Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
  • A61K8/73—Polysaccharides
  • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
• • A—HUMAN NECESSITIES
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  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/728—Hyaluronic acid
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  • A61K31/74—Synthetic polymeric materials
  • A61K31/765—Polymers containing oxygen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/545—Heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
  • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
  • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
  • A61K8/447—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof containing sulfur
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
  • A61K8/81—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions involving only carbon-to-carbon unsaturated bonds
  • A61K8/8141—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
  • A61K8/8147—Homopolymers or copolymers of acids; Metal or ammonium salts thereof, e.g. crotonic acid, (meth)acrylic acid; Compositions of derivatives of such polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
  • A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
  • A61K8/86—Polyethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/87—Application Devices; Containers; Packaging
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/91—Injection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/95—Involves in-situ formation or cross-linking of polymers

Definitions

• the present disclosure provides a variety of insights relating to provision of crosslinked materials (e.g., gels or other high molecular weight materials) in or on a target site of interest (e.g., in situ).
• crosslinked materials e.g., gels or other high molecular weight materials
• the present disclosure provides an insight that lipophilicity of a crosslinkable entity (e.g., comprising a polymer moiety and a crosslink moiety) can be tuned to achieve a desired rate and/or extent of penetration after application to a site (e.g., on a skin surface or otherwise to a skin site above a target site of interest).
• a crosslinkable entity e.g., comprising a polymer moiety and a crosslink moiety
• the present disclosure provides an insight that low viscosity preparations of crosslinkable entities have desirable characteristics for certain embodiments, e.g., permitting the crosslinked material(s) they generate to better fill spaces and/or structures (e.g., microcavities on surfaces) therein, and/or to make better contacts with tissues and/or structures thereon.
• desirable characteristics e.g., permitting the crosslinked material(s) they generate to better fill spaces and/or structures (e.g., microcavities on surfaces) therein, and/or to make better contacts with tissues and/or structures thereon.
• the present disclosure provides an insight that a crosslinked material as provided by the present disclosure may exhibit improved durability/persistence relative to its initial crosslinkable entities (e.g., separate from one another and/or prior to their crosslinking to generate the relevant crosslinked material) in a relevant biological system.
• the present disclosure provides an insight that ensuring first and second crosslinkable entities are separate (i.e., chemically, physically, and/or spatially) on administration (e.g., while being administered), can be beneficial and/or otherwise desirable in certain embodiments.
• the present disclosure provides embodiments in which one or more of a set of crosslinkable entities that, by crosslinking with one another generate a crosslinked material, is characterized by a degree of lipophilicity as described herein.
• the present disclosure also provides embodiments in which one or more crosslinkable entities is utilized as a low viscosity preparation.
• the present disclosure also provides embodiments in which individual crosslinkable entities are maintained separate from one another upon administration (e.g., up to and/or during administration).
• the present disclosure further provides combinations of such embodiments, as well as insights with respect to particular context(s) in which one or more such embodiments might be particularly useful or effective.
• provided insights permit selection and/or utilization of crosslinkable entities or components (e.g., polymer and/or crosslink moieties) or combinations thereof not previously contemplated or utilized for in situ crosslinking systems, and/or provide new methodologies for administration of crosslinkable entities to achieve an in situ crosslinked material.
• crosslinkable entities or components e.g., polymer and/or crosslink moieties
• the present disclosure provides systems comprising components that together can be used to provide in situ crosslinked materials.
• provided systems comprise first and second crosslinkable entities, at least one (and, in many embodiments, both) of which has lipophilicity as described herein, which first and second entities crosslink with one another to form a crosslinked material (e.g., a gel) with material and/or functional characteristics as described herein at or on a site of interest (e.g., in situ).
• first and second crosslinkable entities are separately and serially administered to a site (e.g., on a skin surface); in some such embodiments, the first crosslinkable entity is first administered, and the second crosslinkable entity is administered after a period of time.
• provided devices maintain the first and second crosslinkable entities in physically separate locations, compartments, and/or containers.
• Figure 1 illustrates parts of human skin including epidermis and dermis.
• Figure 2 illustrates 'H NMR of HA-gly-CBT in D2O from Example 1.
• FIG. 3 illustrates gel permeation chromatography (GPC) analysis of modified HA.
• Refractive index reading of unmodified HA (with average molecular weights of 10, 50, and 250 kDa) prior to conjugation is illustrated in Figure 3A
• UV absorbance reading (326 nm) of modified HA (HA-gly-CBT, with average molecular weights of 10, 50, and 250 kDa) is illustrated in Figure 3B.
• Figure 4 illustrates the chemical structure of certain derivatives of CBT and HA (A and B, respectively).
• Figure 5 illustrates 3 ⁇ 4 NMR of Cys-PEG-Cys in deuterated acetone from Example 1.
• Figure 6 illustrates 3 ⁇ 4 NMR of Cys-EDA-Cys in deuterated acetone from Example 1.
• Figure 7 presents an exemplary packaging of a first and second crosslinkable entity, which are dynamically blended on use.
• the illustrated packaging shows that the two entities are mixed in a chamber just prior to dispensing
• Figure 8 presents an exemplary packaging of a first and second crosslinkable entity, which can be individually dispensed.
• Figure 9 illustrates resistance to degradation by hyaluronidase of the crosslinked HA- gly-CBT.
• the plot in Figure 9A depicts fraction of HA-gly-CBT remaining as an insoluble gel and high molecular weight polymer after incubation with hyaluronidase for both crosslinked and uncrosslinked materials.
• Figure 9B is an image of the crosslinked HA-gly-CBT gel after a 24-h incubation with hyaluronidase.
• Figure 10 illustrates bright field and fluorescent (DAPI filter) images of porcine skin sections after topical application of PBS (A) and HA-gly-CBT (B) to intact porcine skin and HA- gly-CBT (C) to tape stripped porcine skin.
• A PBS
• B HA-gly-CBT
• C HA- gly-CBT
• Figure 11 illustrates GPC analyses of skin extracts after topical application of HA- gly-CBT.
• Figure 11A shows a comparison of GPC signal from HA-gly-CBT to GPC signals measured for stratum corneum (SC) extracts and epidermis/dermis extracts, averaged over six samples.
• Figure 11B illustrates average GPC signal for SC extracts with associated error bars.
• Figure 11C illustrates average GPC signal for epidermis/dermis extracts with associated error bars.
• Figure 12 illustrates bright field and fluorescent (DAPI filter) images of porcine skin sections after injection of HA-gly-CBT (A) or HA-gly-CBT followed by Cys-PEG-Cys (B). Sections were imaged both directly after sectioning and after washing to extract soluble HA-gly- CBT.
• DAPI filter bright field and fluorescent
• Figure 13 presents photographs of skin two days after application of the provided system, as compared to the baseline and application of a placebo, illustrating improvement in skin smoothness.
• Figure 14 illustrates bright field and fluorescent (Cy5 filter) images of human skin sections after topical application of PBS (A) and HA-gly-CBT tagged with AlexaFluor 647 (B and C) to disrupted human skin. Skin was tape stripped prior to application in one sample (B) and treated with a dermaroller containing 500 pm needles in another (C).
• Figure 15 presents the exemplary sites of administration from Example 6.
• Figure 16 presents images at time points post administration of the system as described in Example 6.
• Figure 17 presents images of biopsies post administration of the system as described in Example 7.
• Figure 18 illustrates topical delivery of HA-gly-CBT (50 kDa, tagged with IR dye) into human skin treated with a dermaroller containing 500 pm needles.
• Figure 18A presents fluorescent, microscopy images of skin samples obtained with a Cy5 filter.
• Figure 18B presents quantitive data of HA-gly-CBT to the SC, epidermis, and dermis.
• an analog refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance.
• an“analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways.
• an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance.
• an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance.
• an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.
• Two events or entities are“associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other.
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc
• two or more entities are physically“associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
• two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
• Biocompatible refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. 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 less than or equal to 20% cell death, and/or their administration in vivo does not induce significant inflammation or other such adverse effects.
• Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
• comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable.
• the term“corresponding to”, designates the position/identity of a structural element, e.g ., of an amino acid residue, a nucleotide residue, or a chemical moiety, in a compound or composition through comparison with an appropriate reference compound or composition.
• a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
• residues in a polymer may be designated using a canonical numbering system based on a reference related polymer, so that a residue“ corresponding to” one at position 190 of a reference polymer, for example, need not actually be the l O* 11 residue in a polymer of interest, but rather refers to the residue that corresponds to the residue found at position 190 in the reference polymer; those of ordinary skill in the art readily appreciate how to identify“ corresponding ⁇ residues in polymers (e.g., using commercially available sequence comparison software for polypeptide and nucleic acid polymers; optionally manually for other polymers).
• the term“designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents.
• Dosage form may be used to refer to a physically discrete unit of an agent (e.g., a therapeutic, diagnostic or cosmetic agent) for administration to a subject.
• agent e.g., a therapeutic, diagnostic or cosmetic agent
• each such unit contains a predetermined quantity of agent.
• such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial (e.g., therapeutic and/or cosmetic) outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
• such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a regimen that has been determined to correlate with a desired or beneficial cosmetic outcome (e.g., provides visible and/or tactile improvement to skin) when administered to a relevant population.
• a desired or beneficial cosmetic outcome e.g., provides visible and/or tactile improvement to skin
• the total amount of a composition or agent administered to a particular subject is determined by one or more attending professionals (e.g., physicians, nurses, or other licensed professionals) and may involve administration of multiple dosage forms.
• a dosage form may be provided in a formulation that is or comprises a cream, gel, liquid, lotion, mist, mask, matrix, particle, paste, patch, powder, serum, solid, spray (or collection thereof), or a combination thereof.
• Dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
• a given agent has a recommended dosing regimen, which may involve one or more doses.
• a dosing regimen comprises a plurality of doses each of which is separated in time from other doses.
• individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
• all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population. [40] Excipient: as used herein, refers to an inactive (e.g., not a therapeutic active such as a cosmetic active) agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.
• an inactive e.g., not a therapeutic active such as a cosmetic active
• an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
• an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
• in situ refers to a location at, in, or on a target site.
• crosslinkable entities crosslink in situ when they react to form a crosslinked material at, in, or on a target site of interest, for example, on a tissue surface or within a tissue; in many embodiments, a relevant tissue is skin.
• Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
• isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
• a substance is "pure” if it is substantially free of other components.
• a substance may still be considered “ isolated " or even " pure ", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.
• Linker as used herein, is used to refer to that portion of a multi-element agent that connects different elements to one another.
• a marker refers to an entity or moiety whose presence or level is a characteristic of a particular state or event. In some embodiments, presence or level of a particular marker may be characteristic of presence, state, or stage of a disease, disorder, or condition.
• Subject refers to any organism to which a provided system is or may be administered, e.g., for experimental, diagnostic, prophylactic, 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, a subject is a human. In some embodiments, a subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a subject displays one or more symptoms of a disorder or condition. In some embodiments, a subject has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or presence of one or more tumors.
• the subject is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.
• a subject refers to a human seeking cosmetic benefit and/or improvement, such as an improvement of appearance and/or feel of skin.
• Physiological conditions As used herein, has its art-understood meaning referencing conditions under which cells or organisms live and/or reproduce. In some embodiments, the term refers to conditions of the external or internal mileu that may occur in nature for an organism or cell system. In some embodiments, physiological conditions are those conditions present within the body of a human or non-human animal, especially those conditions present at and/or within a target site of interest. Physiological conditions typically include one or more of, e.g., a temperature within the range of 20 - 40 °C (and specifically about 37 °C), atmospheric pressure of 1, pH of 6-8, glucose concentration of 1-20 mM, oxygen concentration at atmospheric levels, and gravity as it is encountered on earth.
• Reference As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
• sample typically refers to an aliquot of material obtained or derived from a source of interest.
• a source of interest is a biological or environmental source.
• a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human).
• a source of interest is or comprises biological tissue or fluid.
• a sample is a“primary sample” obtained directly from a source of interest by any appropriate means.
• sample refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample.
• a“processed sample” may comprise, for example, materials extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as chromatography, extraction, precipitation, etc.
• the term“substantial structural similarity” refers to presence of shared structural features at particular positions.
• the term“substantial structural similarity” refers to presence and/or identity of structural elements such as, for example: loops, sheets, helices, H-bond donors, H-bond acceptors, glycosylation patterns, salt bridges, disulfide bonds, and combinations thereof.
• the term“substantial structural similarity” refers to three dimensional arrangement and/or orientation of atoms or moieties relative to one another (for example: distance and/or angles between or among them between an agent of interest and a reference agent).
• therapeutic agent in general refers to any agent that elicits a desired pharmacological effect (which may, in some embodiments, be or comprise a cosmetic effect) when administered to an organism.
• an agent is considered to exhibit an effect (i.e., to be a therapeutic agent) if it demonstrates a statistically significant effect across an appropriate population.
• the appropriate population may be a population of model organisms.
• an appropriate population may be defined by particular criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc, or combinations thereof.
• a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
• a therapeutic agent is one that achieves a cosmetic effect (i.e., is a cosmetic agent).
• a therapeutic agent can be used to achieve improvement of appearance and/or feel of skin, and/or another cosmetic benefit.
• Treat refers to partial or complete alleviation, amelioration, delay of onset of, inhibition, prevention, relief, and/or reduction in incidence and/or severity of one or more symptoms or features of a disease, disorder, and/or condition, or achievement of another desired physiological effect (e.g., a desired cosmetic effect such as improvement of appearance and/or feel of skin, such as visible and/or tactile improvement to skin.
• treatment comprises administration of an agent which results in a physiological effect.
• treatment comprises a cosmetic treatment which upon administration improves physical appearance in manner described herein.
• treatment may be administered to a subject who does not exhibit signs or features of a disease, disorder, and/or condition (e.g., may be prophylactic). In some embodiments, treatment may be administered to a subject who exhibits only early or mild signs or features of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits established, severe, and/or late-stage signs of the disease, disorder, or condition.
• the present disclosure provides certain technologies relating to administration of crosslinkable entities to a subject, and particular to in situ crosslinking of such crosslinkable entities at, in, or on a target site which, for example, may be a site in or on skin, for example on, at, in, or below the epidermis, dermis or underlying hypodermis.
• the present disclosure provides technologies for administering a system that comprises first and second crosslinkable entities, selected and/or designed to achieve formation of an in situ crosslinked material.
• first and second crosslinkable entities selected and/or designed to achieve formation of an in situ crosslinked material.
• crosslinked material will typically be characterized by one or more different physical properties, such as rheology, and/or chemical properties, such as molecular weight, relative to its parent cross-linkable entities.
• different property(ies) may be or include improved durability/persistence, e.g., in situ and/or otherwise in a relevant biological system.
• first and second crosslinkable entities are characterized by an ability, when contacted with one another, to react with one another to form the crosslinked material in situ , e.g., absent administration of a catalyst or other non-participating agent.
• At least one of the crosslinkable entities will comprise a polymer moiety linked with a crosslink moiety. In some embodiments, at least one of the crosslinkable entities will not comprise a polymer moiety. In some embodiments, a pair of crosslinkable entities that react with one another (e.g., absent administration of a catalyst or other non-participating agent) each comprise a polymer moiety linked with a crosslink moiety.
• a pair crosslinkable entities that react with one another comprises a first crosslinkable entity that comprises a polymer moiety linked with a crosslink moiety and a second crosslinkable entity that does not comprise a polymer moiety.
• a crosslinkable entity comprises a polymer moiety and a plurality of crosslink moieties, which may be the same or different.
• the present disclosure provides insights and technologies relevant to achieving penetration of crosslinkable entities to a target site in skin (e.g., on, at, in, or below the epidermis, dermis or underlying hypodermis).
• the present disclosure teaches that lipophilicity within a particular range may permit desirable (e.g., enhanced) penetration of a crosslinkable entity, and particularly of a crosslinkable entity comprising a polymer moiety and a crosslinkable moiety.
• human skin is multi-layered, comprising an external epidermis (which itself is a layered structure comprising the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale), a dermis, and an underlying hypodermis.
• the skin acts as a barrier to separate and protect the body from its environment.
• a major challenge in the cosmetic and dermatological fields is the development of technologies to facilitate penetration of compounds or agents of interest across skin. See, for example, Ng (2015) Skin Deep: The Basics of Human Skin Structure and Drug Penetration. In: Dragicevic N., Maibach H. (eds) Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement. Springer, Berlin, Heidelberg
• the present disclosure provides technologies relating to penetration across or through skin layer(s) and specifically relates to such penetration by crosslinkable entities that react to generate a crosslinked material that is present at a target site.
• the target site may be on, at, in or below the epidermis, dermis or underlying hypodermis.
• the present disclosure provides a teaching that crosslinkable entities with improved skin penetration characteristic(s) can be designed and/or prepared by modulating lipophilicity.
• the present disclosure teaches that rate and/or extent of skin penetration by a particular agent (and specifically by an agent that is or comprises a polymer moiety and/or otherwise has a number molecular weight (e.g., a number average molecular weight) above 500 daltons, and even within a range of 10-500 kDa) can be enhanced by increasing lipophilicity of the agent, for example by attaching one or more hydrophobic moieties to the agent.
• a number molecular weight e.g., a number average molecular weight
• hydrophobic moieties that increase lipophilicity may be or comprise crosslink moieties; alternatively or additionally, in some embodiments, such hydrophobic moieties may not necessarily be or comprise crosslink moieites and/or may not necessarily participate in crosslinks (e.g., that are in and/or that help generate a crosslinked material as described herein)
• the present disclosure provides technologies relating to providing a crosslinked material in a target site in or on a tissue (and particularly in or on skin), so that contacts between the material and surfaces of the target site are maximized.
• such surfaces may include one or more cavities or irregularities, which may, in some embodiments, be micro- or even nano-scale structures.
• the present disclosure provides preparations of crosslinkable moieties that have flow characteristics that facilitate such contacts.
• a crosslinkable entity for use in accordance with the present disclosure comprises a polymer moiety linked with a crosslink moiety, where the crosslink moiety imparts increased lipophilicity to the conjugate (i.e., to the crosslinkable entity) as compared with the polymer moiety alone; as described herein, in some embodiments, such a crosslink moiety can also improve skin penetration by the crosslinkable entity as compared with that of the polymer moiety alone.
• depth of penetration of a crosslinkable entity will be assessed and/or described in terms of the absolute distance (e.g., in microns) below the surface of the skin. In some embodiments, depth of penetration of a crosslinkable entity will be assessed and/or described in terms of number of cells below the surface of the skin.
• a crosslinkable entity as described herein can and/or does (e.g., when administered as described herein) penetrate to a specified depth into the skin, for example within a particular time period.
• a such a specified depth may be, for example, at least 50 microns, at least 100 microns, at least 200 microns, or more; alternatively or additionally, in some embodiments, such specified depth may be at least 2 cell layers, at least 3 cell layers, at least 4 cell layers, at least 5 cell layers, at least 6 cell layers, at least 7 cell layers, at least 8 cell layers, at least 9 cell layers, at least 10 cell layers or more, and/or such time period may be, for example, within 1 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 less.
• At least 1% of a crosslinkable entity administered to a skin surface penetrates to a target site on, at, in, or below the epidermis, dermis or underlying hypodermis, for example within a time period of 1 day.
• skin penetration characteristic(s) of a crosslinkable entity as described herein are observed in the absence of any chemical or physical abrading or disrupting agent and/or of electrical current or magnetic field, etc (e.g., absent a penetration enhancer as understood in the art).
• a crosslinkable entity is characterized by lipophilicity (log P ) within a range of about -4 and about 2.
• crosslinkable entities described herein comprise a crosslinkable moiety, which is lipophilic.
• lipophilicity of a crosslinkable moiety may be determined independently from that of a polymer moiety, and/or of a crosslinkable entity comprising the polymer moiety and one or more crosslinkable moieties.
• a crosslinkable moiety is characertized by lipophilicity (log P ) within a range of about 0 and about 6. For example, in some embodiments, lipophilicity of a crosslinkable moiety is tested through examination of the crosslinkable moiety prior to association and/or linking with molecule polymer moiety to form a crosslinkable entity.
• lipophilicity for a particular entity or moiety is determined by its partition co-efficient ( P ) relative to a standard solvent (e.g. octanol) and water or solution thereof:
• a standard solvent e.g. octanol
• log P of an entity or moiety (e.g., of a crosslinkable entity and/or of a crosslink moiety or other hydrophobic moiety) useful in accordance with the present disclosure is greater than 0.
• a crosslinkable entity (e.g., a crosslinkable entity with a lipophilicity as described herein) has a molecular weight (e.g., a number average molecular weight) above 500 daltons. In some embodiments, a crosslinkable entity has a molecular weight (e.g., a number average molecular weight) within a range of 1-1000 kDa. In some embodiments, a system comprises a crosslinkable entity that is about 10-250 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) within a range of about 10-150 kDa.
• a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) within a range of about 10-100 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) within a range of about 10-40 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular (e.g., a number average molecular weight) weight of about 10 kDa.
• a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) of about 20 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) of about 30 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) of about 40 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) of about 50 kDa.
• a molecular weight e.g., a number average molecular weight
• a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) of less than 10 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) of less than 100 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) of less than 250 kDa. In some embodiments, a system comprises a crosslinkable entity that has a molecular weight (e.g., a number average molecular weight) of less than 500 kDa.
• a molecular weight e.g., a number average molecular weight
• the crosslink moiety contributes lipophilicity to the crosslinkable entity.
• the identity and/or number and/or density of crosslink moieties linked to a particular polymer moiety in a crosslinkable entity is/are selected so that the crosslinkable moiety has characteristic(s) as described herein.
• a more lipophilic crosslink moiety (and/or a larger number/higher density of lipophilic crosslink moieties) will be linked to a polymer moiety when the polymer moiety is particularly hydrophilic, particularly long, and/or is characterized by particularly poor skin penetration in its unlinked state. .
• crosslinkable entity may be desirable to select a particular combination of polymer moiety and crosslink moiety (and/or number and/or density thereof) so as to achieve three-dimensional packing of the crosslinkable entity within a certain volume, e.g., which may facilitate transfer through one or more skin structures or layers (e.g., rendering the crosslinkable entity sufficiently “slippery” to pass through).
• intra- and/or inter-molecular interactions may contribute to three-dimensional packing of a crosslinkable entity.
• three-dimensional packing of a particular cross-linkable entity as described herein occurs by self- assembly (e.g., without requiring addition of another agent), at least under certain environmental conditions (e.g., physiological conditions).
• a crosslinkable entity is biocompatible.
• administration of a crosslinkable entity as described herein does not cause significant irritation and/or inflammation (e.g., at the site of administration and/or at the target site).
• a crosslinkable entity is utilized in accordance with the present invention in a form or preparation having a viscosity suitable for topical application - e.g., with sufficient viscosity to maintain the applied crosslinkable entity in contact with the administration site for long enough to permit penetration and/or to otherwise permit distribution to the target site rather than simply dribbling off; in some such embodiments, the viscosity is within a range that permits the applied crosslinkable entity to flow into microstructures on the skin surface.
• a crosslinkable entity is utilized in accordance with the present invention in a form or preparation having a viscosity suitable for administration by injection.
• a suitable viscosity is a viscosity wherein the crosslinkable entity can be applied topically without rapidly running off, and/or can be rubbed into skin.
• the present disclosure provides a system comprising two or more crosslinkable entities, that interact with one another to form a crosslinked material (e.g., to provide a crosslinked material in situ at a target site on, at, in or below the epidermis, dermis or underlying hypodermis).
• At least one crosslinkable entity of a provided system comprises a polymer moiety linked with a crosslink moiety.
• at least one crosslinkable entity of the system does not comprise a polymer moiety linked with a crosslink moiety (i.e., comprises or consists of a crosslink moiety but not a polymer moiety); in some such embodiments, only one crosslinkable entity of a provided system comprises a polymer moiety linked with a crosslink moiety.
• each crosslinkable entity of a provided system comprises a polymer moiety linked with a crosslink moiety; in some such embodiments, each crosslinkable entity of a provided system comprises the same polymer moiety.
• Different crosslinkable entities of a provided system comprise complementary crosslink moieties so that the crosslinkable entities react to form a crosslinked material; in some embodiments, such crosslinking occurs absent any added catalyst.
• At least one crosslinkable entity of a provided system has a molecular weight (e.g., a number average molecular weight) above 500 daltons as described herein (e.g., has a molecular weight (e.g., a number average molecular weight) within a range of 1-1000 kDa). In some embodiments, at least one crosslinkable entity of a provided system has a molecular weight (e.g., a number average molecular weight) within a range of 1-10,000 kDa as described herein.
• At least one crosslinkable entity of the system does not have a molecular weight (e.g., a number average molecular weight) above 500 daltons as described herein (e.g., within a range of 1-400 daltons); in some such embodiments, only one crosslinkable entity of a provided system has such a high molecular weight (e.g., a number average molecular weight). Alternatively, in some embodiments, each crosslinkable entity of a provided system has such a high molecular weight (e.g., a number average molecular weight).
• a molecular weight e.g., a number average molecular weight
• the present invention encompasses a crosslinkable entity, which comprises a polymer moiety and a crosslink moiety.
• one or more of the polymer moieties are glycosaminoglycans or polysaccharides.
• polysaccharides include dextran, starch or pectin.
• one or more of the polymer moieties is dextran.
• one or more of the polymer moieties is starch.
• one or more of the polymer moieties is pectin.
• “glycosaminoglycans” includes hyaluronic acid (HA), heparin sulfate, chondroitin sulfate, dermatan sulfate and keratin sulfate.
• a polymer is hyaluronic acid.
• one or more of the polymer moieties is a synthetic polymer.
• a“synthetic polymer” includes PEG, PEG-diamine, polyacrylic acid, N-(2- Hydroxypropyl) methacrylamide (HPMA), polycaprolactone (PCL) or poly(lactic-co-glycolic acid) (PLGA).
• a synthetic polymer is PEG.
• a synthetic polymer is PEG-diamine.
• a synthetic polymer is polyacrylic acid.
• a synthetic polymer is HPMA.
• the synthetic polymer is PCL.
• the synthetic polymer is PLGA.
• one or more of the polymer moieties is a polypeptide (e.g., a protein).
• a useful polypeptide is or comprises collagen, gelatin, elastin, or a functional fragment thereof.
• a polypeptide is or comprises collagen or a functional fragment thereof.
• a polypeptide is gelatin.
• a polypeptide is elastin.
• one or more polymer moieties included in a system as described herein is or comprises HA. In some embodiments, one or more polymer moieties included in a system as described herein is or comprises a glycosaminoglycan. In some embodiments, one or more polymer moieties included in a system as described herein is or comprises a polypeptide (e.g., a non-natural polypeptide and/or a synthetic or recombinant polypeptide).
• a polypeptide e.g., a non-natural polypeptide and/or a synthetic or recombinant polypeptide.
• a provided system includes a plurality of different crosslinkable entities comprising polymer moieties; in some such embodiments, the polymer moieties are selected from the group consisting of HA, proteoglycans, polypeptides, and combinations thereof.
• the present disclosure provides systems that include two or more crosslinkable entities that are compatible with one another in that they react to form a crosslinked material in situ.
• crosslinkable entities of the system comprise a crosslink moiety.
• a“crosslink moiety” is capable of participating in a click reaction.
• a click reaction may be a reaction of two or more moieties which brings two or more substrates together and occurs under physiological pH.
• a click reaction exhibits suitable kinetics (e.g. second- order rate constant ⁇ ki) is about 9 M _1 s _1 ).
• crosslink chemistries can include, but is not limited to, cycloadditions, nucleophilic substitution reactions, condensation reactions and nucleophilic addition reactions.
• a click reaction is a [3+2] cycloaddition, a [4+2] cycloaddition or a [4+1] cycloaddition.
• a click reaction is an azide-alkyne cycloaddition, a nitrone-olefin cycloaddition or a Diels-Alder reaction.
• a click reaction is a Schiff reaction, a Michael-type addition, a nucleophilic substitution reaction on a haloacetate, a formation of a disulphide linkage, a free radical polymerization, a Huysgen reaction, phenols (tyramines) that spontaneously cross link after their enzymatic oxidation to catechols or a reaction between cyanobenzothiazole (CBT) and D- cysteine (CYS).
• a click reaction is a reaction between CBT and D- cysteine.
• a click reaction is a reaction between CBT and L-cysteine.
• CBT can include analogs of CBT, e.g., isotopically labelled CBT. In some embodiments, CBT includes substituted analogs of CBT. In some embodiments, CYS can include analogs of CYS, e.g., isotopically labelled CYS. In some embodiments, CYS includes substituted analogs of CYS.
• crosslink moieties may be particularly useful in in situ crosslinking contexts as described herein.
• the present disclosure utilizes one or more crosslink moieties characterized by a desired degree of lipophilicity, for example, when linked with a particular polymer moiety.
• CBT represents a crosslinkable moiety that can be linked with a polymer moiety in a useful crosslinkable entity as described herein.
• lipophilicity of a crosslinkable entity comprising a particular polymer moiety may be adjusted through linkage of a plurality of hydrophobic moieties (e.g., hydrophobic crosslink moieties), which may be the same or different (and need not all be crosslink moieties).
• a crosslink moiety is or comprises azide, alkyne, nitrone, olefin, diene, tetrazine, isocyanate, Michael acceptor, enone, aldehyde, amine, a-halo carbonyl moiety, maleimide, thiol, CBT, D-cysteine, acrylic residues, phenol, tyramine or catechol.
• a crosslink moiety is or comprises D-cysteine.
• a crosslink moiety is or comprises L-cysteine.
• a reactive moiety is or comprises CBT.
• At least one crosslink moiety included within a provided system may be utilized without linkage to a polymer moiety. That is, in some embodiments, at least one crosslinkable entity included within a provided system may consist of a crosslink moiety, or may comprise a crosslink moiety and at least one other moiety that is not a polymer moiety (which other moiety, as is clear from the present disclosure may, in some embodiments, be or comprise a hydrophobic moiety).
• a provided system may utilize a crosslink moiety that is or comprises, for example, a diamine, peptide, dithiol or dihydrazide.
• a diamine may be an ethylene diamine, e.g., a polyethylene glycol (PEG) diamine, etc; in some such embodiments, a dihydrazide may be an oxalic dihydrazide, malonic dihydrazide, succinic dihydrazide, glutaric dihydrazide, adipic dihydrazide or pimelic dihydrazide, etc.
• crosslink moiety or as comprising one or more crosslink moieties (e.g., each of which may be a single reactive atom or a small number of atoms), and a polymer moiety that may include only a very small (e.g., 2 or 3) number of“monomers” (and, in some instances where each such“monomer” might consist of only a few atoms).
• crosslink moieties e.g., each of which may be a single reactive atom or a small number of atoms
• polymer moiety that may include only a very small (e.g., 2 or 3) number of“monomers” (and, in some instances where each such“monomer” might consist of only a few atoms).
• a crosslinkable entity as described herein may comprise one or more moieties other than a polymer moiety or crosslink moiety, which other moiety(ies) may, in some embodiments, be covalently associated with a polymer moiety and/or with a crosslink moiety.
• an“other” moiety may be releasably associated with the crosslinkable entity (e.g., via a cleavable bond; in some such embodiments, such cleavable bond may be cleaved at the target site).
• a crosslinkable entity may comprise a drug moiety (which may, in some embodiments, itself be in a pro-drug form).
• a drug moiety is or comprises a synthetic or natural small molecule or biomolecule (e.g., a carbohydrate, a lipid, a nucleic acid, a polypeptide, or an analog or combination thereof).
• a synthetic or natural small molecule or biomolecule e.g., a carbohydrate, a lipid, a nucleic acid, a polypeptide, or an analog or combination thereof.
• a drug moiety may be one that improves the appearance of the skin in one or more ways.
• a drug moiety may brighten skin; clear breakouts; firm; improve cellular activity within skin; improve collagen synthesis; improve healing profile of a wound; improve pigmentation; improve skin barrier function; normalize healthy micoflora via topical pre-biotics; prevent damage (e.g., due UV exposure); reduce the appearance of scars; reduce inflammation; reduce itching; reduce redness; seal wounds; smooth, or treat bums or combinations thereof.
• a drug moiety may comprise an alpha-hydroxy acid (e.g., lactic, tartaric, or citric acid), an antioxidant (e.g., glutathione, an isoflavone, a polyphenol (e.g., resveratrol), or selenium), a beta-hydroxy acid (e.g., salicylic acid), a polyhydroxyl acid (e.g., gluconolactone or lactobionic acid), hydroquinone, natural skin lightening agents (e.g., Kojic acid), retinoids (e.g.
• an alpha-hydroxy acid e.g., lactic, tartaric, or citric acid
• an antioxidant e.g., glutathione, an isoflavone, a polyphenol (e.g., resveratrol), or selenium
• a beta-hydroxy acid e.g., salicylic acid
• a polyhydroxyl acid e.g., gluconolactone or lac
• retinoic acid retinol, tretinoin or derivatives thereof
• ceramide a peptide, an amino acid, a curcuminoid
• vitamins (and derivatives thereof) e.g., L-Ascorbic acid, vitamin B, niacinimide, and vitamin K
• a sunscreen agent e.g., oxybenzone, avobenzone, octisalate, octocrylene, homosalate, octinoxate, Meroxyl (SX and XL) or metallic oxides
• coloring agents pigments, or natural botanicals or combinations or prodrugs thereof.
• a drug moiety may comprise an anti-inflammatory (e.g., corticosteroids, non-steroidal anti-inflammatory drugs (crisaborole)), an antibiotic (e.g, clindamycin or ketoconazole), an antifungal (e.g., clotrimazole or ketoconazole), an anti-acne agent (e.g.
• an anti-inflammatory e.g., corticosteroids, non-steroidal anti-inflammatory drugs (crisaborole)
• an antibiotic e.g, clindamycin or ketoconazole
• an antifungal e.g., clotrimazole or ketoconazole
• an anti-acne agent e.g.
• retinoids or salicylic acid an analgesics
• an anticancer or antiproliferative agent an agent which treats erythema (e.g., oxymetazoline hydrochloride), an agent which reduces subdermal fat (e.g., deoxycholate), a hair growth agent (e.g., finasteride), or combinations or prodrugs thereof.
• erythema e.g., oxymetazoline hydrochloride
• an agent which reduces subdermal fat e.g., deoxycholate
• a hair growth agent e.g., finasteride
• a target site is or comprises (e.g., is on or within) a bodily tissue.
• Described herein are technologies for applying materials to an application site, such that a crosslinked material is provided (e.g., forms) at the target site.
• a bodily tissue is or comprises epithelial tissue.
• bodily tissue is or comprises connective tissue.
• bodily tissue is or comprises nerve tissue.
• a bodily tissue is or comprises muscle tissue.
• a bodily tissue is or comprises tissue of the eye, tissue of the skin or subcutaneous tissue.
• bodily tissue is or comprises subcutaneous fat, corneal epithelium or a mucous membrane.
• materials are administered to an application site such that the crosslinkable material is provided in situ at the target site (e.g., forms at the target site).
• a target site is a site that is reached after application to a surface, e.g., a tissue surface.
• a tissue surface is a surface of a tissue (e.g., skin, eye, or certain mucous membranes) that is exposed on a surface of an organism.
• a tissue surface is a surface of an internal tissue that may, for example, be accessed or exposed by performance of a procedure (e.g., a medical procedure such as a surgical procedure including, for example, an orthoscopic procedure) or process applied to an organism.
• a procedure e.g., a medical procedure such as a surgical procedure including, for example, an orthoscopic procedure
• process applied to an organism e.g., a surgical procedure including, for example, an orthoscopic procedure
• a target site is a target site on, at, in, or below the stratum corneum. In some embodiments, a target site is a site on, at, in, or below the epidermis. In some embodiments, a target site is a target site on, at, in, or below the dermis.
• a target site may be selected to be or comprise skin that has been damaged (e.g., due to a disease, disorder, or condition, or to a surgical, cosmetic, or dermatological treatment).
• damaged skin may be or comprise laser (e.g., fractional laser) treated skin, intense-pulse-light-treated skin, microneedled skin, skin subjected to chemical peel treatment, skin exposed to dermabrasion, traumatically injured skin (including by surgery), skin suffering from a medical condition such as skin atrophy, extreme dryness, etc.
• a target site may be selected to treat skin that is wrinkled (e.g., crow’s feet or other facial wrinkles), or otherwise shows signs of aging, or may be skin with respect to which additional plumpness and/or resilience is desired.
• wrinkled e.g., crow’s feet or other facial wrinkles
• skin of interest may be or comprise skin at a site selected from the group consisting of lips, lower lip, upper lip, tear troughs or other sites around the eyes (e.g., crow’s feet), nasolabial folds, forehead, cheeks back of hands, ear lobes, knees, neck, decolletage, arms, legs, torso, buttocks, feet, and combinations thereof, including, for example, full face, etc.
• a site selected from the group consisting of lips, lower lip, upper lip, tear troughs or other sites around the eyes (e.g., crow’s feet), nasolabial folds, forehead, cheeks back of hands, ear lobes, knees, neck, decolletage, arms, legs, torso, buttocks, feet, and combinations thereof, including, for example, full face, etc.
• a target site may be or comprise adipose tissue (e.g., subcutaneous adipose tissue).
• a target site may be or comprise a gland, such as a sebaceous gland and/or a serous (e.g., sweat) gland and/or a target site may be or comprise a vessel (e.g., a blood vessel) or duct (e.g., a sweat duct).
• a vessel e.g., a blood vessel
• duct e.g., a sweat duct
• a target site may be or comprise one or more specific cell populations, for example, that may be localized within one or more skin layers; for example, in some such embodiments, a relevant cell population may be or comprise particular stem cells or cell populations associated with a state of disease or injury.
• a target site may be or comprise pigment producing cells.
• a target site may be or comprise one or more hair follicles.
• a target site may be or comprise one or more nerve cells or nerves.
• the present disclosure provides systems that include at least first and second crosslinkable entities, which systems are administered to a skin site.
• both crosslinkable entities are administered to the same site; in some embodiments, each may be administered to a different site so long as both ultimately arrive at a target site of interest, so that a crosslinked material, generated by reaction of the first and second crosslinkable entities, is formed or otherwise provided in situ at the target site.
• At least one crosslinkable entity is administered topically (i.e., to a skin surface). In some embodiments, at least one crosslinkable entity is administered parenterally (e.g., by injection into a target site or to a location from which it penetrates into the target site.
• a provided system is administered to a subject’s face (e.g., full face and/or specific targets of a subject’s face such as to lips, lower lip, upper lip, tear troughs, crow’s feet, nasolabial folds, forehead, cheeks or combinations thereof).
• a provided system is administered to a non-facial site (e.g., knees, neck, decolletage, legs, arms, torso, buttocks or feet).
• a provided system is administered to hands (e.g., to the back of a hand).
• a provided system is administered to ear lobes.
• first and second crosslinkable entities are administered simultaneously.
• first and second crosslinkable entities may be combined prior to or as they are administered.
• such combination may be referred to as a pre-mix, and may be prepared a period of time prior to administration; typically, such a pre-mix is prepared within about 1 minute of administration.
• site of administration is prepared prior to administration of a crosslinkable entity.
• site of administration is prepared by washing site with tepid water and soap.
• site of administration is prepared through tape stripping.
• tape stripping comprises application of Scotch semi transparent tape to site of administration.
• tape stripping further comprises removal of the previously applied of Scotch semi-transparent tape from site of administration.
• tape stripping is repeated until site of administration glistens. In some embodiments, tape stripping is repeated at least 40 times.
• first and second crosslinkable entities are administered sequentially; in some such embodiments, a period of time separates administration of the first and second crosslinkable entities.
• a period of 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 hours, about 6 hours, about 12 hours, or more.
• such a period of time is about 2 hours.
• such a period of 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.
• site of application is covered after application of a crosslinkable entity. In some embodiments, site of application is covered with TegadermTM type film after application of a crosslinkable entity.
• a period of time between administration of first and second crosslinkable entities is sufficient to permit substantial penetration of the first-administered crosslinkable entity before the second crosslinkable entity is administered.
• the period of time is sufficient so that 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 has penetrated prior to administration of the second-administered crosslinkable entity.
• the period of time is sufficient such that the first agent has not degraded and/or been cleared from skin.
• Kinetics and/or extent of penetration for a particular crosslinkable entity may be determined, for example, as described herein, including through use of a model system (e.g., porcine skin); the relevant period of time may be selected in light of such determination.
• skin will be treated with water after administration of a system.
• a system is administered daily. In some embodiments, a system is administered at least once daily. In some embodiments, a system is administered at least twice daily. In some embodiments, a system is administered a 1-5 times daily. In some embodiments, a system is administered a 3-5 times daily. In some embodiments, a system is administered every 3 days. In some embodiments, a system is administered every 7 days. In some embodiments, a system is administered about every 15 days. In some embodiments, a system is administered about every 30 days. In some embodiments, a system is administered about every 60 days. In some embodiments, a system is administered about every 90 days.
• a provided system includes a chemical entity which improves skin penetration for a crosslinkable entity.
• the chemical entity is administered simultaneously with a crosslinkable entity.
• the chemical entity and the crosslinkable entity are administered at different times.
• a provided system comprises one or both of a penetration inhibitor (e.g., which may interact with and/or otherwise retard penetration of a crosslinkable entity) and a cross-link inhibitor (e.g., which may block one or more features of a crosslinkable entity or crosslink moiety, or otherwise interfere with reaction of crosslink moieties to generate a crosslink); in some such embodiments, such an inhibitor may be removed (e.g., via diffusion, washing, degradation) prior to, during, upon or after administration of the crosslinkable entity.
• a penetration inhibitor e.g., which may interact with and/or otherwise retard penetration of a crosslinkable entity
• a cross-link inhibitor e.g., which may block one or more features of a crosslinkable entity or crosslink moiety, or otherwise interfere with reaction of crosslink moieties to generate a crosslink
• such an inhibitor may be removed (e.g., via diffusion, washing, degradation) prior to, during, upon or after administration of the crosslinkable entity.
• one or more crosslinkable entities is administered as or in a sustained-release formulation.
• one or more crosslinkable entities is encapsulated within a matrix or particle (e.g., a nanoparticle).
• one or more crosslinkable entities is provided as or in an emulsion or dispersion.
• crosslinkable entities whose crosslink moieties interact to form crosslinks in the crosslinked material are administered in relative amounts (e.g., are formulated for administration in relative amounts) so that complementary crosslink moieties are present in stoichiometric amounts.
• such crosslinkable entities are administered in relative amounts so that one of a pair of complementary crosslink moieties is present in molar excess relative to the other; in some such embodiments, such molar excess is within a range of 1.1 : 1 - 10,000: 1.
• molar excess of the relative amounts of crosslinkable entities is within a range of 1.1 : 1 - 2: 1.
• molar excess of the relative amounts of crosslinkable entities is within a range of 1.1 : 1 - 10: 1. In some embodiments, molar excess of the relative amounts of crosslinkable entities is within a range of 1.1 :1 - 100: 1. In some embodiments, molar excess of the relative amounts of crosslinkable entities is within a range of 1.1 : 1 - 1,000: 1.
• a formulation comprises about 0.001% w/w to about 5.00% w/w of a crosslinkable entity. In some embodiments, a formulation comprises about 0.01% w/w to about 5.00% w/w of a crosslinkable entity. In some embodiments, a formulation comprises about 0.1% w/w to about 5.00% w/w of a crosslinkable entity. In some embodiments, a formulation comprises about 1% w/w to about 5.00% w/w of a crosslinkable entity. In some embodiments, a formulation comprises about 1 % w/w to about 3% w/w of a crosslinkable entity. In some embodiments, a formulation comprises about 2% w/w of a crosslinkable entity. In some embodiments, a formulation comprises PBS and about 2% w/w of a crosslinkable entity.
• At least one crosslinkable entity of a provided system is provided topically, e.g., to a site on skin, e.g., on a skin surface.
• a formulation for topical administration may be or comprise a cream, gel, liquid, lotion, mask, matrix, mist particle, paste, patch, powder, serum, solid, spray (or collection thereof), or a combination thereof.
• a formulation for topical administration may be or comprise a mask or serum.
• a formulation for topical administration may comprise antioxidants, buffers, chelating agents, clarity enhancers, emollients, emulsifiers, gelling agents, humectants, lubricants, moisturizers, pH adjusters, preservatives, protectants, prebiotics, probiotics, rheological modifiers, sensory modifiers, soothing agents (e.g., soothing plant serums), stabilizers, sunscreens (e.g., inorganic broadspectrum sunscreen), or thickening agents.
• antioxidants e.g., buffers, chelating agents, clarity enhancers, emollients, emulsifiers, gelling agents, humectants, lubricants, moisturizers, pH adjusters, preservatives, protectants, prebiotics, probiotics, rheological modifiers, sensory modifiers, soothing agents (e.g., soothing plant serums), stabilizers, sunscreens (e.g., inorganic broadspectrum sunscreen), or thickening
• topical application does not involve administration or application of penetration enhancer (e.g., a chemical or physical abrading or disrupting agent and/or an electrical current and/or an electrical or magnetic field, etc).
• penetration enhancer e.g., a chemical or physical abrading or disrupting agent and/or an electrical current and/or an electrical or magnetic field, etc.
• the first and second crosslinkable entities are separated by chemical means.
• a crosslinkable entity is encapsulated in, for example, micelles, liposomes, etc.
• the system is administered and the crosslinkable entity is released over a period of time.
• the system comprises a transdermal patch formulated to facilitate time release of a crosslinkable entity.
• a transdermal patch can be single layer agent-in patch, multilayer agent-in patch, reservoir, or matrix.
• a patch may comprise microneedle(s) (some or all of which may, in some embodiments, as is known in the art, be solid protrusions, and some or all of which may, in some embodiments, as is known in the art, be hollow).
• delivery may be achieved or enhanced by topical administration to a site that is or has been damaged, e.g., through exposure to one or more of laser (e.g., fractional laser) treatment, intense-pulse-light-treatment, microneedling, chemical peel treatment, dermabrasion, etc, each of which may, in some embodiments, be or have been administered before, during, or after, topical application of a system as described herein.
• laser e.g., fractional laser
• At least one crosslinkable entity is administered by injection.
• injection is below the surface of skin.
• injection is into a target site.
• injection is into a site from which the injected crosslinkable entity penetrates to the target site.
• injection may comprise application of force or pressure (e.g., via a syringe) so that delivery is achieved, e.g., through a needle, cannula, or other passage.
• force or pressure e.g., via a syringe
• injection may be via microneedles (e.g., that may be associated with a patch).
• one or more crosslinkable entities, or combinations thereof may be administered through microneedles (e.g., as a liquid); alternatively or additionally, in some embodiments, one or more crosslinkable entities, or combinations thereof, may be disposed within one or more microneedles, which may dissolve or degrade upon or after application.
• dissolving or degrading microneedle(s) may be particularly useful or desirable for delivery of relatively viscous materials (e.g., partially or wholly formed crosslinked material as described herein).
• one or more crosslinkable entities may be disposed in a microneedle; e.g., in a lumen (e.g., bore) thereof; in some such embodiments, the one or more crosslinkable entities (or a crosslinked material formed therefrom) may be released by injection through the lumen, and/or by dissolution or degradation of the microneedle (e.g., of its walls).
• one or more crosslinkable entities may be integrally formed with a microneedle; in some such embodiments, the microneedle may be otherwise formed of a material that dissolves or degrades upon or after application, thereby releasing the one or more crosslinkable entities, or a crosslinked material formed therefrom.
• injection may be by a dual bore syringe or needle.
• first and second crosslinkable entities are maintained 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 maintained separately during administration (i.e., each is separately administered, optionally at times separated by a time period as described herein).
• injection is via a needle having a bore size about 26 gauge to about 30 gauge.
• skin has been pretreated prior to (e.g., promptly or immediately prior to) administration of a system.
• skin pretreatment is accomplished by administration or application of a permeabilizing agent or device.
• skin pretreatment comprises one or more of application of an abrasive cleanser or chemical peel, dermablation, electroporation, iontophoresis, low-frequency sonophoresis, mirconeedling.
• skin is abraded prior to administration of a system.
• skin is abraded with microneedles and/or fraction lasers prior to administration of the system.
• a crosslinkable entity will be administered after (e.g., promptly or immediately after) pretreatment of skin with microneedles.
• kits that comprise systems described herein.
• a provided kit comprises a plurality of containers or vessels, including containers that separately house first and second crosslinkable entities.
• a provided kit comprises at least one container or vessel that includes a plurality of separate compartments (e.g., a dual-bore syringe or needle - or dual chamber package with a mixing chamber prior to dispensing); in some embodiments first and second crosslinkable entities are separately housed in such compartments.
• the present disclosure provides one or more containers, vessels, or compartments in which a first or second crosslinkable entity as described herein is disposed.
• the disposed crosslinkable entity is present in dry form; in some such embodiments, the crosslinkable entity is present in liquid form.
• the disposed crosslinkable entity has been stored for a period of time (e.g., for at least 1 day, 1 week, 1 month, 3 months, 6 months or more); in some such embodiments, the stored composition has been stable over the period of storage time, in that at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the crosslinkable entity remains undegraded. In some embodiments, the stored composition has been stable over the period of storage time, in that at least about 50%, 65%, 70%, 75% or more of the crosslinkable entity remains undegraded.
• first and second crosslinkable entities of a system when combined in vitro under physiological conditions, the first and second crosslinkable entities react with one another within a time period less than about 60 minutes at 37 °C to form a crosslinked material.
• Properties of such crosslinked material can be modulated through selection of the crosslinkable entities that generate it, and can represent characteristic traits of the particular crosslinked material provided by the present invention.
• a storage modulus of a crosslinked material is within a range of 50 Pa - 10 kPa. In some embodiments, a storage modulus of a crosslinked material is within a range of 50 Pa - 1 kPa.
• a storage modulus of a crosslinked material is within a range of 50 Pa - 500 Pa. In some embodiments, a storage modulus of a crosslinked material is within a range of 50 Pa - 100 Pa. In some embodiments, a storage modulus of a crosslinked material is within a range of 100 Pa - 10 kPa. In some embodiments, a storage modulus of a crosslinked material is within a range of 500 Pa - 10 kPa. In some embodiments, a storage modulus of a crosslinked material is within a range of 1 kPa - 10 kPa. In some embodiments, a storage modulus of a crosslinked material is within a range of 5 kPa - 10 kPa.
• a storage modulus of a crosslinked material is within a range of 500 Pa - 5 kPa. In some embodiments, a storage modulus of a crosslinked material is within a range of 500 Pa - 1 kPa.
• mass loss of a crosslinked material by degradation of less than 20% over 3 days in physiological buffer In some embodiments, mass loss of a crosslinked material by degradation of less than 10% over 3 days in physiological buffer. In some embodiments, mass loss of a crosslinked material by degradation of less than 5% over 3 days in physiological buffer.
• a degree of crosslinked material swelling upon hydration is characteristic property of a crosslinked material.
• Q m S woiien / mdry.
• a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 50-1000.
• a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 100-1000.
• a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 500-1000.
• a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 750-1000. In some embodiments, a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 50-100. In some embodiments, a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 50- 250. In some embodiments, a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 50-500. In some embodiments, a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 50- 750.
• a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 250-750. In some embodiments, a ratio between dried crosslinked material and fully swollen crosslinked material will be within a range of 400- 600.
• time in which formation of the crosslinked material occurs will be determined by the time from when the first and second crosslinkable entities of the system when are combined until the dynamic storage modulus (G’) becomes larger than the loss modulus (G”).
• a first and second clickable entity will form a crosslinked material within 1 second.
• a first and second clickable entity will form a crosslinked material within 10 seconds.
• a first and second clickable entity will form a crosslinked material within 1 minute.
• a first and second clickable entity will form a crosslinked material within 10 minutes.
• a first and second clickable entity will form a crosslinked material within 30 minutes.
• first and second clickable entity will form a crosslinked material within 60 minutes.
• characterization may involve application of a provided system to a model skin system (e.g., to porcine skin).
• a model skin system e.g., to porcine skin.
• characterization may involve monitoring one or more features of skin penetration.
• amount of a crosslinkable entity which penetrates the skin 0.5-2 days after topical application on porcine skin will be quantified, for example, after solubilizing the skin using enzymes and/or other solubilizing agents.
• amount of a crosslinkable entity which penetrates the skin at least 0.5 days after topical application on porcine skin will be quantified after solubilizing the skin using enzymes and/or other solubilizing agents.
• amount of a crosslinkable entity which penetrates the skin less than 2 days after topical application on porcine skin will be quantified after solubilizing the skin using enzymes and/or other solubilizing agents.
• amount of a crosslinkable entity that comprises a polymer moiety and a crosslink moiety which penetrates after topical application on porcine skin exceeds the amount of an entity which comprises the same polymer moiety and does not comprise the crosslink moiety.
• a crosslinkable entity comprises HA and the amount of crosslinkable entity which penetrates after topical application on porcine skin will exceed the amount of natural HA at the same molecular weight (e.g., number average molecular weight) which penetrates the skin.
• the depth of penetration of crosslinked material within the porcine skin is measured by shining ultraviolet light on the target site.
• the presence of crosslinked material in the porcine skin will be determined by observation of fluorescence upon shining ultraviolet light on the target site.
• presence of the crosslinked material at the target site will be observable after a period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or more days). In some embodiments, presence of the crosslinked material at the target site will be observable after about 3.5 days. In some embodiments, presence of the crosslinked material at the target site will be observable after about 9 days.
• a period of time e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or more days.
• attributes of the target site and/or site of administration i.e. skin
• skin attribute includes wrinkles, radiance, firmness, moisture content, skin thickness, elasticity, and skin smoothness.
• improvement includes repair to atrophic skin or scars.
• administration of the system results in fibroblast activation and/or enhanced collagen synthesis within the target sight of the skin.
• GAIS Global Aesthetic Improvement Scale
• a majority of subjects will observe > 2 improvement on the GAIS. In some embodiments, a majority of subjects will observe > 3 improvement on the GAIS. In some embodiments, a majority of subjects will observe about 1 to about 2 improvement on the GAIS. In some embodiments, a majority of subjects will observe about 1 to about 3 improvement on the GAIS. In some embodiments, a majority of subjects will observe about 1 improvement on the GAIS. In some embodiments, a majority of subjects will observe about 2 improvement on the GAIS. In some embodiments, a majority of subjects will observe about 3 improvement on the GAIS.
• MFWS Modified Fitzpatrick Wrinkle Scale
• a subject’s wrinkles will decrease at least 0.5 on the MFWS. In some embodiments, a subject’s wrinkles will decrease between 1 and 0.5 on the MFWS. In some embodiments, a subject’s wrinkles will decrease between 1.5 and 0.5 on the MFWS. In some embodiments, a subject’s wrinkles will decrease between 2 and 0.5 on the MFWS. In some embodiments, a subject’s wrinkles will see a decrease of between 2.5 and 0.5 on the MFWS. In some embodiments, a subject’s wrinkles will decrease between 3 and 0.5 on the MFWS.
• subject’s skin will be assessed for improvements in smoothness, radiance or firmness.
• moisture content of the skin can be monitored. In some embodiments, moisture content of skin will be higher as compared to untreated skin as determined by methods known in the art. In some embodiments, moisture content of skin will be determined by Corneometer CM 825. In some embodiments, moisture content of skin will be >20% higher after administration of the system at day 7.
• a provided system is used to treat a person exhibiting abnormal transepidermal water loss (TEWL).
• TEWL transepidermal water loss
• a person of skill in the art will understand that abnormal TEWL can be the result of skin damage caused by, for example, burns, certain chemicals, pathological conditions (e.g. eczema), physical abrasion, tape stripping, ultraviolet radiation, or combinations thereof.
• the level of TEWL of treated skin will be lower as compared to untreated skin as determined by methods known in the art.
• the level of TEWL of treated skin will be statistically lower as compared to a baseline level as understood by methods known in the art.
• TEWL will be measured by Tewameter TM 300 meter (Courage-Khazaka Electronics). In some embodiments, TEWL will be measured by Tewameter TM Nano (Courage-Khazaka Electronics). In some embodiments, TEWL will be measured by Tewameter Triple TM 330T (Courage-Khazaka Electronics). In some embodiments, TEWL will be measured by Invitro Tewameter VT310 (Courage-Khazaka Electronics). In some embodiments, TEWL of treated skin will be >10% lower after administration of the system at day 7.
• skin will assessed for improvements in skin smoothness.
• skin after administration of the system will be smoother than skin prior to administration of the system.
• skin’s smoothness will be assessed using Surface Evaluation of Living Skin (SELS).
• SELS Surface Evaluation of Living Skin
• PRIMOS phaseshift rapid in vivo measurement of skin
• skin will be assessed by 3D Skin profilometry via Canfield Primos.
• skin will be assessed using the Lemperle wrinkle scale.
• skin will be assessed using a 7-point subject satisfaction scale.
• skin will be assessed using the Oral commissures.
• skin will be assessed using Allergan Skin Roughness scale.
• skin will be assessed to determine presence and/or extent of crosslinked material within the skin.
• presence and/or of crosslinking material within the skin will be measured using near infrared (NIR) spectroscopy, confocal microscopy, a integrating sphere spectrophotometer, or will be determined using Viscoelastic deformation (VED, mm), elastic deformation (ED, mm), ultimate deformation (UD, mm), and pressure-deformation ratio methods.
• NIR near infrared
• VED Viscoelastic deformation
• ED elastic deformation
• UD ultimate deformation
• pressure-deformation ratio methods e.g., pressure-deformation ratio methods.
• presence and/or of crosslinked material within the skin is measured by shining ultraviolet light on the target site.
• presence and/or of crosslinked material in the skin will be determined by observation of fluorescence upon shining ultraviolet light on the target site.
• presence of the crosslinked material at the target site will be observable after a period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or more days). In some embodiments, presence of the crosslinked material at the target site will be observable after about 2 days. In some embodiments, presence of the crosslinked material at the target site will be observable after about 9 days. In some embodiments, presence of the crosslinked material at the target site will be observable after about 11 days. In some embodiments, presence of the crosslinked material at the target site will be observable after about 20 days. In some embodiments, presence of the crosslinked material at the target site will be observable after about 30 days.
• a period of time e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or more days.
• the present Examples describe, among other things, certain strategies that may be used to characterize and/or assess crosslinkable entities (and/or components and/or combinations thereof) as described herein, and/or the crosslinked materials formed by their reaction. Such strategies (or their equivalents as will be appreciated by those skilled in the art reading the present disclosure) may be used to assess potential crosslinkable entities, components (e.g., moieties), or combinations thereof, and/or crosslinked materials (e.g., gels) they form, for suitability for use in accordance with the present disclosure. In some embodiments, therefore, the present disclosure provides technologies for characterizing and/or selecting useful moieties, crosslinkable entities, and/or combinations thereof.
• a mixture of Boc-glycine (1.4 equiv.), DIPEA (2.1 equiv.), and TBTU (1.4 equiv.) in anhydrous DMF( ⁇ 40 mg Boc-glycine / mL) is stirred for 30 minutes at room temperature.
• a solution of CBT (1 equiv.) in anhydrous DMF (-100 mg CBT/mL) and DIPEA (1.4 equiv.) is added to the mixture. The reaction is stirred at room temperature under nitrogen for 4 h.
• HA-gly-CBT was synthesized with different degrees of CBT functionalization by changing stoichiometry of the reactants. Further, HA-gly-CBTs of different molecular weights were synthesized using the two-step synthesis described herein, by starting with HA of different molecular weights (-10, 50, and 250 kDa purchased from Creative PEG works). Gel permeation chromatography (GPC) was used to verify the synthesis of the crosslinkable entity with different molecular weights.
• GPC Gel permeation chromatography
• a first crosslinking entity can be an HA derivative functionalized with a CBT derivative.
• a CBT derivative (other than 6-amino-2-cyanobenzothiazole) can be conjugated to HA, such as 6- hydroxybenzothiazole-2-carbonitrile or another chemical derivative of the structure shown in Fig. 4a.
• this CBT derivative can be further derivatized prior to conjugation to HA with a chemical linker to facilitate conjugation to HA.
• this chemical linker can be an amino acid among other chemical groups.
• the CBT derivative can be conjugated to the carboxylic acid (Rl), alcohols (R2), or another chemical group on HA as shown in Fig. 4b.
• lipophilicity and/or skin penetration of an HA-CBT derivative can be increased by conjugating one or more other chemical moieties to an HA backbone, for example using one or more CBT derivatives that may be more hydrophobic than is CBT itself (and/or than is the HA itself), among other chemical modifications.
• an HA-CBT comprises ( e.g ., is conjugated with) one or more chemical moieties (e.g, one or more hydrophobic moieties) such as for, example, a fatty acid.
• one or more moieties may be conjugated to an alcohol (R2) on the backbone of HA.
• R2 an alcohol
• HA before or after such conjugation, HA can be oxidized, converting a fraction of the alcohols (R2) to aldehydes.
• conjugation of a moiety to HA may be via a linker; in some such embodiments, the linker can be a hydrophobic amino acid, or other hydrophobic chemical moiety.
• one or more moieties that is conjugated to an HA (or other polymer moiety) as described herein may be modified (e.g., functionalized) before or after such conjugation.
• some or all of (e.g, a fraction of) CBT or CBT derivative moieties conjugated to HA can be further functionalized, e.g, by reacting the nitrile group with a hydrophobic cysteine derivative or another aminothiol, such as cysteamine.
• Cys-PEG-Cys is synthesized by conjugating the carboxylic acid on cysteine (Cys) to the amines on PEG diamine.
• a mixture of N-Boc-S-trityl-D-cysteine (2.4 equiv.) and 1,1'- Carbonyldiimidazole (CDI, 3.0 equiv.) in anhydrous DMF ( ⁇ 55 mg N-Boc-S-trityl-D-cysteine / mL) is stirred for 30 minutes at room temperature.
• a solution of PEG diamine (-2000 Da; 1 equiv.) in anhydrous DMF (-125 mg PEG diamine / mL) is then added to the reaction mixture.
• a lower molecular weight Cys-based crosslinkable entity can be prepared using ethylene diamine as the linker (Cys-EDA-Cys). Such a material was prepared in an analogous procedure to the Cys-PEG-Cys described above.
• a second crosslinking entity contains 2 or more aminothiols (i.e., 2 or more aminothiol crosslinkable moieties) each of which may, in at least some embodiments, be conjugated via linker moiety.
• such an aminothiol is a 1,2-, 1,3-, or l,4-aminothiol or combination thereof.
• an aminothiol is D-cysteine, L-cysteine, a derivative of cysteine, or combination thereof.
• an aminothiols is capped with another chemical moiety, which for example may be released to expose the aminothiol under physiological conditions.
• a linker may be or comprise a polymer, such as PEG, a diamine or a polypeptide.
• a linker may be or comprise a small molecule or amino acid, such as ethylene diamine or lysine.
• the present Example describes certain ex vivo strategies that may be used to characterize or assess crosslinkable entities (and/or components and/oror combinations thereof) as described herein, and/or the crosslinked materials formed by their reaction.
• preparations of individual crosslinkable moieties for use as described herein may have a viscosity low enough to permit delivery by injection (e.g., via microneedles).
• crosslinkable entities may be assessed for their usefulness as described herein based in whole or in part on one or more rheological properties of the crosslinked material they generate.
• one or more rheological properties of a crosslinked material may be adjusted based on polymer moiety and/or crosslink moiety molecular weight (e.g., number average molecular weight), concentration, degree of substitution, as well as on density and/or length of crosslinks within the material, etc.
• rheological assessments may be performed with a rheometer AR-G2.
• it may be desirable to minimize prevent solvent evaporation for example by thermally isolating (solvent trap) material to be assessed and checking its weight before and after the experiment (difference in weights should desirably be less than 1 %).
• analyses will be performed under constant temperature (37°C).
• gelation kinetics may be monitored through time sweeping tests, and storage (G 1 ) and loss (G") moduli may be measured as function of, for example time at 10 rad/s frequency and 1% stress strain.
• first and second crosslinkable entities may be pre-mixed (e.g., for a short time such as ⁇ 5 min), loaded, and measured at the test temperature. Either or both of frequency and flow sweeping tests can be performed after crosslinking between them occurs. In some embodiments, frequency sweeping tests can be conducted isothermally, for example in the frequency range 0.01 ⁇ w ⁇ 100 rad/s, while maintaining stress strain, for example at 1 %. Normal force can desirably be maintained to a constant value as well. In some embodiments, it will be desirable to take care to ensure that residual stress due to loading is relaxed. In some embodiments, a crosslinked material may be allowed a soak time to equilibrate at the test temperature.
• flow sweep experiments can be made isothermally in the shear rate range 0.0005 ⁇ g ⁇ 100 s 1 to determine the zero-shear viscosity and/or an intensive shear thinning regime.
• data points may be collected once every 100 s.
• a crosslinked material as described herein will desirably have a storage modulus within the range of 50 Pa - 10 kPa. In some embodiments, a crosslinked material as described herein will desirably have a storage modulus within the range of 10 Pa - 100 Pa. In some embodiments, a crosslinked material as described herein will desirably have a storage modulus within the range of 100 Pa -300 Pa. In some embodiments, a crosslinked material as described herein will desirably have a storage modulus within the range of 300 Pa - 1000 Pa. In some embodiments, a crosslinked material as described herein will desirably have a storage modulus within the range of 1 kPa -10 kPa.
• a crosslinked material as described herein will desirably have a storage modulus within the range of 5 kPa -10 kPa. In some embodiments, a crosslinked material as described herein will desirably have a storage modulus greater than 10 kPa.
• desirable crosslinkable entities interact with one another to generate a crosslinked material with appropriate kinetics as described herein under physiologically relevant conditions (e.g., at 37 °C and/or in the presence of physiologically relevant salts and/or other agents).
• physiologically relevant conditions e.g., at 37 °C and/or in the presence of physiologically relevant salts and/or other agents.
• it may be useful to assess gelation kinetics in vitro for example by combining first and second crosslinkable entities together under such physiologically relevant conditions and monitoring their interaction, for example, by assessing dynamic storage modulus (G’) and loss modulus (e.g., G”).
• G dynamic storage modulus
• loss modulus e.g., G
• the first and second crosslinkable entities are mixed in a vessel (e.g., a test tube); a period of time elapses; the test tube is inverted and the resultant mixture does not flow from the vessel.
• a vessel e.g., a test tube
• the period of time is 10, 30, 60, 90 or 360 seconds.
• desirable crosslinkable entities are those for which gelation time, determined as described herein when they are combined ex vivo , is less than about 30 minutes.
• an appropriate or desired gelation time will depend on the mode or manner of administration. For example, if the first and second crosslinkable moieties will be combined or otherwise contacted with one another at a site (e.g., within a syringe or at a site of administration) other than the target gelation site, it will typically be desirable that the gelation time be sufficiently long to ensure that significant crosslinking does not occur away from the target gelation site.
• crosslinkable entities, components or combinations thereof may be assessed for usefulness in accordance with the present disclosure by determining one or more swelling attributes of the crosslinked material they create.
• degree of swelling may be measure based on mass ratio between crosslinked material in a dry form vs a fully swollen form (e.g., that has reached equilibrium with an external aqueous buffer: Q - Mswoiien/Mdry) .
• one or more swelling attributes of a crosslinked material may be adjusted based on polymer moiety and/or crosslink moiety molecular weight (e.g., number average molecular weight), concentration, degree of substitution, as well as on density and/or length of crosslinks within the material, etc.
• a crosslinked materials as described herein may have a swelling ratio within the range of 50-1000.
• crosslinking can increase the resistance to degradation of biopolymers, resulting in increased durability in situ.
• an HA gel was formed ex vivo and exposed to hyaluronidase, an enzyme naturally present in the body that degrades HA.
• HA-gly-CBT 50 pL, 15 mg/mL, PBS
• PBS 20 pL
• Cys- PEG-Cys 20 pL, 20 mg/mL
• the mixture of HA-gly-CBT and PBS remained a viscous liquid, while the mixture of HA-gly-CBT and Cys-PEG-Cys formed a clear gel.
• bovine testes hyaluronidase 5 pL
• the mixtures were vortexed gently to obtain final enzyme concentrations of 100 units/mL.
• samples were centrifuged to remove insoluble, gel components and an aliquot of each of the supernatants was analyzed. Resistance to degradation was assessed by summing the fraction of remaining soluble HA with a high molecular weight (determined by GPC analysis of the supernatant) with the fraction of remaining insoluble HA (determined by GPC analysis of the supernatant with plate reader).
• the extent of crosslinking in a crosslinked material is sufficient to impart in situ durability.
• the crosslinked material exhibits an increased durability relative to an appropriate reference, such as the corresponding uncrosslinked material.
• Example 3 Formulations of a Crosslinkable Entities
• sections 3.1-3.11 provide exemplary formulations, each of which comprise at least one crosslinkable entity.
• the component labelled“crosslinkable entity” may be substituted for a different suitable crosslinkable entity as provided herein.
• the system comprises a first formulation and a second formulation of sections 3.1-3.11 which are paired together so long as the crosslinkable entity of the first formulation can react with crosslinkable entity of the second formulation to form a crosslinked material.
• a serum (e.g., that may be utilized, for example, as an anti-aging serum), is prepared by combining part A and B as outlined in Table 3.1 and subsequently adding part C until the pH of the serus is about 5.0 - about 5.5.
• exemplary components may, in some embodiments, participate functionally in a serum preparation as suggested below.
• a lotion formulation (e.g., that may be used as a soothing and/or moisturizing lotion) is prepared as outlined herein.
• Part A is prepared by combining the ingredients summarized in table 3.2 and heating with mixing to 75 °C.
• part B is prepared by combining the ingredients summarized in table 3.2 and heating with mixing to 75 °C.
• part B is added to part A and mixed at 75 °C.
• the formulation is allowed to cool with moderate mixing.
• part C is mixed until uniform. pH is adjusted to 5.0 ⁇ 0.5. with the addition of part D.
• exemplary components may, in some embodiments, participate functionally in a lotion preparation as suggested below.
• a lotion formulation (e.g., that may be used as a natural sun-protecting face lotion) is prepared as outlined herein. Part A is prepared by combining the ingredients summarized in table 3.3 and heating 85 °C. Part B is prepared by combining the ingredients summarized in table 3.2 and heating to 85 °C. Part C is added to part A with stirring and temperature is maintained. Part B is slowly added to part A/C with high shear stirring. The mixture is homogenize. The formulation is stirred and cooled to room temperature, adding part D below 40 °C.
• exemplary components may, in some embodiments, participate functionally in a lotion preparation as suggested below.
• An emulsion formulation (e.g., that may be used as a soothing w/o cream) is prepared as outlined herein.
• Part A is prepared by mixing until dissolved the ingredients summarized in table 3.4.
• the ingredients from Part B are then added to Part A with high shear to afford the formulation.
• exemplary components may, in some embodiments, participate functionally in an emulsion preparation as suggested below.
• hydrogel mask formulaion (e.g., that may be used as a solid fiber mask with impregnated serum) is prepared as outlined herein.
• Part A is prepared by dissolving Tetrasodium EDTA in water under propeller mixing. Gum are pre-dispersed in glycerin to make slurry. The slurry is added to the Tetrasodium EDTA solution under propeller mixing, mixing until uniform and lump-free.
• Part A is heated to 85-90 °C to hydrate the gums. While Part A is hot, part B as summarized in Table 3.5 is added with mixing.
• Part C ingredients as summarized in Table 3.5 are premixed and added to parts A and B.
• the hot solution is immediately poured in molds and allowed to set undisturbed. Once the gel is cooled and is set, the formulation is packed suitably.
• exemplary components may, in some embodiments, participate functionally in a hydrogel mask preparation as suggested below.
• a serum formulation (e.g., that may be used as a BB (Beauty Balm) Powder to Cream Soothing Serum) is prepared as outlined herein. Procedure: Add all ingredients of part A as summarized in table 3.6 and mix with high shear until a homogeneous mixture is obtained. Add part B as summarized in table 3.6 powder ingredients and mix until free flowing powder is obtained.
• exemplary components may, in some embodiments, participate functionally in a serum preparation as suggested below.
• a solid formulation (e.g., that may be used as a targeted deep wrinkle solid stick) is prepared as outlined herein. Procedure: Heat part A as described in table 3.7 to 95 °C and mix until dissolved, then add to Phase B as described in table 3.7 heated to 95 °C, cool to 75 °C and add phase C. Mix to uniform and pour into molds or containers.
• exemplary components may, in some embodiments, participate functionally in a solid preparation as suggested below.
• a mist formulation (e.g. that may be used as a revitalizing mist with pH 5 antioxidant) is prepared as outlined herein.
• the ingredients as summarized in Table 3.8 are combined to afford part A.
• C The ingredients as summarized in Table 3.8 are combined to afford part B.
• Part A is added to part B while mixing until uniform.
• Part C is added to the mixture of parts A and B.
• the resultant mixture is mixed until uniform.
• the pH is adjusted to pH to 4.0-5.0 for stability.
• the formulation is packaged in a spray bottle. Table 3.8
• exemplary components may, in some embodiments, participate functionally in a serum preparation as suggested below.
• Exemplary Powder [211] A powder formulation (e.g., that may be used as a powder to cream formulation with inhibitors to slow/prevent cross linking in product and until within skin) is prepared as outlined herein. The ingredients of part A as summarized in Table 3.10 are combined and mixed with high shear until a homogeneous mixture is obtained. Part B powder ingredients are added and mixed until free flowing powder is obtained. Part C is folded in and mixed until uniform.
• exemplary components may, in some embodiments, participate functionally in a serum preparation as suggested below.
• a lotion (e.g., that may be used as a liquid crystal solid nanoparticle dual action lotion) is prepared as outlined herein.
• the components in part A as summarized in table 3.11 are mixed, and heated to 50 °C.
• the components of part B are added, and the mixture is heated to 65 °C.-70 °C.
• the components of Phase C as summarized in table 3.11 are mixed at room temperature.
• the heated components of parts A and B are then mixed together in a vessel under lamellar flow conditions, which create a lyotropic liquid-crystalline mixed phase (AB) with a mean particle size of l50nm.
• the lyotropic liquid-crystalline mixed parts A and B are then mixed with part C to form a dispersion.
• Phase D is added, pH adjusted through the addition of Phase E, with gentle mixing, then cooled to room temperature.
• exemplary components may, in some embodiments, participate functionally in a serum preparation as suggested below.
• the present Example describes use of porcine skin studies to characterize or assess crosslinkable entities (and/or components and/or combinations thereof) as described herein, and/or the crosslinked materials formed by their reaction.
• a system comprising first and second crosslinkable entities is applied to porcine skin.
• application of the first crosslinkable entity is separated in time (i.e., by a period of time) from application of the second crosslinkable entity.
• multiple different separating periods of time may be assessed.
• the period of time may be dependent on desired depth of penetration, skin type and target location.
• multiple different separating periods of time as a function of desired dept of penetration skin type and target location may be assessed.
• degree of penetration of the first and/or second crosslinkable entities into the porcine skin is assessed at one or more time points after administration of one or both of the crosslinkable entities.
• assessment(s) may be made at one or more time points selected from the group consisting of 1, 4, 12, 24 and 48 hours.
• one or more porcine skin samples may be thawed, for example at room temperature (e.g., for 30 min or more) prior to use.
• hair may be clipped from such samples (e.g., using scissors), and samples may be rinsed, e.g., using 0.9% saline solution.
• subcutaneous in situ gelation may be assessed by administering first and second crosslinkable entities to a site or sites on or in the sample.
• solutions of first and second crosslinkable entities may be injected (e.g., separately or as a mixture, optionally after a premix period, and optionally at one or more different concentration ratios) into the sample.
• porcine skin samples may be excised until their underlying dermis layer is reached, and first and second crosslinkable moieties can be administered to fill the void.
• skin samples to which first and second crosslinkable moieties have been administered may be maintained under stable conditions (e.g., stored at room temperature in a Petri Dish) for a period of time to permit crosslinking (e.g., gelation) to occur in situ.
• stable conditions e.g., stored at room temperature in a Petri Dish
• crosslinking e.g., gelation
• degree of crosslinking may be assessed at one or more time points after administration (e.g., after injection of a mixture or of the first or, more likely, the second, crosslinkable moiety), for example by sectioning the sample(s) (e.g., using a scalpel). In some embodiments, such sectioning involves preparing small (e.g., about 0.4 cm x 0.4 cm) squares, which can be frozen (e.g., by immersion in a isopentane/dry ice bath) and stored, for example at -80°C.
• small e.g., about 0.4 cm x 0.4 cm
• thin (e.g., about 20 mih) skin sections can be obtained (e.g., using a cryostat), and can be imaged (e.g., using a confocal microscope).
• the skin surface was washed three times in the Franz cell with PBS, and then a small region in the center of the application area was excised with a surgical scalpel.
• the skin tissue was placed into OCT in a biopsy mold and snap frozen by placing the mold into a methylpentane/dry ice slurry.
• the frozen skin was sectioned on a cryostat microtome into 20 pm sections.
• a drop of ProLong Gold was placed on each section, and covered with a coverslip prior to imaging. Fluorescent images were taken with a lOx objective on a ZeissAxioPlan2 using a DAPI filter (Zeiss filter 49) to visualize the location of HA-gly-CBT.
• HA-gly-CBT 15 mg/mL, 300 pL, DI water containing 0.5% Tweemo was applied topically to 6 separate, porcine skin pieces in Franz diffusion cells. The samples were incubated for 18h at 37°C in the sealed donor compartment and the skin surfaces washed three times with PBS. The stratum corneum was isolated from the samples using tape strips, then the epidermis was scraped off the dermis, and the dermis cut into small pieces.
• HA-gly-CBT was extracted from each of the skin sections (top 8 layers of SC, bottom 8 layers of SC, epidermis, and dermis) by incubation in 1 : 1 PBS:methanol at 37 °C overnight.
• the volume of the extracts was reduced with rotary evaporation, and the concentrated solutions analyzed by GPC (using the same conditions as described in Example 1, mobile phase of 9: 1 DTMeOH, 0.2 M NaNOi).
• HA-gly- CBT (30 pL, 5 mg/mL, PBS) was injected into two porcine skin samples with a 3 l-gauge insulin syringe right below the skin surface.
• Cys-PEG-Cys (20 mg/mL, PBS) was then injected (using the same technique) into one of the skin samples 1 h after the HA-gly-CBT injection. Both skin samples were incubated at 37 °C overnight in a humidified oven. Following incubation, the samples were frozen and sectioned, as described above.
• the present Example describes administration of a system as described herein to a human, and assessment of one or more skin characteristics in light of such administration.
• first and second crosslinkable entities are administered to a skin site.
• both crosslinkable entities are administered to the same site; in some embodiments, each may be administered to a different site so long as both ultimately arrive at a target site of interest, so that a crosslinked material, formed by reaction of the first and second crosslinkable entities, is formed or otherwise provided in situ at the target site.
• one or both of the first and second crosslinkable entities is administered to the target site (e.g., by injection).
• one or both of the first and second crosslinkable entities is administered to a site above (i.e., external) to the target site, so that the crosslinkable entity(ies) must penetrate skin tissue in order to arrive at the target site.
• the present disclosure provides crosslinkable entities having particular characteristics (e.g., lipophilicity and/or molecular weight (e.g., number average molecular weight), etc) that facilitate such penetration.
• administration of the first crosslinkable entity is separated in time (i.e., by a period of time) from that of the second crosslinkable entity.
• the period of time is sufficient to permit substantial penetration of the first- administered crosslinkable entity toward and/or into the target site prior to administration of the second-administered crosslinkable entity.
• multiple administrations are performed for at least one of the cross-linkable entities; in some embodiments, multiple administrations are performed for each of the cross-linkable entities.
• administration of both crosslinkable moieties is considered a“dose” of a provided system.
• administration of one crosslinkable entity to a subject who has previously received administration of the other crosslinkable entity is considered a“dose” of a provided system.
• multiple doses are administered.
• a crosslinkable entity is a formulation of Example 3. In some embodiments the formulation of the first crosslinkable entity and the formulation of the second crosslinkable entity are the same. In some embodiments the formulation of the first cross linkable entity and the formulation of the second crosslinkable entity are different.
• one or more characteristics of skin is assessed after the system has been administered (e.g., at one or more time points selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31 days or more after the administration - for example after administration of any particular dose or of a complete dosing regimen).
• one or more characteristics of skin is assessed after the system has been administered (e.g., at one or more time points selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 months or more after the administration - for example after administration of any particular dose or of a complete dosing regimen).
• one or more characteristics of skin is assessed about 3.5 days after the system has been administered.
• one or more characteristics of skin is assessed about 9 days after the system has been administered.
• one or more visual features of skin is assessed after system administration (e.g., as compared with prior to such administration).
• visual features are assessed by one or more expert graders with reference to the Global Aesthetic Improvement Scale (GAIS) which, as is known in the art, is a validated, standardized 5-point scale for measurement of response to cosmetic procedures, which has been shown to have good intra- and inter-rater reliability, and intra-class correlation coefficients generally in the range of 0.85-0.95 (see, for example, Carruthers & Carruthers, J. Cosmet Laser Therp. 12:235, 2010)
• GAIS Global Aesthetic Improvement Scale
• administration of a system as describe herein achieves at least a 1 -grade improvement in GAIS scale.
• one or more skin features may be assessed with an established scale such as, for example, the Lemperle wrinkle scale, a Subject Satisfaction scale (typically 7-point), the Allergan skin roughness scale, or any other appropriate scale.
• an established scale such as, for example, the Lemperle wrinkle scale, a Subject Satisfaction scale (typically 7-point), the Allergan skin roughness scale, or any other appropriate scale.
• any validated scale that includes subjective and/or objective assessment of one or more physical, functional, or visual attributes (e.g., smoothness, radiance, firmness, elasticity, volume, etc) of skin may be utilized.
• one or more skin features may be or relate to enhanced mechanical support of dermal extracellular matrix, fibroblast elongation and/or spreading, up- regulation of type II TGF-beta receptor or downstream targets thereof (e.g., type I collagen synthesis), up-regulation of connective tissue growth factor, and/or combinations thereof (see, for example, Quan et al., J Invest Dermatol. 133:658, 2013).
• type II TGF-beta receptor or downstream targets thereof e.g., type I collagen synthesis
• connective tissue growth factor e.g., connective tissue growth factor, and/or combinations thereof
• skin hydration may be assessed after administration of a provided system.
• skin hydration is measured, for example with a corneometer (e.g., a corneometer CM825, which provides a conductance measure of the relative moisture content within skin).
• administration of a system as described herein achieves a statistically significant improvement in skin hydration; in some such embodiments, such improvement is at least 10%, 15%, 20%, 25% or more, for example when measured at a time that is 12, 24, 36 or 48 hours after administration as described herein.
• a 20 mg/ml HA-gly-CBT solution was prepared in deionized water, polysorbate 20 & glycerin.
• a second solution of Cys-PEG-Cys in PBS (20 mg/mL) was prepared and the pH adjusted to 5.5-6.5 with NaOH.
• the HA-gly-CBT solution (20m1) and a solution of the commercial HA benchmark serum (20m1) were applied to 3 cm x 3 cm areas on the volar forearm immediately following baseline readings. Additional Corneometer readings were measured at 0.5 and 6 hours.
• the Cys-PEG-Cys solution (20m1) was applied to the HA-gly-CBT test site and a phosphate buffer solution (20m1) was applied to the commercial HA benchmark serum site. Additional Corneometer readings were then measured at 12 hr, 24 hr, 48 hr, 72 hr and 96 hr. The results are tabulated in Table 3.12.
• the benchmark commercial HA (Hyaluronic Acid) serum was a blend of high and low molecular weight hyaluronic acids, with 1.5% pure hyaluronic acid, described by the manufacturer as capable of replenishing moisture to produce plumped, youthful, supple skin.
• the ingredient list for the commercial HA Serum benchmark includes water, glycerin, hydroxyethylpiperazine ethane sulfonic acid, sodium hyaluronate, PEG-60 hydrogenated castor oil, cereale seed extract/rye seed extract, calcium pantothenate, dipeptide diaminobutyroyl benzylamide diacetate, ascorbyl glucoside, disodium EDTA, pentylene glycol, phenoxyethanol, and chlorphenesin.
• skin smoothness may be assessed after administration of a provided system.
• skin smoothness is measured, for example, by 3D skin profilometry, for example using a Canfield Promos device and/or by silicon/epoxy cast scanning.
• administration of a system as described herein achieves a statistically significant increase in skin smoothness, and/or increasing smoothness over time (with or without additional administrations).
• visual improvement to skin smoothness after administration of the provided system was demonstrated using a 7-point skin smoothness improvement scale. Two days after application of the system, a two-grade improvement was noted as depicted in Figure 13.
• skin thickness may be assessed after administration of a provided system.
• skin thickness may be assessed, for example by ultrasound profiling (see, for example, Van Mulder et al. Vaccine 35: 1810, 2017).
• administration of a system as described herein achieves a statistically significant increase in skin thickness.
• relevant skin may have been damaged (e.g., due to a disease, disorder, or condition, or to a surgical, cosmetic, or dermatological treatment), and improvements to such damaged skin are assessed.
• damaged skin may be or comprise laser (e.g., fractional laser) treated skin, intense-pulse-light-treated skin, microneedled skin, skin subjected to chemical peel treatment, skin exposed to dermabrasion, traumatically injured skin (including by surgery), skin suffering from a medical condition such as skin atrophy, extreme dryness, etc.
• relevant skin may be skin that is wrinkled or otherwise shows signs of aging, or may be skin with respect to which additional plumpness and/or resilience is desired.
• skin of interest may be or comprise skin at a site selected from the group consisting of full face and/or specific targets of a subject’s face such as lips, lower lip, upper lip, tear troughs or other sites around the eyes (e.g., crow’s feet), nasolabial folds, forehead, cheeks back of hands, ear lobes, knees, neck, decolletage, arms, legs, torso, buttocks feet, and combinations thereof, including, for example, full face, etc.
• a site selected from the group consisting of full face and/or specific targets of a subject’s face such as lips, lower lip, upper lip, tear troughs or other sites around the eyes (e.g., crow’s feet), nasolabial folds, forehead, cheeks back of hands, ear lobes, knees, neck, decolletage, arms, legs, torso, buttocks feet, and combinations thereof, including, for example, full face, etc.
• a biopsy is obtained of a site to which a provided system has been administered or delivered, and histology on the biopsy is performed to assess degree (e.g., amount) and/or depth of penetration.
• degree e.g., amount
• crosslinkable entity(ies) and/or crosslinked material is detected in the epidermis, dermis or underlying hypodermisor below.
• an amount of crosslinkable entity(ies) is detected within the skin in the biopsy (e.g., on, at, in, or below the epidermis, dermis or underlying hypodermis and/or otherwise at the target site) that is a meaningful (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) percentage of that administered (e.g., to a surface of the skin and/or to a site external to the target site).
• a meaningful e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more
• the penetration of the crosslinking entity into human skin can be increased through skin barrier disruption techniques.
• skin barrier disruption techniques For example, the ex vivo penetration of HA-gly-CBT (tagged with AlexaFluor 647 hydrazide) into human skin, which was pretreated with different techniques was assessed with microscopy. Prior to application of the material, one skin sample was tape stripped twenty -five times to remove the majority of the stratum corneum. In a second sample, HA-gly-CBT was applied to the surface of the skin, and the skin was then treated with a dermaroller (500 pm needle), rolling over the product 40 times. Both samples were incubated in Franz cells in a humidified oven at 37 °C for 20 h after topical application. The samples were sectioned to 20 pm and imaged on a fluorescent microscope using a Cy5 filter, as described above ( Figure 14).
• HA-gly-CBT (50 kDa, tagged with AlexaFluor 647 hydrazide) was applied topically to human skin, rolled twenty times with a dermaroller (500 pm needles). The skin was transferred to a Franz cell and incubated at 37 °C for 20 h. The skin was frozen, sectioned, and imaged on a fluorescent microscope with a Cy5 filter to observe depth of penetration. (Fig 18 A). Two additional human skin samples were treated similarly.
• the stratum corneum was removed from the samples with tape stripping (20 x) and the epidermis and dermis isolated with a scalpel. After extracting HA-gly-CBT (overnight at 37 °C) from each skin layer into 1 : 1 PBS:Methanol, the concentration of HA-gly-CBT was quantified by measuring the fluorescence of the AlexaFluor dye in each extract on a plate reader (Fig 18 B).
• HA-gly-CBT 50 kDa was detected in both the epidermis and dermis after treatment with a dermaroller. Observed permeation of HA-gly-CBT suggests feasibility of topical delivery of a stable, HA-based crosslinked material deep into the dermis when coupled with subsequent, concurrent, or previous topical delivery of a cysteine-based crosslinkable entity.
• Sites of administration on the skin of the left volar forearm (see Figure 15) of the subject were prepared for administration of the crosslinkable entites.
• the sites were prepared by cleaning with mild soap and tepid water for 30 seconds, rinsing the sites for an additional 30 under tepid water and patting the sites dry.
• Semi-transparent Scotch Tape was repeatedly applied to each site and removed until each of the sites glistened slightly (about 40 cycles).
• a first formulation was administered to each of the sites:
• each site was covered with about 3 cm 3 of TegadermTM type film. A period of 2 hours was allowed to elapse. The TegadermTM paper was removed. The sites were allowed to equilibrate for 10 minutes. Visual appearance of sites were photographed under both visible light and ultraviolet light (-365 nm).
• a second crosslinkable formulation were administered to each of the sites:
• each site was covered with about 3 cm 3 of TegadermTM type film. The sites were left covered overnight. The TegadermTM paper was removed. The sites were allowed to equilibrate for 10 minutes. At which time, the sites of administration were examined using visible and ultraviolet (365 nm) light. Recordable fluorescent signal was not observed at sites A and B. Fluorescent signal was clearly visible at the site C (see Figure 15) indicating crosslinked material had penetrated the skin.
• TegadermTM was applied on top of the application site between and after each administration of a crosslinkable entity. The administration process was repeated daily for 2 days, for a total of 4 administrations of both HA-gly-CBT and Cys-PEG-Cys. On the third morning, a 4 mm biopsy was taken in the center of the application square, after washing and sterilizing the site. The biopsy was immediately frozen in OCT.

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