000 μm. In some embodiments, the macroemulsion has a droplet size distribution of about 500 nm to about 5000 μm.

[0041] Microneedle: As used herein, the term "microneedle" generally refers to an elongated structure of a length, diameter, and shape suitable for penetrating the skin. In some embodiments, the microneedle is positioned and constructed (by itself or within a device) to create an efficient pathway for drug delivery while minimizing contact with nerves when inserted into the skin. In some embodiments, the microneedle has a diameter that is constant along the length of the microneedle. In some embodiments, the microneedle has a diameter that varies along the length of the microneedle. In some embodiments, the microneedle has a diameter that tapers along the length of the microneedle. In some embodiments, the diameter of the microneedle is narrowest at the tip where it penetrates the skin. In some embodiments, the microneedle can be solid. In some embodiments, the microneedle can be hollow. In some embodiments, the microneedle can be tubular. In some embodiments, the microneedle can be sealed at one end. In some embodiments, multiple microneedles are utilized. In some embodiments, multiple microneedles are utilized in an array format. In some embodiments, the microneedles may have a length ranging from about 1 μm to about 4,000 μm. In some embodiments, the microneedles may have a length ranging from about 1 μm to about 2,000 μm. In some embodiments, the microneedles may have a length ranging from about 50 μm to about 400 μm. In some embodiments, the microneedles may have a length ranging from about 800 μm to about 1,500 μm.

[0042] Pressing of a microneedle array: As used herein, the term "pressing of a microneedle array" refers to microneedle pressing achieved by pressing a microneedle and / or a microneedle array onto the skin and then removing it from the skin. In some embodiments, the microneedle array can be stamped onto the skin (e.g., using a microneedle array stamp). In some embodiments, the microneedle array can be rolled onto the skin (e.g., using a microneedle array roller).

[0043] Microneedle density: As used herein, the term "microneedle density" refers to the number of microneedles per area measurement (e.g., square centimeter). In some embodiments, the microneedle density is measured as the number of microneedles per area of the microneedle array; in some embodiments, the microneedle density is measured as the number of microneedle punctures per area of the microneedled site; in some embodiments, the microneedle density is measured as the number of microneedles per area that simultaneously achieves the maximum or near maximum possible skin penetration for the microneedles in the array. In any case, those skilled in the art will understand that the microneedle density can be expressed regardless of whether the relevant area is flat (e.g., a microneedle array stamp), curved (e.g., a microneedle array roller), or irregular. Those skilled in the art will understand that assessing microneedle density as microneedle punctures per area of the microneedled site can be particularly useful, for example, when the array has needles of different lengths and / or when the site to be microneedled has topological diversity such that not all needles are able to actually pierce the skin when the array is applied to the site.

[0044] Microneedle puncture size: As used herein, the term "microneedle puncture size" or "microneedle puncture hole size" refers to the calculated puncture area made by each microneedle of a microneedle array after the microneedle and / or microneedle array is pressed onto the skin and removed from the skin. In many embodiments, the microneedle puncture size is calculated as the area of the base of the microneedle.

[0045] Nanoemulsion: As used herein, the term "nanoemulsion" refers to an emulsion in which at least some droplets have diameters in the nanometer size range. As will be understood by those skilled in the art, nanoemulsions are characterized by droplets with diameters of 300 nm or less. In some embodiments, nanoemulsion compositions utilized in accordance with the present disclosure include one or more large drugs or one or more bioactive substances. In some embodiments, the large drug included in the nanoemulsion composition can be a bioactive substance. It will be understood by those skilled in the art that nanoemulsion compositions for use in accordance with the present 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 within the range of about 1 nm to about 300 nm. In some embodiments, the nanoemulsion has a droplet size distribution of about 1 nm to about 300 nm.

[0046] Nanoparticle: As used herein, the term "nanoparticle" refers to a solid particle having a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, nanoparticles have a diameter of less than 100 nm, as defined by the National Institutes of Health.

[0047] Patient: As used herein, the term "patient" refers to any organism to which a provided composition is or can be administered, for example, for experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals, such as mice, rats, rabbits, non-human mammals, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes the presence of one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular therapy to diagnose and / or treat a disease, disorder, or condition.

[0048] Penetration enhancer: As used herein, the term "penetration enhancer" refers to a substance whose presence or level correlates with increased penetration of a target drug across the skin compared to that observed in its absence. In some embodiments, a penetration enhancer is characterized by its ability to degrade and / or destroy skin structure. In some embodiments, a penetration enhancer is or includes a chemical agent (e.g., a chemical or enzyme). For example, chemical agents that can damage, destroy, and / or decompose one or more components of the stratum corneum can include, for example, alcohols, such as short-chain alcohols, long-chain alcohols, or polyalcohols; amines and amides, such as urea, amino acids or esters thereof, amides, AZONE®, AZONE® derivatives, pyrrolidone or pyrrolidone derivatives; terpenes and terpene derivatives; fatty acids and esters thereof; macrocyclic compounds; surfactants; or sulfoxides (e.g., dimethyl sulfoxide (DMSO), decyl methyl sulfoxide, etc.); surfactants, such as anionic, cationic, and nonionic surfactants; polyols; essential oils; and / or hyaluronidase. In some embodiments, a penetration enhancer may be irritating in that it causes inflammatory and / or allergic reactions when applied to the skin. In some embodiments, a penetration enhancer is not irritating. In some embodiments, a penetration enhancer may be or include a chemical agent that does not damage, destroy, or degrade skin structure, but whose presence or level nevertheless correlates with increased penetration of a target drug across the skin compared to that observed in its absence. In some embodiments, copeptides, carrier molecules, and carrier peptides may be penetration enhancers that do not damage, destroy, and / or degrade skin structure. In some embodiments, copeptides, carrier molecules, and carrier peptides may be penetration enhancers that do not irritate the skin. The term "penetration enhancer" does not include mechanical devices (e.g., needles, scalpels, etc.) or their equivalents (e.g., other damaging procedures).Those skilled in the art will also understand that structures such as nanoparticles or emulsions are not chemical agents and therefore are not chemical penetration enhancers, even though their presence may correlate with improved skin penetration of a target drug that may be associated with the structure.

[0049] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active substance is formulated with one or more pharmaceutically acceptable carriers.In some embodiments, the active substance is present in a unit dose suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.In some embodiments, the pharmaceutical composition can be specially formulated for administration in solid or liquid form, including those adapted for local administration, such as gel, cream, ointment, or controlled-release patch or spray that is applied to the skin, lungs, or oral cavity; for example, as a pessary, cream, or foam that is applied to the vagina or rectum; sublingual; intraocular; transdermal; or applied to the nose, lungs, and other mucous membranes, as a sterile liquid or suspension, or sustained-release formulation.

[0050] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" as applied to carriers, diluents, or excipients used to formulate the compositions described herein, means the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0051] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid excipient, diluent, additive, or solvent encapsulating a material, involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject or patient. Some examples of substances which can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; additives such as cocoa butter and suppository wax; 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 glycerin, 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; ethyl alcohol; pH buffers; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0052] Premix: As used herein, the term "premix" refers to a combination of ingredients that are subsequently used to form an emulsion composition (e.g., a nanoemulsion composition). For example, in some embodiments, a premix is a collection of ingredients that, when subjected to high shear, produces a nanoemulsion useful in accordance with the present disclosure. In some embodiments, a premix is a collection of ingredients that, when subjected to high shear, produces a uniform nanoemulsion composition. A premix often contains a liquid dispersion medium and sufficient other ingredients to produce a nanoemulsion within the dispersion medium. According to some embodiments of the present disclosure, one or more large drugs may be included in the premix. According to some embodiments of the present disclosure, one or more biological drugs may be included in the premix. According to the present invention, a botulinum toxin may be included in the premix. According to the present invention, one or more antibodies may be included in the premix. In some embodiments, a premix may include one or more surfactants, penetration enhancers, and / or other substances. In some embodiments, a premix comprises a solution. In some embodiments in which the premix comprises a botulinum toxin, an antibody, another bioactive agent, and / or a penetration enhancer, the botulinum toxin, the antibody, another bioactive agent, and / or the penetration enhancer is in solution before applying high shear force to the premix.

[0053] Prevent or prophylaxis: As used herein, when used in reference to the occurrence of a disease, disorder, and / or condition, prevents or prophylaxis refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete when the onset of the disease, disorder, or condition has been delayed for a certain period of time.

[0054] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked together by peptide bonds). Proteins may contain moieties other than amino acids (e.g., glycoproteins, proteoglycans, etc.) and / or may be processed or modified in other ways. Those of skill in the art will understand that a "protein" can be an entire polypeptide chain as produced by a cell (with or without a signal sequence), or a characteristic portion thereof. Those of skill in the art will understand that a protein may comprise multiple polypeptide chains, for example, linked by one or more disulfide bonds or linked by other means. 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 contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0055] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three amino acids. Those of skill in the art will appreciate that the term "polypeptide" is intended to encompass not only polypeptides having the complete sequences described herein, but also polypeptides representing functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Furthermore, those of skill in the art will appreciate that protein sequences generally tolerate some substitutions without destroying activity. Thus, polypeptides that retain activity and share at least 30-40%, often greater than 50%, 60%, 70%, or 80% overall sequence identity with polypeptides of the same class, and further include at least one region of much higher identity, often greater than 90%, and even greater than 95%, 96%, 97%, 98%, or 99%, in one or more highly conserved regions, usually comprising at least 3-4, and often 20 or more amino acids, are encompassed by the related term "polypeptide" as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0056] Reference: As used herein, a reference describes a standard or control against which a comparison is made. For example, in some embodiments, a drug, animal, individual, population, sample, regimen, sequence, or value of interest is compared with a drug, animal, individual, population, sample, regimen, sequence, or value of a reference or control. 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, and is optionally embodied in a tangible medium. Typically, as understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. Those skilled in the art will understand when there is sufficient similarity to determine reliance and / or comparison on a particular possible reference or control.

[0057] Self-administration: As used herein, the term "self-administration" refers to a situation in which a subject has the ability to administer a composition to himself or herself without the need for medical supervision. In some embodiments of the present invention, self-administration can be performed outside of a clinical setting. For example, in some embodiments of the present invention, a facial cosmetic cream can be administered by a subject in his or her own home.

[0058] Small molecule: Generally, a "small molecule" is understood in the art to be an organic molecule less than about 5 daltons (Kd) in size. In some embodiments, a small molecule is less than about 3 Kd, 2 Kd, or 1 Kd. In some embodiments, a small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not a protein, peptide, or amino acid. In some embodiments, a small molecule is not a nucleic acid or nucleotide. In some embodiments, a small molecule is not a saccharide or polysaccharide.

[0059] Subject: As used herein, "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments prenatal human forms). In some embodiments, the subject is suffering from the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a person with one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is being and / or has been diagnosed and / or treated.

[0060] Substantially: As used herein, the term "substantially" refers to the qualitative condition of the total or near-total extent or degree of a desired characteristic or property. Those skilled in the art of biology understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0061] Therapeutic Agent: As used herein, the term "therapeutic agent" generally refers to any drug that induces a desired pharmacological effect when administered to an organism. In some embodiments, a drug is considered to be a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, the appropriate population may be defined by various criteria, such as a specific age group, sex, genetic background, pre-existing clinical condition, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, mitigate, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is a drug that has been approved or needs to be approved by a government agency before it can be sold for administration to humans. In some embodiments, a "therapeutic agent" is a drug that requires a prescription for administration to humans. In some embodiments, a drug is not considered to be a "therapeutic agent" if it merely enhances the delivery of a different drug that actually achieves a desired effect.

[0062] Therapeutically effective amount: As used herein, a therapeutically effective amount refers to an amount that produces the desired effect to which it is administered. In some embodiments, the term refers to an amount that is sufficient when administered to a population suffering from or susceptible to a disease, disorder, and / or condition according to a therapeutic dosing regimen to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount reduces the incidence and / or severity of one or more symptoms of the disease, disorder, and / or condition, and / or delays the onset. Those skilled in the art will understand that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a specific individual. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, provides a specific desired pharmacological response in a significant number of subjects. In some embodiments, a reference to a therapeutically effective amount may refer to the amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or body fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will appreciate that in some embodiments, a therapeutically effective amount of a particular drug or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective drug may be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0063] Therapeutic regimen: As used herein, the term "therapeutic regimen" refers to a dosing regimen whose administration across a relevant population can be correlated with a desired or beneficial therapeutic outcome.

[0064] Treatment: As used herein, the term "treatment" ("treat" or "treating") refers to any administration of a therapy that partially or completely relieves, improves, alleviates, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be for subjects who do not exhibit signs of the associated disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be for subjects who exhibit one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be for subjects who have been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be for subjects who are known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the associated disease, disorder, and / or condition.

[0065] Uniform: The term "uniform," as used herein with respect to nanoemulsion compositions, refers to a nanoemulsion composition in which the individual droplets have a particular range of droplet diameter sizes. For example, in some embodiments, a uniform nanoemulsion composition is one in which the difference between the smallest and largest diameters is no more than about 300, 250, 200, 150, 100, 90, 80, 70, 60, 50 nm, or less. In some embodiments, the droplets (e.g., large drug-containing droplets) within the uniform large drug nanoemulsion compositions of the invention have diameters of less than about 300, 250, 200, 150, 130, 120, 115, 110, 100, 90, 80 nm, or less. In some embodiments, the droplets (e.g., large drug-containing droplets) within the uniform large drug nanoemulsion compositions of the invention have diameters within the range of about 10 nm to about 300 nm. In some embodiments, droplets within the uniform large drug nanoemulsion compositions of the present invention have diameters within the range of about 10-300, 10-200, 10-150, 10-130, 10-120, 10-115, 10-110, 10-100, or 10-90 nm. In some embodiments, droplets (e.g., large drug-containing droplets) within the large drug nanoemulsion compositions of the present invention have an average droplet size of less than about 300, 250, 200, 150, 130, 120, 115, 110, 100, or 90 nm. In some embodiments, the average droplet size is within the range of about 10-300, 50-250, 60-200, 65-150, 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, a majority of the droplets (e.g., large drug-containing droplets) within a uniform nanoemulsion composition of the invention have diameters less than a specified size or within a specified range, i.e., 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, a uniform nanoemulsion composition is achieved by microfluidization of the sample.

[0066] Variant: As used herein, the term "variant" refers to an entity that exhibits significant structural identity with a reference entity, but is structurally different from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a variant is also functionally different from the reference entity. Generally, whether a particular entity is properly considered to be a "variant" of a reference entity is based on the degree of structural identity with the reference entity. As will be understood by those skilled in the art, every biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To give some examples, small molecules can have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic pendant moieties, such that variants of the small molecule share the core structural element and characteristic pendant moieties but differ in other pendant moieties and / or linkage types within the core (single vs. double, E vs. Z, etc.); polypeptides can have characteristic sequence elements composed of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or that contribute to a particular biological function; and nucleic acids can have characteristic sequence elements composed of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. For example, variant polypeptides can differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the variant polypeptide exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% overall sequence identity with the reference polypeptide, optionally excluding conservative amino acid substitutions. Alternatively or additionally, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more biological activities of the reference polypeptide.In some embodiments, a mutant polypeptide lacks one or more biological activities of a reference polypeptide. In some embodiments, a mutant polypeptide exhibits a reduced level of one or more biological activities compared to a reference polypeptide. In many embodiments, a target polypeptide is considered to be a "variant" of a parent or reference polypeptide when it has an amino acid sequence identical to that of the parent but with a few sequence changes at specific positions. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the mutant are substituted compared to the parent. In some embodiments, a mutant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residues compared to the parent. In many cases, a mutant has a very small number (e.g., less than 5, 4, 3, 2, or 1) of substituted functional residues (i.e., residues responsible for a specific biological activity). Furthermore, a mutant typically has no more than 5, 4, 3, 2, or 1 additions or deletions, and often no additions or deletions compared to the parent. Furthermore, any additions or deletions are typically less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and generally less than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature.

[0067] Detailed Description of Specific Embodiments Transdermal Drug Delivery In some embodiments, the present disclosure provides techniques for improving the transdermal delivery and / or bioavailability of large drugs (e.g., botulinum toxin, antibodies). In some embodiments, the present disclosure teaches that particularly advantageous results are achieved when microneedling technology is combined with an emulsion composition. In some embodiments, microneedling technology is combined with a lotion, cream, or liquid composition, and in some embodiments, such a composition may in turn be or include an emulsion composition (e.g., a macroemulsion composition, a microemulsion composition, and / or a nanoemulsion composition). Alternatively, in some embodiments, the provided technology combines microneedling technology with transdermal delivery that does not utilize a nanoemulsion, a microemulsion, a macroemulsion, or any emulsion. In some embodiments, the provided technology does not utilize a penetration enhancer. In some embodiments, the provided technology does not utilize a chemical penetration enhancer that damages, disrupts, and / or degrades the skin. In some embodiments, the provided technology does not utilize a chemical penetration enhancer.

[0068] Human skin comprises the dermis and the epidermis, which has several layers of tissue: the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale (identified from the outer surface of the skin inwards).

[0069] The stratum corneum presents the greatest hurdle in transdermal delivery of large drugs in general, and perhaps especially. The stratum corneum is typically about 10-15 μm thick and consists of flattened keratinized cells (keratinocytes) arranged in several layers. The spaces between the keratinocytes are filled with lipid structures, which may play an important role in the penetration of substances through the skin (Bauerova et al., 2001, European Journal of Drug Metabolism and Pharmacokinetics, 26:85).

[0070] The remaining epidermis, below the stratum corneum, is approximately 150 μm thick. The dermis, located below the epidermis, is approximately 1–2 mm thick and is innervated by various capillaries and nerve processes.

[0071] Transdermal administration has been the subject of research to provide an alternative route of administration that does not generally involve the undesirable consequences associated with injections and oral delivery. For example, needles often cause local pain and can expose the patient receiving the injection to blood-borne diseases. Oral administration can suffer from poor bioavailability of pharmaceuticals due to the highly acidic environment of the patient's stomach.

[0072] In an attempt to overcome these drawbacks by providing non-invasive administration, efforts have been made to develop transdermal administration techniques for certain pharmaceuticals. Generally, transdermal administration is desirable to minimize damage to the patient's skin. Thus, transdermal administration can reduce or eliminate the pain associated with injections, reduce the possibility of blood contamination, and improve the bioavailability of drugs when incorporated into the system.

[0073] Traditionally, attempts at transdermal administration have focused on the destruction and / or decomposition of the stratum corneum. Some attempts include the use of chemical penetration enhancers. Penetration enhancers can function to decompose and / or destroy skin structures. In some embodiments, the penetration enhancer is or includes a chemical agent (e.g., a chemical or enzyme that can destroy and / or decompose one or more components of the stratum corneum). In some embodiments, the penetration enhancer can be irritating in that it can cause inflammatory and / or allergic reactions when the drug is applied to the skin.

[0074] "However, a major limitation of penetration enhancers is that their effectiveness is often closely correlated 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 stratum corneum." Pharmaceutics. 7:438-470 (2015). Penetration enhancers tend to have poor efficacy and safety profiles. "They do not achieve the desired skin breakdown, and their ability to increase transport across the skin is low and variable." Ibid.

[0075] Some attempts have involved the use of mechanical devices to bypass or ablate portions of the stratum corneum. Others have involved the use of ultrasound or iontophoresis to enhance the penetration of pharmaceuticals through the skin. In most cases, the goal is to allow drugs, typically small molecules, to pass into the dermal capillary bed, where they 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.

[0076] Microneedling technology has been shown to improve the transdermal delivery of a variety of small drugs, such as calcein (approximately 623 Da), desmopressin (approximately 1070 Da), diclofenac (approximately 270 Da), methyl nicotinate (approximately 40 Da), bischloroethylnitrosourea (approximately 214 Da), insulin (approximately 5.8 kDa), bovine serum albumin (approximately 66.5 kDa), and ovalbumin (approximately 45 kDa), but until the present disclosure, improving the delivery and / or bioavailability of larger drugs, especially those above 100 kDa, remained problematic.

[0077] Transdermal delivery of large drugs It is recognized that transdermal delivery of large drugs (e.g., macromolecules) poses significant challenges. The present application has demonstrated that microneedling, and in particular microneedle skin preconditioning using a relatively low microneedle density and / or a relatively small microneedle puncture size (e.g., puncture size per microneedle), surprisingly influences and / or is effective in transdermal administration of large drugs (e.g., botulinum toxin) (see, e.g., U.S. Patent Application Nos. 62 / 774,677, 62 / 789,407, and 62 / 808,274). This demonstration was surprising in light of the state-of-the-art, including studies using solid microneedles to deliver four hydrophilic peptides: low-molecular-weight tetrapeptide-3 (456.6 Da); hexapeptide (498.6 Da); acetylhexapeptide-3 (889 Da); and oxytocin (1007.2 Da), as well as L-carnitine (161.2 Da). This study demonstrated that while microneedle pretreatment significantly increased the penetration of each test peptide, the skin penetration of the peptide depended on its molecular weight and decreased as the molecular weight increased. Zhang, S., et al., "Enhanced delivery of hydrophilic peptides in vitro by transdermal microneedle pretreatment." Acta Pharmaceutica Sinica B. 4(1):100-104 (2014).

[0078] Additionally, when sandpaper abrasion, tape stripping, and single-puncture hypodermic needle models of MSCs were compared to study the effect of molecular size on transdermal delivery of larger FITC (fluorescein isothiocyanate) conjugate molecules, all methods were found to be similar, and when tested on untreated skin, transdermal drug delivery again decreased as the size of the test molecule increased (4.3, 9.6, and 42.0 kDa FITC conjugates). Tape stripping was found to be the most effective technique, while sandpaper abrasion was found to be the most damaging to the skin. Wu, X., et al., "Effects of pretreatment of needle puncture and sandpaper abrasion on the in vitro skin permeation of fluorescein isothiocyanate (FITC)-dextran." International Journal of Pharmaceutics. 316:102-108 (2006).

[0079] 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). Microneedles of various lengths (300, 550, 700, or 900 μm) were used to puncture human skin at the epidermal level and assess the diffusion of each of these compounds. Transport of each compound was observed across all but the 300 μm microneedle array, although degradation of DCB and FITC-Dex was observed.

[0080] Prior to the work of the present applicant, it was understood in the art that as molecular size increased, transdermal penetration using MSCs ("microneedle skin conditioning") decreased to a minimum, or even to the point of nonexistence. Even when some minimal penetration was observed, larger molecules were observed to degrade and become biologically inactive. Research by the present applicant (including, for example, International Application No. PCT / US17 / 53333, published as WO2018 / 093465) demonstrated that combining emulsion and microneedling techniques for the transdermal delivery of large drugs of interest can achieve various advantages; in some embodiments, these techniques have shown that they can achieve particularly surprising improvements in the transdermal delivery of large molecular structures without the use of mechanical or chemical penetration enhancers. For example, in some embodiments, these techniques achieved transdermal delivery of Clostridium botulinum, which is approximately 150 KDa, more than twice the size of FITC-Dex.

[0081] The present disclosure provides certain techniques that have surprising properties and / or achieve unexpected benefits that are useful in the administration (eg, via transdermal delivery) of macromolecular drugs, particularly botulinum toxins.

[0082] Those skilled in the art will be aware of various protein drugs that have been approved for therapeutic use by relevant regulatory authorities. For example, the U.S. Food and Drug Administration maintains a list of approved biological therapeutics, organized by the 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 may be applicable to various such drugs. Of particular interest are those intended and / or formulated for local administration, including, for example, those that may be undergoing or have undergone clinical trials (e.g., may be approved or in the process of being approved by the U.S. Food and Drug Administration or an equivalent agency in another jurisdiction, or may be involved in a clinical trial, e.g., may be included in the list at www.clinicaltrials.gov, or may be on file with one or more clinical institution's Institutional Review Boards or their equivalents).

[0083] Those skilled in the art reading this disclosure will understand that in some embodiments, the large drug to which the teachings relate can be or include an antibody drug.

[0084] In some embodiments, the antibody drug may be suitable for treating skin conditions. In some embodiments, the antibody drug may be a fusion protein. In some embodiments, the antibody drug may be conjugated to another moiety. In some embodiments, the antibody drug may be conjugated to polyethylene glycol. In some embodiments, the antibody may be multispecific (e.g., bispecific) and can bind to two or more different target antigens or epitopes.

[0085] In some embodiments, the antibody drug targets TNFα (e.g., comprises an epitope-binding element found in anti-TNFα antibodies, such as infliximab, adalimumab, golimumab, etanercept, etanercept-szzs, and / or certolizumab pegol). In some embodiments, the antibody drug targets CD2 (e.g., comprises an epitope-binding element found in anti-CD2 antibodies, such as siplizumab). In some embodiments, the antibody drug targets CD4 (e.g., comprises an epitope-binding element found in anti-CD4 antibodies, such as zanolimumab).

[0086] In some embodiments, the antibody drug targets IL-12 (e.g., comprises the epitope-binding element found in anti-IL-12 antibodies, such as briakinumab). In some embodiments, the antibody drug targets IL-17 (e.g., comprises the epitope-binding element found in anti-IL-17 antibodies, such as secukinumab and / or brodalumab). In some embodiments, the antibody drug targets IL-22 (e.g., comprises the epitope-binding element found in anti-IL-22 antibodies, such as fezakinumab). In some embodiments, the antibody drug targets IL-23 (e.g., comprises the epitope-binding element found in ustekinumab and / or guselkumab).

[0087] Those skilled in the art reading this disclosure will understand that in some embodiments, the macro-drugs to which the teachings relate can be or include prophylactic agents such as vaccines. In some embodiments, vaccines can include isolated proteins or peptides, inactivated organisms and viruses, killed organisms and viruses, genetically modified organisms or viruses, and cell extracts. In some embodiments, prophylactic agents can be combined with interleukins, interferons, cytokines, and adjuvants, such as cholera toxin, alum, and Freund's adjuvant.In some embodiments, the prophylactic agent is selected from the group consisting of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, and the like. interrogans), Borrelia burgdorferi, Campylobacter jejuni, and other bacterial organisms; smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E Antigens may include antigens of viruses such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and other fungi, protozoa, and parasites. In some embodiments, these antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

[0088] Those skilled in the art will recognize that the preceding paragraphs provide an exemplary, non-exhaustive list of drugs that can be delivered using the techniques according to the present invention; they will understand the application of the techniques provided to the administration of other drugs.

[0089] Botulinum toxin treatment Those skilled in the art know that botulinum toxins are potent and effective inhibitors of acetylcholine release. Acetylcholine is a neurotransmitter that is active, for example, at neuromuscular junctions and other specific synapses (e.g., in the central nervous system and in ganglia, particularly in the visceromotor system). Botulinum toxins are useful in the treatment and / or prevention of various diseases, disorders, and conditions, including, in particular, those associated with the release and / or activity of acetylcholine.

[0090] Botulinum toxins are naturally produced by the gram-positive anaerobic bacterium Clostridium botulinum. Botulinum toxins are classified into one of seven antigenically distinct but structurally similar classes: A, B, C (C1 or C2), D, E, F, and G. All seven are understood to inactivate (by enzymatic cleavage) proteins required for the docking and fusion processes involved in acetylcholine release.

[0091] Botulinum toxins are naturally produced as single polypeptides (approximately 150 kD) that form intrapeptide disulfide bonds and are cleaved to produce dipeptide toxins in which the chains are linked to each other by disulfide bonds. The light chain (approximately 50 kD) possesses endopeptidase activity; the heavy chain binds to presynaptic receptors and also facilitates the translocation of the light chain across the endosomal membrane.

[0092] Botulinum toxins are released by Clostridium bacteria as complexes containing a 150 kDa botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the BTX-A complex is produced by Clostridium bacteria as 900 kDa, 500 kDa, and 360 kDa forms. Botulinum toxin types B and C1 are apparently produced only as the 500 kDa complex. Botulinum toxin type D is produced as both a 300 kDa and a 500 kDa complex. Finally, botulinum toxin types E and F are produced only as approximately the 300 kDa complex.

[0093] A variety of botulinum toxin therapeutic agents have been approved by the U.S. Food and Drug Administration, including, for example: a) abobotulinumtoxin A (marketed as Dysport® and approved for use in the treatment of achalasia, blepharospasm associated with dystonia, cervical dystonia (spasmodic torticollis), chronic anal fissures, detrusor overactivity (detrusor hyperreflexia) or detrusor-sphincter dyssynergia due to spinal cord injury or disease, hand dystonia, hand tremor, hemifacial spasm, hyperhidrosis including gustatory sweating (Frey's syndrome), moderate to severe glabellar lines in adults, oromandibular dystonia, hypersalivation, spastic dystonia (laryngeal dystonia), spasticity associated with cerebral palsy or multiple sclerosis or neuromyelitis optica or stroke or other traumatic disease or tumor of the brain or spinal cord, strabismus, lingual dystonia, torsional dystonia, and spasticity of the upper and lower limbs including lower limb spasticity in children 2 years of age and older, and tremor of the voice); b) incobotulinumtoxinA (sold as Xeomin® and approved for use in treating blepharospasm, cervical dystonia, chronic hypersalivation, moderate to severe glabellar lines, cerebral palsy or multiple sclerosis or neuromyelitis optica or spasticity associated with stroke or other damaging diseases or tumors of the brain or spinal cord); c) onabotulinumtoxinA (sold as Botox® and approved for use in achalasia, blepharospasm, cervical dystonia, chronic anal fissures, chronic migraine headaches, neurogenic detrusor overactivity, hand dystonia, hand tremor, hemifacial spasm, hyperhidrosis including gustatory sweating (Frey's syndrome), oromandibular dystonia, overactive bladder, hypersalivation, spastic dystonia (laryngeal dystonia), severe primary axillary hyperhidrosis, spasticity associated with cerebral palsy or multiple sclerosis or neuromyelitis optica or stroke or other traumatic disease or tumor of the brain or spinal cord, strabismus, lingual dystonia, torsion dystonia, and voice tremor; Botox Cosmetic® is approved for use in moderate to severe epicanthal lines, also known as crow's feet and moderate to severe glabellar lines); d) Prabotulinum toxin A-xcfs (Jeuveau TM and is approved for use in the temporary improvement of the appearance of moderate to severe glabellar wrinkles associated with corrugator supercilii and / or procerus muscle activity in adults; and e) Rimabotulinumtoxin B (sold as Myobloc® and approved for use in cervical dystonia, detrusor overactivity (detrusor hyperreflexia), excessive salivation, spasticity associated with cerebral palsy or multiple sclerosis or neuromyelitis optica or stroke or other traumatic disease or tumor of the brain or spinal cord, etc.).

[0094] Those skilled in the art will be aware of the standard and / or approved administration regimens for such commercially available botulinum toxin compositions and, upon reading this disclosure, will understand how and to what extent such compositions and / or regimens may be utilized in conjunction with the microneedling technology (e.g., particularly MSCs) described herein. Alternatively or additionally, those skilled in the art reading this disclosure will understand how such approved products and / or regimens may be considered appropriate benchmarks by which the timing of peak effect and / or duration of response may be assessed as described herein.

[0095] Use of botulinum toxin is considered cosmetic when it is associated with the improvement of a feature of appearance or when there is no physiological dysfunction that is expected to be improved by administration of the toxin; use is considered therapeutic (in the presence of such a dysfunction) and is considered prophylactic when administered before the onset of significant symptoms or features of the relevant cosmetic feature of the physiological dysfunction.

[0096] Specifically for wrinkle treatment, the U.S. Food and Drug Administration (FDA) has provided draft guidance for industry, recommending that "maximal contraction measurements should be used to assess the efficacy of botulinum toxin drug products and demonstrate paralytic effects," and that "success should be defined as a 2-point improvement from baseline on both [investigator assessment] and [subject self-assessment] measures to confirm clinical significance." See www.fda.gov / downloads / Drugs / GuidanceComplianceRegulatoryinformation / Guidances / UCM407983.pdf.

[0097] Botulinum toxin is a complex protein, and three regions or functional parts must be intact for the protein to be biologically active. Therefore, damage to any one of the three regions of the protein renders the protein biologically inactive. According to Johnson, E. et al., "Botulinum toxin is highly susceptible to denaturation due to surface denaturation, heat, and alkaline conditions." U.S. Patent Application Publication No. 5,512,547. Many traditional microneedling conditions (e.g., those described by Wu) are expected to risk significant levels of degradation and inactivation of botulinum toxin. The findings presented in this disclosure—for example, that administering a nanoemulsion composition in combination with microneedle skin conditioning can delay peak effects and / or extend response duration—are particularly surprising in light of this known disadvantage of botulinum toxin.

[0098] Those skilled in the art reading this disclosure will understand that the teachings may be applicable to the administration of botulinum toxin polypeptides and / or botulinum toxin complexes, and / or any portion or fragment or variant of a botulinum toxin protein or complex that retains relevant activity.

[0099] In some embodiments, the botulinum toxin utilized in accordance with the present 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, e.g., having one or more structural mutations compared 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 lifespan of biological activity than an appropriate equivalent reference form (e.g., wild-type form). In some embodiments, the botulinum toxin exists as 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).

[0100] In some embodiments, the botulinum toxin provided and / or utilized in accordance with the present invention may be or may include one or more approved or developmental botulinum products, such as abobotulinumtoxinA, daxibotulinumtoxinA, Hengri, incobotulinumtoxinA, Medy-Tox, Neuronox, NT-201, onabotulinumtoxinA, prabotulinumtoxinA-xcfs, PurTox, rimabotulinumtoxinB, etc.

[0101] Some embodiments of the present invention contemplate pharmaceutical compositions comprising a stabilized botulinum toxin for transdermal delivery to a human patient. The botulinum toxin may be selected from botulinum toxin types A, B, C1, D, E, F, and G, isolated and / or purified (i.e., approximately 150 kDa) botulinum toxin, and natural or recombinantly produced botulinum toxin. In some embodiments, the composition may contain and / or deliver a unit amount of botulinum toxin, which may be from about 1 unit to about 100,000 units, and / or the composition may contain and / or deliver a sufficient amount of botulinum toxin to achieve a therapeutic effect lasting from about 1 month to about 5 years.

[0102] Response duration and peak effect As explained in a recent review, "time to response onset and duration of response are important measures of botulinum toxin efficacy that have a substantial impact on patient satisfaction." See Nestor et al. Aesthetic Surg J. 37:S20, 2017. The review also reports, "Typically, some patients notice improvement in wrinkles within one day of treatment, and recovery of muscle function is generally thought to occur 3 to 6 months after treatment. Patients who undergo multiple treatment sessions may notice a longer duration of effect, which allows for longer intervals 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 J Oral 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.

[0103] Many studies have concluded that the appropriate clinical effect diminishes significantly and prematurely, so multiple such treatments may be necessary to achieve the longer duration of effect reported. For example, Carruthers, a leading researcher in botulinum toxin research, explains that "in most individuals, the clinical effect of botulinum toxin A begins to appear within 1-2 days, peaks within 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 of injectable botulinum toxin for the treatment of crow's feet lines found that the median duration of action for patients who were responders at day 30 of the trial was approximately 4.5 months (Carruthers A, et al. "Efficacy and Safety of OnabotulinumtoxinA for the Treatment of Crow's Feet Lines: A Multicenter, Randomized, Controlled Trial" Dermatol Surg 2014;40:1181-1190). TM The package insert itself states that the effects of injectable botulinum to treat glabellar wrinkles last for approximately 3 to 4 months (Botox Cosmetic Package Insert).

[0104] While many in the community report an expectation that a rapid onset of response is desirable, reporting, for example, that "patients desire to see the effects of their treatment as soon as possible after the procedure" (see Nestor et al. Aesthetic Surg J. 37:S20, 2017), the present disclosure understands that too rapid an onset can have drawbacks including, for example, increasing the risk of a "frozen face" effect and / or causing unwanted social commentary or awkwardness if immediate, noticeable changes in facial features are observable by others.

[0105] Thus, among other things, the present disclosure identifies a problem with certain available approaches to botulinum toxin therapy, in that they are designed to achieve a rapid onset of effect (typically within about 1 day, e.g., about 1-3 days, of administration) and / or a rapid peak effect (typically within 1 month of administration). The present disclosure recognizes that, particularly for treatments (including cosmetic treatments) with visible effects, it may be desirable to design and / or administer botulinum therapy with a delayed onset and / or peak effect. In some embodiments, such onset or peak effect is assessed for (e.g., each) a single dose; in some embodiments, such onset or peak effect is assessed for the end of multiple doses in a regimen.

[0106] Those skilled in the art will understand that when multiple doses are administered, the time to peak effect for later doses administered to a particular individual may be shorter than the time to peak effect for earlier doses administered to the same individual due to the presence of already weakened muscles. Those skilled in the art will be familiar with the expected variability in the time to peak effect for administration to a given subject and will therefore be able to evaluate and understand the relevant effects described herein in comparison to comparable doses (e.g., doses administered to subjects who have equivalently received earlier doses).

[0107] In some embodiments, provided botulinum toxin treatment regimens achieve a peak effect (e.g., for one or more doses within the regimen) after administration more than 1 month, and in some embodiments more than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 months, or more than 1 year.

[0108] In particular, the present disclosure provides the discovery that the delay in peak effect described herein may have particular advantages, particularly in certain patient populations. For example, many subjects, particularly those undergoing cosmetically or otherwise visually significant botulinum toxin treatment (such as wrinkle reduction), may prefer a relatively slow onset and / or rate of effect, either or both of which may be embodied and / or expressed in a delay in peak effect such that the fact that a treatment has been received is not immediately apparent or alarming to bystanders. In some embodiments, the delay in peak effect described herein allows for a sufficiently gradual onset of action that bystanders may not recognize that a subject is receiving botulinum toxin treatment.

[0109] Alternatively or additionally, the present disclosure provides the discovery that the delayed peak effect described herein may present a reduced risk of developing a "hard look" or another potentially undesirable side effect of certain other botulinum toxin treatments.

[0110] Furthermore, the present disclosure demonstrates the surprising efficacy of certain botulinum toxin treatments described herein (e.g., delayed peak effect treatments) in that they exhibit median durations of response longer than those reported for various approved botulinum toxin treatments.

[0111] Without wishing to be bound by any particular theory, the present disclosure observes that the delayed peak effect demonstrated herein accounts for the altered pharmacokinetics and / or pharmacodynamics achieved by the provided treatments compared to certain other botulinum toxin treatments (including, for example, injectable treatments). One of skill in the art reading this disclosure will appreciate that such alterations in pharmacokinetics and / or pharmacodynamics also likely underlie the observed increased duration of effect described herein.

[0112] As previously noted herein, currently marketed botulinum toxin products have a median duration of response of approximately 3-4 months (e.g., as reported for the treatment of wrinkles according to approved regimens), and typically have a maximum duration of response of less than 6 months.

[0113] For example, provided botulinum toxin treatments comprising administering botulinum toxin in combination with microneedle skin conditioning are characterized by a median duration of effect (e.g., a single dose of botulinum toxin) of greater than 6 months. In some embodiments, such treatments are provided that are characterized by a median duration of response of greater than 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more.

[0114] Thus, in some embodiments, provided botulinum toxin treatments (e.g., comprising administering botulinum toxin in combination with microneedle skin conditioning) comprise administering two or more individual doses separated from one another by periods of more than about 6, 7, 8, 9, 10, 11 months, or 1 year or more. In some embodiments, different pairs of doses within a provided regimen are separated from one another by different such periods; in some embodiments, some or all pairs may be separated from one another by the same such period.

[0115] Those skilled in the art know that increasing the dose size may extend the duration of the effect of a particular treatment. However, the extended duration of effect provided herein may not be due solely to a change in dose size. Those skilled in the art know that increasing the dose extends the duration of effect without affecting the timing of the peak effect. For example, studies have shown that increasing the dose of injected botulinum by a factor of three increases the median duration of effect; however, such an increase in dose does not affect the timing of the peak effect. That is, the peak effect of all doses was achieved within four weeks after injection, consistent with other studies of injected botulinum (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).

[0116] In sharp contrast, as described herein, the treatments provided (e.g., combining certain topical botulinum treatments with MSP) result in an extended duration of effect and a delayed peak effect (i.e., a peak effect that is much later than 4 weeks). As noted above, without wishing to be bound by any particular theory, it is believed that these combined effects reflect changes in pharmacokinetics and / or pharmacodynamics achieved by the technology provided herein.

[0117] Thus, the present disclosure provides techniques for both delaying the peak effect and extending the duration of response of an administered large drug (e.g., botulinum toxin), e.g., by combining topical administration (e.g., emulsion formulations, e.g., nanoemulsion formulations) with MSCs, e.g., as described and / or exemplified herein.

[0118] Microneedling In some embodiments, microneedling (e.g., microneedle skin conditioning) according to the present disclosure is performed with a microneedle (MN) array that features or shares these features of a minimally invasive system. In some embodiments, the microneedling techniques useful according to the present invention avoid and / or overcome one or more drawbacks commonly associated with the use of hypodermic and / or subcutaneous needles, improving patient comfort and compliance. Such drawbacks include, for example, the possibility of needle tip misplacement with hypodermic needles because medical professionals cannot accurately visualize where the needle is going; such needle misplacement can be particularly problematic for the administration of botulinum toxin, because, for example, when botulinum is injected incorrectly into the face, it can cause adverse reactions such as drooping eyelids ("ptosis"). Microneedling techniques are less prone to such problems. Other advantages of microneedling technology include: it does not cause bleeding, minimizes the introduction of pathogens through the holes made by microneedles, and / or eliminates the variability of transdermal administration.Other advantages include the possibility of self-administration, reduces the risk of accidental needlestick injury, reduces the risk of infectious disease transmission, and is easy to dispose of.In some embodiments, microneedles are a plurality of microscopic protrusions assembled on one side of a support such as a patch or device (for example, a stamp, a roller, an array, an applicator, a pen).

[0119] In some embodiments, microneedles for use according to the present disclosure can be designed and / or constructed in an array, which can, for example, improve skin contact and / or promote penetration into the skin. In some embodiments, the microneedles utilized are of a length, width, and shape suitable for creating an efficient pathway for drug delivery while minimizing contact with nerves when inserted into the skin. 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).

[0120] In some embodiments, suitable microneedles can be solid, coated, porous, dissolving, hollow or hydrogel microneedles.Solid microneedles create tiny holes in the skin, thereby increasing the transport of drug formulations (e.g., the "poke and patch" method).Coated microneedles allow the coated drug to rapidly dissolve into the skin (e.g., the "coat and poke" method).Dissolving microneedles allow the drug incorporated into the microneedle to be rapidly and / or controlled released.Hollow microneedles can be used to puncture the skin and release the composition after active injection or diffusion of the formulation through the microneedle holes (e.g., the "poke and flow" method).

[0121] In the case of dissolving microneedles, the microneedles may function as a drug depot, holding the drug composition until released by dissolution in the case of dissolving microneedles, or swelling in the case of hydrogel microneedles (e.g., a "poke and release" method). However, as previously described herein, in many embodiments, large drugs are not delivered by injection through one or more microneedles. That is, in many embodiments, any microneedles utilized in accordance with such embodiments are not coated with, loaded with, or fabricated with large drugs in any manner that achieves delivery of the large drug.

[0122] Alternatively or additionally, in some embodiments, the microneedles described herein (in MSCs or otherwise) utilized in accordance with the present disclosure may contain and / or deliver large drugs when the large drugs are formulated into the macro- or nanoemulsions described herein. Thus, as will be understood by those skilled in the art upon reading this specification, treatment of the skin with microneedles that deliver large drugs (e.g., by injection through the microneedles, by release of a microneedle coating, or by release from dissolved microneedles) is not microneedle skin conditioning.

[0123] In some embodiments, the microneedle has a diameter that is constant throughout the length of the microneedle. In some embodiments, the diameter of the microneedle is greatest at the base end of the microneedle. In some embodiments, the microneedle tapers to a point at the end distal to the base of the microneedle. In some embodiments, the microneedle can be solid. In some embodiments, the microneedle can be hollow. In some embodiments, the microneedle can be tubular. In some embodiments, one end of the microneedle can be sealed. In some embodiments, the microneedle is part of an array of microneedles.

[0124] In some embodiments, the microneedles may be from about 1 μm to about 4,000 μm in length. In some embodiments, the microneedles may be from about 1 μm to about 2,000 μm in length. In some embodiments, the microneedles may be from about 50 μm to about 400 μm in length. In some embodiments, the microneedles may be from about 50 μm to about 500 μm in length. In some embodiments, the microneedles may be from about 50 μm to about 600 μm in length. In some embodiments, the microneedles may be from about 50 μm to about 700 μm in length. In some embodiments, the microneedles may be from about 50 μm to about 800 μm in length. In some embodiments, the microneedles may be from about 800 μm to about 1,500 μm in length. In some embodiments, the microneedles may be less than about 1,400 μm in length. In some embodiments, the microneedles may be less than about 1,100 μm in length. In some embodiments, the microneedles may be less than about 1,000 μm in length. In some embodiments, the microneedles may be less than about 800 μm in length, hi some embodiments, the microneedles may be from about 100 μm to about 800 μm in length.

[0125] In some embodiments, the microneedling described herein comprises applying to the skin multiple microneedles of a common length (e.g., a microneedle array); the microneedling described herein comprises applying to the skin multiple microneedles of different lengths (e.g., a microneedle array).

[0126] Microneedles of various lengths can be used in the microneedling techniques described herein. In some embodiments, the length of the microneedles used in the MSCs described herein is adjusted based on the thickness of the skin at the treatment site.

[0127] In some embodiments, the microneedles or microneedle arrays comprise microneedles that are 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 in length.

[0128] In some embodiments, the microneedle or microneedle array comprises a plurality of needles. In some embodiments, the microneedle or microneedle array comprises a plurality of needles. 2 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

[0129] Microneedles of any shape can be used in the microneedling techniques described herein. In some embodiments, the microneedles can have a circular cross-section. In some embodiments, the microneedles can have a triangular cross-section. In some embodiments, the microneedles can have a rectangular cross-section. In some embodiments, the microneedles can have a square cross-section. In some embodiments, the microneedles can have a quadrangular cross-section. In some embodiments, the microneedles can have a pentagonal cross-section. In some embodiments, the microneedles can have a hexagonal cross-section. In some embodiments, the microneedles can have a heptagonal cross-section. In some embodiments, the microneedles can have an octagonal cross-section. In some embodiments, the microneedles can have a nonagonal cross-section. In some embodiments, the microneedles can have a decagonal cross-section.

[0130] Microneedles of various cross-sectional areas can be used in the microneedling techniques described herein. The cross-sectional area of each microneedle in the MN array used for MSCs ("microneedle skin conditioning") described herein can then define the puncture size (e.g., puncture size per microneedle) of the microneedle in the MN array used for MSCs. In some embodiments, the puncture size of the microneedles ranges from about 100 to about 60,000 μm. 2 In some embodiments, the puncture size of the microneedle may range from about 100 to about 30,000 μm. 2 / Microneedle range.

[0131] In some embodiments, the microneedles or microneedle arrays comprise needles having multiple microneedle puncture sizes. In some embodiments, the microneedles or microneedle arrays comprise needles having at least two different microneedle puncture sizes. In some embodiments, the microneedles or microneedle arrays comprise needles having at least three different microneedle puncture sizes. In some embodiments, the microneedles or microneedle arrays comprise needles having at least four different microneedle puncture sizes. In some embodiments, the microneedles or microneedle arrays comprise needles having at least five different microneedle puncture sizes. In some embodiments, the microneedles or microneedle arrays comprise needles having at most 10 different microneedle puncture sizes. In some embodiments, the microneedles or microneedle arrays comprise needles having at least 11 different microneedle puncture sizes. In some embodiments, the microneedles or microneedle arrays comprise needles having at least 12 different microneedle puncture sizes. In some embodiments, the microneedle or microneedle array comprises needles having a puncture size of at most one microneedle.

[0132] In some embodiments, microneedles or microneedle arrays comprising microneedles of various microneedle puncture sizes can be used in the microneedling techniques described herein. In some embodiments, the microneedles or microneedle arrays can be 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, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 7500, about 8000, about 8500, about 9000, about 9500, about 10000, about 10500, about 11000, about 11500 or about 12000 μm 2 In some embodiments, the microneedles or microneedle arrays have a microneedle puncture size of 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 per microneedle. 2 The microneedles include microneedles having a microneedle puncture size of less than 1 / 4.

[0133] In some embodiments, the microneedles utilized may be solid; in some embodiments, the microneedles utilized may be hollow; in some embodiments, the microneedles utilized may be uncoated; in some embodiments, the microneedles utilized may be coated (e.g., with a composition that may be or include a large drug as described herein).

[0134] In some embodiments, MNs for use according to the present disclosure can be fabricated from different materials using techniques including, but not limited to, micromolding processes or lasers. In some embodiments, MNs can be fabricated using various types of biocompatible materials, including polymers, metals, ceramics, semiconductors, organics, composites, or silicon. Unless they are designed to penetrate and dissolve in the skin, in some embodiments, microneedles have the mechanical strength to remain intact and deliver drugs or collect biological fluids while inserted into the skin and / or removed from the skin after insertion. In some embodiments, MNs can remain in place for up to several days before being completely removed. In some embodiments, microneedles can be sterilized using standard techniques. In some embodiments, MNs are biodegradable. In some embodiments, MNs comprise polymeric materials. In some embodiments, the polymeric material comprises poly-L-lactic acid, poly-glycolic acid, polycarbonate, polylactic-co-glycolic acid (PLGA), polydimethylsiloxane, polyvinylpyrrolidone (PVP), copolymer of methyl vinyl ether and maleic anhydride, sodium hyaluronate, carboxymethylcellulose, maltose, dextrin, galactose, starch, gelatin, or a combination thereof.

[0135] In some embodiments, the MSCs described herein comprise one impression of MNs or MN arrays. In some embodiments, the MSCs comprise two impressions of MNs or MN arrays. In some embodiments, the MSCs comprise three impressions of MNs or MN arrays. In some embodiments, the MSCs comprise four impressions of MNs or MN arrays. In some embodiments, the MSCs comprise five impressions of MNs or MN arrays. In some embodiments, the MSCs comprise six impressions of MNs or MN arrays. In some embodiments, the MSCs comprise seven impressions of MNs or MN arrays. In some embodiments, the MSCs comprise eight impressions of MNs or MN arrays. In some embodiments, the MSCs comprise nine impressions of MNs or MN arrays. In some embodiments, the MSCs comprise ten impressions of MNs or MN arrays. In some embodiments, the MSCs comprise eleven impressions of MNs or MN arrays. In some embodiments, the MSCs comprise 12 impressions of MNs or MN arrays. In some embodiments, the MSCs comprise thirteen impressions of MNs or MN arrays. In some embodiments, the MSCs comprise 14 presses of MNs or MN arrays. In some embodiments, the MSCs comprise 15 presses of MNs or MN arrays. In some embodiments, the MSCs comprise 16 presses of MNs or MN arrays. In some embodiments, the MSCs comprise 17 presses of MNs or MN arrays. In some embodiments, the MSCs comprise 18 presses of MNs or MN arrays. In some embodiments, the MSCs comprise 19 presses of MNs or MN arrays. In some embodiments, the MSCs comprise 20 presses of MNs or MN arrays. In some embodiments, the MSCs comprise rolling the microneedles or microneedle array on the skin one or more times. In some embodiments, the MN array is rotated between presses. In some embodiments, the MN array is not rotated between presses. In some embodiments, the presses are performed on the same site.In some embodiments, the pressing is performed at overlapping sites. In some embodiments, the pressing is performed at different sites. In some embodiments, the pressing is performed by stamping the MN array. In some embodiments, the pressing is performed by rolling a microneedle roller over the site one or more times. In accordance with established MN practice, in some embodiments, the pressing of the MN array against the skin lasts less than 1 second, or in some embodiments, lasts for 1 second or more, e.g., 30 seconds or more, 60 seconds or more, 2 minutes or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, etc.

[0136] In some embodiments, the present disclosure recognizes that the bioavailability of large drugs in emulsions applied to the skin increases as the total surface area of the skin pierced by the microneedles decreases. See, for example, U.S. Patent Application No. 62 / 789,407. Thus, in some embodiments, a relatively smaller force may be preferred. In some embodiments, when the large drug administered in conjunction with microneedle skin conditioning is in a topical formulation that is not (or does not contain) an emulsion (e.g., an emulsion containing a drug), a smaller force of the microneedle array may be preferred. In some embodiments, a shorter microneedle length may be preferred. In some embodiments, when the large drug administered in conjunction with microneedle skin conditioning is in a topical formulation that is not (or does not contain) an emulsion (e.g., an emulsion containing a drug), a relatively shorter microneedle length may be preferred.

[0137] Furthermore, in some embodiments, the present disclosure recognizes that the application of a relatively reduced amount (e.g., volume and / or dose) of a product containing a biologically active substance (e.g., a large drug) in combination with MSCs can achieve a greater biological effect. See, for example, U.S. Patent Application No. 62 / 808,274. Thus, in some embodiments, a relative reduction in the volume of a product containing an active substance (e.g., a large drug) may be preferred. In some embodiments, when the large drug administered in combination with microneedle skin conditioning is in a topical formulation that is or contains an emulsion (e.g., a nanoemulsion), a relatively smaller product volume may be preferred. In some embodiments, when the large drug administered in combination with microneedle skin conditioning is in a topical formulation that is not (or does not contain) an emulsion (e.g., a drug-containing emulsion), a relatively smaller product volume may be preferred. MN arrays and MSC devices suitable for use in combination with compositions containing large drugs for transdermal delivery of large drugs include devices such as those described in U.S. Patent Nos. 6,334,856; 6,503,231; 6,908,453; 8,257,324; and 9,144,671.

[0138] In some embodiments, MSC of the site is performed before (e.g., before the specific application and / or each application) of a formulation (e.g., including an emulsion composition, such as a nanoemulsion composition) that contains and / or delivers a large drug to the site. In some embodiments, MSC of the site is performed after the application of such a formulation to the site. In some embodiments, MSC of the site and the application of such a formulation to the site occur substantially simultaneously.

[0139] In some embodiments, the formulation is not injected through one or more microneedles. In some embodiments, the microneedle is part of an array of microneedles. In some embodiments, the microneedle can be about 1 μm to about 4,000 μm in length. In some embodiments, the microneedle can be about 1 μm to about 2,000 μm in length. In some embodiments, the microneedle can be about 50 μm to about 400 μm in length. In some embodiments, the microneedle can be about 800 μm to about 1,500 μm in length.

[0140] Emulsion Composition In some embodiments, the present disclosure provides and / or utilizes emulsion compositions (e.g., comprising or otherwise utilized with large drugs such as botulinum toxin). In some embodiments, the formulations described herein that comprise and / or deliver large drugs can be or comprise emulsions (e.g., emulsions that include large drugs).

[0141] The present disclosure encompasses the recognition that emulsion technology can provide stabilization benefits to drugs of interest, including large drugs as described herein, and particularly including botulinum toxin and / or antibody drugs.

[0142] Furthermore, the present disclosure recognizes that certain liquid nanoemulsion technologies have been shown to provide superior transdermal delivery properties, even for extremely large molecules such as botulinum and / or antibody drugs. See, e.g., U.S. Patent Application Publication Nos. 2012 / 0328701, 2012 / 0328702, 8,318,181, and 8,658,391, the disclosures of which are incorporated herein by reference in their entireties. These liquid nanoemulsions are far superior to solid nanoparticle drug delivery, particularly transdermal drug delivery, because, as noted by Gomaa, solid nanoparticles cannot penetrate the skin and only accumulate in hair follicles. These liquid nanoemulsions are stable for at least 34 months, making them commercially viable.

[0143] The present disclosure provides specific techniques whereby administering an emulsion composition in conjunction with microneedling achieves surprising results, such as delayed peak effect and / or increased duration of response.

[0144] A particular emulsion composition of interest may be or may 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 comprising an aqueous phase in which oil droplets are dispersed).

[0145] In some embodiments, the emulsion may be or comprise a macroemulsion, characterized by a droplet size ranging from about 300 nm to about 5,000 μm in diameter, for example, hi some embodiments, the emulsion may be or comprise a nanoemulsion, characterized by a droplet size ranging from about 1 nm to about 300 nm in diameter, for example.

[0146] In some embodiments, the use of nanoemulsions may achieve, for example, greater and / or deeper transdermal penetration, which may be at least partially due to the nanoemulsion itself (e.g., compared to that achieved with another composition, including, in some embodiments, a macroemulsion).

[0147] In some embodiments, useful emulsions may be characterized by one or more of an aqueous dispersion medium to oil ratio ranging from about 0.01:1 to about 20:1; an oil to surfactant ratio ranging from about 0.1 to about 40; and / or a zeta potential ranging from about -80 mV to about +80 mV.

[0148] In some embodiments, provided emulsion (e.g., nanoemulsion) compositions comprise an oil and a surfactant in a ratio of about 0.1:1 to about 2:1. In some embodiments, provided emulsion compositions comprise an oil and a surfactant in a ratio of about 0.1:1 to about 1:1. In some embodiments, provided emulsion compositions comprise an oil and a surfactant in a ratio of about 0.5:1 to about 1:1. In some embodiments, provided emulsion compositions comprise an oil and a surfactant in a ratio of about 0.5:1 to about 1:1.5. In some embodiments, provided emulsion compositions comprise an oil and surfactant in a ratio of about 0.1:1, about 0.15:1, about 0.2:1, about 0.25:1, about 0.3:1, about 0.35:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, or about 1:1. In some embodiments, provided emulsion compositions comprise an oil and surfactant in a ratio of about 0.67:1.

[0149] In some embodiments, the aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) and surfactant are utilized in a ratio ranging from 0.01 to 20. In some embodiments, the aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) and surfactant are utilized in a ratio ranging from 0.1 to 20. In some embodiments, the aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) and surfactant are utilized in a ratio ranging from 0.5 to 10. In some embodiments, the aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) and surfactant are utilized in a ratio ranging from 0.5 to 1. In some embodiments, the ratio of aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) to surfactant is about 0.01:1, about 0.02:1, about 0.03:1, about 0.04:1, about 0.05:1, about 0.06:1, about 0.07:1, about 0.08:1, about 0.0:1, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments, the ratio of surfactant to water is about 0.5:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, or about 20:1. In some embodiments, the aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) and surfactant are utilized in a ratio ranging from 0.5 to 2. In some embodiments, the ratio of aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) to surfactant is about 0.5:1, about 1:1, or about 2:1. In some embodiments, the ratio of surfactant to aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) is about 0.5:1, about 1:1, or about 2:1. In some embodiments, the ratio of aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) to surfactant is about 1: 1. In some embodiments, compositions utilizing such a ratio of aqueous dispersion medium (e.g., water, buffer, salt solution, etc.) to surfactant comprise a water-in-oil emulsion.

[0150] In some embodiments, the droplets in the nanoemulsion composition have a diameter (e.g., average diameter and / or median diameter) within the range of about 10 nm to about 300 nm, about 10 nm to about 200 nm, about 10 nm to about 150 nm, about 10 nm to about 130 nm, about 10 nm to about 120 nm, about 10 nm to about 115 nm, about 10 nm to about 110 nm, about 10 nm to about 100 nm, or about 10 nm to about 90 nm. In some embodiments, the droplets in the nanoemulsion composition have a diameter (e.g., average diameter and / or median diameter) within the range of 1 nm to 300 nm, 1 nm to 200 nm, 1 nm to 150 nm, 1 nm to 120 nm, 1 nm to 100 nm, 1 nm to 75 nm, 1 nm to 50 nm, or 1 nm to 25 nm. In some embodiments, the droplets within the nanoemulsion composition have a diameter (eg, mean diameter and / or median diameter) of 1 nm to 15 nm, 15 nm to 200 nm, 25 nm to 200 nm, 50 nm to 200 nm, or 75 nm to 200 nm.

[0151] In some embodiments, the entire droplet distribution falls within the specified range of droplet diameter sizes. In some embodiments, less than 50%, 25%, 10%, 5%, or 1% of the entire droplet distribution is outside the specified range of droplet diameter sizes. In some embodiments, less than 1% of the entire droplet distribution is outside the specified range of droplet diameter sizes.

[0152] In some embodiments, the nanoemulsion composition is substantially free of droplets having a diameter greater than 300 nm, 250 nm, 200 nm, 150 nm, 120 nm, 100 nm, 75 nm, 50 nm, or 25 nm, hi some embodiments, less than 50%, 25%, 10%, 5%, or 1% of the total droplet distribution have a diameter greater than 300 nm, 250 nm, 200 nm, 150 nm, 120 nm, 100 nm, 75 nm, 50 nm, or 25 nm.

[0153] In some embodiments, the droplets in the nanoemulsion composition have an average droplet size of 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 less than about 50 nm. In some embodiments, the average droplet size is within the range of about 10 nm and about 300 nm, about 50 nm and about 250 nm, about 60 nm and about 200 nm, about 65 nm and about 150 nm, or about 70 nm and about 130 nm. In some embodiments, the average droplet size is about 80 nm and about 110 nm. In some embodiments, the average droplet size is about 90 nm and about 100 nm.

[0154] In some embodiments, the emulsion (e.g., nanoemulsion) droplets have a zeta potential in the range of -80 mV to +80 mV. In some embodiments, the emulsion droplets have a zeta potential in the range of -50 mV to +50 mV. In some embodiments, the emulsion droplets have a zeta potential in the range of -25 mV to +25 mV. In some embodiments, the emulsion droplets have a zeta potential in the range of -10 mV to +10 mV. In some embodiments, the emulsion droplets have a zeta potential of about -80 mV, about -70 mV, about -60 mV, 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 emulsion droplets have a zeta potential of about +50 mV, about +40 mV, about +30 mV, about +25 mV, about +20 mV, about +15 mV, about +10 mV, or about +5 mV. In some embodiments, the emulsion droplets have a zeta potential of about 0 mV.

[0155] Among other things, the present disclosure recognizes that the use of emulsion compositions can provide stability to large drugs, such that, for example, the relevant drug may remain intact to a greater extent and / or retain activity to a greater extent and / or for a longer period of time when maintained in an emulsion composition than when maintained under comparable conditions in an otherwise comparable composition.

[0156] The present disclosure recognizes that, despite certain reports in the art that microneedle skin conditioning is useful for facilitating the transdermal delivery of small compounds, the transdermal delivery of large drugs (e.g., botulinum toxin) can be significantly enhanced by using emulsion compositions in combination with microneedle skin conditioning as described herein, including achieving unexpected results such as delayed peak effect and / or extended duration of response. The present disclosure provides the finding that such techniques are particularly useful in certain situations (e.g., treating certain subjects and / or sites thereof, particularly subjects suffering from and / or sites reflecting certain disease disorders or conditions where delayed peak effect and / or extended duration of response may be particularly desirable).

[0157] The present disclosure particularly recognizes that emulsion compositions combined with microneedling can achieve surprisingly effective transdermal delivery, given reports that microneedle conditioning combined with the encapsulation of small molecule drugs in solid nanoparticles (e.g., 105±2.92 nm) provides only a small amount of penetration 6 hours after administration, and no substantial penetration was observed up to 24 hours after administration. For example, Gomaa et al. described a study in which a solution of rhodamine dye (molecular weight 479 Da) encapsulated in PLGA nanoparticles was applied to preconditioned skin with microneedles to evaluate skin penetration. See Gomaa, Y., et al., "Effect of microneedle treatment on the skin permeation of a nanoencapsulated dye." J Pharm Pharmacol. 2012 November; 64(11): 1592-1602. The data showed that very little dye began to penetrate the skin after six hours of continuous application; no significant increase in penetration was observed until the skin was continuously treated for 24 hours. The researchers explained, "There is an emerging consensus that NPs [nanoparticles] can be well deposited in hair follicles, but generally cannot penetrate the stratum corneum." Thus, prior to the present disclosure, one skilled in the art would have expected that the use of microneedling technology with vehicles significantly larger than 105 nm would be unable to effectively deliver even small molecule drugs (e.g., rhodamine dyes) transdermally; indeed, delivery of large drugs would have been considered impossible. However, the present disclosure teaches that microneedling can significantly improve transdermal delivery of large drugs, especially when utilized in combination with emulsion technology, particularly when involving macroemulsion technology, which contains particles or droplets substantially larger than those used by Gomaa and is therefore expected to be less able to achieve transdermal delivery.

[0158] Among other things, the present disclosure recognizes that the provided technology can enhance transdermal delivery (e.g., of large drugs, particularly from nanoemulsion compositions) when other disruptive agents (i.e., chemical penetration enhancers and other technologies that disrupt or puncture skin structures) are not utilized. Thus, in some embodiments, the provided technology can achieve effective delivery without the inflammation, irritation, and / or allergic reactions often associated with the use of skin disruptive agents.

[0159] Previous studies of transdermal delivery of agents similar in size to botulinum toxin (i.e., approximately 150 kDa) using microneedles have reported that delivery fails unless additional treatments are applied to disrupt the skin. For example, U.S. Patent Application Publication No. 2010 / 0196445 reports that botulinum toxin is not effectively delivered from pre-coated microneedles unless skin digestive enzymes are also applied so that skin structure is disrupted at the site of microneedling.

[0160] The present disclosure recognizes, inter alia, that the provided techniques can achieve transdermal delivery (e.g., of large drugs, particularly from macroemulsion and nanoemulsion compositions) when no coating or loading of microneedles is utilized and / or when the microneedles are not designed to be left in the skin. In particular, as previously noted, the present disclosure recognizes that such coating or loading of microneedles may not be commercially viable due to the instability of at least the coating or material loaded thereon (e.g., large drugs, such as botulinum toxin). For example, Johnson, E. et al., states, "Botulinum toxin is highly susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. Freeze-drying of botulinum toxin is the most economically sound and practical method of distributing the product in a form that is stable and easily used by clinicians." U.S. Pat. No. 5,512,547.

[0161] Furthermore, as will be understood by those skilled in the art upon reading this specification, the techniques described herein have certain advantages, including the fact that the microneedles do not need to remain in or be attached to tissue. For example, those skilled in the art will understand that leaving microneedles in the skin risks skin irritation, inflammation, allergic reactions, and / or cosmetically undesirable scarring. In contrast to the present invention, techniques such as those described in U.S. Patent Application Publication No. 2017 / 0209553 utilize microneedle arrays designed to load needles with botulinum and allow the microneedles to penetrate the skin (see U.S. Patent Application Publication Nos. 2017 / 0209553 and 2016 / 0263362; International Publication No. WO / 2018 / 151832).

[0162] The present disclosure surprisingly provides effective technology for the transdermal delivery of large drugs (e.g., botulinum toxin, antibodies, etc.). In particular, the present disclosure teaches that the transdermal delivery of such drugs can be significantly improved by the use of microneedling technology without other disruptive strategies. Thus, the provided technology can achieve effective delivery without the inflammation, irritation, and / or allergic reactions often associated with the use of skin-disrupting agents. As will be understood by those skilled in the art upon reading this specification, the present disclosure teaches that the transdermal delivery of such large drugs can be significantly improved by the use of the provided technology, even when they are not loaded into, coated on, and / or manufactured as part of, microneedles. Similarly, as will be understood by those skilled in the art upon reading this specification, the present disclosure teaches that the delivery of large drugs described herein can be significantly improved by the use of the provided technology (particularly the use of MSCs) without leaving microneedles in the skin (e.g., by breaking them and / or otherwise maintaining and / or degrading them in situ). For example, those skilled in the art will understand that the provided technology can avoid challenges with long-term stability of large drugs necessary for commercially viable products and can achieve effective delivery without the inflammation, irritation, and / or allergic reactions that can result from skin disrupting agents and / or microneedles left in the skin. Indeed, in the Examples and elsewhere, the present disclosure expressly teaches that MSCs implemented with microneedles that do not contain botulinum toxin facilitate transdermal delivery of botulinum toxin from topical (e.g., creams, ointments) compositions, particularly from compositions that include macro- or nano-emulsions.

[0163] In some embodiments, the present disclosure teaches that particularly advantageous results are achieved when microneedling technology is combined with an emulsion composition. In some embodiments, the microneedling technology is combined with a lotion, cream, or liquid composition, which in turn may be or may include an emulsion composition. In some embodiments, the provided technology does not utilize skin-disrupting technology, such as chemical penetration enhancers.

[0164] In some embodiments, the present invention utilizes emulsion compositions containing large drugs that are particularly effective and / or useful in medical settings, for example, for therapeutic purposes. In some embodiments, certain emulsion compositions are particularly effective and / or useful for topical administration of drugs to subjects in need thereof. In some embodiments, the emulsion composition may contain one or more large drugs.

[0165] In some embodiments, the emulsion can be formulated into a composition suitable for topical administration on the skin, hi some embodiments, the composition suitable for topical administration can be a lotion, cream, powder, ointment, liniment, gel, or eye drops.

[0166] In some embodiments, the emulsion formulation comprises water, medium chain triglycerides, Span 65, polysorbate 80, methylparaben, and propylparaben. In some embodiments, the macroemulsion formulation comprises water, medium chain triglycerides, Span 65, and polysorbate 80.

[0167] formulation In some embodiments, large drugs (e.g., botulinum toxin) may be provided and / or utilized in accordance with the present disclosure in compositions formulated for appropriate administration (e.g., topical administration, e.g., to the surface of the skin to achieve transdermal delivery, or, in some embodiments, parenteral administration).

[0168] In some embodiments, the large drug composition (eg, a botulinum toxin composition) can be or can include an emulsion composition, such as a nanoemulsion composition.

[0169] In some embodiments, the large drug composition (e.g., a botulinum toxin composition) may be formulated as a cream, eye drop, foam, gel, liniment, liquid, lotion, ointment, powder, spray, etc. (e.g., in some embodiments, may be or include an emulsion composition, e.g., a nanoemulsion composition).

[0170] It is understood by those skilled in the art that the composition for topical administration can be formulated as, for example, emollient, nourishing lotion type emulsion, cleansing lotion, cleansing cream, skin milk, emollient lotion, massage cream, emollient cream, makeup base, facial pack or facial gel, cleaner formulation, for example, shampoo, rinse, body cleanser, hair tonic or soap, or skin composition, for example, lotion, ointment, gel, cream, patch or spray.In some embodiments, the composition for topical administration is not formulated for administration to mucosa (for example, it is not suitable for application to mucosa and / or is not formulated to deliver an appropriate amount of large drug to or across mucosa).

[0171] In some embodiments, formulations for use (e.g., topical administration) according to the present disclosure may include one or more of purified water, methylparaben, mineral oil, isopropyl myristate, white petrolatum, emulsifying wax, and propylparaben; in some embodiments, formulations for use (e.g., topical administration) according to the present disclosure may include one or more of purified water, mineral oil, isopropyl myristate, white petrolatum, and emulsifying wax.

[0172] Generally, formulations for use according to the present disclosure can be prepared by any suitable method, e.g., known or hereafter developed in the art of pharmacology. Generally, such preparations include combining a provided composition with one or more excipients, and then, if necessary and / or desired, shaping and / or packaging the composition into a suitable form for administration, e.g., single or multiple dose units.

[0173] In some embodiments, useful compositions can be manufactured, packaged and / or sold in bulk as one single unit dose and / or multiple single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of provided composition.In some embodiments, the formulation contains and / or delivers a single dose of relevant active substance (e.g., large drug, such as botulinum toxin), or a convenient portion thereof.

[0174] In some embodiments, additives suitable for use in a composition (e.g., a pharmaceutically and / or cosmetically acceptable composition) may include, for example, one or more additives, such as solvents, dispersion media, granulation media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants and / or emulsifiers, isotonicity agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, disintegrants, binders, preservatives, buffers, etc., as appropriate for the particular dosage form desired. In some embodiments, additives such as cocoa butter and / or suppository wax, colorants, coating agents, sweeteners, flavors and / or fragrances may be utilized. Remington's The Science and Practice of Pharmacy, 21 st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2005; incorporated herein by reference) discloses various additives used in the known art for formulating and manufacturing pharmaceutical compositions.

[0175] In some embodiments, suitable additives (e.g., pharmaceutically and / or cosmetically acceptable additives) are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the additives are approved by the U.S. Food and Drug Administration. In some embodiments, the additives are pharmaceutical grade. In some embodiments, the additives meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other international pharmacopoeias.

[0176] Certain exemplary formulations can be prepared as, for example, cosmetic preparations, such as skin softeners, nourishing lotion-type emulsions, cleansing lotions, cleansing creams, skin milks, emollient lotions, massage creams, emollient creams, makeup bases, facial packs or facial gels, cleaner preparations, such as shampoos, rinses, body cleansers, hair tonics or soaps, or skin compositions, such as lotions, ointments, gels, creams, liniments, patches, deodorants or sprays.In some embodiments, the composition for topical administration is not formulated for administration to mucosa (e.g., it is not suitable for application to mucosa and / or is not formulated to deliver an appropriate amount of large-sized drugs to or across mucosa).

[0177] In some embodiments, the present invention provides certain cream and / or lotion formulations 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 emulsifying wax. In some embodiments, the provided cream and / or lotion formulations comprise propylparaben. In some embodiments, the provided cream and / or lotion formulations are paraben-free. In some embodiments, the provided cream and / or lotion formulations are methylparaben-free. In some embodiments, the provided cream and / or lotion formulations are propylparaben-free.

[0178] An exemplary lotion formulation is provided in Table 1. [Table 1]

[0179] In some embodiments, the cream and / or lotion formulations may be useful for topical and / or transdermal administration.The present invention encompasses the recognition that the provided cream and / or lotion formulations may be particularly useful for delivering drugs to the dermal 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 thereof.In some embodiments, the provided cream and / or lotion formulations are administered to a subject in need thereof by topical and / or transdermal delivery.

[0180] In some embodiments, provided compositions are formulated with cosmetically acceptable ingredients.For example, in some embodiments, provided compositions are formulated with water and any cosmetically acceptable solvent, particularly monoalcohols, such as alkanols having 1-8 carbon atoms (for example, ethanol, isopropanol, benzyl alcohol and phenylethyl alcohol), polyalcohols, such as alkylene glycols (for example, glycerin, ethylene glycol and propylene glycol), and glycol ethers, such as mono-, di- and tri-ethylene glycol monoalkyl ethers, such as ethylene glycol monomethyl ether and diethylene glycol monomethyl ether (used alone or in mixture).Such ingredients can be present, for example, in a proportion of up to 60 wt%, 70 wt%, 80 wt% or 90 wt% based on the total weight of the composition.

[0181] In some embodiments, the provided compositions for topical administration include one or more cosmetically acceptable ingredients that impart desirable or suitable appearance characteristics to the subject to which the composition is applied (e.g., a matte appearance that may be particularly desirable or suitable for administration to subjects with oily skin).

[0182] In some embodiments, provided compositions are formulated with at least one cosmetically acceptable filler material to achieve a mattifying product, which may be particularly desirable, for example, for individuals with oily skin.

[0183] In some embodiments, a botulinum toxin composition formulated for administration (e.g., as a cream or lotion) contains from about 1 to about 200,000 units of botulinum toxin per mL, or from about 1 to about 100,000 units of botulinum toxin per mL, or from about 1 to about 50,000 units of botulinum toxin per mL, or from about 500 to about 20,000 units of botulinum toxin per mL, or from about 100 to about 2,000 units of botulinum toxin per mL, or from about 50 to about 500 units of botulinum toxin per mL, or from about 25 to about 400 units of botulinum toxin per mL. In some embodiments, the botulinum toxin composition formulated for administration (e.g., as a cream or lotion) contains from about 2 to about 40,000 units of botulinum toxin per mL, or about 12,000 units of botulinum toxin per mL, or from about 100 to about 2,000 units of botulinum toxin per mL, or from about 50 to about 1,000 units of botulinum toxin per mL.

[0184] Those skilled in the art will understand that the units herein refer to units that are biologically equivalent or bioactively equivalent to units defined by commercial manufacturers of botulinum toxin.

[0185] In some embodiments, the present invention provides topical formulations of botulinum toxin that avoid potential complications, including, but not limited to, systemic toxicity or botulism. In some embodiments, the dosage of botulinum toxin (including botulinum types A, B, C, D, E, F, or G, or botulinum toxin genetically engineered or chemically modified to have a longer or shorter duration of action than botulinum toxin serotype A) can range from as low as about 1 unit to as high as about 50,000 units, while minimizing the risk of adverse side effects. Specific dosages can vary depending on the condition being treated and the therapeutic regimen being utilized. For example, treatment of subcutaneous overactive muscles may require a high transdermal dose of botulinum toxin (e.g., 1,000 units to 20,000 units). In comparison, treatment of neurogenic inflammation or overactive sweat glands may require a relatively low transdermal dose of botulinum toxin (e.g., about 1 unit to about 1,000 units).

[0186] In some embodiments, one large drug composition can be formulated and delivered in combination with the MSCs described herein to achieve systemic delivery; in some embodiments, provided compositions can be formulated and / or delivered to achieve local, but not systemic, delivery.

[0187] In some embodiments, multiple large drug compositions can be formulated and delivered in combination with the MSCs described herein to achieve systemic delivery; in some embodiments, provided compositions can be formulated and / or delivered to achieve local, but not systemic, delivery.

[0188] In some embodiments, the composition suitable for topical formulation contains a penetration enhancer. In some embodiments, the penetration enhancer degrades, destroys, and / or damages the skin structure and / or skin. In some embodiments, the penetration enhancer does not degrade, destroy, and / or damage the skin structure and / or skin. In some embodiments, the penetration enhancer is irritating. In some embodiments, the penetration enhancer is not irritating.

[0189] The present disclosure specifically demonstrates effective and efficient delivery of therapeutic agents (particularly large biological drugs, e.g., botulinum toxin and / or antibody drugs and / or prophylactic agents, e.g., vaccines) to the dermis using the compositions provided in combination with the MSCs described herein. For example, in some embodiments, the present invention provides methods comprising administration of the compositions described herein without clinically significant side effects. By way of example, when local delivery is contemplated, clinically significant side effects include, but are not limited to, undesirable systemic side effects, damage to nerve tissue underlying the dermis (e.g., nerve paralysis), undesirable effects on muscles (e.g., muscle paralysis), and / or undesirable blood levels of the therapeutic agent.

[0190] Those skilled in the art who read this disclosure will understand that in some embodiments, provided compositions can be incorporated into devices such as patches.Various transdermal patch structures are known in the art; those skilled in the art will understand that provided compositions can be easily incorporated into any of these various structures.In some embodiments, the transdermal patch can include multiple needles extending from one side of the patch that is applied to the skin, where the needles extend from the patch and 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 deep enough to reach nerves in the dermis of the skin.

[0191] In some embodiments, the transdermal patch comprises an adhesive.Some examples of adhesive patches are well known (e.g., U.S. Patent No. 296,006; and U.S. Patent Nos. 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 generally have an adhesive layer that is applied to the patient's skin, a depot or reservoir for holding the provided composition, and an outer surface that prevents the provided composition from leaking from the depot.The outer surface of the patch can be non-adhesive.

[0192] According to some embodiments, the large drug composition can be a patch; in some embodiments, the incorporated large drug remains stable for extended periods of time. For example, in some embodiments, the provided compositions can be incorporated into a polymer matrix that stabilizes the large drug and allows the drug to diffuse from the matrix and the patch. In some embodiments, the large drug composition is incorporated into the adhesive layer of the patch so that when the patch is applied to the skin, the provided composition can diffuse through the skin. In some embodiments, the adhesive layer can be heat-activated, where a temperature of about 37°C slowly liquefies the adhesive so that the drug can diffuse through the skin. The adhesive can remain tacky when stored below 37°C, and when applied to the skin, the adhesive loses its tack as it liquefies.

[0193] In some embodiments, the large drug composition can be provided in a depot within the patch, so that pressure applied to the patch will force the provided composition through the microneedle and the stratum corneum to the outside of the patch.Exemplary embodiments of microneedles are described above.Devices suitable for intradermal administration of the provided compositions include, for example, 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 can be administered by devices that limit the effective penetration length of needles into the skin, such as those described in WO 99 / 34850 and their functional equivalents.

[0194] In some embodiments, it may be desirable to slow the absorption of the provided composition into the skin, for example, to prolong the effect of a large drug composition. In some embodiments, this can be achieved by using a liquid suspension of a crystalline or amorphous material with low water solubility. The absorption rate of the provided composition then depends on its dissolution rate, which in turn may depend on the crystal size and crystalline morphology. 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. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).

[0195] In some embodiments, provided formulations comprise a mixture of a 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 a provided nanoemulsion composition and / or a saline solution.

[0196] In some embodiments, the provided compositions comprise the provided nanoemulsion compositions. In some embodiments, the provided compositions are in cream and / or lotion formulations. In some embodiments, the provided cream and / or lotion formulations comprise the nanoemulsion compositions. In some embodiments, the compositions comprise the provided nanoemulsion compositions but are not cream and / or lotion formulations. In some embodiments, the suitable compositions are formulated into creams and / or lotions but comprise the nanoemulsion compositions.

[0197] In some embodiments, provided compositions comprise a mixture of provided nanoemulsion compositions and one or more pharmaceutically acceptable excipients, e.g., for topical and / or transdermal administration (e.g., via lotions, creams, powders, ointments, liniments, gels, drops, etc.).

[0198] In some embodiments, for nanoemulsion compositions containing known therapeutic agents and / or independently active bioactive agents, such nanoemulsion compositions are configured, constructed, and administered in combination with MSCs to deliver a sufficient amount of therapeutic agent to the desired target site (e.g., the epidermal and / or dermal structures) to treat a condition or disorder. In some embodiments, provided nanoemulsion compositions are configured and constructed (e.g., by drug selection and / or combination, composition structure, etc.) to achieve a desired therapeutic effect upon administration to the skin. In some embodiments, provided nanoemulsion compositions are configured and constructed so as not to induce undesirable clinical effects inside and / or outside the desired site of action (e.g., the skin surface, the dermis, etc.). In some embodiments, provided nanoemulsion compositions are configured, constructed, and administered in combination with MSCs to have a systemic effect.

[0199] In some embodiments, provided compositions can be formulated and delivered in combination with MSCs to achieve systemic delivery; in some embodiments, provided compositions can be formulated and / or delivered to achieve local, but not systemic, delivery.

[0200] The present disclosure specifically demonstrates effective and efficient delivery of therapeutic agents (particularly large biological drugs, such as botulinum toxin or antibody drugs) to the dermis using the provided compositions in combination with MSCs. For example, in some embodiments, the present invention provides methods comprising administering the compositions described herein without clinically significant side effects. By way of example, when local delivery is contemplated, clinically significant side effects include, but are not limited to, undesirable systemic side effects, damage to nerve tissue underlying the dermis (e.g., nerve paralysis), undesirable effects on muscles (e.g., muscle paralysis), and / or undesirable blood levels of the therapeutic agent.

[0201] In some embodiments, the present invention provides topical formulations of large drugs (such as botulinum toxin or antibody drugs) that allow the drug to penetrate the skin of a subject without penetrating blood vessels in significant amounts. For example, in some embodiments of the present invention, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the drug present in the formulation penetrates blood vessels upon application according to the present disclosure.

[0202] Those skilled in the art will appreciate that the compositions of the present invention that achieve transdermal administration of a botulinum toxin or antibody drug can be incorporated into a device, such as a patch, roller, pen, stamp, or the like.

[0203] Purpose In some embodiments, the present disclosure provides macrodrug treatments (e.g., botulinum toxin treatments) comprising administration of one or more doses of an associated macrodrug composition (e.g., a botulinum toxin composition) in combination with microneedling (e.g., microneedle skin conditioning) according to a regimen shown to achieve a delayed onset of effect and / or delayed peak effect described herein (e.g., detectable onset after 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 after about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 months, or more than one year) for one or more doses within the regimen.

[0204] The present disclosure provides the discovery that such techniques are particularly useful in certain circumstances (e.g., in certain subjects and / or sites thereof, particularly subjects suffering from and / or sites reflecting certain disease disorders or conditions where a delayed peak effect and / or extended duration of response may be particularly desirable).

[0205] The present disclosure demonstrates that certain dosing regimens (e.g., with extended times between administrations in light of the extended duration of effect shown herein) can be surprisingly effective and useful in certain circumstances (e.g., when administered to subjects and / or sites afflicted with diseases, disorders, or conditions where a delayed peak effect and / or extended duration of response may be particularly desirable, including for reasons described herein).

[0206] The present invention provides, inter alia, techniques for administering large drugs, such as botulinum toxin or antibody drugs, improving transdermal delivery and / or improving the bioavailability of such large drugs by incorporating one or more large drugs into one or more emulsion compositions that are then administered in combination with MSCs as described herein, which techniques surprisingly provide a delayed peak effect and / or an extended duration of action compared to certain other techniques for delivering the relevant large drug. The inventors have surprisingly found that not only is the transdermal penetration and bioavailability of botulinum toxin or antibody drugs incorporated into the emulsion composition dramatically improved when used in combination with MSCs using microneedles or microneedle arrays with a relatively low microneedle density or a relatively small microneedle puncture size (e.g., puncture size per microneedle, cross-sectional area of each microneedle), but also that an advantage of the present invention is the ability to administer such large drugs intradermally while minimizing irritation or damage to the skin. Furthermore, because certain unexpected results can be achieved, the present disclosure teaches that topical application of emulsion compositions containing and / or delivering large drugs in combination with MSCs is particularly useful in certain situations (e.g., when administered to certain subjects and / or sites). Furthermore, the present disclosure establishes specific administration regimens (e.g., individual administrations of formulations, including emulsion compositions, e.g., nanoemulsions, containing and / or delivering large drugs, are separated by extended periods of time, e.g., in light of extended duration of effect).

[0207] In some embodiments, the large drug is a botulinum toxin. In some embodiments, a botulinum toxin emulsion composition (e.g., in a formulation described herein) is applied directly to the skin and for absorption through the epithelial layer prior to the MSCs. In some embodiments, such a botulinum toxin emulsion composition is applied directly to the skin and for absorption through the epithelial layer after the MSCs. In some embodiments, the botulinum toxin emulsion composition is applied directly to the skin and for absorption through the epithelial layer substantially simultaneously with the MSCs.

[0208] In some embodiments, a botulinum toxin emulsion (e.g., nanoemulsion) composition combined with MSCs can penetrate the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, without the use of penetration enhancers. In some embodiments, a botulinum toxin emulsion composition combined with MSCs can penetrate the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, without the use of disintegrating, irritating, and / or abrasive agents.

[0209] In some embodiments, the antibody drug emulsion composition in combination with MSCs can penetrate the uppermost layers of the skin, including the stratum corneum, skin pores, and / or skin glands, without the use of a penetration enhancer. In some embodiments, the large drug is an antibody drug. In some embodiments, the antibody drug emulsion composition is applied directly to the skin and for absorption through the epidermal layer before the MSCs. In some embodiments, the antibody drug emulsion composition is applied directly to the skin and for absorption through the epidermal layer after the MSCs. In some embodiments, the antibody drug emulsion composition is applied directly to the skin and for absorption through the epidermal layer substantially simultaneously with the MSCs. In some embodiments, the antibody drug emulsion composition is applied directly to the skin and for systemic absorption.

[0210] In some embodiments, the antibody drug emulsion composition can penetrate the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, without the use of disintegrating, irritating, and / or abrasive agents.

[0211] Diseases, Disorders and Conditions The technology provided by this disclosure is useful for treating and / or preventing any of a variety of diseases, disorders and / or conditions, including, in particular, certain systemic or skin diseases, disorders or conditions.

[0212] In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders, or conditions associated with sweat and / or sebaceous gland activity. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders, or conditions associated with infection. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders, or conditions associated with inflammation. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders, or conditions associated with cancer. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders, or conditions that are systemic. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders, or conditions that are autoimmune. In some embodiments, the present invention provides techniques for treating and / or preventing diseases, disorders, or conditions associated with the epithelium and / or dermal level of the skin. In some embodiments, the present invention provides techniques for treating and / or preventing ocular diseases, disorders, or conditions.

[0213] In some embodiments, the present invention provides a method for treating acne, unwanted sweating, body odor, hyperhidrosis, bromhidrosis, chromhidrosis, rosacea, alopecia, psoriasis, actinic keratosis, eczematous dermatitis (e.g., atopic dermatitis, etc.), disorders of excessive sebum production (e.g., seborrhea, seborrheic dermatitis, etc.), burns, Raynaud's phenomenon, lupus erythematosus, hyperpigmentation disorders (e.g., melasma, etc.), hypopigmentation disorders (e.g., plaques, etc.), skin cancer (e.g., squamous cell skin carcinoma, basal cell skin carcinoma, etc.), skin infections (e.g., bacterial infections, viral infections, fungal infections, etc.), facial wrinkles (e.g., wrinkles involving the forehead, glabellar, rhytid, and / or periorbital areas), headaches, eye problems, and eye problems. Techniques are provided for treating and / or preventing one or more of: facial pain (e.g., due to overactivity of the underlying facial musculature), neck wrinkles, hyperfunctional facial wrinkles, hyperkinetic facial wrinkles, platysma bands, décolleté wrinkles, neuromuscular diseases and conditions involving muscle spasms and / or contractures (including facial paralysis, cerebral palsy, blepharospasm, various forms of facial contractures), dystonia, benign prostatic hyperplasia, headaches, strabismus, hemifacial spasm, tremors, spasticity, such as that caused by multiple sclerosis, retroorbital muscles, various ophthalmologic and urological conditions (e.g., penile and / or bladder disorders and penile and scrotal wrinkles), and / or combinations thereof.

[0214] In certain embodiments, the provided techniques may be particularly useful for treating wrinkles, including, for example, wrinkles involving the forehead, glabellar, rhytid and / or periorbital area (including crow's feet), unsightly facial expressions (e.g., due to overactivity of the underlying facial musculature), neck wrinkles, hyperactive facial wrinkles, hyperkinetic facial wrinkles, platysma bands, décolleté wrinkles, hand wrinkles, foot wrinkles, chest wrinkles, penile wrinkles, and scrotal wrinkles.

[0215] 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.

[0216] 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.

[0217] In some embodiments, the present invention provides techniques for treating and / or preventing pulmonary disorders. In some embodiments, the pulmonary disorder can be asthma or chronic obstructive pulmonary disorder.

[0218] In some embodiments, the present invention provides techniques for treating and / or preventing amyloidosis. In some embodiments, the amyloidosis is systemic or cutaneous.

[0219] In some embodiments, the present invention provides techniques for treating and / or preventing cancer, hi some embodiments, the cancer is of the skin, blood, breast, colon, or lung.

[0220] In some embodiments, the present invention provides techniques for treating and / or preventing dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia.

[0221] In some embodiments, the present invention provides techniques for treating and / or preventing an infection, hi some embodiments, the infection is or is caused by C. difficile or Staphylococcus aureus.

[0222] 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.

[0223] In some embodiments, the present invention provides techniques for treating and / or preventing a neurological condition. In some embodiments, the neurological condition is Alzheimer's disease, Parkinson's disease, or stroke.

[0224] In certain embodiments, the provided technology is useful for the treatment and / or prevention of one or more diseases, disorders and conditions, such as certain skin conditions (e.g., acne, rosacea), certain eye disorders (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 disorders (e.g., urinary incontinence, overactive bladder (including in subjects who cannot tolerate side effects associated with other treatments), urinary urgency, etc.), migraines, sweat disorders (e.g., bromhidrosis, chromhidrosis, hyperhidrosis, etc.), wrinkles, etc.

[0225] In some embodiments, the disclosure includes administration according to a dosing regimen sufficient to achieve a reduction in the severity and / or prevalence of the associated skin condition by at least about 20%; 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%; 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%, or %, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90% or more reduction.

[0226] In some embodiments, the invention comprises administration of at least one provided composition administered in combination with MSCs according to a dosing regimen sufficient to achieve a reduction in the severity and / or prevalence of the associated skin condition by at least about 20% in a specified percentage of a patient population to which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve a reduction by at least about 25% in a specified percentage of a patient population to which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve a reduction by at least about 30% in a specified percentage of a patient population to which the composition is administered; in some embodiments, according to a dosing regimen sufficient to achieve a reduction by at least about 31%, about 32%, Approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, 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% %, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90% or more reduction. In some embodiments, the specified percentage of the patient population to which the composition is administered is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. To give some examples, in some embodiments, the invention involves administration of at least one provided composition according to a dosing regimen sufficient to achieve a reduction in the severity and / or prevalence of the associated skin condition in at least about 20% of the patient population to which the composition is administered.In some embodiments, the invention involves administration of at least one provided composition according to a dosing regimen sufficient to achieve a reduction in the severity and / or prevalence of the associated skin condition in at least about 30% of a patient population to which the composition is administered.

[0227] The present invention provides techniques for treating and / or preventing skin diseases, comprising administering a composition in combination with MSCs to a subject suffering from, susceptible to, and / or exhibiting symptoms of a skin disease. In some embodiments, the provided compositions for treating the skin diseases described herein are formulated for any of the administration routes described herein. In some embodiments, the provided compositions are formulated for topical administration. In some embodiments, the provided compositions are formulated as creams, liniments, lotions, gels, shampoos, conditioners, sunscreens, deodorants and / or antiperspirants (e.g., as roll-ons, solid sticks, gels, creams, aerosols, etc.), etc., depending on the condition to be treated.

[0228] In some embodiments, such provided compositions are administered locally in combination with MSCs to the affected area (e.g., the axilla, hands, feet, scalp, hair follicles, face, neck, back, arms, chest, legs, groin, groin, etc., depending on the particular condition being treated). In some embodiments, local administration is achieved by topical administration in combination with MSCs.

[0229] combination In some embodiments, the techniques described herein can be utilized to administer a large drug (eg, a botulinum toxin) in combination with another drug and / or treatment.

[0230] In some embodiments, the provided technology can be utilized to administer multiple large drugs (eg, a botulinum toxin in combination with one or more other large drugs, such as one or more antibodies).

[0231] In some embodiments, the provided technology can be utilized in combination with one or more penetration enhancing technologies (e.g., one or more penetration enhancers); in some embodiments, no penetration enhancers are utilized. In some embodiments, the provided technology can be utilized with one or more penetration enhancers that are non-irritating and / or do not degrade, disrupt, and / or damage the skin structure and / or skin. In some embodiments, the non-irritating penetration enhancer can be selected from, for example, a copeptide, a carrier molecule, and a carrier peptide. In some embodiments, the carrier molecule is positively charged. In some embodiments, the carrier molecule can be a copeptide. In some embodiments, the carrier molecule can be a long-chain positively charged polypeptide or a positively charged non-peptidyl polymer, such as a polyalkyleneimine. In some embodiments, the carrier peptide can be a cationic peptide. In some embodiments, the carrier peptide has the sequence RKKRRQRRRG-(K) 15 -GRKKRRQRRR. In some embodiments, the carrier molecule can be one disclosed in U.S. Patent Application Publication No. 2010 / 0168023 or U.S. Patent Application Publication No. 2009 / 0247464, the contents of which are incorporated herein by reference in their entirety.

[0232] In some embodiments, the provided technology can be used in combination with one or more treatments that act on or within the skin and / or have therapeutic and / or cosmetic effects.In some embodiments, the bioactive substance used in combination with the antibody drug described herein can be a drug that acts on or within the skin and / or has therapeutic and / or cosmetic effects.For example, the provided technology can be used in combination with one or more therapeutic drugs, such as anesthetics (e.g., lidocaine), steroids (e.g., hydrocortisone) and / or retinoids (e.g., retinoin), cosmetic drugs, such as dermal fillers (e.g., hyaluronic acid or other elastic materials), collagen, and / or silicone.

[0233] Target The technology of the present disclosure is suitable for both human and veterinary use.The subjects that can benefit from treatment with the technology described herein include, in particular, subjects suffering from the diseases, disorders or conditions described herein.In certain embodiments, the subject suffers from diseases, disorders or conditions that include cosmetic and / or visually obvious features, effects or characteristics.Other contributions of the present disclosure include the finding that for certain such subjects, delaying peak effect and / or prolonging effect duration may be particularly desirable.

[0234] In some embodiments, the administration site is the skin overlying a 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 in 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 décolleté. In some embodiments, the site is in the armpit. In some embodiments, the site is in the axilla. 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.

[0235] In some embodiments, the site is affected by skin disease. In some embodiments, the site is the skin covering the muscle or muscle group affected by neuromuscular condition. In some embodiments, the length of the microneedle used with MSCs is adjusted based on the thickness of the skin at the treatment site.

[0236] In many embodiments, the subject is afflicted with wrinkles and / or the techniques described herein are applied to an area of wrinkles (eg, an area of wrinkled skin).

[0237] In some embodiments, the provided technology demonstrates that certain compositions described herein, in combination with the MSCs described herein, can achieve efficient and specific, controlled and / or improved delivery of active agents to biologically relevant target sites (e.g., specific tissues, locations within the skin, cells, etc.). In some embodiments, the present invention demonstrates controlled delivery and / or achievement of therapeutic effects at specific, biologically relevant target sites without the significant side effects associated with delivery to other areas.

[0238] In some embodiments, the provided technology can efficiently improve the delivery and / or bioavailability of active substances and specific delivery to the dermis, and / or can have cosmetic and / or therapeutic effects when administered to the skin of a subject. In some embodiments, the present invention demonstrates improved delivery and / or bioavailability through the dermis and / or the achievement of therapeutic effects without significant side effects associated with delivery to other areas (e.g., subdermal or extradermal structures and / or tissues other than the dermis). In some embodiments, the provided technology can improve the transdermal delivery and / or bioavailability of active substances, such as therapeutic agents (e.g., botulinum toxin, antibody drugs, etc.).

[0239] In some embodiments, large drugs administered in accordance with the present disclosure penetrate the skin within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes of administration. In some embodiments, large drugs penetrate the skin within about 5 to about 60 minutes of administration. In some embodiments, large drugs penetrate the skin within about 5 to about 12 minutes of administration. In some embodiments, large drugs penetrate the skin within about 5 to about 15 minutes of administration. In some embodiments, large drugs penetrate the skin within about 15 to about 30 minutes of administration. In some embodiments, large drugs penetrate the skin within about 1 hour of administration. In some embodiments, large drugs penetrate the skin within about 2 hours of administration. In some embodiments, large drugs penetrate the skin within about 3 hours of administration. In some embodiments, large drugs penetrate the skin within about 4 hours of administration. In some embodiments, large drugs penetrate the skin within about 5 hours of administration. In some embodiments, large drugs penetrate the skin within about 6 hours of administration.

[0240] In some embodiments, the large drug penetrates into skin layers within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes of administration. In some embodiments, the large drug penetrates into skin layers within about 5 to about 60 minutes of administration. In some embodiments, the large drug penetrates into skin layers within about 5 to about 12 minutes of administration. In some embodiments, the large drug penetrates into skin layers within about 5 to about 15 minutes of administration. In some embodiments, the large drug penetrates into skin layers within about 15 to about 30 minutes of administration. In some embodiments, the large drug penetrates into skin layers within about 1 hour of administration. In some embodiments, the large drug penetrates into skin layers within about 2 hours of administration. In some embodiments, the large drug penetrates into skin layers within about 3 hours of administration. In some embodiments, the large drug penetrates into skin layers within about 4 hours of administration. In some embodiments, the large drug penetrates into skin layers within about 5 hours of administration. In some embodiments, the large drug penetrates into skin layers within about 6 hours of administration.

[0241] In some embodiments, the large drug penetrates the top layer of the skin within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 5 to about 60 minutes of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 5 to about 12 minutes of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 5 to about 15 minutes of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 15 to about 30 minutes of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 1 hour of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 2 hours of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 3 hours of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 4 hours of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 5 hours of administration. In some embodiments, the large drug penetrates the top layer of the skin within about 6 hours of administration.

[0242] In some embodiments, the large drug penetrates into the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes of administration. In some embodiments, the large drug penetrates into the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 5 to about 60 minutes of administration. In some embodiments, the large drug penetrates into the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 5 to about 12 minutes of administration. In some embodiments, the large drug penetrates into the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 5 to about 15 minutes of administration. In some embodiments, the large drug penetrates into the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 15 to about 30 minutes of administration. In some embodiments, the large drug penetrates into the top layers of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 1 hour of administration. In some embodiments, the large drug penetrates into the top layer of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 2 hours of administration. In some embodiments, the large drug penetrates into the top layer of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 3 hours of administration. In some embodiments, the large drug penetrates into the top layer of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 4 hours of administration. In some embodiments, the large drug penetrates into the top layer of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 5 hours of administration. In some embodiments, the large drug penetrates into the top layer of the skin, including the stratum corneum, skin pores, and / or skin glands, within about 6 hours of administration.

[0243] kit In some embodiments, the present invention provides a pharmaceutical package or kit, comprising one or more emulsion compositions and one or more microneedle devices for use according to the present invention.In some embodiments, the pharmaceutical package or kit comprises a formulation or pharmaceutical composition, comprising a composition provided in one or more containers, optionally filled with one or more additional components of the pharmaceutical composition.In some embodiments, the pharmaceutical package or kit comprises an additional approved therapeutic agent for use in combination therapy (for example, benzoyl peroxide for the treatment of acne; aluminum compounds for the treatment of hyperhidrosis, etc.).In some embodiments, the container can optionally be associated with a notice in the form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, and this notice reflects the agency's approval of manufacture, use, or sale for human administration.

[0244] In some embodiments, a kit containing therapeutic reagent is provided.As a non-limiting example, provided composition can be provided as a topical preparation and can be administered as a treatment combined with the use of microneedling device.Medicinal dosage or the instructions for self-administration thereof can be provided in the kit for administering to individuals who suffer from or are at risk of conditions or disorders, such as those related to the dermis level of the skin.

[0245] In some embodiments, the kit may include (i) a provided composition; and (ii) at least one pharmaceutically acceptable excipient; and (iii) at least one device for microneedling of the skin; and (iv) instructions for use. In some embodiments, at least one device has a relatively low microneedle density (e.g., about 2 microneedles / cm). 2 ~approximately 50 microneedles / cm 2In some embodiments, for example, at least one device may include microneedles having a relatively small microneedle puncture size (e.g., a puncture size of about 100 μm per microneedle). 2 / Microneedle ~ approx. 30,000 μm 2 / Microneedle range, puncture size per microneedle is approximately 100 μm 2 / Microneedle ~ approx. 60,000 μm 2 / microneedle range). [Example]

[0246] Example 1 : Effect of MSC preconditioning on the timing of peak therapeutic effect and duration of therapeutic effect in reducing crow's feet wrinkles This example describes a study that evaluated 1) the timing of peak therapeutic effect and 2) the duration of therapeutic effect for topically administered botulinum toxin (in a nanoemulsion formulation) in combination with microneedle skin preconditioning on the reduction of crow's feet wrinkles.

[0247] The study included two test groups, Group A and Group B, including subjects with moderate to severe crow's feet. Each subject's crow's feet area was treated topically once with a botulinum toxin emulsion (e.g., nanoemulsion) formulation (Group A) or its vehicle placebo control (Group B). Subjects in Group A (N=9) and Group B (N=9) underwent skin preconditioning with an identically matched set of microneedle arrays. Administration of the topical formulation to the skin took approximately 5 minutes, by which time the topical formulation was completely absorbed into the skin. All subjects were preconditioned with eight microneedle strokes of the microneedle array before application of the botulinum toxin formulation.

[0248] The expected effect of botulinum toxin treatment is a reduction in crow's feet at the site of botulinum treatment. Wrinkle severity is measured by both the investigator and the subject using a 5-point wrinkle scale (Wrinkle Scale) with 0 = none, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe. As generally accepted in the field, a subject was considered a "responder" for purposes of peak effect when both the investigator and the subject rated a 2-point or greater reduction in wrinkle severity at contraction compared to baseline, and a "responder" for purposes of duration of effect when the investigator rated a 1-point or greater reduction in wrinkle severity at contraction compared to baseline.

[0249] The study described in this example showed that at baseline, the mean severity of crow's feet wrinkles, as measured by the wrinkle scale, was approximately equal between Groups A and B.

[0250] In terms of peak efficacy, at 1 month after treatment, Group A had an 11% responder rate and Group B had a 0% responder rate; at 2 months after treatment, Group A had an 11% responder rate and Group B had a 0% responder rate; at 3 months after treatment, Group A had a 33% responder rate and Group B had a 0% responder rate.

[0251] Regarding the duration of treatment effect, at 6 months after treatment, Group A had a 56% responder rate. In comparison, literature reports of studies using commercially available botulinum preparations administered by injection into the crow's feet have documented a 15% responder rate at approximately 6 months using this same responder metric; other studies have found a median duration of treatment effect of commercially available botulinum preparations to be 3.5 to 4 months.

[0252] Figure 1 shows a comparison of the results achieved (specifically, the observed responder rates) at specific time points (specifically, 18 and 28 weeks) in Group A of this study compared to literature reports of an approved botulinum toxin injection formulation administered according to its approved regimen for treating crow's feet. As shown in Figure 1, at 18 weeks, the responder rate for the injectable formulation was less than 50%, while the responder rate for the technology described herein was greater than 80%. Furthermore, the provided technology achieved a higher responder rate (i.e., greater than 70%) at 26 weeks than the responder rate for the injectable formulation at 18 weeks.

[0253] The studies described in this example demonstrate the unexpected results described herein that topical administration of botulinum toxin (e.g., in a nanoemulsion formulation) in conjunction with microneedle skin preconditioning can extend the time to peak therapeutic effect and further extend the duration of the therapeutic effect of botulinum toxin treatment (e.g., for wrinkles).

[0254] Example 2 : Effect of MSC preconditioning on the timing of peak therapeutic effect compared with commercially available injectable botulinum toxin This example describes a study comparing the timing of peak therapeutic effect in reducing crow's feet wrinkles for topically administered botulinum toxin (in a nanoemulsion formulation) in combination with microneedle skin preconditioning as described herein to reports describing the timing of peak therapeutic effect in crow's feet wrinkles for botulinum toxin administered by injection.

[0255] As described above, Group A (N=9) subjects with moderate to severe crow's feet were treated once topically with a botulinum toxin emulsion in combination with skin preconditioning with a microneedle array (specifically, eight microneedle presses of the microneedle array prior to application of the botulinum toxin formulation). Administration of the topical formulation to the skin took approximately 5 minutes, by which time the topical formulation was completely absorbed into the skin.

[0256] The results of this study were compared to literature reports describing the treatment of subjects receiving approved botulinum toxin injection therapy. Specifically, Group B (N=48) was treated with a first commercially available botulinum toxin injection, and Group C (N=222) was treated with a different second commercially available botulinum toxin injection.

[0257] Figure 2 compares the results of these studies. The expected effect of botulinum toxin treatment is a reduction in crow's feet wrinkles at the site of botulinum treatment. For Group A, wrinkle severity was measured by each investigator using a 5-point wrinkle scale (Wrinkle Scale): 0 = none, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe. For Groups B and C, wrinkle severity was measured by each investigator using a 4-point wrinkle scale (Wrinkle Scale): 0 = none, 1 = mild, 2 = moderate, and 3 = severe. Subjects were considered "responders" for purposes of peak efficacy when the investigators rated the wrinkle score as mild or a more favorable score (e.g., none).

[0258] As is evident from reference to Figure 2, all three treatments ultimately achieve comparable peak effects; therefore, one skilled in the art will understand that the related regimens can be considered equivalent despite the fact that they may involve different absolute "doses" (whether considered in terms of units of botulinum toxin and / or amounts (e.g., ng) of botulinum toxin) contained in a single administered dose.

[0259] With respect to peak efficacy, Figure 2 shows that at one month post-treatment, Group A had a responder rate of 44%, Group B had a responder rate of 60%, and Group C had a responder rate of 67%; at two months post-treatment, Group A had a responder rate of 44%, Group B had a responder rate of 54%, and Group C had a responder rate of 56%; and at three months post-treatment, Group A had a responder rate of 56%, Group B had a responder rate of 30%, and Group C had a responder rate of 39%. See Figure 1. A peak efficacy was observed for Group A within a time range of three months after administration of the botulinum toxin dose; for Groups B and C, a peak efficacy was observed at one month post-administration.

[0260] The studies described in this example demonstrate the unexpected result described herein that topical administration of botulinum toxin (e.g., in a nanoemulsion formulation) in conjunction with microneedle skin preconditioning can extend the time to peak therapeutic effect. As described herein, those skilled in the art will also understand that this demonstration of delayed peak effect also indicates an extended duration of response.

[0261] Example 3 Effect of MSC preconditioning on the timing of peak therapeutic effect compared with local treatment without MSC preconditioning This example describes a study that evaluated the timing of peak therapeutic effect in reducing crow's feet wrinkles for topically administered botulinum toxin (in a nanoemulsion formulation) in combination with microneedle skin preconditioning compared to a group treated with topically administered botulinum toxin (in a nanoemulsion formulation) without microneedle skin preconditioning.

[0262] The study included two test groups, Group A and Group B, which included subjects with moderate to severe crow's feet. Each subject's crow's feet area was topically treated once with an equivalent dose of a botulinum toxin emulsion (e.g., nanoemulsion) formulation. Subjects in Group A (N=26) did not receive microneedle skin preconditioning, while subjects in Group B (N=9) received skin preconditioning with a microneedle array. Administration of the topical formulation to the skin took approximately 5 minutes, by which time the topical formulation was completely absorbed into the skin.

[0263] The expected effect of botulinum toxin treatment is a reduction in crow's feet at the site of botulinum treatment. Wrinkle severity is measured by the investigator and the subject using a 5-point wrinkle scale (wrinkle scale) with 0 = none, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe. As generally accepted in the field, a subject was considered a "responder" for purposes of peak effect when the investigator assessed a 2-point or greater reduction in wrinkle severity at contraction compared to baseline.

[0264] Group A was found to have a peak response rate at 1 month after treatment; Group B was found to have a peak response rate at 6 months after treatment.

[0265] The studies described in this example demonstrate the unexpected result described herein that topical administration of botulinum toxin in a nanoemulsion formulation in conjunction with microneedle skin preconditioning can extend the time to peak therapeutic effect compared to topical administration of botulinum toxin in a nanoemulsion formulation without microneedle skin preconditioning.

[0266] Example 4 : Effect of needle length on timing of peak effect U.S. Patent Application No. 62 / 808,274 describes a single-dose topical study of the bioavailability of botulinum toxin in humans after topical administration of a botulinum nanoemulsion formulation in combination with microneedle skin conditioning. The study examined the effect of varying the length of the microneedles on improving the bioavailability of botulinum toxin in humans by measuring the reduction in skin wrinkles after topical treatment with this formulation following skin conditioning with a microneedle array.

[0267] The study included three groups, Group A, Group B, and Group C, each of which included subjects with moderate to severe crow's feet. Each subject's crow's feet area was treated once with a topical botulinum emulsion formulation, specifically a nanoemulsion. Group A (N=9) subjects underwent skin preconditioning using a 500 μm needle length, Group B (N=9) subjects underwent skin preconditioning using an 800 μm needle length, and Group C (N=9) subjects underwent skin preconditioning using a 1400 μm needle length.

[0268] Administration of the topical formulation to the skin in this particular study took approximately 5 minutes, at which point the topical formulation was completely absorbed by the skin. All subjects were preconditioned with the same number of microneedle impacts of the microneedle array prior to application of the botulinum formulation. The dose of botulinum used in Groups A, B, and C was matched across subjects.

[0269] The expected effect of botulinum nanoemulsion treatment is a reduction in crow's feet wrinkles at the site of botulinum nanoemulsion treatment; such reduction was assessed for the various treatments applied in this example. Wrinkle severity is measured by both the investigator and the subject using a 5-point wrinkle scale (wrinkle scale): 0 = none, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe. Responders in this study were subjects with a reduction in wrinkle severity of 2 or more points compared to baseline, as assessed by both the investigator and the subject.

[0270] This study found that at baseline, the mean severity of crow's feet wrinkles, as measured by the wrinkle scale, was approximately equal in Groups A, B, and C. At 12 weeks post-treatment, the responder rate for Group A was 36%, the responder rate for Group B was 14%, and the responder rate for Group C was 13%. This study demonstrated that using shorter microneedles during microneedle skin preconditioning unexpectedly increased the bioavailability of topical large drug nanoemulsions, particularly those where the large drug included botulinum toxin.

[0271] This example further establishes that the use of such shorter microneedles surprisingly prolongs the peak effect compared to that observed with longer microneedles. Without the present disclosure, it would have been reasonable to predict that the length of the microneedles would have no effect on the timing of the peak effect, or if it had an effect, such effect would be to accelerate (rather than delay) the peak effect.

[0272] The above tests were performed using shorter (e.g., 500 μm long) versus longer (e.g., 1500 μm long) microneedles, with peak effects observed substantially later (e.g., about 4-8 weeks later) with the shorter microneedles.

[0273] Example 5 Exemplary emulsion compositions In some embodiments, an exemplary macroemulsion can be: [Table 2]

[0274] In some embodiments, a nanoemulsion can be produced from a premix composition (e.g., by subjecting it to shear forces, such as by microfluidization, which in some embodiments can be single-pass microfluidization). In some embodiments, an exemplary premix composition can include: [Table 3]

[0275] Exemplary nanoemulsion formulations, which are not meant to be limiting, are provided in Table 3. [Table 4]

[0276] Exemplary formulations of botulinum nanoemulsion premixes, which are not meant to be limiting, are provided in Table 4. [Table 5]

[0277] (Equivalent) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but is instead set forth in the following claims.

Claims

1. A method of treating a subject in need of treatment, comprising administering multiple doses of a composition that delivers a large drug having a molecular weight of 100,000 Da or greater to a site on the skin of the subject in combination with microneedle skin conditioning (MSC) according to a dosing regimen in which at least two consecutive doses are separated from each other by a period of at least one month.

2. 10. The method of claim 1, wherein the period is at least two months.

3. 10. The method of claim 1, wherein the period is at least four months.

4. 10. The method of claim 1, wherein the period is at least six months.

5. 10. The method of claim 1, wherein the period is at least 8 months.

6. 10. The method of claim 1, wherein the period is at least 10 months.

7. 10. The method of claim 1, wherein the period is at least 12 months.

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

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

10. 9. The method of claim 8, wherein the average duration is at least 4 months.

11. 9. The method of claim 8, wherein the average duration is at least 6 months.

12. 9. The method of claim 8, wherein the average duration is at least 8 months.

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

14. 9. The method of claim 8, wherein the average period is at least 12 months.

15. 9. The method of claim 8, wherein each period is the same.

16. 16. The method of claim 15, wherein each period is at least one month.

17. 16. The method of claim 15, wherein each period is at least two months.

18. 16. The method of claim 15, wherein each period is at least four months.

19. 16. The method of claim 15, wherein each period is at least six months.

20. 16. The method of claim 15, wherein each period is at least 8 months.

21. 16. The method of claim 15, wherein each period is at least 10 months.

22. 16. The method of claim 15, wherein each period is at least 12 months.

23. 23. The method of any one of claims 1 to 22, wherein administering comprises topically applying the composition to the site.

24. 24. The method of any one of claims 1 to 23, wherein the composition comprises a nanoemulsion.

25. The method of any one of claims 1 to 24, wherein the composition comprises a macroemulsion.

26. 26. The method of any one of claims 1 to 25, further comprising the step of administering a non-irritating penetration enhancer.

27. 27. The method of claim 26, wherein the non-irritating penetration enhancer is selected from a carrier peptide and a copeptide.

28. The non-irritating penetration enhancer has the sequence RKKRRQRRRG-(K) 15 The method according to any one of claims 26 to 27, wherein the cationic peptide is selected from cationic peptides having the formula -GRKKRRQRRR and positively charged carriers.

29. 29. The method of any one of claims 1 to 28, wherein the administering process comprises administering MSCs prior to administering the composition to the site.

30. 30. The method of any one of claims 1 to 29, wherein the administering step comprises administering the composition to the site followed by administering MSCs.

31. 31. The method of any one of claims 1 to 30, wherein the administering step comprises administering the MSCs simultaneously with administering the composition to the site.

32. 32. The method of any one of claims 1 to 31, wherein the large drug is a botulinum toxin.

33. 33. The method of claim 32, wherein the administration process is further combined with a cosmetic or therapeutic agent.

34. 34. The method of claim 33, wherein the cosmetic or therapeutic agent is selected from the group consisting of anesthetics, collagen, fillers, retinoids, silicones, steroids, and combinations thereof.

35. 35. The method of claim 34, wherein the cosmetic or therapeutic agent is selected from the group consisting of hydrocortisone, retin A, lidocaine, and combinations thereof.

36. 36. The method of any one of claims 1 to 35, wherein the large drug is an antibody drug.

37. The method of claim 36, wherein the antibody drug is selected from the group consisting of an anti-TNFα antibody, an anti-CD2 antibody, an anti-CD4 antibody, an anti-IL-12 antibody, an anti-IL-17 antibody, an anti-IL-22 antibody, and an anti-IL-23 antibody.

38. 38. The method of any one of claims 36-37, wherein the antibody drug is selected from the group consisting of antibodies having an epitope binding element found in one or more of infliximab, adalimumab, golimumab, etanercept, etanercept-szzs, certolizumab pegol, siplizumab, zanolimumab, briakinumab, secukinumab, brodalumab, fezakinumab, ustekinumab and / or guselkumab.

39. The method of any one of claims 36 to 38, wherein the administration process is further combined with a cosmetic or therapeutic agent.

40. 40. The method of any one of claims 36 to 39, further comprising administering a non-irritating penetration enhancer.

41. 41. The method of claim 40, wherein the non-irritating penetration enhancer is selected from a copeptide and a carrier peptide.

42. 42. The method of any one of claims 1 to 41, wherein the MSC is achieved with a device comprising at least one microneedle (MN).

43. The method of claim 42, wherein the device comprises a plurality of MNs.

44. 44. The method of claim 42 or 43, wherein the device is a patch, roller, stamp, or pen.

45. 45. The method of any one of claims 1 to 44, wherein the site is a skin surface overlying a muscle or muscle group.

46. The method of any one of claims 1 to 45, wherein the site is a skin surface containing sweat glands.

47. The method according to any one of claims 1 to 46, wherein the site is a skin surface containing a sebaceous gland.

48. 48. The method of any one of claims 1 to 47, wherein the site is a skin surface containing hair follicles.

49. 48. The method of any one of claims 42 to 47, wherein the MNs have a length sufficient to protrude through the stratum corneum of the skin.

50. 50. The method of any one of claims 42 to 49, wherein the MNs have an insufficient length to reach nerves in the dermis of the skin.

51. The method of any one of claims 42 to 50, wherein the MN is composed of a biocompatible material.

52. 52. The method of any one of claims 42 to 51, wherein the MN is composed of a metal.

53. The method of any one of claims 42 to 51, wherein the MN is composed of at least one soluble polymer.

54. 54. The method of any one of claims 1 to 53, wherein the MSC comprises administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 microneedle (MN) impacts, each impact occurring during a period of continuous contact between the site and the device comprising one or more MNs.

55. 55. The method of claim 54, wherein the device is or includes a stamp containing one or more MNs.

56. 55. The method of claim 54, wherein the device is or comprises a roller containing one or more MNs.

57. 55. The method of claim 54, wherein the device is or comprises a patch containing one or more MNs.

58. 58. The method of any one of claims 54 to 57, wherein the device comprises a plurality of MNs, the plurality of MNs being arranged in a geometric pattern.

59. 55. The method of claim 54, wherein the MSCs comprise multiple push administrations.

60. 60. The method of claim 59, wherein the two or more compressions are applied to approximately the same area.

61. 60. The method of claim 59, wherein the two or more pressings are applied at overlapping locations.

62. 60. The method of any one of claims 54 to 59, wherein each pressing is performed at a different site.

63. 63. The method according to any one of claims 54 to 62, wherein the pressing is performed by stamping.

64. 63. The method of any one of claims 54 to 62, wherein the pressing is performed by rolling.

65. 65. The method of any one of claims 1-64, wherein the administration process achieves delivery of the large drug to the skin within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

66. 66. The method of any one of claims 1-65, wherein the administration process achieves delivery of the large drug to the skin within 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.

67. 67. The method of any one of claims 1-66, wherein the administration process achieves delivery of the large drug to the skin within about 1, 2, 3, 4, 5, or 6 hours.

68. 68. A method of treating a skin disease, disorder or condition, comprising the method of any one of claims 1 to 67.

69. 69. The method of any one of claims 1-68, wherein the subject is suffering from or susceptible to a skin disease, disorder or condition, and wherein the administration achieves amelioration of one or more characteristics or symptoms of the skin disease, disorder or condition.

70. 70. The method of claim 68 or 69, wherein the skin disease, disorder or condition is selected from acne, actinic keratosis, body odor, bromhidrosis, burns, chromhidrosis, skin infections, eczematous dermatitis, excessive sebum production disorders, facial wrinkles, hair loss, hyperfunctional facial wrinkles, hyperhidrosis, hyperkinetic facial wrinkles, hyperpigmentation disorders, hypopigmentation disorders, keloids, linear scleroderma, lupus erythematosus, neck wrinkles, platysma bands, psoriasis, Raynaud's syndrome, rosacea, scleroderma, skin cancer, unsightly facial expressions, unwanted sweating, and / or combinations thereof.

71. 71. The method of claim 1, further comprising administering to a subject a therapeutically effective amount of ... Disorders (e.g., bromhidrosis, chromhidrosis, skin infections, discoid lupus, drug-induced lupus, eczematous dermatitis, excessive sebum production disorders, facial wrinkles, hair loss, hyperfunctional facial wrinkles, hyperhidrosis, hyperkinetic facial wrinkles, hyperpigmentation disorders, hyperplasia (e.g., prostatic hyperplasia), hypopigmentation disorders, inflammatory bowel disease, keloids, linear scleroderma, lupus erythematosus, neck wrinkles, neonatal lupus, osteoarthritis, platysma bands, psoriasis, psoriatic arthritis a method of treating or preventing a disease, disorder or condition selected from arthritis, pulmonary disorders, Raynaud's syndrome, rheumatoid arthritis, rosacea, scleroderma, skin cancer, systemic amyloidosis, systemic lupus, ulcerative colitis, unsightly facial expression, unwanted sweating, dyslipidemia, hypercholesterolemia, infection, C. difficile infection, staphylococcal infection, dystonia, headache, pain, arthritis-related pain, rheumatoid arthritis-related pain, psoriatic arthritis-related pain, osteoarthritis-related pain, certain ophthalmological conditions, certain urological conditions, neuromuscular diseases, conditions involving muscle spasms and / or contractures, strabismus, hemifacial spasm, tremor, spasticity, such as that caused by multiple sclerosis, retroorbital muscles, neurological conditions, migraine or other headaches, Alzheimer's disease, Parkinson's disease, or stroke, and / or combinations thereof.

72. 72. The method of any one of claims 1 to 71, wherein the composition is formulated as a lotion, cream, powder, ointment, liniment, gel, or drops.

73. A method of treating a subject in need of treatment, comprising administering to a site on the skin of the subject a composition that delivers a large drug having a molecular weight of 100,000 Da or greater in combination with microneedle skin conditioning (MSC) according to a dosing regimen that achieves a peak effect of the large drug at about one month or later after administration.

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