CN1322129A - Electrically controlled transport of charged penetrants across barriers - Google Patents

Abstract

It is an object of the invention to provide a preparation comprising penetrants formed by single molecules or by arrangements of molecules, siad 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. It is another object of the invention to provide a method for effecting the electrically driven transport of said penetrants and associated molecules through the pores in a barrier.

Description

Electrically controlled transport of charged penetrants across barriers

field of invention

The present invention relates to a formulation containing a penetrant, formed by a single molecule or an arrangement of molecules, capable of penetrating the pores of a barrier due to its good fit, even when the pores of said barrier The pores are also permeable when the pores are smaller than the average diameter of the penetrant, and the penetrant is capable of transporting the drug through the pores, or allowing the drug to permeate the pores after the penetrant enters the pores; The average diameter and suitability of the penetrant, and the penetrant and/or the drug have sufficient charge so that the penetrant enables and/or holds the drug under the influence of an appropriate electrical driving force Transport through the pore, or drug permeation through the pore after the penetrant enters the pore, is selected while maintaining sufficient penetration stability. The present invention also relates to methods of achieving electro-driven transport of said penetrants and related molecules across barrier pores.

Background of the invention

A charged body can spontaneously migrate from a high potential to a low potential unless it is hindered by a barrier, such as a barrier. The electric driving force is proportional to the total charge of the charged body and the potential difference. The size of the stream also depends on the resistance of the system to said movement. Thus, electrically driven transport across a barrier is influenced by the number, width and properties of pores in the barrier, which all define the permeability of the barrier, and P, which in turn determines the resistance of the barrier. An example of such a porous barrier is skin, which typically contains pores on the order of a few angstroms in diameter (in the non-expanded state).

Any transdermal potential that drives ion flow across the skin has a tendency to widen certain hydrophilic channels in the organ. This usually occurs at the least densely populated sites between cells, where the role of facilitating transport is most likely to occur.

Permeation of the skin can be achieved with a suitable vehicle for the same purpose without the use of auxiliary devices or external energy sources (Schatzlein, A.; Cevc, G. (1998): Heterogeneous porous filling of the stratum corneum and the permeability barrier function of intact skin: using High-resolution confocal laser scanning electron microscopy studies of highly deformed vesicles (transporters), Br. J. Dermatol. 138: 583-592). Prior to treatment, only small molecules such as water are allowed to pass through the skin's hydrophilic pathways (pores). However, these pores can be opened into wider channels by adding a sufficiently potent penetrant.

Potential differences can drive charged penetrants across barriers, such as skin; in addition, electroosmotic facilitation widens at least some of the hydrophilic channels in the organ. This occurs only in the stratum corneum of the skin (Stratum corneun), which constitutes most of the skin's permeability barrier. Depending on the pore size of the newly enlarged transdermal channel, it is commonly called electrophoresis (iontophoresis) or electroporation, for the electrically induced flow of ions through narrow channels and for the widening of wider pathways, respectively.

For example, a current of about 0.4 mAcm ^{- 2} or below will activate a small fraction of narrow (~0.5 nm) hydrophilic channels that pre-exist between skin cells. The opening of such channels can then persist for many hours (Green, PG; Hinz, RS; Kim, A.; Szoka, FCJr.; Guy, RH (1991), Iontophoretic delivery of a series of tripeptides across the skin in vitro, Pharm.Res.Sep.; 8:1121-7), but when the transdermal voltage is kept in the physiologically tolerable range (less than <3V for ^1cm2 paste), the channel is still relatively narrow (<3nm, in nude mice ).

The widest channel is internally female. The neutral channel is only half as wide and the positive channel is twice smaller (Pikal, M.J.; Shah, S. (1990b), Transport Mechanisms of Iontophoresis II: Electroosmotic Flow and Transfer in Nude Mouse Skin Quantitative Assays, Pharm. Res. 7:213-21). So far, the widest channels that exist during low-voltage electrokinesis have been reported to be only 20 nm or less (Aguillelle, V.; Kontturi, K.; Murtomaeki, L.; Ramirez, P. (1994), Human cadaver skin Evaluation of Mesopore Size and Charge Density, J. Contr. Rel., 32: 249-257).

Therefore, the standard electro-skin permeability enhancement method (electrophoresis) can only improve the transport of relatively small (<2 nm) charged molecules across the organ. It was concluded that electrophoresis across the skin is feasible for some polypeptides but practically ineffective for the delivery of proteins or other large penetrants (for review, see Green, P.G.; Hinz, R.S.; Kim, A.; Szoka , F.C.Jr.; Guy, R.H. (1991), Electrophoretic in vitro delivery of a series of tripeptides across the skin, Pharm. Res. Sep.; 8: 1121-7; Green, P.G.; Flanagan, M.; Shroot, B.; Guy, R. (1993), Electrophoretic drug delivery in enhancement of drug skin penetration (Walters, K. and Hadgraft, J., eds.) Marcel Dekker, New York, 297-319; Heith, M.C.; Williams, P.L; Jayes , F.L.; Chang, S.K.; Riviere, J.E. (1993) Iontophoretic peptide delivery in the skin: in vitro and in vivo, a study of luteinizing hormone release, J.Pharm.Sci.82:240-3; Singh, S.; Singh, J. (1993) A review of transdermal drug delivery by passive diffusion and iontophoresis: Med. Res. Rev. 13:569-621; Singh, J.; Bhatia, K.S. (1996), Drug delivery by local iontophoresis: Routes, Principles, Factors, and Skin Irritation, Med. Res. Rev. 16:285-96).

Transdermal channels opened by low voltage and tolerable small currents cover only 0.005% of the total treated area, although the number of channels appears to be large (<3×10 ⁸ cm ^-2 ) (Pikal, MJ (1990), Transport Mechanisms in Iontophoresis. I, A Theoretical Model of the Effect of Electroosmotic Flow on Flow Facilitation in Transdermal Iontophoresis, Pharm. Res. 7: 118-26). Larger localized skin perforations by higher voltages (>150V) are rarer and generally result from skin lesions that have persisted for several days.

The size of the transdermal channel is influenced by various parameters. For example channel widening with increasing potential also widens with increasing base electrolyte concentration, but variations of the latter are practically only possible within relatively narrow limits. Also, there is as yet no data on how to use such principles to improve transport across barriers. 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 iontophoretic delivery device for transdermal delivery of peptide/protein drugs, Pharm. Res. 10:697-702), and less sensitive to changes in the lipophilicity of drugs (Green, P.G.; Hinz, R.S.; Kim, A.; Szoka, F.C.Jr; Guy, R.H. (1991), iontophoretic delivery in vitro A series of tripeptides cross the skin, Pharm. Res. Sep;8:1121-7).

Repeated electroosmotic delivery through the same skin area results in discrete, but often large fluxes across the barrier, which creates difficulties in data interpretation and evaluation (Heith, M.C.; Williams, P.L.; Jayes, F.L.; Chang, S.K.; Riviere, J.E. (1993) Transdermal delivery of peptides by iontophoresis: in vitro and in vivo studies using luteinizing hormone-releasing hormone, J. Pharm. Sci. 82:240-3). Other complications stem from the passage of electrical currents through appendages in the skin, such as hair follicles.

The electrical expansion of the skin's channels is reflected by the following contributions to the skin's permeability,

P _i.el = permeability of the skin to ions = (c _i Z _i F/RT) D _i /d _s This permeability needs to be added to that observed in the absence of electricity acting on the barrier. In addition to opening the pores, the transcutaneous potential gradient (ΔΨ _el ) activates those electromotive forces that drive charged penetrants through the channel. This gives another parameter in the Fick transport equation for modeling barrier (e.g. transdermal) transport (see further discussion):

j _i =...a c _i +P _i.el Δψ _el c _i is the volume concentration of substance i, Z _i F is the molar charge (valence multiplied by Faraday constant), RT is the molar thermal energy. D _i is the diffusion coefficient of substance i and d _s is the thickness of the skin. The current corresponding to i is obtained by the product of i-flow and Z _i F , but the total current includes all the various effects and is therefore obtained by the sum of these effects.

Electrophoresis represents the directional flow of charged molecules under the electric field of electrodes. Therefore, the drug molecule must be placed on an electrode with the same charge as the drug. In this case, the magnitude of the flux is proportional to the net charge of the various migrating molecules and the applied potential. Other important factors are the drug concentration and diffusion coefficient in the barrier or skin (see equation given below).

Due to the absolute difference in concentration, the electrophoretic current usually also includes the effect of basic electrolyte ions (such as Na ⁺ , Cl ^- ). This makes the effect of the drug less, accounting for only a small fraction of the effect of the measured current. Thus, increasing the ion concentration under the electrodes reduces the useful fraction of the electrophoretic current (Pikal, MJ; Shah, S. (1990b), Transport Mechanisms of Ion Electrophoresis II. Determination of Electroosmotic Flow and Transfer Quantities in Nude Mouse Skin, Pharm .Res.7: 213-21; Pikal, MJ; Shah, S (1990c), The Transport Mechanism of Iontophoresis III. A laboratory study of the role of electroosmotic flow and permeability changes for the transport of low and high molecular weight solutes, Pharm .Res.7:222-9), which can be seen from simple differential calculations.

It is thus the case of the prior art that physical conditions limit the maximum achievable or tolerable electrophoretic current that can penetrate the skin: the current flowing through the channels that have been opened consumes electrical energy; Large (as above), thereby reducing the amount of material that can be transported. In addition, open pores are limited due to the adverse effects of skin energy depletion (skin itching and erosion).

Electroosmotic flux , the flux of water bound to transport ions carrying uncharged species, also depends on the hydrophilic channels in the skin and the applied potential. The flux under the anode generally exceeds the cathode flux, perhaps due to the difference in the average size of the positive and negative charge channels. During 10 hours of galvanostatic iontophoresis (0.36mAcm ^-2 ), the steady-state average flux level was several times higher than at the beginning (<3 μL h ^-1 cm ^-2 ) (Kim, A.; Green, PG ; Rao, G.; Guy, RH (1993), Convective solvent flow through the skin during iontophoresis, Pharm. Res., 10: 1315-20).

Electrically evoked skin changes are greatest during the first hour of electrophoresis (Pikal, MJ; Shah, S. (1990b), Transport Mechanisms of Iontophoresis II. Electroosmotic Flow and Quantitative Determination of Nude Mouse Skin, Pharm.Res Flinterman, NH; Verhoef, J.; Junginger, HE; Bodde, HE (1994), In vitro iontophoresis of peptides across human skin Model: Effects of iontophoresis protocol, pH and ionic strength on peptide flux and skin impedance, Pharm. Res. Sep; 11: 1296-300). During this time, the resistance decreased from >20 KΩ cm ^-2 to about 10% of this initial value.

Skin pretreatment with ethanol either attenuates (Brand, R.M.; Iversen, P.L. (1996) Iontophoretic delivery of telomeric oligonucleotides, Pharm. Res. 13:8514-4) or enhances (Srinivasan, V.; Higuchi , W.I.; Sims, S.M.; Ghanem, A.H.; Behl, C.R. (1989) Iontophoretic transdermal delivery of drugs: Mechanistic analysis and application of peptide delivery, J.Pharm.Sci. 78:370-5) Electrophoresis by organ transport. Most chemical skin penetration enhancers increase the electrical conductivity of the skin and thus also enhance electrically driven transdermal transport (Green, P.G.; Hinz, R.S.; Kim, A.; Szoka, F.C.Jr.; Guy, R.H. (1991) , iontophoresis to deliver a series of tripeptides through the skin in vitro, Pharm.Res.Sep.8: 1121-7); also adjusting the pH, especially lowering the pH can also improve the electrical conductivity of the skin. This effect is partly due to electrophoresis and partly to electroosmosis, but the increase is usually small.

Electrophoretic enhancement of the movement of molecules across the skin has so far been only partially successful (for a recent survey of products and development, see: Cevc, G. (1997), Drug Delivery Through the Skin, Exp. Opin. Invest, Drugs 6: 1887-1937) . Particularly poor results were obtained with macromolecules (see, for example, reviews by Siddiqui, O.; Chien, Y.W., Parenteral 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 Iontophoretic Delivery Devices for Transdermal Delivery of Peptide/Protein Drugs, Pharm. Res. 10:697-702). For example, for insulin, the efficiency of iontophoretic transport is 4%/hour under optimal conditions, usually less than 3%/hour; part of the observed transport may be due to skin damage (Siddiqui, O.; Chien, Y.W. Review of, Parenteral Administration of Peptide and Protein Drugs, Crit. Rev. Therap, Drug Carrier Syst. 1987, 3: 195-208).

This problem is due in part to the high molecular weight of the macromolecules, but also to the hydrophilic nature of most macromolecules, both of which present great difficulties for the general use of conventional skin penetration enhancement techniques.

Other large penetrants fared less well. To date, only one document addresses the problem of driving large lipid aggregates, liposomes, through the skin, but no solution has been found (see further discussion).

It should therefore be said that prior to the present invention there was no known method for ensuring electroosmosis of large penetrants through microporous barriers such as mammalian skin. In addition, no one has proposed a general method for opening the large pores of the skin so far. This is unfortunate given the need for transdermal release of macromolecules such as peptides and proteins, and the long-held desire to control the penetration of aggregates across any type of transport barrier.

The extent and mechanism by which aggregates penetrate biological barriers such as the stratum corneum are highly debated (Cevc, G. (1996) Lipid Suspensions on Skin. Penetration Facilitation, Capsule Penetration and Transdermal Delivery of Drugs, CritRev. Therap.Drug Carrier Systems.13:257-388), there is also great controversy about the penetration route through the skin. We have discussed more than once that hydrotaxis is the most important factor in the transport of surface-hydrophilic, highly deformable vesicles across biological barriers such as skin (Cevc, G. (1996). Lipid suspensions on skin. Penetration facilitation, vesicular Capsule penetration and transdermal drug delivery, Crit. Rev. Therap. Drug Carrier Systems. 13: 257-388; Cevc, G. (1997), Drug delivery across the skin, Exp. Opin. Invest, Drugs 6: 1887 -1937). Our contention is that diffusion is not a good way to transport large aggregates (ie lipid vesicles) across such barriers.

The first reason for this is that the permeability (P _a ) of any large aggregate of useful mass is very low, and the permeability is generally proportional to the number of aggregates ( _na ). Because the P _a value is related to the diffusion constant (D _a ), the permeability and flow rate of such large substances will decrease linearly with the gradual increase of aggregate size. (P _a is proportional to D _a ~ D _l /n _a , where D _l is the diffusion constant of the monomer).

A second reason for the low diffusivity of aggregates is the small difference in the maximum concentration that aggregates can reach across the barrier (Δc _a =Δc _l /n _a , where c _l is the saturated monomer concentration). As calculated by the first Fick's law, the flow rate j _m =P _a Δc _a is proportional to D _a Δc _a ~D _l Δc _l /n _a ² .

From the two phenomena mentioned above, it follows that _Da (n _a >> 1) → 0 and Δc _a (n _a >> 1) → 0, which contribute to the negligibly small barrier permeability of vesicle transport : P _a (n _a >> 1) → 0.

The first part of the permeability problem is solved by utilizing the water activity gradient ( _Δaw ), ie, hydrotaxis, to drive transport across the barrier. Our explanation for this is that aggregates that do not depend on the gradient of water activity exhibit similar attractive forces for all polar molecules in the aggregate; this force increases the pressure on the various aggregates proportionally, ΔP _hyd.a ~ Δa _w RTn _a , or a corresponding increase in the force driving aggregate transport across the barrier, F _hvd . Both of these forces are much greater on aggregates than on single molecules. This compensates for the effect of small differences in aggregate concentrations, as can be seen from the general Fick equation: j _a = P _a Δc _a + P _a ” Δa _w RTn _a

~P' _{a na} F _hyd.l F _hyd.l represents the force acting on each monomer in the aggregate, and RT is heat energy.

To maximize access to an "external" transport driving force (which is independent of penetrant/penetrant concentration), deformable aggregates as described in PCT/EP 91/01596 can be used. This reduces the increase in transport resistance with increasing aggregate number by pushing it below the set linear dependence (see Figure 1).

An example of this is a vesicle composed of a sufficiently flexible membrane with low deformation energy. This is especially true when the bladder is subjected to strong anisotropic (ideally unidirectional) stresses, "forces" or pressures. Under corresponding conditions, the combination of molecular aggregation and membrane softness can lead to passage of capsules through barriers even when the pores of such barriers are smaller than the diameter of the capsules. This allows a distinct flow to flow in the desired direction.

The same is considered for each external force (F _ext ) or pressure (Δp _ext ), thus independent of concentration. This is provided that the increase in the force dependence of aggregate size - or resulting in an increase in the pressure differential across the barrier - exceeds the decrease in permeability (P _a ) as aggregate size increases. This gives the net translocation across the barrier. Figure 1 depicts this relationship. Net transport can eventually be obtained if the driving force increases linearly with the number of charges on each moving object and provided that the transport resistance of such objects increases rapidly below the driving force (full line). In this case the pore shape is suitable for penetrants. When the penetrant becomes large enough, the driving force exceeds the transport resistance and efficient transport occurs. But if the penetrant cannot fit into the pores of the barrier, the resistance of the barrier will inevitably exceed the driving force when the average size of the "penetrant" exceeds the average diameter of the pores (dashed line).

The above reasoning applies as long as the transport driving force is constant or as long as the increase in transport resistance does not exceed the size-dependent increase in the driving force. Highly deformable aggregates subjected to a sufficiently high external pressure (Δp _ext,a ) are examples of this; the opposite occurs for general, essentially non-deformable aggregates at lesser similar pressures, since these aggregates in Easily breaks when crossing barriers.

In the case of an external electric force (F _el ), this can lead to so-called electrophoresis, the principle of which is basically similar, but more complex and not previously recognized.

Assuming that in the simplest case, where the aggregate contains _n charged molecules, each of charge Ze ₀ , and the corresponding counterion is the only other charged species in the system, the rate of aggregate transport varies with aggregate size or The number increases linearly as well as the potential (E) gradient across the barrier. As long as the transport resistance remains constant, which is the case, then the driving force is given by F _el =n _a Ze ₀ E, and the P _a value is considered constant. However, if the transport resistance is dependent on aggregate size and also increases with decreasing _na , pa _values , the transport rate sensitivity to changes in the number of aggregates can be eliminated. Since the increase rate of the transport resistance is higher than the linear increase rate, when the decrease of P _a value is faster than the linear rate, the transport rate decreases with the increase _{of na} .

Thus, in the first of the three cases above, one would expect only one efficient electrophoresis. This tends to cause more problems in practice. Almost all suspensions of charged aggregates contain, in addition to the anionic aggregate components, small ions of the same charge as the aggregates. These small, applied charges interact with the aggregate's electric field and invade the barrier in the same direction. As a result, a parasitic current of "small charges" starts to flow, eventually canceling the potential across the barrier. If the transport resistance of such "opportunistic charges" is less than that of useful aggregates (as is often the case), aggregate transport may eventually stop.

Another anticipated complication in large object electrophoresis is that the applied potential may preferentially push individual charged molecules of the aggregate across the barrier rather than transporting the entire aggregate. A size-dependent transport resistance is expected to be consistent with this, especially for relatively more water-soluble charged aggregate components. Highly variable aggregates composed of substances of different solubility (PCT/EP 91/01596) fully meet this requirement. This calls into question the suitability of the corresponding superdeformed capsules for electrophoresis.

In fact, only one research group has published very rigorous data using liposomes as "penetration enhancers" and reported results on electromotive transport of substances through the skin.

The first report on the combination of liposome and iontophoresis for transdermal drug delivery should be the closest prior art to the present invention. Vutla et al. (Vutla, N.B.; Betageri, G.V.; Banga, A.K. (1996) Transdermal iontophoretic release of enkephalins formulated in liposomes, J.Pharm.Sci., 85:5-8) , reported the following.

Liposomes contain dimyristoylphosphatidylcholine/cholesterol 2/1 mol/mol mixed with unspecified amounts of cationic stearylamide or anionic phosphatidylserine, as appropriate, to make charged vesicles. They were freshly prepared by extrusion with a size of 110 nm. The release of neutral and negative liposomes was higher than positive capsules.

A current at a density of 0.5 mAcm ^-2 co-transports [Leu5]enkephalins across the skin from either the cathode or the anode, depending on the charge on the molecule.

After 12 hours of iontophoresis, liposome-derived material was found in the skin at approximately 2.5%, 0.75%, and 1.5% for positive, negative, and neutral cysts; 0.8% of substances derived from neutral liposomes. No liposome derivatives were recovered from the subject's body fluids.

Transdermal delivery of enkephalins was not facilitated by the use of negatively charged sacs. Positive liposomes even reduced delivery compared to controls.

For neutral capsules used in combination with electrophoresis, the highest amount of polypeptide was delivered into the skin (4.2%), followed by such capsules without the use of electric current (passive delivery: 2.7%); negative and positive capsules delivered subcutaneously using iontophoresis The amount of the drug was much lower, 0.5% and 0.7%, respectively.

Vutla's research clearly shows that conventional liposomes, whether charged or uncharged, are poor delivery media for electrically driven material transport into the skin.

Thus, for large lipid aggregates (such as vesicles) or other large units for the purpose of transdermal delivery, the substitution of electromotive forces for hydrotaxis has not been conceived so far.

Based on the aforementioned reasons, it is an object of the present invention to provide a formulation containing a penetrant, which is formed by a single molecule or molecular arrangement, even when the average diameter of the barrier pores is smaller than that of said penetrant , can also permeate through the pores of the barrier because the penetrant fits the pores, and the penetrant is capable of transporting the drug through the pores, or, after the penetrant enters the pores, enables Drugs pass through the pores.

Another object of the present invention is to provide a method for electrically driving the transport of penetrants and molecules bound thereto through barrier pores by applying a potential across the barrier.

Description of the invention

We have surprisingly found that charged ultradeformable aggregate capsules can be forced through artificial and natural nanoporous barriers by an externally applied potential difference. The condition is that the bladder has sufficient stress, which must be sufficient to deform the bladder. This is only achievable using capsules with highly flexible membranes. The method involves deformation of the entire aggregate, the suitability of the capsule size to the penetration of the pores is influenced by the average capsule/pore diameter ratio.

We also found that the electrical motion of charged aggregates is sensitive to electrolyte concentration. Surprisingly, the measured dependencies deviate both qualitatively and quantitatively from the baseline of known electrical drive of non-deformable small penetrants through the barrier. The electrically driven movement of highly deformable vesicles through "bounded" pores differs from conventional electrophoresis, thus providing a new method for drug delivery across various barriers, including biological barriers.

Apparently, non-obstructive permeation of the superdeformed aggregate suspension by the skin (subsequent application of a transdermal potential) increases the final electrophoretic flux. We speculate that this may be due to the opening of channels in the skin by non-electrical methods. The increased overall permeability of the organ due to the opening of efficient, non-electrical channels can be used to deliver charged sacs across the barrier. The capsule is pushed through the pretreated skin by a transdermal electrical potential applied under occlusion.

Last but not least is the hypothesis that seems to be contrary to previous beliefs: macromolecules can be effectively delivered through the skin. Their delivery can be carried out by conjugation of macromolecules to charged super-deformable carriers, including transdermal electroactuation of this class of normally incompatible carrier protein transporters.

We also discuss the properties of molecular aggregates/associated species suitable for use in conjunction with electrophoresis. We focused only on complexes that could overcome the transport barrier under the influence of a trans-barrier potential difference of sufficient size. We describe fundamental experiments related to this phenomenon and explain their results. The conclusions we draw apply to the application of the general concepts asserted in this study. Of particular interest, but not exclusively, is that our new approach can be used in both human and veterinary medicine.

In the context of the present invention, osmosis refers to concentration-driven diffusional movement of molecules across a barrier. Penetration refers to the passage of large penetrants through a barrier by non-diffusion motion; methods related to penetration generally induce a decrease in barrier resistance (widening of pores or opening of channels).

Thus, a penetrant is any substance that contains single molecules or aggregates of molecules that are impermeable through a barrier. Penetrants, on the other hand, are also substances that are permeable through (semi-permeable) barriers. The driving force of the penetrant by the external electric field is proportional to the nominal penetrant size and the applied electric field. This force can push the penetrant through the barrier if it is strong enough to deform the penetrant or widen the passage of the barrier enough to overcome a barrier the size of the penetrant, or both. . In skin, for example, the transport driving force must first insert the penetrant into the cell to form a channel wider than the size of the effective penetrant. (To obtain a high rate of penetrant transport, the effective penetrant size should be smaller than the nominal penetrant size). Optimal targeting is achieved using controllable and stress-dependent deformable penetrants. The average diameter of the penetrant, its charge and/or its suitability to the shape or size of the pore are selected such that it produces electrically driven movement.

Unless otherwise stated, a potential gradient (through a barrier) refers to the signal or magnitude of any potential difference. Specifically, the electrodes that generate the potential need not be placed directly on the barrier; any location for the electrodes that generate the gradient across the barrier is acceptable. "Potential difference" is a synonym for "potential gradient".

For other definitions, especially those involving highly deformable complexes (aggregates) and their mechanism of action, as well as selected drugs, are detailed in our published or pending patents (DE 4107152, PCT/EP91/01596, PCT/EP91/01596, PCT/ EP 96/04526, DE 4447287). These patents also describe in detail the basic properties and characteristics of such aggregates.

In short, lipid aggregates should be able to compensate the local stress (deformation energy) generated by their deformation to be extremely deformable. This objective can be achieved by tailoring its local composition to such stresses, and is only possible if the aggregates contain at least two components. The carrier components are routinely chosen so that the components that better hold the deformation accumulate while the less conforming components in the areas of greatest stress are diluted. This leads to temporary instability (metastability), which should exist short enough not to compromise the integrity of the aggregate. The design of the highly deformable vesicles known as transfersomes in the above mentioned patent (application) should especially meet this need and comply with the requirements of ultra-deformability of aggregates.

The breakthrough temperature is usually in the range of 0-95°C. Preferably in the temperature range of 18-70°C, more preferably in the range of 15-45°C. The measured skin temperature is generally 32°C. However, it is worth mentioning that for systems containing freezable or non-volatile components, cold or heat stabilizers, other temperature ranges are also possible.

The carrier formulation, with or without active ingredient, can be refrigerated (eg, 4°C) if necessary to maintain the integrity and desired properties of the individual system components. Production and storage under an inert atmosphere, such as a nitrogen atmosphere, may also sometimes be intentional. The shelf-life of (drug-containing) carrier formulations can be extended by the use of unsaturated substances, such as the addition of antioxidants, chelators and other stabilizers, or by special preparations from freeze-dried or dried mixtures.

In most cases, the invention is performed at room temperature. Both lower and higher temperatures and applied potentials are possible using suspensions. This has particular significance for formulations comprising synthetic substances that are rigid at temperatures between room temperature and the temperature of the skin or other barrier.

Formulations used in conjunction with electrophoresis can be prepared at the site of application. Lipid vesicles, charged or uncharged, are given in: our previous German patent application and "Liposome" handbook (Gregoriadis, G., Hrsg., CRC Press, Boca Raton, Fl., Vols 1-3 , 1987), the monograph "Liposomes as Drug Carriers" (Gregoriadis, G., Hrsg., John Wiley & Sons, New York, 1988) or the laboratory manual "Liposomes: A Handbook for Use" (New, R., Oxford-Press, 1989). Suspensions of drug may be diluted or concentrated (by ultracentrifugation or ultrafiltration) immediately before use, if desired; additives may also be added at or before this time. After any system operation, the characteristics of the carrier should be checked and, if necessary, readjusted.

The present invention relates to a formulation containing a penetrant formed by a single molecule or an arrangement of molecules, wherein the penetrant even when the average diameter of the barrier pores is smaller than the average diameter of the penetrant, because The penetrant is adapted to the pores and is also capable of penetrating through the pores of the barrier. In this way, the penetrant is capable of transporting the drug through the pores of the barrier. When the penetrant enters the pore, the penetrant widens the pore or opens the channel. Or the drug penetrates through the (already opened or widened) pores in the barrier to infiltrate the substance into said pores. selecting the average diameter and suitability of the penetrant so that the penetrant and/or the drug are sufficiently charged that, under the influence of an appropriate electrical driving force, the drug or control drug is The transport of the penetrant through the pore, or alternatively the drug permeation through the pore after the penetrant has penetrated the pore, should be chosen while maintaining sufficient penetration stability.

According to the present invention, it is preferred that the penetrant has sufficient charge at least when it is combined with the drug, and the penetrant cannot easily penetrate the pores of the barrier without the action of an electric driving force; the charged penetrant or The choice of mean diameter, charge type and amount and/or suitability of the charged conjugate of penetrant and drug should control drug transport across the barrier under the influence of the electrical driving force.

It is also preferred if the penetrant is sufficiently charged, at least when bound to the drug, to pass through the pores of the barrier in the absence of an electrical driving force; the charged penetrant or penetrant The choice of average diameter, charge type and amount and/or suitability of the charged conjugate with the drug should control the transport of the drug across the barrier under the influence of the electrical driving force.

It is also preferred that the penetrating agent is capable of penetrating the pore under the influence of a suitable driving force, which may be an electrical driving force when the penetrating agent is suitably charged, and Once the penetrating agent enters the pores, it causes or controls the permeation of the drug with sufficient charge through the pores of the barrier.

In a particular version of the invention, the formulation is characterized in that the charged penetrant is formed by charged single molecules or an arrangement of charged molecules, and that the penetrant is combined with one or several charged or uncharged drug molecules .

Alternatively, the above-mentioned penetrants are formed by a neutral monomolecule or an arrangement of neutral molecules and combined with at least one charged drug, the amount of charge should be sufficient for transport.

In a preferred embodiment of the present invention, the penetrant is suspended or dispersed in a liquid medium, and the molecular arrangement contained therein is a layer of at least two types or forms of amphiphilic substances with a tendency to aggregate. Or the form of small droplets surrounded by several layers of film-like coatings, the solubility of the at least two substances in the liquid medium, preferably water, differs by at least 10 times (factor), so that the average diameter of the homogeneous aggregate of relatively soluble substances may include two The mean diameter of heteroaggregates of such species is smaller than the mean diameter of homoaggregates of less soluble species.

It is advantageous if the more soluble substance is the drug that will be transported across the barrier and has a tendency to form larger co-structures with the less soluble substance. The common structure may comprise a physical or chemical complex.

According to the invention, it is preferred if the more soluble substance dissolves small droplets of penetrant and the substance is present in an amount up to 99 mol % of the concentration required to dissolve said droplets or is saturated with the concentration in undissolved droplets. Concentrations correspond to 99 mol%, whichever is higher.

Preferably the content of the more soluble material is less than 50%, more preferably 40%, most preferably 30% of the corresponding dissolved concentration of said material.

According to the invention, the content of the more soluble substance is less than 99%, preferably 80%, most preferably 60% of the saturation concentration of said substance in the droplets.

It is advantageous if the less soluble self-aggregating substance is a lipid substance and the more soluble substance is a surfactant.

Preferably, the average diameter of the penetrant is 40-500 nm, preferably 50-250 nm, more preferably 55-150 nm, especially preferably 60-120 nm.

It is also preferred that the average diameter of the penetrant is 2 to 25 times larger than the average diameter of the pores of the barrier, preferably 2.25 to 15 times larger, more preferably 2.5 to 8 times larger, most preferably 3 to 8 times larger than the average diameter of the pores. 6 times.

According to the present invention, it is also advantageous that the average net charge density on the surface of the droplet is 0.05Cbm ^-2 (coulomb/square meter) ~ 0.5Cbm ^-2 , preferably 0.075Cbm ^-2 ~ 0.4Cbm ^-2 , especially preferably 0.10Cbm ^-2 ~ 0.35Cbm ^-2 .

Preferably, the weight of the liquid droplets in the formulation for human or animal skin is preferably 0.01-40 wt%, especially 0.1-30 wt%, more preferably 5-20 wt% of the total substance of the formulation.

It is also preferred that the weight of the liquid droplets in the preparation for human or animal mucous membrane is 0.0001 to 30% by weight.

Drugs in preferred embodiments disclosed in the present invention are corticotropic hormones, antiadrenaline agents, androgens and antiandrogens, parasiticides, anabolic agents, anesthetics or analgesics, tonic agents, antiallergic agents, anticardiac agents Arrhythmics, antiarteriosclerotics, antiasthmatics and/or bronchial spasmolytics, antibiotics, antidepressants and/or antipsychotics, antidiabetics, antidotes, antiemetics, antiepileptics, antifibrinolytics antispasmodic or anticholinergic, enzyme, coenzyme or corresponding enzyme inhibitor, antihistamine, antihypertensive, antihypotensive, anticoagulant, antimycotic, antimyasthenic, Drugs against Alzheimer's disease or Parkinson's disease, anti-inflammatory agents, antipyretic agents, antirheumatic agents, antibacterial agents, respiratory stimulants or respiratory stimulants, bronchitis agents, cardiotonic agents, chemotherapy drugs, coronary vasodilators , cytostatics, diuretics, genglium-blockers, glucocorticoids, anti-influenza drugs, haemostatics, hypnotics, immunoglobulins or fragments thereof or any other immunoactive substance (e.g. immunomodulators, cellular factors, etc.), biologically active carbohydrates (derivatives), contraceptives, anti-migraine drugs, corticosteroids, muscle relaxants, sedatives, neurotherapeutic agents, (poly)nucleotides, neuroleptics, neurotransmitters, ( Poly)peptides (derivatives), opiates, ophthalmic drugs, (para)sympathomimetic drugs, anti (para)sympathetic drugs, protein (derivatives), psoriasis/neurodermatitis drugs, mydriatic drugs, psychostimulants , nasal medicines, hypnotics, sedatives, antispasmodics, antituberculosis medicines, urology medicines, vasoconstrictors or vasodilators, antiviral agents, wound healing agents, inhibitors of the activity of any of the foregoing or combinations thereof (antagonist) or enhancer (agonist).

It is particularly advantageous to select the properties of the liquid medium, especially the concentration and composition of the base electrolyte, to control efficient transport of the penetrant through the barrier pores.

According to the invention, the base electrolyte, especially the buffer, is selected among monovalent (1:1) substances or other low-price electrolytes with a volume concentration preferably below 150 mM, more preferably below 100 mM, most preferably below 50 mM, and especially Preferably not higher than 10 mM.

Furthermore, the present invention provides methods of effecting the electro-driven transport of the above-mentioned penetrants and bound molecules through the pores of the barrier, characterized in that a sufficient electrical potential is used to cross the barrier.

According to the invention, the electrodes for generating a potential across the screen are located on opposite sides or on the same side of the barrier and are arranged to ensure that the majority of the current generated flows through the barrier.

The applied potential is preferably lower than 30V, usually lower than 15V, more preferably lower than 10V per square centimeter of barrier surface. Advantageously, the electric potential driven across the barrier is physiologically tolerable, usually below 2 mAcm ^-2 , preferably below 1 mAcm ^-2 , more preferably below 0.6 mAcm ^-2 , most preferably below 0.4 mAcm ^{-2 2} .

The size of the electrodes is preferably less than 200 cm ² , more preferably less than 100 cm ² , especially less than 50 cm ² , most preferably less than 10 cm ² , or even less than 5 cm ² .

According to the invention, the conductive material of the electrodes comprises at least one metal, especially selected from noble metals, such as silver and palladium, and/or biocompatible salts or chemical complexes of such metals, preferably biocompatible chlorides, Silver chloride is most preferred.

Advantageously at least one of the electrode chambers contains a charged penetrant.

It is also advantageous that the electrodes are installed or pre-installed at the application site.

The electrodes are preferably installed immediately before use, preferably within 360 minutes before use, more preferably within 60 minutes or even 30 minutes before use.

In a preferred embodiment of the invention, the electrodes are provided with charged penetrants pre-bound with molecules to be transported, especially (bioactive) drugs.

In another preferred embodiment of the invention, the electrodes are provided with a penetrant and the molecule to be transported, especially a drug, is bound thereto during or after loading.

In a preferred version of the present invention, one or more programmed, preferably small portable or self-contained (such as wristwatch) disposable or reusable devices are used to control the polarity and magnitude of the applied potential and/or time dependence.

It is best to choose differently treated surfaces to control this transport.

In another preferred embodiment of the invention, a suitable penetrant is applied non-obstructively to a modifiable barrier, especially a barrier formed by human or animal skin, prior to initiating the electrically driven transport of the charged penetrant , whereby the barrier is pretreated to increase the number or width of penetrable pores in the barrier for subsequent electro-driven transport across the pretreated skin barrier.

The charged or uncharged penetrant used to precondition the barrier is preferably similar or identical to the penetrant used subsequently for electro-driven transport.

Advantageously, the charged or uncharged penetrant is applied non-occlusively for up to 24 hours, even longer, usually up to 12 hours, prior to inducing electro-driven transport of the charged penetrant and/or penetration of the penetrant through the barrier. hours, especially up to 3 hours, more preferably less than 1.5 hours, even less than 30 minutes. It should be emphasized that it is a feature of the present invention that electro-driven transport of an osmolyte (ie, any substance permeable through the pores of the barrier) can be enhanced by pretreatment of the barrier as described above prior to initiating electro-driven transport of the osmolyte.

Advantageously, the rate of transport of the charged penetrant through the pores of the barrier, ie the flux, is measured as a function of the potential or current across the barrier. The function thus obtained can be used to optimize the formulation or its application.

Several illustrative examples of the systems and methods of the invention are given below: it should be clear that they are not intended to define or limit the invention. Unless otherwise indicated, all temperatures are in degrees Celsius, support sizes are in nanometers, and ratios and percentages are in moles. All others use standard SI units.

EXAMPLES General Experimental Protocol and Sample Preparation

The experiment was to study highly adaptive aggregates in most cases, including the anionic dioleoylphosphatidylglycerol (DOPG). Other lipids with detergent or surfactant properties (typically the nonionic Tween 80) can be incorporated into the lipid bilayer to enhance membrane stretchability. Vesicular aggregates are more easily deformed by increasing the surfactant-to-lipid ratio, up to concentrations at which membrane stability is not negatively affected by detergents.

Unless otherwise stated, the total lipid concentration is typically 5% by weight, typically diluted to 0.5%. The bulk phase included buffer components (10 mM) and, in some cases, dilute electrolytes (NaCl).

Laboratory-made platinum electrodes were used in commercially available glass vessels (Crown, Glass, Inc, USA) which were secured with metal clips. The vessel was closed tightly (sealed with a hollow metal O-ring) with a hard plastic lid with two openings for addition and sampling. During the experiment, one of the two pores was always open to allow the gases produced by hydrolysis to escape. Breakage of the membrane/container connection should also be avoided.

The freshly cleaned electrode is separated from the receiving liquid by a microporous membrane (eg, 10 nm on the blank side and 30 nm on the test side). On the donor side, the filled volume (1.2 L) was much higher than on the blank side (14.5 mL), where the electrodes were brought as close as possible to the barrier. The container stirs the received liquid in a horizontal position. Stirring is performed with a small magnetic bar that rotates above the test barrier. This porous barrier was used as a surrogate or "artificial" skin for this study.

Determine the power limit condition, and maintain a balanced current power supply (Phoresor: Iomed, Salt Lake City, USA; general error: 0.1mA) or a balanced voltage power supply (Siemens, Munich, Germany; general error: 1mV). The test suspension is brought into contact with the anode and the blank sample liquid is brought into contact with the cathode.

The receiving liquid contained a charged polymer (alginic acid: 0.25% by weight). The buffer polymer was first dissolved in anhydrous saline (purchased from Elgastat Purification Units (ELGA, UK)) and then titrated with 0.01N sodium hydroxide to adjust the pH to the desired range (7-7.3). To avoid variations in the composition of the mixed lipid vesicles, the liquid in the receptor compartment also contained 10-5 M of the most soluble cystic component, ie, TW80 of the critical micelle concentration. Benzyl alcohol (0.5% by volume) was added to prevent microbial contamination during the experiment. A screw pump is used to circulate the receptor solution through the cuvette (placed in the fluorometer) and through the sample cell into which the pH electrode is inserted. All experiments were performed at 37°C.

Read data continuously. Use the XT-IBM microcomputer equipped with AD-converter and self-editing software to convert the data into information that can be analyzed and stored electrically.

For data comparison, we focus on the data for the start period, during which brownout conditions changed by only a few percent. These variations have been estimated as carefully as possible to determine the errors shown in some figures.

To avoid false positives, the integrity of the labeled aggregate conjugates was confirmed early in the experiment. To determine the relative amount of surfactant-solubilized markers in mixed lipid micelles or other type complexes, various suspensions were tested. The test is performed by pushing the suspension towards a membrane with 10 nm pores. The labeled DPH suspension obtained very little flux. However, the corresponding flow value is still subtracted from the final sac flow (that does not pass through the 10nm pores). In experiments performed with epidermis, Rho-DHPE was used as a fluorescent marker. Rho-DHPE is still highly lipophilic, but more soluble than DPH. Thus, the background signal of the former marker is higher than that of the DPH-labeled vesicles, which can be seen directly from the figure without the need for subtraction of other data.

Examples 1-2: Aggregate charge effects Uncharged highly deformable vesicles:

274mg Phosphatidylcholine (SPC)

226mg Tween 80 (Tw 80)

0.1mol% DPH (relative to SPC)

99.5mL Phosphate Buffer, 10mM, pH7-7.3

Capsule/Aperture Ratio: 3.3 Charged Highly Deformable Capsules:

274mg Phosphatidylglycerol ( ^DOPG- , as above)

226mg Tween 80 (Tw 80)

0.1mol% DPH (relative to SPG)

99mL Phosphate Buffer, 10mM, pH7

Capsule/pore ratio: 3.7 Current: 1.2 mA (current density: 0.279 mA/cm ² ) Preparation of test suspension: The liquid mixture was suspended in dilute electrolyte. The sterile glass container containing the crude liquid suspension was tightly capped and magnetically stirred at room temperature for 3 days. To narrow the capsule size distribution, the suspension was sequentially extruded through Necleopore-type polycarbonate membranes with nominal pore sizes of 400 nm, 100 nm, and 10 nm, respectively. This operation is performed at least 20 times. The capsule suspension was then frozen at -70°C and thawed at 50°C five times. To obtain the desired final capsule size, the suspension was re-extruded 4 times through a 100 nm filter at a pressure of 0.7 MPa. Finally, the suspension of hyperdeformable capsules was filter sterilized with a 200 nm pore sterile filter and stored at 4°C. Electrophoretic assay: First, the background diffusion of labeled molecules is measured. The assay is carried out for several hours without applying a potential. Subsequently, a constant current is set and maintained across the barrier. In this second phase, the pH of different parts of the test system is checked. A digital pH meter was used in the recipient chamber and a pH probe was used in the donor chamber. Always estimate and record the potential difference across the screen. At the same time, the resistance of the barrier is calculated (calculated from the measured potential and current data using Ohm's law). An increase in fluorescence is continuously measured as the liquid flows through the cuvette. This increase in fluorescence is consistent with the amount of substance transported. This experiment was performed with results obtained from a separate calibration assay, during which a known amount of labeled suspension was added directly to the receiver chamber. The flux of a lipophilic fluorescent marker (DPH) across the barrier was considered to indicate electrically driven sac movement across the barrier. Therefore, the transport data presented in Figure 1 correspond to the cumulative effect of sacs across the barrier. The measured data revealed large differences in the passage of charged and uncharged mixed lipid vesicles through the "restricted" pores of the barrier. This clearly demonstrates the electrically driven transfer of the aggregates discovered and explored in this work.

Although no uncharged vesicles were transported, the applied potential across the screen did drive charged small molecules (mainly ions) across the barrier, as seen from maintaining constant current conditions. Figure 2: In the case of a potential difference of 1.2V (producing a cross-screen current of 0.279mAcm ^-2 ), the material and capsule

Time dependence of transport across the barrier. Use of charged and uncharged, amphoteric lipid vesicles

experimenting.

Examples 3-4: Aggregate Deformability Effect Conventionally Charged Vesicles, Liposomes:

500mg Phosphatidylglycerol (DOPG)

(prepared from soybean phosphatidylcholine)

0.1mol% DPH (relative to DOPG)

99.5mL Phosphate Buffer, 10mM, pH7

Capsule/Aperture Ratio: 2.9 Highly Deformable Charged Capsule:

274 mg phosphatidylglycerol (DOPG, above)

226mg Tween 80

0.1mol% DPH

99mL Phosphate Buffer, 10mM, pH7

Capsule/aperture ratio: 3.5 Current: 1.6mA (current density: 0.381mA/cm ² )

The results obtained with conventional vesicles are completely different from those measured with highly deformable vesicles: under the influence of an electrical (or indeed any other) driving force, the charged liposomes do not cross the 30 nm pores of the barrier. The conclusion was supported by assay experiments carried out for at least 6 hours that there was no appreciable movement of the labeled molecules across the barrier. In contrast, well-fitting sacs with highly stretchable and deformable membranes tend to move through narrow pores in the barrier when their right direction is driven by a sufficiently strong potential difference across the screen.

Since ordinary lipid vesicles (liposomes) can only pass through pores much larger than their own diameter, aggregates passing through openings much smaller than the average vesicle diameter should be negligible. Figure 1 contains unexpected and unprecedented data that are open to discussion and interpretation with new concepts.

Results such as those shown in Figures 1 and 2 can be explained by summarizing the highly deformable aggregate permeation model described in this application (see, eg, Crit. Rev. Therapeutic Carrier Syst., 1997). The introductory part of this application presents the basic idea of improving this model. Figure 3: Generation of DPH fluorescence associated with vesicles, inferring the translocation (permeation) of vesicles through a microporous barrier as a function of time. The data indicate that liposomes 3 times larger than the pores cannot pass through these barriers, whereas the opposite is true for larger, but more deformable lipid vesicles mixed with a component that makes their membranes more deformable.

Examples 5-10: Effect of capsule size and potential difference across the screen Suspension properties:

Total lipids in an amount of 0.5% by weight, including:

274mg Phosphatidylglycerol (DOPG)

0.1mol% DPH (relative to DOPG)

226mg Tween 80

99mL Phosphate Buffer, 10mM, pH7

Capsule/Aperture Ratio: 5.2 Electrical Driving Force or Parameters:

Current: 1.8mA, 2.3mA, 2.5mA, 2.8mA, 3mA, 4mA

Current density: 0.429mA/cm ² , 0.547mA/cm ² , 0.595mA/cm ² , 0.666mA/cm ² , 0.714mA/cm ² , 0.952mA/cm ²

The experimental method is as described in Examples 1-4. But in this series of experiments, the effect of the potential difference was investigated. This is the first study using larger capsules more than five times the average pore size.

The results of this series of experiments shown in Figure 3 demonstrate the necessity of applying a potential difference of at least 1 V across the screen, with 1.1 V to 1.2 V being sufficient to transport highly deformed vesicles through a 30 nm pore. For larger quantities of species to be transported across the barrier, the potential across the screen needs to exceed 1.5V. Potential differences of this magnitude can generate rather high [opportunistic] currents (higher than 0.5 mAcm ^-2 ).

Thus, it is clear that the electrical passage of highly deformable vesicles through the barrier is qualitatively different from simple electrophoresis or bulk transport of vesicles. According to Ohm's law, the driving current can be expected to increase linearly with the potential driving the transport, and the same is true for conductivity/reverse resistance. This linear dependence and constant resistance was indeed observed during conventional electrophoresis. However, in this study, a strong non-linear relationship was found. This would not be the result of changing the nature of the barrier. The data presented in Figure 3 therefore demonstrate that application of an electrical potential enhances the ability of the capsule to penetrate the barrier. Similar reports of hydration-driven transport of highly deformable vesicles through microporous barriers have been previously reported, which could be explained by the mechano-sensitivity of highly deformable mixed lipid membranes. Figure 4: Temporary properties (upper panel) and potential sensitivity (lower panel) of capsules with an aggregate/pore size ratio of approximately 5.2,

Penetrates the transport barrier under the influence of an external transport driving potential.

Examples 11-16: Suspension properties:

As in Example 5-10, the difference is that the

Capsule/pore ratio: 4.6. Electric driving force or parameters:

Current: 1.4mA, 1.6mA, 1.8mA, 2mA, 2.5mA, 3mA

Current density: 0.333mA/cm ² , 0.381mA/cm ² , 0.429mA/cm ² , 0.476mA/cm ² , 0.595mA/cm ² , 0.714mA/cm ²

Experiments and analyzes were performed as described in Examples 1-11. The difference to note is that the capsules are smaller, while also reducing the minimum cross-screen potential required for the capsules to cross the barrier to 1.2V (see Figure 4). Even with the highest potential difference to date (1.7 V), the potential-dependent transport was increased and invasion was not significantly reduced. However, apparently smaller transport fluxes were measured using potential differences higher than 0.8V. Figure 5: Permeability characteristics of hyperdeformable capsules through pores 4.6 times narrower than the average aggregate diameter over time

Changes in (upper panel) and potential sensitivity (lower panel).

Examples 17-28: Suspension properties:

Total lipids in an amount of 0.5% by weight, including:

274mg Phosphatidylglycerol (DOPG)

226mg Tween 80

0.1mol% DPH (relative to DOPG)

99mL Phosphate Buffer, 10mM, pH7

Capsule/Aperture Ratio: 3.5 Electrical Driving Force or Parameters:

Current: 0.4mA, 0.6mA, 0.8mA, 1.2mA, 1.4mA, 1.5mA, 1.6mA, 2.3mA, 3mA, 3.5mA, 4mA

Current density: 0.095mA/cm ² , 0.143mA/cm ² , 0.190mA/cm ² , 0.286mA/cm ² , 0.333mA/cm ² , 0.357mA/cm ² , 0.381mA/cm ² , 0.547mA/cm ² , 0.714mA/cm ² , 0.833mA/cm ² , 0.952mA/cm ²

Results: Proper hole penetration was observed when the potential difference across the screen was at least 1 V. However, the potential difference exceeds 0.8V, and the measured flow rate is small.

A cross-screen potential above 1.3 V makes the flux of DPH (via perturbation, sac transport) less sensitive to changes in the cross-screen driving potential. It is not yet entirely clear whether measurements with the highest exploratory potential difference are no longer increasing the penetration state to identify potential-dependent changes in vesicle transport (see Examples 29-35), or are simply due to experimental non-repeatability. The data presented in the middle panel of accompanying drawing 5 support the former interpretation: if this transport is analyzed as a function of time, rather than a function of the time required for a certain number of unrestricted ions to pass through the barrier, all curves Both were measured with this set of drive potentials above 1V. Figure 6: Effect of potential difference on passage of highly deformed, medium-sized capsules through 30 nm pores. Above:

  Flow rate versus time measured under constant current conditions; middle panel: data as above, but

Normalize the time axis with a given current; Bottom: Highly deformable capsules passed through

The ability to drive through a fixed size hole.

Examples 29-35: Suspension properties:

As in Example 17-28, the difference is:

Capsule/Aperture Ratio: 2.6 Electrical Driving Force or Parameters:

Current: 0.25mA, 0.4mA, 1mA, 1.2mA, 1.4mA, 1.8mA, 2.3mA

Current density: 0.060mA/cm ² , 0.095mA/cm ² , 0.238mA/cm ² , 0.286mA/cm ² , 0.333mA/cm ² , 0.429mA/cm ² , 0.548mA/cm ²

The experimental conditions for this series of studies were poor for exudation of lipid vesicles moving through the barrier with a vesicle/pore size ratio of 2.6. (Known from our previous literature (Cevc et al., Biochim. Biophys. Acta 1368, 201-215, 1998)), others believe that when the penetrant/pore size ratio exceeds 2, the exudation effect of size starts to dominate Transport through microporous barriers. Therefore, in this series of experiments, the flow of vesicle-bound markers was biphasic. Normalization of the time axis (see Figures 5 and 6 and middle panel) did not bring the curves together but spread them more evenly.

The initial slope of the mass transport curve measured at different potentials and currents across the screen is approximately constant. The bottom-most graph illustrates this situation, which gives the slope of the normalized curve shown in the upper graph. The "front part" of the assay curve accounts for the less strong, if any, potential dependence. In contrast, later time-flow characteristics (after about 1 hour) show changes in the properties of the system, which can be seen when the potential or current across the screen is sufficiently high (0.7 V and 0.225 mA cm ⁻² , respectively). The time lag was observed to be rather insensitive to current, as shown in the upper panel of Figure 2, but the lag time did become somewhat shorter with increasing current/potential.

Changing the voltage did not significantly increase the permeability of the barrier. Therefore, it is more likely that the aforementioned later changes in permeability are due to the increased ability of lipid aggregates to pass through the barrier. We interpret this difference as a sign of a modest increase in the fit of the sac to the pore narrowing. On the other hand, the initial barrier transport may be due to simple electrophoresis of relatively small sacs. Apparently many of these sacs are small enough to pass through the pores of the barrier, and perhaps this is the electrically mediated (or supported) "diffusion" process.

The penetrability data shown in the lower panel of Figure 6 confirms the complete suitability of the sac (maximum membrane stretch), as can be seen from the several high potential cases to be essentially the same. Figure 7: Electrical actuation of relatively small, highly deformable capsules through the 30 nm pores of the barrier. Pictured above: by

Absolute penetration of capsules from DPH flow calculations; middle panel: same data as above

As a function of normalized time; lower panel: experimental system (=DPH obtained sac flow/unit

Potential) relative penetration. It can be seen that two different transport rates (Φ1 and Φ2) represent two

The phenomenon of transport in different substrates.

Examples 36-40: Effect of electrolyte concentration Suspension properties:

Total Lipids (TL) at a content of 0.5% by weight, comprising:

274mg Phosphatidylglycerol (DOPG)

226mg Tween 80

0.1mol% DPH (relative to DOPG)

Buffer as in previous examples

NaCl concentration (final concentration): 1mM, 10mM, 20mM, 50mM, 100mM

Capsule/pore ratio: 3.3

Current: 1.2mA: Current density: 0.286mA/cm ²

In this series of experiments, we found that increasing the base electrolyte concentration greatly affects the efficiency of electromotive transport across microporous barriers. High electrolyte concentrations generally favor salt transport, but reduce aggregate transport across screens. We believe that some of the threshold concentrations above are related to the barrier and penetrant properties, and the addition of salt prevents the transport of large aggregates.

Comparing the mass flux, ion flux, and driving potential data measured in this experiment with those in previous experiments provides clues to explain the salt-dependent inhibition of the electrical drive of aggregates through narrow pores. The relative magnitude of the effect of small ions (in this case Cl ⁻ ) flowing across the barrier is directly proportional to the volumetric concentration of the salt. Therefore, a lower driving potential is capable of maintaining a constant current through the barrier at higher salt concentrations (see lower panel of Figure 7). At the same time, lower driving potentials tend to reduce and thus may reduce the suitability and penetration ability of large penetrants (see upper right panel). But the latter is the condition for efficient flow of aggregates through narrow pores. Below a certain fitness value (fitness is influenced by the penetrant/pore size ratio), the deformability of the aggregates thus becomes so low that only insignificant transport takes place.

Thus, the addition of salt to suspensions of deformable complex aggregates with a strong stress drive can adversely affect trans-screen transport. Figure 8: Electrically driven transport of charged highly deformable vesicles across an artificial barrier of 30 nm pores in the presence of different salt solutions. Upper left panel: cross-screen flux of DPH-labeled vesicles; upper right panel: penetrability of complex aggregates; lower left panel: potential to drive constant flux across the barrier as a function of time; lower right panel: transport driving potential as a function of NaCl volume concentration function.

Examples 41-50 Variation of Barrier Properties and Test System Properties Suspension Properties:

The same electric driving force or parameters as in Embodiment 17-28:

Same as Example 17-28

Figure 8 shows that the resistance to electrophoretic movement across the barrier increases almost linearly with applied potential, but only within a certain range. The material flow was stable at the beginning, and then rose rapidly (see the comparison diagram of accompanying drawing 5). This indicates that the lag time decreases with increasing potential across the screen. Below this "linear" range (starting at about 1 V for the test suspensions), only insignificant transport of vesicles was observed. The tiny flux of aggregated species is therefore hardly affected by applying a potential or changing the current.

During this series of experiments, the pH of the receiving cavity decreased by about 1.5-1.8, almost independently of the applied voltage. After the first hour of electrophoresis, the pH change was less than 1. (The change, if any, has little, if any, effect on the electrical actuation of the capsule). As the flow leveled off, the pH of the donor cavity became correspondingly more alkaline. The latter change does not change the charge on the mixed lipid vesicles, since the ionic PG in the membrane has a lower pK = 2.9. At least during the first part of the experiment, no significant degradation of the lipids was thought to be present (lipids degrade rapidly when the charged membrane deviates from the optimum pH ~7.1). Figure 9: Electrically driven transport of charged highly deformable vesicles through an artificial barrier with 30 nm pores.

  Upper left: resistance of the barrier; upper right: potential required for a constant current to pass through the barrier; left

Bottom panel: pH value in recipient lacunae containing alginic acid; Bottom right panel: present in donor lacunae

The pH of the highly elastic capsule suspension in .

Interpretation of the data: When the rate of vesicle transport across the barrier is high, the potential difference required to draw a constant current through the pore initially decreases rapidly with time and eventually increases with time. We believe that the former phenomenon is due to the redistribution of highly mobile ions in the system, especially those in front of the electrodes and near the barrier. In the second case, it appears to us that its rise is much slower, mainly due to the gradual blocking of the pores by larger, less deformable sacs that accumulate in front of the barrier.

Variations on and around the electrodes can partly explain the second variation in barrier penetration/driving potential values. Especially when using high currents, we can often see species (alginate?) precipitate near the reference electrode. During the experiment, the surface of the electrodes always turned brown; this became more pronounced the higher the applied potential and the greater the current generated. Last but not least, hydrogen and oxygen emissions from the solution occasionally mitigate the foaming of the suspension and may also affect the aforementioned changes in barrier resistance.

Examples 51-53: Transport characteristics of skin (epidermis) Electrical parameters: as shown in Figure 9

(Currents given in the diagram)

Electrical drive through the epidermis in vitro: can be used to study some properties of electrophoresis in vivo. The condition is that a sufficiently large fragment of intact skin with a functional barrier is used. Such skin slices must be as thin as possible in order to obtain at least semi-quantitatively reliable data. Ideally, this sheet of skin acts only as a barrier, ie with only the stratum corneum. In practice, this is not possible due to the fragility of the horny skin layer. Therefore, the best articles are thin and stretchable skin sheets.

Electrical resistance of the epidermis: is a good indicator of skin integrity. It can also identify any changes in organ barrier performance.

Figure 9 gives an example of skin variable resistance as a function of time during transepidermal electrophoresis. Figure 10: Skin electrical resistance during a series of electrophoretic experiments performed in vitro.

The specific resistance of the ablated skin (originally slightly above 10 kOhm cm ^-2 ) always decreased with the cumulative current flowing through the skin to about 10-20% of the initial value. The observed value and the initial specific resistance value are compared with those recorded in the literature. The specific resistance value of human and rat skin given in the literature is 20 kOhm cm ^-2 lower. This slightly higher resistance down to about 10% of the starting value may be due to the difference in total accumulated current.

We observed no significant difference in electrical resistance, or its temporal and current changes, between the tested human and porcine skin samples.

Examples 54-57: Effect of Barrier (Skin) Thickness Suspension Properties:

Total Lipids (TL) at a content of 0.5% by weight, comprising:

274mg Phosphatidylglycerol (DOPG)

226mg Tween 80

0.1mol% DPH (relative to DOPG)

Buffer as in previous examples

Part A: capsule/pore ratio: 3.3

Current: 0mA, 1.2mA (current density: 0.286mAcm ^-2 )

Potential difference: 0V, 2.0V

Part B: capsule/pore ratio: 2.8

Current: 1.2mA (current density: 0.286mAcm ^-2 )

Potential difference: 3.7V, 5.4V and 7V

Results: In the first experiment (part A), it was shown that a current of about 0.3 mAcm ^-2 co-transported only a small amount of fluorescently labeled hyperdeformable cysts through a thick epidermis that was detached by heating and subjected to 2 hours of pancreatic Prepared by protease action; in about 4 hours, only about 6 micrograms per square centimeter pass through. The greatly reduced skin resistance disrupts the skin barrier while increasing the flow of cargo through the (possibly) perforated organ.

Another experiment (Part B) was performed with two skin preparation methods. For this, thicker (5.4V; 3.7V) or thinner (7V) samples were obtained, respectively, using 2 or 7 hours of enzymatic action. Also, a slightly smaller capsule than that used in the part A experiment was used. Despite the latter discrepancy, repeated experiments confirmed the trends observed in the experiments in Part A. They also reveal the importance of skin thickness for the effective flow of sacs across the barrier. As with the fluorescently labeled flux assay in Part B, the transport of hyperdeformable vesicles through the thin skin was considerable after a lag time of approximately 22 minutes (0.4 μg/cm ⁻² /min or approximately 25 μg/cm ^{−2 2} /hour, see accompanying drawing 11). In contrast, smaller fluxes were measured with the two thicker epidermal samples. However, wetting was observed even at higher flow rates. This may be due to blocking the limited number of pores available in the skin, especially under the conditions used in this trial. Figure 11: A) Electrically driven transport of superdeformable vesicles through human epidermis (upper panel), resistance of the barrier (middle panel)

and the pH in the recipient cavity during the electrophoresis experiment (bottom).

B) Transport of charged sacs through thin or thick epidermal samples to which an electric potential is applied.

Example 58: Suspension properties:

Total Lipids (TL) at a content of 0.5% by weight, comprising:

274mg Phosphatidylglycerol (DOPG)

226mg Tween 80

3H-DPPC (relative to DOPG)

Buffer as in previous examples

Capsule/pore ratio: 3.3 Electrical properties:

Current: 0mA, 0.2mA (current density: 0, 0.048mAcm ^-2 )

Potential difference: 0.5V

Barriers for this experiment were prepared by trypsinizing samples of heated isolated murine epidermis for 7 hours. Radioactive phospholipids are used instead of the fluorescent labels used. Therefore, samples were drawn manually from the body fluid and read with a beta-counter. Other differences between this test and the previous ones are the use of rat, rather than human epidermis, and the use of relatively low electrical currents.

After a lag time of approximately 30 minutes, constant vesicular transport was initially observed. The rate of lipid transfer across the barrier calculated from the subsequent 90 min linear time was approximately 4 µg/hr/cm ^-2 . Afterwards, no significant transport was observed according to other measurements with non-radioactive labels.

This experiment clearly demonstrates that a charged carrier (here due to the presence of DOPG) can be used to electrotransport an uncharged species (here ³ H-DPPC) across a barrier with an applied potential difference.

Figure 12: Electrically controlled transport of uncharged molecules bound to charged superdeformable capsules through mouse epidermis in vitro.

Claims (39)

1. preparation that contains penetrating agent, this penetrating agent is formed by unimolecule or molecules align, because it is fine adaptive with the hole of barrier energy, so can see through the hole of barrier, even when the hole of described barrier also can see through during less than the average diameter of described penetrating agent, and described penetrating agent can make medicine see through the hole and transport, and perhaps makes medicine see through the hole after penetrating agent enters described hole; Select the average diameter and the suitability of described penetrating agent, and make described penetrating agent and/or described medicine have enough electric charges, so that described penetrating agent can make and/or control transport of drug by described hole under the influence of suitable electrical drive power, perhaps medicine sees through described hole after penetrating agent enters described hole, carries out described selection when keeping enough penetrating stability.

2. according to the preparation of claim 1, it is characterized in that described penetrating agent has enough electric charges at least when combining with medicine, but under the influence that does not have electrical drive power, penetrating agent is difficult for the penetration barriers hole; Select the average diameter of the charged coalition of charged penetrating agent or penetrating agent and medicine, kind and the quantity and/or the suitability of electric charge, transport by barrier so that control is described under the influence that is implemented in electrical drive power.

3. according to the preparation of claim 1, it is characterized in that described penetrating agent has enough electric charges at least when combining with medicine, and described penetrating agent can pass the hole of barrier under the influence that does not have electrical drive power; Select the average diameter of the charged coalition of charged penetrating agent or penetrating agent and medicine, kind and the quantity and/or the suitability of electric charge, make it under the influence of electrical drive power, to control medicine and transport by barrier.

4. each preparation of claim 1-3, described penetrating agent can pass described hole under the influence of suitable driving force, when penetrating agent has suitable electric charge, described driving force can be an electrical drive power, and described medicine has enough electric charges, and described penetrating agent makes medicine and/or controls the hole of drug osmotic by barrier after entering described hole by means of electrical drive power.

5. each preparation of claim 1-4, the average diameter that it is characterized in that this charged penetrating agent or penetrating agent and the charged coalition of medicine than barrier hole average diameter at least greatly to 2 times.

6. each preparation of claim 1-5 is characterized in that this penetrating agent is formed by the arrangement of charged unimolecule or charged molecule, and or uncharged drug molecule charged with one or more combines.

7. each preparation of claim 1-5 is characterized in that this penetrating agent is formed by electroneutral unimolecule or electric neutrality molecules align, and combines with at least a charged medicine that the amount of this electric charge should be enough to and can transport.

8. claim 6 or 7 preparation, it is characterized in that described penetrating agent is suspended in or is scattered in the liquid medium, and comprise the molecules align of assembling the droplet form that one or more layers class membrane coat of the amphiphilic substance of tendency surrounds by having of at least two classes or two kinds of forms, the dissolubility of described at least two kinds of materials in the liquid medium of preferred water differs at least 10 times, like this than the average diameter of the equal aggregation of lyotrope matter or the average diameter of assorted aggregation that comprises two kinds of described materials less than the average diameter of the equal aggregation of the relatively poor material of dissolubility.

9. the preparation of claim 8 is characterized in that being transported medicine by barrier than lyotrope matter, and has and the relatively poor material of dissolubility forms the tendency of big common structure.

10. the preparation of claim 9 is characterized in that described common structure comprises the physics or the chemical complex of these materials.

11. claim 8,9 or 10 preparation, the content that it is characterized in that being tending towards dissolving than lyotrope matter penetrating agent droplet and this material is up to the 99mol% of the required concentration of this drop of dissolving or is up to the 99mol% that does not dissolve the saturated concentration in the drop, with higher being as the criterion.

12. the preparation of claim 11 is characterized in that being lower than 50% of the corresponding concentration of ordinary dissolution of described material than the content of lyotrope matter, and more preferably 40%, most preferably 30%.

13. the preparation of claim 11, it is characterized in that content than lyotrope matter be lower than material described in the drop saturated concentration 99%, preferred 80%, most preferably 60%.

14. each preparation of claim 8-13 is characterized in that the relatively poor accumulative material of dissolubility own is a lipid material, and is surfactant than lyotrope matter.

15. each preparation of claim 8-14, the average diameter that it is characterized in that this penetrating agent is 40～500nm, preferred 50～250nm, and more preferably 55～150nm is especially preferably at 60～120nm.

16. each preparation of claim 8-14 is characterized in that the average diameter of this penetrating agent is bigger 2～25 times than the average diameter in barrier hole, preferred 2.25～15 times, and more preferably 2.5～8 times, most preferably 3～6 times.

17. each preparation of claim 8-16 is characterized in that the average net charge density in drop surface is 0.05Cbm ^-2(coulomb/square metre)～0.5Cbm ^-2, preferred 0.075Cbm ^-2～0.4Cbm ^-2, especially preferred 0.10Cbm ^-2～0.35Cbm ^-2

18. each preparation of claim 8-17, the weight that it is characterized in that being used for the preparation drop of human or animal's skin are 0.01～40 weight %, especially 0.1～30 weight % of total preparation material, more preferably 5～20 weight %.

19. each preparation of claim 8-17, the weight that it is characterized in that being used for the preparation drop of human or animal's mucosa is 0.0001～30 weight %.

20. each preparation of claim 8-19 is characterized in that described medicine is a thyroliberin, antiadrenergic agent, androgen and androgen antagonist, parasiticide, anabolic agent, anesthetis or analgesic, analeptic, antiallergic agent, antiarrhythmics, the arteriosclerosis agent, antiasthmatics and/or bronchial spasmolytic agent, antibiotic, antidepressant and/or psychosis, antidiabetic drug, antidote, antiemetic, town's epilepsy agent, the antifibrin distintegrant, spasmolytic or anticholinergic, enzyme, coenzyme or corresponding enzyme inhibitor, hydryllin, hypotensive agent, the hypotension agent, anticoagulant, antimycotic agent, antimyasthenic, sick or the parkinsonian medicine of Kang Aercihaimoshi, antiinflammatory, antipyretic, rheumatism, antibacterial, respiratory stimulant or respiratory stimulant, the bronchitis agent, cardiac tonic, chemotherapeutics, the coronary dilation agent, cytostatics, diuretic, ganglionic block agents, glucocorticoid, antiviral drug, hemorrhage, sleeping pill, immunoglobulin or its fragment or any other immunologic active material be immunomodulator for example, cytokine etc., biological activity carbohydrate or derivant, contraceptive, antimigraine, corticosteroid, muscle relaxant, tranquilizer, the Neurotherapeutic agent, (many) nucleotide, psychosis, neurotransmitter, (many) peptides (derivant), Opiate, the eye medication, intend (pair) sympathetic nerve medicine, anti-(pair) sympathetic nerve medicine, protein (derivant), psoriasis/neurodermatitis medicine, mydriatic, psychostimulant, the medication of nose section, somnifacient, tranquilizer, spasmolytic, antituberculotic, the urology department medication, vasoconstrictor or vasodilation, antiviral agent, Wound-healing agent, inhibitor of any active said medicine or its compositions (antagonist) or promoter (agonist).

21. each preparation of claim 8-20 is characterized in that characteristic, the especially basic electrolytical concentration of the liquid medium selected and forms should making this penetrating agent or controlling this penetrating agent and effectively transport by the barrier hole.

22. the preparation of claim 8-21, it is characterized in that described basic electrolyte, especially buffer agent is electrolyte or other low price electrolyte that is selected from unit price (1: 1), its volumetric concentration preferably is lower than 150mM, more preferably less than 100mM, most preferably be lower than 50mM, and especially preferably be not higher than 10mM.

23. the electricity of aforementioned any claim definition of influence drives transhipment penetrating agent and the binding molecule method by the barrier hole, it is characterized in that applying enough screen electromotive forces of striding.

24. the method for claim 23 is characterized in that being used to produce the electrode of striding the screen electromotive force and is positioned at the relative both sides of barrier or can guarantees that with being arranged on one side and with it most electric currents that produced flow through barrier.

25. the method for claim 23 or 24 is characterized in that being lower than 30V for the electromotive force that every square centimeter barrier surface applies, and is usually less than 15V, more preferably less than 10V.

26. the method for claim 23 or 24 is characterized in that applying that electromotive force produces that to stride the screen drive current be that physiology can tolerate, and is usually less than 2mAcm ^-2, preferably be lower than 1mAcm ^-2, more preferably less than 0.6mAcm ^-2, most preferably be lower than 0.4mAcm ^-2

27. each method of claim 23-26, the size that it is characterized in that electrode is less than 100cm ², be more preferably less than 100cm ², especially less than 50cm ², most preferably less than 10cm ², perhaps even less than 5cm ²

28. each method of claim 23-27 is characterized in that conductive material of electrodes comprises at least a metal, especially can be selected from noble metal, as silver and palladium, and/or the biocompatibility salt or the chemical complex of this metalloid, preferred biocompatibility chloride, most preferably silver chloride.

29. each method of claim 23-28 is characterized in that at least one electrode chamber fills charged penetrating agent.

30. the method for claim 29 is characterized in that electrode is in application site installation or installation in advance.

31. the method for claim 29 or 30 is characterized in that using preceding installing electrodes facing, and preferably in facing with preceding 360 minutes, is more preferably facing with preceding 60 minutes, even is installing in 30 minutes.

32. claim 29,30 or 31 method is characterized in that electrode is equipped with to combine in advance to treat the especially charged penetrating agent of biologically active drug of transport molecule.

33. claim 29,30 or 31 method is characterized in that electrode is equipped with penetrating agent and the bonded with it transport molecule for the treatment of, especially loading days or afterwards with the bonded medicine of penetrating agent.

34. each method of claim 23-33, it is characterized in that with one or more sequencing, preferably little portable or Self-carried type is polarity, size and/or the time dependence of the electromotive force that applied of the disposable or reusable device control of watch style for example.

35. each method of claim 23-34 is characterized in that selecting the surfaces of different treatments to control transhipment.

36. each method of claim 23-35, it is characterized in that barrier initially carries out will carrying out pretreatment before the electricity driving transhipment at charged penetrating agent, promptly by suitable penetrating agent is added on the transformable barrier in the unblock mode, especially the barrier that forms by human or animal's skin, pretreatment can increase the quantity or the width in penetrable hole in the barrier, and this barrier is exactly to be used for the described pretreated skin barrier that electricity drives transhipment.

37. the method for claim 36, to drive the penetrating agent of transhipment similar or identical with being used for electricity subsequently for the charged or uncharged penetrating agent that it is characterized in that being used for the pretreatment barrier.

38. the method for claim 36 or 37, it is characterized in that charged or uncharged penetrating agent drives before transhipment sees through barrier initially carrying out electricity, the nonobstructive load time can reach 24 hours or even the longer time, normally at the most 12 hours, especially at the most 3 hours, more preferably less than 1.5 hours, even less than 30 minutes.

39. each method of claim 23-38, it is characterized in that the transport velocity of charged penetrating agent by the barrier hole is that flow is measured as the function of the electromotive force that applies or passes through the function of the electric current of barrier, and the function that records thus is used to make preparation or uses optimization.

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