Classifications

• • 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
  • A61K9/703—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms characterised by shape or structure; Details concerning release liner or backing; Refillable patches; User-activated patches
  • A61K9/7038—Transdermal patches of the drug-in-adhesive type, i.e. comprising drug in the skin-adhesive layer
  • A61K9/7046—Transdermal patches of the drug-in-adhesive type, i.e. comprising drug in the skin-adhesive layer the adhesive comprising macromolecular compounds
  • A61K9/7053—Transdermal patches of the drug-in-adhesive type, i.e. comprising drug in the skin-adhesive layer the adhesive comprising macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds, e.g. polyvinyl, polyisobutylene, polystyrene
  • A61K9/7061—Polyacrylates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
  • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/26—Iron; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/30—Zinc; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/34—Copper; Compounds thereof
• • 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
  • A61K9/703—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms characterised by shape or structure; Details concerning release liner or backing; Refillable patches; User-activated patches
  • A61K9/7084—Transdermal patches having a drug layer or reservoir, and one or more separate drug-free skin-adhesive layers, e.g. between drug reservoir and skin, or surrounding the drug reservoir; Liquid-filled reservoir patches

Definitions

• Zinc, iron, and vitamin D are essential nutrients that are actively absorbed and transported in the body. Despite the availability of zinc, iron, and vitamin D in several foods, inadequate intake, malabsorption, lifestyle and environmental factors which reduce exposure to sunlight, or increased requirements for zinc, iron, and Vitamin D can all cause deficiencies in one or more of these essential nutrients.
• Zinc, iron, and vitamin D supplements are available commercially alone or in combination with other minerals in the form of oral tablets or capsules. There are a number of barriers to efficient oral uptake of zinc, iron, and vitamin D, however. For example, zinc readily forms coordination complexes with hydroxide ions in the small intestines. These complexes can hinder the metal’s delivery and its biological activity.
• Existing vitamin D supplements have limited absorption of vitamin D via oral administration. Absorption of iron by oral supplementation is not efficient (typically between 10-15%) and often cause gastric side-effects and this renders them unpopular with many subjects in need.
• a patch for transdermal delivery of a compound including: a backing layer; an active agent layer including at least one compound; and a permeable- membrane release layer configured to transfer the at least one compound to a skin surface.
• a method of delivering a compound to a subject in need including: applying a patch for transdermal delivery of a compound, including: an outer backing layer; an intermediate active agent layer including at least one compound; and a permeable-membrane release layer configured to transfer the at least one compound to a skin surface.
• a method of synthesizing a nutrient-counter ion salt for transdermal delivery including: providing at least one nutrient; providing at least one counter ion; solubilizing the at least one nutrient and the at least one counter ion in at least one solvent to produce a solution; optionally adjusting a pH of the solution; and removing the solvent from the solution to produce a nutrient-counter ion salt.
• a patch for transdermal delivery of a compound including: an outer backing layer including polyester; an intermediate active agent layer including a supersaturated solution of zinc ethyl maltol and PEG400: PEG 1000 (8%w/w: 2%w/w); and a permeable-membrane release layer configured to transfer the at least one compound to a skin surface, wherein the permeable-membrane release layer is nylon.
• a patch for transdermal delivery of a compound including: an outer backing layer including polyester; an intermediate active agent layer including a solution of 18 mg/mL iron maltol in polyethylene glycol; and a permeable- membrane release layer configured to transfer the at least one compound to a skin surface, wherein the permeable membrane release layer is nylon.
• a patch for transdermal delivery of a compound including: an outer backing layer including polyester; an intermediate active agent layer including: a compound including cholecalciferol phosphate sodium; a penetration enhancer including polyethylene glycol; and an adhesive including acrylates copolymer; and a permeable-membrane release layer configured to transfer the at least one compound to a skin surface, wherein the permeable-membrane release layer is ethylene vinyl acetate.
• FIG. 1 A is a top view of a non-limiting example transdermal patch that may be used in accordance with the present disclosure.
• FIG. IB is an exploded side perspective view of the transdermal patch of FIG. 1A.
• FIG. 2 is a flowchart of non-limiting example steps of a method for optimization of a transdermal patch.
• FIG. 3 depicts structures for non-limiting example zinc complexes that may be used in accordance with the present invention.
• FIG. 4 depicts a schematic representation of a Franz cell.
• FIG. 5 shows the effect of different post-column reagent (PAR) flow rates on zinc acetate samples' absorbance in HPLC. The number of injections was 3 for each flow rate.
• FIG. 10 depicts a schematic of the work-flow for the development of drug in adhesive patch.
• FIG. 11 shows a photograph of (A) inner side and (B) outer side of reservoir based on supersaturated solution of zinc ethyl maltol in PEG400:PEG1000 (60 mg/mL).
• FIG. 13 shows a schematic representation of in vitro and ex vivo permeation studies using Franz cells.
• FIG. 14 depicts structures for non-limiting example iron complexes that may be used in accordance with the present invention.
• FIG. 15 shows skin necrosis post iron chloride hexahydrate permeation.
• FIG. 16 shows iron chloride hexahydrate skin permeation and deposition after
• FIG. 17 shows iron ethyl maltol skin permeation and deposition after 24 h.
• FIG. 18 shows iron oleate permeation and deposition after 24 h.
• FIG. 19 shows permeation and deposition of iron (ferric) propionate and iron maltol solutions.
• FIG. 20 shows photographs of skin after the 24 h permeation study, a) iron propionate, b) iron maltol (made at pH 4) in 7.5: 1.5: 1 : 1 water: PG: dimethicone: ethanol, c) iron maltol (made at pH 4) in PG, and d) iron maltol (made at pH 1.6) in Transcutol.
• FIG. 21 shows workflow for the development of drug in adhesive patch.
• FIG. 22 a 4% loading (2 mg FeEM, 45 mg 9301 and 2 mg PG), b) 7.5% loading
• FIG. 23 shows an inner face of a 34 mm diameter patch based on iron ethyl maltol in acrylate adhesive.
• FIG. 24 shows adhesive patch deposition of iron using iron (ferric) complexes.
• FIG. 25 shows an example of reservoir patch R3 including 11 mg/mL iron maltol in PG using a Durapore membrane).
• FIG. 26 shows reservoir patches permeation and deposition.
• R1 FeProp is iron propri onate dissolved in propylene glycol
• R2 included 18 mg/mL iron maltol complex in PG using a nylon rate controlling membrane
• R3 included 11 mg/mL iron maltol in PG using a Durapore membrane
• R4 included 16 mg/mL iron maltol in 7.5: 1.5: 1 : 1 water: PG: dimethicone using a Durapore membrane.
• FIG. 27 shows a photograph of a patch (left) and skin (right) post 24 hours permeation study; a) Patch R2, b) Patch R3, and c) Patch R4.
• FIG. 28 shows a summary of formulations showing the best solution (iron maltol made at pH 4 in PG), formulating it into a nylon membrane reservoir patch, the best iron maltol adhesive patch and an existing commercial iron transdermal patch.
• FIG. 29 shows chemical synthesis of cholecalciferol phosphate disodium from cholecalciferol.
• FIG. 32 shows an example of optimizing vitamin D phosphate concentration in Oppanol-based patch (F2).
• FIG. 36 shows stability of cholecalciferol phosphate acid over 24 hours in the donor, extraction and receiver solvents.
• transdermal delivery refers to the delivery of at least one compound through skin (z.e., epidermis, dermis, hypodermis) with the intent for the compound to be pass through skin layers and exert a systemic effect on a subject in need.
• Transdermal delivery may involve transferring (z.e., exposing, bringing into contact with, adsorbing into, permeating, or otherwise making available for systemic effect) a compound to a skin surface.
• a patch for transdermal delivery of a compound including a backing layer, an active agent layer including at least one compound, and a permeable- membrane release layer configured to transfer the at least one compound to a skin surface.
• a transdermal patch 100 is shown in a top view (FIG. 1 A) and an exploded side perspective (FIG. IB).
• the transdermal patch may include a backing layer 104.
• the backing layer 104 may be configured to provide structural support for the transdermal patch 100, to protect the contents of an active agent layer 102, to provide for an aesthetic surface when the transdermal patch 100 is placed on the skin of a subject.
• the backing layer 104 may be a carrier foil layer.
• the backing layer may include films of polyesters, polyolefins, polyurethanes, or any combinations thereof. Suitable commercially available backing layer may include 3MTM ScotchpakTM Polyester Backing Film Laminate 9730.
• An active agent layer 102 may be formed between backing layer 104 and a permeable-membrane release layer 106 and may contain at least one compound 108.
• the permeable-membrane release layer 106 may be a release foil or may act as a protecting layer between the skin of a subject and the transdermal patch 100.
• the permeable- membrane release layer may be permeable or may include openings to allow transfer between the at least one compound 108 and the skin of a user.
• the permeable-membrane release layer may include nylon, regenerated cellulose, hydrophobic polyvinylidene fluoride (PVDF), ethylene vinyl acetate, or fluoropolymer on polyester film.
• PVDF polyvinylidene fluoride
• Suitable commercially available permeable-membrane release layer may include regenerated cellulose (12-14 k molecular weight cut-off Medical International), Whatman nylon (0.20 pm), and 3MTM CoTran 9728.
• the patches disclosed herein include an active agent layer (102) including at least one compound (108) (z.e., a composition for transdermal delivery).
• the at least one compound may include zinc, iron, vitamin D (z.e., vitamin DI as a mixture of ergocalciferol with lumisterol, vitamin D2 as ergocalciferol, vitamin D3 as cholecalciferol, vitamin D4 as 22- dihydroergocalciferol, vitamin D5 as sitocalciferol, and any derivatives thereof, including cholecalciferol phosphate), copper, selenium, magnesium, or any combinations thereof.
• the at least one compound is a salt including a nutrient and at least one counter ion.
• the nutrient may be Zn + , Zn 2+ , Fe 2+ , Fe 3+ , Fe 4+ , Fe 6+ , cholecalciferol PCU 2- , or any combinations thereof.
• Counter ions may be selected based upon their ability to neutralize the charge of the nutrient.
• the counter ion may include ethyl maltol, maltol, kojic acid, propionate, hexanoate, oleate, chloride, phosphate, or any combination thereof.
• the at least one compound may be a nutrient-counter ion salt including iron ethyl maltol, iron maltol, iron kojic acid, iron propionate, iron hexanoate, iron oleate, zinc ethyl maltol, zinc maltol, zinc kojic acid, zinc propionate, or any combinations thereof.
• a non-limiting example patch or composition configured for vitamin D delivery may include vitamin D that has been phosphorylated to provide for permeation through the skin.
• the synthesis and resulting compound may be optimized for skin delivery.
• an active agent layer may contain zinc and iron, zinc and vitamin D, iron and vitamin D, or zinc and iron and vitamin D. It may be desirable to include combinations of compounds or compositions which are synergistically absorbed or have a particular health benefit when administered at the same time, such as a patch which contains a combination of vitamin D and calcium.
• the patches disclosed herein include an active agent layer (102) including at least one compound (108).
• the active agent layer includes at least one compound or composition, and further includes a solvent. These embodiments may also be referred to as ‘reservoir patches.’
• the solvent may be selected and optimized for solubilizing the at least one compound or composition. In some cases, it may be desirable to fully solubilize the at least one compound or composition. In other cases, it may be desirable to partially solubilize the at least one compound or composition (z.e., some solids remain undissolved). It may be desirable for the active agent layer to include a supersaturated solution of the at least one compound or composition in a solution.
• a supersaturated solution is a solution which contains more than the maximum concentration of solute (z.e., the at least one compound or composition) than is capable of being dissolved in a given volume of solvent at a given temperature.
• Suitable solvents may include propylene glycol, methanol, ethyl acetate, ethanol, hexane, polyethylene glycol, diethylene glycol monoethyl ether, glycerol, triacetin, miglyol, liquid paraffin, sodium laureth sulfate, Tween 85, Span 20, Kolliphore®, Natrsol M250, dimethicone, or any combination thereof.
• a non-limiting example patch configured as a zinc ethyl maltol reservoir patch may be made from a composition of 60 mg of zinc ethyl maltol dissolved in 3 mL of methanol, followed by adding 1 mL of PEG 400: PEG1000 (8%w/w: 2%w/w).
• Significant transdermal permeation may be provided by a supersaturated solution of PEG400:PEG1000.
• a supersaturated solution based on PEG400/PG6000 may be used to achieve 28.8 mg/mL of elemental zinc.
• a non-limiting example patch configured as an iron delivery patch or composition may include a range of suitable excipients and solvents to formulate a solution of high concentration that could be loaded into the patch reservoir.
• Potential solvents include: ethanol, methanol, PG, PEG400, SDS, Tween 85, Span 20, Kolliphore®, Natrsol M250 and dimethicone.
• the reservoir patch or composition may be made via heat sealing a packing material onto a controlled release membrane.
• the patch backing (3MTM ScotchpakTM Polyester Backing Film Laminate 9730, UK) may be placed on the surface of a heat press machine (HP230B, China), a 200 pL solution of the iron nutrient-counterion complex composition may be loaded onto the backing and then may be sealed using heat onto the controlled release membrane.
• a 200 pL solution of the iron nutrient-counterion complex composition may be loaded onto the backing and then may be sealed using heat onto the controlled release membrane. Examples of iron reservoir patches and their performance as described herein are provided throughout the Examples and particularly in FIG. 28.
• the patches disclosed herein include an active agent layer (102) including at least one compound (108) (i.e., a composition for transdermal delivery).
• the active agent layer includes at least one compound or composition and further includes an adhesive. These embodiments may also be referred to as ‘drug-in-adhesive’ or ‘adhesive’ patches.
• the at least one compound or composition is solubilized within the adhesive and is dried in the adhesive without crystallization (i.e., crystals, precipitates, particulates, solids).
• Suitable adhesives may be selected and optimized for solubilizing the at least one compound or composition.
• adhesives may include polyisobutene, acrylates copolymer, amine-compatible silicone, polymethacrylate, or any combination thereof.
• the patches, methods, compounds, and compositions disclosed herein may be formulated to further include one or more polymers, penetration enhancers, stabilizers, plasticizers, pH regulators, thickeners, colorants, desiccants, solubility enhancers i.e., solubilizer), or any combinations thereof.
• the penetration enhancer may include polyethylene glycol.
• the patch may further include one or more antioxidants.
• the antioxidant may be vitamin E, vitamin E derivatives, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), L-cysteine, and propyl gallate.
• the solubility enhancer may be Tween, such as Tween-85, or dodecyl amine.
• the patches, methods, compounds, and compositions disclosed herein may also be further formulated to include one or more binding agents, filling agents, lubricating agents, suspending agents, flavoring agents, preservatives, buffers, wetting agents, disintegrants, buffers, bacteriostats, and effervescent agents.
• Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PHI 01 and Avicel® PHI 02, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCCTM).
• Suitable lubricants may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
• preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.
• the transdermal patch 100 may be placed on a skin surface of a subject in need.
• a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal.
• a “subject in need” may include a subject having a disease, disorder, or condition that is responsive to therapy with a patch for transdermal delivery of at least one compound, the patch including a backing layer, an active agent layer including at least one compound, and a permeable-membrane release layer configured to transfer the at least one compound to a skin surface.
• the at least one compound may include any one of or any combination of the presently disclosed compounds described above and below.
• a “subject in need” may include a subject having inadequate nutritional intake, malabsorption, lifestyle and environmental factors which reduce exposure to sunlight, or increased requirements for the at least one compound.
• the subject in need may have a disease associated with a nutrient deficiency.
• the subject in need may have a disease which has at least one symptom, or at least one root cause, alleviated by transdermal delivery of at least one compound or composition.
• the subject in need may also include a subject for which there is a desire to increase levels of at least one compound (i.e., a composition for transdermal delivery), independent of whether the subject has a particular disease, disorder, or condition.
• the phrase “effective amount” or a “therapeutically beneficial amount” means that compound or composition dosage that provides the specific pharmacological response or amount delivered for which the compound or composition is administered in a significant number of subjects in need of such treatment.
• An effective amount of at least one compound or composition that is administered to a particular subject in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.
• the compounds and compositions utilized in the patches and methods disclosed herein may be formulated to include: (a) a therapeutically effective amount of at least one compound as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents.
• the methods, patches, and compositions may be administered to provide the one or more compounds at a daily dose of about 0.05mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.5 mg, about 1.0 mg, about 1.5mg, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg over 24 hours from approximately a 1 inch x 1 inch or 3.1 x 3.1 cm patch.
• the transdermal patch may position on the subject in need in any suitable placement, such as on an arm, leg, or trunk of the subject in need. Preparation of the area of skin where the patch will be placed may be prepared in order to optimize the adherence or contact of the patch to the skin. Skin preparations may include, cleansing, shaving, softening, dermaplaning (sometimes referred to as ‘dermatoming’), soaking, or any combinations thereof. [0064] Disclosed herein is a method of delivering a compound or composition to a subject in need, including applying a patch for transdermal delivery of a compound, as described above and below.
• Applying may include adhering, placing, laying, smoothing, or otherwise contacting the patch with the skin so that the permeable-membrane release layer, which permits the release of the at least one compound or composition out of the patch, lays in close proximity with an area of skin on the subject in need. It may be necessary for the skin of the subject in need to remain in contact with the patch for a duration of time in order for a therapeutically beneficial amount of the at least one compound or composition to transfer onto the skin.
• the patch may remain in contact with the skin of the subject in need for about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hour, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, or about 24 hours.
• compositions and methods of synthesizing a nutrientcounter ion salt for transdermal delivery including providing at least one nutrient, providing at least one counter ion, solubilizing the at least one nutrient and the at least one counter ion in at least one solvent to produce a solution, optionally adjusting a pH of the solution, and removing the solvent from the solution to produce a nutrient-counter ion salt.
• Suitable nutrients include those previously disclosed above and in Examples below, including zinc and iron.
• Suitable counter ions include those disclosed above and in Examples below.
• the methods described herein may also include synthesis of a mixture of two or more nutrients with the same counter ion. Alternatively, the synthesis may include the same nutrient and a mixture of two or more counter ions.
• Solubilizing the at least one nutrient and the at least one counter ion in at least one solvent includes at least partially solvating the at least one nutrient and the at least on counter ion.
• Suitable solvents may be chosen based upon the individual solubilities of the one or more nutrients and one or more counter ions.
• Suitable solvents may include aqueous solvent and non-aqueous solvents. In some cases, the solvent is water. In other cases, the solvent is combination of water and methanol.
• the pH of the solution z.e., a composition for transdermal delivery
• a composition for transdermal delivery containing at least one nutrient and at least one counter ion.
• Methods of measuring pH already well established in the art such as indicator methods, metal-electrode methods, glass-electrode methods, or semiconductor sensor methods, may be used to measure the pH of the solution.
• the pH of the solution may be adjusted with methods already established in the art, including providing a source of OH" or providing a source of H + .
• Suitable sources of OH" include sodium hydroxide.
• Suitable sources of H + include hydrochloric acid.
• the pH may be adjusted to between about 1.0 to about 8.0.
• the pH may be adjusted to about 1.6, about 4.0, or about 7.4.
• the pH of the as-combined solution of nutrient and counterion and solvent may already be the desired pH and no pH adjustment is desired.
• the solvent is removed from the solution to produce a nutrient-counter ion salt (z.e., a composition for transdermal delivery).
• a nutrient-counter ion salt z.e., a composition for transdermal delivery.
• the nutrient-counter ion salt produced as a result of the solvent removal may include any combination of nutrients and counter ions disclosed herein, including iron ethyl maltol, iron maltol, iron kojic acid, iron propionate, iron hexanoate, iron oleate, zinc ethyl maltol, zinc maltol, zinc kojic acid, zinc propionate, or any combinations thereof.
• the nutrient-counter ion salt may be a solid (z.e., crystalline, nanocrystalline, amorphous), a liquid, an emulsion (z.e., a microemulsion), a gel, a colloid, or any combinations thereof.
• the at least one nutrient is iron and the at least one counter ion is maltol.
• the iron and maltol may form a solution in water and the pH may be measured and adjusted, if necessary, to about pH 4.
• the water may be removed by lyophilization and iron maltol, a nutrient-counter ion salt, may be produced.
• the pH of the aqueous solution of iron and maltol may be impactful to the stoichiometry of the nutrient-counter ion salt.
• the stoichiometry of the nutrient-counter ion salt may result in optimized solubility of the nutrient-counter ion salt in solvents used in reservoir patches for the transdermal delivery of the nutrient to the skin of a subject in need.
• the stoichiometry of the nutrientcounter ion salt may result in optimized transport of the nutrient-counter ion salt to the skin and/or within the skin.
• a vitamin D3 phosphate and pharmaceutically acceptable salts thereof and pharmaceutical compositions comprising the compound, composition for transdermal delivery, and methods for preparing the same, which are suitable for use in the transdermal patches disclosed herein, is detailed in International Publication Number WO 2022/084669, which is incorporated by reference herein, for any purpose.
• a solubility screening may be performed at step 202 to identify or determine a common solvent system at step 204 for the at least one compound.
• a maximum active loading may be determined at step 206, where active loading may determine the maximum amount of the at least one compound (i.e., a composition for transdermal delivery) that may be included in the patch system.
• a transdermal patch or composition may be optimized for coating thickness or permeation enhancement at step 208. Other optimization may include optimizing adhesive properties, adhesive layer thickness, inclusion/percentage of permeation enhancers, and the like.
• the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
• the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
• the terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims.
• the term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
• Zinc chloride (conforms to Ph Eur, BP, USP) and concentrated hydrochloric acid, (HC1) (37%) were purchased from VWR International (UK).
• Pyridine-2,6-dicarboxylic acid (PDCA), 4-(2-pyridylazol) resorcinol (PAR), DionexTM PC 10 post-column pneumatic delivery packages, and DionexTM lonPacTM CS5A IC column and pre-column were purchased from Thermo Fisher Scientific (UK).
• Helium Gas was supplied by BOC (UK).
• Ethyl maltol (FCC, food-grade), maltol ( FCC, food grade), sodium propionate (> 99%), propylene glycol (99%), and diethylene glycol monoethyl ether (Transcutol) (99%) and deuterated methanol (methanol-d4) were purchased from Sigma Aldrich (UK).
• Kojic acid (99%) was purchased from Alfa Aesar.
• Glycerol was purchased from Fisher Scientific (UK).
• Polyethylene glycol 400 (PEG400) was purchased from Fluka (UK).
• Phosphate buffered saline (Dulbecco A) (PBS) was purchased from Oxoid (UK).
• Liquid paraffin BP was purchased from Pinewood (UK).
• Zinc chloride was used as the zinc donor and it was combined with the organic ligands in excess (molar ratio of 1 :2.5) to ensure it was possible to form the 1 :2 Zinc: coordination complexes (FIG. 3).
• To make zinc ethyl maltol, zinc maltol, zinc kojic acid, and zinc propionate complexes 40 mM of zinc chloride solution was prepared by weighing 545 mg into 100 mL with deionised water. Zinc chloride solution was then mixed with 300 mL of a 33.33 mM solution of the required ligand.
• the 300 mL stock solution at a concentration of 33.33 mM of the ligand was prepared by weighing 1401 mg, 1261 mg, 1421 mg, or 960.60 mg of ethyl maltol, maltol, kojic acid, or sodium propionate, respectively and adding it to 300 ml of deionised water (Error! Reference source not found.).
• the pH of the zinc-ligand solution was adjusted using 1 M NaOH or 0.1 M HC1 to achieve the target pH.
• the pH of zinc ethyl maltol and zinc maltol was adjusted to 7.4, while the pH of zinc kojic acid was not adjusted.
• the pH of the zinc propionate complex was adjusted to 7.4, 6.4, 5.4, 4.4, 3.4, and 2.4. Aliquots of 8 ml of the final pH-adjusted mixtures were loaded into 27 mL glass vials and stored in the freezer for a minimum of 24 h. The samples were then placed in a Lyotrap freeze dryer (LTE Scientific Ltd, Oldham, Greater Manchester, UK) with a freeze-drying temperature of 218 ⁇ 5 K and a pressure of 0.03 mBar using an RV3 vacuum pump (Edwards Ltd, Burgess Hill, West London, UK). The freeze-drying cycle length was 2-3 days. The freeze-dried products were visually checked for complete solvent evaporation and stored in the freezer until used.
• the preparation protocol of Zinc complexes The solution was prepared by mixing 100 mL of zinc chloride (solution A) with 300 mL of solution B (ethyl maltol, maltol, kojic acid, or sodium propionate) to achieve a molar ratio of 1 :2.5 (Zinc chloride: counter ion).
• T H NMR spectra were generated using a DRX 400 (Bruker, UK) with Topspin software for data analysis. All zinc complexes were prepared by dissolving 8 mg in 0.8 mL of deuterated methanol (methanol -D4). Proton NMR used a sweep width of 8250.825 Hz, an acquisition time of 4.0 s, an interpulse delay time of 1 s, and 16 scans.
• a two-eluent system was used for zinc determination with pyridine-2,6- dicarboxylic acid (PDCA) in the eluent buffer (7.0 mM PDCA, 66 mM KOH, 5.6 mM K2SO4, 74 mM HCOOH) employing a 1.2 mL/min flow rate and 4-(2 -Pyridylazo) resorcinol (PAR) in the post-column derivatization buffer (0.2 mM PAR, 1.0 M 2- dimethylaminoethanol, 0.50 M NH4OH, and 0.30 M CH3COOH) at flow rate 0.6 mL/min.
• the PDCA eluent was filtered through a 0.2 pm Whatman filter paper.
• the ion chromatography method utilised the bidentate chelating agent, pyridine-2,6-dicarboxylic acid (PDCA), to form negatively charged 1 :2 complexes with Zn 2+ [1], Once separated, the detection of zinc was performed using the colorimetric reagent, 4-(2- pyridylazo) resorcinol (PAR), mixed in excess with the effluent. PAR formed 1 :2 complexes with Zn 2+ in aqueous and aqueous-organic solutions. To optimise the post-column reaction robustness between Zn 2+ and PAR the effect of different post-column reagent (PAR) flow rates on the absorbance of zinc acetate samples in HPLC was tested.
• PAR post-column reagent
• Zinc acetate standard solutions were prepared by serial dilution in the range 1.25 - 20.0 pg/mL (corresponding to 0.44 - 7.13 pg/mL elemental zinc) and assayed employing five five-point calibration curves (three injections per each calibration point) run on three separate occasions. Linearity and range were determined by plotting the data points from all five calibration curves and calculating the regression line by the method of least squares, to obtain a value for the linear correlation coefficient (R) and the coefficient of determination (R 2 ). The limit of detection (LOD) and the limit of quantitation (LOQ) were determined from all five calibration curves utilising Equations 1 and 2:
• LOQ 10*SB/m (Equation 2) [0099] where m is the slope of the regression line, and SB is the standard error of the regression.
• Intra-assay precision was determined from the relative standard deviations (RSD) of the analyte peak areas from three calibration curves of the same standard preparation run consecutively on the first occasion. Inter-assay precision was determined in the same way except from three calibration curves, each from a different standard preparation and run on three separate occasions.
• Accuracy was determined from five prepared sample concentrations in the range 13.40-19.58 pg/mL zinc. The actual concentrations (x values) of the five sample concentrations were calculated from the response (y -values) and the equation of the regression line. The accuracy was then calculated utilising Equation 3.
• A is the actual concentration (pg/mL)
• T is the theoretical concentration (pg/mL) of the sample.
• the zinc ethyl maltol donor was prepared by dissolving 60 mg of the complex in 3 mL of methanol and 1 mL of 80%w/w PEG400: 20% w/w PEG 1000 then added. The solution was left overnight to allow the evaporation of methanol.
• Each piece of skin was sealed between the two compartments using parafilm (Bemis Company, Neenah, WI).
• a 12 mm magnetic stir bar was placed into the receiver compartment of each Franz cell.
• the receiver compartment was loaded with 50 mM phosphate-buffered saline (pH 7.4).
• the cell sealing was verified by Franz cell inversion (6 times) and monitoring solvent back diffusion.
• the temperature of the Franz diffusion was set at 37 °C in a water bath to reach a skin temperature of 32 °C.
• Each formulation (ImL) was applied in the donor compartment that was then covered with parafilm to avoid evaporation. After 24 h, the receiver fluid was collected and analysed by HPLC.
• the Franz cells were dismantled.
• the donor solutions were collected from each cell and transferred into 5-mL glass bottles containing 2 mL of PDCA as extraction fluid.
• This step was followed by wiping the skin surface and the donor compartment with two wet (wetted with extraction fluid) and then two dry cotton buds.
• the skin's permeation area was then cut into small pieces by scalpels and placed into a 5 mL glass bottle containing 4 mL of extraction fluid. All skin samples were kept at the bench with extraction fluid for 1 hour.
• This step was followed by separating each vial's content (cut skin and extraction fluid) into 2 mL Eppendorf tubes that were then loaded in TissuLyser machine for 20 minutes. Samples were then centrifuged for 15 minutes at 13,000 rpm. The content of each Eppendorf tube was filtered using 0.45 pm syringe filters, collected, and analysed by HPLC.
• the formed film on the surface of the backings was examined for consistency and film clarity (clear films showed the drug was not crystalising in the adhesive).
• the optimised drug-adhesive solutions were cast in 10 x 10 cm 2 trays made of the patch backing (3MTM ScotchpakTM Polyester Backing Film Laminate 9730, 3M, Germany).
• An adequate volume of the adhesive was applied to achieve a target coating level of 8 mg/cm 2 (equivalent to 80 g/m 2 ).
• the adhesive layer was covered by an M3 Release liner (SCOTCHPAK 9755), compressed, and then cut using a 34 mm circular manual die cutter (SCP Super Cutting Press). Table 2. Type of patch adhesives and their properties.
• the zinc ethyl maltol patch was made using the DURO-TAK 87-9301 adhesive (Table 3). Zinc ethyl maltol was dissolved in methanol at a concentration of 16 mg/mL. The adhesive was diluted in ethyl acetate in a ratio of 1 %v/v of adhesive to 2%v/v ethyl acetate. The formulation was prepared by mixing the dry equivalent of 59.2 mg of zinc ethyl maltol, 666 mg of DURO-TAK 87-9301, and 74 mg of Transcutol (Table 3). The patch formulations were then cast into an in-house rectangular tray made from backing film material measuring 10 x 10 cm. The mixture was left to dry overnight. The laminate was then applied to the adhesive, and circles of 34 mm diameter were cut using a dye cutting machine.
• the skin permeation and deposition studies were performed as per Section 2, but the skin was glued to the receiver chamber of the Franz diffusion cell with the stratum comeum facing the donor compartment to allow the patches to be placed on the surface of the skin.
• the receiver fluid was PEG400: saline pH 7.4 (80 %v/v: 20%v/v).
• Liner release membranes were investigated including, regenerated cellulose (12-14 k molecular weight cut-off Medical International UK), nylon (0.20 pm, Whatman, UK), and CoTran 9728 (3M, Germany). A 200 pL aliquot of zinc ethyl maltol complex in solution was applied on the liner release membrane, then covered by the backing layer and all layers thermally sealed (HeatPress machine HP230B).
• the zinc ethyl maltol reservoir patch was made by dissolving 60 mg of zinc ethyl maltol in 3 mL of methanol, followed by adding 1 mL of PEG 400: PEG1000 (8%w/w: 2%w/w). The solution left to evaporate overnight to form a supersaturated solution (Table 4). This solution was then loaded into the reservoir patch.
• NMR analysis was conducted to confirm the structure of the synthesised zinc complexes.
• the NMR of ethyl maltol showed a triplet at 1.23 ppm, quartet at 2.75 ppm, doublet at 6.38 ppm, and a doublet at 7.96.
• peaks around 2 ppm, 3.3 ppm, and 4.7 ppm are related to acetone, methanol D4, and water.
• the NMR spectra of zinc ethyl maltol showed a similar splitting pattern and chemical shifts, but the small downfield shifts of the CH2 and CH groups in the NMR indicated the formation of zinc ethyl maltol complex.
• the NMR spectra of maltol and zinc maltol showed the chemical shifts from 2.36 to 2.45 ppm (singlet), from 6.40 to 6.62 ppm (doublet), and from 7.95 ppm to 8.03 ppm (doublet) that indicated complex formation.
• the NMR spectra of the proposed zinc kojic acid showed no chemical shift and similar chemical pattern, suggesting that the complex was not formed.
• the NMR spectra of sodium propionate and zinc propionate (pH 5.4) showed a chemical shift from 1.08 to 1.10 ppm (triplet) and from 2.15 to 2.25 ppm (quartet). Chemical shifting was also noticed with zinc propionate complexes produced at different pH values (Error! Reference source not found.).
• the NMR spectra confirmed the formation of zinc ethyl maltol, zinc maltol, and zinc propionate complexes only.
• the %RSD values for inter-day were calculated based on the injections of elemental zinc concentration of 2.60 (pg/mL) from the three calibration curves derived from three different standard preparations and run non-consecutively on occasions 1-3.
• the reported %RSD for the interday variability was 1.8% (Table 7).
• the intraday variability was calculated based on the middle elemental zinc concentration (2.60 pg/mL), which reported a %RSD of 0.7% (Table 6).
• Waters HPLC's precision showed % RSD of 1.6 % and 1.3 %, for intraday and interday variability, respectively (Table 8).
• the Accuracy of the 5 prepared samples was 98.82 ⁇ 2.80, which is within the level of the ICH guidelines (Table 9).
• Intra-day variability (repeatability, % CV, ⁇ 2% 0.7 for Zn concentration 2.60 pg/mL)
• Table 10 Illustrated most suitable solvents for each compound.
• the symbol (*) indicates the visual solubility of the compound.
• a new calibration curve of zinc chloride in PG/PDCA and zinc ethyl maltol in PG/PDCA was developed to account for PG: PDCA ratio in the recovery study.
• zinc chloride and zinc ethyl maltol calibrations using the skin matrix was constructed to consider the influence of the skin proteins.
• FIG. 6 When comparing the recovery experiment for zinc chloride and zinc ethyl maltol from porcine skin the results were more consistent when the skin calibration curve was used (FIG. 6).
• Using the skin calibration curve a recovery of 80 ⁇ 2% and 93 ⁇ 10% for zinc chloride and zinc ethyl maltol, respectively. No correction was applied to the data to account for the losses observed in the recovery studies as they were considered not to have a significant impact on the subsequent studies. There was some detection of zinc in the blank samples of the skin matrix also indicates endogenous zinc in the skin.
• a skin permeation study was performed by applying 1 mL of zinc chloride (elemental zinc 2.45 mg/mL) and zinc ethyl maltol (elemental zinc 2.10 mg/mL) in PG solutions. After 24 h, deposition studies showed that both zinc chloride and zinc ethyl maltol showed elemental zinc skin deposition of 83.14 pg/cm 2 and 41.00 pg/cm 2 , respectively (FIG. 7). Similarly, both compounds permeated the skin; however, zinc chloride showed superior transdermal permeation (35.82 pg/cm 2 ) compared to zinc ethyl maltol (4.85 pg/cm 2 , FIG. 13).
• DURO-TAK 9301 suitability screening. Drug loading Zinc ethyl DURO-TAK Transcutol Solubility
• Zinc maltol and zinc ethyl maltol complexes were generated, and their formation was confirmed using NMR and UV spectroscopy.
• Solutions of zinc ethyl maltol in PG (2.1 mg/mL) and PEG (6 mg/mL) allowed the permeation of 41 and 173.6 pg/cm 2 of elemental zinc respectively.
• PG 2.1 mg/mL
• PEG 6 mg/mL
• a novel supersaturated solution based on PEG400/PG6000 was engineered to achieve 28.8 mg/mL of elemental zinc.
• porcine skin 237.93 pg/cm 2 of elemental zinc passed through the skin.
• Two types of formulations were generated, a drug in adhesive patch and a reservoir patch.
• Iron chloride hexahydrate C13Fe.6H2O (99%) and sodium hexanoate (99%) were purchased from Acros Organic (UK).
• Ascorbic acid, ammonium acetate, glycerol, acetic acid and oleic acid were purchased from Fisher Scientific (UK).
• Concentrated hydrochloric acid, HC1 (37%) was purchased from VWR Chemicals (UK).
• Ferrozine® ethyl maltol, maltol, sodium propionate (> 99%), sodium oleate (> 95%), propylene glycol, PG (99%), diethylene glycol monethyl ether (Transcutol®) (99%), Kolliphore® EL, Tween 85, Span 20, ethyl acetate, ethanol (absolute > 99.8%), methanol HPLC grade (> 99.9%), chloroform (> 99.8%), and n-hexane (> 95%) were purchased from Sigma Aldrich (UK). Phosphate buffered saline (Dulbecco A) (PBS) was purchased from Oxoid (UK).
• PBS Phosphate buffered saline
• Oxoid UK
• a 100 mL stock solution of 46.7 mM counterion was prepared by weighing 654 mg, 588 mg, or 664 mg of ethyl maltol, maltol or kojic acid respectively, adding the solids to a 100 mL volumetric and solubilising in deionised water.
• the whole 100 ml of the iron chloride solution was mixed with 60 mL of the counterion solutions and the pH was adjusted to the required value (either pH 1.6, 4 or 7.4) using 1 M NaOH.
• the iromcounterion mixture was then split into 4-8 mL aliquots in glass vials of and placed in the freezer for a minimum of 24 h.
• the samples were then placed in a Lyotrap freeze dryer (LTE Scientific Ltd, Oldham, Greater Manchester, UK) with a freeze-drying temperature of 218 ⁇ 5 K. Once the drying chamber was in place and the draining valve closed, the internal pressure of the drying chamber was reduced to 0.03 mBar using a RV3 vacuum pump (Edwards Ltd, UK). The samples were freeze-dried for 2-3 days after which the vacuum was released and the generated powder was stored in the freezer until used.
• a Lyotrap freeze dryer LTE Scientific Ltd, Oldham, Greater Manchester, UK
• Iron hexanoate and iron oleate were produced in a 2-phase liquid extraction system. Iron chloride hexahydrate (1.08 g equivalent to 4 mM) and 1.7 g of sodium hexanoate (equivalent to 12 mM) were added to 60 mL water and pH adjusted to either 4 or 7 using 1 M NaOH. Then 80 mL ethanol and 140 mL //-hexane was added to the iromcounterion solution.
• Ferrozine® was used to quantify iron due to its sensitivity and low interference with a range of biological matrices [3], In the assay Ferrozine® reacts with the divalent iron to form a stable magenta complex in aqueous solution at pH 3-6 [3,4], The method of the assay was developed by Carter 1971 [5], The visible absorption spectrum of the ferrous complex of ferrozine exhibits a single sharp peak with maximum absorbance (A ma ⁇ ) at 562 nm [3], To conduct the assay a reducing agent was prepared by dissolving 20 mg ascorbic acid in a small quantity of water, to which 1.67 mL of concentrated hydrochloric acid was added and diluted to 100 mL.
• a 10% w/v ammonium acetate buffer was prepared, and its pH was reduced to 4-5 using acetic acid.
• a Ferrozine® solution was prepared by dissolving 60 mg of Ferrozine® in small volume of water containing 1 drop of concentrated HC1 and made up to 20 mL with water.
• a 70.2 pg/mL elemental iron stock solution was prepared by weighing 27 mg of iron chloride hexahydrate (ChFe 6H2O) and making it up to 100 mL with phosphate buffered saline. Standards were prepared in phosphate buffered saline via serial dilutions in the range 0- 17.6 pg/mL elemental iron.
• Test solutions of 885 mg/mL iron chloride hexahydrate in PG or 2.6 mg/mL iron ethyl maltol (complex made at pH 7.4) in PG were used to spike 10 pL or 100 pL respectively to each skin layer. This was incubated for 1 h, then 4 mL of extraction fluid (50:50 ethanol: PBS) was added to the stratum corneum vials and 2 mL to the epidermis and dermis vials. The stratum corneum samples were placed on vibrating plate for a minimum of 2 h and then vortexed prior assay.
• the epidermis and dermis samples were transferred to Eppendorf tubes, one stainless steel 5 mm bead was added then the tubes were loaded into the TissuLyser II (Qiagen, UK) for 15 min at 30Hz. The samples were centrifuged (5 min at 13,000 rpm) and the supernatant was assayed for iron content. Note: the calibration solvent was 100% PBS and therefore a new calibration was drawn in 50:50 ethanol: PBS solution to quantify those samples.
• the skin was defrosted.
• the subcutaneous fat removed (2.5 mm thick) and for the dermatomes skin studies the tissued was dermatomed to a 1 mm thickness.
• the tissue was cut into the appropriate size sections (2.19 ⁇ 0.21 cm 2 ) and mounted between donor and receiver compartments of Franz diffusion cells with the SC facing the donor compartment. Each cell was sealed using parafilm and 12 mm magnetic stir bars were placed into the receiver compartment.
• the receiver compartment was filled with the desired receiver solution and the Franz cells were placed in a stirring water bath at 37°C.
• An 80:20 PEG400: water receiver was used for iron propionate solutions, for which a complementary calibration (iron chloride in PEG400: water) was established for the assay of the samples.
• Iron propionate was dissolved in PEG400 (300 pL of 135 mg/mL) and iron propionate was dissolved in Transcutol® (300 pL of 92 mg/mL) due to the optimal solubility of the complexes in these vehicles.
• a 80:20 PG: water receiver was used along with a complementary calibration for iron assay (iron chloride in PG: water).
• the donor solution for the iron maltol made at pH 1.6 was 200 pL of 100 mg/mL complex in Transcutul®, for iron maltol made at pH 4 the donor solution was 200 pL of 23 mg/mL complex in PG and for iron maltol made at pH 4 the donor solution was 150 pL of 16 mg/mL complex in 7.5: 1.5: 1 : 1 water: PG: dimethicone: ethanol.
• the donor solutions were collected into bottles containing the extraction solution. This was followed by skin surface cleaning using four cotton buds, the first two were wetted by the extraction fluid and the second two were used dry. Then two tape striping procedures were performed using 2 tapes to remove any iron on the skin surface with the tape being added to the donor solution and cotton buds in the donor recover vial.
• the Stratum corneum, the dermal and the epidermal layers were isolated in the same way as the recovery method (Section 2.3.2.1).
• Extraction fluid 50:50 PBS: ethanol
• the Stratum corneum samples were placed on vibrating plate for a minimum of 2 h to extract the iron and then vortexed prior assay.
• the epidermis, dermis or dermatomed samples however were incubated with the extraction fluid for 1 h after which were transferred to Eppendorf tubes, one stainless steel 5 mm bead was added and the samples were loaded into the TissuLyser II (Qiagen, Germany) for 30-15 min at 30Hz. The samples were then centrifuged (5 min at 13,000 rpm) and supernatant assayed.
• Adhesive patches were produced by mixing the required volume of adhesive and ethyl acetate then adding the complex dissolved in methanol. For most patches PG was added at the end of the process to act as a penetration enhancer when placed on the skin (Table 16). To produce the patch a sufficient volume of the formulation was applied to the patch backing to achieve a target coating of 8 mg/cm 2 . The patch formulations were casted on a 10 x 10 cm square tray made of the backing (3MTM ScotchpakTM Polyester Backing Film Laminate 9730, UK). Following the complete drying of the solvents, the adhesive layer was covered by M3 Release liner (SCOTCHPAK 9755, UK), compressed, and then cut using 34 mm circular manual die cutter (SCP Super Cutting Press, UK).
• M3 Release liner SCOTCHPAK 9755, UK
• dermatomed skin (1 mm) was glued onto the receiver compartment of the Franz cell that contained 80:20 PG: water.
• the patches that were tested were A2, A3, A4 (Table 16) as well as the existing commercial iron transdermal patch.
• the receiver fluid was assayed using the quantitative iron assay and a compatible calibration in the same medium (80:20 PG: water) was used.
• the skin deposition was determined by removing the dermatomed skin from the donor compartment when the 24 h was completed, chopping into smaller pieces using scissors and incubating with the extraction fluid, ethanol.
• the samples were then transferred to Eppendorf tubes containing one stainless steel 5 mm bead and loaded into the TissuLyser II (Qiagen, Germany) for 30 min 30Hz. The samples were centrifuged (5 min at 13,000 rpm) and supernatant assayed for iron content.
• the reservoir patch was made via heat sealing a packing material onto a controlled release membrane.
• the patch backing (3MTM ScotchpakTM Polyester Backing Film Laminate 9730, UK) was placed on the surface of the heat press machine (HP230B, China), a 200 pL solution of complex was loaded onto the backing and then it was sealed using heat onto the controlled release membrane.
• a circular stainless-steel template (outer circle of 5.3 cm and inner circle of 3.2 cm) was used to form the seal at 90 °C for 1.5 min.
• the controlled release membranes that were used were M3 CoTran 9728, Whatman Nylon (0.45 pm nylon membrane, 47 mm diameter) and Millipore Durapore (0.22 pm GVHP membrane).
• Table 17 summarises the reservoir patches that were made.
• the skin deposition was determined by removing the dermatomed skin from the donor compartment when the 24 h experiment was completed, chopping it into smaller pieces and incubating with the extraction fluid, ethanol. The samples were then transferred to Eppendorf tubes containing one stainless steel 5 mm bead and loaded into the TissuLyser II (Qiagen, Germany) for 30 min at 30Hz. The samples were then centrifuged (5 min at 13,000 rpm) and supernatant assayed for iron content.
• Iron propionate (made at pH 7) 54% Red/brown flakes
• FIG. 2 illustrates the structures of the complexes.
• a calibration curve was constructed using both a UV spectrophotometer and UV plate reader.
• the plate reader did detect concentrations ⁇ 2.8 pg/mL of elemental iron and hence a higher concentration range than that used for spectrophotometer was used on the plate reader. Both techniques were verified as suitable to detect iron using the Ferrozine approach (Table 19).
• the limits of detection (LOD) and limits of quantification (LOQ) were significantly different between the two detection methods.
• the spectrophotometer had a LOD and a LOQ of 0.26 and 0.86 pg/mL elemental iron respectively. While the plate reader had higher values of 0.94 and 3.12 pg/mL elemental iron respectively (Table 19).
• Iron complex formation confirmation using UV scanning spectrophotometry [0220] For iron complexed with ethyl maltol, maltol and kojic acid the lateral shift in the kmax compared to the iron confirmed complex formation. All complexes were scanned at the same concentration. Iron ethyl maltol and iron maltol complexes were made at both pH 4 and 7.4 while iron kojic was only made at pH 7.4.
• IR Infrared
• Ci C 2 1609 1566 1603 1607
• Iron propionate also showed a difference in solubility when it was made at different pH values (Table 29) with the complexes made at pH 7 shown to be insoluble in the tested solvents.
• Iron hexanoate on the other hand showed a poor solubility ( ⁇ 20mg/mL visual solubility of complex) when was made at pH 4 and 7 and tested in PG, PEG4000 and Transcutul.
• Iron oleate was made without any pH adjustments (at pH 2.5) and had a substantial solubility in Miglyol 810N and liquid paraffin (Table 30).
• Iron ethyl maltol solubility screen (*visual solubility). Solvent Iron ethyl maltol made at pH 7.4 Iron ethyl maltol made at pH 8.
• Iron chloride hexahydrate had a high solubility in PG allowing the dosing of 370 mg elemental iron, of which 180 mg permeated (48%) the skin after 24 h through the 2 cm 2 permeation area of the experiment. While that dose was 40 times above the desired target (4.6 mg), it led to the necrosis of the skin (FIG. 15), making it inappropriate candidate for skin delivery.
• the amount permeated and deposited in each skin layer is illustrated in FIG. 16.
• the patch development process was performed using a systematic process (FIG. 21). Initially a common solvent for both drug and polymers was identified. This was followed by optimising drug loading, adhesive properties, adhesive layer thickness, and inclusion/percentage of permeation enhancers.
• Iron ethyl maltol was only compatible with DURO-TAK 9301.
• the concentration of the stock solution of iron ethyl maltol in methanol had a major influence on the complex loading achieved in the adhesive patch. For example, initially a 4% loading was not achieved when the stock concentration was 20 mg/mL however when it was reduced to 10 mg/mL a 4% loading was achieved (Table 32).
• Adhesive (mg) .
• the four reservoir patches showed better skin permeation and deposition compared to the previously assessed adhesive patches (FIG. 26).
• the ferric propionate reservoir patch formulated using a PG solution gave approximately the same deposition into the skin compared to the same complex applied to the skin as a PG solution (8 pg /cm 2 ) (FIG. 19 vs. FIG. 26), but neither the patch nor the solution allowed iron to pass through the skin.
• the two iron maltol patches that used the Durapore rate controlling membrane showed similar delivery of iron into the skin as the iron propionate patch (5 ⁇ 2 pg/cm 2 for the dimethacone patch and 7 ⁇ 2 pg/cm 2 for the iron maltol patch.
• the iron maltol durapore patches gave a significantly lower deposition into the skin compared to the previously tested solutions applied directly to the skin (see FIG. 19) and they did not show any transdermal penetration.
• the iron maltol patch using the nylon membrane gave the best penetration into the skin at 5 ⁇ 3 pg/cm 2 and the best transdermal penetration at 22 ⁇ 8 pg:cm 2 .
• images of the skin after the application of patch show that the patch did not apply the solution to the skin evenly suggesting that there is scope for further patch development and potential to improve the delivery further.
• the iron maltol reservoir system delivered 53 times more iron into the skin (iron maltol total delivery was 27pg/cm 2 vs the existing commercial iron transdermal adhesive patch 0.51 pg/cm 2 ).
• iron ethyl maltol, iron maltol, iron propionate and iron oleate were manufactured, and their formation was confirmed using IR and UV spectroscopy.
• solubility and skin compatibility of the formed complex was dependent on the pH of complex solution prior to lyophilisation, which is a novel finding that may make the patches patentable.
• the iron maltol complex produced at pH 4 demonstrated a high solubility (18 mg/mL in PG and PEG400) without inducing skin necrosis. Hence, it was selected for further development.
• Two types of product prototypes were manufactured: a drugin-adhesive and a reservoir patch.
• the iron complexes in the polyacrylate adhesive (Duro-TAK 9301) transdermal patch did not delivery clinically meaningful quantities of iron into the skin.
• the existing commercial iron transdermal adhesive patch showed limited skin delivery (4.9 pg of elemental iron / 3.1 x 3.1 patch) and no permeation.
• a novel solution of iron maltol (made at pH 4) in PG (18 mg/mL), formulated into a reservoir patch prototype with the aid of a nylon membrane was the best system assessed in this work.
• the patch was scaled up to a 3.1 cm x 3.1 cm reservoir patch then based on its delivery in this study per cm 2 of skin it would deliver 360 pg of iron maltol into porcine skin and 1650 pg through porcine skin over 24 hours, which would provide a 2 mg of iron maltol over 24 hours from 3.1 x 3.1 cm patch.
• images of the reservoir patch and skin after delivery demonstrate that the contact between the patch and the skin could be optimised further and the consequences of the orange colour of the complex needs to be investigated.
• n-Hexane fractions ethyl acetate, sodium hydroxide (NaOH), hydrochloric acid (HC1), absolute ethanol and trifluoroacetic acid (TFA) HPLC grade (> 99 %) were purchased from Fisher Scientific Ltd UK.
• Deuterated chloroform (CDCh) 99.8 %, 0.03 % TMS was purchased from Merck, Germany.
• Phosphate buffered saline (Dulbecco A) (PBS) was purchased from Oxoid, and formic acid was purchased from Honeywell.
• a range of cosmetic grade adhesives were used, polyisobutene (Oppanol B12 and Oppanol N100) were supplied by BASF (Germany), Acrylates copolymer (DURO-TAK 87- 9301, 87-2516 and 87-2052) were purchased from Henkel (USA), and amine-Compatible Silicone (BIO-PSA 7-4302) were purchased form Dupont (USA). Porcine ear skin was purchased from the local butcher Ginger Pig, UK. All chemicals were reagent grade unless otherwise stated.
• the vit-D THF solution was added dropwise to a POCh solution in THF (POCI3 (0.510 mL, 5.46 mmol, 1,4 eq) over a period of 40 min, (15 mL).
• POCI3 0.510 mL, 5.46 mmol, 1,4 eq
• This reaction was allowed to stir at room temperature under nitrogen for 2.5 h to generate the intermediate, phosphorochloridate.
• Thin layer chromatography (TLC) analysis (6:4, hexane: ethyl acetate) using Merck TLC aluminium sheets silica 60-F254 to confirm the reaction was complete.
• the main reaction by-product, solid white triethylamine hydrochloride powder was removed by suction filtration using a Buchner filter and 1 pm Whatman filter paper.
• Carbon NMR used a sweep width of 24038.461 Hz, an acquisition time of 1.4 sec, an interpulse delay time of 2 sec and 1000 scans all within a zgpg30 pulse program.
• Phosphorus NMR used a sweep width of 64724.918 Hz, an acquisition time of 0.5 sec, an interpulse delay time of 2 sec and 16 scans all within a zgpg30 pulse program.
• cholecalciferol phosphate disodium analysis was carried out using a 1100 series HPLC with a G1322A Degasser (Agilent, USA) attached to a mass spectrometer (LCQ DECA XP, USA) using a Phenomenex Luna C18 (50 x 2 mm, 2.5 pm particle size, 100 A diameter).
• the ion trap was operated in positive electrospray ionisation (ESI) mode.
• the sample concentration was 1 mg/mL, prepared in 45 % acetonitrile (ACN), 45 % water, 0.1 % formic acid, and 10.7% methanol.
• the mobile phase was carried out as a gradient between 0.1%TFA in water and 0.1% TFA in ACN, starting with a 50:50 volume ratio and ending with a 98:2 by the end of a run time of 14.5 min.
• the column temperature was 45°C, a flow rate of 0.2 mL/min, and ion source temperature of 350 °C.
• the cholecalciferol phosphate acid form was analysed on a Waters Xevo TQ LC-MS/MS.
• the liquid chromatography utilised a biphenyl microbore 50 mm length x 2.1 mm i.d. 5 pm Phenom enex Kinetix column.
• the ion trap was operated in positive electrospray ionisation (ESI) mode.
• the sample was prepared at 1 mg/mL in 90: 10 volume ratio of methanol: formic acid 0.1%.
• a gradient method was employed using mobile phase A containing 70 % acetonitrile, 10 % water, 0.1 % formic acid and 20% methanol and mobile phase B containing 80 % water, 0.1 % formic acid and 20% methanol.
• the sample injection volume was 10 pL and the runtime was 18 min.
• the autosampler was at 8 DC, column oven temperature of 40 °C, a flow rate of 0.3 mL/min, and ion source temperature of 350 °C.
• HPLC analysis was carried out using an HPLC system consisting of an autosample injector (Jasco AS-4050, Japan), a pump (Jasco PU-4180, Japan), and a photodiode array detector (Jasco MD-4017, Japan).
• the data was collected and analysed using the ChromNAV software.
• the HPLC column was a Phenomenex C18 Synergi Hydro-RP-column (250 x 4.6 mm, 4 pm particle size, 80 A diameter) with a Phenomenex-AQ C18 guard a for cholecalciferol and a Phenomenex Kinetex Biphenyl column (250 x 4.6 mm, 5 A diameter) for cholecalciferol phosphate.
• the mobile phase was ACN: MeOH (55:45) for cholecalciferol and MeOH: PBS pH 3.2 (90: 10) for cholecalciferol phosphate (disodium and acid). Both mobile phases were filtered through a nylon membrane filter and degassed using a sonicator.
• the injection volume was 20 pL, the flow rate for cholecalciferol was 2.0 mL/min and it was 1.0 mL/min for cholecalciferol phosphate (disodium and acid).
• a UV detection wavelength of 265 nm was employed with run times of 10 min for cholecalciferol and cholecalciferol phosphate disodium salt and 15 min for cholecalciferol phosphate acid.
• the Franz cell methodology was also used to study how the vitamin D is deposited into the skin with extraction from the three layers of the skin being verified using a recovery measurement using spiked samples.
• test solutions (1 mg/mL of cholecalciferol or cholecalciferol phosphate disodium in PG) were spiked (500 pL) into a sample bottle containing the SC and (200 pL) into the sample bottles containing the epidermis and dermis.
• the samples containing the spiked test agents were left for 1 h for the agents to absorb into the tissues.
• donor solutions were prepared by adding 2.5 mg/mL of cholecalciferol or cholecalciferol phosphate disodium in 100% PG. Note the acid form of cholecalciferol phosphate disodium was not tested in this experiment.
• the skin was defrosted, and the subcutaneous fat was manually removed using a scalpel. The tissue was cut into the appropriate size sections (0.25 mm thick, 2.19 ⁇ 0.21 cm 2 ) and mounted with the SC facing the donor compartment between donor and receiver compartments of Franz diffusion cells.
• Each pieces of skin was sealed between the two compartments using parafilm (Bemis Company, Neenah, WI, and 12 mm magnetic stir bars were placed into the receiver compartment.
• the receiver compartment was filled with 50:50 PG: mobile phase (the mobile phase was made of 55:45 acetonitrile: ethanol for cholecalciferol and 90: 10 methanol: water pH 3.2 for cholecalciferol phosphate disodium).
• the cell sealing was confirmed by cell inversion and monitoring solvent back diffusion. Sink conditions were maintained throughout the transport assays as the drug concentration did not exceed 10% of its statured solubility.
• the temperature of the Franz diffusion (2.2 cm 2 ) was maintained at 37 °C in a water bath, to achieve a skin temperature 32 °C.
• the Franz cells were dismantled, and the donor solutions were collected into bottles containing 10 mL of extraction fluid; PG: EtOH (50:50) for cholecalciferol and MeOH: water (90: 10) for cholecalciferol phosphate disodium. This was completed by wiping the surface of the skin and inside of the donor compartment with two wet and then two dry cotton buds.
• the SC was removed from the epidermis by tape stripping (as mentioned above). The first two pieces of tape were added into the donor solution, as the first 2 strips were considered as part of the applied formulation and its removal was part of the formulation wash off.
• the receiver compartment was filled with either 50:50 PG: mobile phase (90: 10 methanol: water pH 3.2), or 80:20 PG: distilled water, or 75 mM pH 7 saline.
• the cell sealing was confirmed by cell inversion and monitoring solvent back diffusion. Sink conditions were maintained throughout the transport assays because the active concentration did not exceed 10% of its saturated solubility in the receiver fluid throughout the experiments.
• the temperature of the Franz diffusion was maintained at 37 °C in a water bath, to set up the membrane temperature to 32 °C.
• the laminate was then added to the adhesive and circles of 34 mm diameter were cut using a dye cutting machine and the release of the cholecalciferol was assessed using the Franz diffusion cells fitted with a nylon membrane over 24 h.
• a nylon membrane (0.20 pm) was glued to the receiver chamber and no donor chamber was employed to allow the patch to be placed directly on the surface of the nylon membrane.
• a cholecalciferol phosphate disodium saturated PG solution was used as a comparator with the standard Franz cell set-up previously described.
• the receiver fluid was 80% PG 20% water for both release testing from the solution and the patch.
• Patch Ingredient Ingredient Ingredient Dry weight Theoretical conversion (per 100 mg) Patch
• the laminate was then added to the adhesive and circles of 34 mm diameter were cut using a dye cutting machine and the release of the cholecalciferol was assessed using the Franz diffusion cells fitted with a cellulose membrane over 24 h as described above for the sodium form of the active.
• a regenerated cellulose membrane was employed in the Franz cells for the cholecalciferol phosphate acid release testing as the drug saturated solution experiments had demonstrated that it allowed more cholecalciferol to pass through it compared to nylon thus making the release experiment more sensitive.
• the regenerated cellulose membrane was heated to 60°C for 30 minutes after which the double layer was separated and mounted onto the Franz cell ( Figure 4) by sealing between the two compartments using parafilm, and 12 mm magnetic stirrers were placed into the receiver compartment that contained the receiver fluid, 50:50 PG: mobile phase (90: 10 Methanol: water pH 3.2), which was modified from the disodium release studies to facilitate more cholecalciferol to pass the membrane and thus make the testing more sensitive.
• the temperature of the Franz diffusion was maintained at 37 °C in a water bath, to set up the membrane temperature to 32 °C. Receiver fluid was collected after 24 hours and analysed by the developed HPLC method.
• Patch Ingredient Weight Ingredient Theoretical z . e conversion Patch
• Oppa-Double Oppanol B12 1,200 mg 1,200 mg 95%
• Thickness VDP acid 65 mg 65 mg 5%
• Enhancer VDP acid 65 mg 65 mg 5%
• dermatomed skin 500 pm was mounted into Franz cells by sealing between the two compartments using parafilm, and 12 mm magnetic stirrers were placed into the receiver compartment that contained the receiver fluid, 80:20 PG: water.
• the temperature of the Franz diffusion was maintained at 37 °C in a water bath, to set up the skin temperature to 32 °C.
• Receiver fluid was collected at 2,5,21,22 and 24 hours and analysed by HPLC method.
• the skin deposition was determined by removing the dermatomed skin from the donor compartment when the 24 h was completed and incubating it with 2 mL of extraction fluid (MeOH: water, 90: 10).
• the samples were then transferred to Eppendorf tubes with the addition of one metal ball and loaded into the TissuLyser II (Qiagen) for 15 minutes at 30Hz. The samples were then centrifuged (5 minutes at 13 k rpm) and supernatant assayed.
• the UV absorbance maximum for cholecalciferol phosphate in the optimised mobile phase was 265 nm, which was used as the detection wavelength throughout the study.
• the calibration curves were linear over the concentration range of 1-100 pg/mL and 3.1-2000 pg/mL for cholecalciferol and cholecalciferol phosphate disodium, respectively.
• Limits of detection (LoD) and quantification (LoQ) for cholecalciferol were calculated at 0.59 pg/mL and 1.98 pg/mL, respectively.
• LoD and LoQ were calculated at 13.81 pg/mL and 46.06 pg/mL for cholecalciferol phosphate disodium, respectively (Table 43).
• the solvent used to extract the test agents from the skin were 50:50 PG: ethanol for cholecalciferol and 90: 10 methanol: water for cholecalciferol phosphate respectively because the actives showed good solubility in these solvents.
• the receiver solutions of 50:50 PG: mobile phase (90: 10 methanol: water pH 3.2) for cholecalciferol phosphate disodium was selected as the active showed good solubility in this solution and it was thought more compatible with the skin compared to the extraction fluid.
• the chemical stability of cholecalciferol in the receiver fluid was not assessed as it was not expected that this agent would pass through the skin.

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