Transdermal delivery

Abstract

Offering transdermal delivery. [Solution] This application describes the delivery, formulation, and use of a system containing a drug that can penetrate the skin. This disclosure provides various insights into the transdermal delivery of drugs (including, for example, crosslinkable entities) and / or the techniques for realizing cosmetic or therapeutic activity or benefits in the skin. In some embodiments, this disclosure provides insights into the fact that certain advantages can be realized when using microneedling techniques. For example, in some embodiments, microneedling techniques can enhance or realize, for example, the delivery of a drug to a specific target site.

Description

[Technical Field]

[0001] Cross-reference of related applications The benefit of priority is claimed against U.S. Provisional Application No. 63 / 106,623, filed on 28 October 2020 (28.10.2020). The entire contents of this document are incorporated herein by reference in accordance with PCT Rule 20.6. [Background technology]

[0002] Various systems have been developed to establish or generate crosslinking materials at or on specific target sites, for example, at sites of surgical or traumatic injury (e.g., organs, connective tissue, muscles, tendons and / or membranes; see, e.g., Seal et al. Mater Sci. Eng 34:147, 2001). Some of these materials have shown promise in improving wound healing, for example, by effectively sealing internal wounds and / or enabling tissue access (e.g., Ruel-Gariepy). See et al. Eur.J.Pharm.Biopharm.58:409,2004. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Seal et al. Mater Sci. Eng 34:147,2001 [Non-Patent Document 2] Ruel-Gariepy et al.Eur.J.Pharm.Biopharm.58:409,2004 [Overview of the project]

[0004] This disclosure provides various insights into technologies for transdermal delivery of drugs (including, for example, crosslinkable entities) and / or technologies for realizing cosmetic or therapeutic activity or benefits in the skin. In some embodiments, this disclosure provides insight into the certain advantages that can be realized when using microneedling techniques. For example, in some embodiments, microneedling techniques can enhance or enable, for example, the delivery of a drug to a specific target site. In some embodiments, the microneedling described herein can improve the degree of delivery (e.g., the total amount delivered, the amount delivered per unit time, and / or the degree of penetration (e.g., depth)).

[0005] In some embodiments, the Disclosure provides insight that a desired rate and / or degree of drug penetration and / or in situ formation can be achieved by controlling one or more specific parameters. In particular, the Disclosure illustrates specific embodiments of one or more parameters relating to a preparation (e.g., drug properties and / or one or more characteristics, e.g., molecular weight, lipophilicity, density of functional portion, viscosity, pH, etc.), one or more parameters relating to microneedling (e.g., needle dimensions [e.g., diameter and / or length], needle density, etc.), and / or a method of administration, which provides useful results regarding the delivery of a drug(s) (e.g., crosslinkable entities) and / or the in situ formation of a crosslinkable material(s) (e.g., the position and / or timing of microneedling relative to the application of the drug(s), the manner and / or timing of drug administration relative to and / or microneedling).

[0006] In various embodiments, the provided technology enables the formation and retention of desired crosslinkable material(s) at target sites(s) within the skin. In particular, this disclosure specifies particularly useful sizes and / or concentrations, molar ratios, combination conditions (e.g., pH, formulation additives, timing) of crosslinkable entities(s), microneedle properties, and combinations thereof that enable transdermal delivery of crosslinkable entities and / or in situ generation of crosslinkable material(s) (including large and / or complex crosslinkable material(s)).

[0007] Among the specific teachings provided herein is that a low pH (e.g., lower than physiological pH) may, in some embodiments, contribute to increased in situ formation of crosslinked material (e.g., large and / or complex crosslinked material) after local application of the crosslinkable entity(s) described herein. Without being bound by any particular theory, this disclosure proposes that such a low pH may allow the crosslinkable entity(s) to penetrate before crosslinking is complete (and thus before a crosslinked material too large to penetrate is formed), because such a low pH may slow the reaction time between crosslinked portions and / or allow penetration before crosslinking is otherwise complete.

[0008] In one embodiment, the present disclosure provides a method for establishing a crosslinked material at a target site in the skin, comprising: (i) applying first and second crosslinkable entities that react to form a crosslinked material to a skin site; and (ii) microneedling the skin site after applying at least one of the crosslinkable entities to ensure that the crosslinked material is present at the target site in the skin.

[0009] In some embodiments, the first and second crosslinkable entities are in a pH range of about 3 to about 5.

[0010] In some embodiments, the presence of the crosslinking material can be determined at a target site within the skin. In some embodiments, the crosslinking material becomes present over a period of time from 1 minute to 1 hour.

[0011] In some embodiments, the first crosslinkable entity comprises a polymer portion, the weight-average molecular weight of the polymer portion prior to the application step being in the range of 1 kDa to 500 kDa. In some embodiments, the weight-average molecular weight of the polymer portion prior to the application step is in the range of 5 to 20 kDa.

[0012] In some embodiments, the polymer portion is a hyaluronic acid ("HA") polymer. In some embodiments, the first crosslinkable entity comprises another molecule that reacts with either the -SH or NH2 group of CBT, a CBT mimetic, or Cys (e.g., D-Cys, L-Cys, or a combination thereof).

[0013] In some embodiments, the method includes applying the first and second crosslinkable entities to a skin location.

[0014] In some embodiments, the first and second crosslinkable entities are applied simultaneously. In some embodiments, the method further includes mixing the first and second crosslinkable entities before the application step. In some embodiments, the mixing step is performed 0 to 30 minutes before the application step.

[0015] In some embodiments, the second crosslinkable entity is applied after the first crosslinkable entity.

[0016] In some embodiments, microneedling is performed by a microneedling apparatus having microneedles. In some embodiments, the microneedling apparatus has a microneedle density of approximately 20 to 150 microneedles / cm³. 2 It is within the range. In some embodiments, the microneedle device has 1 to 100,000 microneedles. In some embodiments, the microneedles have a length of about 100 μm to about 1,000 μm. In some embodiments, the microneedle device is a derma roller.

[0017] In some embodiments, the internal dermal layer is the epidermis (e.g., stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, stratum basale) or the dermis.

[0018] In some embodiments, the concentration of the first crosslinkable entity is in the range of 0.1 to 100 mg / mL.

[0019] In some embodiments, the second crosslinkable entity is selected from the group consisting of cysteine-ethylenediamine-cysteine ​​(CEC), cysteine-lysine-cysteine ​​(CKC), cysteine-PEG-cysteine, and combinations thereof.

[0020] In some embodiments, the concentration of the second crosslinkable entity is in the range of 0.1 to 100 mg / mL.

[0021] In some embodiments, the molecular ratio of the first crosslinked portion to the second crosslinked portion is in the range of 1:1 to 5:1.

[0022] In some embodiments, the first crosslinkable entity includes a first crosslinking portion, where 1 to 20 mol% of the first crosslinkable entity comprises the first crosslinking portion.

[0023] In some embodiments, the crosslinking material is characterized in that the weight-average molecular weight of the crosslinking material at the target site in the skin is (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 times) greater than the weight-average molecular weight of the first crosslinkable entity.

[0024] In another embodiment, the present disclosure provides a method for establishing a crosslinked material at a target site in the skin, comprising: (i) applying first and second crosslinkable entities to a skin site at a pH in the range of about 3 to about 5; and (ii) microneedling at the skin site to ensure that the crosslinked material is present at the target site in the skin.

[0025] In some embodiments, the presence of the crosslinking material can be determined at a target site within the skin. In some embodiments, the crosslinking material becomes present over a period of time from 1 minute to 1 hour.

[0026] In some embodiments, microneedling is performed after the application step. In some embodiments, microneedling is performed before the application step. In some embodiments, microneedling is performed before or after the application step.

[0027] In some embodiments, the first crosslinkable entity comprises a polymer portion, the weight-average molecular weight of the polymer portion prior to the application step being in the range of 1 kDa to 500 kDa. In some embodiments, the weight-average molecular weight of the polymer portion prior to the application step is in the range of 5 to 20 kDa.

[0028] In some embodiments, the polymer portion is a hyaluronic acid ("HA") polymer. In some embodiments, the first crosslinkable entity comprises another molecule that reacts with either the -SH or NH2 group of CBT, a CBT mimetic, or Cys (e.g., D-Cys, L-Cys, or a combination thereof).

[0029] In some embodiments, the method further includes applying the first and second crosslinkable entities to a skin location.

[0030] In some embodiments, the first and second crosslinkable entities are applied simultaneously. In some embodiments, the method further includes mixing the first and second crosslinkable entities before the application step. In some embodiments, the mixing step is performed 0 to 30 minutes before the application step.

[0031] In some embodiments, the second crosslinkable entity is applied after the first crosslinkable entity.

[0032] In some embodiments, microneedling is performed by a microneedling apparatus having microneedles. In some embodiments, the microneedling apparatus has a microneedle density of approximately 20 to 150 microneedles / cm³. 2 It is within the range. In some embodiments, the microneedle device has 1 to 100,000 microneedles. In some embodiments, the microneedles have a length of about 100 μm to about 1,000 μm. In some embodiments, the microneedle device is a derma roller.

[0033] In some embodiments, the internal dermal layer is the epidermis (e.g., stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, stratum basale) or the dermis.

[0034] In some embodiments, the concentration of the first crosslinkable entity is in the range of 0.1 to 100 mg / mL.

[0035] In some embodiments, the second crosslinkable entity is selected from the group consisting of cysteine-ethylenediamine-cysteine ​​(CEC), cysteine-lysine-cysteine ​​(CKC), cysteine-PEG-cysteine, and combinations thereof.

[0036] In some embodiments, the concentration of the second crosslinkable entity is in the range of 0.1 to 100 mg / mL.

[0037] In some embodiments, the molecular ratio of the first crosslinked portion to the second crosslinked portion is in the range of 1:1 to 5:1.

[0038] In some embodiments, the first crosslinkable entity includes a first crosslinking portion, where 1 to 20 mol% of the first crosslinkable entity comprises the first crosslinking portion.

[0039] In some embodiments, the crosslinking material is characterized in that the weight-average molecular weight of the crosslinking material at the target site in the skin is (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 times) greater than the weight-average molecular weight of the first crosslinkable entity.

[0040] In another aspect, the present disclosure provides a method for establishing a crosslinked material in the interior of a skin by local application of first and second crosslinkable entities, the improvement comprising bringing the first and second crosslinkable entities into contact with each other at a pH in the range of about 3 to about 5.

[0041] In another embodiment, the present disclosure provides a method for establishing a crosslinked material in a subcutaneous location by applying first and second crosslinkable entities, wherein at least one of them is applied locally to a skin location, the improvement being a method comprising microneedling at the skin location after the local application of at least one of the crosslinkable entities.

[0042] In another embodiment, the present disclosure provides a combination of first and second crosslinkable entities having a pH in the range of about 3 to about 5. [Brief explanation of the drawing]

[0043] [Figure 1] This figure shows the linear HA-CBT concentration delivered when hyaluronic acid-6-amino-2-cyanobenzothiazole (HA-CBT) is administered to tape-exposed skin via microneedling. A shows the results from HA-CBT with an HA molecular weight of 10 kDa (HA10CBT). B shows the results from HA-CBT with an HA molecular weight of 20 kDa (HA20CBT).

[0044] [Figure 2] This figure shows the concentrations of HA-CBT in the stratum corneum and epidermis, including the dermis, when HA-CBT is administered via microneedling.

[0045] [Figure 3] This figure shows the concentrations of HA-CBT in the upper stratum corneum, lower stratum corneum, epidermis, and dermis. HA10CBT was administered before and after microneedling (e.g., rotary derma roller).

[0046] [Figure 4] This figure shows the HA-CBT concentration in the epidermis, including the stratum corneum and dermis. HA10CBT was administered in combination with microneedling and incubated at 37°C for 1 hour or overnight.

[0047] [Figure 5] These are gel permeation chromatography (GPC) chromatography images of HA10CBT after crosslinking with CKC at different concentrations. In A, the crosslinked structure was formed by mixing HA10CBT at different concentrations with stoichiometric equivalents of CKC. In B, the crosslinked structure was formed by mixing HA10CBT at different concentrations with stoichiometric equivalents of CKC. In C, the crosslinked structure was formed by combining a constant concentration of HA10CBT (1 mg / mL) with CKC at different molar ratios (R).

[0048] [Figure 6] This is a GPC chromatography of HA10CBT after crosslinking with CKC in the presence of human skin homogenate. In A, HA10CBT (1 mg / mL) was incubated with or without skin (± skin), and then mixed with CKC (±XL) at specified time points (0, 30, and 90 minutes). In B, HA10CBT (2 mg / mL) was incubated with skin (+ skin), and then mixed with CKC (±XL) at specified time points (0 and 90 minutes).

[0049] [Figure 7] This is a GPC chromatography of HA10CBT with or without the crosslinking agent cysteine-ethylenediamine-cysteine ​​(CEC) in phosphate-buffered saline (PBS, pH=7.4) and citrate buffer (25 mM, pH=4.6). HA10CBT (20 mg / mL) was mixed with CEC (equimolar CBT: Cys concentration), and the crosslinking reaction was performed on a GPC after dilution of the polymer concentration to 1 mg / mL and rapid cooling at specified time points.

[0050] [Figure 8]This is a GPC chromatography of epidermal (A) and dermal (B) skin extracts after microneedling with HA10CBT and CEC (ecomoleric CBT: Cys concentration) at different polymer concentrations: 20 mg / mL and 50 mg / mL. Each formulation was treated with acidic buffer: 25 mM citrate, pH=4.6, 0.2% ethylenediaminetetraacetic acid (EDTA).

[0051] [Figure 9] This is a GPC chromatography of epidermal (A) and dermal (B) skin extracts after microneedling of HA10CBT (50 mg / ml) and CEC (ecomoleric CBT: Cys concentration) into the skin. HA10CBT and CEC were mixed in acidic buffer (25 mM citrate, pH=4.6, 0.2 wt% EDTA) for S1 and S2 immediately before topical application. The two molecules were mixed 1 hour prior to application in acidic buffer (25 mM citrate, pH=4.6, 0.2 wt% EDTA) for S3 and S4, and in neutral buffer (PBS) for S5 and S6.

[0052] [Figure 10] This is a GPC chromatography of epidermal (A) and dermal (B) skin extracts after microneedling of HA10CBT (50 mg / ml) and CEC (equomolar CBT: Cys concentration) into the skin using different microneedle sizes (250, 500, and 1000 μm).

[0053] [Figure 11] This is a GPC chromatograph after simultaneous (A) and sequential (B) injection of HA10CBT and CEC into the skin.

[0054] [Figure 12] This figure shows the concentrations of HA-CBT in the upper sclera, lower sclera, epidermis, and dermis when administered via microneedling at concentrations of 10 mg / mL and 50 mg / mL in PBS.

[0055] [Figure 13]This figure shows the concentrations of HA-CBT in the upper sclera, lower sclera, epidermis, and dermis when administered using 250 μm and 500 μm derma rollers.

[0056] [Figure 14] These are microscopic images visualizing the difference in local delivery of HA-CBT and CEC using derma rollers with different needle lengths.

[0057] [Figure 15] This GPC chromatograph shows the detection of HA-CBT in skin extracts when HA-CBT is applied using a derma roller without CEC 11 days after application (A), and the detection of high molecular weight material in skin extracts when HA-CBT is applied with CEC via injection using a derma roller (B).

[0058] [Figure 16A] This figure shows H&E staining of excised pig skin 11 days after application of buffer with a derma roller. [Figure 16B] This figure shows H&E staining of excised pig skin 11 days after intradermal injection of HA-CBT and CEC. [Figure 16C] This figure shows H&E staining of excised pig skin 11 days after application of HA-CBT and CEC using a derma roller.

[0059] [Figure 17] This figure shows H&E staining of excised pig skin on day 28, after HA-CBT and CEC were applied using a 500 μm derma roller on day 0 and day 14. HA (shown in blue and indicated by arrows) is observed in the superficial dermis. [Modes for carrying out the invention]

[0060] definition When used herein in relation to a value, the term "about" refers to a value that is similar to the reference value in the given context. In general, a person skilled in the art familiar with the context will understand the reasonable degree of difference that "about" encompasses in that context. For example, in some embodiments, the term "about" may encompass a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 1% of the reference value.

[0061] Administration: As used herein, the term “administration” generally refers to administering a composition to a subject or system to achieve delivery of the composition or any drug contained in that composition. Those skilled in the art will recognize the various routes that may be used to administer to a subject (e.g., a human) in appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial droplet), buccal, percutaneous (e.g., one or more of the following, including topical, intradermal, interdermal, transdermal, etc., to the dermis), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal droplet), vaginal, vitreous, etc. In some embodiments, administration may consist of only a single dose. In some embodiments, administration may consist of the application of a fixed number of doses. In some embodiments, administration may include medication that is intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses spaced apart by a common time). In some embodiments, administration may include continuous medication (e.g., perfusion) over at least a selected period of time.

[0062] Analogue: As used herein, the term “analogue” refers to a substance that shares one or more specific structural features, elements, components, or parts with a reference substance. Typically, an “analogue” exhibits significant structural similarity to the reference substance, for example, sharing a core or consensus structure, but differing in a certain distinct manner. In some embodiments, an analogue is a substance that can be produced from a reference substance, for example, by chemical manipulation of the reference substance. In some embodiments, an analogue is a substance that can be produced through the performance of a synthetic process that is substantially similar to the process used to produce the reference substance (for example, sharing several steps). In some embodiments, an analogue is produced or can be produced through the performance of a synthetic process different from the one used to produce the reference substance.

[0063] Drugs: In general, the term “drugs” as used herein may refer to compounds or entities of any chemical classification, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. Where appropriate, as will be apparent to those skilled in the art, the term may also refer to entities that are cells or organisms, or fractions, extracts, or components thereof, or entities containing them. Alternatively or in addition thereto, as will be apparent to the context, the term may also refer to natural products found in nature and / or obtained from nature. Where appropriate, as will also be apparent to the context, the term may also refer to one or more entities that are artificial in the sense that they are designed, engineered and / or produced and / or not found in nature through human activity. In some embodiments, drugs may be used in isolated or pure form. In some embodiments, drugs may be used in crude form. In some embodiments, potential drugs may be provided as aggregates or libraries that can be screened, for example, to identify or characterize the active drug within. In some cases, the term “agent” may refer to a compound or entity that is or contains a polymer. In some cases, the term may refer to a compound or entity that contains one or more polymer portions. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer, and / or a compound or entity that is substantially free of any polymer, and / or a compound or entity that is substantially free of one or more specific polymer portions. In some embodiments, the term may refer to a compound or entity that has no polymer portions or is substantially free of any polymer portions.

[0064] Associated: Two events or entities are “associated” with each other if, as this term is used herein, the presence, level, and / or form of one correlate with that of the other. For example, a particular entity (e.g., polypeptide, gene signature, metabolite, microorganism, etc.) is considered associated with a particular disease, disorder, or condition if its presence, level, and / or form correlate with the incidence and / or susceptibility to disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are “associated” with each other if they interact directly or indirectly so that they are in and / or maintain a state of physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently bonded to each other. In some embodiments, two or more entities that are physically associated with each other are not covalently bonded to each other but are non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0065] Biocompatibility: The term "biocompatibility," as used herein, refers to compatibility with living tissues, for example, in This refers to materials that, when placed in contact with and positioned in vivo, do not cause significant harm to the tissue in question. In certain embodiments, materials are "biocompatible" if they are not toxic to cells. In certain embodiments, materials are "biocompatible" if their addition to cells in vitro results in no more than 20% cell death, and / or their administration in vivo does not induce significant inflammation or other such adverse effects.

[0066] Designed: As used herein, the term “designed” means (i) a drug whose structure is selected or chosen by human hands, (ii) a drug produced by a process that requires human hands, and / or (iii) a drug that is different from natural substances and other known drugs.

[0067] In situ: As used herein, the term "in situ" refers to an event occurring within a tissue or tissue layer at least one cell layer below the surface of that tissue or tissue layer.

[0068] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment rather than within a multicellular organism, such as in a test tube, reaction vessel, or cell culture.

[0069] In vivo: As used herein, this refers to events occurring within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, this term means events occurring within living cells (for example, in It can also be used to refer to events that occur (not in a vitro system).

[0070] “Improve,” “Increase,” “Inhibit,” or “Decrease”: As used herein, the terms “improve,” “increase,” “inhibit,” “decrease,” or their grammatical equivalents, refer to a value compared to a baseline or other reference measure. In some embodiments, a suitable reference measure may be or include a measurement in a particular system (e.g., in a single individual) under inherently comparable conditions (e.g., before and / or after) in the absence of a particular drug or treatment, or in the presence of a suitable comparable reference drug. In some embodiments, a suitable reference measure may be or include a measurement in a comparable system known or expected to respond in a particular manner in the presence of the relevant drug or treatment.

[0071] Physiological state: As used herein, the term has the meaning understood in the art, referring to the state in which a cell or organism is surviving and / or regenerating. In some embodiments, the term refers to the state of the external or internal environment that can occur in nature for an organism or cell system. In some embodiments, the physiological state is the state present in the body of a human or non-human animal, in particular, the state present in and / or within the target site of interest. The physiological state typically includes, for example, one or more of the following: temperature in the range of 20 to 40°C (specifically about 37°C), atmospheric pressure of 1, pH 6 to 8, glucose concentration of 1 to 20 mM, oxygen concentration at atmospheric levels, and gravity encountered on Earth.

[0072] Subject: As used herein, the term “Subject” refers to any organism to which the provided system is administered or may be administered for, for example, experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the subject is human. In some embodiments, the subject has or is susceptible to one or more disorders or conditions. In some embodiments, the subject exhibits one or more symptoms of a disorder or condition. In some embodiments, the subject has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes the presence of cancer or one or more tumors. In some embodiments, the subject is or has received a particular therapy to diagnose and / or treat a disease, disorder, or condition. In some embodiments, the subject refers to a human being seeking cosmetic benefits and / or improvements, such as improved appearance and / or feel of the skin.

[0073] Therapeutic agent: As used herein, the term “therapeutic agent” generally refers to any agent that, when administered to an organism, induces a desired pharmacological effect (which in some embodiments may be or include a cosmetic effect). In some embodiments, an agent is considered effective (i.e., therapeutic agent) if it exhibits a statistically significant effect across a suitable population. In some embodiments, a suitable population may be a population of model organisms. In some embodiments, a suitable population may be defined by certain criteria, such as a particular age group, sex, genetic background, pre-existing clinical condition, or a combination thereof. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, reduce, inhibit, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent is one that achieves a cosmetic effect (i.e., a cosmetic). In some embodiments, a therapeutic agent can be used to improve the appearance and / or feel of the skin, and / or achieve another cosmetic effect.

[0074] To treat: As used herein, the terms “treat,” “treatment,” or “treating” mean the partial or complete relief, improvement, delay, inhibition, prevention, mitigation, and / or reduction of incidence and / or severity of one or more symptoms or characteristics of a disease, disorder, and / or condition, or the achievement of another desired physiological effect (e.g., a desired cosmetic effect, e.g., improvement of the appearance and / or feel of the skin, e.g., improvement of the visible and / or tactile sensation of the skin). In some embodiments, treatment includes the administration of a drug that produces a physiological effect. In some embodiments, treatment includes a cosmetic treatment that, when applied, improves physical appearance in the manner described herein. In some embodiments, treatment may be applied to subjects that do not show any signs or characteristics of a disease, disorder, and / or condition (e.g., it may be preventive). In some embodiments, treatment may be applied to subjects that show only early or mild signs or characteristics of a disease, disorder, and / or condition, for the purpose of reducing the risk of developing a condition associated with the disease, disorder, and / or condition. In some embodiments, the treatment may be administered to subjects exhibiting established, severe, and / or late signs of disease, disorder, or condition.

[0075] Detailed description of a particular embodiment The following statements are for illustrative purposes only and are not intended to limit the disclosure to any specific embodiments described herein. Unless otherwise defined, technical and scientific terms have the same meanings as those widely understood by those skilled in the art to which this disclosure pertains. All references cited herein (including patent applications and publications) are incorporated by reference throughout.

[0076] Human skin is multilayered and consists of the outer epidermis, dermis, and the subcutaneous tissue beneath it. The epidermis has several layers of tissue, namely the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale (identified in order from the outer surface of the skin to the inside). The skin functions as a barrier that separates and protects the body from its environment. The stratum corneum, the outermost layer of the skin, has strong barrier properties that limit the success of drug delivery. The stratum corneum is usually about 10-15 μm thick and consists of several layers of flat, keratinized cells (keratinocytes). The remainder of the epidermis beneath the stratum corneum is approximately 150 μm thick. The dermis is about 1-2 mm thick and is located below the epidermis. The dermis is innervated by various capillaries and neurites.

[0077] It is understood in the art that human skin restricts the transport of compounds with a molecular weight (e.g., number or weight-average molecular weight) greater than approximately 500 daltons (Bos and Meinardi, Experimental Dermatology, 9:165, 2000). Even for such small compounds, effective transport often requires further manipulation, such as the administration of chemical or physical abrasives or destructive agents and / or electric currents or magnetic fields.

[0078] According to one or more embodiments, the present disclosure provides specific techniques for the penetration of a drug into a target site, which may be, for example, a site within or on the skin, such as the epidermis (e.g., the stratum corneum), the dermis, or a site on the subcutaneous tissue below therein, a site within them, or a site below them. The techniques provided enable desired delivery of the drug across the skin surface and / or into the target site, such as desired penetration depth / volume, skin area per dose, pain reduction, recovery time, treatment time, etc. In some embodiments, the drug may be administered in combination with microneedling.

[0079] In some embodiments, certain aspects of the provided techniques make them particularly useful and / or effective for agents comprising one or more crosslinkable entities or capable of forming a crosslinked material in situ. In some embodiments, the present disclosure provides techniques for applying first and second crosslinkable entities selected and / or designed to achieve the formation of a crosslinked material in situ. As those skilled in the art will see, the formation of this crosslinked material can induce changes in physical properties such as rheology and / or changes in chemical properties such as an increase in molecular weight. As those skilled in the art will see, these physiological and chemical changes can result in advantageous benefits for the crosslinked material compared to the original crosslinkable entity, such as improved durability / sustainability in biological systems.

[0080] Bridgeable entities This disclosure provides certain techniques relating to the administration of a crosslinkable entity to a target, in particular, in a target site which may be, for example, a site within or on the skin, such as a site in the epidermis, dermis, or subcutaneous tissue below therein, a site within or below therein, in the crosslinkable entity within or on the target site Specific techniques for crosslinking in situ are provided.

[0081] In some embodiments, the present disclosure provides techniques for administering a system comprising first and second crosslinkable entities selected and / or designed to achieve in situ crosslinking of a crosslinkable material. As those skilled in the art will see, the formation of this crosslinkable material can induce changes in physical properties such as rheology and / or changes in chemical properties such as an increase in molecular weight. As those skilled in the art will see, these physiological and chemical changes can result in advantageous benefits for the crosslinkable material compared to the original crosslinkable entity, such as improved durability / sustainability in a biological system.

[0082] As described herein, such first and second crosslinkable entities are characterized by their ability to react with each other in situ to form a crosslinked material when in contact with each other, for example, without the administration of a catalyst or other non-involved agent.

[0083] In many embodiments, at least one of the crosslinkable entities includes a polymer portion bonded to a crosslinked portion. In some embodiments, at least one of the crosslinkable entities does not include a polymer portion. In some embodiments, each of a pair of crosslinkable entities reacting with each other (e.g., without the administration of a catalyst or other non-involved agent) includes a polymer portion bonded to a crosslinked portion. In some embodiments, a pair of crosslinkable entities reacting with each other (e.g., without the administration of a catalyst or other non-involved agent) includes a first crosslinkable entity including a polymer portion bonded to a crosslinked portion and a second crosslinkable entity not including a polymer portion.

[0084] In some embodiments, the crosslinkable entity comprises a polymer portion and multiple crosslinkable portions, which may be the same or different. In particular, the disclosure provides insights and techniques relating to achieving the penetration of a crosslinkable entity into a target site in the skin (e.g., a target site on the epidermis, dermis, or subcutaneous tissue below therein, a target site within them, or a target site below them). For example, in some embodiments, the disclosure teaches that microneedling can enable the desired (e.g., enhanced) penetration of a crosslinkable entity, particularly a crosslinkable entity comprising a polymer portion and crosslinkable portions.

[0085] In particular, the Disclosure provides teaching that microneedling can improve the delivery of crosslinkable entities before crosslinking reactions in the skin. Such improved delivery of crosslinkable entities can be designed and / or prepared by adjusting lipophilicity and / or molecular weight. For example, in some embodiments, the Disclosure teaches that the rate and / or degree of skin penetration by a particular agent (specifically, by an agent that is or contains a polymer moiety and / or otherwise has a weight-average molecular weight greater than 500 daltons and even in the range of 1 to 500 kDa) can be enhanced by reducing the molecular weight of the agent or increasing the lipophilicity of the agent, for example by attaching one or more hydrophobic moieties to the agent.

[0086] Alternatively, or in addition thereto, in some embodiments, the present disclosure provides techniques for providing a crosslinkable material within or on tissue (particularly within or on skin) a target site such that contact between the material and the surface of the target site is maximized. In some embodiments, such a surface may contain one or more cavities or irregularities, which in some embodiments may even be microscale or nanoscale structures. In some embodiments, the present disclosure provides preparations of crosslinkable portions having flow properties that facilitate such contact.

[0087] In some specific embodiments, the crosslinkable entity for use by this disclosure comprises a polymer portion bonded to a crosslinked portion, the crosslinked portion imparting increased lipophilicity to the complex (i.e., the crosslinkable entity) compared to the polymer portion alone. As described herein, in some embodiments, such a crosslinked portion can also improve skin penetration by the crosslinkable entity compared to that of the polymer portion alone.

[0088] In some embodiments, the penetration depth of the crosslinkable entity is evaluated and / or described in terms of the absolute distance (e.g., microns) below the skin surface. In some embodiments, the penetration depth of the crosslinkable entity is evaluated and / or described in terms of the number of cells below the skin surface.

[0089] In some embodiments, the crosslinkable entities described herein (when administered, for example, as described herein) can penetrate and / or penetrate into the skin to a specified depth, for example, within a certain time. In some embodiments, such specified depth may be, for example, at least 50 microns, at least 100 microns, at least 200 microns, or deeper. Alternatively or in addition thereto, in some embodiments, such specified depth may be at least two cell layers, at least three cell layers, at least four cell layers, at least five cell layers, at least six cell layers, at least seven cell layers, at least eight cell layers, at least nine cell layers, at least ten cell layers, or more, and / or such time may be, for example, within one day, within 18 hours, within 12 hours, within 11 hours, within 10 hours, within 9 hours, within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, or shorter.

[0090] In some embodiments, at least 1% of a crosslinkable entity administered to the skin surface penetrates, for example, within a day, into target sites on the epidermis, dermis, or subcutaneous tissue, target sites within them, target sites within them, or target sites below them. In some embodiments, the skin penetration properties of the crosslinkable entity(s) described herein are observed in the absence of any chemical or physical abrasives or destructive agents and / or electric currents or magnetic fields, etc. (e.g., in the absence of penetration enhancers as understood in the art).

[0091] In some embodiments, the crosslinkable entity is characterized by a lipophilicity (log P) in the range of about -4 to about 2. In some embodiments, the crosslinkable entity described herein includes a crosslinkable portion that is lipophilic. In some embodiments, the lipophilicity of the crosslinkable portion may be determined independently of that of the polymer portion and / or that of the crosslinkable entity comprising a polymer portion and one or more crosslinkable portions. In some embodiments, the crosslinkable portion is characterized by a lipophilicity (log P) in the range of about 0 to about 6. For example, in some embodiments, the lipophilicity of the crosslinkable portion is tested through inspection of the crosslinkable portion before association and / or bonding with the molecular polymer portion to form the crosslinkable entity.

[0092] In some embodiments, lipophilicity to a particular entity or part is determined by its partition coefficient (P) in a standard solvent (e.g., octanol) and water or a solution thereof.

number

[0093] In some embodiments, the log P of a useful entity or part of the present disclosure (e.g., a crosslinkable entity and / or crosslinkable part) is greater than 0.

[0094] In some embodiments, the crosslinkable entities (e.g., lipophilic crosslinkable entities as described herein) have a molecular weight in the range of 1 to 1000 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight of about 10 to 250 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) in the range of about 10 to 150 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) in the range of about 1 to 500 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) in the range of about 10 to 40 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of about 10 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of about 20 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of about 30 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of about 40 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of about 50 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of less than 10 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of less than 100 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of less than 250 kDa. In some embodiments, the system includes crosslinkable entities having a molecular weight (e.g., weight-average molecular weight) of less than 500 kDa. In some embodiments, the crosslinkable entities have a molecular weight (e.g., weight-average molecular weight) greater than 500 Da. In some embodiments, the crosslinkable entities have a molecular weight (e.g., weight-average molecular weight) of less than 500 Da.

[0095] In some embodiments of a crosslinkable entity comprising a polymer portion and a crosslinked portion, the crosslinked portion imparts lipophilicity to the crosslinkable entity. In some embodiments, the properties and / or number and / or density of the crosslinked portions bonded to a particular polymer portion within the crosslinkable entity are selected such that the crosslinkable entity has the features(s) described herein. For example, in some embodiments, if the polymer portion is characterized by being particularly hydrophilic, particularly long, and / or having particularly poor skin penetration in its unbonded state, more lipophilic crosslinked portions (and / or more numerous / higher density lipophilic crosslinked portions) are bonded to the polymer portion. In some embodiments, the crosslinkable entity may include further portions that do not participate in crosslinking but contribute to the lipophilicity of the crosslinkable entity and improve its penetration properties.

[0096] While not bound by any particular theory, we propose that in some embodiments, it may be desirable to select a specific combination of polymer portions and crosslinked portions (and / or their number and / or density) to achieve three-dimensional filling of a particular volume of crosslinkable entities, which may, for example, facilitate movement through one or more skin structures or layers (e.g., becoming sufficiently "slippery" to pass through the crosslinkable entities). In some embodiments, intramolecular and / or intermolecular interactions may contribute to the three-dimensional filling of the crosslinkable entities. In some embodiments, the three-dimensional filling of the particular crosslinkable entities described herein occurs by self-assembly (e.g., without the need for the addition of other agents) at least under certain environmental conditions (e.g., physiological conditions).

[0097] Typically, crosslinkable entities are biocompatible. For example, in some embodiments, administration of the crosslinkable entities described herein does not cause significant irritation and / or inflammation (e.g., at the administration site and / or target site).

[0098] In some embodiments, the crosslinkable entity is used in a form or preparation having a viscosity suitable for topical application (for example, sufficient viscosity to maintain contact between the applied crosslinkable entity and the administration site for long enough to allow penetration and / or distribution to the target site, rather than simply dripping). In some such embodiments, the viscosity is within a range that allows the applied crosslinkable entity to flow into the microstructure on the skin surface. In some embodiments, the crosslinkable entity is used in a form or preparation having a viscosity suitable for injectable administration. In some embodiments, a preferred viscosity is one that allows the crosslinkable entity to be applied topically without rapidly flowing down and / or rubbed into the skin.

[0099] In some embodiments, the present disclosure provides a system comprising two or more crosslinkable entities that interact with each other to form a crosslinkable material (for example, providing a crosslinkable material in situ at target sites on the epidermis, dermis, or subcutaneous tissue thereunder, at target sites thereunder, at target sites within them, or at target sites thereunder).

[0100] In some embodiments, at least one crosslinkable entity of the provided system includes a polymer portion bonded to a crosslinked portion. In some such embodiments, at least one crosslinkable entity of the system does not include a polymer portion bonded to a crosslinked portion (i.e., it includes or consists of a crosslinked portion but does not include a polymer portion). In some such embodiments, only one crosslinkable entity of the provided system includes a polymer portion bonded to a crosslinked portion. Alternatively, in some embodiments, each crosslinkable entity of the provided system includes a polymer portion bonded to a crosslinked portion. In some such embodiments, each crosslinkable entity of the provided system includes the same polymer portion.

[0101] The different crosslinkable entities in the provided system include complementary crosslinking portions such that the crosslinkable entities react to form a crosslinked material. In some embodiments, such crosslinking occurs without any added catalyst.

[0102] In some embodiments, at least one crosslinkable entity of the provided system has a molecular weight (e.g., weight-average molecular weight) greater than 500 daltons as described herein (e.g., molecular weight (e.g., weight-average molecular weight) in the range of 1 to 1000 kDa). In some embodiments, at least one crosslinkable entity of the provided system has a molecular weight (e.g., weight-average molecular weight) in the range of 1 to 10,000 kDa as described herein. In some embodiments, at least one crosslinkable entity of the provided system has a molecular weight (e.g., weight-average molecular weight) greater than 10,000 kDa. In some such embodiments, at least one crosslinkable entity of the system does not have a molecular weight (e.g., weight-average molecular weight) greater than 500 daltons as described herein (e.g., in the range of 1 to 400 daltons). In some such embodiments, only one crosslinkable entity of the provided system has such a high molecular weight (e.g., weight-average molecular weight). Alternatively, in some embodiments, each crosslinkable entity of the provided system has such a high molecular weight (e.g., weight-average molecular weight).

[0103] Polymer part In some embodiments, the present invention encompasses a crosslinkable entity comprising a polymer portion and a crosslinked portion. In some embodiments, one or more of the polymer portions are glycosaminoglycans or polysaccharides. In some embodiments, the “polysaccharide” includes dextran, starch, or pectin. In some embodiments, one or more of the polymer portions are dextran. In some embodiments, one or more of the polymer portions are starch. In some embodiments, one or more of the polymer portions are pectin. In some embodiments, the “glycosaminoglycan” includes hyaluronic acid (HA), heparin sulfate, chondroitin sulfate, dermatan sulfate, and keratin sulfate. In some embodiments, the polymer is hyaluronic acid.

[0104] In some embodiments, one or more of the polymer portions are synthetic polymers. In some embodiments, the "synthetic polymer" includes PEG, PEG-diamine, polyacrylic acid, N-(2-hydroxypropyl)methacrylamide (HPMA), polycaprolactone (PCL), or poly(lactic acid-co-glycolic acid) (PLGA). In some embodiments, the synthetic polymer is PEG. In some embodiments, the synthetic polymer is PEG-diamine. In some embodiments, the synthetic polymer is polyacrylic acid. In some embodiments, the synthetic polymer is HPMA. In some embodiments, the synthetic polymer is PCL. In some embodiments, the synthetic polymer is PLGA.

[0105] In some embodiments, one or more polymer moieties are polypeptides (e.g., proteins). In some embodiments, the useful polypeptide is collagen, gelatin, elastin, or functional fragments thereof, or comprises them. In some embodiments, the polypeptide is collagen or functional fragments thereof, or comprises them. In some embodiments, the polypeptide is gelatin. In some embodiments, the polypeptide is elastin.

[0106] In some embodiments, one or more polymer portions included in the system described herein are or contain HA. In some embodiments, one or more polymer portions included in the system described herein are glycosaminoglycans or contain glycosaminoglycans. In some embodiments, one or more polymer portions included in the system described herein are polypeptides (e.g., unnatural polypeptides and / or synthetic or recombinant polypeptides) or contain polypeptides. In some embodiments, the provided system includes a plurality of different crosslinkable entities comprising polymer portions. In some such embodiments, the polymer portions are selected from the group consisting of HA, proteoglycans, polypeptides, and combinations thereof.

[0107] bridging part As described herein, the present disclosure provides a system comprising two or more crosslinkable entities that are compatible with one another in that they react to form a crosslinkable material in situ.

[0108] Those skilled in the art are familiar with various chemical crosslinking systems. In many embodiments, the present disclosure employs crosslinking chemistry that does not require the addition of a catalyst.

[0109] In some embodiments, one or more of the crosslinkable entities of the system include a crosslinking portion. In some embodiments, the “crosslinking portion” can participate in a click reaction.

[0110] As those skilled in the art will know, a click reaction can be a reaction of two or more parts that occur when two or more substrates are brought together and under physiological pH. In some embodiments, click reactions exhibit favorable reaction rates (for example, a second-order rate constant (k²) of about 9 M⁻¹ s⁻¹).

[0111] For example, in some embodiments, crosslinking chemistry may include, but is not limited to, cyclization, nucleophilic substitution, condensation, and nucleophilic addition. In some embodiments, the click reaction is [3+2] cyclization, [4+2] cyclization, or [4+1] cyclization. In some embodiments, the click reaction is azide-alkyne cyclization, nitrone-olefin cyclization, or Diels-Alder reaction. In some embodiments, the click reaction is a Schiff reaction, Michael-type addition, nucleophilic substitution on a haloacetate, disulfide bond formation, free radical polymerization, huisgen reaction, spontaneous crosslinking of phenol (tyramine) after its enzymatic oxidation to catechol, or a reaction between cyanobenzothiazole (CBT) and D-cysteine ​​(CYS). In some embodiments, the click reaction is a reaction between CBT and D-cysteine. In some embodiments, the click reaction is a reaction between CBT and L-cysteine. In some embodiments, the click reaction is a reaction between CBT and a mixture of D and L-cysteine. In some embodiments, CBT may include analogues of CBT, such as isotopically labeled CBT. In some embodiments, CBT may include substituted analogues of CBT. In some embodiments, CYS may include analogues of CYS, such as isotopically labeled CYS. In some embodiments, CYS may include substituted analogues of CYS.

[0112] In some embodiments, the present disclosure describes a particular crosslinking portion as described herein. Understand that this can be particularly useful in the context of situ crosslinking.

[0113] For example, in some embodiments, the Disclosure utilizes one or more crosslinked moieties that, when bonded to a particular polymer moiety, exhibit a desired degree of lipophilicity. To give just one example, CBT represents a crosslinkable moiety that can bond to a polymer moiety within a useful crosslinkable entity described herein. Those skilled in the art will understand, upon reading the Disclosure, that in some embodiments, the lipophilicity of a crosslinkable entity containing a particular polymer moiety can be tuned through the bonding of multiple hydrophobic moieties (e.g., hydrophobic crosslinked moieties). These multiple hydrophobic moieties may be the same or different (and not all of them must be crosslinked moieties).

[0114] Furthermore, in some embodiments, the crosslinked moiety is or contains azides, alkynes, nitrones, olefins, dienes, tetrazines, isocyanates, Michael receptors, enones, aldehydes, amines, α-halo-bonyl moieties, maleimides, thiols, CBTs, D-cysteine, acrylic residues, phenols, tyramines, or catechols. In some embodiments, the crosslinked moiety is or contains D-cysteine. In some embodiments, the crosslinked moiety is or contains L-cysteine. In some embodiments, the reactive moiety is or contains CBTs.

[0115] In some embodiments, at least one crosslinked portion included in the provided system may be used without being bonded to the polymer portion. That is, in some embodiments, at least one crosslinkable entity included in the provided system may consist of a crosslinked portion or may include a crosslinked portion and at least one other portion that is not a polymer portion.

[0116] In some embodiments, the provided system may use a crosslinked moiety that is or contains, for example, a diamine, peptide, dithiol, or dihydrazide. In some such embodiments, the diamine may be ethylenediamine, e.g., polyethylene glycol (PEG) diamine, lysine, etc. In some such embodiments, the dihydrazide may be oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, or pimelic acid dihydrazide, etc. Without wanting to delve into semantics, it should be noted that certain such compounds can be described as comprising a crosslinked moiety or a polymer moiety that comprises one or more crosslinked moieties (e.g., each of which may consist of a single reactive atom or a small number of atoms) and a very small number (e.g., two or three) "monomers" (in some cases, each of which may consist of only a small number of atoms). In any case, those skilled in the art will read this disclosure and understand that these compounds can be used in various embodiments together with other crosslinking entities with complementary crosslinking portions as described herein.

[0117] Other parts In some embodiments, the crosslinkable entities described herein may include one or more polymer portions or non-crosslinkable portions, and the other portions may, in some embodiments, be covalently associated with the polymer portion and / or crosslinkable portion. In some embodiments, the “other” portions may be release-releasably associated with the crosslinkable entity (for example, via cleavable bonds; in some such embodiments, such cleavable bonds may be cleaved at a target site).

[0118] For example, in some embodiments, the crosslinkable entity may include a drug portion (in some embodiments, it may itself be in the form of a prodrug).

[0119] In some embodiments, the drug portion is a synthetic or natural small molecule or biomolecule (e.g., a carbohydrate, lipid, nucleic acid, polypeptide, or analogue thereof, or a combination thereof), or comprises such molecules.

[0120] In some embodiments, the drug portion may improve the appearance of the skin in one or more ways. For example, in some such embodiments, the drug portion may: brighten the skin; remove blemishes; firm up; improve cellular activity within the skin; improve collagen synthesis; improve the wound healing profile; improve hyperpigmentation; improve skin barrier function; normalize a healthy microbiome via topical prebiotics; prevent damage (e.g., from UV exposure); reduce the appearance of scars; reduce inflammation; reduce itching; reduce redness; seal wounds; smooth or treat burns, or a combination thereof.

[0121] In some embodiments, the drug portion may include the following: Alpha hydroxy acids (e.g., lactic acid, tartaric acid, or citric acid), antioxidants (e.g., glutathione, isoflavones, polyphenols (e.g., resveratrol), or selenium), beta hydroxy acids (e.g., salicylic acid), polyhydroxy acids (e.g., gluconolactone or lactobionic acid), hydroquinone, natural skin whitening agents (e.g., kojic acid), retinoids (e.g., retinoic acid, retinol, tretinoin, or their derivatives), ceramides, peptides, amino acids, curcuminoids, vitamins (and their derivatives) (e.g., L-ascorbic acid, vitamin B, niacinimide, and vitamin K), sunscreens (e.g., oxybenzone, avobenzone, octisalate, octocrylene, homosalate, octinoxate, meloxyl (SX and XL) or metal oxides), colorants, pigments, or natural botanicals, or combinations thereof or prodrugs.

[0122] In some embodiments, the drug portion may include: anti-inflammatory drugs (e.g., corticosteroids, nonsteroidal anti-inflammatory drugs (crisabolol), antibiotics (e.g., clindamycin or ketoconazole), antifungal drugs (e.g., clotrimazole or ketoconazole), antiacne agents (e.g., retinoids or salicylic acid), analgesics, anticancer or antiproliferative agents, erythematous agents (e.g., oxymetazoline hydrochloride), subcutaneous fat-reducing agents (e.g., deoxycholic acid), hair growth agents (e.g., finasteride), or combinations thereof or prodrugs.

[0123] Microneedling Microneedling is a developed technology that describes a collection of techniques that use micron-sized "needles" to improve the appearance or health of the skin and / or deliver active ingredients into the skin. Traditionally used as a collagen induction therapy for facial scarring and skin rejuvenation, it is also widely used as a transdermal delivery system for drugs (e.g., therapeutic drugs and vaccines). Microneedling has been shown to enhance drug permeability to the skin in vitro, ex vivo, or in vivo.

[0124] Microneedling offers advantages over conventional injections using subcutaneous needles or cannulas. In particular, it can reduce patient anxiety associated with conventional needles or cannulas. Unlike injections, microneedling allows for faster treatment of large areas of skin. Faster treatment times can improve revenue for providers and enhance patient convenience. Furthermore, microneedling can provide more uniform treatment than individual injections. Depending on the application, microneedling may reduce the demands on specific skills and techniques compared to conventional injections. Certain types of "consumer-grade" microneedling devices are sold directly to consumers who have no special skills in microneedling. The depth to which microneedles penetrate the skin is partially controlled by the length of the microneedle. Shorter microneedles have been shown to cause less pain than conventional injections. Microneedling allows for the delivery of medication to both the epidermis and dermis in a more uniform manner compared to conventional injections. This is especially true for applications where the drug is intended to be delivered to the more superficial layers of the skin (e.g., the epidermis and upper dermis). In contrast, intradermal injection using subcutaneous injection requires careful placement of the needle within the skin. For maximum effectiveness, a specific drug must be delivered within a limited depth. Microneedling also produces less hazardous waste and is easier to use than needles. Furthermore, microneedling can give people with limited ability to undergo hospitalization the ability to safely and comfortably administer medication at home. Microneedling reduces the rate of microbial invasion into the delivery site because it damages the skin to a depth of 10-15 μm, making it difficult for bacteria to enter the bloodstream and reducing the size of the wound that the body needs to heal.McConville A, Hegarty C, Davis J (June 2018). “Mini-Review: Assessing the Potential Impact of Microneedle Technologies on Home Healthcare Applications”. Medicines. 5(2):50.

[0125] In some embodiments, the microneedles according to this disclosure are solid or hollow. In some embodiments, solid microneedles may be used to create pores in the skin (e.g., the stratum corneum) to increase the skin penetration of a topically applied formulation. In some embodiments, a drug (e.g., a crosslinkable entity) is coated onto a solid microneedle. After the microneedle is removed, the drug remains deposited in the skin. In some embodiments, a solid microneedle may be rolled or pressed over a formulation on the skin (e.g., to deposit the formulation in the skin). In some embodiments, a hollow microneedle has a hollow hole in the center of the needle. In some embodiments, a hollow microneedle is used to inject a drug directly into the skin. In some embodiments, the hollow hole transports the drug through the inside of the needle (e.g., by diffusion or pressure-driven flow).

[0126] In some embodiments, the microneedle apparatus according to this disclosure includes one or more microneedles. In some embodiments, the microneedle apparatus is individual microneedles, patches, or rollers.

[0127] In some embodiments, the microneedle device according to the present invention includes flat or circular microneedles.

[0128] In some embodiments, the microneedles according to this disclosure include materials selected from the group consisting of silicon, metals (e.g., stainless steel, titanium, palladium, nickel, platinum, alloys, gold), glass, ceramics (e.g., alumina, calcium phosphate, calcium sulfate), polymers (e.g., hydroxypropyl methylcellulose, hyaluronic acid, carboxymethylcellulose (CMC), alginates, poly(e.g., methyl vinyl ether / maleic anhydride), polystyrene, polyvinyl alcohol, polyvinylpyrrolidone (PVP), polylactic acid, polyglycolic acid, and their copolymers (poly(lactic acid-co-glycolic acid) [PLGA])), sugars (e.g., maltose, trehalose, raffinose, mannitol, xylitol, galactose), and combinations thereof. While not bound by any particular theory, general properties essential for a material to be suitable for microneedles include inertness, non-immunogenicity, high tensile strength, non-brittleness, excellent mechanical strength, low corrosion rate, biocompatibility, stability, ease of use, and low cost.

[0129] In some embodiments, the microneedles include biodegradable materials (e.g., polymers, sugars, etc.). In such embodiments, the microneedles may dissolve and / or decompose at the administration site (e.g., no removal is required). Such microneedles may be attached to the applicator before application, or removed from the applicator after application.

[0130] In some embodiments, the microneedles according to the present disclosure have lengths in the range of approximately 10 μm to approximately 1000 μm, approximately 10 μm to approximately 750 μm, approximately 10 μm to approximately 500 μm, approximately 100 μm to approximately 1000 μm, approximately 100 μm to approximately 750 μm, approximately 100 μm to approximately 500 μm, approximately 100 μm to approximately 250 μm, approximately 250 μm to approximately 750 μm, or approximately 250 μm to approximately 500 μm.

[0131] In some embodiments, the microneedles according to the present disclosure have a microneedle density in the range of about 10 to 15,000 needles / cm 2 、about 10 to 10,000 needles / cm 2 、about 10 to 5,000 needles / cm 2 、about 10 to 1,000 needles / cm 2 、about 50 to 15,000 needles / cm 2 、about 50 to 10,000 needles / cm 2 、about 50 to 5,000 needles / cm 2 、or about 20 to 1,000 needles / cm 2 within the range of.

[0132] In some embodiments, the microneedles according to the present disclosure have a base width in the range of about 10 μm to about 500 μm, about 10 μm to about 400 μm, about 10 μm to about 300 μm, about 10 μm to about 200 μm, about 25 μm to about 500 μm, about 25 μm to about 400 μm, about 25 μm to about 300 μm, about 25 μm to about 200 μm, about 50 μm to about 500 μm, about 50 μm to about 400 μm, about 50 μm to about 300 μm, or about 50 μm to about 200 μm.

[0133] In some embodiments, the microneedles according to the present disclosure have a tip diameter in the range of about 0.1 μm to about 100 μm, about 0.1 μm to about 50 μm, about 0.1 μm to about 25 μm, about 1 μm to about 100 μm, about 1 μm to about 50 μm, or about 1 μm to about 25 μm.

[0134] In some embodiments, the hollow microneedles according to the present disclosure have a lumen diameter in the range of about 10 μm to about 100 μm, or about 10 μm to about 50 μm.

[0135] While not bound by any particular theory, the design of microneedles may be intended to minimize pain. Certain microneedles with a length of approximately several hundred microns have been reported to be painless. Int J Pharm Tech 2010;2(3):329-344. It has been reported that increasing the needle length 13-fold (i.e., 500-1500 microns) increases pain 7-fold (i.e., 5-35% of that with a subcutaneous injection needle). If the length remains constant, increasing the number of microneedles (i.e., 620 microns in length) 5-50-10-fold also increases pain 3-fold.

[0136] Administration This disclosure provides a technology for establishing intradermal crosslinking materials.

[0137] Those skilled in the art will know of the various contexts in which it is desirable to achieve such establishment of intradermal crosslinking materials. For example, intradermal crosslinking materials may be used for facial rejuvenation, including restoring three-dimensional facial volume, rebalancing facial proportions and symmetry, and reducing fine lines and wrinkles. Crosslinking can enhance the stability and durability of clinical implants.

[0138] Furthermore, those skilled in the art are aware of the various problems associated with establishing intradermal crosslinking materials, particularly when the material may have large and / or complex structures. For example, large and complex structures cannot penetrate the skin on their own. Efforts to achieve placement by intradermal injection may present significant challenges, including the difficulty of achieving precise localization (i.e., specifically between the epidermis and subcutaneous tissue). Reports have described very specific requirements regarding the injection angle (e.g., specifically 5-15 degrees) and / or the needle used. Doyle, GR, & McCutcheon, JA (2015). Clinical Procedures for Safer Patient Care. Victoria, BC: BCcampus., Chapter 7.3. In addition, injections may be uncomfortable for the subject and may trigger a wound healing response that can lead to unpleasant and / or unattractive results (e.g., scarring).

[0139] In particular, this disclosure builds upon the insights provided in International Patent Application Publications WO2016 / 201382, WO2020 / 093022, and U.S. Patent Application Publication US2018 / 0186900. These documents describe certain techniques for local application of crosslinkable entities to achieve the establishment of intradermal crosslinking materials. This disclosure provides techniques in which local application is combined with microneedling. This disclosure also provides insights on particularly effective local application techniques (e.g., concerning pH, concentration [e.g., relative concentration], and / or the timing of contact between crosslinkable entities and / or skin). For example, insights that are particularly useful in combination with microneedling, and insights on particularly effective such combinations of microneedling (e.g., concerning the relative timing of application of crosslinkable entities compared to microneedling and / or one or more parameters [e.g., needle length]).

[0140] In some embodiments, at least one crosslinkable entity is administered in combination with microneedling. In some embodiments, at least one crosslinkable entity (e.g., a first crosslinkable entity including a polymer moiety) is applied topically before microneedling. In some embodiments, at least one crosslinkable entity is applied topically after microneedling. In some embodiments, at least one crosslinkable entity is applied topically before and after microneedling. This disclosure provides insight that application before microneedling may help deliver at least one crosslinkable entity to deeper target sites (e.g., below the stratum corneum, in the dermis). While not bound by any particular theory, microneedling can impart physical force (e.g., downward pressure) to the crosslinkable entity if one is present during microneedling.

[0141] In some embodiments, a first crosslinkable entity (e.g., including a polymer portion) is applied locally (e.g., before and / or after microneedling). In some embodiments, a second crosslinkable entity is applied via microneedles (e.g., by coating the microneedles or incorporating the second crosslinkable entity into the hollow pores of the microneedles).

[0142] In some embodiments, the first and second crosslinkable entities are administered simultaneously. For example, in some embodiments, the first and second crosslinkable entities may be combined before or at the time of administration. In some embodiments, such a combination may be called premixing and may be prepared at some time before administration. Typically, such premixing is prepared within about 30 minutes of administration.

[0143] In some embodiments, the first and second crosslinkable entities are administered sequentially. In some embodiments, the first crosslinkable entity is administered first (for example, in combination with microneedling). In some embodiments, the second crosslinkable entity is administered first (for example, in combination with microneedling).

[0144] In some such embodiments, a certain time separates the administration of the first and second crosslinkable entities. In some embodiments, the time between the administration of the first and second crosslinkable entities is sufficient to allow substantial penetration of the first-administered crosslinkable entity before the administration of the second crosslinkable entity. In some embodiments, such time is about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 6 hours, about 12 hours, or longer. In some embodiments, such time is about 2 hours. In some embodiments, such time is less than about 12 hours, less than about 6 hours, less than about 1 hour, less than about 20 minutes, less than about 10 minutes, less than about 5 minutes, less than about 2 minutes, or less than about 1 minute. In some embodiments, the time is sufficient for at least about 1%, at least about 2%, at least about 5%, at least about 10%, or at least about 20% of the first-administered crosslinkable entity to penetrate before the administration of the second-administered crosslinkable entity. In some embodiments, the time is sufficient to prevent the first agent from being degraded and / or removed from the skin. The reaction rate and / or degree of penetration to a particular crosslinkable entity may be determined, for example, by using a model system (e.g., pig skin), as described herein. The relevant time may be selected with such determination in mind.

[0145] This disclosure provides insight that certain pH ranges (e.g., lower than physiological pH) may enable increased delivery of crosslinkable entities and / or in situ formation of crosslinked materials. For example, certain crosslinkable entities (e.g., a first crosslinkable entity (e.g., HA-CBT), a second crosslinkable entity (e.g., CEC, CKC)) react rapidly at physiological pH (e.g., as shown in Example 2). In some embodiments, a rapid reaction may increase the size of the crosslinkable entities (e.g., molecular weight, physical dimensions), so that the crosslinkable entities may not have enough time to penetrate into the target site (e.g., below the stratum corneum, epidermis, dermis). In some embodiments, pH lower than physiological pH may slow down and / or delay the crosslinking reaction. In some embodiments, the first and second crosslinkable entities may be administered at pH ranges of 2-7, 3-6, 3-5, 4-6, or 4-5. In some embodiments, the presence of further additives may further slow down and / or delay the crosslinking reaction.

[0146] In some embodiments, the preparation containing the crosslinkable entity includes a buffer. In some embodiments, the buffer includes citrate, acetate, MES, alpha hydroxy acid (glycolic acid, lactic acid, tartaric acid), or any other buffer that can maintain the desired pH. In some embodiments, the preparation containing the crosslinkable entity includes citrate at a concentration in the range of 10 to 100 mM.

[0147] In some embodiments, the provided techniques enable the delivery of a sufficient amount / concentration of a crosslinkable entity at a target site so that the crosslinkable entity can form a larger molecule and / or structure than the original crosslinkable entity. Smaller molecules are generally easier to transport than larger molecules. However, larger molecules are preferred because they are retained at the target site for a longer period. For example, larger molecules may take longer to degrade and / or diffuse from the target site than smaller molecules. Delivery of smaller (e.g., lower molecular weight) crosslinkable entities is undesirable because insufficient amounts / concentrations of smaller crosslinkable entities may not form an in situ crosslinkable material with a larger structure that is retained at the target site for a sufficient time. This disclosure provides the remarkable insight that administration in combination with microneedling is particularly useful for the delivery of crosslinkable entities containing polymer moieties having a specific molecular weight range (e.g., 50 kDa, 40 kDa, 30 kDa, 20 kDa, less than 15 kDa). While not bound by any particular theory, when administered in combination with microneedling, a sufficient amount / concentration of crosslinkable entities containing a polymer moiety with a specific molecular weight can be delivered to the target site. In some embodiments, a sufficient concentration of crosslinkable entities to form an in situ crosslinkable material with a large structure that is retained at the target site for a sufficient time (e.g., 24 hours, 3 days, 7 days, 30 days, 60 days, or 90 days) is approximately 5 μg / cm³. 2 ~Approx. 300μg / cm 2 5 μg / cm³ 2 ~Approx. 100μg / cm 2 5 μg / cm³ 2 ~about 90μg / cm 2 5 μg / cm³ 2 ~about 80μg / cm 2 5 μg / cm³ 2 ~about 70μg / cm 2 5 μg / cm³ 2 ~about 60μg / cm 2 5 μg / cm³ 2 ~Approx. 50μg / cm 2 5 μg / cm³ 2 ~about 40μg / cm 2、5µg / cm 2 ∼30µg / cm 2 、5µg / cm 2 ∼20µg / cm 2 、5µg / cm 2 ~10µg / cm 2 、10µg / cm 2 ~100µg / cm 2 、10µg / cm 2 ∼90µg / cm 2 、10µg / cm 2 ∼80µg / cm 2 、10µg / cm 2 ∼70µg / cm 2 、10µg / cm 2 ∼60µg / cm 2 、10µg / cm 2 ∼50µg / cm 2 、10µg / cm 2 ∼40µg / cm 2 、10µg / cm 2 ∼30µg / cm 2 、10µg / cm 2 ∼20µg / cm 2 、20µg / cm 2 ~100µg / cm 2 、20µg / cm 2 ∼90µg / cm 2 、20µg / cm 2 ∼80µg / cm 2 、20µg / cm 2 ∼70µg / cm 2 、20µg / cm 2 ∼60µg / cm 2 、20µg / cm 2 ∼50µg / cm 2 、20µg / cm 2 ∼40µg / cm 2 It contains 20µg / cm 2 ∼30µg / cm 2 である。

[0148] This disclosure provides insight that certain concentration ranges of crosslinkable entities may enable increased delivery of the crosslinkable entity and / or in situ formation of crosslinked material. For example, in some embodiments, high concentrations of the crosslinkable entity may increase the viscosity of the crosslinkable entity and / or decrease its penetration. In some embodiments, high concentrations of the crosslinkable entity may not penetrate into the target site (e.g., the dermis, below the epidermis (e.g., the stratum corneum)) by conventional administration / delivery. In some embodiments, the provided technique (e.g., administration in combination with microneedling) enables the penetration of high concentrations of the crosslinkable entity into the target site (e.g., the dermis, below the epidermis (e.g., the stratum corneum)). In some embodiments, low concentrations of the crosslinkable entity may not yield a sufficient amount of crosslinkable entity to form a crosslinked material in situ. In some embodiments, the concentration of the crosslinkable portion is in the range of 0.01 to 100 mg / mL, 0.1 to 100 mg / mL, 1 to 100 mg / mL, or 1 to 10 mg / mL.

[0149] In some embodiments, the provided crosslinkable entity is administered to the face of the subject (e.g., the entire face and / or specific targets on the face of the subject, e.g., lips, lower lip, upper lip, tear ducts, crow's feet, nasolabial folds, forehead, cheeks, or a combination thereof). In some embodiments, the provided crosslinkable entity is administered to a non-facial area (e.g., knee, neck, décolleté, legs, arms, torso, buttocks, or feet). In some embodiments, the provided crosslinkable entity is administered to the hand (e.g., back of the hand). In some embodiments, the provided crosslinkable entity is administered to the earlobe.

[0150] In some embodiments, the administration site is prepared before administering the crosslinkable entity. In some embodiments, the administration site is prepared by washing the site with lukewarm water and soap. In some embodiments, the administration site is prepared by removing tape.

[0151] In some embodiments, the application site is covered after the application of the crosslinkable entity. In some embodiments, the application site is covered with a Tegaderm® type film after the application of the crosslinkable entity.

[0152] In some embodiments, the skin is treated with water after the system is administered.

[0153] In some embodiments, the crosslinkable entity is administered daily. In some embodiments, the crosslinkable entity is administered at least once daily. In some embodiments, the crosslinkable entity is administered at least twice daily. In some embodiments, the crosslinkable entity is administered 1 to 5 times daily. In some embodiments, the crosslinkable entity is administered 3 to 5 times daily. In some embodiments, the crosslinkable entity is administered every 3 days. In some embodiments, the crosslinkable entity is administered every 7 days. In some embodiments, the crosslinkable entity is administered approximately every 15 days. In some embodiments, the crosslinkable entity is administered approximately every 30 days. In some embodiments, the crosslinkable entity is administered approximately every 60 days. In some embodiments, the crosslinkable entity is administered approximately every 90 days.

[0154] In some embodiments, a chemical substance that improves skin penetration to the crosslinkable entity may be administered. In some embodiments, the chemical substance is administered simultaneously with the crosslinkable entity. In some embodiments, the chemical substance and the crosslinkable entity are administered at different times.

[0155] In some embodiments, one or both of the following inhibitors may be administered: one that interacts with the crosslinkable entity and / or otherwise slows the penetration of the crosslinkable entity and / or a crosslinking inhibitor that blocks one or more feature parts of the crosslinkable entity or crosslinking portion, or otherwise interferes with the reaction of the crosslinking portion that generates the crosslink. In some such embodiments, such inhibitors may be removed before, during, at, or after administration of the crosslinkable entity (e.g., by diffusion, washing, or decomposition).

[0156] In some embodiments, one or more crosslinkable entities are administered as or within a sustained-release formulation. In some embodiments, one or more crosslinkable entities are encapsulated within a matrix or particles (e.g., nanoparticles). In some embodiments, one or more crosslinkable entities are provided as or within an emulsion or dispersion. In some embodiments, additives are added to the formulation to slow the degradation of the crosslinkable entities.

[0157] In some embodiments, crosslinkable entities whose crosslinking portions interact to form crosslinks within the crosslinking material are administered in relative amounts such that complementary crosslinking portions are present in stoichiometric amounts (for example, prescribed to be administered in relative amounts). Alternatively, in some embodiments, such crosslinkable entities are administered in relative amounts such that one of a pair of complementary crosslinking portions is present in molar excess relative to the other. In some such embodiments, such molar excess is in the range of 1.1:1 to 10,000:1. In some embodiments, the molar excess of the relative amount of the crosslinkable portion is in the range of 1.1:1 to 2:1. In some embodiments, the molar excess of the relative amount of the crosslinkable portion is in the range of 1.1:1 to 10:1. In some embodiments, the molar excess of the relative amount of the crosslinkable portion is in the range of 1.1:1 to 100:1. In some embodiments, the molar excess of the relative amount of the crosslinkable portion is in the range of 1.1:1 to 1,000:1.

[0158] In some embodiments, the formulation contains about 0.001% w / w to about 5.00% w / w of crosslinkable entities. In some embodiments, the formulation contains about 0.01% w / w to about 5.00% w / w of crosslinkable entities. In some embodiments, the formulation contains about 0.1% w / w to about 5.00% w / w of crosslinkable entities. In some embodiments, the formulation contains about 1% w / w to about 5.00% w / w of crosslinkable entities. In some embodiments, the formulation contains about 1% w / w to about 3% w / w of crosslinkable entities. In some embodiments, the formulation contains about 2% w / w of crosslinkable entities. In some embodiments, the formulation contains PBS and about 2% w / w of crosslinkable entities.

[0159] In some embodiments, the crosslinkable entity may be administered via a microneedle. For example, in some embodiments, the crosslinkable entity is coated onto the microneedle. In some embodiments, the crosslinkable entity is contained within the hollow pores of the microneedle. In such embodiments, the crosslinkable entity may also be administered topically. In some embodiments, the first crosslinkable entity may be administered topically, and the second crosslinkable entity may be administered via a microneedle. In some embodiments, the second crosslinkable entity may be administered topically, and the first crosslinkable entity may be administered via a microneedle.

[0160] In some embodiments, the microneedling device is a derma roller. In some embodiments, the technique provided involves rolling the derma roller. In some embodiments, each roll includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 passes. In some embodiments, the derma roller is rolled 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the derma roller is rolled over the same area in multiple passes in different directions.

[0161] In some embodiments, one or more crosslinkable entities may be injected via a microneedle (for example, which may be associated with a patch). For example, in some embodiments, one or more crosslinkable entities, or a combination thereof, may be administered through a microneedle (for example, as a liquid). Alternatively or in addition thereto, in some embodiments, one or more crosslinkable entities, or a combination thereof, may be placed within one or more microneedles that can be dissolved or degraded at the time of or after application. As those skilled in the art will see, the use of dissolving or degrading microneedles may be particularly useful or desirable for the delivery of relatively viscous materials (for example, partially or fully formed crosslinkable materials as described herein). In some embodiments, one or more crosslinkable entities may be placed within a microneedle, for example, within its lumen (for example, a hole). In some such embodiments, one or more crosslinkable entities (or a crosslinkable material formed therefrom) may be released by injection through the lumen and / or by dissolution or degradation of the microneedle (for example, its wall). In some embodiments, one or more crosslinkable entities may be formed integrally with the microneedle. In some such embodiments, the microneedles may be formed of a material that otherwise releases one or more crosslinkable entities, or crosslinked material formed therefrom, by dissolving or decomposing during or after application.

[0162] In some embodiments, the skin is pre-treated before (e.g., immediately before or just before) the administration of one or more crosslinkable entities. In some embodiments, skin pre-treatment is achieved by administration or application of a permeation agent or device. In some embodiments, skin pre-treatment includes one or more of the following: application of abrasive cleaners or chemical peels, skin excision, electroporation, iontophoresis, low-frequency sonophoresis, and microneedling. In some embodiments, the skin is scraped before administration of the system. In some such embodiments, the skin is scraped with microneedles and / or a fractional laser before administration of the system. In some embodiments, the crosslinkable entities are administered after (e.g., immediately after or just after) skin pre-treatment with microneedles.

[0163] In some embodiments, one or more crosslinkable entities may be injected by a dual-bore syringe or needle. In some such embodiments, the first and second crosslinkable entities are kept in separate compartments of the syringe or needle at least until administration. In some embodiments, they are combined during administration. In other embodiments, they are kept separately during administration (i.e., each is administered separately at times separated by an optional time interval as described herein). In some embodiments, the dual-bore syringe or needle further includes an acid reservoir. In some embodiments, the acid reservoir may provide a pH in the range of 3 to 5 before, during, and / or after administration of one or more crosslinkable entities.

[0164] In some embodiments, the provided technique includes preparations of crosslinkable entities. In some embodiments, the preparations are made by dissolving a dry crosslinkable entity in a solvent or solvent system. In some embodiments, the preparations are made by dissolving a crosslinkable entity in water. In some embodiments, the preparation of the aqueous solution also includes pH adjustment (e.g., using buffer solutions, NaOH, etc.) and / or the application of destructive energy and / or force, such as sonic treatment and / or homogenization.

[0165] Characterization Those skilled in the art will understand, upon reading this specification, that it may be desirable to characterize one or more features of the crosslinkable entities and / or components or combinations thereof and / or the crosslinkable material(s) they produce, for example, when designing (e.g., selecting its appropriate components) or manufacturing the provided system and / or when monitoring or evaluating its preparation. Alternatively or in addition, in some embodiments, it may be desirable to evaluate one or more features of the provided system at administration, for example, to monitor the subject or treatment thereof.

[0166] In some embodiments, when first and second crosslinkable entities are combined in vitro under physiological conditions, the first and second crosslinkable entities react with each other to form a crosslinked material. The properties of such a crosslinked material can be tuned through the selection of the crosslinkable entities that produce it, and can represent characteristic features of a particular crosslinked material provided by the present invention. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 1 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 500 Pa. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 100 Pa. In some embodiments, the storage modulus of the crosslinked material is in the range of 100 Pa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 500 Pa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 1 kPa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 5 kPa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 500 Pa to 5 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 500 Pa to 1 kPa. In some embodiments, the molecular weight (e.g., weight-average molecular weight) of the crosslinked material is greater than the molecular weight (e.g., weight-average molecular weight) of the first crosslinkable entity. In some embodiments, the molecular weight (e.g., weight-average molecular weight) of the crosslinked material is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than the molecular weight (e.g., weight-average molecular weight) of the first crosslinkable entity. In some embodiments, the molecular weight (e.g., weight-average molecular weight) of the crosslinked material is greater than the molecular weight (e.g., weight-average molecular weight) of the second crosslinkable entity. In some embodiments, the molecular weight (e.g., weight-average molecular weight) of the crosslinked material is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than the weight of the molecular weight (e.g., weight-average molecular weight) of the second crosslinkable entity. In some embodiments, the molecular weight (e.g., weight-average molecular weight) of the crosslinked material is measured by gel permeation chromatography (GPC) analysis.

[0167] In some embodiments, the mass loss of the crosslinked material due to degradation is less than 20% over 3 days in physiological buffer. In some embodiments, the mass loss of the crosslinked material due to degradation is less than 10% over 3 days in physiological buffer. In some embodiments, the mass loss of the crosslinked material due to degradation is less than 5% over 3 days in physiological buffer. In some embodiments, the decrease in molecular weight of the crosslinked material is slower (2, 3, 4, 5, 6, 7, 8, 9, or 1 / 10th slower) than that of the first or second crosslinkable entity when exposed to degradation conditions (i.e., hyaluronidase or oxidative stress).

[0168] In some embodiments, the degree to which the crosslinking material swells during hydration is a characteristic property of the crosslinking material. In some embodiments, the degree of swelling of the crosslinking material is measured based on the mass ratio between the dry crosslinking material and the fully expanded crosslinking material that has reached equilibrium with an external aqueous buffer (Q = mswollen / mdry). In some embodiments, the ratio between the dry crosslinking material and the fully expanded crosslinking material is in the range of 50 to 1000. In some embodiments, the ratio between the dry crosslinking material and the fully expanded crosslinking material is in the range of 100 to 1000. In some embodiments, the ratio between the dry crosslinking material and the fully expanded crosslinking material is in the range of 500 to 1000. In some embodiments, the ratio between the dry crosslinking material and the fully expanded crosslinking material is in the range of 750 to 1000. In some embodiments, the ratio between the dry crosslinking material and the fully expanded crosslinking material is in the range of 50 to 100. In some embodiments, the ratio between the dry crosslinking material and the fully expanded crosslinking material is in the range of 50 to 250. In some embodiments, the ratio between the dry crosslinking material and the fully expanded crosslinking material is in the range of 50 to 500. In some embodiments, the ratio of dry crosslinking material to fully expanded crosslinking material is in the range of 50 to 750. In some embodiments, the ratio of dry crosslinking material to fully expanded crosslinking material is in the range of 250 to 750. In some embodiments, the ratio of dry crosslinking material to fully expanded crosslinking material is in the range of 400 to 600.

[0169] In some embodiments, the first and second crosslinkable entities form a crosslinked material (e.g., a gel) when the dynamic storage modulus (G') is greater than the loss modulus (G''). In some embodiments, the first and second clickable entities form a crosslinked material within 1 second. In some embodiments, the first and second clickable entities form a crosslinked material within 10 seconds. In some embodiments, the first and second clickable entities form a crosslinked material within 1 minute. In some embodiments, the first and second clickable entities form a crosslinked material within 10 minutes. In some embodiments, the first and second clickable entities form a crosslinked material within 30 minutes. In some embodiments, the first and second clickable entities form a crosslinked material within 60 minutes.

[0170] In some embodiments, the first and second crosslinkable entities, when combined in vitro under physiological conditions, react with each other to form a crosslinked material. The properties of such a crosslinked material can be tuned through the selection of the crosslinkable entities that produce it, and can represent characteristic features of the particular crosslinked material provided by the present invention. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 10 kPa.

[0171] In vitro characterization In some embodiments, characterization may involve applying the provided system to a model ex vivo skin system (e.g., pig skin or human skin).

[0172] In some embodiments, characterization may include monitoring one or more features of skin penetration. In some embodiments, the amount of crosslinkable entity that penetrates the skin 30 minutes to 2 days after topical application to excised skin is quantified after solubilizing the skin, for example, with an enzyme and / or other solubilizer. In some embodiments, the amount of crosslinkable entity that penetrates the excised skin is quantified at least 30 minutes after topical application to excised skin after solubilizing the skin with an enzyme and / or other solubilizer. In some embodiments, the amount of crosslinkable entity that penetrates the skin less than 2 days after topical application to excised skin is quantified after solubilizing the skin with an enzyme and / or other solubilizer. In some embodiments, the amount of a crosslinkable entity including a polymer portion and a crosslinkable portion that penetrates after topical application to excised skin exceeds the amount of an entity that includes the same polymer portion but does not include the crosslinkable portion. In some embodiments, the crosslinkable entity includes HA, and the amount of crosslinkable entity that penetrates after topical application to excised skin exceeds the amount of natural HA of the same molecular weight (e.g., weight-average molecular weight) that penetrates the skin. Preferably, in some embodiments, the amount of crosslinkable entities containing HA that penetrate after topical application to excised skin exceeds the natural amount of HA already found in the skin (natural amount of HA already found in pig skin = approximately 100-800 μg / g dry tissue).

[0173] In some embodiments, the penetration depth of the cross-linking material within the excised skin is measured by illuminating the target site with ultraviolet light. In some embodiments, the presence of cross-linking material within the skin of a pig is determined by observing fluorescence when the target site is illuminated with ultraviolet light.

[0174] In some embodiments, the presence of crosslinking material at the target site is observable after a certain period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 days, or longer). In some embodiments, the presence of crosslinking material at the target site is observable after approximately 3.5 days. In some embodiments, the presence of crosslinking material at the target site is observable after approximately 9 days.

[0175] In some embodiments, the presence of cross-linking material at the target site is observed by frozen sectioning followed by fluorescence microscopy. In some embodiments, the presence of cross-linking material at the target site is observed by histological staining.

[0176] in human characterization After administering the system described herein, the attributes of the target site and / or administration site (i.e., skin) are evaluated for improvement over an appropriate period of time compared to the skin attributes before administration of the system. In some embodiments, skin attributes include wrinkles, radiance, firmness, moisture content, skin thickness, elasticity, and skin smoothness. In some embodiments, the improvement includes repair of atrophied skin or scars. In some embodiments, administration of the system results in activation of fibroblasts and / or enhanced collagen synthesis within the target site of skin.

[0177] In some embodiments, skin improvement is measured by the Global Aesthetic Improvement Scale (GAIS). As those skilled in the art will see, the GAIS can be determined using the following evaluation scale. • 1 = greatly improved •2 = Greatly improved 3 = Improved 4 = No change 5 = Worse

[0178] In some embodiments, most targets show an improvement of >1 on GAIS. In some embodiments, most targets show an improvement of >2 on GAIS. In some embodiments, most targets show an improvement of >3 on GAIS. In some embodiments, most targets show an improvement of about 1 to 2 on GAIS. In some embodiments, most targets show an improvement of about 1 to 3 on GAIS. In some embodiments, most targets show an improvement of about 1 on GAIS. In some embodiments, most targets show an improvement of about 2 on GAIS. In some embodiments, most targets show an improvement of about 3 on GAIS.

[0179] In some embodiments, improvement in the skin is measured using the Modified Fitzpatrick Wrinkle Scale (MFWS). As those skilled in the art will see, the MFWS can be determined using the following evaluation scale. • Class 0 - Wrinkle-free. No visible wrinkles. Continuous skin lines. • Class 0.5 - Very shallow but visible wrinkles. • Class 1 - Minor wrinkles. Visible wrinkles and slight indentations. • Class 1.5 - Visible wrinkles and noticeable indentations. Wrinkle depth is 1 mm. • Class 2 - Moderate wrinkles. Clearly visible wrinkles, 1-2 mm deep. • Class 2.5 - Noticeable, visible wrinkles. Wrinkle depth is between 2mm and 3mm. • Class 3 - Deep wrinkles. Deep, groove-like wrinkles. Wrinkle depth exceeds 3mm.

[0180] In some embodiments, the wrinkles on the target surface are reduced by at least 0.5 on the MFWS. In some embodiments, the wrinkles on the target surface are reduced by 1 to 0.5 on the MFWS. In some embodiments, the wrinkles on the target surface are reduced by 1.5 to 0.5 on the MFWS. In some embodiments, the wrinkles on the target surface are reduced by 2 to 0.5 on the MFWS. In some embodiments, the wrinkles on the target surface are reduced by 2.5 to 0.5 on the MFWS. In some embodiments, the wrinkles on the target surface are reduced by 3 to 0.5 on the MFWS.

[0181] In some embodiments, the skin in question is evaluated for improvements in smoothness, shine, or firmness.

[0182] In some embodiments, skin moisture content can be monitored. In some embodiments, skin moisture content is higher than that of untreated skin, as determined by methods known in the art. In some embodiments, skin moisture content is determined by a CM825 corneometer. In some embodiments, skin moisture content is >20% higher on day 7 after administration of the system.

[0183] In some embodiments, the provided system is used to treat a person exhibiting abnormal transepidermal water loss (TEWL). As those skilled in the art will know, abnormal TEWL may result from skin damage caused, for example, burns, certain chemicals, pathological conditions (e.g., eczema), physical abrasion, tape stripping, ultraviolet radiation, or a combination thereof. In some embodiments, the level of TEWL in treated skin is lower than that of untreated skin, as determined by methods known in the art. In some embodiments, the level of TEWL in treated skin is statistically lower than the baseline level, as understood by methods known in the art. In some embodiments, TEWL is measured by a Tewameter TM300 meter (Courage-Khazaka Electronics). In some embodiments, TEWL is measured by a Tewameter TM Nano (Courage-Khazaka TEWL is measured by a Tewameter Triple TM330T (Courage-Khazaka Electronics). In some embodiments, TEWL is measured by an Invitro Tewameter VT310 (Courage-Khazaka Electronics). In some embodiments, the TEWL of the treated skin is >10% lower on day 7 after administration of the system.

[0184] In some embodiments, the skin is evaluated for improvement in skin smoothness. In some embodiments, the skin is smoother after administration of the system than before administration of the system. In some embodiments, skin smoothness is evaluated using living skin surface evaluation (SELS). In some embodiments, the skin is evaluated using rapid in vivo measurement of skin phase shift (PRIMOS). In some embodiments, the skin is evaluated by 3D skin profilometry using Canfield Primos. In some embodiments, the skin is evaluated using the Remperl wrinkle scale. In some embodiments, the skin is evaluated using a 7-point subject satisfaction scale. In some embodiments, the skin is evaluated using oral commissure. In some embodiments, the skin is evaluated using the Allergan skin roughness scale.

[0185] In some embodiments, the skin is evaluated to determine the presence and / or extent of cross-linked material within the skin. In some embodiments, the presence of cross-linked material within the skin is measured using near-infrared (NIR) spectroscopy, confocal microscopy, integrating sphere spectrophotometer, or determined using viscoelastic deformation (VED, mm), elastic deformation (ED, mm), ultimate deformation (UD, mm), and pressure deformation ratio methods. In some embodiments, the presence and / or of cross-linked material within the skin is measured by illuminating the target site with ultraviolet light. In some embodiments, the presence of cross-linked material within the skin is determined by observing fluorescence when the target site is illuminated with ultraviolet light.

[0186] In some embodiments, the presence of crosslinking material at the target site is observable after a certain period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 days, or longer). In some embodiments, the presence of crosslinking material at the target site is observable after about 2 days. In some embodiments, the presence of crosslinking material at the target site is observable after about 9 days. In some embodiments, the presence of crosslinking material at the target site is observable after about 11 days. In some embodiments, the presence of crosslinking material at the target site is observable after about 20 days. In some embodiments, the presence of crosslinking material at the target site is observable after about 30 days.

[0187] kit In some embodiments, this disclosure provides a kit comprising the system described herein.

[0188] In some embodiments, the provided kit includes a plurality of containers or vessels, each containing a separate container for housing the first and second crosslinkable entities.

[0189] In some embodiments, the provided kit includes at least one container or vessel containing a plurality of separate compartments (e.g., a dual-bore syringe or needle, or a dual-chamber package with a mixing chamber before dispensing). In some embodiments, the first and second crosslinkable entities are housed separately within such compartments.

[0190] In some embodiments, the present disclosure provides one or more containers, vessels, or compartments in which the first or second crosslinkable entities described herein are placed. In some such embodiments, the placed crosslinkable entities exist in a dry form. In some such embodiments, the crosslinkable entities exist in a liquid form. In some embodiments, the placed crosslinkable entities are stored for a period of time (for example, at least one day, one week, one month, three months, six months, or longer). In some such embodiments, the stored composition is stable over the storage period, i.e., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the crosslinkable entities remain undegraded. In some embodiments, the stored composition is stable over the storage period, i.e., at least about 50%, 65%, 70%, 75%, or more of the crosslinkable entities remain undegraded. [Examples]

[0191] In this embodiment, in particular, certain strategies that may be used to characterize and / or evaluate the penetrants (and / or components and / or compositions or combinations thereof) described herein are described. Using such strategies (or equivalents thereto, as will be apparent to those skilled in the art who have read this disclosure), penetrants, components (e.g., parts), compositions, or combinations thereof may be evaluated for their suitability for use according to this disclosure. Thus, in some embodiments, this disclosure provides techniques for characterizing and / or selecting useful parts, linkers, penetrants, and / or components, compositions, and / or combinations thereof.

[0192] Example 1: Enhanced delivery of HA-CBT by microneedling This embodiment demonstrates an improvement in the transdermal delivery of a particular crosslinkable entity (i.e., two CBT-bound HAs of different molecular weights) when microneedling the skin, especially when microneedling is performed after topical application of the crosslinkable entity. In particular, this embodiment demonstrates an improvement achieved even with tape stripping.

[0193] Hyaluronic acid (HA) was functionalized with a 6-amino-2-cyanobenzothiazole (CBT) derivative (e.g., bound to glycine). HA-gly-CBT was further derivatized with an IR dye (CF647 amine, Sigma-Aldrich) for quantitative study. 10kDa(HA) 10 CBT and HA 10 CBT IR ) and 20kDa(HA 20 CBT IR Both HA derivatives were synthesized by initiating HA modification with HA of different molecular weights.

[0194] Human skin tissue (obtained from the abdomen of a female donor aged approximately 40-50 years) was purchased from ZenBio (North Carolina). The skin surface was washed with PBS and soapy water, and then thoroughly rinsed again with PBS. Next, the tissue was examined to ensure a surface area of ​​at least 2 cm². 2 The polymer was cut into small pieces and hydrated in warm PBS for at least 30 minutes. The hydrated pieces were then dried in ambient air for approximately 10 minutes. Each skin section was stretched on a rigid base and fixed with a needle before microneedling using a derma roller with a microneedle length of 250 μm. The concentration, volume, and formulation used for the polymer in each example are shown in Table 1. The formulation to be tested was applied to the skin surface before and after treating the skin with a derma roller. [Table 1]

[0195] Then, any excess solution remaining on the surface was rubbed into the skin using a spatula. In the example where the skin surface was peeled off with tape before topical application, adhesive tape was applied to the skin surface under pressure and peeled off 20 times in a row to remove most of the stratum corneum. Then, skin sections were prepared with a diffusion area of ​​1 cm². 2 The cells were mounted on a Franz diffusion cell (PermeGear, Bethlehem, PA). 100 μl of the formulation was filled into the donor chamber and sealed with Parafilm. The receiver compartment was filled with 6 mL of PBS buffer (pH 7.4). Care was taken to remove bubbles from under the skin and in the receptor solution. Depending on the study, the cells were maintained at 37°C for 1 hour or overnight.

[0196] After incubation, each skin sample was removed from the Franz diffusion cell and rinsed with PBS. The sections were then subjected to tape stripping. The stratum corneum was peeled off using adhesive tape (Scotch® Clear Tape, 3M Corporate, St. Paul, MN). The first 10 strips were called the upper stratum corneum (USC), and the next 10 strips were called the lower stratum corneum (LSC). The epidermis was then separated from the dermis using a sterile surgical blade. The remaining dermis was cut into smaller pieces. The USC and LSC were collected and placed in separate glass vials with 3 ml of PBS / methanol (1:1) mixture. The epidermis and dermis were collected and placed in separate glass vials with 1 ml of PBS:methanol (1:1) mixture. The vials containing the separated layers were then shaken overnight at 37°C to extract the "IR" labeled polymer (HA-CBT). IR The tissue extract was then extracted. The tissue extract was centrifuged to remove tissue residue, and the supernatant was collected for analysis using a fluorescence plate reader (Tecan) at excitation emission wavelengths of (630 nm / 665 nm).

[0197] Study 1. Topical delivery of HA-CBT by surface skin exfoliation versus microneedling. 200 μl of HA 10 CBT IR or HA 20 CBT IRwas applied topically to the skin stripped with tape. In the microneedling group, 50 μl of HA 10 CBT IR or HA 20 CBT IR was applied each time before two rotations with 5 passes. Subsequently, 100 μl of the formulation was applied topically to the microneedled skin. All groups were incubated overnight at 37 °C. Compared with the skin stripped with tape, microneedling enhanced the delivery of HA 10 CBT IR by approximately 9.5-fold in the stratum corneum and epidermis and approximately 8-fold in the dermis, as shown in Figure 1A. For HA 20 CBT IR in the case of 、 microneedling enhanced its delivery by approximately 4-fold in the stratum corneum and epidermis and approximately 7-fold in the dermis, as shown in Figure 1B.

[0198] Study 2. Local delivery of HA-CBT of different sizes by microneedling The effect of molecular weight (10 kDa vs 20 kDa) on HA-CBT delivery by microneedling was investigated. 50 μl of HA-CBT was applied each time before two rotations with 5 passes. Following this step, 100 μl of the formulation was applied topically to the microneedled skin. All groups were incubated overnight at 37 °C. The skin levels of low molecular weight HA-CBT (HA 20 CBT IR ) increased by approximately 4-fold as shown in Figure 2 compared with microneedling of high molecular weight HA-CBT (HA 10 CBT IR ).

[0199] Study 3. Application of HA-CBT formulation before or after microneedling HA 10 CBT IRThe formulation was applied to the skin in two different ways. In the first study group, the skin surface was first micro-needled in a total of 10 passes using a 250-μm derma roller. Then, 100 μl of the formulation was topically applied to the micro-needled skin. In the second study group, 50 μl of the formulation was applied each time in 5 passes before 2 rotations. Then, 100 μl of the formulation was topically applied to the micro-needled skin. All groups were incubated at 37 °C for 1 hour. HA was applied before micro-needling 10 CBT IR When applied, as shown in Figure 3, the accumulation in the stratum corneum increased by about 4-fold and in the epidermis and dermis by about 2-fold. These results indicate that a significant amount of material is being pushed into the skin during the micro-needling process.

[0200] Study 4. 1-hour incubation vs overnight incubation 50 μl of HA 10 CBT IR The formulation was applied each time before micro-needling 2 times in 5 passes. Following this step, 100 μl of the formulation was topically applied to the micro-needled skin. One group was incubated at 37 °C for 1 hour and the other group was incubated overnight. Similar HA-CBT accumulations in the stratum corneum and epidermis were obtained for overnight incubation and 1-hour incubation. As shown in Figure 4, a 3-fold increase in the delivery of HA-CBT was detected in the dermis for the polymer group incubated overnight. Although the incubation was less than overnight, a significant amount of HA-CBT was delivered even with 1-hour incubation.

[0201] Study 5. Effect of HA-CBT concentration on delivery by micro-needling HA 10 CBT IRThe HA-CBT was applied to the skin by microneedling at concentrations of 10 mg / mL and 50 mg / mL in PBS. The details of the application were consistent with the procedure described in Example 1 and Study 4, which included overnight incubation. As shown in Figure 12, HA-CBT delivery was 5 times higher in the sclera and epidermis with higher concentrations. Similarly, HA-CBT delivery into the dermis was 8 times higher. These results demonstrate that the amount of HA-CBT delivered when microneedling is applied can be manipulated using HA-CBT concentration as a variable.

[0202] Example 2: Parameters for HA-CBT crosslinking This embodiment demonstrates the effect of the concentration of the crosslinkable entity on the generation and size of the crosslinked material produced in vitro. In particular, this embodiment specifies particularly useful concentrations (e.g., of the crosslinkable entity) and molar ratios (e.g., of the crosslinked portion and / or the crosslinkable entity).

[0203] In this embodiment, the relationship between concentration and the timing of exposure (e.g., contact) of crosslinkable entities (and / or crosslinked portions) to each other is further shown with respect to the degree and / or size of the crosslinked material formed in situ.

[0204] Furthermore, this embodiment also shows the effect of the pH to which crosslinkable entities (and / or crosslinked portions) are exposed to each other on the degree and / or size of the crosslinked material formed in situ, as well as the relationship between such pH and the concentration and / or time to which the relevant entities (and / or portions) are exposed.

[0205] In this example, the degree of crosslinking was characterized by measuring the increase in molecular weight of the starting HA-CBT using gel permeation chromatography (GPC). GPC analysis was performed on an Agilent 1100 HPLC with a diode array detection (DAD) detector (326 nm). An Agilent PL AquaGel-OH MIXED-M column (8 μm 300 x 7.5 mm) and an Agilent PL AquaGel-OH20 column (8 μm 300 x 7.5 mm) were connected in series. A mobile phase of 0.2 M NaNO3 in 9:1 water:methanol was used at a flow rate of 1 mL / min.

[0206] Study 1. Effects of HA-CBT and crosslinking agent concentration HA 10 CBT was mixed with the crosslinking agent molecule cysteine-lysine-cysteine ​​(CKC) in PBS at different concentrations and molar ratios. The two components were incubated at 37°C for 30 minutes to induce crosslinking. After incubation, all samples were further diluted in PBS to a concentration of 1 mg / mL, unless the starting concentration had already fallen below that, and then subjected to GPC.

[0207] In Figures 5A and 5B, HA 10 The CBT concentration was varied from 0.1 to 10 mg / mL, while the CKC concentration was linearly varied to maintain a 1:1 CBT:Cys molar ratio. As the polymer concentration increased, the chromatograph shifted to the left, indicating the formation of larger crosslinked structures. HA 10 When the CBT concentration exceeds 20 mg / mL, insoluble gels with higher molecular weights may form. As shown in Figure 5B, the retention time shift is much less in samples crosslinked at less than 1 mg / mL, compared to HA above 1 mg / mL. 10 This indicates that a CBT concentration is preferable for forming larger cross-linking structures.

[0208] In Figure 5C, HA 10The concentration of CBT was kept constant at 1 mg / mL and mixed with CKC at different concentrations to obtain CBT:Cys molar ratios of R=0.5, 1, 2, and 10. At R=1, the chromatograph shifted to the leftmost position, indicating that an equimolar ratio of Cys:CBT is desirable for obtaining the largest cross-linking structure. A 2x molar excess of Cys (R=2) resulted in a much larger structure than a 2x molar excess of CBT (R=0.5), and HA 10 An excess of CKC relative to CBT indicates that this is favorable for effective in situ crosslinking.

[0209] Study 2. Effects of skin on HA-CBT crosslinking HA 10 CBT (1 mg / mL or 2 mg / mL) was incubated with human skin explant homogenate (50 mg skin / 100 μL solution) at 37°C. A crosslinking agent (CKC) was added to the mixture at the specified time to induce crosslinking. Following the addition of the crosslinking agent, the sample was centrifuged to remove skin fragments, and the supernatant was subjected to GPC as described above.

[0210] As shown in Figure 6A, skin homogenate (+HA, +Skin, -XL) and HA after incubation. 10 The CBT spectrum is pure HA 10 CBT spectra (+HA, -Skin, -XL) were compared, and the intensity increased at the same retention time. This result may indicate that a reaction occurred with soluble aminothiols (e.g., free cysteine) in the homogenate solution immediately after mixing the polymer with skin (+HA, +Skin, +XL). 0分 When the crosslinking agent was added to the polymer / skin solution at 30 minutes (+HA, +Skin, +XL), the spectrum shifted to the left, indicating that crosslinking occurred in the presence of skin homogenate. However, when the crosslinking agent was added at 30 minutes (+HA, +Skin, +XL), the spectrum shifted to the left, indicating that crosslinking occurred in the presence of skin homogenate. 30分 When added after 90 minutes of incubation (+HA, +Skin, +XL), the shift was less noticeable, indicating that fewer crosslinks occurred. 90分Crosslinking was almost nonexistent in the sample (+HA, +Skin, -XL). This is evident from the similarity of the spectrum to that of the sample without the crosslinking agent (+HA, +Skin, -XL). Conversely, as shown in Figure 6B, crosslinking was still evident in the polymer incubated with skin homogenate for 90 minutes at a higher polymer concentration of 2 mg / mL (+HA, +Skin, +XL). 90分 ).

[0211] These results indicate that HA 10 This indicates that CBT has a finite time to be active and react with the crosslinking agent CKC after incubation with skin. However, if the polymer concentration is sufficiently high (e.g., higher than 1 mg / mL HA-CBT in the skin), the polymer can remain active for a much longer time. These results suggest that HA 10 This illustrates the importance of delivering CBT and the crosslinking agent in close proximity in time to ensure efficient in situ crosslinking between the two.

[0212] Study 3. Effect of pH on HA-CBT crosslinking To deliver HA-CBT and the crosslinking agent molecule locally through the skin, it is preferable that the two molecules do not crosslink until they reach the target site. However, to increase the degree of crosslinking at the target site (as shown in Figure 6), it is advantageous to deliver the two molecules simultaneously. Simultaneous delivery also allows for the application of a single product. To achieve simultaneous application without crosslinking only on the skin surface, the crosslinking reaction rate may be reduced using pH.

[0213] To demonstrate the effect of pH on reaction rate, HA 10 CBT was mixed with the crosslinking agent molecule cysteine-ethylenediamine-cysteine ​​at various pH levels. Specifically, HA 10 CBT (20 mg / mL) was mixed with CEC (Cys:CBT molar ratio = 1) in both neutral pH (PBS, pH = 7.4) and acidic buffer (25 mM citrate, pH = 4.6). As shown in Figure 7, immediately after combining the two molecules, they reacted in PBS to form a larger cross-linked structure (+XL).0min、pH=7.4 ). However, the GPC spectrum of the combination system at pH=4.6 (+XL 0分、pH=4.6 The spectrum (-XL) of the polymer alone appears to have a similar molecular weight, and it is evident that the two do not react when mixed at pH=4.6. The combination system at pH=4.6 provides time to apply the two products to the skin before crosslinking. Even after incubating the two components at 25°C and 37°C for 20 minutes, the final structure had a molecular weight that was much smaller than that of the mixture immediately crosslinked at neutral pH.

[0214] Study 4. In situ crosslinking within the skin. To demonstrate the ability to crosslink within the skin, HA 10 CBT and CEC were simultaneously injected into the skin, separated at 30°C, and then injected sequentially. As a control, HA was used. 10 CBT was also injected alone. As shown in Figure 11A, when the two materials were injected simultaneously, a significant amount of cross-linking structure was present (HA marked by green arrows). 10 (Leftward shift of CBT). When there was a 30-minute delay between HA-CBT and CEC injections, fewer cross-linking structures were observed (Figure 11B), demonstrating that better cross-linking is achieved when the two materials are delivered in close proximity in time.

[0215] Example 3: Delivery of HA-CBT by microneedling and formation of crosslinked structures in situ This embodiment demonstrates the effect of the concentration of the crosslinkable entity on the formation and size of the resulting in situ crosslinked material. In particular, this embodiment specifies particularly useful concentrations (e.g., of the crosslinkable entity) and molar ratios (e.g., of the crosslinked portion and / or the crosslinkable entity).

[0216] This embodiment also demonstrates the effect of needle length on the degree and / or size of crosslinking material formation in situ.

[0217] The procedure was the same as in Example 1. Briefly, the surface of a human skin section obtained from the abdomen of a female donor aged 40 - 50 years was washed with a solution of PBS and soap. Then, a 2 cm 2 skin section was hydrated with warm PBS for about 30 minutes. The hydrated skin section was stretched and fixed with needles and kept taut during micro-needling using a derma roller. Details regarding the concentrations, volumes, and solvents used for the polymer and cross-linker in each study are shown in Table 2. The final formulation to be tested was applied to the skin surface before micro-needling. Derma rollers with different needle lengths (such as 250 μm, 500 μm, 1000 μm, etc.) were tested for local delivery and in situ cross-linking.

Table 2

[0218] After incubation, each skin piece was removed from the Franz diffusion cell and rinsed with PBS. Then, as described above, the sections were subjected to the tape stripping method. The upper stratum corneum (USC) and lower stratum corneum (LSC) were collected and placed in separate glass vials with a 3 ml PBS / deionized water (1:1) mixture. The epidermis and dermis were collected and placed in separate glass vials with a 1 ml PBS:deionized water (1:1) mixture. Next, the vials with the separated layers were shaken at 37 °C overnight to extract HA-CBT. The obtained tissue extracts were centrifuged to remove tissue debris, and the collected supernatant was concentrated by rotary evaporation and resuspended in 125 μl of deionized water. The resuspended concentrate was centrifuged again at 5000 g for 3 minutes, and 90 μl of these purified supernatants were analyzed by GPC as described above.

[0219] Study 1. Influence of HA-CBT concentration on in situ cross-linking HA-CBT formulations at concentrations of 20 mg / mL and 50 mg / mL, accompanied by equimolar amounts of crosslinking agent CEC, were applied topically in combination with microneedling. GPC chromatography of epidermal and dermal extracts for the high-concentration group (50 mg / mL) showed initial uncrosslinked HA 10 Compared to CBT, there is a significant leftward shift. The low-concentration group (20 mg / mL) also showed a shift in the GPC chromatogram for epidermal extracts, but the shift was much larger in the high-concentration group. Increasing the HA-CBT concentration from 20 mg / mL to 50 mg / mL induced qualitative changes in the GPC spectra of epidermal and dermal extracts, indicating that crosslinking was far more effective in the 50 mg / mL group. Data from ex vivo crosslinking studies (e.g., Figures 5A-5C) support the idea that there is a threshold concentration that can be achieved in situ to achieve effective crosslinking. This experiment shows that HA concentrations greater than 20 mg / mL 10 This suggests that applying CBT concentration locally in combination with microneedling (e.g., a 250 μm derma roller) can achieve effective crosslinking in situ.

[0220] Study 2. Evidence of crosslinking in situ HA 10 CBT and the crosslinking agent cysteine-ethylenediamine-cysteine ​​(CEC) were applied locally using microneedling in both non-crosslinked and pre-crosslinked forms. For the "non-crosslinked" formulation, HA 10 CBT and CEC were mixed immediately before application and applied to an acidic buffer (25 mM citrate, pH=4.6, 0.2% EDTA). The non-crosslinked formulation showed a significant decrease in crosslinking reaction. For the "pre-crosslinked formulation," the two components were mixed 1 hour before application to the acidic buffer and neutral buffer (PBS). After applying the formulation using microneedling, the skin samples were further incubated in Franz cells at 37°C for 1 hour, and HA-CBT was extracted from each skin layer as described above.

[0221] As shown in Figures 9A and 9B, extracts from skin sections microneedled with a non-crosslinked formulation revealed a leftward shift in the GPC spectrum. The spectrum indicates the formation of high molecular weight crosslinked HA-CBT in the epidermis and dermis (S1, S2). However, the GPC spectra obtained from extracts of skin sections treated with pre-crosslinked formulations in acidic buffer (S3 and S4) and neutral buffer (S5 and S6) were similar to the background tissue signal (blank). The absence of a signal for the pre-crosslinked formulation highlights the advantage of using a non-crosslinked HA-CBT / Cys crosslinking agent system to construct a network of higher molecular weight crosslinked HA in situ. 10 Unlike pre-crosslinked formulations that are too large to penetrate the skin, CBTs can react after penetrating the skin to form larger structures in situ.

[0222] Study 3. Effect of needle size HA-CBT and CEC were delivered locally by microneedling using derma rollers of different needle lengths (250 μm, 500 μm, and 1000 μm). As shown in Figures 10A and 10B, extracts from skin sections revealed that the GPC spectrum shifted further to the left and its intensity increased with increasing needle size. Microneedling with 500 μm (S3 and S4) and 1000 μm (S5 and S6) derma rollers delivered significantly more HA-CBT in both the epidermis and dermis compared to microneedling with a smaller 250 μm derma roller, forming larger in situ cross-linked structures (the spectrum shifted further to the left).

[0223] IR labeled material (HA 10 CBT IR) was also delivered using 250 μm and 500 μm derma rollers, similar to CEC. A fluorescence plate reader (Tecan) was used to quantify material delivery, as described elsewhere in this specification. As shown in Figure 13, consistent with the GPC results, the use of a larger derma roller (500 μm) resulted in significantly more HA-CBT being delivered into the epidermis and dermis.

[0224] HA-CBT and CEC were delivered locally by microneedling using derma rollers with different needle lengths. Microscopy was also used to visualize the differences in delivery using derma rollers with different needle lengths. (IR-labeled material (HA)) 10 CBT IR ) and CEC were delivered using the same application and incubation procedures described immediately prior to this. After incubation, skin sections were snap-frozen in OCT and then thinned on a cryostat microtome. Microscopic images were then taken on a Zeiss AxioPlan2 microscope using a 10x objective lens and Cy5 filter to visualize the location of the IR dye. As shown in Figures 14A-C, much greater fluorescence was observed in the dermis and epidermis compared to the group treated with larger microneedles.

[0225] Example 4: In vivo study This example describes the local delivery of in situ crosslinked HA-CBT and the resulting persistence and safety. This example also demonstrates the effect of derma roller size on HA-CBT delivery and persistence.

[0226] HA-CBT and a cross-linking agent (CEC) were administered to a miniature pig model either (i) topically using a derma roller or (ii) intradermally by injection. Three Yorkshire miniature pigs were used (provided by CBSET (a contract research organization (CRO) (Lexinton, MA)) and raised under standard environmental conditions with access to feed and water). All animal experiments were reviewed and approved by the CBSET Animal Experiments Committee.

[0227] The test groups for the proposed research are listed below. For groups 4, 5, and 6, the test formulations were treated with HA immediately before application. 10 An equimolar solution of CBT and the crosslinking agent molecule cysteine-ethylenediamine-cysteine ​​(CEC) (CBT to Cys) was prepared by mixing them in an acidic buffer (25 mM citrate buffer and 0.2% EDTA, pH 4.6). For Group 3 (2 negative controls), HA 10 Prepare CBT in an acidic buffer and apply it without the crosslinking molecule CEC. Apply 50 μl of the formulation. Roll the derma roller 10 times vertically to push the formulation into the skin. Repeat this 4 times vertically to apply a total of 200 μl of the formulation vertically and cover the entire skin area. Similarly, apply 50 μl of the formulation perpendicular to the previous direction. Roll the derma roller 10 times horizontally. Repeat this 4 times to apply a total of 200 μl of the formulation horizontally. As a result, a total of 400 μl of the formulation is applied to the entire skin area to be treated. Exam group: Group 1. Intradermal injection of HA-CBT (50 mg / mL) and CEC (2.43 mg / mL) (positive control) Group 2. Local application of a 500 μm derma roller using PBS (negative control 1) Group 3. Local use of a 500 μm derma roller with HA-CBT (2 negative controls) Group 4. Topical use of a 250 μm derma roller for simultaneous delivery of HA-CBT (50 mg / ml) and the crosslinking agent molecule CEC (2.43 mg / ml) (Test Group 1) Group 5. Local application of a 500 μm derma roller for simultaneous delivery of HA-CBT (50 mg / ml) and the crosslinking agent molecule CEC (2.43 mg / ml) (Test Group 2) Group 6. Local application of a 1000 μm derma roller for simultaneous delivery of HA-CBT (50 mg / ml) and the crosslinking agent molecule CEC (2.43 mg / ml) (Test Group 3)

[0228] Each animal has six parts (25-36 cm) on each side of its back (side A and side B).2 There are two application sites ( / area). On day 0, six different test groups are applied once to each site on side A and side B, for a total of 12 application sites / animal. The application sites are covered with Tegaderm tape for 1 hour, after which it is removed.

[0229] On day 1 after application, three biopsies are taken from each of the six "A" application areas (N=18 biopsies / pig) and used to analyze and measure the amount of cross-linked HA-CBT in the skin. Analytical techniques (including UV plate reader and GPC) are used to quantify the amount of HA-CBT and characterize the size of HA-CBT in the skin. Since CBT is fluorescent and HA-CBT is also labeled with IR dye, microscopy is also used to visualize the location of HA-CBT in the skin. The animals are then left for 10 days without further application, during which time irritation / redness, swelling / edema, peeling, cracking, etc., at each site are evaluated. Digital photographs of the application sites are taken daily.

[0230] On day 10, three biopsies are taken from each of the six "B" application areas (N=18 biopsies / pig) and used to analyze and measure the amount of HA-CBT remaining in the skin. The biopsies may also be fixed and sent for histological examination to understand the resistance of cross-linked HA-CBT in the pig skin (data not shown).

[0231] Study 1. In vivo delivery of HA-CBT and CEC and in situ crosslinking. Eleven days after the application of HA-CBT and CEC, the pigs were euthanized. Skin samples were excised from each treatment group. A portion of the skin was fixed and preserved for histology, and another portion was used for GPC analysis (after extraction with buffer, as described above). Figure 15A shows that HA-CBT was detected in the skin 11 days after application when CEC was not used. However, the molecular weight was significantly reduced. In contrast, high molecular weight material was detected in the skin 11 days after the application of HA-CBT and CEC (Figure 15B). This indicates that the material was cross-linked in the skin and remained in a high molecular weight state in the skin during the study period.

[0232] Figure 16 shows H&E staining of the skin 11 days after treatment using (A) Group 2, (B) Group 1, and (C) Group 5. Figure 16A shows that when a buffer was dermarolled into porcine skin using a 500 μm dermaroller, no obvious side effects such as inflammation were observed 11 days after treatment. Figure 16B further shows that no inflammation was observed after direct injection of HA-CBT and CEC into the skin. In addition, Figure 16C shows that no inflammation was observed after applying HA-CBT and CEC using a dermaroller.

[0233] Study 2. Evidence of HA in the superficial dermis A follow-up study was conducted using the same procedure as described in Study 1, except that the formulation was applied repeatedly (on days 0 and 14). Figure 17 shows that when crosslinked HA-CBT and CEC were dermarolled into porcine skin twice (on days 0 and 14) using a 500 μm dermaroller, HA (shown in blue and indicated by arrows) was observed in the superficial dermis of biopsy samples taken from the animals on day 28. In certain embodiments, for example, the following are provided: (Item 1) A method of establishing a crosslinking material at a target site within the skin, comprising: (i) applying a first crosslinkable entity and a second crosslinkable entity that react to form the crosslinking material to a skin location; and (ii) after applying at least one of the crosslinkable entities, micro-needling the skin location to cause the crosslinking material to be present at the target site within the skin. The method as described above. (Item 2) The method according to Item 1, wherein the first crosslinkable entity and the second crosslinkable entity are at a pH within the range of about 3 to about 5. (Item 3) The method according to any preceding item, wherein the presence of the crosslinking material can be determined at the target site within the skin The method as described above. (Item 4) A method for any preceding item, wherein the crosslinking material is present for a period of time from 1 minute to 1 hour. (Item 5) The method according to any prior art, wherein the first crosslinkable entity comprises a polymer portion, and the weight-average molecular weight of the polymer portion prior to the application step is in the range of 1 kDa to 500 kDa. (Item 6) The method according to item 5, wherein the weight-average molecular weight of the polymer portion prior to the application step is in the range of 5 to 20 kDa. (Item 7) The method according to items 5-6, wherein the polymer portion is a hyaluronic acid ("HA") polymer. (Item 8) The method according to any prior item, wherein the first crosslinkable entity comprises another molecule that reacts with either the -SH or NH2 group of CBT, a CBT mimetic, or Cys (e.g., D-Cys, L-Cys, or a combination thereof). (Item 9) The method of any prior item, further comprising applying the first crosslinkable entity and the second crosslinkable entity to the skin location. (Item 10) A method for any prior item, wherein the first crosslinkable entity and the second crosslinkable entity are applied simultaneously. (Item 11) The method of item 10, further comprising mixing the first crosslinkable entity and the second crosslinkable entity before the application step. (Item 12) The method according to item 11, wherein the mixing step is performed 0 to 30 minutes before the application step. (Item 13) A method for any preceding item, wherein the second crosslinkable entity is applied after the second crosslinkable entity. (Item 14) The method according to any prior item, wherein the microneedling is performed by a microneedling device having microneedles. (Item 15) The aforementioned microneedle device has a microneedle density of approximately 20 to 150 microneedles / cm³. 2 The method described in item 14, which is within the range. (Item 16) The microneedle device having 1 to 100,000 microneedles, according to the method described in items 14 to 15. (Item 17) The microneedle is the method described in items 14 to 16, wherein the length is approximately 100 μm to approximately 1000 μm. (Item 18) The microneedle device is a derma roller, as described in items 14-17. (Item 19) The method according to any preceding item, wherein the dermal layer is the epidermis (e.g., stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, stratum basale) or the dermis. (Item 20) The method according to any prior item, wherein the concentration of the first crosslinkable entity is in the range of 0.1 to 100 mg / mL. (Item 21) The method according to any preceding item, wherein the second crosslinkable entity is selected from the group consisting of cysteine-ethylenediamine-cysteine ​​(CEC), cysteine-lysine-cysteine ​​(CKC), cysteine-PEG-cysteine, and combinations thereof. (Item 22) The method according to any prior item, wherein the concentration of the second crosslinkable entity is in the range of 0.1 to 100 mg / mL. (Item 23) The method according to any prior item, wherein the molecular ratio of the first cross-linked portion to the second cross-linked portion is in the range of 1:1 to 5:1. (Item 24) The method according to any prior item, wherein the first crosslinkable entity comprises a first crosslinking portion, and 1 to 20 mol% of the first crosslinkable entity comprises the first crosslinking portion. (Item 25) The method according to any prior art, characterized in that the crosslinking material has a weight-average molecular weight at the target site within the skin that is (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 times) greater than the weight-average molecular weight of the first crosslinkable entity. (Item 26) A method for establishing a cross-linking material at a target site within the skin, (i) The step of applying the first crosslinkable entity and the second crosslinkable entity to a skin site at a pH in the range of approximately 3 to approximately 5, (ii) Microneedling at the skin location, The method comprising the step of ensuring that the crosslinking material is present at a target site within the skin. (Item 27) The method of item 26, wherein the presence of the crosslinking material can be determined at the target site within the skin. (Item 28) The method according to items 26-27, wherein the crosslinking material is present for a period of time from 1 minute to 1 hour. (Item 29) The method according to items 26-28, wherein the microneedling is performed after the application step. (Item 30) The method according to items 26-28, wherein the microneedling is performed before the application step. (Item 31) The method according to items 26 to 28, wherein the microneedling is performed before and after the application step. (Item 32) The method according to items 26-31, wherein the first crosslinkable entity comprises a polymer portion, and the weight-average molecular weight of the polymer portion prior to the application step is in the range of 1 kDa to 500 kDa. (Item 33) The method according to item 32, wherein the weight-average molecular weight of the polymer portion prior to the application step is in the range of 5 to 20 kDa. (Item 34) The aforementioned polymer portion is hyaluronic acid ("HA") polymer, as described in items 32-33. The method. (Item 35) The method according to items 26-34, wherein the first crosslinkable entity comprises another molecule that reacts with either the -SH or NH2 group of CBT, a CBT mimetic, or Cys (e.g., D-Cys, L-Cys, or a combination thereof). (Item 36) The method according to items 26-35, further comprising applying the first crosslinkable entity and the second crosslinkable entity to the skin location. (Item 37) The method according to items 26-36, wherein the first crosslinkable entity and the second crosslinkable entity are applied simultaneously. (Item 38) The method according to item 37, further comprising mixing the first crosslinkable entity and the second crosslinkable entity before the application step. (Item 39) The method according to item 38, wherein the mixing step is performed 0 to 30 minutes before the application step. (Item 40) The method according to items 26-39, wherein the second crosslinkable entity is applied after the second crosslinkable entity. (Item 41) The method according to items 26 to 40, wherein the microneedling is performed using a microneedling device having microneedles. (Item 42) The aforementioned microneedle device has a microneedle density of approximately 20 to 150 microneedles / cm³. 2 The method described in item 41, which is within the range. (Item 43) The microneedle device having 1 to 100,000 microneedles, according to the method described in items 41 to 42. (Item 44) The microneedle is the method described in items 41 to 43, having a length of approximately 100 μm to approximately 1000 μm. (Item 45) The microneedle device is a derma roller, as described in items 41-44. (Item 46) The method according to items 26-45, wherein the dermal layer is the epidermis (e.g., stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, stratum basale) or the dermis. (Item 47) The method according to items 26 to 46, wherein the concentration of the first crosslinkable entity is in the range of 0.1 to 100 mg / mL. (Item 48) The method according to items 26 to 47, wherein the second crosslinkable entity is selected from the group consisting of cysteine-ethylenediamine-cysteine ​​(CEC), cysteine-lysine-cysteine ​​(CKC), cysteine-PEG-cysteine, and combinations thereof. (Item 49) The method according to items 26 to 48, wherein the concentration of the second crosslinkable entity is in the range of 0.1 to 100 mg / mL. (Item 50) The method according to items 26 to 49, wherein the molecular ratio of the first cross-linked portion to the second cross-linked portion is in the range of 1:1 to 5:1. (Item 51) The method according to items 26 to 50, wherein the first crosslinkable entity comprises a first crosslinking portion, and 1 to 20 mol% of the first crosslinkable entity comprises the first crosslinking portion. (Item 52) The method according to any prior art, characterized in that the crosslinking material has a weight-average molecular weight at the target site within the skin that is (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 times) greater than the weight-average molecular weight of the first crosslinkable entity. (Item 53) In a method for establishing a crosslinked material within the skin by local application of a first crosslinkable entity and a second crosslinkable entity, the improvements are as follows: The method comprising bringing the first crosslinkable entity and the second crosslinkable entity into contact with each other at a pH in the range of about 3 to about 5. (Item 54) A method for establishing a crosslinking material in the subcutaneous tissue by applying a first crosslinkable entity and a second crosslinkable entity, wherein at least one of them is applied locally to the skin site, the improvements are: The method comprising applying at least one of the crosslinkable entities locally, and then microneedling at the skin location. (Item 55) A combination of a first crosslinkable entity and a second crosslinkable entity, wherein the pH of the combination is in the range of approximately 3 to approximately 5.

Claims

[Claim 1] The invention described in the specification.

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