WO2024259323A2 - Compositions pharmaceutiques pour administration ciblée d'agents bioactifs à biodisponibilité améliorée et méthodes d'utilisation - Google Patents

Compositions pharmaceutiques pour administration ciblée d'agents bioactifs à biodisponibilité améliorée et méthodes d'utilisation Download PDF

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WO2024259323A2
WO2024259323A2 PCT/US2024/034128 US2024034128W WO2024259323A2 WO 2024259323 A2 WO2024259323 A2 WO 2024259323A2 US 2024034128 W US2024034128 W US 2024034128W WO 2024259323 A2 WO2024259323 A2 WO 2024259323A2
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pharmaceutical composition
amino acid
composition
subject
acid
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WO2024259323A3 (fr
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Scott Shapiro
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'

Definitions

  • the field of the invention relates to pharmaceuticals and medicine. More particularly, the field of the invention relates to the targeted delivery of bioactive agents, such as topical or transdermal delivery with improved drug bioavailability.
  • bioactive agents such as topical or transdermal delivery with improved drug bioavailability.
  • BACKGROUND Topical administration of biologically active agents has become an important method for treating a variety of skin conditions. Carlin, Cosmetic Dermatology, February 2001, pp. 35-38 teaches topical administration of vitamin C to reduce erythema of acne rosacea. Greco, Plastic and Reconstructive Surgery 105: 464-465 (2000) suggests the use of topical vitamin C in the treatment of fine wrinkles and as a stimulant for wound healing. Norman and Nelson, Skin and Aging, February 2000, pp.
  • the invention provides pharmaceutical compositions that allow efficient delivery (e.g., transdermal or topical) of effective amounts of bioactive agents for absorption (e.g., percutaneous).
  • these bioactive agents can include traditionally hard to deliver large hydrophilic molecules such as proteins, peptides and nucleic acids including short interfering RNAs (siRNAs).
  • the formulations according to the invention are generally non-irritating to the skin or tissue.
  • the invention provides a pharmaceutical composition for topical application for delivery of a bioactive agent to a subject, wherein the composition comprises i) an anhydrous carrier; ii) an effective amount of a bioactive agent; and iii) a penetration enhancer selected from the group consisting of an amino acid derivative compound, a cell penetrating peptide (CPP), an antimicrobial peptide, a lipid nanoparticle composition, and combinations thereof.
  • CPP cell penetrating peptide
  • the composition is applied topically, but the targeted site of action of the bioactive agent is not necessarily located at the topical site of application.
  • the bioactive agent is absorbed into the systemic circulation and acts distally to the topical site of application.
  • the composition is for topical (e.g., local skin or other tissues), transdermal (systemic/bloodstream), ocular (local eye), intra-articular (local muskculoskeletal, e.g., tumors, inflammation), intra-tympanic (local ear), or transungual (local through nail plate) delivery.
  • the invention also provides methods of administering the compositions by one or more of these routes to treat a disease or condition in a subject.
  • the anhydrous carrier comprises one or more esters, amides, ethoxylated fats, mineral oil, petrolatum, vegetable oils, animal fats, triglycerides, polyols (e.g., glycerol), glycerin, propylene glycol, sorbitol, isopropyl myristate and combinations thereof.
  • the anhydrous carrier comprises glycerin in a concentration of 20-40% (by weight).
  • the anhydrous carrier comprises capric/caprylic triglyceride, glycerol, beta hydroxy acid, and one or more diethylene glycol monoethyl ether, dimethyl isosorbide, propylene glycol, oleic acid, epidermal enzymes, ethanol and dimethylsulfoxide.
  • the anhydrous carrier comprises a combination of glycerin, isopropyl myristate, oleic acid, capric/caprylic triglyceride and diethylene glycol monoethyl ether.
  • the composition comprises the amino acid derivative compound, wherein the amino acid derivative compound has the following configuration: (AA) ---- (L) ---- (HP), wherein AA is an amino acid or a group of two amino acid molecules linked together by a peptide bond or an alkylene chain linking group having up to six carbon atoms, wherein L is a linker group, and wherein HP is a hydrophobic moiety.
  • HP is an alkyl or alkenyl hydrocarbon chain having a length of from 10 to 20 carbon atoms.
  • L is selected from the group consisting of a single bond, a carbonyl group, -C(O)-O-CH2, -C(O)-O-CH2CH2-, -C(O)-NH-CH2- and – C(O)-NH-CH2CH2-.
  • the amino acid(s) are selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, 6-(dimethylamino)hexanoic acid, derivatives thereof, and combinations thereof.
  • the amino acid derivative is selected from a compound identified in Table 1, or a combination thereof.
  • the composition comprises a cell penetrating peptide.
  • the peptide consists of six to twenty amino acid residues. In some embodiments, at least 50% of the amino acid residues of each peptide are positively charged at physiological pH. In some embodiments, the positively charged amino acid residues are selected from arginine and lysine. In some embodiments, at least one of the permeation enhancer peptide is an antimicrobial peptide. In particular embodiments, the antimicrobial peptide can be magainin or a derivative of magainin. In some embodiments, the derivative of magainin can be magainin substituted by a dipeptide. In some embodiments, the dipeptide derivative of magainin can be magainin substituted by Gly-Ala.
  • the dipeptide derivative of magainin can be magainin substituted by Lys-Leu.
  • the antimicrobial peptide is a magainin peptide.
  • the cell penetrating peptide is selected from the group consisting of any of SEQ ID NOS:1-49 and combinations thereof.
  • the composition comprises a lipid nanoparticle composition.
  • the lipid nanoparticle (LNP) composition comprises one or more cationic lipids.
  • the bioactive agent is selected from a biologic, protein, vaccine, macromolecule, and a small molecule. In some embodiments, the bioactive agent is an antibody.
  • the LNPs can be employed in combination with amino-based peptides for transdermal delivery of protein drugs, biologics and vaccines.
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject’s skin an effective amount of a topical pharmaceutical composition as provided herein.
  • effective amounts of the bioactive agent are absorbed into the subject’s blood circulation.
  • the method further comprises exfoliating the skin of the subject prior to the administering step.
  • the present invention provides a composition for facilitating transdermal delivery of biologics, proteins, macromolecules, nucleic acids, or small molecules, the composition comprising an anhydrous topical cream delivery formulation for percutaneous absorption comprising one or more of i) capric/caprylic triglyceride, glycerol, beta hydroxy acid, and one or more azone, urea, pyrrolidones, essential oils, terpenes and terpenoids, oxazolidinones, propylene glycol, epidermal enzymes, oleic acid, dimethyl isosorbide, sulphoxides, dimethylsulfoxide, dimethylsulfone, ethanol, diethylene glycol monoethyl ether, hyalauronic acid, chitin, mucopolysaccharides, fatty acids, linoleic acid, alpha linoleic acid, cod liver oil, menthol, menthol derivatives, squal
  • the diameters of the lipid nanoparticles can be within a range of from about 10 nm to about 60 nm.
  • the compositions comprise an anhydrous carrier medium, a an effective amount of a bioactive agent, a penetration enhancer and optionally an exfoliant. Such formulations are free of any occlusive agent that prevents percutaneous absorption.
  • the invention provides methods for using the formulations according to the invention to treat a dermatologic condition, the methods comprising applying therapeutically effective amounts of the formulations according to the invention to the skin.
  • the invention provides a method for treating a dermatologic condition, the method comprising exfoliating the skin and applying to the skin a topical pharmaceutical composition as described herein.
  • the composition comprises an exfoliant.
  • the invention provides a method for introducing a bioactive agent into the blood circulation of a subject. The method according to this aspect of the invention comprises applying therapeutically effective amounts of the topical pharmaceutical composition according to the invention to the subject’s skin.
  • the invention provides a method for introducing a bioactive agent into the blood circulation of a subject. The method according to this aspect of the invention comprises exfoliating the skin of a subject and applying to the skin a topical pharmaceutical composition according to the invention.
  • compositions according to the invention can comprise an anhydrous carrier medium, an effective amount of bioactive agent and a skin permeation enhancer material.
  • the invention provides a method for introducing a bioactive agent into a limited local region of a subject’s body.
  • the method according to this aspect of the invention comprises applying therapeutically effective amounts of the topical pharmaceutical composition according to the invention to the subject’s skin.
  • the bioactive agent acts locally in the subject’s deep tissues including, without limitation, tumors or the musculoskeletal system, such as muscles, joints, tendons, ligaments, or bone.
  • the invention provides a method for introducing a bioactive agent into a limited local region of a subject’s body.
  • the method according to this aspect of the invention comprises exfoliating the skin of a subject and applying to the skin a therapeutically effective amount of the topical pharmaceutical composition according to the invention.
  • compositions according to the invention can comprise an anhydrous carrier medium, an effective amount of bioactive substance and a skin permeation enhancer material.
  • FIG.1 Anatomy of the human skin and possible routes for cutaneous drug delivery. Diagrammatic representation of the 3 compartments of the skin as they relate to drug delivery: surface, stratum (Str.), and viable tissues. After application of a drug to the surface, evaporation and structural/compositional alterations occur that determine the drug’s bioavailability. The stratum corneum limits diffusion of compounds into the viable skin and body.
  • FIG. 4 Schematic illustration of the skin in mouse and humans. Left: murine skin structure. Mouse skin has a high density of fibroblasts (blue and purple). The panniculus carnosus is under the hypodermis. Right: human skin structure. Human skin structure differs from that of mouse.
  • the epidermis is thicker and forms ingrowths called rete ridges (RR). Hair follicle density in human skin is lower than in mouse.
  • APM arrector pili muscle
  • BM basement membrane
  • DP dermal papillae
  • DS dermal sheath
  • DWAT dermal white adipose tissue
  • EP epidermis
  • ESG eccrine sweat gland
  • HD hypodermis
  • HF hair follicle
  • PC panniculus carnosus
  • PD papillary dermis
  • RD reticular dermis
  • SG sebaceous gland
  • transdermal drug delivery Compared to the commonly used method of drug delivery by injection, transdermal drug delivery generates lower amounts of hazardous medical waste and presents a lower risk of disease transmission by needle re-use.
  • Patient compliance with transdermal drug delivery methods is generally good, and drug administration can be sustained over a long period of time and/or varied in a desired way over time.
  • the present invention provides compositions and methods for topical application for targeted delivery of bioactive agents with improved drug bioavailabilty. More effective drug delivery to target tissues results in a greater therapeutic effect. Drugs with a molecular mass larger than 500 Da are hindered primarily by low skin permeability.
  • the present invention provides anhydrous carriers in combination with certain permeation enhancers selected from amino acid derivatives, cell penetrating peptides, or lipid nanoparticles, or combinations thereof, to increase drug flux through the skin or other tissues into the systemic circulation.
  • bioactive form with respect to a bioactive agent or drug refers to a form in which it is capable of performing its physiological or therapeutic role.
  • antiimicrobial peptide refers to a peptide, generally of 10 to 100 amino acid residues, that differentiates between bacterial cells and mammalian cells in its ability to alter cell membrane permeability and cause cell lysis and death.
  • Cathelicidine refers to a class of amphiphilic antimicrobial peptides that kills bacterial pathogens by disintegrating, damaging, or puncturing cell membranes. Cathelicidines are natural peptides that have been isolated from a variety of mammalian species.
  • magainin refers to a member of a class of cationic antimicrobial peptides that have been isolated from the African clawed frog, Xenopus laevis.
  • percutaneous drug delivery refers to penetration of the stratum corneum by a bioactive agent. As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used.
  • nucleic acid and “polynucleotide,” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties.
  • polypeptide polypeptide
  • peptide and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • sequence relates to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded; and also can include an amino acid sequence of any length.
  • identity relates to an exact nucleotide-to-nucleotide or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity.
  • Calculations of homology or sequence identity between two sequences are performed as follows.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • a “therapeutically effective amount” or “effective amount” refers to a minimal amount of therapeutic agent which is necessary to impart therapeutic benefit to a subject.
  • a “therapeutically effective amount” to a mammal is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder.
  • treating and “treatment” as used herein refer to administering to a subject a therapeutically effective amount of a composition so that the subject has an improvement in the disease or condition.
  • Bioactive agent or “therapeutic agent” refers to a chemical compound or other composition, such as a protein, antigen, antibody, nucleic acid, or small molecule capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.
  • therapeutic agents for cancer include agents that prevent or inhibit development or metastasis of cancer, either acting alone, or in combination with other agents.
  • antibody means an immunoglobulin molecule (or antigen binding sequence thereof) that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments, dual affinity retargeting antibodies (DART)), single chain Fv (scFv) mutants, single domain antibodies (nanobodies), multispecific antibodies such as bispecific and trispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments, dual affinity retargeting antibodies (DART)
  • scFv single chain Fv mutants
  • single domain antibodies single domain antibodies
  • multispecific antibodies such as bispecific and trispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion
  • an antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • An IgM antibody consists of 5 basic heterotetramer units along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain.
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable region (V H ) followed by three constant domains (C H ) for each of the ⁇ and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable region (VL) followed by a constant domain (CL) at its other end.
  • the VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (C H1 ).
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable regions.
  • the pairing of a V H and V L together forms a single antigen-binding site.
  • L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains (C L ).
  • immunoglobulins can be assigned to different classes or isotypes.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ) respectively.
  • the ⁇ and ⁇ classes are further divided into subclasses on the basis of relatively minor differences in C H sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the terms "antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject.
  • the term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
  • a "monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants.
  • monoclonal antibody encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site.
  • monoclonal antibody refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.
  • humanized antibody refers to forms of non-human (e.g.
  • murine antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences.
  • humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536).
  • CDR complementary determining region
  • the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability.
  • the humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability.
  • the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539 or 5,639,641.
  • the terms “subject” and “patient” are used interchangeably herein, and refer to an animal such as a mammal. In general, the terms refer to a human.
  • the terms also includes domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals, goats, apes (e.g. gorilla or chimpanzee), and rodents such as rats and mice.
  • compositions for topical application for delivery of bioactive agents and methods of treating diseases or conditions by administering the same are selected from the group consisting of compositions applied to local skin (and other tissues), transdermal (systemic/bloodstream), ocular (local eye tissues), intra-articular (local joints), musculoskeletal (local muscle, tendons, ligaments, joints), intra-tympanic (local into the ear), transungual (through nail plate into nail bed), and combinations thereof.
  • compositions are formulated as creams, ointments, pastes, lotions, liquid solutions, or liquid suspensions.
  • the invention relates to anhydrous formulations for percutaneous absorption that comprise one or more permeation enhancers as described herein.
  • the invention provides a composition for facilitating topical or transdermal delivery of bioactive agents, such as biologics, proteins, macromolecules, nucleic acids, and small molecules.
  • the invention provides a pharmaceutical composition for topical application for delivery of a bioactive agent to a subject, wherein the composition comprises i) an anhydrous carrier; ii) an effective amount of a bioactive agent; and iii) a penetration enhancer selected from the group consisting of an amino acid derivative compound, a cell penetrating peptide (CPP), an antimicrobial peptide, a lipid nanoparticle composition, and combinations thereof.
  • the composition is for topical, transdermal, ocular, intra- articular, musculoskeletal, intra-tympanic, or transungual delivery.
  • the amount of the anhydrous carrier that is present in the composition is from about 70% to about 99% by weight of the composition. In some embodiments, the amount of the anhydrous carrier that is present in the composition is from about 90% to about 95% by weight of the composition. In some embodiments, the anhydrous carrier is selected from glycerin, oleic acid, capric/caprylic triglyceride diethylene glycol monoethyl ether, and a combination thereof. In some embodiments, the amount of the bioactive agent that is present in the composition is from about 0.001% to about 5% by weight of the composition. In some embodiments, the amount of the bioactive agent that is present in the composition is from about 0.05% to about 2% by weight of the composition.
  • the bioactive agent is a monoclonal antibody or a fragment thereof. In some embodiments, the bioactive agent is an antibody. In some embodiments, the bioactive agent is an antibody-drug conjugate (e.g., antibody conjugated to a cytotoxic molecule). In some embodiments, the amount of the penetration enhancer that is present in the composition is from about 0.3% to about 10% by weight of the composition. In some embodiments, the amount of the penetration enhancer that is present in the composition is from about 1% to about 3% by weight of the composition. In some embodiments, the penetration enhancer comprises a cell penetrating peptide (CPP). In some embodiments, the invention provides formulations that allow efficient delivery of effective amounts of bioactive substances for percutaneous absorption.
  • CPP cell penetrating peptide
  • the formulations according to the invention are generally non-irritating to the skin, which may cause slight tingling of a passive nature due to the heightened activity.
  • the compositions according to the invention provide many advantages over aqueous formulations. For example, acidic or basic biological actives in an aqueous environment will at high concentrations affect the pH of the formulation, thus rendering it irritating to the skin. In an anhydrous environment, ionization does not occur, so high concentrations of such actives may be achieved in a formulation that does not irritate the skin. This is advantageous because one of the governing factors for percutaneous absorption is the concentration of the biological active, with higher concentrations leading to increased percutaneous absorption.
  • the bioactive agents are highly stable in the anhydrous compositions.
  • amino acid derivatives and/or cell-penetrating peptides have a hydrophobic “tail” attached to an amino acid “head” via an ester linkage. Ester bonds can be hydrolyzed (i.e. broken) by water.
  • the anhydrous formulation with no water present is favorable in terms of stability (preserving compound integrity over time during storage), compared to aqueous formulations.
  • the combination of the anhydrous carrier and permeation enhancer achieves a synergistic effect on delivery of the bioactive agent compared with either the carrier or permeation enhancer alone.
  • the compositions provide a further advantage of providing the biological active agent in a particle size in proportion to the molecular size of the biological active.
  • anhydrous compositions according to the invention is increased stability of the biological active. Hydrophilic biological actives are frequently labile when exposed to water. They are prone to oxidation, hydrolysation and decomposition. In the formulations according to the invention, they are not. Also, in some embodiments of the invention, this stability is further enhanced by coating the molecular-sized particles with protective oils. This allows non-derivatized (i.e., not covalently modified) biological actives to be used and prevents coalescence of particles.
  • anhydrous compositions according to the invention are efficiently partition hydrophilic biological actives for percutaneous absorption, because the hydrophilic molecules prefer the hydrophilic environment of the dermis to the hydrophobic environment of the anhydrous formulation.
  • an additional advantage provided by the compositions according to the invention is that they can exfoliate the skin as they are applied. By removing dead skin cells of the stratum corneum without damaging underlying keratinocytes and fibroblasts, percutaneous absorption is further enhanced.
  • the invention provides formulations that are capable of efficient percutaneous absorption of high concentrations of hydrophobic, hydrophilic or amphoteric bioactive substances.
  • a great variety of bioactive substances may be included in the formulations according to the invention.
  • the formulations according to the invention comprise an anhydrous carrier medium, an effective amount of micronized or nanosized bioactive substance, an exfoliant, and a penetration enhancer herein.
  • Such formulations are preferably free of any occlusive agent that prevents percutaneous absorption, such as silicones.
  • an “anhydrous carrier medium” is a substance that is free of water.
  • Preferred anhydrous carriers include, without limitation, esters, amides, ethoxylated fats, mineral oil, petrolatum, vegetable oils, animal fats, triglycerides, polyols (e.g., glycerol), glycerin, propylene glycol, isopropyl myristate and sorbitol. Glycerin in concentrations of 5-40%, 25-35% and about 35% are among the preferred embodiments.
  • the exfolient is non-irritating.
  • the bioactive substance is non-derivitized.
  • the compositions may be manufactured by standard “dry” micronization or nanosizing (particle size reduction) processes.
  • the bioactive substance preferably in powder form is subjected to a “wet” micronization or nanosizing process, as made available by Microniser Pty. Ltd. of Dandenong, Australia/Micronisers of Australia of Melbourne, Australia.
  • Wet micronization or nanosizing prevents overheating of the active, the coating prevents coalescence of particles and protects against oxidation, reduction and hydrolysis.
  • This process preferably involves the grinding of the powder, suspended in or otherwise in the presence of a non-aqueous liquid, preferably an oil (hereinafter, the “suspending medium”).
  • a non-aqueous liquid preferably an oil
  • the process is preferably conducted in an abrasion-resistant container in the presence of a grinding medium, using sufficiently high rpm for a sufficiently long duration, and a suitable stirrer.
  • the resulting suspension may be separated from the grinding medium by suction filtration of the powder.
  • This micronization or nanosizing process is capable of producing particles of bioactive substance having a mean particle size corresponding to the molecule size of the bioactive substance.
  • the grinding may be conducted in the presence of 0.1 to 30%, and preferably 0.5 to 15% by weight, of a grinding aid such as an alkylated vinylpyrrolidone polymer, a vinylpyrrolidone-vinylacetate copolymer, an acylglutamate, an acrylate-tert.- octylpropenamide copolymer, a ditolylether sulphonic acid-formaldehyde condensate, a carbomer, a commercial mixture of fatty acid esters comprising a nonionic precursor such as tristyrylphenol ethoxylate or, in particular, a phospholipid, as described in U.S. Pat. No. 5,869,030.
  • a grinding aid such as an alkylated vinylpyrrolidone polymer, a vinylpyrrolidone-vinylacetate copolymer, an acylglutamate, an acrylate-tert.- octylpropenamide copolymer
  • the suspending medium is most preferably a vegetable oil, which promotes (along with the physical micronization or nanosizing process, as described above) breaking the bioactive substance into ultrafine particles and, at the same time, coating the particles with the oil, which promotes maximum absorbance and stability of the bioactive substance in the formulation.
  • the micronized bioactive substance particles used preferably exhibit a mean particle size of no more than approximately 5 ⁇ m, and preferably a mean particle size of in the range of from about 0.01 to about 2 ⁇ m, and most preferably from about 0.05 to about 1.5 ⁇ m, and especially from about 0.1 to about 1.0. ⁇ m.
  • Oils most preferable and therefore most suitable for use include, without limitation, caprylic triglycerides, capric triglycerides, isostearic triglycerides, adipic triglycerides, propylene glycol myristyl acetate, lanolin oil, polybutene, isopropyl palmitate, isopropyl myristate, diethyl sebacate, diisopropyl adipate, hexadecyl stearate, cetyl oleate, oleyl alcohol, hexadecyl alcohol, wheatgerm oil, vegetable oils such as castor oil, corn oil, cottonseed oil, olive oil, palm oil, coconut oil, palm kernel oil, canola oil, sunflower oil, safflower oil, meadow foam oil, jojoba oil, hydrogenated vegetable oils, and mineral oil.
  • caprylic triglycerides such as castor oil, corn oil, cottonseed oil, olive oil,
  • the bioactive substance is micronized in the presence of capric/caprylic glycerides in which the bioactive substance is present at high concentration.
  • the anhydrous carrier comprises one or more esters, amides, ethoxylated fats, mineral oil, petrolatum, vegetable oils, animal fats, triglycerides, polyols (e.g., glycerol), glycerin, propylene glycol, sorbitol, isopropyl myristate and combinations thereof.
  • the anhydrous carrier comprises glycerin in a concentration of about 20-40%.
  • the anhydrous carrier comprises one or more of capric/caprylic triglyceride, glycerol, beta hydroxy acid, and one or more azone, urea, pyrrolidones, essential oils, terpenes and terpenoids, oxazolidinones, propylene glycol, epidermal enzymes, oleic acid, dimethyl isosorbide, sulphoxides, dimethylsulfoxide, dimethylsulfone, ethanol, diethylene glycol monoethyl ether, hyalauronic acid, chitin, mucopolysaccharides, fatty acids, linoleic acid, alpha linoleic acid, cod liver oil, menthol, menthol derivatives, squalene, glycerol derivatives, and glycerol monoethers,
  • the present invention provides a composition for facilitating transdermal delivery of biologics, proteins, macromolecules and
  • the bioactive agent in the composition and which can be administered to a subject is not limiting.
  • the bioactive agent(s) is one or more biologics, proteins (e.g., insulin), vaccines, antibodies (such as a monoclonal or humanized antibodies), macromolecules, nucleic acids (such as mRNAs, siRNAs, antisense RNAs, shRNAs, or oligonucleotides), or small molecules (e.g., chemotherapeutics).
  • the bioactive agent is an antibody.
  • the bioactive agent is a therapeutic oligonucleotide, including antisense oligos (ONs), siRNA, and microRNA mimics and inhibitors.
  • the bioactive agent is one or more biologics selected from ADAMTS13, Albumin (Human), Alpha ⁇ 1 ⁇ Proteinase Inhibitor (Human), Standardized Cat Hair allergen, standardized Cat Pelt allergen, Anthrax Immune Globulin (Human), Anthrax Vaccine Adsorbed, Adjuvanted, Anti ⁇ Human Globulin, Anti ⁇ thymocyte Globulin, Antibody to Hepatitis B Surface Antigen, Antihemophilic Factor (Human), Antihemophilic Factor (Recombinant), Antihemophilic Factor (Recombinant), Fc Fusion protein, Antihemophilic Factor (Recombinant), Full Length, Antihemophilic Factor (Recombinant), GlycoPEGylated ⁇ exei, Antihemophilic Factor (Recombinant), PEGylated, Antihemophilic Factor (Recombinant), Plasma/Albumin Free, Antihemophilic Factor (Recombinant), Plasma/Albumin Free,
  • the bioactive agent is selected from one or more of Abciximab (Reopro), Rituximab (MabThera, Rituxan), Basiliximab (Simulect), Daclizumab (Zenapax),,Etanercept (TNFR2 ECD, 1998), Alefacept (LFA3 ECD, 2003), Abatacept (CTLA4 ECD, 2005) , Rilonacept (IL-1RI/IL-1RacP ECD, 2008), Romiplostim (41aa thrombopoietin (TPO) analogue peptide, 2008), Belatacept (CTLA4 ECD, 2011), Insulin, Pramlintide acetate, Growth hormone GH, Pegvisoman, Mecasermin, Factor VIII, Factor IX, Protein C concentrate, ⁇ 1-proteinase inhibitor, Erythropoietin, Filgrastim, Sargramostim , Oprelvekin, Human
  • the bioactive agent is one or more nucleic acids, such as an oligonucleotide or modified oligonucleotide that is long acting or resistant to nucleases.
  • the nucleic acid bioactive agent is selected from one or more of fomivirsen, mipomersen, inotersen, eteplirsen, golodirsen, nusinersen, patisiran, givosiran or pegaptanib. See, e.g., Roberts, T.C., Langer, R. & Wood, M.J.A. Advances in oligonucleotide drug delivery.
  • the bioactive agent is a GLP-1 agonist.
  • the GLP-1 agonist is selected from one or more of tirzepatide. dulaglutide, exenatide, semaglutide, liraglutide, or lixisenatide.
  • the bioactive agent is selected from one or more agents that can be useful for treating cancer in a subject, comprising Cetuximab, Leuprolide, Pegfilgrastim, Sargramostim, Thyrotropin Alfa, Trastuzumab, Capromab, Filgrastim, Bevacizumab, Pertuzumab, Denosumab, Buserelin, Aflibercept, Abarelix Satumomab Pendetide, Ancestim, Filgrastim-sndz, Pembrolizumab, Necitumumab, Sipuleucel-T, Capromab pendetide, Galiximab, Ranpirnase, Oregovomab, LErafAON, M40403, Tigapotide, IGN311, Adecatumumab, Labetuzumab, Matuzumab, Bavituximab, Keyhole limpet hemocyanin, Girentuximab, Cintrede
  • the bioactive agent for treating cancer is selected from the group consisting of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Apre
  • the bioactive agent is selected from one or more immune checkpoint inhibitors, e.g., useful for treating cancer in a subject.
  • the agent is selected from one or more of ipilimumab, pembrolizumab, nivolumab, cemiplimab, atezolizumab, durvalumab, or tiragolumab.
  • the bioactive agent is an antibody.
  • the bioactive agent is an antibody-drug conjugate.
  • the bioactive agent is any of the antibodies as provided herein conjugated with any of the bioactive agent drugs (e.g., chemotherapeutic drugs) as provided herein.
  • the bioactive agent comprises one or more agents that can be useful for treating infectious disease in a subject, including Gramicidin D, Interferon Alfa-2a, Recombinant, Daptomycin, Filgrastim, Teicoplanin, Filgrastim-sndz, Tuberculin Purified Protein Derivative, Obiltoxaximab, Hepatitis A Vaccine, Human Varicella-Zoster Immune Globulin, Lenograstim, Erythropoietin, Interferon alfa-2a, Bavituximab, Ciliary neurotrophic factor, QS-21, CPG 10101, PRO-542, Motavizumab, Ibalizumab, Bezlotoxumab, Besilesomab, Elapegademase, Atoltivimab, Maftivimab, Odesivimab, Imdevimab, Casirivimab, P113D, LC16M8, MVA3000, V
  • the bioactive agent comprises one or more agents that can be useful for treating an immunological disorder in a subject, including immunoglobulin, Alefacept, Daratumumab, Immune Globulin Human, Vedolizumab, Siltuximab, Dinutuximab, Sipuleucel-T, Human immunoglobulin G, Somatotropin, Caplacizumab, Bovine type I collagen, Rabies immune globulin, human, Inebilizumab, Ibalizumab, Pepsin, Elapegademase, Belantamab mafodotin, Cilgavimab, Tixagevimab, and rhMBL
  • the bioactive agent comprises one or more agents that can be useful for treating a cardiac disorder in a subject, including Abciximab, Erythropoietin, Pexelizumab, Coagulation factor VII human, FX06, ACY001
  • the bioactive agent comprises one or more agents that can
  • the bioactive agent comprises one or more agents that can be useful in treating a respiratory disorder in a subject, including Palivizumab, Lucinactant, Beractant, Beractant, Poractant alfa, and Albumin human. In some embodiments, the bioactive agent comprises one or more agents that can be useful in treating a hematological disorder in a subject, including Emapalumab and Tagraxofusp.
  • the bioactive agent comprises one or more agents that can be useful in treating an eye disorder in a subject, including Gramicidin D, Botulinum Toxin Type A, Ciliary neurotrophic factor, Teprotumumab, and Teprotumumab
  • the bioactive agent comprises one or more agents that can be useful in treating a bone disorder in a subject, including Burosumab.
  • the bioactive agent comprises one or more agents that can be useful in treating a metabolic disorder in a subject, including Insulin detemir.
  • the bioactive agent comprises one or more agents that can be useful in treating a malabsorption disorder in a subject, including Teduglutide.
  • the bioactive agent comprises a system for achieving genomic modification in cells, such as a CRISPR/Cas9 system.
  • one or more targeted "nucleases” e.g. CRIPSR/Cas9, TALEN or ZFN, can create a double-stranded break in the target sequence (e.g., cellular chromatin) at a predetermined site.
  • a "target site” or “target sequence” is a nucleic acid sequence that defines a general region of a nucleic acid to which a binding molecule may bind, provided sufficient conditions for binding exist.
  • the CRISPR/Cas system comprises (a) a gRNA molecule comprising a targeting domain which is complementary with a target domain sequence of a gene of interest; (b) a Cas9 molecule or variant thereof, or nucleic acid encoding the same; and (c) optionally a template nucleic acid sequence that harbors the desired genomic modification.
  • the bioactive agent is a vitamin or a micronutrient.
  • Preferred vitamins or micronutrients include, without limitation, vitamin C, vitamin E, vitamin B12, vitamin B6, folic acid, beta carotene and niacin or combinations thereof.
  • a combination of vitamin C and vitamin E is one preferred embodiment.
  • One embodiment may further include niacin.
  • Niacin in combination with an anti-inflammatory agent is another preferred embodiment.
  • Preferred anti-inflammatory agents include, without limitation, corticosteroid medicines such as cortisone, hydrocortisone, prednisone and methylprednisolone and non- steroidal anti-inflammatories (NSAIDs) wherein the NSAID is selected from the group consisting of aspirin (acetylsalicylic acid), diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen and naproxen sodium, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin, celecoxib and acetaminophen, provided, however, that when the NSAID
  • the bioactive agent is an antimicrobial or antiviral antibiotic.
  • Preferred antimicrobial antibiotics include antibacterial antibiotics antiparasitic antibiotics and antifungal antibiotics.
  • Preferred fungicides include, without limitation, butocouazole nitrate, haloprogen, clotrimazole and other azoles.
  • Preferred antivirals include, without limitation, O-[(2-hydroxyetoxy)- methyl]guanine and other herpes treatment medications, and tee tree oil (oil of Melaleuca spp.).
  • the bioactive agent is an antioxidant selected from, without limitation, urocanic acid and other imidazoles; D,L-carnosine, D-carnosine, L-carnosine, anscrine and other peptides; alpha-carotine, beta-carotine, lycopine and other carotines; carotenoids; dihydrolipoic acid and other lipoic acids; aurothioglucose, propylthiouracil, thioredoxin, glutathion, cysteine, cystine, cystamine and other thiols; dilauryl thioproponate; distearyl thiopropionate; thiopropionate; thiopropionic acid; butathione- sulfoxamines, homocysteine-sulphoxamine, butathione-sulphones, penta-, hexa- and heptathioninesulphomimine and other sulfoxamine compounds; al
  • the bioactive agent is a sugar, an amino acid or a small peptide, including, without limitation carnosine, acetyl-L-carnithine, N-acetyl-carnithine, N-acetyl-cysteine, N-acetyl-D-glucosamine, B/D/L-alanine, alpha-keto-glutarates, arginates, L-arganine base/HCl, L-arginine-pyroglutamate, ascorbates, L-aspargine monohydrate, aspartame, aspartates, L-aspartic acid, L-carnithine base, L-carnithine fumarate, L-carnithine/HCl, L carnithine bitartrate, L-carnithine-L-tartrate, chelates, L- citruline, creatin monohydrate, creatin phosphate, creatine pyruvate, L-cysteine base, L- cysteine
  • the antioxidant may be an antioxidant derivative.
  • Preferred derivatives include, without limitation, esters, ethers, peptides, lipids, nucleotides and nucleosides of such antioxidants. Preferred derivatives also include glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl lauryl, palmitoyl, oleyl, gamma-linoloyl, cholesteryl and glyceryl esters of such antioxidants.
  • the bioactive agent is a biological additive.
  • biological additive indicates any compound obtained from a natural source, including plants, animals, bacteria and yeast, which has a medicinal or otherwise beneficial effect when topically applied to the skin.
  • biological additives include, without limitation, oil of Melaleuca spp. (tea tree oil), oil of Lavandula angustifolia, Carica papaya extract, Echinacea angustifolia extract, Mimosa tenuiflora extract, Hydrocotyl (centella) asiatica extract, gingko biloba extract, Matricaria chamomila (chamomile oil) extract, Hypericum perforatum extract, Aloe barbedensis extract, and the like.
  • biological additive may also include, but are not limited to the following: Aloe Vera, (e.g., Aloe Barbedensis); Arnica, (e.g., Arnica Montana); Bladderwrack (seaweed), (e.g., Fucus Vesciculosis); Birch, (e.g., Betula Alba) (Pendula); Chamomile, (e.g., Matricaria Chamomila, Chamomila Recutita); Marsh Mallow, (e.g., Althea Officinalis); Meadow Sweet, (e.g., Spirea Ulmaria) (Filipendula); Mint/Lemon Balm, (e.g., Melissa Officinalis); Mimosa, (e.g., Mimosa Tenuiflora); Myrrh Tincture, (e.g., Commiphor Myrrha); Neem, (e.g., Melia Azadirachta); Nettle (stinging), (
  • Johns Wort (e.g., Hypericum Perforatum); Strawberry, (e.g., Fragaria Vesca); Thea Sinensis (green tea), (e.g., Camelia Sinensis); Walnut, (e.g., Juglans Regia); Witchhazel (dist/extr), (e.g., Hamamelis Virginiana); Yarrow, (e.g., Achillea Millefolium); Wild Yam, (e.g., Dioscorea Villosa); Hawthorn, (e.g., Crataegus Monogina/Oxyantha); Herma (black/rod), (e.g., Lawsoma Ehemus); Hops, (e.g., Humulus Lupulus); Horse Chestnut, (e.g., Aesculus Hippocastanum); Horse Tail, (e.g., Equisitum Arvense); Ivy, (e.g., Hedera Helix); Linden/Lime Tree
  • a particularly preferred biological additive is tea tree oil.
  • one or more biological additive is present in the formulation in a combined amount of from about 1% to 10% by weight, more preferably from about 2% to 8% by weight, and most preferably from about 4% to 6% by weight.
  • the bioactive agent is a local anesthetic.
  • Preferred local anesthetics include, without limitation, lidocaine and procaine. See also Martindale, The Extra Pharmacopia, Twenty-eighth Edition, The Pharmaceutical Press, London (1982), pp. 899-923.
  • the total dose of the bioactive agents that can be administered to a subject can be in amounts, for example, from 0.01 to 25 mg/kg body weight, or more usually from 0.1 to 15 mg/kg body weight.
  • Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
  • treatment regimens according to the present invention comprise administration to a human or other mammal in need of such treatment from about 1 mg to about 1000 mg of the bioactive substance(s) or in a single dose of from 1 mg, 5 mg, 10 mg, 100 mg, 500 mg or 1000 mg.
  • the compositions can be administered once, twice, or multiple times.
  • the compositions are administered, daily, weekly, monthly, every few months, or as needed or prescribed.
  • the composition comprising the bioactive agent useful for treating a disease or condition is administered alone.
  • the composition is administered in combination with one or more additional therapies, either together in the same composition, in separate compositions with the same or different routes of administration, either at the same time or at different times.
  • bioactive agents useful for treating cancer such as one or more checkpoint inhibitors are administered in combination with one or more chemotherapeutic agents.
  • the bioactive agent is poorly soluble or generally insoluble.
  • the bioactive agent has a particle size at the nanoscale level to increase the rate of dissolution of poorly soluble drugs and increase percutaneous absorption of insoluble drugs by enlarging the effective surface area.
  • the composition comprises an exfoliant.
  • an “exfoliant” is a substance that eliminates dead cells from the stratum corneum without killing underlying living skin cells.
  • the exfoliants are chemical exfoliants, such as alpha hydroxy acids (AHAs) and beta hydroxy acids (BHAs).
  • the exfoliating agents are enzymes .
  • Preferred enzymes include, without limitation, serine proteases, macramidase, penicillinase, pepsin, plasmin, bromelain, papain, streptokinase, sutilains, trypsin, urokinase, keratinase, amylase, hyaluronidase, cholic acid, chymopapain, chymotrypsin, cynara, brinolase and chenodeoxycholic acid. See also Martindale, The Extra Pharmacopia, Twenty-eighth Edition, The Pharmaceutical Press, London (1982), pp. 644-661.
  • the composition comprises an amino acid derivative compound as a permeation enhancer.
  • the amino acid derivatives comprise a hydrophobic “tail” attached to an amino acid “head” via a biodegradable linkage, e.g., an ester bond.
  • a biodegradable linkage e.g., an ester bond.
  • an amphiphilic enhancer is incorporated into the stratum corneum lipid barrier and disrupts the tight arrangement of the membrane lipids.
  • its labile bond is hydrolyzed.
  • Acyclic amino acid-based permeation enhancers include derivatives of glycine (e.g., dodecyl-N,N-dimethylamino acetate (DDAA)), alanine (e.g., dodecyl-2-(dimethylamino)propionate (DDAIP)), sarcosine (e.g., lauroylsarcosine), proline (e.g., dodecyl-N-acetylprolinate (e.g., L-Pro2)), and 6- aminohexanoates (e.g., transkarbam 12 (T12) and undecyl 6-(dimethylamino)hexanoate (DDAK)).
  • glycine e.g., dodecyl-N,N-dimethylamino acetate (DDAA)
  • DDAIP dodecyl-2-(dimethylamino)propionate
  • sarcosine
  • amino acid derivative skin permeation enhancers can take the following form: (AA) ---- (L) ---- (HP), wherein AA is an amino acid or a group of two amino acid molecules linked together by a peptide bond or an alkylene chain linking group having up to six carbon atoms, wherein L is a linker group, and wherein HP is a hydrophobic moiety.
  • the makeup of the HP portion is not particularly limiting and can be any moiety having substantial hydrophobic character.
  • HP can be a branched or unbranched alkyl or alkenyl hydrocarbon chain.
  • the alkyl or alkenyl hydrocarbon chain can have 10 to 20 carbon atoms.
  • HP can be an unbranched alkyl chain having 10-12 carbon atoms or an unbranched alkenyl chain having 18-20 carbon atoms.
  • the function of the linker group L is to tie the hydrophilic “head group,” here represented by an amino acid moiety, to the hydrophobic “tail” to form an amphiphilic skin permeation enhancer.
  • the constitution of linker group L is not particularly limiting.
  • linker group L can be an ester or amide group.
  • an ester linking group L is especially useful because it can readily be hydrolyzed after delivery of a drug into products that are less toxic than the enhancer molecule, where removal of the enhancer assists with return of the skin to its natural permeability.
  • the linker group L can be a single bond, a carbonyl group, -C(O)-O-CH2, -C(O)-O-CH2CH2-, -C(O)-NH-CH2- and –C(O)-NH-CH2CH2-.
  • the AA moiety can comprise any natural amino acid or any of a variety of artificial amino acids.
  • the amino acids can be natural L-amino acids or D-amino acids or a combination thereof.
  • the AA moiety can be an amino acid molecule or a group of two amino acid molecules linked together by a peptide bond, wherein the amino acid molecules are selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, 6- (dimethylamino)hexanoic acid and derivatives thereof.
  • the amino acid molecules are selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalan
  • Amino acids can be derivatized, for example, by alkyl groups including methyl, ethyl, n-propyl, or isopropyl, by alkoxy groups including methoxy or ethoxy, or by other substituent groups including hydroxy, amino, or alkylamino such as methylamino.
  • the amino acid derivative is selected from a compound identified in Table 1, or a combination thereof. Table 1. Polar Head Amino E nhancer Flux Rate Donor A cid Structure ER / ⁇ g ⁇ cm ⁇ 2 ⁇ h ⁇ 1 Drug Conditions Cytotoxicity
  • CPPs Cell-penetrating peptides
  • AMPs antimicrobial peptides
  • CPPs are generally amphiphilic peptides of up to 30 amino acids derived from natural or unnatural protein sequences, mostly composed of positively charged amino acids, like arginine and lysine.
  • CPPs are commonly linked to the drug (cargo) through covalent or electrostatic bonds and can penetrate the SC corneocyte cells by destabilizing the intercellular matrix and increasing permeability and therefore enabling the transport of their cargoes across the skin.
  • the topical pharmaceutical composition comprises a cell penetrating peptide.
  • the peptides are positively charged.
  • at least 50% of the component amino acid residues of each peptide are either arginine or lysine.
  • At least 50% of the component amino acid moieties of each peptide can be arginine, lysine, proline, or histidine. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90%, or 100% of the amino acid residues of each cell penetrating peptides is either arginine, lysine, or proline. In some embodiments, the cell penetrating peptide can be a homopolymer of arginine, lysine or ornithine. See, e.g., R.E.
  • the peptide consists of six to twenty amino acid residues.
  • the peptides comprise up to 30 amino acid moieties derived from natural or unnatural sequences.
  • at least 50% of the amino acid residues of each peptide are positively charged at physiological pH.
  • the positively charged amino acid residues are selected from arginine and lysine.
  • the cell penetrating peptide is covalently attached to the bioactive agent, e.g., a fusion protein.
  • the cell penetrating peptide is non-covalently associated with the bioactive agent.
  • Cell penetrating peptides include protein transduction domains (PTDs) that are short modular motifs, which, when attached to heterologous proteins, can transfer proteins across cell membranes. These short motifs, generally rich in positively charged amino acids, permit transfer of proteins across plasma membrane, without requiring any receptors for their internalization.
  • Viral and cellular proteins- such as the HIV-TAT, herpes simplex viral VP22, the homeodomain protein antennapedia, lactoferrin and fibroblast growth factor contain such domains, which can be modularly attached to other proteins.
  • PTDs are also called cell delivery domain or cell transduction domains.
  • the cell penetrating peptide sequence is not limiting, provided it encodes a peptide sequence that enhances uptake of a functional polypeptide by cells.
  • the cell penetrating peptide comprises RRRRRRRRRPSASYPYDVPDYA (SEQ ID NO:1).
  • the cell penetrating peptide comprises one or more variants of TAT protein from HIV selected from GRKKRRQRRR (SEQ ID NO: 2), YGRKKRRQRRR (SEQ ID NO: 1), or GRKKRRQ (SEQ ID NO: 4). Alternate forms of TAT can also be used.
  • Non-limiting examples of cell penetrating peptides which can be used in the present invention are shown in Table 2. Table 2. Cell penetrating peptides SEQ ID NO: RQLRIAGRRLRGRSR 16 INLKALAALAKKIL 39
  • a linker may be used to connect one or more cell penetrating peptides and the bioactive agent.
  • the cell penetrating peptides is fused or linked in frame to the N-terminal and/or C-terminal end of any one of the bioactive agents.
  • the bioactive agent sequence is located downstream from the cell penetrating peptide sequence, i.e., the cell penetrating peptides sequence is N- terminal to the bioactive agent sequence.
  • the permeation enhancer is an antimicrobial peptide. B.H. Gan, et al., The multifaceted nature of antimicrobial peptides: current synthetic chemistry approaches and future directions, Chemical Society Reviews 2021(13).
  • Zhang, et al. report that 3791 antimicrobial peptides are known and that they come from six kingdoms including bacteria, archaea, protozoal, fungal, plants and animals.
  • Zhang, et al. Antimicrobial peptides: mechanism of action, activity and clinical potential, Military Med. Res.2021, 8: 48, p.1, col.1.
  • most antimicrobial peptides have a net charge of +2 to +9 and include 10 to 100 amino acid moieties (p. 3, col. 1, last paragraph).
  • Positively charged antimicrobial peptides differentially target bacterial cell membranes because they include several negatively charged materials, while mammalian cell membranes include materials having a neutral charge instead (p. 4, col.
  • Cancer cells can exhibit negatively charged cell membranes as compared with normal cells, giving antimicrobial peptides a role in cancer research.
  • One group of positively charged antimicrobial peptides known as cathelicidins, are amphiphilic by virtue of including at least 50% hydrophobic amino acids and are ⁇ - helical.
  • the cathelicidins are the magainins.
  • the antimicrobial peptide can be magainin 2, an ⁇ -helical peptide isolated from the African clawed frog Xenopus laevis.
  • Magainin 2 (GIGKFLHSAKKFGKAFVGEIMNS) (SEQ ID NO:48) has been noted to have significantly differing effects on bacterial versus mammalian cell membranes, forming pores of differing sizes in the membranes.
  • the peptide can be magainin or a derivative of magainin.
  • the derivative of magainin can be magainin substituted by a dipeptide.
  • the dipeptide derivative of magainin can be magainin substituted by Gly-Ala.
  • the dipeptide derivative of magainin can be magainin substituted by Lys-Leu.
  • the permeation enhancer is a peptide that comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% or more identical to any of SEQ ID NOS:1-49.
  • the composition comprises a lipid nanoparticle composition.
  • the lipid nanoparticle (LNP) composition comprises one or more cationic lipids.
  • the diameters of the lipid nanoparticles can be within a range of from about 10 nm to about 60 nm.
  • the nanoparticles can facilitate sustained drug release for a long period of time.
  • the nanoparticles can protect encapsulated materials from chemical degradation.
  • the nanoparticles can be prepared from natural or synthetic polymers.
  • the lipid nanoparticles can comprise chitosan.
  • the lipid nanoparticles can comprise synthetic polymers including polyalkylcyanoacylates, poly-lactic acid, poly-caprolactone, poly-glycolic acid, or their copolymers, e.g., poly- lactic-co-glycolic acid. See, e.g., Desai, et al., Interaction of nanoparticles and cell- penetrating peptides with skin for transdermal drug delivery, Mol. Membr. Biol.
  • the LNPs comprise a cationic lipid.
  • an LNP composition further comprises a helper lipid that contributes to their stability and delivery efficiency.
  • Helper lipids with cone-shape geometry favoring the formation hexagonal II phase such as dioleoylphosphatidylethanolamine (DOPE), can promote endosomal release.
  • DOPE dioleoylphosphatidylethanolamine
  • cylindrical-shaped lipid phosphatidylcholine can provide greater bilayer stability, which is important for in vivo application of LNPs.
  • Cholesterol is often included as a helper that improves intracellular delivery as well as LNP stability in vivo.
  • PEGylating lipid can enhance LNP colloidal stability in vitro and circulation time in vivo but may reduce uptake and inhibit endosomal release at the cellular level. This problem can be addressed by choosing reversible PEGylation in which the PEG moiety is gradually released in blood circulation.
  • pH-sensitive anionic helper lipids such as fatty acids and cholesteryl hemisuccinate (CHEMS)
  • CHEMS cholesteryl hemisuccinate
  • the LNPs can be employed in combination with amino- based peptides for transdermal delivery of protein drugs, biologics and vaccines.
  • the LNP comprises a mixture of multiple components and excipients including ionizable lipid, helper lipid, PEG-lipid, and cholesterol.
  • Excipients that can optionally be used in certain embodiments of the present invention, but which are not required, can include lipid nanoparticles, helper lipids, poly(ethylene glycol) lipids and cholesterol.
  • Helper lipids can include, for example, 1,2-dioleoyl-sn-glycero-3- polyethylene (DOPE), or the O-pegylated derivative of the N,N-dimyristylamide of 2- hydroxyacetic acid (ALC-0159), available from BroadPharm, San Diego, CA.
  • DOPE 1,2-dioleoyl-sn-glycero-3- polyethylene
  • ALC-0159 2- hydroxyacetic acid
  • the composition comprises an additional penetration enhancer, which can include, without limitation azone, urea, pyrrolidones, essential oils, terpenes and terpenoids, oxazolidinones, propylene glycol, epidermal enzymes, oleic acid, dimethyl isosorbide, sulphoxides, dimethylsulfoxide, dimethylsulfone, ethanol, diethylene glycol monoethyl ether, hyaluronic acid, chitin, mucopolysaccharides, fatty acids, linoleic acid, alpha linoleic acid, cod liver oil, menthol, menthol derivatives, squalene, glycerol derivatives, glycerol monoethers; chamomile flavones apigenin, lutrolin, and 7-O-beta- glucoside.
  • an additional penetration enhancer can include, without limitation azone, urea, pyrroli
  • such penetration enhancers act through one or more of the following mechanisms: increasing the fluidity of the stratum corneum lipids and reducing the diffusional resistance to permeants; removing intercellular lipids and dialation between adherent cornified cells; increasing the thermodynamic activity of drugs in vehicles; exfoliating stratum corneum cell membranes; dissociating adherent cornified cells and elimination of the barrier function.
  • the pharmaceutical composition further comprises an effective amount of one or more vasodilators or vasoconstrictors.
  • Vasodilators and vasoconstrictors can enable targeted drug delivery.
  • Vasodilators act to expand or dilate the capillaries and other blood vessels in and beneath the dermal layer, resulting in increased blood flow at the site of system application. Capillary vasodilation increases the rate of diffusion of the therapeutic drug across the capillary wall and into the systemic circulation.
  • Vasodilators that can be added include, without limitation, acetylcholine, amrinone, bamethan sulphate, bencyclane fumarate, benfurodil hemisuccinate, benzyl nicotinate, buflomedil hydrochloride, buphenine hydrochloride, butalamine hydrochloride, cetiedil citrate, ciclonicate, cinepazide maleate, cyclandelate, di-isopropylammonium dichloroacetate, ethyl nicotinate, hepronicate, hexyl nicotinate, ifenprodil tartrate, inositol nicotinate, isoxsuprine hydrochloride, kallidinogenase, methyl nicotinate, methyl salicylate, naftidrofuryl oxalate, nicametate citrate, niceritrol, nicoboxil, nicofuranose
  • vasodilators are centrally acting agents, including, without limitation, clonidine, quanaberz and methyl dopa. Some vasodilators are alpha-adrenoceptor blocking agents, including, without limitation, indoramin, phenoxybenzamine, phentolamine and prazosin. Some vasodilators are adrenergic neuron blocking agents, including, without limitation, bedmidine, debrisoquine and guanethidine.
  • vasodilators are ACE inhibitors, including, without limitation, benazepril, captopril, cilazapril, enalapril, fosinopril, lisinopril, perindopril, quinapril and ramipril.
  • Some vasodilators are ganglion-blocking agents, including, without limitation, pentolinium and trimetaphan.
  • Some vasodilators are calcium channel blockers, including, without limitation, amlodipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nimodipine and verapamil.
  • vasodilators are prostaglandins, including, without limitation, prostacyclin, thromboxane A2, L-arginine, leukotrienes, PGA, PGA1, PGA2, PGE1, PGE2, PGD, PGG and PGH.
  • Some vasodilators are Angiotension II analogs including, without limitation, saralasin.
  • Other vasodilators including, without limitation, nitroglycerin, labetalol, thrazide, isosorbide dinitrate, pentaerythritol tetranitrate, digitalis, and diazoxide can serve exclusively as vasodilation agents, or may also serve another function to the delivery complex such as the penetration agent or the active drug agent.
  • vasodilators or chemically modified vasodilators may be used in the delivery complex at any one time for one formulation for the purpose of transdermally delivering an active drug molecule or agent.
  • the delivery complex may contain one or more different vasodilators in the same complex to achieve varying and different degrees and modes of vasodilation.
  • Vasoconstrictors optimize local drug delivery and restrict systemic drug absorption as a result of intense subcutaneous vasoconstriction, as well as direct hydrostatic compression of capillaries and veins.
  • Vasoconstrictors include, without limitation, catecholamines, norepinephrine, epinephrine, isoproterenol, dopamine, ephedrine, phenylisopropylamines, phenylephrine, amphetamine, metraminol, methoxamine, lysergic acid, lisergic acid diethylamine.
  • vasoconstrictor refers to a composition of matter or mixture that narrows the lumen of blood vessels and, hence, reduces peripheral blood flow.
  • vasoconstrictors include, without limitation, amidephrine, cafaminol, cyclopentamine, deoxyepinephrine, epinephrine, felypressin, indanazoline, metizoline, midodrine, naphazoline, nordefrin, octodrine, ornipressin, oxymethazoline, phenylephrine, phenylethanolamine, phenylpropanolamine, propylhexedrine, pseudoephedrine, tetrahydrozoline, tramazoline, tuaminoheptane, tymazoline, vasopressin, xylometazoline and mixtures thereof.
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as provided herein.
  • the composition is administered to the skin and is a topical or transdermal composition.
  • effective amounts of the bioactive agent are absorbed into the subject’s blood circulation and treat the disease or condition systemically.
  • the bioactive agent acts locally in the subject’s skin.
  • the bioactive agent acts locally in the subject’s deep tissues including, without limitation, the musculoskeletal system.
  • the method further comprises exfoliating the skin of the subject prior to the administering step.
  • the administration results in a bioavailability (e.g., amount present in the systemic circulation following administration) of the bioactive agent of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% compared with the amount of the bioactive agent administered in the composition.
  • the composition is administered using microneedles.
  • the microneedles are arranged in an array format, and comprise a patch that contacts the skin.
  • the microneedles have a length of about 50- 1000 ⁇ m.
  • the composition is coated onto the microneedle array and administered to the skin, for passive diffusion directly into the epidermis.
  • the composition penetrates the stratum corneum, and enables further penetration into deeper skin tissues and eventually the systemic circulation.
  • the distal part of the microneedles ( ⁇ 25-50 ⁇ m) penetrate the stratum corneum.
  • the disease or condition is not limiting. In some embodiments, the disease or condition is selected from a cancer, an infectious disease, an immunological disorder a cardiac or circulatory disorder, a hormonal disorder, a neurological disorder, adjunct therapy, a genetic disorder, a respiratory disorder, a hematological disorder, an eye disorder, pain, a bone disorder, a metabolic disorder, or a malabsorption disorder.
  • the disease or condition to be treated is a dermatological condition.
  • Dermatological conditions include, without limitation, acne, bruises, burns, eczema, mycoses, pruritis, psoriasis, seborrhea, scabs, shingles, tineapedis, wounds, wrinkles and erythema of acne rosacea.
  • Preferred agents for treating dermatological conditions include, without limitation, anti-acne preparations; anti-inflammatory agents; monobenzone and other depigmenting agents; amcinonide, diflorasone diacetate, hydrocortisone and other dermatitis relief agents; methylbenzethonium chloride and other diaper rash relief agents; mineral oil, PEG-4 dilaurate, lanolin oil, petrolatum, mineral wax and other emolients and moisturizers; alclometasone dipropionate, betamethasone valerate, isopropyl myristate MSD and other pruritic medications; anthralin, methoxsalen, coal tar and otherpsoriasis, seborrhea and scabicide agents; 2-(acetyloxy)-9-fluoro-1′,2′3′4′- tetrahydro-11-hydroxypregna-1,4-dieno[16,17b]napthalene-3,20-dione,
  • the invention provides a method for treating a dermatologic condition, the method comprising exfoliating the skin and applying to the skin a composition comprising an anhydrous carrier medium, a bioactive agent and a permeation enhancer as described herein.
  • the method may optionally include in the composition an exfoliant, preferably a non-irritating exfoliant.
  • Exfoliation of the skin may be chemical, mechanical or enzymatic. Mechanical exfoliation may be carried out by any mechanical frictional force, including, without limitation, brushing, washing or particle pressure.
  • the formulation contains a wet-micronized bioactive agent.
  • the method comprises preventing the formulation according to the invention from escaping to the exterior of the skin.
  • cancer refers to a pathophysiological condition whereby cells are characterized by dysregulated and/or proliferative cellular growth and the ability to induce said growth, which includes but is not limited to, carcinomas and sarcomas, such as, for example, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical cancer, AIDS-related cancers, AIDS-related lymphoma, anal cancer, astrocytoma (including, for example, cerebellar and cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor (including, for example, ependymoma, meduUoblastoma, supratentorial primitive neuroectodermal,
  • carcinomas and sarcomas such as, for example, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical cancer, AIDS-related cancers, AIDS-related lymphoma, anal cancer
  • the pharmaceutical composition can be useful for treating or preventing a disease or condition in a subject and comprises about 20-40% (w/w) glycerin, about 5-15% (w/w), caprylic capric triglycerides (CCT), about 2-5% (w/w) salicylic acid (SA), about 2-15% (w/w) ceatearyl alcohol and polysorbate 60, about 1-6% (w/w), beeswax, about 0.1%-1% (w/w) cell penetrating peptide (cpp), about 10-30% (w/w) oleic acid (oa), about 1-5% (w/w) azone, about 5-30% (w/w) diethylene glycol monoethyl ether (degee) and about 0.1-6% (w/w) semaglitude.
  • glycerin about 5-15% (w/w), caprylic capric triglycerides (CCT), about 2-5% (w/w) salicylic acid (SA), about 2-15%
  • the composition is a topical cream that is applied to the skin of the subject.
  • the pharmaceutical composition can be useful for treating metabolic disorders, diabetes or obesity in a subject.
  • the pharmaceutical composition can be useful for treating or preventing a disease or condition in a subject and comprises about 5-20% (w/w) isopropyl mysistate (ipm), about 20-40% (w/w) glycerin, about 5-15% (w/w) caprylic capric triglycerides (CCT), about 2-6% (w/w) salicylic acid (SA), about 5-10% (w/w) ceatearyl alcohol and polysorbate 60, about 1-10% (w/w) DMSO, about 0.1-6% (w/w) cell penetrating peptide (cpp), about 5-30% (w/w) oleic acid (oa), about 1-6% (w/w) terpenes, about 5-30% (w/w) diethylene glycol monoethyl
  • ipm isopropyl
  • the composition is a topical cream that is applied to the skin of the subject.
  • the pharmaceutical composition can be useful for generating an immune response (including a protective immune response) in a subject.
  • the pharmaceutical composition can be useful for treating or preventing a disease or condition in a subject and comprises about 1-10% (w/w) chemotherapeutic(s), about 0.25%-2% (w/w) cell penetrating peptide (cpp), about 5-40% (w/w) glycerin, about 2-30% (w/w) l-ascorbic acid, about 5-25% (w/w) caprylic capric triglycerides (CCT), about 5-30% (w/w) isopropyl mysistate (ipm), about 5-30% (w/w) diethylene glycol monoethyl ether (degee), about 2-15% (w/w) ceatearyl alcohol and polysorbate 60, about 1-6% (w/w) beesw
  • chemotherapeutic(s)
  • the composition is a topical cream that is applied to the skin of the subject.
  • the pharmaceutical composition can be useful for treating cancer, such as breast cancer, in a subject.
  • the pharmaceutical composition can be useful for treating or preventing a disease or condition in a subject and comprises about 5-30% (w/w) glycerin, about 5-20% (w/w) isopropyl mysistate (ipm), about 2-5% (w/w) salicyclic acid, 0.1%-1% (w/w) cell penetrating peptide (cpp), about 2-20% (w/w) oleic acid (oa), about 2-30% (w/w) diethylene glycol monoethyl ether (degee), about 1-10% (w/w) DMSO, and about 0.1-3% (w/w) monoclonal antibody.
  • the composition is applied to the skin of the subject and the antibody is absorbed into the systemic circulation.
  • the pharmaceutical composition can be useful for treating cancer in the subject.
  • the antibody is a checkpoint inhibitor antibody (e.g., as described herein).
  • the antibody is conjugated to a chemotherapeutic agent (e.g., one or more of the chemotherapeutic agents described herein).
  • the pharmaceutical composition can be useful for treating or preventing a disease or condition in a subject and comprises about 5-30% (w/w) glycerin, about 5-20% (w/w) isopropyl mysistate (ipm), about 2-5% (w/w) salicyclic acid, 0.1%-1% (w/w) cell penetrating peptide (cpp), about 2-20% (w/w) oleic acid (oa), about 2-30% (w/w) diethylene glycol monoethyl ether (degee), and about 0.1-2% (w/w) oligonucleotide (such as siRNA or antisense oligonucleotide).
  • oligonucleotide such as siRNA or antisense oligonucleotide
  • the composition is a liquid that is useful for targeting ocular tissues and application to the eye to treat a disease or condition.
  • All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Any discrepancy between an incorporated reference and the present description shall be resolved in favor of the present description.
  • EXAMPLES Example 1. Transdermal delivery of Antibody FAB fragment in Rats. Currently, it is impossible to deliver therapeutic agents like small-to-medium size proteins (referred to as biologics) through the skin. However, more and more therapeutic agents are biologics aiming at reaching a single endogenous, often pathological, target. An example of such biologics are antibodies that are used more and more in daily medical practice.
  • antibodies will be incorporated in a transdermal vehicle cream for delivery into the bloodstream. There are challenges associated with this delivery method that relate to the anatomy of the skin.
  • a therapeutic agent needs to cross multiple anatomical barriers before reaching blood vessels to allow systemic delivery to organs and tissues.
  • the first barrier is the stratum corneum, which is hydrophobic, thus requires specific drug delivery methods to go through and reach the epidermis.
  • At the bottom of the epidermis is a thick basement membrane that is also a strong barrier limiting access to blood vessels.
  • This overview of skin anatomy shows that, to successfully deliver antibodies to the bloodstream via the transdermal route, it is necessary to manipulate the biophysical properties of the skin, which is permitted by the special cream made in accordance with embodiments herein.
  • the first step in the process will involve some exfoliation to thin up the stratum corneum.
  • the antibody may be necessary to puncture the stratum corneum wit mechanical devices such as microneedle arrays, which are already used in patients for the delivery of non-protein therapeutics.
  • the cream will include a vasodilator that will transiently open up the underlying blood vessels to let physiological liquids extravasate and enter the deep skin tissues. The local accumulation of liquid mimics a local inflammatory event. Biophysically, it will increase the space between the cells in the epidermis. Plus, the antibody will be able to diffuse more easily in a microenvironment with more biofluids, allowing this antibody to reach and enter dilated blood vessels.
  • Rats will be housed individually to prevent licking of the cream across animals, thus prevent cross-activation of immune responses between rats as much as possible. Plus, a male rat could display aggressive behaviors when housed with other male rats, resulting in injuries. Our single housing procedure will proactively manage this possible issue.
  • Wistar rats to not have too many animals growing beyond 400- 450 gr. The bigger rats are, the more difficult they are to handle.
  • Wistar rats in the weight range of 150-200 grams, i.e. they will be of young age (4-8 weeks). For the present protocol, we will carry out experiments only on rats that are >250 grams.
  • the rats will continue to grow and likely reach 250+ grams at the age of 6-10 weeks, per growth chart provided by Charles River Labs.
  • the rats will be anesthetized to prevent the stress associated with restraint and cream application operations.
  • One week prior to each cream application all rats will be anesthetized for 10-15 min, and 1-1.5 mL of blood will be collected from the tail vein into EDTA tubes. These blood samples are to be used for reference/control during antibody detection experiments.
  • the anesthesia will last 40- 45 minutes. For practical issues, we will handle 1-2 rats per day, thus spread the cream application procedure over 3-4 days.
  • the duration of anesthesia is chosen to allow most of the cream to penetrate the stratum corneum and epidermis, which should take approximately 30-60 min. This will prevent the rats from removing too much of the cream when licking their hips upon waking up.
  • the hips area to apply the cream to prevent rats from scratching behaviors in case of irritation.
  • the cream used in the example has soothing properties and will not develop irritations by itself, but rather prevent irritations. Then the rats will be replaced in their cage.
  • each batch of 5 rats will receive a maximum of 5 cream applications at a frequency of once every 4 weeks.
  • We will alternate rat batches over a 2 week period i.e. batch #1 will start at week 2, then week 6, 10, etc.; batch #2 will start at week 4, then week 8, 12, etc.).
  • the second application will be on the left hip, and so on. As explained above, the delivery of antibodies through the skin will likely require exfoliation and vasodilation procedures. The chemicals used to carry out these steps will be integrated into the vehicle cream itself. Repeated cream applications to the same animals for up to 5 applications with a gap of 4 weeks between each application can be performed. As indicated in Table 2, each cream application will be done using a different batch of cream and we will attempt to detect the antibody in the blood before testing a different batch. If the antibody is detected in a batch of animals before the 5th cream application, then the experiment/protocol will stop at the second positive presence of antibody in the plasma. This could happen, after 1, 2, or more formulations of cream have been tested.
  • the antibody is not detectable in the blood after 10 different formulations of cream have been tests. Furthermore, if the cream alone is not sufficient for the successful delivery of the antibody after the 6th attempt, we will use a microneedle array to facilitate the puncture of the stratum corneum. After the 5th application to an individual rat, the animal will be euthanized and their carcasses disposed of per federal, state, and local regulations. For the non-immunoreactive and non-toxic antibody, we can share the exact product with other IACUC members, but, if possible, we would like the experimenter (Mr. Kirsh, member of the IACUC) to be blind to the antibody product used for our tests to limit experimental bias.
  • Mr. Kirsh member of the IACUC
  • the commercial antibody will be made in mice (close species) and bear a tag that allows for its easy detection in biological samples. Such antibody is frequently used for immunostaining and ELISA procedures, thus is high quality research grade.
  • the antibody will be mixed with the cream before delivery to the rats.
  • the cream is pharmaceutical grade and, as indicated above, has soothing properties. Based on current medical practice with antibodies to treat human diseases, it is possible that rats develop a very mild immune reaction with the production of endogenous antibodies against our test antibody after several applications. However, this reaction is normal and will not endanger the life of the rats. Also unlikely is the possible development of adverse reactions to the antibody, including pain, redness at the site of cream delivery, swelling, and rash.
  • microneedle arrays to facilitate the penetration of the antibody at the surface of the skin.
  • Each device will include a matrix of 15 x 15 rows or microneedles.
  • Each microneedle will be 75 ⁇ m in length, since the thickness of the stratum corneum in rats is in the range of 4-10 ⁇ m (Wei et al., 2017). Microneedle arrays have been used in many experiments with rats.
  • microneedle arrays are adapted to the animal species used in the present protocol and to the biochemical mechanisms activated by the inventive cream. A summary of the study procedure is presented below. Cream applications will take place for sure at weeks 3 and 5.
  • Rat Batch #1 (N 5) - WK02: Collect 1-1.5 mL of tail vein blood for control + Isolate plasma by centrifugation - WK03: Shave the right hips + Cream application #1 + Collect 1-1.5 mL of blood after 22-26h + Isolate plasma by centrifugation - WK06: Collect 1-1.5 mL of tail vein blood for control - WK07: Shave the left hips + Cream application #3 (if required) + Collect 1-1.5 mL of blood after 22-26h + Isolate plasma by centrifugation - WK10: Collect 1-1.5 mL of tail vein blood for control - WK11: Shave the right hips + Cream application #5 (if required) + Collect 1-1.5 mL of blood after 22-26h + Isolate plasma by centrifugation - WK14: Collect 1-1.5 mL of tail vein blood for control - WK15: Shave the left hips + Cream application #7 (with microarray if required) + Collect 1-
  • Rat Batch #2 (N 5) - WK04: Collect 1-1.5 mL of tail vein blood for control - WK05: Shave the right hips + Cream application #2 + Collect 1-1.5 mL of blood after 22-26h + Isolate plasma by centrifugation - WK08: Collect 1-1.5 mL of tail vein blood for control - WK09: Shave the left hips + Cream application #4 (if required) + Collect 1-1.5 mL of blood after 22-26h + Isolate plasma by centrifugation - WK12: Collect 1-1.5 mL of tail vein blood for control - WK13: Shave the right hips + Cream application #6 (if required) + Collect 1-1.5 mL of blood after 22-26h + Isolate plasma by centrifugation - WK16: Collect 1-1.5 mL of tail vein blood for control - WK17: Shave the left hips + Cream application #8 (with microarray if required) + Collect 1-1.5 mL of blood after 22-26
  • transdermal cream is pharmaceutical grade. Given the antibody quantity will be limited, overall the cream batches applied on the skin of the rats will be very close to pharmaceutical grade. Either the antibody is detected in the blood of the rats, or we reach up to 5 delivery attempts without success, whichever is achieved first. If a rat shows any signs of distress it will also be euthanized. This includes the possible development of pain and/or distress at the site of cream delivery, such as pain, redness, swelling, and rash, or due to adverse reactions to the antibody such as trouble breathing, an allergic reaction, uncontrollable fever, uncontrollable changes in blood pressure, and large swelling of the face and paws.
  • Additional endpoints include >20% weight loss, hunched posture, lethargy with impaired righting reflex or labored breathing, ulcerated skin >2.0 cm.
  • Literature cited Abd E Yousef SA, Pastore MN, Telaprolu K, Mohammed YH, Namjoshi S, Grice JE, Roberts MS (2016) Skin models for the testing of transdermal drugs. Clin Pharmacol 8:163-176.
  • Schmook FP Meingassner JG, Billich A (2001) Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absorption. Int J Pharm 215:51-56.
  • CCT Caprylic Capric Triglycerides
  • SA Salicylic Acid
  • CPP Cell Penetrating Peptide
  • OA Oleic Acid
  • DEGEE Diethylene Glycol Monoethyl Ether
  • Target systemic Topical formulation applied to skin (amounts by weight) Isopropyl Mysistate (IPM) 5-20% Glycerin 20-40% Caprylic Capric Triglycerides (CCT) 5-15% Salicylic Acid (SA) 2-6% Ceatearyl Alcohol and polysorbate 60 5-10% DMSO 1-10% Cell Penetrating Peptide (CPP) 0.1-6% Oleic Acid (OA) 5-30% Terpenes 1-6% Diethylene Glycol Monoethyl Ether (DEGEE) 5-30% mRNA 0.1-5% Example 5.
  • IPM Isopropyl Mysistate
  • CCT Caprylic Capric Triglycerides
  • SA Salicylic Acid
  • CPP Cell Penetrating Peptide
  • OA Oleic Acid
  • DEGEE Diethylene Glycol Monoethyl Ether
  • Target breast tumor Topical cream applied to skin CHEMOTHERAPEUTICS 1-10% CELL PENETRATING PEPTIDE 0.25-2% GLYCERIN 5-40% L-ASCORBIC ACID 2-30% CAPRIC/CAPRYLIC TRIGYLCERIDE 5-25% ISOPROPYL MYRISTATE 5-30% DIETHYLENE GLYCOL MONOETHYL E THER 5-30% CEATEARYL ALCOHOL (and) P OLYSORBATE 60 2-15% BEESWAX (white) BENTONE GEL GTCC V SALICYCLIC ACID 1-3% BHT 0.25-2% PARABENS 0.50% TRIHYDROXYSTEARIN (THIXCIN R) 0.50% Example 6.
  • Target systemic/bloodstream Topical formulation applied to skin (amounts by weight) Glycerin 5-30% Isopropyl Mysistate (IPM) 5-20% Salicylic Acid (SA) 2-5% Cell Penetrating Peptide (CPP) 0.1%-1% Oleic Acid (OA) 2-20% Diethylene Glycol Monoethyl Ether (DEGEE) 2-30% DMSO 1-10% mAbs 0.1-3% Example 7.
  • Oligonucleotide Pharmaceutical Composition applied to skin (amounts by weight) Glycerin 5-30% Isopropyl Mysistate (IPM) 5-20% Salicylic Acid (SA) 2-5% Cell Penetrating Peptide (CPP) 0.1%-1% Oleic Acid (OA) 2-20% Diethylene Glycol Monoethyl Ether (DEGEE) 2-30% DMSO 1-10% mAbs 0.1-3%
  • Example 7 Oligonucleotide Pharmaceutical Composition.
  • Target eye tissues Topically applied liquid to the eye (amounts by weight) Glycerin 5-30% Isopropyl Mysistate (IPM) 5-20% Salicylic Acid (SA) 2-5% Cell Penetrating Peptide (CPP) 0.1%-1% Oleic Acid (OA) 2-20% Diethylene Glycol Monoethyl Ether (DEGEE) 2-30% siRNA 0.1-2% While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

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Abstract

L'invention concerne des compositions thérapeutiques et pharmaceutiques pour une application topique et une administration ciblée d'agents bioactifs ayant une biodisponibilité de médicament améliorée à un sujet, la composition comprenant i) un support anhydre ; ii) une quantité efficace d'un agent bioactif ; et iii) un activateur de pénétration choisi dans le groupe constitué par un composé dérivé d'acide aminé, un peptide de pénétration cellulaire (CPP), un peptide antimicrobien, une composition de nanoparticules lipidiques et des combinaisons de ceux-ci.
PCT/US2024/034128 2023-06-14 2024-06-14 Compositions pharmaceutiques pour administration ciblée d'agents bioactifs à biodisponibilité améliorée et méthodes d'utilisation Pending WO2024259323A2 (fr)

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US20080233053A1 (en) * 2005-02-07 2008-09-25 Pharmalight Inc. Method and Device for Ophthalmic Administration of Active Pharmaceutical Ingredients
CN105491982B (zh) * 2013-08-12 2019-09-10 3M创新有限公司 用于增强透皮递送的肽
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