WO2000012060A1 - Transport induit electriquement de penetrants charges a travers des barrieres - Google Patents

Transport induit electriquement de penetrants charges a travers des barrieres Download PDF

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Publication number
WO2000012060A1
WO2000012060A1 PCT/EP1998/005539 EP9805539W WO0012060A1 WO 2000012060 A1 WO2000012060 A1 WO 2000012060A1 EP 9805539 W EP9805539 W EP 9805539W WO 0012060 A1 WO0012060 A1 WO 0012060A1
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Prior art keywords
barrier
penetrants
pores
penetrant
transport
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PCT/EP1998/005539
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English (en)
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WO2000012060A8 (fr
Inventor
Gregor Cevc
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Idea Innovative Dermale Applikationen Gmbh
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Application filed by Idea Innovative Dermale Applikationen Gmbh filed Critical Idea Innovative Dermale Applikationen Gmbh
Priority to BR9816014-1A priority Critical patent/BR9816014A/pt
Priority to CN98814268A priority patent/CN1322129A/zh
Priority to AU97404/98A priority patent/AU9740498A/en
Priority to EP98951338A priority patent/EP1107729A1/fr
Priority to PCT/EP1998/005539 priority patent/WO2000012060A1/fr
Priority to JP2000567180A priority patent/JP2002523442A/ja
Priority to KR1020017002688A priority patent/KR20010106462A/ko
Priority to MXPA01002149A priority patent/MXPA01002149A/es
Priority to CA002345355A priority patent/CA2345355A1/fr
Publication of WO2000012060A1 publication Critical patent/WO2000012060A1/fr
Publication of WO2000012060A8 publication Critical patent/WO2000012060A8/fr

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    • 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
    • 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
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • 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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

  • This invention relates to a preparation comprising penetrants formed by single molecules or by arrangements of molecules, said penetrants being capable of penetrating the pores of a barrier even when the average diameter of said barrier pores is less than the average diameter of said penetrants, since the penetrants are adaptable to the pores, and said penetrants being capable of transporting agents through said pores, or enabling agent permeation through said pores after the penetrants have entered said pores; the average diameter and the adaptability of said penetrants being selected, and said penetrants and / or said agents being provided with sufficient electrical charges, to enable and / or control agent transport through said pores by said penetrants, or agent permeation through said pores after penetrant entry into said pores, under the influence of a suitable electrical driving force, said selection at the same time maintaining sufficient penetrant stability.
  • This invention also relates to a method for effecting the electrically driven transport of said penetrants and associated molecules through the pores in a barrier.
  • Charged entities may migrate spontaneously from the high to the low electrostatic potential site, unless prevented from doing so by an obstacle, such as a barrier.
  • the driving electrostatic force is proportional to the total charge on an entity and to the electrostatic potential difference.
  • Material flow also depends on the system's resistance to resulting motion. Consequently, the electrically driven transport across a barrier is sensitive to the number, width and characteristics of pores in a barrier, which together define the barrier permeability, P, and its inverse, the barrier resistance.
  • P barrier permeability
  • One example for such pourous barrier is the skin, which typically contains pores (in the unwidened state) with the diameter of a few Angstroms, approximately. Any transcutaneous electric potential that drives an ion flux across the skin tends to widen some hydrophilic channels in the organ. This typically happens at the worst packed sites between the cells, where the biggest opportunity for the transport enhancement resides.
  • Electrophoresis electrophoresis
  • electroporation for the electrically induced flow through narrow passages and for the widening of more extended passages, respectively.
  • an electric current of approximately 0.4 mA cm "2 or less will activate a small proportion of narrow (-0.5 nm) hydrophilic channels pre-existing between the cells in the skin. Such channels then remain open for many hours (Green, P.G.; Hinz, R.S.; Kim, A.; Szoka, F.C. Jr.; Guy, R.H. (1991) Iontophoretic delivery of a series of tripeptides across the skin in vitro. Pharm. Res. Sep; 8: 1 121-7), but remain narrow ( ⁇ 3 nm, in nude mice), when a transcutaneous voltage remains in the physiologically tolerable range ( ⁇ 3 V for a 1 cm ⁇ patch). The widest channels are negative inside.
  • Electrophoresis across the skin, consequently, is feasible for certain polypeptides but is practically useless for the delivery of proteins or other large penetrants (for reviews see refs. Green, P.G.; Hinz, R.S.; Kim, A.; Szoka, F.C. Jr.; Guy, R.H. (1991) lontophoretic delivery of a series of tripeptides across the skin in vitro. Pharm. Res. Sep; 8: 1121-7; Green, P. G.; Flanagan, M.; Shroot, B.; Guy, R.
  • Transcutaneous channels opened by the low voltages and tolerably small currents only cover some 0.005 % of the total treated area, despite their seemingly high number
  • Transcutaneous channels size is affected by a number of parameters. For example, a channel widens with increasing electrostatic potential as well as with decreasing supporting electrolyte concentration, but the latter variation is practically only possible within relatively narrow limits. Moreover, no teaching was given to date on how to improve the transport across a barrier by using such principles. Perhaps, this is due to the fact that the electrophoretic channels in the skin are charge and molecular weight selective (Banga, A.K.; Chien, Y.W. (1993) Hydrogel-based iontotherapeutic delivery devices for transdermal delivery of peptide/protein drugs. Pharm. Res.
  • transcutaneous electrical potential gradient ( ⁇ ) also activates the electromotive forces which try to drive charged penetrants through the channels. This gives rise to an additional term in Fick's transport equation used to model transbarrier (e.g. transcutaneous) transport (see further discussion):
  • Electrophoresis represents the direct flow of charged molecules in an electric field under the electrode. Drug molecules must therefore be placed at electrodes having a polarity of the same charge as the agent. Under such circumstances, the flux magnitude is proportional to the net number of charges on each migrating molecule and to the applied potential. Further important factors are drug concentration and diffusivity in the barrier or skin (see equation given later in the text).
  • electrophoretic current normally also comprises contributions from the supporting electrolyte ions (e.g. Na + , Cl"). These are often diminant making the drug contribution only a minor part of the measured current. Increased ion concentration under the electrodes therefore lowers the useful part of electrophoretic flow (Pikal, M.J.; Shah, S. (1990b) Transport mechanisms in iontophoresis.
  • III An experimental study of the contributions of electroosmotic flow and permeability change in transport of low and high molecular weight solutes. Pharm. Res. 7: 222-9), as can be see from simple differential calculation.
  • Electro-osmotic flux that is, the flow of water associated with the transported ions carrying uncharged species, also relies on hydrophilic channels in the skin and on the applied potential. Flux under an anode typically exceeds the cathodal values, probably due to the different average size of positively and negatively charged channels. The average flux value reaches a steady-state during 10 hours of constant-current iontophoresis (0.36 mA cm ⁇ 2) at the level a few times higher than at the beginning ( ⁇ 3 ⁇ L h"l cm"2: Kim, A.; Green, P.G.; Rao, G.; Guy, R.H. (1993) Convective solvent flow across the skin during iontophoresis. Pharm. Res. 10: 1315-20).
  • Electrophoretic enhancement of molecular motion across the skin was only partly successful to date (for a recent survey of products and developments see: Cevc, G. (1997) Drug Delivery Across the Skin. Exp. Opin. Invest. Drugs 6: 1887-1937). Particularly poor results were achieved with macromolecules (see, e.g. the reviews by Siddiqui, O.; Chien, Y. W. Nonparenteral administration of peptide and protein drugs. Crit. Rev. Therap. Drug Carrier Syst. 1987, 3: 195-208 and Banga, A.K.; Chien, Y.W. (1993) Hydrogel-based iontotherapeutic delivery devices for transdermal delivery of peptide/protein drugs. Pharm. Res.
  • F hyd denotes the force acting on each monomer in the aggregate and RT is the thermal energy.
  • vesicles with a membrane sufficiently flexible to result in a low vesicle deformation energy are subject to strongly anisotropic (ideally: unidirectional) stress, 'force' or pressure.
  • strongly anisotropic stress, 'force' or pressure may lead to vesicle motion through a barrier even when the pores in such barrier are smaller than the vesicle diameter.
  • Significant material flow in the desired direction can result from this.
  • a further expected complication in electrophoresis of large objects is the possibility that an applied electrical potential tends preferentially to pull individual charged molecules from an aggregate over the barrier, instead of transporting the whole aggregate.
  • the expected size-dependence of transport resistance nourishes this notion, especially for the relatively strongly water soluble, charged aggregate components.
  • the highly deformable aggregates which consist of substances of different solubility (according to PCT/EP91/01596), fulfill such requirement. This rises the doubt about the suitability of the corresponding ultradeformable vesicles for electrophoresis.
  • Liposomes comprised dimyristoylphosphatidylcholine /cholesterol 2/1 mol/mol mixture with an unspecified amount of cationic stearylamine or anionic phosphatidylserine, when appropriate, to make the vesicles charged. They were prepared fresh by extrusion and had a size of 110 nm. The release was much higher from neutral and negative liposomes than from the positive vesicles.
  • [3H]enkephalin across the skin from anode or cathode, depending on the charge on the molecule.
  • liposome derived material was found in the skin at the level of approx. 2.5%, 0.75%, and 1.5% for the positive, negative and neutral vesicles, respectively; in the absence of electrical current 0.8% of material from neutral liposomes was found in the skin. No liposome derived material was recovered from receiver fluid.
  • the polypeptide delivery into the skin was the highest (4.2%) for the neutral vesicles used in conjunction with electrophoresis (sic!), followed by the same kind of vesicles used in the absence of electrical current (passive delivery: 2.7%); anionic and cationic vesicles used with iontophoresis mediated much lower intracutaneous drug delivery of 0.5% and 0.7%, respectively.
  • penetrants formed by single molecules or by arrangements of molecules, said penetrants being capable of penetrating the pores of a barrier even when the average diameter of said barrier pores is less than the average diameter of said penetrants, since the penetrants are adaptable to the pores, and said penetrants being capable of transporting agents through said pores, or enabling agent permeation through said pores after the penetrants have entered said pores.
  • permeation denotes diffusive, concentration driven motion of molecules across a barrier.
  • Penetration describes the non-diffusive motion of large penetrants across a barrier; the process typically being associated with a penetration induced decrease in the barrier resistance (pore widening or channel opening).
  • a penetrant consequently, is any entity comprising a single molecule or an arrangements of molecules too big to permeate through the barrier.
  • a permeant is an entity that can permeate through the (semi-permeable) barrier.
  • a penetrant in an external field experiences driving force proportional to the nominal penetrant size and to the applied field. Such a force may push the penetrant through the barrier, such as the skin, if the force is strong enough either to deform the penetrant or else to widen the passages in the barrier sufficiently to elude the problem of size exclusion, or both.
  • a transport-driving force must first intercalate the penetrant between cells to form channel wider than the effective penetrant size.
  • the effective penetrant size should be much smaller than the nominal penetrant size.
  • This goal is best achieved by the penetrants that are controllably and stress-dependently deformable.
  • the average diameter, the electrical charge and/or the adaptability in shape or size to the pores of the penetrant is selected so as to enable electromotion.
  • Electrical potential gradient (across a barrier) means an arbitrary potential difference of any sign or magnitude, unless otherwise specified. Specifically, it is not necessary to place the potential generating electrodes directly on the barrier; any placement resulting in trans-barrier gradient is acceptable. "Potential difference” is used as a synonym for "potential gradient”.
  • lipid aggregates should be able to compensate their deformation-induced, local stress (deformation energy) in order to be extremely deformable. This can be accomplished by adjusting their local composition to such a stress, which is only possible if aggregates comprise at least two components.
  • the carrier ingredients are conventiently chosen so that the component which can sustain the deformation better is accumulated while the less adaptable component is diluted at the maximally stressed site. This results in a transient instability (metastability) which must be sufficiently short-lived not to compromise the aggregate integrity.
  • Highly deformable vesicles named Transfersomes in the above mentioned patents (applications) were designed specifically to meet this need and to comply with the requirements for aggregate ultradeformability .
  • Preparation temperature is normally chosen in the 0 to 95 °C range. Preferrably, one works in the temperature range 18-70 °C, most frequently at temperatures between 15 and 45 °C. On the skin, 32 °C are normally measured. Other temperature ranges are possible, however, most notably for the systems containing freezable or non-volatile components, cryo- or heat-stabilizers, etc.
  • carrier formulations can be stored in cold (e.g. at 4°C), with or without an associated active. Manufacturing and storage under an inert atmosphere, e.g. under nitrogen, is also possible and sometimes sensible.
  • the shelf-life of (drug-loaded) carrier formulation moreover, can be extended by using little unsaturated substances, by the addition of antioxidants, chellators, and other stabilizing agents, or by the ad hoc preparation from a freeze dried or dry mixture.
  • Formulations for the use in conjunction with electrophoresis can be processed at the site of application.
  • lipid vesicles both charged and uncharged, examples are given in our previous german patent application and in the handbook on 'Liposomes' (Gregoriadis, G., Hrsg., CRC Press, Boca Raton, FI., Vols 1-3, 1987), in the monography 'Liposomes as drug carriers' (Gregoriadis, G., Hrsg., John Wiley & Sons, New York, 1988), or in the laboratory manual 'Liposomes.
  • 'Liposomes' Gregoriadis, G., Hrsg., CRC Press, Boca Raton, FI., Vols 1-3, 1987
  • monography 'Liposomes as drug carriers'
  • Gregoriadis, G., Hrsg., John Wiley & Sons, New York, 1988 or in the laboratory manual 'Lipos
  • any suspension of drugs can also be diluted or concentrated (e.g. by ultracentrifugation or ultrafiltration) just before application; additives can also be given into a formulation at this time or before. After any system manipulation, the carrier characteristics should be checked and, if required, readjusted.
  • This invention concerns a preparation comprising penetrants formed by single molecules or by arrangements of molecules, in which said penetrants are capable of penetrating the pores of a barrier even when the average diameter of said barrier pores is less than the average diameter of said penetrants, since the penetrants are adaptable to the pores.
  • the penetrants are capable of transporting agents through the pores in the barrier.
  • the average diameter and the adaptability of said penetrants are selected, and said penetrants and / or said agents are provided with sufficient electrical charges, to enable and / or control agent transport through said pores by said penetrants, or in the alternative case agent permeation through said pores after penetrant entry into said pores, under the influence of a suitable electrical driving force, said selection at the same time maintaining sufficient penetrant stability
  • said penetrant is provided with sufficient electrical charges, at least when associated with an agent, and the penetrant could, in the absence of an electrical driving force, not readily penetrate the barrier pores; the average diameter, the kind and amount of electrical charges and / or the adaptability of the electrically charged penetrants or the charged associations of penetrant and agent, being selected to achieve, and in case, control said transport through the barrier under the influence of the electrical driving force.
  • said penetrant is provided with sufficient electrical charges, at least when associated with an agent, and the penetrant could penetrate the barrier pores in the absence of an electrical driving force; the average diameter, kind and amount of electrical charges and / or the adaptability of the electrically charged penetrants or the charged associations of penetrant and agent being selected to provide control of the agent transport through the barrier under the influence of an electrical driving force
  • said penetrant is capable of penetrating said pores under the influence of a suitable driving force, which may be an electrical driving force when the penetrant is suitably electrically charged, and the agents being sufficient electrically charged to enable and / or control their permeation through the pores of the barrier subsequent to entry of said penetrant into said pores.
  • a suitable driving force which may be an electrical driving force when the penetrant is suitably electrically charged, and the agents being sufficient electrically charged to enable and / or control their permeation through the pores of the barrier subsequent to entry of said penetrant into said pores.
  • Said electrically charged penetrants or the charged association of penetrant and agent have preferably an average diameter which is greater (by at least the factor of 2) than the average diameter of the pores of the barrier.
  • a preparation which is characterized by the fact that the electrically charged penetrant is formed by an electrically charged single molecule or an arrangement of electrically charged molecules and is associated with one or several charged or uncharged agent molecules.
  • the above mentioned penetrant is formed by an electrically neutral single molecule or an arrangement of electrically neutral molecules and is associated with at least one electrically charged agent, the quantity of electrical charges being sufficient to enable transport.
  • said penetrants are suspended or dispersed in a liquid medium and comprise arrangements of molecules in the form of minute fluid droplets surrounded by a membrane-like coating of one or several layers of at least two kinds or forms of amphiphilic substances with a tendency to aggregate, said at least two substances differing by at least a factor of 10 in solubility in the, preferably aqueous, liquid medium, such that the average diameter of homo-aggregates of the more soluble substance or the average diameter of hetero-aggregates comprising both said substances is smaller than the average diameter of homo-aggregates of the less soluble substance.
  • the common structure may comprise a physical or chemical complex of the substances.
  • the more soluble substance tends to solubilize the penetrant droplet and the content of this substance is up to 99 mol% of the concentration required to solubilize the droplet, or else corresponds to up to 99mol% of the saturating concentration in the unsolubilized droplet, whichever is higher.
  • he content of the more soluble substance is below 50 %, especially below 40 % and most preferably below 30 %, of the respective solubilizing concentration of said substance.
  • the content of the more soluble substance is below 99 %, preferably below 80 % and most preferably below 60 % of the saturation concentration of said substance in the droplet.
  • the less soluble self-aggregating substance is a lipid-like substance and the more soluble substance is a surfactant.
  • the average diameter of the penetrant is between 40 nm and 500 nm, preferably between 50 nm and 250 nm, even more preferably between 55 nm and 150 nm and particularly preferably between 60 nm and 120 nm.
  • the average diameter of the penetrant is 2 to 25 times bigger than the average diameter of the pores in the barrier, preferably between 2.25 and 15 times bigger, even more preferably between 2.5 and 8 times bigger and most preferably between 3 and 6 times bigger than said average pore diameter.
  • the average net surface charge density on a droplet is between 0.05 Cb m '2 (Coulomb per square meter) and 0.5 Cb m "2 , preferably between 0.075 Cb m "2 and 0.4 Cb m “2 , and particularly preferably between 0.10 Cb m "2 and 0.35 Cb m "2 .
  • the weight amount of droplets in formulations for use on human or animal skin is 0.01 to 40 weight-% of the total preparation mass, in particular between 0.1 and 30 weight-%, and particularly preferably between 5 and 20 weight-%.
  • the weight amount of droplets in formulations for the use on human or animal mucosa is 0,0001 to 30 weight-%.
  • the agent is an adrenocorticostaticum, an adrenolyticum, an androgen or antiandrogen, an antiparasiticum, an anabolicum, an anaestheticum or analgesicum, an analepticum, an antiallergicum, antiarrhythmicum, antiarteroscleroticum, antiasthmaticum and/or bronchospasmolyticum, an antibioticum, antidrepressivum and/or antipsychoticum, an antidiabeticum, an antidot, antiemeticum, antiepilepticum, antifibrinolyticum, anticonvulsivum or anticholinergicum, an enzyme, coenzyme or a corresponding enzyme inhibitor, an antihistaminicum, antihypertonicum, an antihypotonicum, anticoagulant, antimycoticum, antimyasthenicum, an agent against Morbus Alzheimer or Parkinson, an antiph
  • liquid medium characteristics especially the concentration and the composition of the supporting electrolyte, are selected so as to enable and / or control the rate or the efficiency of transport of the penetrant through the pores of the barrier.
  • the supporting electrolyte in particular a buffer, is selected among monovalent (1 :1) or other low valency electrolytes, with the bulk concentration preferably below 150 mM, more preferably below 100 mM, even more preferably below 50 mM, and particularly preferably up to 10 mM.
  • a method for effecting the electrically driven transport of said penetrants and associated molecules through the pores in a barrier is provided which is characterized by the fact that a sufficient electrical potential is applied across the barrier.
  • the electrodes used to generate the electrical potential across the barrier are located on opposite sides or on the same side of the barrier and are arranged so as to ensure that most of the resulting electrical current will flow across the barrier.
  • the applied electrical potential value is chosen to be below 30 V, more often below 15 V, and even more preferably below 10 V, per cm 2 of the barrier surface. It is advantageous if the current driven across the barrier by the applied electrical potential is in the physiologically tolerable range, typically below 2 mA cm “2 , preferably below 1 mA cm '2 , more preferably below 0.6 mA cm “2 and most preferably up to 0.4 mA cm “2 .
  • the electrode size is less than 200 cm 2 , more preferably below 100 cm 2 , especially below 50 cm 2 , most preferably below 10 cm 2 , or even below 5 cm 2 .
  • the electrically conductive material on or of the electrodes comprises at least one metal, in particular selected from precious metals, such as silver or palladium, and/or biocompatible salts or chemical complexes of such metals, preferably the biocompatible chlorides, and most preferably silver chloride.
  • At least one the electrode compartment is loaded with electrically charged penetrants.
  • the electrode is loaded shortly before application, preferably within 360 min, more preferably within 60 min and even more preferably within 30 min.
  • the electrode is loaded with the electrically charged penetrant pre-associated with molecules to be transported, in particular (biologically active) agents.
  • the electrode is loaded with the penetrant and the molecules to be transported, in particular agents, that associate therewith during or after said loading.
  • one or more programmable, preferably small, hand-held or self-supported, for example wrist-watch like, devices for single or repeated use are employed to control the polarity, magnitude and / or time-dependence of applied electric potential.
  • the barrier is pretreated before initiating he electrically driven transport of charged penetrants, by a non-occlusive application of suitable penetrants on the modifiable barrier, especially formed by human or animal skin, to increase the number or width of penetratable pores in the barrier subsequently to be used for the electrically driven transport across said pre-treated skin barrier.
  • the charged or uncharged penetrants used to pre-treat the barrier are similar or identical with those employed for the subsequent electrically driven transport.
  • the charged or uncharged penetrants are non-occlusively applied for up to 24 hours or even longer, typically for up to 12 hours, especially up to 3 hours, or more preferably for less than 1.5 hours, and in case even for less than 30 min, prior to the initiation of electrically driven transport of charged penetrants and/or permeants across the barrier.
  • the electrically driven transport of permeants i.e. any entity being capable to permeate through the pores in the barrier, may be enhanced by a pre-treatment of the barrier as above described before initiating the electrically driven transport of the permeant.
  • the transportation rate, i.e. the flux, of charged penetrants through the barrier pores is determined as a function of the applied electrical potential or of the electrical current across the barrier, and the function thus found is then employed to optimize the preparation or application.
  • Highly adaptable charged aggregates in the majority of cases studied in this work comprised anionic dioleoylphosphatidylglycerol (DOPG). Additional lipids with detergent or surfactant-like properties (typically the non-ionic Tween 80) were incorporated into lipid bilayers to increase the membrane flexibility. Increasing surfactant-to-lipid ratio made the vesicular aggregates more and more deformable, up to the concentration at which membrane stability was negatively affected by the detergent.
  • DOPG anionic dioleoylphosphatidylglycerol
  • the total lipid concentration was typically 5 w-% and typically diluted to 0.5 %, unless stated otherwise.
  • the bulk phase included buffering ingredients (10 mM) as well as, in some cases, dilute electrolyte (NaCl).
  • Freshly cleaned electrodes were separated from the receiving fluid with a microporous membrane (10 nm on the blank side and, for example, 30 nm on the test side). On the donor side, the filling volume was substantially bigger (1.2 mL) than on the blank side (14.5 mL), where the electrode was kept as close to the barrier as possible.
  • the holder was used in a horizontal position to permit stirring of the receiver fluid. Stirring was achieved with a small magnetic bar that revolved on top of the tested barrier.
  • the microporous barrier served as a surrogate or "artificial" skin, for the purposes of this study.
  • test suspension was in contact with the cathode, whereas the blank sample volume was contacted by the anode.
  • the receiving fluid contained charged polymers (alginic acid: 0,25 w-%). These buffering polymers were first dissolved in salt-free water from an Elgastat purification unit (ELGA, UK) and then adjusted to the desired pH range (between 7 and 7.3) by titration with 0.01 N sodium hydroxide. To avoid changes in the mixed lipid vesicles composition, the fluid in the receiver compartment also contained 10 ⁇ 5 M of the most soluble vesicle component, that is, the critical micelle concentration of Tween 80. Benzyl alcohol (0.5 volume %) was added to prevent microbial system contamination during the experiments. The receiver fluid was forced by a peristaltic pump to circulate through the cuvette (placed in a fluorimeter) and to pass through the sampling cell into which a pH electrode was inserted. All experiments reported here were run at 37 degrees Celsius.
  • ELGA Elgastat purification unit
  • Readings were taken continuously.
  • the data were transformed into an electronically analysable and storable file using a XT-IBM micro-computer, equipped with an AD- converter and our own dedicated software.
  • DOPG phosphatidylglycerol
  • test suspension Preparation of test suspension.
  • the lipid mixture was suspended in dilute electrolyte.
  • a sterile glas container containing crude lipid suspension was covered tightly and stirred magnetically for 3 days at room temperature.
  • the suspension was sequentially extruded through polycarbonate membranes of Nucleopore type with a nominal pore size of 400 nm, 100 nm and 50 nm, respectively. This was done at least 20 times.
  • Vesicle suspension was then frozen and thawed 5 times at -70°C and + 50°C, respectively.
  • suspension was re-extruded, 4 times through a 100 nm filter at 0.7 MPa.
  • the suspension of highly deformable vesicles was sterilized by filtration through a sterile filter with 200 nm pores (Millipore) and stored at 4 °C.
  • Electrophoretic measurements First, the background diffusion of label molecules was determined. This was done for several hours without applying an electrostatic potential. Next, constant electric current was set and maintained across the barrier. During this second period, pH in various parts of the test system was monitored. In receiver compartment a digital pH meter was used whereas in donor compartment dipsticks were employed. Electrical potential difference across the barrier was permanently assessed and recorded as well. Concurrently, the electrical barrier resistance was calculated (from the measured potential and current data using Ohm's law). Fluorescence increase in the flow-through cuvette was monitored continuously. Fluorescence increase was identified with the transported amount of material. This was done by using results from separate calibration measurements, during which known amounts of labelled suspensions were added directly into the receiving compartment.
  • the flow of lipophilic fluorescent label (DPH) across the barrier is believed to be representative of the electrically driven vesicles motion through the barrier.
  • the transport data given in figure 1 consequently, correspond to cummulative effect of vesicle penetration through the barrier.
  • the measured data reveal dramatic differences in the transport of charged and uncharged mixed lipid vesicles through "confining" pores in the barrier. This clearly demonstrates the electromotive nature of aggregate transfer discovered and explored in this work.
  • Figure 2 Time dependence of material and vesicle transport across a barrier with an applied electrical potential difference of 1.2 V, which gives rise to the trans- barrier electrical current of 0,279 mA cm ⁇ 2. Charged and uncharged, zwitterionic, lipid vesicles were tested.
  • phosphatidylglycerol prepared from from soy-bean phosphatidylcholine
  • DPH relative to DOPG
  • DOPG phosphatidylglycerol
  • results obtained with conventional vesicles differ completely from the data measured with highly deformable vesicles: simple charged liposomes do not cross 30 nm pores in the barrier under the influence of an electrical (or, in fact, any other) driving force. The fact that no significant motion of the labelled molecules across the barrier is detected for at least 6 hours supports this conclusion. Conversely, the vesicles with a highly flexible and deformable, and thus better adaptable, membrane tend to move through the narrow pores in a barrier, when they are driven in the right direction by sufficiently strong transbarrier electrical potential difference.
  • results shown in figures 1 and 2 can be interpreted, for example, by generalizing the model of ultradeformable aggregate penetration described by the applicant (see e.g. Crit. Rev. Therapeutic Carrier Syst., 1997). The basic considerations for making such model modification are given in the introductory part of this application.
  • Figure 3 Vesicle transport (penetration) across a microporous barrier, deduced from the delivery of vesicle-associated DPH fluorescence, as function of time. Data suggests that liposomes that are ⁇ 3 times bigger than the pores cannot pass these obstacles, in contrast to the comparabaly large, but much more deformable, mixed lipid vesicles with composition that renders their membranes more flexible.
  • Total lipid (TL) content 0.5 w-% comprising:
  • DOPG phosphatidylglycerol
  • Figure 4 Temporal characteristics (upper panel) and potential sensitivity (lower panel) of the vesicles with an aggregate/pore size ratio of approximately 5.2, penetrating the transport barrier under influence of an external, transport driving electrical potential.
  • Figure 5 Characteristic time course (upper panel) and potential sensitivity (lower panel) of ultradeformable vesicle penetrating through the pores nearly 4.6 narrower than the average aggregate diameter.
  • Total lipid (TL) content 0.5 w-% comprising:
  • DOPG phosphatidylglycerol
  • Transbarrier potential difference bigger than approximately 1.3 V appears to make the flux of DPH (and by inferrence, the transport of vesicles) less sensitive to changes in the electrical transbarrier driving potential. It is not entirely clear whether or not the diminished increase in penetration capability, measured with the highest explored potential difference, is diagnostic of saturation of the potential dependent changes in the vesicle transport (see examples 29-35), or else is simply due to the experimental irreproducibility. Data given in the middle panel of figure 5 circumstantially support the former interpretion: if the transport is not analyzed as a function of time but rather as a function of time required to bring certain number of non-confined ions across the barrier, all the curves measured with driving potentials higher than 1 V group closer together.
  • Figure 6 Effect of electrostatic potential difference on the transport of highly deformable, intermediate size vesicles passing 30 nm pores.
  • Upper panel time course of flux measured under the constant current conditions; middle panel: data as above, but with the time-axis normalized with relatively to the given electrical current; lower panel: capability of ultradeformable vesicles to penetrate pores of fixed-size by electromotion.
  • Test conditions in this experimental series were such that the exclusion criterium for the lipid vesicles motion across a barrier with the vesicle/pore size ratio of 2.6 was very weak. (It is known from previously published work by us (Cevc et al., Biochim. Biophys. Acta 1368, 201-215, 1998) and the others that size exclusion begins to govern the transport across microporous barriers when the penetrant/pore size ratio exceds the value of 2. The flux of vesicle-associated label, consequently, was biphasic in this test series (cf. figure 6). Normalization of the time axis (see the middle panel of figures 5 and 6) does not group the curves together. Rather than this it makes the spread more uniform.
  • Barrier penetrability can not increase significantly upon changing the applied voltage. It is therefore more than probable that the above mentioned late penetrability change results from increased capability of lipid aggregates to pass the barrier. We interpret this difference as a sign of a moderately increased vesicle adaptability to pore narrowness.
  • the earlier transbarrier transport is likely to be due to simple electrophoresis of relatively tiny vesicles. Obviously, many such vesicles are small enough to cross the pores in a barrier, probably in the process of an electrically mediated (or supported) "diffusion".
  • Penetration capability data illustrated in the lower panel of figure 6 are diagnostic of complete vesicle adaptability (maximum membrane flexibility), as can be seen from the fact that several high potential values are nearly the same.
  • Figure 7 Elektromotion of relatively small, highly deformable vesicles through 30 nm pores in a barrier.
  • Two different transport rates ( ⁇ l and ⁇ 2) are seen, indicative of two different unerlaying transport phenomena.
  • Total lipid (TL) content 0.5 w-% comprising: 274 mg phosphatidylglycerol (DOPG) 226 mg Tween 80 0.1 mol-% DPH (relative to DOPG) Buffer as in previous examples NaCl concentration (final): 1 mM, 10 mM, 20 mM, 50 mM, 100 mM Vesicle/pore size ratio: 3.3
  • Figure 8 Electrically driven transport of charged, highly deformable vesicles across an artificial barrier with 30 nm pores in the presence of different salt solutions.
  • Upper left transbarrier flow of DPH labelled vesicles; upper right: penetration capability of complex aggregates; lower left: electrical potential that drives the constant current across a barrier as a function of time; lower right: transport driving electrostatic potential as function of bulk NaCl concentration.
  • Figure 8 shows that the electrical resistance to electrophoretic motion across a barrier increases nearly linearly with the applied electrical potential, but only in certain range. The onset of material flux, in parallel, gets faster (see figure 5 for comparison). This means that the lag-time becomes shorter with increasing transbarrier potential difference. Below the "linear" range, which commences at approximately 1 V for the tested suspension, only insignificant vesicle transport is observed. The tiny flow of aggregate material is then hardly affected by the applied potential or by the changing electrical current.
  • Figure 9 Electrically driven transport of charged, highly deformable vesicles across an artificial barrier with 30 nm pores.
  • Upper left barrier electrical resistance
  • upper right electrical potential required to drive constant electrical current across the barrier
  • lower left pH value in the receiver compartment containing alginic acid
  • lower right pH of suspension of highly flexible vesicles present in the donor compartment.
  • Electromotion through the epidermis in vitro can be used to study someof the characteristics of electrophoresis in vivo.
  • the proviso for this is the use of sufficiently large and intact skin segments with a functional barrier.
  • skin piecee In order to obtain at least semi- quantitatively reliable data, such skin piecee must also be as thin as possible.
  • Electrical resistance of the epidermis is a good marker for the skin intactness. It is also diagnostic of any major changes in the barrier properties of the organ.
  • variable electrical resistance of the skin as a function of time during transepidermal electriophoresis is given in figure 9.
  • Figure 10 Electrical resistance of epidermis during serial electrophoresis experiments done in vitro.
  • Total lipid (TL) content 0.5 w-% comprising: 274 mg phosphatidylglycerol (DOPG) 226 mg Tween 80 0.1 mol-% DPH (relative to DOPG) Buffer as in previous examples
  • Part A Vesicle/pore size ratio: 3.3; electrical current: 0 mA, 1.2 mA (current density 0.286 mA cm" 2 ) electrical potential: difference: 0 V, 2,0 V
  • Part B Vesicle/pore size ratio: 2.8 electrical current: 1.2 mA (current density 0.286 mA cm" 2 ) electrical potential: difference: 3.7 V, 5.4 V, and 7 V
  • part B Further experiments (part B) were done with two skin preparation methods. 2 hours and 7 hours of enzymatic action were used for this purpose, which gave rise to rather thick (5,4 V; 3,7 V) or thin (7 V) specimen, respectively. Moreover, slightly smaller vesicles were used than in part A. This latter difference notwithstanding, the results from repeat experiments have confirmed the trend observed in part A experiment. They also revealed the importance of skin thickness on the effective vesicle flux across the barrier. After a lag-time of approximately 22 min the transport of ultradeformable vesicles across thin skin, as assessed by means of fluorescent label flux determination in part B, was substantial (0.4 microgramms cm" 2 min ⁇ l or approx. 25 microgramms cm “2 h”s see figure 11).
  • Figure 11 A) Electrically driven transport of ultradeformable vesicles across human epidermis (upper panel), electrical resistance of the barrier (middle panel) and pH in receiver compartment (lower panel) during an electrophoretic experiment.
  • Total lipid (TL) content 0.5 w-% comprising: 274 mg phosphatidylglycerol (DOPG) 226 mg Tween 80
  • DOPG can be used to electro-transport uncharged substances (here -1H-DPPC) across a barrier with an applied electrical potential difference.
  • Figure 12 Electro-transport of uncharged molecules associated with charged, ultradeformable vesicles across murine epidermis in vitro.

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Abstract

L'invention porte sur une préparation comportant des pénétrants constitués de molécules isolées ou en arrangements capables de pénétrer les pores d'une barrière même lorsque le diamètre moyen desdits pores est inférieur au diamètre moyen desdits pénétrants. Les pénétrants peuvent s'adapter aux pores et transporter des agents à travers eux ou en permettre la perméation à travers ces mêmes pores après que les pénétrants les aient traversé. Le diamètre moyen et l'adaptabilité des pénétrants doivent être sélectionnés et les pénétrants et/ou les agents doivent présenter des charges électriques suffisantes pour permettre et contrôler le transport des agents ou assurer la perméation des agents à travers les pores après que les pénétrants les aient traversé sous l'influence d'une force électrique d'entraînement idoine. Ladite sélection doit assurer en même temps une stabilité suffisante des pénétrants. L'invention porte également sur le procédé de transport induit électriquement desdits pénétrants et des molécules associées à travers les pores d'une barrière.
PCT/EP1998/005539 1998-09-01 1998-09-01 Transport induit electriquement de penetrants charges a travers des barrieres WO2000012060A1 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
BR9816014-1A BR9816014A (pt) 1998-09-01 1998-09-01 Transporte eletricamente controlado de penetrantes carregados através de barreiras
CN98814268A CN1322129A (zh) 1998-09-01 1998-09-01 带电穿透剂通过屏障的电控转运
AU97404/98A AU9740498A (en) 1998-09-01 1998-09-01 Electrically controlled transport of charged penetrants across barriers
EP98951338A EP1107729A1 (fr) 1998-09-01 1998-09-01 Transport induit electriquement de penetrants charges a travers des barrieres
PCT/EP1998/005539 WO2000012060A1 (fr) 1998-09-01 1998-09-01 Transport induit electriquement de penetrants charges a travers des barrieres
JP2000567180A JP2002523442A (ja) 1998-09-01 1998-09-01 バリヤを横切る荷電浸透物の電気制御輸送
KR1020017002688A KR20010106462A (ko) 1998-09-01 1998-09-01 배리어를 가로지른 대전 침투제의 전기적으로 제어된수송
MXPA01002149A MXPA01002149A (es) 1998-09-01 1998-09-01 Transporte electricamente controlado de penetrantes cargados a traves de barreras.
CA002345355A CA2345355A1 (fr) 1998-09-01 1998-09-01 Transport induit electriquement de penetrants charges a travers des barrieres

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WO2010140061A3 (fr) * 2009-06-03 2011-07-28 John Charles Mayo Formulations destinées au traitement de la douleur de tissus profonds
US9452179B2 (en) 2009-08-21 2016-09-27 Sequessome Technology Holdings Limited Vesicular formulations
US9555051B2 (en) 2012-03-29 2017-01-31 Sequessome Technology Holdings Limited Vesicular formulations
US10744090B2 (en) 2015-06-30 2020-08-18 Sequessome Technology Holdings Limited Multiphasic compositions

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Publication number Priority date Publication date Assignee Title
WO2007071448A2 (fr) * 2005-12-23 2007-06-28 Partnership & Corp. Technology Transfer Peptides synthetiques utilises comme inhibiteurs de la secretion des neurotransmetteurs et comme inducteurs de la relaxation cellulaire

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FR2552666A1 (fr) * 1983-09-29 1985-04-05 Kao Corp Compositions vesiculaires a base de sel de phosphate et d'agent tensio-actif
EP0475160A1 (fr) * 1990-08-24 1992-03-18 Gregor Prof. Dr. Cevc Préparation pour l'application d'un principe actif sous forme de gouttelettes miniscules
WO1994017792A2 (fr) * 1993-01-27 1994-08-18 Affymax Technologies N.V. Compositions et procedes d'administration transdermique de medicaments

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010140061A3 (fr) * 2009-06-03 2011-07-28 John Charles Mayo Formulations destinées au traitement de la douleur de tissus profonds
AU2010255391B2 (en) * 2009-06-03 2016-06-02 Sequessome Technology Holdings Limited Formulations for the treatment of deep tissue pain
AU2010255391C1 (en) * 2009-06-03 2016-10-27 Sequessome Technology Holdings Limited Formulations for the treatment of deep tissue pain
EA029132B1 (ru) * 2009-06-03 2018-02-28 Секвессам Текнолоджи Холдингс Лимитед Составы в виде везикул для лечения боли, связанной с остеоартритом
US9452179B2 (en) 2009-08-21 2016-09-27 Sequessome Technology Holdings Limited Vesicular formulations
US9555051B2 (en) 2012-03-29 2017-01-31 Sequessome Technology Holdings Limited Vesicular formulations
US10744090B2 (en) 2015-06-30 2020-08-18 Sequessome Technology Holdings Limited Multiphasic compositions
US11547665B2 (en) 2015-06-30 2023-01-10 Sequessome Technology Holdings Limited Multiphasic compositions

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