WO2001035998A1 - Compositions pour administration transdermique et transmuqueuse d'agents therapeutiques - Google Patents

Compositions pour administration transdermique et transmuqueuse d'agents therapeutiques Download PDF

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Publication number
WO2001035998A1
WO2001035998A1 PCT/CA2000/001323 CA0001323W WO0135998A1 WO 2001035998 A1 WO2001035998 A1 WO 2001035998A1 CA 0001323 W CA0001323 W CA 0001323W WO 0135998 A1 WO0135998 A1 WO 0135998A1
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Prior art keywords
composition
amino acid
insulin
skin
ifn
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PCT/CA2000/001323
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English (en)
Inventor
Marianna Foldvari
Sam K. Attah-Poku
Martin King
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Pharmaderm Laboratories, Ltd.
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Priority to EP00974224A priority Critical patent/EP1265638A1/fr
Priority to AU12625/01A priority patent/AU1262501A/en
Publication of WO2001035998A1 publication Critical patent/WO2001035998A1/fr

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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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7023—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

Definitions

  • the present invention relates to compositions and methods for administration of therapeutic agents across the skin or mucosa
  • the invention relates to acylated amino acid compounds tor transdermal and transmucosal administration of macromolecules such as polypeptides and proteins
  • the skin provides an appealmg site for the noninvasive entry of drugs into the Delivery of drugs through the skm for a systemic effect or transdermal de verv has become a conventional and recognized approach for small, lipophihc molecules, such as nitroglyce ⁇ n and nicotine
  • hydrophilic and macromolecular drugs such as
  • transdermal and transmucosal admimstration has been less successful
  • One approach for transdermal administration of proteins and other macromolecues is lontophoretic delivery Electroporation has also been described for use in achieving transdermal delivery of compounds While these approaches have met with some success a passive method capable of delivering sufficient amounts of peptides. proteins, or
  • transdermal and transmucosal route of admimstration of proteins offers several advantages Since these compounds have short half-lives, the continuous mode of transdermal administration derives maximum therapeutic benefits
  • the skin is also very low in proteolytic activity, as compared to, for example, the oral route of admimstration so that 5 degradation at the administration site is reduced Additionally, a drug absorbed transdermally bypasses the hepatic circulation, avoiding another major site of potential degradation
  • the stratum corneum serves as the primary barrier to absorption of substances coming mto contact with the skm
  • the stratum corneum consists of dead, flattened keratm- filled cells, corneocytes, in a lip id matrix Each corneocyte is bounded by a thick, proteinaceous envelope with rough fibrous protein (keratm) as the mam component
  • keratm proteinaceous envelope with rough fibrous protein
  • the intercellular spaces are filled with broad multiple lamellae of lipids, primarily ceramides. cholesterol, fatty acids, and cholesteryl esters Human skm is 2-3 mm thick, and the stratum corneum over most of the body is 15 ⁇ m thick
  • the invention includes a composition for transdermal or transmucosal administration composed of a therapeutic agent and an acylated amino acid, wherein application of the composition to the skin is effective to achieve enhanced uptake of the s therapeutic agent when compared to transdermal uptake of the therapeutic agent alone, that is, in the absence of the acylated amino acid
  • the composition further comprises a suspension of hposomes
  • the therapeutic agent and/or the acylated ammo acid in other embodiments, is entrapped in the hposomes
  • the hposomes can have an aqueous inner core or an oil-in water 0 emulsion in hposomes' central compartment
  • the acylated amino acid in a preferred embodiment, is an acylated amino acid represented by the formula
  • R 1 is an acyl group having from 1-20 carbons
  • R 2 is hydrogen or an alkyl ⁇ group
  • R 3 corresponds to a modified or unmodified R group of a selected amino acid
  • the therapeutic agent can be virtually any compound, and in preferred embodiments in a macromolecule such as a peptide, protein, or nucleic acid Interferons. mterleukins. and insulin are exemplary agents
  • the invention includes a method for administering a therapeutic n agent to a subject
  • the method includes preparing a composition comprised of a therapeutic agent and an acylated amino acid, as describe above, and administering the composition transdermally to the subject Administration of the composition is effective to achieve a concentration of the agent in the blood sufficient tor therapy
  • the invention includes a method for selecting a composition for therapeutic transdermal admimstration of a macromolecule
  • the invention includes a transdermal delivery device comprising, (1) a suspension comprised of hposomes. an acylated amino acid, and a macromolecular therapeutic agent, (n) a reservoir adapted to retain the suspension and adapted for release of lipid vesicles therefrom, and (in) means tor affixing the device to a subject for transdermal administration of said agent
  • the invention includes an interferon- ⁇ composition
  • biphasic lipid vesicles comprised of (1) a lipid bilaver comprising a phospho pid and a fatty acylated amino acid, (n) an oil-in-water emulsion entrapped in the biphasic lipid vesicles, where the oil-in-water emulsion is stabilized by a surfactant, and (in) interferon- ⁇ entrapped in the vesicles
  • the composition when applied to the skin of a subject is effective to administer a therapeutically effective amount of interferon- ⁇
  • the acylated ammo acid in one embodiment, is selected to achieve dermal administration of mterferon- ⁇ for treatment of a local, topical condition In another embodiment, the acylated amino acid is effective to achieve transdermal administration of mterferon- ⁇
  • R 2 in the acylated amino acid is ((CO)C 19 H 39 )
  • the amino acid is serine or threomne
  • Another preferred acylated amino acid is monolauroyl lysine
  • the oil-m-water emulsion in the biphasic lipid vesicles further comprises a fatty alcohol
  • the fatty alcohol can have between about 8-24 carbon atoms.
  • the oil-m- water emulsion further comprises a triglyceride, such as pharmaceutically-acceptable oil, such as canola oil and olive oil
  • the oil-in-water emulsion is further comprised of a fatty glyce ⁇ de dispersed in the water phase and stabilized by the surfactant
  • a fatty glyce ⁇ de can be, for example, glycerol monostearate.
  • the lipid bilayer of the vesicles can further comprise of a sterol
  • the invention includes a composition for administration ot interferon- ⁇ , comprising biphasic lipid vesicles comprised of (1) a lipid bilayer comprised of a phospholipid and an amino acid acylated.
  • an oil-in-water emulsion entrapped in the biphasic lipid vesicles where the oil-in-water emulsion is comprised of a triglyceride that is dispersed in a water phase containing a fatty alcohol and that is stabilized by a surfactant, and (in) interferon- ⁇ entrapped in the vesicles
  • the composition when applied to the skin of a subject is effective to administer a therapeutically effective amount of interferon- ⁇
  • the invention includes a method of administering a therapeutically effective amount of interferon- ⁇ to a subject, comprising preparing biphasic lipid vesicles as described above The biphasic lipid vesicles are then contacted with the skin of a subject for transdermal or dermal delivery, depending on the selected acylated amino acid
  • the invention includes a method of treating human papilloma virus in a subject
  • the method includes preparing biphasic lipid vesicles as described above and contacting the biphasic lipid vesicles with the skin of a subject or more preferably, applying the lipid vesicles to the site of the infection
  • the invention includes a composition for transdermal administration of insulin
  • the composition mcludes biphasic lipid vesicles comprising (l) a lipid bilayer comprised of a phospholipid and a fatty acylated amino acid, (n) an oil-in- water emulsion entrapped in the biphasic lipid vesicles, where the oil-in-water emulsion is comprised of a triglyceride dispersed in a water phase and stabilized by a surfactant, and (in) insulin entrapped in said vesicles
  • the composition when applied to the skin of a subject is effective to administer a therapeutically effective amount of insulin
  • the lipid bilayer of the biphasic lipid vesicles is further comprised of a sterol
  • the acylated amino acid is an acylated lysine
  • One preferred acylated lysine is N ⁇ -capryloyl-N ⁇ lauroyl L-lysine ethyl ester (PDM27)
  • the triglyceride in the lipid vesicles is a natural oil, such as canola or olive oil
  • the invention includes a composition for transdermal administration of insulin, comprising biphasic lipid vesicles comprised of (l) a lipid bilayer comprised ot a phospholipid and a fatty acylated lysine compound, (n) an oil-in- water emulsion entrapped in the biphasic lipid vesicles, where the oil-in-water emulsion is comprised of a triglyceride dispersed in a water phase and stabilized by a surfactant, and (in) insulin entrapped in said vesicles
  • the surfactant is a cationic phospholipid
  • the invention includes a composition for transdermal administration of insulin, comprising biphasic lipid vesicles comprised of (1) a lipid bilayer comprised of a phospholipid and N -capryl
  • the invention includes a method of administering a therapeutically effective amount of insulin to a subject, comprising preparing biphasic lipid vesicles as described above and contacting the biphasic lipid vesicles with the skin of a subject
  • Fig 1A is a bar graph showing the antiviral activity in serum taken from guinea pigs after transdermal admimstration of IFN- ⁇ from biphasic lipid vesicles contaimng IFN- ⁇ and monolauroyl lysine (MLL), N-eicosanoyl-L-se ⁇ ne (PDM3) or N-eicosanoyl threonine (PDM4) entrapped in the lipid vesicles
  • Fig IB is a bar graph showing the antiviral activity in skm homogenates prepared from the skin of guinea pigs after transdermal admimstration of IFN- ⁇ from biphasic lipid vesicles contaimng IFN- ⁇ and monolauroyl lysine (MLL), N-eicosanoyl-L-se ⁇ ne (PDM3) or N-eicosanoyl threonine (PDM4) entrapped in the lipid vesicles
  • Figs 2A-2C are graphs showing the antiviral activity in skm biopsy samples taken from human subjects after transdermal administration of IFN- ⁇ from biphasic lipid vesicles containing monolauroyl lysine and IFN- ⁇ at dosage levels of 5 MU. 15 MU, and 40 MU entrapped in the lipid vesicles.
  • the Y-axis shows the individual's initials and the numbers above each bar are the fold increase over each individual's respective untreated skin sample. ; Figs.
  • 3A-3C are graphs showing IFN- ⁇ concentration as pg/mg protein in skin homogenate prepared from samples taken from human subjects after transdermal administration of IFN- ⁇ from biphasic lipid vesicles containing monolauroyl lysine and IFN- ⁇ at dosage levels of 5 MU. 15 MU. and 40 MU entrapped in the lipid vesicles.
  • the Y-axis shows the individual's initials and the numbers above each bar are the fold increase i c over each individual's respective untreated skin sample.
  • Figs. 4A-4C are graphs showing the antiviral activity in serum samples taken from human subjects after transdermal administration of IFN- ⁇ from biphasic lipid vesicles containing monolauroyl lysine and IFN- ⁇ at dosage levels of 5 MU, 15 MU, and 40 MU entrapped in the lipid vesicles.
  • the Y-axis shows the individual's initials and the numbers 15 above each bar are the fold increase over each individual's respective untreated skin sample.
  • 5A-5C are graphs showing the 2-5A synthetase enzyme activity in serum samples taken from human subjects after transdermal administration of IFN- ⁇ from biphasic lipid vesicles containing monolauroyl lysine and IFN- ⁇ at dosage levels of 5 MU, 15 MU, and 40 MU entrapped in the lipid vesicles.
  • the Y-axis shows the individual's initials and 0 the numbers above each bar are the fold increase over each individual's respective untreated skin sample.
  • Fig. 6 shows the serum insulin concentration in a diabetic rat following subcutaneous injection of insulin (1 mg).
  • Fig. 7 shows blood glucose levels, in mmol/L, as a function of time, in hours, in rats 5 treated with transdermally administered insulin from biphasic lipid vesicles containing insulin and the acylated amino acid PDM27 (open triangles).
  • the dosage applied was 200 mg of the biphasic lipid vesicle formulation having 50 mg insulin/mg formulation.
  • the blood glucose levels of non-diabetic, healthy, untreated rats (solid triangles) and of diabetic, untreated rats (open circles) are also shown. 0 Fig.
  • FIG. 8 shows the change in blood glucose levels, in mmol/L, as a function of time, in hours, in rats treated with transdermally administered insulin from biphasic lipid vesicles containing insulin and the acylated amino acid PDM 27 (open triangles).
  • the dosage applied was 1000 mg of the biphasic lipid vesicle formulation having 10 mg insulin/mg formulation.
  • the blood glucose levels of non-diabetic, healthy, untreated rats (solid triangles) and of diabetic rats treated with 1 mg insulin administered subcutaneously (open circles) are also shown.
  • Fig. 9 is a bar graph showing the duration of response to transdermally (dotted bars) and subcutaneously (lined bars) administered insulin.
  • the insulin administered subcutaneously at a dose of 28 U was in the form of a saline solution.
  • the insulin administered at a dose of 140 U and 280 U. both transdermally and subcutaneously. was in the form of biphasic lipid vesicles containing insulin.
  • amino acid refers to an amino acid modified at one or more amine groups with an acylating agent that reacts with an amine group.
  • amino acid refers to any carboxylic acid having at least one free amine group, including naturally-occurring and synthetic amino acids.
  • Dermat as used herein intends transport of an agent across the stratum corneum and into the viable epidermis for treatment of a topical skin disorder that responds to local, non-systemic administration of an agent. It will be appreciated that some of the agent intended for dermal therapy may be transdermally administered, however typically not in an amount sufficient for therapy.
  • Fatty-acylated amino acid refers to an acylated amino acid where the acyl group has more than about 8 carbon atoms.
  • Interferon-alpha is abbreviated herein as "IFN- ⁇ " and intends all of the known and unknown subtypes including but not limited to IFN- ⁇ ,.
  • R group of an amino acid refers to the R group in the general structure [(COOH)CHNH 2 (R)] , where R represents the moiety that individualizes each amino acid.
  • R is selected from H (glycine), CH 3 (alanine), CH 2 OH (serine) CH 2 COOH (aspartic acid), (CH 2 ) 3 NHC(NH)NH 2 (arginine), CH(CH 3 ) 2 (valine), CHOH(CH 3 ) (threonine), CH 2 CH 2 COOH (glutamic acid), CH 2 CH(CH 3 ) 2 (leucme), CH 2 SH (cysteine), CH 2 (C(NH)(CH)(N)(CH)) (histidine), (CH 2 ) 4 NH 2 (lysine), CH(CH 3 )CH 2 CH 3 (isoleucine), CH 2 (C 6 H4)OH (tyrosine), CH 2 (CONH 2 ) (asparagme), (CH 2 ) 2 SCH
  • “Therapeutically effective amount” intends an amount of a therapeutic agent sufficient to reduce the symptoms associated with a disease or condition and/or lessen the severity of the disease or condition
  • the condition can be a topical, local condition, such as genital warts, or a more pervasive condition, such as a viral infection or diabetes
  • a therapeutically effective amount is understood to be in context to the condition being treated where the actual effective amount is readily discerned by those of skill in the art
  • Transdermal as used herein intends transport of an agent across the stratum corneum for introduction into systemic circulation
  • compositions of the invention are comp ⁇ sed of a therapeutic agent and an acylated amino acid
  • the two components are placed in a selected earner vehicle for administration transdermally or transmucosally
  • the acylated amino acid, the therapeutic agent and the earner vehicles will now be descnbed 1 Acylated Amino Acids
  • Tables 1 A- I F show exemplary acylated amino acids for use as absorption promoters in the compositions of the present invention
  • the acvlated ammo acid compounds are represented by the schedulecture X-CO-A here X is an aliphatic hydrocarbon group, an aryl- substituted lower hydrocarbon or an aromatic hydrocarbon group, each of which may optionally be substituted, and A is an ammo acid residue which may optionally be substituted
  • the acylated ammo acids are represented by the general formula
  • R 1 is typically an acyl group having from 1-20 carbons
  • R 2 is hydrogen or a lower O 01/35998 alkyl
  • R 3 corresponds to the R group of the selected ammo acid.
  • R ! includes an amino group which can be acylated. such as when R 3 is lysine. arginine. or alu t amine. and compounds PDM5 and PDM17 in Table 1A are exemplary.
  • the preferred naturally occurring ⁇ -amino acids for use in the invention are alanine. arginine. asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine. histidine. isoleucme. leucine, lysine. methionine. ornithine, phenylalanine. proline. se ⁇ ne. threonine. tryptophan. tyrosine. valine. More preferred amino acids are lysine, threonine. serine, glycine, cysteine and glutamine.
  • the therapeutic agent to be used in the present mvention is not limited, and includes ordinary pharmaceuticals, water-soluble and non-water-soluble compounds, low molecular weight compounds, high molecular weight compounds and macromolecules. such as peptides. proteins and protein hormones
  • the therapeutic agent is a macromolecule. by which is meant a therapeutic agent having a molecular weight above about 500 Daltons. more preferably above 1000 Daltons.
  • Exemplary macromolecules include proteins, including but not limited to insulin, somatostatin calcitonin, angiotensm, secretin, parathhryoid hormone, granulocyte colony-stimulation factor, glucocerebrosidase, rhDNase, somatotropin. interferons, rFactor VIII. interleukins. erythropoietm, growth factors, growth hormones, LHRH analogues, and the like 3 Carrier Vehicle
  • the composition of the invention includes a carrier vehicle for application ot the acylated amino acid and therapeutic agent to the skin or mucosa
  • the carrier vehicle can take the form of a simple aqueous-based solution, a water-in-oil emulsion, an oil-in-water emulsion or a pharmaceutically acceptable solvent, such as polyethylene glycol or polypropylene glycol
  • the pH of the carrier vehicle can be adjusted to alter the lonization state of the therapeutic agent and/or the acylated amino acid
  • the tonization can have an effect on the skin penetration rate, thus, can be optimized for maximum penetration
  • the earner vehicle takes the form of a suspension of hposomes
  • the hposomes can be small unilamellar vesicles or mululamellar vesicles, and a variety of techniques, such as those detailed by Szoka, (Ann Re ⁇ Biophys Bioeng 9 467 (1980)) Mululamellar vesicles (MLVs) can be formed by simple lipid-film hydration techniques In this procedure, a mixture of hposome-forming lipids of the type described below are dissolved in a suitable organic solvent which is evaporated in a vessel to form a thin film. which is then covered by an aqueous medium The lipid film hydrates to form MLVs
  • the hposome-forming lipid components necessarily include a vesicle-forming lipid. by which is meant an amphipathic lipid having a hydrophobic tail and a head group which can form spontaneously into bilayer vesicles in water
  • the vesicle-forming lipids are preferably ones having two hydrocarbon chains, typically acyl chains, and where the head group is either polar or nonpolar
  • phospho pids which include phosphatidylcholine, phosphatidylethanolamine. phosphatidylse ⁇ ne. phosphatidyhnositol. phosphatidic acid, and sphingomyelin, where ic the two hydrocarbon chains are typically between about 14-22 carbon atoms in length. and have varying degrees of unsaturation
  • the lipid vesicles of the present invention can include other lipid components capable of being stably incorporated into
  • lipid bilayers with their hydrophobic moieties in contact with the interior, hydrophobic region of the bilayer membrane, and their polar head groups oriented toward the exterior, polar surface of the membrane
  • hydrophobic moieties in contact with the interior, hydrophobic region of the bilayer membrane, and their polar head groups oriented toward the exterior, polar surface of the membrane
  • glycohpids, ceramides and sterols such as cholesterol, coprostanol.
  • cholestanol and cholestane long chain fatty acids (C 16 to C 22 ), such as stea ⁇ c acid, can be incorporated into the lipid bilayer
  • C 16 to C 22 long chain fatty acids
  • lipid components that may be used include fatty amines, fatty acylated proteins, fatty acylated peptides. oils, fatty alcohols, glyce ⁇ de esters, petrolatum and waxes Specific examples of preferred lipids include beeswax, glyceryl stearate, cetyl alcohol, myristyl myristate, and cetyl palmitate
  • a cationic lipid can also be used in the lipid components of the hposomes
  • Cationic lipids also have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, yet the lipid has an overall net positive charge
  • the head group of the lipid carries the positive charge
  • Exemplary cationic lipids include l,2-d ⁇ oleyloxy-3- (t ⁇ methylammo) propane (DOTAP), N-[l-(2,3,-d ⁇ tetradecyloxy)propyl]-N,N-d ⁇ methyl- N-hydroxyethylammonium bromide (DMRIE), N-[l-(2,3,-d ⁇ oleyloxy)propyl]-N,N-
  • DOTAP l,2-d ⁇ oleyloxy-3- (t ⁇ methylammo) propane
  • DMRIE N-[l-(2,3,-d ⁇ tetradecyloxy)propyl]-N,N-d ⁇ methyl- N-hydroxyethylammonium bromide
  • DMRIE N-[l-
  • N,N,N-tr ⁇ methylammon ⁇ um chloride (DOTMA), 3 ⁇ [N-(N',N'-d ⁇ methylam ⁇ noethane) carbamoly] cholesterol (DC-Choi), and dimethyldioctadecylammonium bromide
  • the cationic vesicle-forming lipid may also be a neutral lipid, such as
  • DOPE dioleoylphosphatidyl ethanolamme
  • an amphipathic lipid such as a phospholipid
  • a cationic lipid such as polylysine or other polyamine lipids
  • the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid
  • the carrier vehicle takes the form of lipid vesicles having an oil-in-water emulsion incorporated into the central core compartment of the vesicles and between the lipid bilayers
  • lipid vesicles having an oil-in-water emulsion incorporated into the central core compartment of the vesicles and between the lipid bilayers
  • biphasic lipid vesicles Such biphasic lipid vesicles have been described for example, in PCT Publication Nos WO 95/03787, WO 99/11247 and in U S Patent No 5.853.755
  • Biphasic lipid vesicles are prepared from a selected lipid composition comprised of one or more lipids
  • the composition will include at least one vesicle-forming lipid. described above
  • the biphasic lipid vesicles can further include other lipid components capable of being stably incorporated into lipid bilayers For example, glycolipids.
  • ceramides and sterols such as cholesterol coprostanol, cholestanol and cholestane, long chain fatty acids (C 16 to C 22 ), such as stearic acid
  • lipid components that may be used include fatty amines, fatty acylated proteins, fatty acylated peptides. oils, fatty alcohols glyce ⁇ de esters, petrolatum and waxes
  • a skin permeation enhancer, such as an acylated amino acid, can be included as part ot the lipid components of the lipid vesicle
  • the lipid vesicles include between about 1-40% of vesicle-forming hpid. more preferably from about 5-25 % (percentages are weight percentages based on the total lipid vesicle composition, including the oil-in-water emulsion phase described below)
  • the hydrophilic solvent other than water typically constitutes between 1-15% of the hposome, and the acylated amino acid constitutes between 0 1-5 % Cholesterol or other sterol. when added, is typically in the 1-10% range
  • the biphasic lipid vesicles include an oil-in-water emulsion entrapped in the vesicles ' aqueous spaces
  • the oil-m-water emulsion is comprised of
  • the oil-in-water emulsion is one having water as the continuous phase and the lipophilic component as the dispersed phase.
  • the surfactant serves to stabilize the emulsion, and. during formation of the emulsion, it is added to either the water phase or the lipophilic. oil phase. depending on the hydrophilic-lipophilic balance (HLB) of the surfactant.
  • HLB hydrophilic-lipophilic balance
  • the surfactant is mixed with the water and this mixture is added to the oily lipohpilic phase for homogenization and formation of the emulsion.
  • the stabilizing surfactant is other than a vesicle-forming lipid, e.g. , the surfactant is one which does not spontaneously form lipid bilayers.
  • the oil-in-water emulsion is stable by virtue of the oil droplets in the dispersed phase being surrounded by the surfactant. That is, the hydrophilic portion of each surfactant molecule extends into the aqueous phase of the emulsion and the hydrophobic portion is in contact with the lipophilic droplet.
  • the vesicle-forming lipids may act to first stabilize the emulsion rather than form lipid bilayers around the oil-in-water emulsion.
  • Surfactants suitable for formation of the oil-in-water emulsion are numerous, including both cationic. anionic and nonionic or amphoteric surfactants.
  • the preferred surfactant is a cationic surfactant, such as linoleamidopropyl propylene glycol-dimonium chloride phosphate, cocamidopropyl propylene glycol- dimonium chloride phosphate and stearamido propylene glycol-dimonium chloride phosphate.
  • a cationic surfactant such as linoleamidopropyl propylene glycol-dimonium chloride phosphate, cocamidopropyl propylene glycol- dimonium chloride phosphate and stearamido propylene glycol-dimonium chloride phosphate.
  • These are synthetic phospholipid complexes commercially available from DEBRO (Mississauga. Ontario, Canada) sold under the tradenames Phospholipid EFATM, Phospholipid SNTM and Phospholipid SVCTM, respectively.
  • Exemplary anionic surfactants include acylglutamates. such as triethanolamine - cocoyl glutamate. sodium lauroyl glutamate. sodium hydrogenated tallow glutamate and sodium cocoyl glutamate.
  • nonionic surfactants include naturally derived emulsifiers. such as polyethyleneglycol-60 almond glycerides, avocado oil diethanolamine. ethoxylated jojoba oil (polyethyleneglycol-40 jojoba acid and polyethyleneglycol-40 jojoba alcohol); polyoxyethylene derivatives, such as polyoxyethylene-20 sorbitan monooleate and polyoxythethylene-20 sorbitan monostearate; lanolin derivatives, such as polychol 20 (LA ⁇ ETH 20) and polychol 40 (LA ⁇ ETH 40); and neutral phosphate esters, such as polypropyleneglycol-cetyl ether phosphate and diethanolamine oleth-3 phosphate.
  • naturally derived emulsifiers such as polyethyleneglycol-60 almond glycerides, avocado oil diethanolamine.
  • ethoxylated jojoba oil polyethyleneglycol-40 jojoba acid and polyethyleneglycol-40 jojoba alcohol
  • polyoxyethylene derivatives such as poly
  • the oil component of the emulsion can be selected from a variety of lipophilic compounds, including natural and synthetic triglycerides. fatty glycerides. solid and semi-solid waxes and mixtures thereof.
  • the primary oil component is a triglyceride selected from synthetic and natural oils, such as olive oil. canola oil, sunflower oil and other oils recited in Col. 12, lines 20-42 of U.S. Patent No. 5,854,755, which is herein incorporated by reference.
  • a preferred fatty glyceride is glycerol monostearate and a preferred wax is beeswax.
  • biphasic lipid vesicles prepared in support of the invention included as the primary oil component a triglyceride. with a fatty glyceride and a wax added as minor components.
  • the oil-in-water emulsion further includes a fatty alcohol. C n H n+2 0. where n is from 2-24. more preferably from 8-24.
  • the fatty alcohol is cetyl alcohol (C l6 H 34 O) or stearyl alcohol (C, 8 H 38 0).
  • the fatty alcohol is typically added to the oil phase prior to homogenization.
  • the oil-in-water emulsion can also include other components, such as antimicrobial agents or preservatives.
  • these agents are admixed with the aqueous phase prior to homogenization with the oil phase.
  • Preferred agents include hydroxy lated benzoate esters, such as methyl paraben, propyl paraben, l-(3-chlorallyl)-3.4,7-triaza-l- azoniaadamantane chloride (DOWICIL, (C 6 H I2 N 4 (CH 2 CHCHC1)C1), butylated hydroxytoluene (BHT). and other antimicrobial agents known to those of skill in the art.
  • the oil-in-water emulsion is generally prepared by mixing the water and the surfactant along with any additional hydrophilic components, such as a fatty alcohol and a preservative, in a first container.
  • the oil components such as a triglyceride and a fatty glyceride. are mixed in a second container.
  • the water phase is added to the oil phase for formation of the emulsion by agitation, such as by homogenization or emulsification, or by a micro-emulsion technique which does not involve agitation.
  • the resulting emulsion preferably has water as the continuous phase and oil as the dispersed phase.
  • the oil droplets in the dispersed oil phase preferably have sizes of less than about 1 ⁇ m. more preferably less than about 0.5 ⁇ m, in diameter. The droplet size, of course, is readily adjusted by mixing conditions, e.g. , shear and time of mixing, etc.
  • the Hposomes include between 1-20% of the surfactant, more preferably between 2-15% (percentages are weight percentages based on the total lipid vesicle
  • composition including the lipid phase described above).
  • the lipophilic oil constitutes typically between 1-10% of the liposome composition.
  • a fatty glyceride when added is typically between 0.1-5% of the composition and a fatty alcohol, when added, is typically between 0.1-5% of the composition.
  • a suitable solvent which in a preferred embodiment, is a pharmaceutically acceptable hydrophilic solvent, such as a polyol. e.g. , propylene glycol, ethylene glycol. glycerol. or an alcohol, such as ethanol. or mixtures of such solvents.
  • a pharmaceutically acceptable hydrophilic solvent such as a polyol. e.g. , propylene glycol, ethylene glycol. glycerol. or an alcohol, such as ethanol. or mixtures of such solvents.
  • the surfactant-stabilized oil-in-water emulsion described above is prepared.
  • a concentrated aqueous solution of interferon- ⁇ is also prepared, and biphasic lipid vesicles are formed by simultaneously mixing the oil-in-water emulsion and the concentrated drug solution with the solubilized lipids.
  • the emulsion and the lipid components are mixed under conditions effective to form multilamellar vesicles having in the central compartment the oil-in-water emulsion.
  • the amount of a therapeutic agent entrapped in the lipid vesicles is readily controlled and varied by the concentration of the solution of therapeutic agent used during lipid vesicle formation.
  • the size of the vesicles is typically between about 0.1-100 ⁇ m.
  • a lipid vesicle size of between about 0.5-5 ⁇ m is preferred, which can be most readily obtained by adjusting the mixing conditions.
  • the composition of lipid vesicles has a consistency similar to a cream without further addition of thickening or gelling agents, and. therefore, are readily applied directly to the skin of a subject for transdermal administration of the entrapped agent.
  • the lipid vesicle composition can be readily incorporated into the reservoir of a transdermal device.
  • Biphasic lipid vesicles having entrapped IFN- ⁇ and an acylated amino acid were prepared for studies in support of the invention. These in vitro and in vivo studies will now be described
  • Biphasic lipid vesicles were prepared as described in Example 20A
  • the lipid vesicles were comprised of IFN- ⁇ and an acylated amino acid selected from PDM,. PDM 3 , PDM 4 . PDM.. PDM 17 . PDM 18 , PDM 27 and MLL (see Tables 1A-1F for chemical name and structure)
  • the composition of the biphasic lipid vesicles is set forth in Table 2
  • 3 ves ⁇ cles were prepared using the following acylated amino acids PDMl , PDM3, PDM4, PDM5, PDM17, PDM 18 PDM27 and MLL
  • the vesicles were prepared by simultaneously mixing the oil m-water emulsion and an aqueous solution containing five million units of IFN- ⁇ with the solubilized lipid phase
  • a propylene glycol-based formulation was prepared by dissolving the same amounts of IFN ⁇ and the selected acylated amino acid in propylene glycol
  • Example 20C The in vitro diffusion of IFN ⁇ into human skin was determined using flow-through diffusion cells, as detailed in Example 20B About 100 mg of each test formulation was applied on the stratum corneum side of the skin and allowed to remain for 24 hours The amount of IFN- ⁇ that penetrated into the skin was assessed using a bioassay that measures interferon antiviral activity (Example 20C)
  • Table 3 shows the average amount of IFN- ⁇ in the skin at the end of the 24 hour test period, as determined by antiviral activity, for each of the test formulations
  • Fig 1A is a bar graph showing the serum antiviral activity for animals treated with the three lipid vesicle test formulations and for control animals that where untreated As seen, the formulation containing PDM 3 achieved a significant increase in transdermal skin penetration
  • Fig IB is a bar graph showing the antiviral activity in skm homogenates prepared from the skin of guinea pigs after transdermal administration of the IFN- ⁇ lipid vesicle formulations As seen, the animals treated with the lipid vesicles contaimng the acylated ammo acids PDM 3 and PDM 4 show the highest antiviral activity at about 7.000 U/mg protem b Humans
  • the lipid vesicles were prepared with three dosages of IFN- ⁇ , 5 MU, 15 MU and 40 MU, by addition of an aqueous solution at the dosage concentration to the lipid phase during vesicle formation, as described in Example 22 A.
  • the human subjects were divided randomly into three groups for treatment with a transdermal patch containing a lipid vesicle compositions at one of the three IFN- ⁇ concentrations.
  • the experimental protocol is set forth in Example 22C. Briefly, in Phase I of the study, a placebo transdermal patch containing the biphasic lipid vesicles described in Table 4 and prepared as described in Examples 20A-20B with no IFN- ⁇ was adhered to the inner upper arm. After treatment with the placebo, skin biopsies and blood samples were collected from each subject.
  • each subject was treated with a transdermal patch containing IFN- ⁇ entrapped in biphasic lipid vesicles at one of the three concentrations: 5 MU/g formulation/patch, 15 MU/g formulation/patch or 40 MU/g formulation/patch.
  • skin biopsies and blood samples were taken and analyzed.
  • the biopsy samples were analyzed by immunohistochemistry. antiviral assay, ELISA and 2-5 A synthetase assays (see Example 20D-20F).
  • Figs. 2A-2C Skin biopsies analyzed by the antiviral assay for each subject are shown in Figs. 2A-2C.
  • the antiviral assay demonstrates delivery of IFN- ⁇ by the presence of antiviral bioactivity due to IFN- ⁇ or by possible induction of other antiviral compounds.
  • Table 5 summarizes the average antiviral activity in skin homogenates for each of the three treatment groups.
  • Figs. 3A-3C show the IFN- ⁇ concentration in the skin homogenates for each individual in each of the three treatment groups.
  • the ELISA assay demonstrates delivery of IFN- ⁇ by a sandwich immunoassay using an IFN- ⁇ -specific antibody and a horseradish peroxidase labelled secondary antibody for detection.
  • the data show for each individual in Figs. 3A-3C is averaged for each treatment group in Table 6.
  • Table 8 shows the results of an anti-proliferation assay performed on skin sections of the test subjects in each treatment group after treatment with the respective dosages of IFN- ⁇ for 48 hours The number of stained cells were counted before and after treatment sections The decreased number of stained cells were expressed as a percentage
  • a method for the long term introduction of insulin into the body in a non-mvasive manner is desirable, as a typical insulin-dependent patient with diabetes patient may live with the disease for more than 40 years over which time 60.000 to 70.000 insulin injections are given Approximately 5 % of the North American population suffer from diabetes resulting in enormous medical costs, and in the U S in 1995 the cost of diabetes both direct medical cost and indirect costs, was approximately $95 billion
  • the iC major problem with transdermal delivery of insulin is the inability ot the protein to normally cross the stratum corneum Recombinant human insulin has a low solublihty at physiological pH and frequently forms high molecular mass hydrophilic hexamers This hexamer (36 kDa) would be unlikely to penetrate the hydrophobic stratum corneum in any significant concentrations without the active assistance of a delivery system ⁇ 5 Accordingly, in one embodiment, the invention provides for non-invasive delivery of insulin
  • Biphasic lipid vesicles having entrapped insulin and an acylated amino acid were prepared for in vivo studies in support of the invention, now to be described
  • compositions for transdermal administration of insulin were prepared as described in 2C
  • Compositions for transdermal administration of insulin were prepared as described in 2C
  • Biphasic lipid vesicles having the composition set forth in Table 9 were prepared
  • the vesicles were prepared by simultaneously mixing the oil-in-water emulsion and an aqueous solution with the solubilized lipid phase
  • the resulting biphasic lipid vesicles formulations had 50 mg porcine insuhn/g formulation
  • One additional formulation was prepared with human insulin at 10 mg insuhn/g formulation and with the acylated ammo acid PDM,-,
  • the biphasic lipid vesicle formulations, which differ only in the acylated ammo acid and in the type/concentration of insulin are summarized in Table 10
  • an insulin dependent diabetes melhtus (IDDM) animal model in rats was used As described in Example 24.
  • a diabetic state was induced in rats by injection of streptozotocin. which destroys pancreatic ⁇ -cells Normal rat blood glucose ranges from approximately 3-7 mM during the day, with the level higher just after feeding Streptozotocm-induced diabetic rats having a higher blood glucose typically greater than 8mM
  • a blood glucose level of between 10-16 mM is considered a mild diabetic state, and a blood glucose of greater than 20 mM being more severe
  • a control group of rats were each injected with citrate buffer Three days after the streptozoticin or citrate buffer injections, the rats were divided into groups for treatment
  • Fig 6 is a plot showing the insulin concentration in the serum following subcutaneous injection of insulin (1 mg) to a diabetic rat that is characteristic of the treatment group
  • Animals in the transdermal test groups received a transdermal device contaimng a test formulation placed on the skin at the start of the test period The device was left in place for 2 days, du ⁇ ng which time the blood was drawn periodically to analyze blood glucose levels After the treatment period, skin samples were taken and analyzed for quantity of insulm, and blood was collected and the amount of insulm in the plasma was determined
  • Fig 7 shows blood glucose levels, in mmol/L, as a function of time, in rats treated with transdermally
  • the blood glucose levels of untreated, diabetic rats fluctuated with feeding over the test period.
  • the fluctuation in blood glucose level for the untreated diabetic rats was more pronounced than for the normal, non-diabetic rats (solid triangles), confirming induction of a diabetic state in the streptozotocin-injected animals.
  • the blood glucose levels fluctuated less compared to the untreated diabetic rats, with the blood glucose level dropping to a "normal" (approximately 3-7 mm) range in the 24-30 hour time frame.
  • Fig. 8 shows the change in blood glucose levels, in mmol/L, as a function of time, in rats treated with a transdermal biphasic lipid vesicle composition containing the acylated amino acid PDM 27 ("PDM 27 /10mg" (see Table 10): open triangles).
  • the transdermal device contained 1000 mg of a 10 mg insulin g formulation.
  • the blood glucose levels of diabetic rats treated with a subcutaneous injection of insulin (open circles) and of normal, healthy rats (solid triangles) are also shown.
  • Fig. 8 shows that transdermal administration of insulin from the biphasic lipid vesicles effectively reduced the blood glucose levels over the 60 hour test period.
  • the animals treated subcutaneously with insulin experienced a reduction in blood glucose for a period of less than 10 hours.
  • Figs. 7 and 8 also show the effect of the insulin concentration in the lipid vesicle formulation on blood glucose levels.
  • a 200 mg amount of vesicles containing 50 mg insulin g formulation resulted in a shorter (6-20 hours), more intense therapeutic response, returning the blood glucose levels to normal baseline (Fig. 7), as compared to a 1000 mg amount of vesicles containing 10 mg insulin g formulation, which provided a more extended, less intense response.
  • Figs. 7 and 8 also show that the composition containing a 200 mg amount of vesicles containing 50 mg insulin g formulation had a lag time of about 16 hours (Fig. 7).
  • the duration of the response to transdermally administered insulin from biphasic lipid vesicles was compared to the duration of the response to subcutaneously administered insulin in solution and entrapped in biphasic lipid vesicles. As shown in Fig.
  • the mean response duration for subcutaneously administered insulin at a does of 28 U in solution form was about 3 8 hours Insulm at a dose of 280 U was admimstered from biphasic lipid vesicles transdermally (dotted bars) and subcutaneously (lined bars)
  • the mean response duration for subcutaneously injected biphasic lipid vesicles contaimng insulin was about 40 hours
  • the mean response duration for topically-applied biphasic lipid vesicles contaimng insulin was about 44 hours
  • topical administered of the biphasic lipid vesicles formulation contaimng insulin achieved the same prolonged response as subcutaneously admimstered biphasic lipid vesicles, without requirmg an injection As noted in Example 23.
  • the pH of the biphasic lipid vesicle formulations was i o adjusted with 1 M NaOH to between 4 5-5 5
  • Other studies (not reported here) with biphasic lipid vesicle formulations having a pH below about 3 5 resulted in little transdermal administration of insulin
  • diabetic animals were treated transdermally with the formulations set forth in Table 10 ("PDM, " , “PDM 4 " "PDM, , ')
  • the animals were prepped and the patches adhered as set forth in Example 24
  • the patches were left in place for three days after which blood and skin samples from the
  • Table 11 shows the amount of insulin in the serum of the test animals as well as the amount of insulin in the skin after treatment
  • the overall efficiency of the transdermally administered insulin was determined by taking the subcutaneously administered route as being 100% efficient, where in one study, subcutaneous administration of insulin decreased the blood glucose 17.2 mmol/L.
  • a transdermal liposome formulation with PDM 27 as the acylated amino acid decreased the blood glucose 16.2 mmol/L.
  • the percent efficiency of the transdermal formulation is then (16.2/17.2)xl00. or 94.2% .
  • Table 12 summarizes the mean blood glucose levels at the 24 hour time point and the average response duration for several of the animals in the control group, in the diabetic, untreated group, in the subcutaneously-treated group, and in the group treated transdermally with biphasic lipid vesicles containing PDM 27 and insulin at a dose of lOmg insulin/g formulation.
  • biphasic lipid vesicle formulations were determined using confocal microscopy (Example 24). Photomicrographs, not shown here, were taken from skin sections of diabetic rats treated with the transdermal biphasic lipid vesicle compositions containing insulin and an acylated amino acid. In the images of the skin treated with the liposome formulation containing PDM 27 . the fluorescent distribution throughout the epidermis and dermis was evident, indicating liposome penetration to these depths. Skin treated with the formulations containing PDM, and PDM 4 showed that the insulin is superficially in the stratum corneum upper epidermis or around the hair shaft and follicles.
  • compositions of the invention can be optimized to achieve primarily transdermal or primarily dermal delivery as desired.
  • albumin (bovine, MW 60kDA)
  • the test composition in accord with the invention was comprised of the acylated amino acid monolauroyl lysine (MLL) and albumin
  • test formulations were applied to the stratum corneum side of skm mounted in flow-through diffusion cells Twenty-four hours after application, the skm was cleansed and sectioned into thicknesses of about 0 5 mm The sections were scanned at 10 ⁇ m depth with a Multiprobe 2001 CLSM with a Nikon Diaphot cross-epifluorescence inverted microscope as described in Example 24B
  • the invention includes a method for selecting a composition for therapeutic transdermal administration of a macromolecule
  • the method includes selecting from a library of acylated amino acids (such as those in Tables 1A-1F), one or more acylated amino acids for analysis
  • a carrier vehicle is selected where the carrier vehicle can be any of those described above, and in particular is selected from an aqueous-based solution, a non-aqueous solution and a suspension of hposomes
  • the acylated amino acid is admixed with the carrier vehicle and the macromolecule or therapeutic agent is also added
  • the transdermal skin penetration of the therapeutic agent is determined, either by in vitro or in vivo methods For example, the ⁇ n vitro penetration method used above to determine penetration of albumin into the skin via microscopy can be employed
  • acylated ammo acid and carrier vehicle combination suitable to achieve a transdermal penetration rate sufficient for therapy is selected as the composition for
  • caprylic acid N-hvdroxysuccinimide ester (Intermediate) Caprylic acid (7.2 g. 50 mmol) was dissolved in 40 mL of dichloromethane and 10 mL of N,N-dimethylformamide. 1-N -Hydroxy succinimide ( 5.75 g , 50 mmol) and N.N-dicyclohexylcarbodiimide (50 mmol, 100 mL, of 0.5 M solution) was added at -50°C and the solution allowed to warm up to room temperature and the reaction mixture stirred at room temperature for 16 hr. Dicyclohexyl urea precipitate was filtered off. The capryloyl NHS ester was purified by crystallization from diethyl ether- n- hexane solution (9.8 g. 82%).
  • Caprylic acid N-hydroxysuccinimide ester was prepared as described in Example 1A. N- ⁇ -Lauroyl-L-lysine methyl ester was added to 1.1 equivalents of caprylic acid N- hydroxysuccinimide ester in dichloromethane and stirred at room temperature for 12 hr. The reaction mixmre was treated with water for 1 hr to hydrolyze the unreacted N-
  • N- ⁇ -Lauroyl- ⁇ -lauroyl-L-lvsine methyl ester was added to 1.1 equivalents of lauric acid N- hydroxysuccinimide ester in dichloromethane and stirred at room temperature for 12 hr.
  • the reaction mixture was treated with water for 1 hr to hydrolyze the unreacted N- hydroxysuccinimide ester.
  • the organic phase was washed sequentially with IN hydrochloric acid, saturated sodium bicarbonate, and brine.
  • the organic phase was 10 separated and dried with anhydrous sodium sulfate, filtered and concentrated.
  • the product was crystallized from ethyl acetate - n-hexane solution. The product was filtered and dried under vacuum.
  • N- ⁇ -lauroyl-L-lysine (10 mmol) was suspended as a slurry in 20 mL of anhydrous ethanol.
  • Thionyl chloride ( 12 mmol) was slowly added at 0°C and stirred for 30 minutes. The resulting solution was stirred at room temperature for 16 hrs. The excess ethanol was evaporated in vacuo on a rotary evaporator.
  • Dichloromethane was added to ? the residue and aqueous saturated sodium bicarbonate solution. The precipitated material was filtered off. The organic phase was separated from the filtrate and dried with anhydrous sodium sulfate, filtered, and concentrated under vacuum. The product was crystallized from dichloromethane-n- hexane solution.
  • N- ⁇ -Lauroyl -L-lysine (10 mmol) was suspended as a slurry in 20 mL of anhydrous methanol.
  • Thionyl chloride (12 mmol) was slowly added at 0°C and stirred for 30 minutes. The resulting solution was stirred at room temperature for 16 hr. The excess 30 ethanol was evaporated under vacuum on a rotary evaporator.
  • Dichloromethane was added to the residue and the product was neutralized with aqueous saturated sodium bicarbonate solution. The precipitated material was filtered off. The organic phase was
  • N- ⁇ -Lauroyl-L-lysine ethyl ester was added to 1 1 equivalents of caprylic acid N- hydroxysuccinimide ester in dichloromethane and stirred at room temperature for 12 hr
  • the reaction mixmre was treated with water for 1 hr to hydrolyze the unreacted N hydroxysuccinimide ester
  • the organic phase was washed sequentially with IN hydrochloric acid, saturated sodium bicarbonate, and brine solution
  • the organic phase was separated and dried with anhydrous sodium sulfate. filtered and concentrated
  • the product was crystallized from ethyl acetate-n-hexane solution The product was filtered and dried under vacuum
  • N- ⁇ -Maleoyl- ⁇ -lauroyl-L-lvsine ethyl ester (PDM 29) N- ⁇ -Lauroyl-L-lysine ethyl ester (5 mmol) was dissolved in dichloromethane 2 equivalents of maleic anhydride dissolved in 1 mL dimethylformamide was added and stirred at room temperature for 3 hr Sodium carbonate (5 mmol) and 200 L of water were added and stirred for 12 hr The reaction mixmre was treated with water for 1 hr to hydrolyze the unreacted anhydride The product was acidified to pH 2 5 with concentrated hydrochloric acid at 0°C The organic phase was washed sequentially with IN hydrochloric acid, water and brine solution The organic phase was separated and dried with anhydrous sodium sulfate. filtered and concentrated The product was crystallized from ethyl acetate n-hexane solution The product was filtered and dried under vacuum
  • N- ⁇ -Lauroyl-L-lysine was suspended as a slurry in n-propanol.
  • Thionyl chloride. 2 equivalents was slowly added at 0°C and stirred for 30 minutes.
  • the resulting solution 5 was stirred at room temperature for 16 hr.
  • the excess propanol was evaporated on a rotary evaporator.
  • Dichloromethane was added to the residue and the product was neutralized with aqueous saturated sodium bicarbonate solution. The precipitated material was filtered off. The organic phase was separated from the filtrate and dried with anhydrous sodium sulfate, filtered, and concentrated under vacuum.
  • the product i " was crystallized from dichloromethane-hexane solution.
  • N- ⁇ -Lauroyl-L-lysine was suspended as a slurry in n-butanol.
  • Thionyl chloride 2 15 equivalents, was slowly added at room temperature and then heated at 60 °C for 4 hr. The resulting solution was allowed to cool to room temperature and diethyl ether was added to precipitate the product. The product was cooled on ice and the precipitate filtered.
  • Dichloromethane was added to the precipitate and the product was neutralized with aqueous saturated sodium bicarbonate solution. The precipitated material was 0 filtered off. The organic phase was separated from the filtrate and dried with anhydrous sodium sulfate. filtered, and concentrated under vacuum. The product was crystallized from dichloromethane-hexane solution.
  • N- ⁇ -Lauroyl -L-lysine was suspended as a slurry in isoamyl alcohol.
  • Thionyl chloride. 2 equivalents was slowly added at room temperature and then heated at 70 °C for 4 hr. The resulting solution was allowed to cool to room temperature.
  • o Dichloromethane was added to the product and aqueous saturated sodium bicarbonate solution added The precipitated material was filtered off. The organic phase was separated from the filtrate and dried with anhydrous sodium sulfate, filtered, and
  • N- ⁇ -Lauroyl -L-lysine was suspended as a slurry in n-dodecanol Thionyl chloride. 2 equivalents, was slowly added at room temperature and then heated at 70 °C for 3 hr The resulting solution was allowed to cool to room temperature and diethyl ether was added to precipitate the product The product was cooled on ice and the precipitate filtered Dichloromethane was added to the precipitate and aqueous saturated sodium bicarbonate solution added The precipitated material was filtered off The organic phase was separated from the filtrate, dried with anhydrous sodium sulfate. filtered, and concentrated under vacuum The product was crystallized from dichloromethane-hexane solution
  • N- ⁇ -lauroyl L-lysme isoamyl ester was added to 1 1 equivalents of caprylic acid N- hydroxysuccinimide ester in dichloromethane and stirred at room temperature tor 12 hr The reaction mixture was treated with water for 1 hr to hydrolyze the unreacted N- hydroxysuccinimide ester The organic phase was washed sequentially with saturated sodium bicarbonate. IN hydrochloric acid, and brine solution The organic phase was separated and dried with anhydrous sodium sulfate, filtered and concentrated The product was crystallized from ethyl acetate-n-hexane solution The product was filtered and dried under vacuum
  • N- ⁇ -lauroyl-L-lysme n-dodecyl ester was added to 1 1 equivalents of caprylic acid N-hydroxysuccinimide ester m dichloromethane and stirred at room temperature for 12 hr The reaction mixmre was treated with water for 1 hr to hydrolyze the unreacted N- hydroxysuccinimide ester The organic phase was washed sequentially with saturated sodium bicarbonate, IN hydrochloric acid, and brine solution The organic phase was separated and dried with anhydrous sodium sulfate, filtered and concentrated The
  • N- ⁇ -Lauroyl-L-lysine dodecyl ester (5 mmol) was dissolved in dichloromethane. 2 equivalents of maleic anhydride dissolved in 1 mL dimethylformamide was added and stirred at room temperature for 3 hr. Sodium carbonate (5 mmol) and 200 L of water were added and stirred for 12 hr. The reaction mixmre was treated with water for 1 hr to hydrolyze the unreacted anhydride. The product was acidified to pH 2.5 with concentrated hydrochloric acid at 0°C The organic phase was washed sequentially with IN hydrochloric acid, water and brine solution. The organic phase was separated and dried with anhydrous sodium sulfate. filtered and concentrated. The product was crystallized from ethyl acetate-n-hexane solution. The product was filtered and dried under vacuum.
  • IFN- ⁇ 2b Intron A was obtained from Schering-Plough. Synthetic beeswax was obtained from Croda. Toronto, Ontario. DOWICIL was obtained from Dow Chemical (Midland, Michigan). Phospholipid EFATM was purchased from PEBRO (Missassauga. Ontario. Canada). Canola oil was obtained from Natural Oils International. Arleta California.
  • An anhydrous lipid gel was prepared by mixing the following components together:
  • the lipids and the solvents were weighted into a glass container and warmed to 65- 75°C by intermittent heating, and gently mixed.
  • An oil-in-water emulsion was prepared by combining the hydrophilic ingredients in a container and combining the lipophilic ingredients in another container:
  • Hvdrophilic Ingredients Amount (% w/w) distilled water q.s. to 100
  • Lipophilic Ingredients canola oil 4.0 glyceryl monostearate 1.0 cetyl alcohol 0.6 synthetic beeswax 0.28
  • 'PEFA noleamidopropyl propylene glycol-dimonium chloride
  • Dowic ⁇ l l-(3-chlorallyl)-3,4,7-t ⁇ aza-l-azon ⁇ aadamantane chloride
  • the oil-in-water emulsion was prepared by adding the lipophilic mixture to the hydrophilic ingredient mixture at 60-80°C in a homogenizer at 20-80 psig for 5-30 minutes to obtain a small droplet size of less than about 0.5 ⁇ m.
  • Biphasic lipid vesicles were prepared by simultaneously adding the oil-in-water emulsion and an aqueous solution containing 5 million units of IFN- ⁇ to the lipid gel. which was warmed to 55°C. The gel, IFN- ⁇ and emulsion were vigorously mixed by vortexing or propeller mixing to achieve the desired particle size.
  • the diffusion of IFN- ⁇ into human skin was investigated using flow-through diffusion cells
  • the diffusion cells are designed such that fluid may be continuously pumped through them m order to maintain sink conditions (flow rate 3 mL/hour)
  • a phosphate buffer (PBS 7 5 mM Na 2 HPO 4 , 2 5 mM NaH 2 P0 4 , 141 2 mM NaCl) lsotomc with body fluids was used and the temperature was maintained at 32 °C by a circulating water bath
  • MEM essential medium for MDBK: 5 x 10 ⁇ PFU in 5 mL supplemented MEM for WISH).
  • the infected flasks were incubated for 60 min at 37 °C and were shaken every 15 min.
  • the medium was poured out and 20 mL supplemented MEM was added to each infected flask and then incubated overnight at 37 °C.
  • the infected flasks were frozen at -70°C.
  • the medium was thawed at room temperature. The supernatants were transferred to a 50 mL centrifuge tube and centrifuged at 500 rpm for 10 minutes.
  • the virus-rich supernatants were pooled and placed on ice.
  • the virus stock was aliquot into 500 L/vial and stored at -70°C. These VSV stocks were used for the antiviral assay.
  • a single cell suspension of MDBK cells was prepared from a confluent culture and the MDBK cells were resuspended at 6-7 x 10 3 cells/mL in supplemented MEM. 50 ⁇ L of supplemented MEM was added to each well of a 96-well flat-bottomed microtiter plate. 50 ⁇ L of IFN standard was added to well 3 of row A and B. 50 ⁇ L of the first sample was added to well 3 of row C and D, and repeated for samples 2 and 3.
  • a 2-fold serial dilution of IFN samples was prepared by gently mixing the contents of well 3 and then transferring 50 ⁇ L from well 3 to well 4, then from 4 to well 5, etc. , through well 12. 50 ⁇ L of sample was discarded from well 12. At this point, each well contained 50 ⁇ L. 50 ⁇ L cells were added to each well and the plate was agitated to ensure the cells were evenly distributed. The plate was incubated overnight at 37°C. The VSV was diluted with supplemented MEM to 10000 PFU/mL. The supernatants were aspirated from each well of the microtiter plate with an 8-channel pipettor. Each well in vertical row 1 had 100 mL supplemented MEM added.
  • 100 mL virus was added to each well, starting with row 2 (1000 PFU/well). The plate was incubated 20-24 hours at 37 °C. The monolayers were examined, prior to washing and fixing the cells, using an inverted microscope. In the cell control wells, uniform monolayers were observed. In the virus control wells, the monolayers were completely destroyed. The supernatants were removed from each well. Each well was washed three times with 100 mL cold HBSS. The final wash was aspirated and replaced with 100 mL of 5% formalin in each well and incubated 10 minutes at room temperature. The formalin was shaken from the plate into a sink with the water running.
  • CytoscreenTM ELISA kit (Medicorp, Montreal, PQ) was used The sensitivity of the assay is ⁇ 25 pg/mL and the range of concentration is 0-500 pg/mL The assay is specific for human IFN- ⁇ with no cross-reactivity with human IFN ⁇ , IFN ⁇ or IFN ⁇ The results are expressed as pg IFN-
  • 2-5A synthetase was determined by a 125 I 2-5A radioim uno assay kit (Eiken Chemical Corp , Tokyo, Japan) using rabbit anti-human 2-5 A antibody and goat antirabbit IgG as secondary antibody Briefly, 2-5A synthetase was extracted from the samples by poly (I) poly (C) agarose for 10 min After the addition of ATP solution (24 ⁇ g/mL) and incubation, 100 ⁇ L I25 I-2-5A was added, followed by the primary and secondary antibody Radioactivity bound was determined by gamma counting 2 5A synthetase is measured as pmol 2-5 A produced/ lOOmL/h and subsequently expressed as nmole enzyme/mg protein 1 h in skin homogenate All samples were run in duplicates
  • the skin was lightly washed 5 with distilled water and patted dry with tissue prior to the addition of the patch.
  • the patch was applied by removing the backing paper from the adhesive foam and firmly pressing the patch to a clean area of skin away from any skin abrasion and located in a position that the animal is unlikely to access.
  • the patch was covered with OPSITETM occlusive dressing and a plastic tape to keep the patch in place for 24 hours.
  • the skin surface was cleaned by wiping the area once with a dry tissue, 4 x with a tissue soaked in 70% ethanol, 4 swabs with 0.5% (v/v) Tween 80 i s in distilled water using a cotton wool swab, finally 4 x with a tissue soaked in 70% ethanol.
  • the section of the skin marked as treatment area was removed using clean sharp scissors, ensuring that only the treated area was sampled. The skin samples were frozen until analysis.
  • the frozen skin samples were weighed and pulverized in liquid N 2 by five blows from a hammer in a tissue pulveriser (preincubated in liquid N 2 ).
  • the pulverized tissue was reweighed to calculate the recovery of material and extracted by moderate vortexing 5 with 5-10 volumes of PBS containing 1 mg/mL leupeptin and 20 mg/mL soybean trypsin inhibitor as protease inhibitors.
  • the skin was resuspended in buffer and then sonicated 3 x 15 seconds, with 1 min intervals on ice, then centrifuged at 500g for 10 min at 4°C to remove undisrupted cells and connective tissue.
  • the resulting supernatants were termed "whole cell homogenates" and were used immediately or aliquoted into 2-300 mL 0 aliquots and stored at -80°C. Skin homogenates were used to determine IFN- ⁇ absorption by antiviral assay described above in the Methods section. Results are shown in Figs. 1A-1B.
  • An anhydrous lipid gel was prepared by mixing the following components together
  • the lipids and the solvents were weighed into a glass container and warmed to 65- 75 °C by intermittent heating, and gently mixed
  • An oil-in-water emulsion was prepared by combining the following hydrophilic ingredients in a container and combimng the following lipophilic ingredients in another contamer
  • Hydrophilic Ingredients Amount (% w/w) distilled water q s to 100
  • Synthetic beeswax 0 28 'PEFA hnoleam ⁇ dopropyl propylene glycol-dimonium chloride
  • the oil-in-water emulsion was prepared by adding the lipophilic mixture to the hydrophilic ingredient mixture at 60-80 °C m a homogemzer at 20-80 psig for 5-30 minutes to obtain a small droplet size of less than about 0 5 ⁇ m
  • aqueous solutions of IFN- ⁇ were prepared having activities of 5 MU, 15 MU and 40 MU Lyophihzed IFN- ⁇ (Intron A) was dissolved in part of the formulation water to the total quantity required for the batch size
  • Biphasic lipid vesicles were prepared by simultaneously adding the oil-in-water emulsion and an IFN- ⁇ aqueous solution at a selected concentration to the lipid gel, which was warmed to 55 °C
  • the lipid gel, IFN- ⁇ solution, and emulsion were vigorously mixed by vortexing or propeller mixing to achieve the desired particle size
  • OPSITE transparent adhesive for added protection After the 48 hour test period, 6 mm punch biopsies and blood samples were collected from each subject for analysis
  • Biopsy samples were used for lmmunohistochemistry and for homogenate preparation for antiviral, ELISA and 2-5 A synthetase assays, and procedures for each are provided above in Example 20 Blood samples were used to prepare serum for the antiviral and ELISA assays, and to extract peripheral blood mononuclear cells (PBMC) for the 2-5 A synthetase assay Results are shown in Figs 2A-2C. 3A-3C and Tables 5 and 6
  • PBMC peripheral blood mononuclear cells
  • the patch was applied to the upper inner arm as described above
  • 6 mm punch biopsies and blood samples were collected as described above from each subject for analysis
  • Results are shown in Figs 4A-4C and 5A-5C and Tables 7 and 8
  • Biphasic lipid vesicles having entrapped insulm were prepared as follows An anhydrous lipid gel was prepared by mixing the following components together
  • Component Amount (% w/w)
  • the lipids and the solvents were weighed into a glass container and warmed to 65 75 °C by intermittent heatmg, and gently mixed
  • An oil-in-water emulsion was prepared by combimng the hydrophilic ingredients in a container and combimng the lipophilic ingredients in another container
  • Lipophilic Ingredients olive oil 0 25
  • the oil-in-water emulsion was prepared by adding the lipophilic mixmre to the hydrophilic mgredient mixture at 60-80 °C in a homogenizer at 20-80 psig for 5-30 mmutes to obtain a small droplet size of less than about 0 5 ⁇ m
  • An aqueous solution of insulin in 3 mM HC1, pH 3-3 5 was prepared at concentrations sufficient to obtain 10 mg insulin 200 mg formulation and 50 mg ⁇ nsuhn/200 mg formulation
  • Biphasic lipid vesicles were prepared by simultaneously adding the oil-m-water emulsion and the aqueous solution contaimng insulin to the lipid gel, which was warmed to 55 °C
  • the gel, insulin, and emulsion were vigorously mixed by vortexing or propeller mixing to achieve the desired particle size
  • the pH of the formulation was adjusted with 1 M NaOH to between 4 5-5 5
  • the biphasic lipid vesicle preparations were placed in transdermal devices constructed from a backing member peripherally joined to a styrofoam adhesive member
  • the square patches were 2.25 cm 2 (1 5 cm x 1 5 cm) in size with an active delivery area of 1 cm 2 200 mg of each biphasic lipid vesicle preparation was placed in the reservoir of each patch, except for the vesicles having 10 mg msuhn/g formulation
  • Sprague-Dawley rats (150-225g) were obtained from Charles River Laboratories
  • Streptozotocin (STZ: mixed anomers: 75 % -anomer) was obtained from Sigma. Insulin was purchased from Cansera (recombinant human insulin) or from Sigma (porcine insulin) . One Touch Profile blood glucose monitoring system and One Touch glucose test strips were obtained from Lifescan. Opsite was obtained from Smith & Nephew (U.K. ) and Insulin ELISA kits were obtained from Mercodia (ALPCO, USA).
  • Insulin was originally dissolved in " 3 mM HC1, pH 3-3.5. before being adjusted to the formulation pH by 1 M - concentrated NaOH.
  • Diabetic rat model An insulin dependent diabetes mellitus (IDDM) animal model was established to study the delivery of insulin after topical application.
  • Sprague-Dawley rats 150-225g were fasted for 24 hours to increase the sensitivity of the pancreatic -cells to destruction by streptozotocin (STZ).
  • STZ streptozotocin
  • the rats were injected with STZ (50-55 mg/kg body weight) in 100 mM sodium citrate buffer, pH 4.5.
  • Control rats were injected with citrate buffer alone.
  • the rats were allowed three days for the diabetic state to stabilize. Blood glucose, body weight, and general health were recorded daily for each rat. Rats receiving topically applied insulin treatment were carefully shaved under halothane anesthesia and the skin allowed to recover for 6-24 hours .
  • Insulin treatment was for three days, at the end of which the rats were sacrificed and blood and skin samples were collected.
  • the skin was washed with distilled water, swabbed 6 x with 0.5 % (v/v) Tween 80 and
  • Plasma and serum were produced by centrifugmg hepa ⁇ mzed blood The serum was used immediately or ahquoted and stored at -20 °C until used
  • ELISA Skin homogenates and serum samples (5-100 ⁇ L) were analyzed for human and porcine insulin content by ELISA (ALPCO, USA)
  • the ELISA kit for human insulin detection had a 500% cross-reactivity with porcine insulin, however, the rat insulin cross-reactivity was 0 5% making this an accurate way to determine the specific absorption of porcine insulin in the rat model
  • the sensitivity of the kit was > 25 pg/mL porcine insulin with a standard curve range of 50-1000 pg/mL Human insulin detection used a superconjugate with a standard curve range of 0 15-100 ⁇ IU/mL (5 36-357 pg msuhn/mL) and 5-25 ⁇ L of sample
  • Blood glucose levels were recorded periodically (approx every 4 hours) after administration of the topical dose of insulin
  • Blood glucose was measured by a LIFESCANTM blood glucose meter using approximately 50 ⁇ L of fresh whole blood obtained from the tail veins of the rats Where blood insulin levels were to be determined.

Abstract

L'invention concerne une composition permettant l'administration transdermique ou transmuqueuse d'un agent thérapeutique. La pénétration de cet agent dans la peau ou dans une muqueuse s'effectue en présence d'un acide aminé acylaté sélectionné de façon à améliorer l'agent à administrer. Selon certains modes de réalisation, un excipient de vecteur liposomal est inclus dans la composition. L'invention concerne également des méthodes d'administration et de sélection d'un acide aminé acylé, de façon à optimiser l'administration transdermique ou transmuqueuse d'un agent sélectionné.
PCT/CA2000/001323 1999-11-12 2000-11-10 Compositions pour administration transdermique et transmuqueuse d'agents therapeutiques WO2001035998A1 (fr)

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WO2003022310A1 (fr) * 2001-09-06 2003-03-20 Nexmed Holdings, Inc. Compositions de prostaglandine et procedes de traitement de troubles de l'erection chez l'homme
FR2834215A1 (fr) * 2001-12-27 2003-07-04 Physica Composes amphiphiles a usage pharmaceutique ou cosmetique
US7105571B2 (en) 2000-01-10 2006-09-12 Nexmed Holdings, Inc. Prostaglandin compositions and methods of treatment for male erectile dysfunction
US7906137B2 (en) * 2004-05-21 2011-03-15 Mediplex Corporation, Korea Delivery agents for enhancing mucosal absorption of therapeutic agents
WO2012140155A1 (fr) 2011-04-14 2012-10-18 Novo Nordisk A/S Acides aminés acylés par un acide gras pour l'administration de peptides par voie orale
WO2014060447A1 (fr) 2012-10-17 2014-04-24 Novo Nordisk A/S Acides d-aminés acylés par un acide gras pour l'administration de peptides par voie orale
WO2014060512A1 (fr) * 2012-10-17 2014-04-24 Novo Nordisk Health Care Ag Acides aminés acylés par un acide gras pour l'administration d'hormone de croissance
US8841342B2 (en) 2002-08-09 2014-09-23 Vital Health Sciences Pty. Ltd. Carrier
WO2015121102A1 (fr) * 2014-02-13 2015-08-20 Basf Se Ester éthylique de capryloylalanine en tant qu'activateur de pénétration
US9168216B2 (en) 2005-06-17 2015-10-27 Vital Health Sciences Pty. Ltd. Carrier comprising one or more di and/or mono-(electron transfer agent) phosphate derivatives or complexes thereof
WO2015162195A1 (fr) * 2014-04-23 2015-10-29 Novo Nordisk A/S Acides aminés acylés par un acide gras pour l'administration de peptides par voie orale
US9314527B2 (en) 2010-03-30 2016-04-19 Phosphagenics Limited Transdermal delivery patch
US9561243B2 (en) 2011-03-15 2017-02-07 Phosphagenics Limited Composition comprising non-neutralised tocol phosphate and a vitamin A compound
US10071030B2 (en) 2010-02-05 2018-09-11 Phosphagenics Limited Carrier comprising non-neutralised tocopheryl phosphate
US10973761B2 (en) 2015-12-09 2021-04-13 Phosphagenics Limited Pharmaceutical formulation
WO2022204308A1 (fr) * 2021-03-24 2022-09-29 Glo Pharma, Inc. Vésicules lipidiques multisomes pour l'administration d'agents cosmétiques
US11753435B2 (en) 2016-12-21 2023-09-12 Avecho Biotechnology Limited Process
US11801221B2 (en) 2019-09-23 2023-10-31 Dds Research Inc. Lipid vesicle compositions with penetration enhancing agents

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Cited By (24)

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Publication number Priority date Publication date Assignee Title
US6693135B2 (en) 2000-01-10 2004-02-17 Nexmed (Holdings) Incorporated Prostaglandin compositions and methods of treatment for male erectile dysfunction
US7105571B2 (en) 2000-01-10 2006-09-12 Nexmed Holdings, Inc. Prostaglandin compositions and methods of treatment for male erectile dysfunction
EA009384B1 (ru) * 2001-09-06 2007-12-28 Нексмед Холдингс, Инк. Композиции простагландина и способы лечения мужской эректильной дисфункции
WO2003022310A1 (fr) * 2001-09-06 2003-03-20 Nexmed Holdings, Inc. Compositions de prostaglandine et procedes de traitement de troubles de l'erection chez l'homme
FR2834215A1 (fr) * 2001-12-27 2003-07-04 Physica Composes amphiphiles a usage pharmaceutique ou cosmetique
WO2003055528A2 (fr) * 2001-12-27 2003-07-10 Physica S.A.R.L. Composes amphiphiles a usage pharmaceutique ou cosmetique
WO2003055528A3 (fr) * 2001-12-27 2004-02-26 Physica S A R L Composes amphiphiles a usage pharmaceutique ou cosmetique
US8841342B2 (en) 2002-08-09 2014-09-23 Vital Health Sciences Pty. Ltd. Carrier
US7906137B2 (en) * 2004-05-21 2011-03-15 Mediplex Corporation, Korea Delivery agents for enhancing mucosal absorption of therapeutic agents
US9168216B2 (en) 2005-06-17 2015-10-27 Vital Health Sciences Pty. Ltd. Carrier comprising one or more di and/or mono-(electron transfer agent) phosphate derivatives or complexes thereof
US10071030B2 (en) 2010-02-05 2018-09-11 Phosphagenics Limited Carrier comprising non-neutralised tocopheryl phosphate
US9314527B2 (en) 2010-03-30 2016-04-19 Phosphagenics Limited Transdermal delivery patch
US10188670B2 (en) 2011-03-15 2019-01-29 Phosphagenics Limited Composition
US9561243B2 (en) 2011-03-15 2017-02-07 Phosphagenics Limited Composition comprising non-neutralised tocol phosphate and a vitamin A compound
WO2012140155A1 (fr) 2011-04-14 2012-10-18 Novo Nordisk A/S Acides aminés acylés par un acide gras pour l'administration de peptides par voie orale
WO2014060447A1 (fr) 2012-10-17 2014-04-24 Novo Nordisk A/S Acides d-aminés acylés par un acide gras pour l'administration de peptides par voie orale
WO2014060512A1 (fr) * 2012-10-17 2014-04-24 Novo Nordisk Health Care Ag Acides aminés acylés par un acide gras pour l'administration d'hormone de croissance
WO2015121102A1 (fr) * 2014-02-13 2015-08-20 Basf Se Ester éthylique de capryloylalanine en tant qu'activateur de pénétration
JP2017505795A (ja) * 2014-02-13 2017-02-23 ビーエーエスエフ ソシエタス・ヨーロピアＢａｓｆ Ｓｅ 浸透促進剤としてのカプリロイルアラニンエチルエステル
WO2015162195A1 (fr) * 2014-04-23 2015-10-29 Novo Nordisk A/S Acides aminés acylés par un acide gras pour l'administration de peptides par voie orale
US10973761B2 (en) 2015-12-09 2021-04-13 Phosphagenics Limited Pharmaceutical formulation
US11753435B2 (en) 2016-12-21 2023-09-12 Avecho Biotechnology Limited Process
US11801221B2 (en) 2019-09-23 2023-10-31 Dds Research Inc. Lipid vesicle compositions with penetration enhancing agents
WO2022204308A1 (fr) * 2021-03-24 2022-09-29 Glo Pharma, Inc. Vésicules lipidiques multisomes pour l'administration d'agents cosmétiques

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