RU2209031C2 - Method for forming micro-pores in human skin for delivering medicaments and carrying out monitiring - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves forming micro-pores and additionally acting with acoustic energy and chemical amplifier. The acoustic energy is modulated with by means of frequency modulation, amplitude modulation, phase modulation and/or their combination. Micro- pore formation is carried out by means of corneal layer destruction and by means of fast local water heating so that water evaporates resulting in this way by cell erosion. Micro-pore formation is carried out by piercing the corneal layer with scalpel of exactly selected size for creating micro-pore of up to 1000 mcm diameter; by means of corneal layer destruction using acoustic energy beam focused upon the mentioned corneal layer; by means of hydraulic puncture of the corneal layer with high pressure liquid jet for building a micro-pore of approximately 1000 mcm diameter; or by piercing the corneal layer with short electric impulses for building a micropore of approximately 1000 mcm diameter. EFFECT: reduced barrier properties of the corneal layer; controlled collection substance for making analysis. 60 cl, 34 dwg, 2tbl

Description

 This application is a partial continuation of the application from Series 08/520547, filed August 29, 1995, which is a partial continuation of the application from Series 08/152442, filed November 15, 1993, now US Patent 5458/140, and Series 08/152174, filed December 8, 1993, now US Pat. No. 5,445,611. This application also claims the benefit of provisional application US 60/0088043, filed October 30, 1995

State of the art
This invention relates generally to the field of monitoring analytes in the body and transdermal drug delivery to the body. More specifically, this invention relates to a method, from minimally invasive to non-invasive, to increase the permeability of the skin by creating micropores in the stratum corneum, moreover, the method can be combined with acoustic energy, chemical permeation enhancers, pressure and the like to selectively enhance the outward flow of analytes from organism to monitor them or to deliver drugs to the body.

 The stratum corneum is mainly responsible for the well-known barrier properties of the skin. Thus, this is the layer that represents the biggest obstacle to the transdermal flow of drugs or other molecules into the body and analytes from the body. The stratum corneum, the outer stratum corneum of the skin, is a complex structure of compact keratinized cell residues separated by lipid fragments. Compared to the mucous membrane of the oral cavity or stomach, the stratum corneum is much less permeable to molecules both outside and inside the body. The stratum corneum is formed from keratinocytes, which make up the majority of epidermal cells, which lose their nuclei and become cornsocytes. These dead cells make up the stratum corneum, having a thickness of only about 10-30 microns and, as noted above, is a very stable waterproof membrane that protects the body from the introduction of external substances and the outward migration of liquids and dissolved molecules. The stratum corneum is constantly updated by exfoliating the cells of the stratum corneum during desquamation and the formation of new cells of the stratum corneum using the keratinization process.

 The flow of a drug or analyte through the skin can be increased by a change in either resistance (diffusion coefficient) or driving force (gradient for diffusion). The flow can be enhanced by the use of so-called penetration enhancers or chemical amplifiers. Chemical enhancers are well known in the art and will be described in more detail below.

 Another way to increase skin permeability for drugs is iontophoresis. Iontophoresis involves the use of an external electric field and the local delivery of an ionized form of a drug or deionized drug carried with a stream of water associated with ion transport (electroosmosis). Although enhancing penetration by iontophoresis was effective, regulation of drug delivery and irreversible skin damage are problems associated with this technique.

 Acoustic energy has also been used to enhance the permeability of the skin and synthetic membranes for drugs and other molecules. Ultrasound was defined as mechanical pressure waves with a frequency above 20 kHz (H. Lutz et al., Mannual of Ultrasound 3-12 (1984). Acoustic energy is generated by vibration of a piezoelectric crystal or other electromechanical element by passing alternating current through the material (R. Brucks et al., 6 Pharm. Res. 697 (1989). The use of acoustic energy to increase the permeability of the skin to drug molecules has been called co-phoresis or phonophoresis.

 Although it was found that enhancing the permeability of the skin would theoretically provide the possibility of transporting molecules from the internal environment of the body through the skin to the external environment of the body for collection and monitoring, the methods applicable in practice were not disclosed. In US patent 5139023 issued by Stanley et al. , a device and method for non-invasive monitoring of blood glucose is disclosed. In this invention, chemical permeation enhancers are used to increase the glucose permeability of the tissue of the mucous membrane or skin. Then there is passive diffusion of glucose through the tissue of the mucous membrane or skin and the capture of glucose by the receiving medium. Measurement of the amount of glucose in the receiving medium and correlation to determine the level of glucose in the blood. However, according to Stanley et al. this method is much more effective when used on mucosal tissue such as buccal tissue, which results in the collection of detectable amounts of glucose in the receiving medium after a delay time of about 10-20 minutes. However, the method proposed by Stanley et al. , depending on the composition of the chemical enhancer used, leads to an extremely long delay time in the range of 2 to 24 hours before detectable amounts of glucose diffusing in vitro through the skin (epidermis separated by thermal exposure) can be determined. These long delay periods can be attributed to the length of the time period required for passive diffusion of chemical permeation enhancers through the skin and to enhance the permeability of the stratum corneum barrier, as well as the length of time required for passive diffusion of glucose through the skin. Thus, Stanley et al. they do not provide a clear description of the non-invasive method for transporting blood glucose or other analytes through the skin in such a way as to enable rapid monitoring, which is required for monitoring blood glucose in patients with diabetes and for many other analytes of the body, such as blood electrolytes.

 Although the use of sound energy for drug delivery is known, the results were largely disappointing because the increase in permeability was relatively small. There is no agreement on the effectiveness of sound energy to increase the flow of a drug through the skin. Although some studies have reported the success of sonophoresis (see J. Davick et al., 68 Phys. Ther. 1672 (1988); J. Griffin et al., 47 Phys. Ther. 594 (1967); J. Griffin & J . Touchstone, 42 Am. J. Phys. Med. 77 (1963); J. Griffin et al., 44 Am. J. Phys. Med. 20 (1965); D. Levy et al., 83 J. Clin. Invest. 2074; D. Bommannan et al., 9 Pharm. Res. 559 (1992)), others received negative results (H. Benson et al., 69 Phys. Ther. 113 (1988); J. McElnay et al. , 20 Br. J. Clin. Pharmacol., 4221 (1985); H. Pratzel et al., 13 J. Phamacol. 1122 (1986)). Systems that used rodent skin gave the most promising results, while systems that used human skin gave disappointing results. Specialists in this field are well aware that the skin of rodents is much more permeable than human skin, and therefore, the above results do not provide specialists in this field with information on how to effectively use sonophoresis when used for transdermal delivery and / or monitoring through the skin person.

 A significant improvement in the monitoring of analytes, as well as the delivery of drugs to the body using ultrasonic energy, is disclosed and claimed in jointly filed applications of Series 08/152442, filed November 15, 1993, currently US Pat. No. 5,458,140 and Series 08/152174, filed 8 December 1993, is currently US patent 54456110, both of which are incorporated into this application by reference. In these inventions, transdermal sampling of analytes or transdermal drug delivery is achieved by using sound energy that is modulated by intensity, phase or frequency, or a combination of these parameters in combination with chemical penetration enhancers. The use of sound energy is also disclosed, optionally with modulation of frequency, intensity and / or phase for controlled pushing, and / or injection of molecules through the stratum corneum through perforations formed by punctures with a needle, hydraulic jet, laser, electroporation, or other methods.

 The formation of micropores (i.e., micropore formation) in the stratum corneum to enhance drug delivery has been the subject of various studies and has led to the grant of patents for such techniques.

 Jacgues et al. 88 J. Invest. Dermatol. 88-93 (1987) describe a method for administering a drug by removing the stratum corneum from the skin using pulsed laser radiation with a wavelength, pulse duration, pulse energy, pulse number and pulse repetition rate sufficient to remove the stratum corneum without significant damage to the subject epidermis and subsequent application of the drug to the area of removal. This work led to the grant of US patent 4775361 Jacgues et al. Skin removal through the use of ultraviolet laser radiation was previously reported by Lane et al., 121 Arch. Dermatol. 609-617 (1985). Jacques et al. limited to using light with several wavelengths and expensive lasers.

 Tankovich, US Patent No. 5,165,418 (hereinafter, “Tankovich '418") discloses a method for producing blood samples by irradiating human or animal skin with one or more laser pulses with an energy sufficient to cause evaporation of the skin tissue so as to form an opening in the skin extending through the epidermis for penetration into the lumen of at least one blood vessel to expel blood through the opening to allow collection. Thus, Tankovich '418 is not suitable for a non-invasive or invasive increase in the permeability of the stratum corneum so that it is possible to deliver the drug to the body or to analyze the analyte from the body.

 Tankovich, US Patent No. 5,423,803 (hereinafter referred to as “Tankovich '803”) discloses a method for laser removal of surface epidermal skin cells in human skin for cosmetic use. The method involves applying a light-absorbing “impurity” to the outer layers of the epidermis and forcing a certain amount of this impurity to penetrate into the intercellular spaces in the stratum corneum and illuminate the infiltrated skin with laser pulses of sufficient intensity so that the amount of energy absorbed by the impurity causes the impurity to explode with sufficient energy to rupture some cells of the epidermis of the skin. Tankovich '803 also instructs that there should be high energy absorption by the impurity at the laser wavelength, that the laser radiation must be pulsed radiation with a duration of less than 1 ms, that the impurity must be forced to penetrate the upper layers of the epidermis, and that the impurity must explode with the release of energy sufficient to rupture epidermal cells after absorption of laser energy. This invention also did not disclose or suggest a method for delivering a drug or collecting analytes.

Raven et al. WO 92/00106 describe a method for selectively removing unhealthy tissue from an organism by introducing into a selected tissue a compound having a high absorbance of infrared radiation with a wavelength of 750-860 nm, and irradiating the region with appropriate infrared radiation at a power sufficient to cause thermal evaporation of the tissue into which the compound was introduced, but not sufficient to cause evaporation of the tissue into which the compound was not introduced. The absorbent compound must be soluble in water or serum, such as green indocyanin, chlorophyll, porphyrins, heme compounds or compounds containing a polyene structure, and power levels are in the range of 50 to 1000 W / cm ² or even higher.

Konig et al., DD 259351 propose a method for heat treatment of tumor tissue, which comprises introducing into the tumor tissue a medium that absorbs radiation in the red and / or close to the red infrared region of the spectrum, and irradiating the infiltrated tissue with laser light with an appropriate wavelength. Absorbing media may include methylene blue, reduced porphyrin or its aggregates, and phthalocyanine blue. Examples are methylene blue, which strongly absorbs at 600–700 nm, and a krypton laser emitting at 647 and 676 nm. The power level must be at least 200 mW / cm ² .

 It was shown that by exfoliating the stratum corneum from a small area of the skin while re-applying and removing the cellophane tape to the same place, it is possible to easily collect the desired amount of interstitial fluid, in which you can then quantify a number of analytes of interest. In a similar manner, it has also been shown that skin-peeled skin is permeable to transdermal delivery of compounds to the body. Unfortunately, “peeling off the tape” leaves an open wound that heals within weeks, and therefore, as well as for other reasons, this method is not considered acceptable for widespread use to enhance transdermal transport.

As discussed above, it has been shown that pulsed lasers such as an excimer laser operating at 193 nm, an erbium laser operating at approximately 2.9 μm, or a CO ₂ laser operating at 10.2 μm can be used to efficiently form small holes in the stratum corneum of human skin. These laser destruction techniques provide an opportunity to create a method for selectively and potentially non-traumatic hole formation in the stratum corneum for feeding and / or sampling. However, due to the unacceptably high costs associated with these light sources, products based on this principle have not been developed on an industrial scale. By defining a method for directly transferring thermal energy to the stratum corneum at a very strictly defined resolution in space and time, the disclosed invention provides the ability to induce the desired microdestruction of the stratum corneum using very cheap energy sources.

 Given the above problems and / or disadvantages, the development of a method for safely enhancing the permeability of the skin for minimally invasive or non-invasive monitoring of the analyzed substances in the body for a shorter period of time would be a significant achievement in this area. Another significant achievement in this area would be the provision of a method of minimally invasive or non-invasive enhancement of the transdermal flow rate of a drug into a selected area of the human body.

SUMMARY OF THE INVENTION
The objective of the invention is to minimize the barrier properties of the stratum corneum using pore formation for the controlled collection of the analyzed substances from the internal environment of the body through perforations in the stratum corneum to provide control over these analytes.

 The objective of the invention is also to provide a method for controlling selected analytes in the body through micropores in the stratum corneum in combination with sound energy, permeability enhancers, pressure gradients and the like.

 Another object of the invention is to provide a method for controlling the rate of transdermal flow of drugs or other molecules into the body and, if desired, into the blood stream through small perforations in the stratum corneum.

 Another objective of the invention is the provision of a method for the delivery of drugs into the body through micropores in the stratum corneum in combination with sound energy, permeability enhancers, pressure gradients and the like.

These and other tasks can be solved by providing a method for controlling the concentration of the analyte in the human body, including the steps of enhancing the permeability of the stratum corneum of a selected area of the body surface of an individual using
a) the formation of pores in the stratum corneum of the selected area using means that form micropores in the stratum corneum without causing serious damage to the underlying tissues, thereby reducing the barrier properties of the stratum corneum to remove the analyte;
(b) collecting a selected amount of an analyte; and
(c) quantifying the collected analyte.

 In a preferred embodiment, the method further comprises exposing the selected region with sound energy at a frequency in the range of about 5 to 100 MHz, in which the sound energy is modulated by an element selected from the group consisting of a frequency modulator, amplitude modulator, phase modulator, and combinations thereof.

 In another preferred embodiment, the method also includes contacting a selected area of the body of the individual with a chemical amplifier with exposure to sound energy to further enhance the removal of the analyte.

 The formation of pores in the stratum corneum is achieved using a means selected from the group consisting of (a) destruction of the stratum corneum by contacting a selected region of the stratum corneum with a transverse dimension of up to about 1000 μm with a heat source so that the temperature of the water and other evaporated tissue substances in the selected area rises above the point of evaporation of water and other evaporated substances, thus removing the stratum corneum in the selected area; (b) piercing the stratum corneum with a microscalpel calibrated to form micropores with a diameter of up to about 1000 microns; (c) destruction of the stratum corneum by focusing a rigidly focused beam of sound energy on the stratum corneum; (d) hydraulic puncture of the stratum corneum with a high-pressure liquid jet to form micropores with a diameter of up to about 1000 microns; and (e) puncture of the stratum corneum with short pulses of electricity to form micropores with a diameter of up to about 1000 microns.

 One preferred embodiment of thermal destruction of the stratum corneum involves treating at least a selected area with an effective amount of a dye that exhibits strong absorption in the emission range of the pulsed light source and focuses the generated series of pulses from the pulsed light source onto the dye so that the dye is heated sufficiently to conduct heat transfer to the stratum corneum to raise the temperature associated with the tissue of water and other evaporated substances in the selected region STI above the vaporization point of water and other substances evaporated. Preferably, the pulsed light source emits at a wavelength that is slightly absorbed by the skin. For example, the pulsed light source may be a laser diode emitting in the range of approximately 630 to 1550 nm, a laser diode optical parametric pump generator emitting in the range of approximately 700 to 3000 nm, or an element selected from the group consisting of arc lamps , incandescent lamps and light emitting diodes. A monitoring system may also be provided to determine when the barrier properties of the stratum corneum are overcome. One preferred monitoring system includes light trapping means for receiving light reflected from a selected area and focusing the reflected light on a photodiode, a photodiode for receiving focused light and for sending a signal to a control unit in which the signal indicates the quality of the reflected light, and a control unit connected with a photodiode and with a pulsed light source for receiving a signal and for turning off the pulsed light source when a previously selected signal is received.

 In another preferred embodiment, the method further comprises cooling the selected region of the stratum corneum and adjacent skin tissues with a cooling agent such that said selected region and adjacent skin tissues are in a selected, pre-cooled, stable state before pore formation.

 In yet another preferred embodiment, the method includes the destruction of the stratum corneum so that the interstitial fluid flows from the micropores, collecting the interstitial fluid and analyzing the analyte in the collected interstitial fluid. After collecting the interstitial fluid, the micropore can be sealed by supplying an effective amount of energy from a laser diode or other light source so that coagulation of the interstitial fluid remaining in the micropore is caused. Preferably, a vacuum is created to enhance the collection of interstitial fluid in a selected pore region.

 In yet another preferred embodiment, the method includes, before pore formation in the keratinized layer, illuminating at least a selected area with unfocused light from a pulsed light source so that the selected area illuminated with light is sterilized.

Another preferred method for forming pores in the stratum corneum involves contacting the selected area with a metal wire so that the temperature of the selected area rises from ambient skin temperature to a level above 100 ^° C in the range of 10 to 50 ms and then returns the temperature of the selected area to approximately the temperature of the surrounding skin in the range of about 30 to 50 ms, in which this cycle of raising the temperature and returning to approximately the temperature of the surrounding skin is repeated several times, which is effective to reduce the barrier properties of the keratinized layer. Preferably, the step of returning to approximately the temperature of the surrounding skin is carried out by removing the wire from contact with the keratinized layer. It is also preferable to provide means for monitoring the total electrical resistance between the wire and the human body through a selected area of the stratum corneum and adjacent skin tissue and means for advancing the position of the wire so that as degradation occurs with a concomitant decrease in resistance, the advancing means advances the wire so that the wire is in contact with the stratum corneum during heating of the wire. It is further preferable to also provide means for removing wire from contact with the stratum corneum, in which the control means is able to detect a change in impedance associated with contacting the epidermis layer lying under the stratum corneum, and supplying a signal to the means for removing wire from contact with the stratum corneum. The wire may be heated using an active heating element, may include a current loop having a high resistance point, at which the temperature of the high resistance point is modulated by passing a modulated electric current through said current loop to affect heating, or may be located in a modulated, alternating magnetic field excitation coils so that the excitation of the excitation coil by alternating current causes the formation of eddy currents sufficient to heat rods using internal active losses.

A method of increasing the transdermal flow rate of an active penetrating substance to a selected area of the human body includes the steps of enhancing the permeability of the stratum corneum of a selected area of the body surface of the individual for the active penetrating substance by
(a) forming a pore in the stratum corneum of a selected area by means of forming micropores in the stratum corneum without causing serious damage to the underlying tissues, and thus reducing the barrier properties of the stratum corneum for the flow of active penetrating substance and
(b) contacting a selected area having a created pore with a composition comprising an effective amount of a penetrating agent such that the flow of the penetrating agent into the body is enhanced.

 In a preferred embodiment, the method further comprises exposing the selected region to sound energy at times over time and at an intensity and frequency effective to create an entrainment flow effect and thereby increase the rate of transdermal flow of the penetrating substance into the body.

Also provided is a method of applying a tattoo to a selected area of the skin on the surface of an individual's body, comprising the steps of:
(a) the formation of pores in the stratum corneum of the selected area using means that form micropores in the stratum corneum, without causing serious damage to the underlying tissues, and thus reducing the barrier properties of the stratum corneum for the flow of active penetrating substance and
(b) contacting a selected area having a created pore with a composition comprising an effective amount of tattoo ink as a penetrating agent such that the flow of said ink into the body is enhanced.

 Further, there is still provided a method of reducing the time delay of diffusion of an analyte from human blood into the interstitial fluid of said person in a selected area of the skin, comprising applying an agent for cooling to said selected area of the skin.

 Still further, a method is provided for reducing evaporation of interstitial fluid and its vapor pressure, wherein said interstitial fluid is collected from micropores in a selected area of the stratum corneum of a person’s skin, comprising applying a cooling agent to said selected skin area.

 A brief description of the drawings in several projections.

 FIG. 1 illustrates a schematic illustration of a system for supplying laser diode radiation and monitoring the pore formation process.

 Figure 2 illustrates a schematic diagram of a closed loop feedback system for controlling pore formation.

 3A illustrates a schematic illustration of an optical system for forming a pore including a cooling device.

 FIG. 3B illustrates a top view of a schematic illustration of an illustrative cooling device in accordance with FIG. 3A.

 FIG. 4 illustrates a schematic illustration of an active heating element with a mechanical starter.

 FIG. 5 illustrates a schematic illustration of a heating element with a current loop having a high resistance point.

 6 illustrates a schematic illustration of a device for modulating heating using induction heating.

 7 illustrates a schematic illustration of a closed-loop impedance monitor using impedance changes to determine the degree of pore formation.

In FIG. 8A-D show cross sections of human skin treated with copper phthalocyanite and then exposed to light radiation with a wavelength of 810 nm and a pulse frequency of 0, 1, 5 and 50, respectively, at an energy density of 4000 J / cm ² for a pulse period of 20 ms.

 Figures 9-11 illustrate a schematic representation of the temperature distribution during the simulation of thermal processes of pore formation using optical pore formation.

 FIG. 12 and 13 illustrate a schematic representation of temperature as a function of time, respectively, in the stratum corneum and viable epidermis during the simulation of thermal processes of pore formation using optical pore formation.

 FIG. 14-16 illustrate a schematic representation of temperature distribution as a function of time in the stratum corneum and temperature as a function of time in a viable epidermis during the simulation of thermal processes of pore formation using optical pore formation in which the tissue was cooled before pore formation.

 FIG. 17-19 illustrate a schematic representation of temperature distribution as a function of time in the stratum corneum and temperature as a function of time in a viable epidermis during the simulation of thermal processes of pore formation in which tissue was heated by a hot wire.

 FIG. 20-22 illustrate a schematic representation of temperature distribution as a function of time in the stratum corneum and temperature as a function of time in a viable epidermis during the simulation of thermal processes of pore formation in which tissue was heated by hot wire and the tissue was cooled before pore formation.

 23 and 24 illustrate a schematic representation of the temperature and temperature distribution, respectively, as a function of time in the stratum corneum during the simulation of pore formation processes in which tissue is heated optically in accordance with Tankovich '803 functional parameters.

 FIG. 25 illustrates a schematic representation of glucose levels in interstitial fluid (CSF (o)) and blood (*) as a function of time.

 FIG. 26 illustrates a graph of the scatter of the differences between the GIS glucose data and the glucose data in FIG. 25.

 FIG. 27 illustrates a histogram of the relative deviation of the ILI relative to blood glucose levels of FIG. 25.

 FIG. 28 illustrates a cross-sectional view of an exemplary delivery device for delivering a drug to a selected area on human skin.

 FIG. 29A-C illustrate a schematic representation of skin areas after lidocaine is delivered to selected areas where a pore has been formed in the stratum corneum (Figs. 29A-B) or not (Fig. 29C).

 FIG. 30 illustrates a graph comparing the amount of interstitial fluid collected from micropores using only suction (ω) and a combination of suction and ultrasound (*).

 FIG. 31-33 illustrate a perspective view of an ultrasonic transducer (a vacuum device for collecting interstitial fluid), respectively a transverse section of the same device and a schematic cross-sectional view of the same device.

 FIG. 34 A-B illustrate a top view of an ultrasound probe respectively held in hands and a side view of its blade end.

Detailed description
Before these methods for increasing the permeability of the stratum corneum to facilitate transdermal drug delivery and sampling of analytes are disclosed and described, it should be understood that this invention is not limited to the specific configurations, process steps, and materials disclosed herein, since such configurations, steps methods and materials may vary to some extent. It should also be understood that the terminology used here is used only to describe certain embodiments and is not intended to be limiting, since the range of claims of the present invention will be limited only by the attached claims and their equivalents.

 It should be noted that used in the present description and the attached claims, articles of a single form, denoting "one" or "this" include the plural, unless the context clearly indicates otherwise. Thus, a reference to a method for delivering a “drug” includes a link to delivering a mixture of two or more drugs, a link to an “analyte” includes a link to one or more such analytes, and a link to a “permeation enhancer” includes a link to a mixture of two or more permeation enhancers.

 In describing and presenting the claims of the present invention, the following terminology will be used in accordance with the following definitions.

 As used herein, the term “pore formation”, “formed micropores” or any similar term means the formation of a small hole or pore in the stratum corneum in a selected area of an individual’s skin to reduce the barrier properties of this skin layer to allow analytes to pass from under the skin surface for analysis or for the passage of active penetrating substances or drugs into the body for medicinal purposes. Preferably, the opening or pore will be no more than about 1 mm in diameter, and more preferably no more than about 100 microns in diameter, and will extend into the stratum corneum enough to destroy the barrier properties of this layer without adversely affecting the underlying tissues.

 As used herein, the term “destruction” refers to the controlled removal of cells due to the kinetic energy released when the vaporized components of the cells are heated to the point where evaporation occurs and the resulting rapid expansion of volume due to a phase change causes the cells and possibly some adjacent cells to “blow out” from the destruction site.

 As used herein, the term “puncture” or “micro-puncture” refers to the use of mechanical, hydraulic, or electrical means to perforate the stratum corneum.

 To the extent that "destruction" and "puncture" achieve the same goal of pore formation, i.e. creating holes or pores in the keratinized layer without significant damage to underlying tissues, these terms can be used interchangeably.

 As used herein, the term “penetration enhancing” or “enhancing permeability” refers to an increase in skin permeability for a drug, analyte, dye, paint or other chemical molecule (also called “penetrating agent”), i.e. in order to increase the rate at which a drug, analyte or chemical molecule penetrates through the stratum corneum and facilitates the formation of pores in the stratum corneum, removal of analytes through the stratum corneum, or drug delivery through the stratum corneum to the underlying tissues. Enhanced penetration achieved through the use of such enhancers can be observed, for example, by observing the diffusion of a dye as a penetrating substance through the skin of an animal or person using a diffuser.

 As used herein, the terms “chemical enhancer”, “penetration enhancer”, “permeation enhancer” and the like include all amplifiers that increase the flow of a penetrating agent, analyte, or other molecule through the skin and are limited only by functionality. In other words, it is intended to include all compounds and solvents and any other chemical enhancers that cause cell wall disorganization.

 The terms “dye”, “paint” and the like used here will be used interchangeably and refer to a biologically suitable chromophore exhibiting strong absorption in the range of light radiation and a pulsed light source used to destroy tissue of the keratinized layer to form micropores in it.

 As used herein, the term “transdermal” or “transdermal” refers to the passage of penetrating substance into and through the skin to achieve effective therapeutic levels in the blood or in the deep tissues of a drug or passage of a molecule present in the body (“analyte”) outward through the skin as so that molecules of the analyte can be collected outside the body.

 As used herein, the terms “penetrating agent”, “drug” or “pharmacologically active agent” or any other similar term refers to any chemical or biological material or compound suitable for transdermal administration using methods known in the prior art and / or using methods disclosed in the present invention, which causes the desired biological or pharmacological effect, which may include (but not limited to): (1) the presence of a prophylactic effect on organ m and preventing undesired biological effect such as preventing an infection, (2) alleviating a condition caused by a disease such as alleviating pain or inflammation caused as a result of disease, and / or (3) alleviating, reducing or eliminating the disease from the organism. The effect may be local, such as providing a local analgesic effect, or it may be systemic.

 This invention does not cause interest in new penetrating substances or in new classes of active substances. It is rather limited by the method of delivery of substances or penetrating substances that exist at the present level of technology or which can be later established as active substances and which are suitable for delivery using the present invention. Such substances include broad classes of compounds typically delivered to the body, including delivery through body surfaces and membranes, including skin. In general, this includes (but is not limited to): anti-infectious agents, such as antibiotics and antiviral agents; analgesics and analgesic combinations; anorexic drugs; anthelmintic; arthritis drugs; anti-asthma drugs; anticonvulsants; antidepressants; antidiabetic agents; anti-diarrhea drugs; antihistamines; anti-inflammatory drugs; anti-migraine drugs; remedies for nausea; antitumor agents; drugs against parkinsonism; anti-itch remedies; antipsychotic drugs; antipyretic drugs; antispasmodics; anticholinergics; sympathomimetic agents; xanthine derivatives; cardiovascular drugs, including potassium and calcium channel blockers, beta blockers, alpha blockers and antiarrhythmic drugs; antihypertensive drugs; diuretics and antidiuretics; vasodilators, including general, coronary, peripheral and cerebral; central nervous system stimulants; vasoconstrictor drugs; preparations for cough and colds, including decongestants; hormones such as estradiol and other steroids, including corticosteroids, hypnotics, immunosuppressive drugs; muscle relaxants; parasympatholytics; psychostimulants and tranquilizers. Using the method of the present invention, both ionized and non-ionized drugs can be delivered, as well as drugs with both high and low molecular weight.

 As used herein, the term “effective” amount of a pharmacologically active substance denotes a sufficient amount of a compound to provide the desired local or systemic effect and action with an acceptable benefit / risk ratio associated with any drug treatment. As used herein, the term “effective” amount of a penetration enhancer or chemical enhancer refers to an amount selected to provide a desired increase in skin permeability and a desired penetration depth, rate of administration, and amount of drug delivered.

 As used herein, the term “carriers” or “excipients” refers to carrier materials without significant pharmacological activity in the amounts used, suitable for administration with other pharmaceutically active materials, and include any such materials known in the art, for example, any liquid, gel, solvent, a liquid diluent, solubilizer or the like, which are non-toxic in the amounts used and do not cause harmful interactions with the drug. Examples suitable for use in this case include water, mineral oil, silicone, inorganic gels, aqueous emulsions, liquid sugars, waxes, petroleum jelly and many other oils and polymeric materials.

 As used herein, the term “biological membrane” is intended to mean membrane material present within a living organism that separates one area of the body from another and, in many cases, which separates the body from its external environment. Thus, the skin and mucous membranes are included.

 As used herein, the term "individual" refers to both humans and animals in which the present invention can be applied.

 As used herein, the term “analyte” means any chemical or biological material or compound suitable for passing through a biological membrane using the method disclosed in the present invention, or using a method known in the prior art, the concentration or activity of which within the body may want know the individual. Glucose is a particular example of an analyte because it is a sugar suitable for passing through the skin, and individuals, such as those with diabetes, may want to know their blood glucose levels. Other examples of analytes include, but are not limited to, substances such as sodium, potassium, bilirubin, urea, ammonia, calcium, tin, iron, lithium, salicylates and the like.

 As used herein, the term “transdermal current velocity” is the rate of outward passage of any analyte through the skin of an individual, human or animal, or the rate of passage of any drug, pharmacologically active dye or pigment in the skin of an individual, human or animal, and through the skin.

 The terms “intensity amplitude”, “intensity” and “amplitude” as used herein are used synonymously and refer to the amount of energy generated by an acoustic energy system.

 As used herein, the term “frequency modulation” or “oscillation” refers to a continuous, gradual, or step change in the amplitude or frequency of an ultrasound over a given period of time. Frequency modulation is a gradual or step change in frequency over a given period of time, for example, 5.4-5.76 MHz in 1 s, or 5-10 MHz in 0.1 s, or 10-5 MHz in 0.1 s, or any other frequency range or time period that corresponds to a particular application. Complex modulation can include both a change in frequency and intensity. For example, FIGS. 4A and 4B of US Pat. No. 5,458,140, respectively, can represent amplitude and frequency modulations simultaneously applied to a single acoustic energy transducer.

 As used herein, the term “phase modulation” means that the signal timing has been changed from its original state shown in FIG. 4C of US Pat. No. 5,458,140. The frequency and amplitude of the signal may remain the same. Phase modulation can be carried out with a different delay so as to selectively delay or give a signal earlier relative to its initial state or relative to another signal.

 Acoustic energy in its various applications, such as modulation in frequency, intensity or phase, or combinations thereof and the use of chemical amplifiers in combination with modulated acoustic energy, as described here, can vary in the frequency range from about 5 kHz to 100 MHz, with the preferred range from about 20 kHz to 30 MHz.

 As used herein, the term “non-invasive” means not requiring the insertion of a needle, catheter, or other invasive medical instrument into a part of the body.

 As used herein, the term “minimally invasive” refers to the use of mechanical, hydraulic, or electrical means that penetrate the stratum corneum to create a small hole or micropore without causing substantial damage to underlying tissues.

Means for forming pores of the stratum corneum
The formation of micropores in the stratum corneum can be achieved using various means of the modern level of technology, as well as certain means disclosed here, which are their improvements.

Use of laser destruction as described by Jacgues et al. in US patent 4775361 and Lane et al. above, they actually provide one means for the destruction of the stratum corneum using an excimer laser. It was found that at a wavelength of 193 nm and a pulse width of 14 ns., Using a laser pulse can be removed from approximately 0.24 to 2.8 μm keratinized layer when exposed to an emitter from approximately 70 to 480 mJ / cm ² . As energy increases
more tissue is removed from the stratum corneum, and a smaller number of pulses is required for complete formation of pores in this layer. The lower threshold for exposure to the energy of the emitter, which must be absorbed by the stratum corneum within the thermal relaxation time in order to induce suitable micro-exposures that will lead to tissue destruction, is approximately 70 mJ / cm ² within a time period of 50 ms. In other words, a total of 70 mJ / cm ² should be delivered within a 50 ms window. This can be done with a single pulse of 70 mJ / cm ² or 10 pulses of 7 mJ / cm ² , or constant lighting at a power of 1.4 W / cm ² for a time of 50 ms. The upper limit of the impact of the energy of the emitter is one that will lead to the destruction of the stratum corneum without damaging the underlying tissue and can be empirically determined by the light source, the length of the light waves and other indicators that are within the experience and knowledge of specialists in this field.

 By "feeding" is meant that the claimed amount of energy is absorbed by the tissue to be destroyed. At an excimer laser wavelength of 193 nm, substantially 100% absorption occurs within the first 1 or 2 μm tissue of the stratum corneum. Given that the stratum corneum is approximately 20 μm thick, at a longer wavelength, such as 670 nm, only approximately 5% of the incident light is absorbed within a 20 μm layer. This means that approximately 95% of the high-energy rays pass into the tissues lying under the stratum corneum, where they are likely to cause significant damage.

 It is ideal to use only the amount of power necessary for perforation of the stratum corneum without causing bleeding, thermal or other damage to the underlying tissues, from which the analyte is supposed to be extracted or into which drugs or other penetrating substances are supposed to be delivered.

 It would be advantageous to use energy sources that are more economical than energy from excimer lasers. Excimer lasers that emit light at a wavelength in the far ultraviolet spectrum are much more expensive to use and maintain than, for example, diode lasers that emit light at a wavelength in the visible and infrared spectra (from 600 to 1800 ni). However, with a longer wavelength, the stratum corneum becomes more transparent and absorption occurs mainly in the underlying tissues.

 The present invention facilitates the implementation of a quick and painless method of eliminating the barrier function of the stratum corneum to facilitate transdermal transport of therapeutic substances into the body with local application or to access the analytes inside the body for analysis. This method uses a procedure that begins with the contact overlay of a heat source with a small area on the target area of the stratum corneum.

A heat source must have several important properties that will now be described. First, the size of the heat source must be selected so that contact with the skin is limited to a small area, usually about 1 to 1000 microns in diameter. Secondly, it should be able to modulate the temperature of the stratum corneum at the point of contact from the surface temperature levels of the surrounding skin (33 ^o ) to a level above 123 ^o C and then return to approximately the temperature of the surrounding skin with a cycle time that minimizes collateral damage to viable tissues and sensations of the exposed individual. This modulation can be created by electronic, mechanical, or chemical means.

In addition, the inherent sign of limiting the depth of the micropore formation process can be facilitated if the heat source has a sufficiently small heat mass and a limited energy source to raise its temperature so that when it is placed in contact with tissues with a water content of more than 30%, the heat dispersion in these tissues is sufficient to limit the maximum heat source temperature to a level of less than 100 ^o C. This feature effectively stops the thermal vaporization process once the heat probe will penetrate Ogove layer to the deeper layers of the epidermis.

When the heat source is placed in contact with the skin, it is carried out through cycles including a series of one or more temperature modulations from the initial point of the temperature of the surrounding skin to a maximum temperature exceeding 123 ^° C, with a return to a temperature approximately equal to the temperature of the surrounding skin. To minimize the sensitive perception of the micropore formation process by the subject, these pulses are limited in duration and the interval between pulses is long enough to allow cooling of the layers of viable tissue in the skin and, in particular, that the innervated dermis tissue reaches an average temperature lower than about 45 ^o C. These parameters are based on the thermal time constants of viable epidermal tissues (roughly 30-80 ms) located between the thermal sensor and the innervated tissue in p appropriate dermis. The result of this application of pulsed thermal energy is that enough energy is carried into the stratum corneum within a very small target area, so that the local temperature of this volume of tissue rises significantly above the evaporation point of the water contained in the stratum corneum. As the temperature rises to a level exceeding 100 ^o C, evaporation of the water contained in the stratum corneum (usually 5 to 15%) within a limited area is caused, and very rapid expansion, causing steam-triggered removal of those corneocytes in the stratum corneum located nearby to this evaporation process. In US patent 4,775,361 it is described that the temperature of the stratum corneum 123 ^o C represents the threshold at which this type of flash evaporation occurs. As subsequent pulses of thermal energy are supplied, additional layers of the stratum corneum are removed until micropores are formed through the entire stratum corneum down to the next epidermis layer of the transparent layer. By limiting the duration of the heat pulse to less than one thermal time constant of the epidermis and allowing any thermal energy to be conducted into the epidermis for dissipation for a sufficiently long time, the temperature increase of the viable layers of the epidermis is minimal. This allows the entire process of micropore formation to be carried out without any sensations for the individual and without damaging the underlying and surrounding tissues.

 The present invention includes a method for painlessly creating microscopic holes, i.e. micropores, ranging in size from 1 to 1000 microns, in the stratum corneum of human skin. The key point for implementing this method is the creation of an appropriate source of thermal energy, or a thermal sensor, which is held in contact with the stratum corneum. The main technical problem in the manufacture of the corresponding thermal sensor is the creation of a device design that has the desired contact with the skin and which can undergo thermal modulation with a sufficiently high frequency.

An appropriate heat sensor can be made by topical application of a suitable light-absorbing compound to the stratum corneum, such as a dye or paint, selected because of its ability to absorb light at the wavelength emitted by the selected light source. In this case, the selected light source may be a laser diode emitting light at a wavelength that would normally be absorbed by skin tissues. By focusing the light source on a small area on the surface of the applied dye layer, temperature modulation of the target area can be performed by changing the intensity of the light flux focused on it. It is possible to use energy from laser sources emitting at a longer wavelength than an excimer laser, first by topically applying a suitable light-absorbing compound to the stratum corneum, such as a dye or paint, selected because of its ability to absorb light at a wavelength emitted by the laser source. The same principle can be applied at any wavelength and you only need to choose the appropriate dye or paint and the length of the light waves. It is only necessary to look at the handbook and find suitable dyes and the wavelength of the maximum optical density of this dye. One such reference is Green, The Sigma-Aldrich Handbook of Stains, Dyes and Indicators, Aldrich Chemical Company, Inc. Milwaukee, Wisconsin (1991). For example, copper phthalocyanine (Pigment Blue 15; FCM) absorbs at about 800 nm, copper phthalocyanine salt of tetrasulfonic acid (Acid Blue 249) absorbs at about 610 nm and Indocyanin Green absorbs at about 775 nm and Cryptocyanin absorbs at about 703 nm. The FCM is particularly well suited for this embodiment for the following reasons: it is a very persistent and inert compound already approved by the US Food and Drug Administration for use as a dye in an implantable suture material; it absorbs very strongly at wavelengths from 750 to 950 nm, which coincides well with many cheap, solid emitters, such as laser diodes and light emitting diodes, and, in addition, this region of the width of the optical spectrum is also not directly absorbed by skin tissues in some or a significant amount; The FTP has a very high evaporation point (> 550 ^o C in vacuum) and directly passes from the solid phase to the vapor phase without a liquid phase; The FTP has a relatively low thermal diffusivity constant, allowing the light energy focused on it to selectively heat an area located right at the focal point with a very small lateral spread of the “hot spot” on the surrounding FTP, thereby helping the spatial orientation of the contact thermal sensor.

 The purpose of this description is not to compile a large list of suitable dyes or paints, because they can be easily installed by experts in this field on publicly available data.

The same is true for any desired specific pulsed light source. For example, this method can be carried out using a mechanically shielded, focused incandescent lamp as a pulsed light source. Various catalogs and sales guides present numerous lasers operating in the spectrum close to ultraviolet, visible and close to infrared. Representative lasers are: Hammamatsu Photonic System Model PLP-02, which operates at an output power of 2 • 10 ^-8 J, at a wavelength of 415 nm; Hammamatsu Photonic Systems Model PLP-05, which operates at an output power of 15 J, at a wavelength of 685 nm; SDL, Inc. , an SDL-3250 Series pulse laser, which operates at an output power of 2 • 10 ^-6 J, at a wavelength of approximately 800-810 nm; SDL, Inc., Model SDL-8630, which operates at an output power of 500 mW at a wavelength of approximately 670 nm; Uniphase Laser Model AR-081-15000, which operates at an output power of 15,000 mW at a wavelength of 790-830 nm; Toshiba America Electronic Model TOLD9150, which operates at an output power of 30 mW at a wavelength of 690 nm; and LiCONIX, Model Diolite 800-50, which operates at a power of 50 mW at a wavelength of 780 nm.

 For the purposes of the present invention, a pulsed laser light source can emit radiation over a wide wavelength range in the range of about 100 to 12,000 nm. Excimer lasers typically emit in the range of about 100 to 400 nm. Excimer lasers with wavelengths in the range of about 193 to 350 nm are currently on sale. Preferably, the laser diode will have a radiation range in the range of about 380 to 1550 nm. The doubled frequency laser diode will have a radiation range of approximately 190 to 775 nm. When using a laser diode optical parametric pump generator, longer wavelengths in the range of about 1300 to 3000 nm can be used. Given the amount of laser technology research in progress, these ranges are expected to expand over time.

 The supplied and absorbed energy does not have to be obtained from the laser, since any light source can be used, regardless of whether it is a laser, a short arc lamp, such as a xenon flash lamp, an incandescent lamp, a light emitting diode (LED), the sun or any other source. Thus, the specific device used to supply electromagnetic radiation is less important than the wavelength and energy associated with them. Within the scope of the claims of the invention, any suitable device capable of supplying the necessary energy at a suitable wavelength, i.e. in the range of from about 100 nm to about 12000 nm. An essential sign is that the energy must be absorbed by the light-absorbing compound in order to cause its localized heating, followed by a sufficient amount of heat to the tissue to be destroyed within the permissible time frame.

 In one illustrative embodiment, the heat sensor itself is made of a thin layer of a biologically inert solid compound, preferably about 5 to 1000 microns thick, which is large enough to cover the area where micropores are to be applied, applied locally to a selected area of an individual's skin. The particular composition of the chemical compound is selected so that it exhibits high absorption in the spectral range of the light source selected to supply energy to the light-absorbing compound. The sensor may be, for example, a sheet of a solid compound, a film treated with an absorbing compound with a high melting point, or direct application of the light-absorbing compound to the skin in the form of a precipitate or suspension in a carrier. Regardless of the configuration of the light-absorbing heat sensor, it should have a sufficiently low coefficient of lateral thermal diffusion so that any local temperature rises will remain spatially limited, and the direct way to heat loss will be direct passage into the stratum corneum through the contact point between the skin and the sensor.

The required change in the temperature of the sensor can be achieved by focusing the light source on the light-absorbing compound and by changing the intensity of this light source. If the energy absorbed inside the illuminated area is high enough, it will cause rapid heating of the light-absorbing compound. The amount of supplied energy and subsequently both the heating rate and the maximum temperature of the light-absorbing compound at the focal point can be easily modulated by changing the pulse width and the maximum power of the light source. In this embodiment, the heat sensor forms only a small amount of a light-absorbing compound heated by the focused energy of the incident light, and an additional light-absorbing compound, which could overlap a larger area than the area of the actual pore formation, is not significant. By using a solid-phase light-absorbing compound with a relatively high melting point, such as copper phthalocyanine (FCM), which remains in its solid phase to temperatures above 550 ^o C, the heat sensor can quickly heat up to a temperature of several hundred degrees Celsius and nevertheless remain in contact with the skin, providing the possibility of conducting thermal energy to the stratum corneum. In addition, this embodiment includes the selection of a light source with a radiation spectrum in which usually very little energy will be absorbed in the skin tissues.

As soon as the light-absorbing compound is located locally on the target area, a thermal sensor is formed when the light source is activated by a focused beam, located to coincide with the surface of the treated area. The light energy density at the focal point of the beam and the amount of absorption that takes place inside the light-absorbing compound are set so as to be sufficient to heat the light-absorbing compound within the region of a small area determined by the focus of the light source to a temperature above 123 ^° C within a few milliseconds. As the temperature of the heat sensor rises, conducting into the stratum corneum delivers energy to these tissues, raising the local temperature of the stratum corneum. When enough energy is supplied to this small area of the stratum corneum in order to cause the local temperature to rise above the boiling point of the water contained in these tissues, this water instantly evaporates, causing degradation of the stratum corneum at this point.

 By turning the light source on and off, it is possible to easily modulate the temperature of the heat sensor, and selective destruction of these tissues can be achieved, making it possible to create holes with very precisely defined sizes that penetrate only through the first 10-30 microns of the skin.

 An additional feature of this embodiment is that by selecting a light source, typically having very low absorption by skin or underlying tissues, and by designing the focus and delivery optics to provide a sufficiently high numerical aperture, a small amount of the light that is not absorbed in thermal sensor, quickly diverges as it penetrates deep into the body. Since very little absorption occurs at the supplied wavelength, essentially no energy is supplied directly from the light source to the skin. This three-dimensional dilution of the bound energy in the tissues due to beam divergence and low absorption in the untreated tissue leads to a completely benign interaction between the light beam and the tissues, thus eliminating damage.

 In one preferred embodiment of the invention, a laser diode with a radiation wavelength of 800 ± 30 nm is used as a light source. A heat sensor can be manufactured by topically applying a transparent adhesive tape that has been treated on the adhesive side with a 0.5 cm stain formed from a precipitate of finely ground copper phthalocyanine (FCM). The FCM exhibits extremely high absorption coefficients in the spectral range of 800 nm, usually absorbing more than 95% of the radiation energy from the laser diode.

 Figure 1 shows a system 10 for supplying light from such a laser diode to a selected area of the individual's skin and for monitoring the progress of the pore formation process. The system includes a laser diode 14 connected to a regulator 18, which controls the intensity, duration and intervals between light pulses. The laser diode emits a beam 22, which is directed to a collecting lens (or lenses) 26, which focuses the beam on the mirror 30. Then the beam is reflected by the mirror on the lens (or lenses) 34 of the lens, which focuses the beam at a preselected point 38. This preselected point coincides with the plane of the coordinate table 42 and its lens aperture 46 so that a selected area of the individual's skin can be irradiated. The coordinate table is connected to the controller so that the position of the coordinate table can be adjusted. The system also includes a monitoring system including a camera on a charge-coupled device 50 connected to a monitor 54. The camera on a charge-coupled device is confocally aligned with the objective lens so that the progress of the pore formation process can be monitored visually on the monitor.

 In another illustrative embodiment of the invention, a system of control photodiodes and collecting optics is provided, which has been confocally combined with a destruction light source. 2 shows a sensor system 60 for use in this embodiment. The system includes a light source 64 for emitting a beam of light 68, which is directed through an optical delivery system 72, which focuses the beam on a pre-selected point 76, such as the surface of the skin of an individual 80. Part of the light in contact with the skin is reflected and other light is emitted from the irradiated area. Part of this reflected and emitted light passes through a filter 84 and then through a collecting optical system 88, which focuses the light on the photodiode 92. The controller 96 is connected to both the laser diode and the photodiode, respectively, to regulate the radiated power of the laser diode and detect light that reaches photodiode. Only selected portions of the spectrum emitted from the skin pass through the filter. By analyzing the shifts of reflected and emitted light from the target area, the system is able to detect the moment of a gap in the stratum corneum, and this feedback is then used to regulate the light source by inactivating the light pulses when micropore formation in the stratum corneum has been achieved. By using this type of active closed loop system with feedback, a self-regulating, universally applicable device is obtained that forms micropores of the same size with minimal power requirements in the stratum corneum, regardless of changes from one individual to another.

In another illustrative embodiment, a cooling device is mounted in the contact surface of the system in contact with the skin. In FIG. 3A is an illustration of a schematic representation thereof. In this system 100, the light source 104 (connected to the controller 106) emits a beam of light 108 that passes through the hole and is focused by the optical delivery system 112. The beam is focused by the optical delivery system at a preselected point 116, such as a selected area of the skin 120 of the individual. Coolant 124 includes a Peltier device or other cooling device that is in contact with the skin to cool its surface. In a preferred embodiment of the cooling device 124 (FIG. 3B) there is a central opening 128 through which a beam of focused light passes to come into contact with the skin. Returning again to FIG. 3A, the heat sink 132 is also preferably placed in contact with the cooling device. By providing a cooling device with a small hole in its center, which coincides with the focus of light, in the entire area where the creation of pores is supposed, skin tissues can be pre-cooled to 5-10 ^o C. Pre-cooling provides a greater safety margin for the system to work in that that, in comparison with the uncooled embodiment, the possible user experience and the possibility of any collateral damage to the epidermis immediately below the site of pore formation are significantly reduced. Furthermore, for control purposes, pre-cooling minimizes evaporation of the interstitial fluid and can also provide superior physical properties, such as reduced surface tension of such interstitial fluid. It is further known that tissue cooling causes a localized increase in blood flow in such a cooled tissue, thereby promoting the diffusion of analytes from the blood into the interstitial fluid.

 The method can also be used for other microsurgical techniques in which a light-absorbing compound (thermal sensor) is superimposed on the area to be degraded and then a light source is used to selectively modulate the temperature of the sensor in the selected target area, acting on the tissue through the evoked evaporation-destruction process.

 A further feature of the invention is the use of a light source to help seal micropores after they are no longer needed. In particular, in the case of control of the internal analyte, micropore is created and a certain amount of interstitial fluid is extracted through this hole. After a sufficient amount of interstitial fluid has been collected, the light source is reactivated with a reduced power level to facilitate rapid clotting or coagulation of the interstitial fluid within the micropore. By stimulating the coagulation or clot formation of fluid in the pore, this hole in the body is effectively sealed, thereby reducing the risk of infection. The use of a light source per se, both for the formation of micropores and for its sealing, is also an inherently sterile procedure with the exception of the physical penetration of any device or device into the body. In addition, heat stroke caused by light energy kills any germs that may be present in the area of destruction.

 This concept of optical sterilization can be extended to include an additional step in which the light source is first used without focusing, covering the target area with a lighted area extending 100 microns or more beyond the actual micropore size, the formation of which is assumed. By selecting the area that is supposed to be affected by an unfocused light beam, the current density can be correspondingly reduced to a level that is significantly below the destruction threshold, but high enough for effective sterilization of the skin surface. After a sufficiently long exposure by a sterilizing beam of light to a wider area, or in one continuous stage, or by a series of pulses, the system is then transferred to the mode of sharply focused destruction, and the process of optical formation of micropores begins.

Another illustrative embodiment of the invention is the creation of the desired thermal sensor from a solid metal, such as a wire of small diameter. As in the previously described embodiment, the contact surface of the heat sensor should be capable of modulating its temperature from ambient skin temperatures (33 ^° C) to temperatures exceeding 123 ^° C, within an acceptable period of time, preferably from about 1 to 50 ms at high temperature (on time) and at least about 10 to 50 ms at low temperature (off time). In particular, the ability to modulate the temperature to a level above 150 ^{° C.} during a turn-on time of approximately 5 ms and a turn-off time of 50 ms causes a very effective thermal degradation with little or no sensation for the individual.

 Several methods of modulating the temperatures of the contact surface of a wire heat sensor can be successfully applied. For example, a short length of wire can be heated to the desired high temperature using an external heating element, such as an active resistance heating element, used at the end of a soldering iron. 4 shows an active resistance heater 140 with a mechanical starter. An active resistance heater includes an active resistance heat source 144 connected to a wire thermal sensor 148. The active resistance heater is also connected by means of an insulating holder 152 to a mechanical modulating device 156, such as a solenoid. In this configuration, a stable state can be achieved in which the tip of the wire sensor stabilizes at some level of temperature balance determined by the physical parameters of the structure, i.e. the temperature of the heater with active resistance, the length and diameter of the wire, the temperature of the air surrounding the wire, and the material contained in the wire. When the desired temperature is reached, by means of a mechanical modulating device, it is possible to directly influence the temperature modulation of the selected skin region of the individual 160 to alternatively place the hot wire tip in contact with the skin, preferably within 5 ms of the on time and then remove it into the air, preferably for 50 ms of the off time.

 In another illustrative example (FIG. 5), a device 170 is shown including a current source 174 connected to a regulator 178. The current source is connected to a current loop 182 including a wire 186 from which the structure is formed so that it represents a high resistance point. Preferably, the wire is fixed in the holder 190, and the insulator 194 separates the various parts of the current loop. The desired temperature modulation is then achieved by simply modulating the current through the wire. If the thermal mass of the wire element is appropriately sized and heat is removed using the electrodes connecting it to the current source is sufficient, the heating and cooling time of the wire element can be achieved within a few milliseconds. Contacting the wire with a selected area of skin 198 heats the stratum corneum to achieve selective destruction.

 In FIG. 6 shows another illustrative example of the formation of pores in the stratum corneum using a hot wire. In this system 200, wire 204 can be placed inside a modulated alternating magnetic field generated by winding wire 200 with an excitation coil. By supplying the excitation coil with alternating current through the controller 212 connected to it, eddy currents of sufficient intensity for direct heating due to internal active losses can be caused in the wire heat probe. This is essentially a miniature version of an inductive heating system, commonly used to heat treat tool tips or induce the removal of gas from electrodes in vacuum tubes or flash tubes. An advantage of the inductive heating method is that the energy supplied to the wire thermal probe can be carefully controlled and easily modulated by electronic regulation of the excitation coil. If the thermal mass of the wire probe itself and the thermal mass of the stratum corneum contacting the probe tip are known, the regulation of the supplied inductive energy can provide very precise temperature control at the point of contact 216 with the skin 220. Since the skin tissue has essentially no magnetic properties at lower frequencies at which inductive heating can be achieved, if correctly selected frequencies are used in the excitation coil, then this alternating electromagnetic field will not affect the skin tissue.

 If mechanically adjustable contact modulation is used, an additional feature can be realized by the inclusion of a simple control system with feedback, in which the total electrical resistance between the probe tip and the skin of the individual is controlled. Thus, the probe can be placed in contact with the skin of the individual, as evidenced by a stepwise decrease in resistance after contact is established, and then held there for the desired “turn-on time”, after which it can be removed. Several types of linear actuators are suitable for this form of feedback control, such as a voice coil mechanism, a simple solenoid, a rotary system with a cam or crank, and the like. The advantage is that as thermal degradation develops, the position of the tip of the heat probe can likewise advance into the skin, always providing reliable contact to facilitate efficient transfer of the required thermal energy. Changing the conductivity properties of the stratum corneum and epidermis can also be used to provide fine verification with feedback that the pore formation process is complete, i.e. when resistance indicates that the epidermis has been reached, and time to stop the pore formation process.

 In FIG. 7 shows an illustrative example of such a feedback impedance monitor. This device 230 has an electrically heated heat source 234 connected to a wire heat probe 238. The heat source is mounted by means of an insulating holder 242 on a mechanical modulator 246. The controller 250 is connected to a wire and skin 254, in which the controller detects changes in resistance in a selected area 258 skin, and when a predetermined level is obtained, the regulator stops the pore formation process.

 In the same line as the hydraulic means of pore formation, microscalpels are adapted specifically for penetration through the stratum corneum to introduce a penetrating substance, such as a drug, through the pore formed or to remove the analyte through the pore for analysis. Such a device is considered to be “minimally invasive” in comparison with devices and / or methods that are non-invasive. The use of microscalpels that penetrate below the stratum corneum to remove blood is well known. Such devices are commercially available from manufacturers such as Becton-Dickinson and Lifescan, and can be used in the present invention by adjusting the depth of penetration. As an example of a micro-scalpel device for collecting biological fluids, reference is made to the PCT International Published Application Erickson et al. WO 95/10223 (published April 20, 1995). This application shows a device for penetrating the dermal layer of the skin without penetrating the subcutaneous tissue to collect body fluids, such as for determining blood glucose levels.

The formation of pores in the keratinized layer can also be carried out using acoustic means. Acoustic pore formation is a type of optical means described above, except that instead of using a light source, a very carefully focused beam of acoustic energy is supplied to the area of the stratum corneum to be destroyed. The same energy levels are required, i.e. nevertheless absorbed energy threshold level should be 70 mJ / cm ^2/50 ms. The same pulsed focused ultrasound transducers, as described in the parent applications of serial numbers 08/152442 and 08/152174, can be used to supply the required energy density for destruction, as those used in the supply of acoustic energy modulated in intensity, phase or frequency, or a combination of these parameters for transdermal sampling of an analyte or transdermal drug delivery. This has the advantage of allowing the use of the same converter. which is used to push the drug through the stratum corneum or to extract biological fluid to the surface for analysis, first to create micropores.

 In addition, electrical pore formation or short bursts or pulses of electric current can be supplied to the stratum corneum with sufficient energy to form micropores. Electrical pore formation is known in the field of pore formation in biological membranes, and devices for electric pore formation are commercially available. Thus, a person skilled in the art can choose a device and conditions for its use without unnecessary experimentation in accordance with the instructions provided here.

 The micropores formed in the stratum corneum using the methods of the present invention enable high flow rates of therapeutic compounds with high molecular weight for transdermal delivery. In addition, these non-traumatic microscopic holes in the body provide access to various analytes within the body, a quantitative analysis of which can be carried out to determine their internal concentration.

Example 1
In this example, skin samples are prepared as follows. The epidermal membrane is separated from the whole skin of a human corpse using the thermal separation method Kligman and Christopher, 88 Arch. Dermatol. 702 (1963), including exposure to the entire skin thickness of temperatures of 60 ^o C for 60 s, after which the stratum corneum and part of the epidermis (epidermal membrane) are carefully peeled from the dermis.

Example 2
Thermally separated samples of the stratum corneum prepared in accordance with the procedure described in example 1 are cut into sections of 1 cm ² size. Then these small samples are attached to the coverslip by placing them on a slide and applying them to a skin sample using a pressure-sensitive adhesive disc with a central hole of 6 mm. Then the samples are ready for experimental research. In some cases, skin samples are hydrated by allowing them to soak for several hours in a neutral solution of phosphate buffer or in pure water.

 As a test of these untreated skin samples, the samples are exposed to an emitted power of several different infrared laser diodes emitting roughly at 810, 905, 1480 and 1550 nm. A guiding optical system designed to form a focused beam with a transverse size of 25 μm through the target lens has a numerical aperture of 0.4. According to the measurement data, the total power delivered to the focused point ranged from 50 to 200 mW for 8810 and 1480 nm laser diodes, which were able to operate in a continuous wave (NV) mode. Laser diodes 905 and 1550 nm were designed to generate pulses with high maximum power lasting roughly from 10 to 200 ns at a repetition rate of up to 5000 Hz. According to the measurement data, the maximum power levels of pulsed lasers were 45 W at 905 nm and 3.5 W at 1550 nm.

 Under these operating conditions, none of the lasers exerted a visible effect on skin samples. The target area was continuously illuminated for 60 s and then examined under a microscope without revealing visible effects. In addition, the sample was placed in a modified Franz cell, usually used to test transdermal delivery systems based on chemical penetration enhancers, and the conductivity from one side of the membrane to the other, measured both before and after laser irradiation, did not reveal a change. Based on these tests, which were conducted on skin samples from four different donors, it was concluded that at these wavelengths, the interaction of optical energy with skin tissues was so small that no effects were detected.

Example 3
To assess the potential sensation of a living subject when illuminated with optical energy in the conditions of Example 2, six volunteers were used, and the radiation of each laser source affected their fingertips, forearms and rear of the hands. In the case of using lasers 810, 905 and 1550 nm, people could not feel when the laser turned on or off. In the case of using a 1480 nm laser, there was some sensation during illumination with a 1480 nm laser operating at 70 mW MB, and a little later a very small bubble was formed under the skin due to the absorption of 1480 nm radiation from one of the water absorption bands. Obviously, the amount of energy absorbed was sufficient to cause the formation of a bubble, but was insufficient to cause destruction of the stratum corneum. In addition, 1480 nm light absorption occurred mainly in the deeper, hydrated (epidermis and dermis water content (water content from 85 to 90%), and not in the relatively dry (keratinized water content (10 to 15% water content) tissues.

Example 4
Having demonstrated that there is no effect on the skin in its natural state (Example 3), a number of chemical compounds were produced in terms of efficiency in absorbing light energy and then transferring this absorbed energy by passing the stratum corneum into the target tissue. The tested compounds included mascara, SHARPIE marking pens with black, blue and red indelible pastes, methylene blue, fuscian red, epolyte # 67, an absorbing compound designed for casting in polycarbonate lenses for laser goggles, tincture of iodine; iodine-polyvinyl-pyrrolidone complex ("BETADINE"), copper phthalocyanine and printing ink.

When using the MB laser diodes described in Example 2, positive degradation results were observed on in vitro samples of the thermally separated stratum corneum prepared in accordance with Example 1 with all of these products, however, some worked better than others. In particular, copper phthalocyanine (FCM) and epolyte # 67 were among the most effective. One of the possible reasons for the best operation of the FCM is its high boiling point in excess of 500 ^o C, and the fact that it retains its solid phase to this temperature.

Example 5
Since copper phthalocyanine has already been approved by the FDA for use in implantable suture material and is listed on the Merck Index as a sufficiently benign and stable molecule for biocompatibility in humans, the next step taken was a combination of topical application of FCM and a focused light source on the skin of healthy volunteers . A suspension of carefully ground FCM in isopropyl alcohol was prepared. The application method used consisted of agitating the solution and applying a small drop to the target area. Since the alcohol evaporated, a thin and uniform coating of the solid phase of the FCM remained on the skin surface.

 Then, on the site where the FCM coating was formed, the device shown in FIG. 1 is placed by placing the selected area of the individual’s skin near the reference plate. The reference plate consists of a thin glass window of approximately 3x3 cm with a 4 mm hole in the center. Then the area covered by the FCM is placed so that it is within the center hole. A confocal video microscope (FIG. 1) is then used to bring a clear focus to the surface of the skin. The placement of the skin to achieve the clearest focus on the video system is also carried out in such a way that the focused point of the laser system coincides with the surface of the skin. Then the operator generates pulses of laser light, while at the same time observing the effects in the target area on the video monitor. The degree of penetration is estimated visually by the operator using a calibration of the quantitative characteristic of defocusing the laser spot in the micropore as the depth of the micropore increases, and this can be dynamically corrected by the operator, essentially following downward destruction of the surface in the tissue by shifting the position of the camera (laser radiation source) along the axis " z "into the skin. At the point when the stratum corneum is removed deep into the epidermis, the appearance of the base of the hole changes noticeably, becoming much wetter and brighter. Seeing these changes, the operator turns off the laser. In many cases, depending on the state of hydration of the subject, as well as other physiological conditions, in response to the elimination of the barrier function of the stratum corneum above this small area, an abundant outflow of interstitial fluid occurred. A video system is used to record this video recording the availability of interstitial fluid in the pore forming region.

Example 6
The procedure of Example 5 is followed except that the FCM is applied to a transparent adhesive tape, which is then glued to a selected area on an individual's skin. The results are essentially the same as in example 5.

Example 7
Histological experiments were performed on the skin of a corpse in accordance with methods well known in the art for determining threshold destruction parameters for these dye mixtures and additional damage information. The upper surface of the skin sample is treated with a solution of copper phthalocyanine (FCM) in alcohol. After the evaporation of alcohol, the local layer of solid phase FCM is distributed over the surface of the skin with an average thickness of 10 to 20 microns. On figa shows a cross section of the full thickness of the skin before laser irradiation, which shows the FCM layer 270, the stratum corneum 274 and the underlying layers of the epidermis 278. On figv shows a sample after a single exposure to a light pulse of 810 nm on a circle with a diameter of 80 μm with a density an energy of 4000 W / cm ² for a pulse period of 20 ms. It should be noted that on the surface of the stratum corneum there was still a significant amount of FCM even in the middle of the destruction crater 282. It should also be noted that laboratory measurements indicate that in reality only about 10% of the incident light energy is absorbed with reflection or back scattering of the remaining 90% . Thus, the effective energy flow delivered to the dye layer, which can cause the desired heating, is only about 400 J / cm ² . On figs shows a sample after exposure to 5 pulses of light 810 nm, in which the keratinized layer barrier was removed without damaging the underlying tissue. These results are a good representation of the “ideal” operation of optically modulated thermal degradation. On fig.8D shows a sample
after exposure to 50 pulses. Damaged tissue 296 was present in the layers of the epidermis due to carbonization of untreated tissue and thermal denaturation of the underlying tissue. 8A-8C illustrate the separation between the stratum corneum and the underlying epidermis layers due to an artifact of dehydration, freezing, and imaging preparations.

Example 8
To study the details of the mechanism of thermal destruction, a mathematical model of skin tissue was constructed, on which it was possible to test various options for implementing the method of thermal destruction. This model calculates the temperature distribution in a layered semi-infinite medium with a certain incoming heat flux locally on the surface and heat removal from the surface at a certain distance to the side, i.e. convection occurs between the two sections. The equation of the axisymmetric axis dependent on the diffusion time is solved in cylindrical coordinates using the method of implied changing direction (PMN). (Note: a constant temperature between the centers is supplied to the lower boundary and serves as z-∞, and a zero radial heat flux is fed to the maximum radial boundary and serves as r-∞; the layers are parallel to the surface and are denoted as: 1 - dye; 2 - stratum corneum; 3 - underlying epidermis and 4 - dermis.For each layer, it is necessary to clarify the depth in a semi-infinite medium and thermal properties, density (rho), specific heat (s) and conductivity (k)).

 First, the heat transfer coefficient, h, is calculated on the skin based on a “constant” “1-D” temperature distribution determined by the ambient temperature, skin surface temperature and dermis temperature. It is assumed that the dye is not there, and the result is the value of "h" on the surface of the skin. The program then allows you to use this value of "h" on the surface of the dye layer or enter another desired value of "h" for the surface of the dye. Then, a “constant” temperature distribution is calculated over all layers (including the dye layer) using the updated value “h” on the dye surface. This temperature distribution is a prerequisite for a time-dependent heating problem. This makes up the "file m" - the original m. The program then calculates the time-dependent temperature distribution using time travel, calculating and displaying the temperature field at each stage.

 Each embodiment of the method described here, in which empirical data were collected, was modeled for at least one set of functional parameters, showing how the destruction of the stratum corneum can be achieved in an accurate and controlled manner. The simulations output data are presented graphically in two different formats: (1) is a sectional view of the skin showing various tissue layers with three isotherms plotted at the top of this projection that define three critical temperature thresholds, and (2) two different dependency graphs temperature over time, one for a point in the middle of the stratum corneum directly below the target site and the second for a point on the border of viable layers of epidermal cells and the lower side of the stratum corneum. These graphs show how the temperature at each point changes with time when heat pulses are applied, as if a microscopic thermocouple was implanted in the tissue. In addition, the application of this model allows us to study the parametric boundaries within which the method can be used to set external limits for two important aspects of the work of these methods. Common cases are presented cases that define the shell within which the method can be used without causing pain or unwanted tissue damage.

 For each given heat source, as described in several different embodiments of the invention, there is a point at which exposure to the skin tissue of the subject becomes suboptimal in the sense that the subject perceives pain or that viable cells in the underlying epidermis and / or dermis are exposed to temperatures which, if maintained for a sufficiently long time, will cause damage to these tissues. Accordingly, the test was simulated using, as an initial method, an embodiment with an optically heated topically applied copper phthalocyanine dye (FCM) to establish how the thermal time constants of various skin tissues essentially define a window within which the method can be applied without pain or damage adjacent layers of fabric.

Figures 9 and 10 schematically show sectional views of a skin and a locally applied dye layer. Each figure depicts three different isotherms: (1) - 123 ^o C, the point at which evaporation of water in the tissue causes tissue destruction; (2) - 70 ^o C, the point at which viable cells will be damaged if the temperature is maintained for several seconds; and (3) - 45 ^o C, the point at which the subject will perceive a sensation of pain. This pain threshold is described in several fundamental guides in physiology, but experience shows that this threshold is subjective to a certain extent. In fact, in repeated trials in the same individual, different areas of pore formation within a few millimeters of each other can show significant differences in the degree of sensation, possibly due to proximity to the nerve ending in relation to the site of pore formation.

The dimensions on the graphs show the different layers of dye and skin, measured in micrometers with their flat borders bounding them. Since actual skin tissues have much more convoluted boundaries in terms of the average sizes involved, the model provides a good approximation of the thermal gradients present in real tissues. The thickness dimensions of the FCM dye and various skin layers used in this and in all subsequent simulations were as follows:
dye - 10 microns; keratinized layer - 30 microns; the underlying epidermis is 70 microns and the dermis is 100 microns.

 Additional conditions imposed on the model for this particular simulation are shown in Table. 1 and 2.

When conducting these simulations, the following cautious assumptions are introduced:
1. Although it can be established that the temperature of some part of the keratinized layer already exceeds the destruction threshold for the thermal evaporation of the contained water, this process is not modeled, and the subsequent energy loss in the tissues due to this evaporation is not taken into account in the simulation. This will cause a slight temperature increase detected in the underlying tissues from a point in the simulation cycle.

2. Similarly, when it is shown that some of the copper phthalocyanine dye (FCM) reached the evaporation point of 550 ^° C, this process is not modeled, but the temperature is simply strictly limited by this level. This will also cause a slight increase in subsequent temperatures in the underlying layers with the development of modeling.

 Even with these simplifications used in the model, there is a pronounced correlation between the predicted work and the empirically observed work, based on both clinical studies and histological studies of donor skin samples. The key data to be noted in FIG. 9 and 10 are the duration of the heat pulse and the location of three different threshold temperatures represented by isotherms.

In Fig. 9, with a pulse duration of 21 ms, the isotherm of 70 ^{° C.} just crosses the boundary separating the stratum corneum and layers of viable cells in the epidermis. In in vitro studies on donor skin samples under these conditions, fifty pulses of thermal energy supplied at 50 ms intervals cause detectable damage to the upper layer of living cells (see FIG. 8D). However, it was also shown in in vitro studies that five pulses of thermal energy with the same functional parameters did not cause any significant damage to these tissues. It seems appropriate that even though the nominal threshold of damage could be exceeded at least in a transient sense, this temperature must be maintained for some period of accumulation time in order to actually cause damage to cells. However, the basic information presented by the simulation is that if the “on-time” is kept at less than 20 ms with a flux density of 400 W / cm ² , then living cells in the underlying epidermis will not be damaged, even though the isotherm the destruction threshold was sufficiently displaced into the stratum corneum. In other words, by using a heat source with a low flux density modulated so that the “on time” is suitably short, destruction of the stratum corneum can be achieved without any damage to adjacent cells in the underlying epidermis (see FIG. 8C). This is largely possible due to the significantly different thermal diffusivities of these two layers of tissue. That is, a stratum corneum containing only about 10 to 20% water has. much lower thermal diffusivity constant 0.00123 J / (S • cm • K) than 0.0421 J / (S • cm • K), epidermis. This allows you to create a temperature in the stratum corneum, while at the same time maintaining a clear spatial limitation by the point at which the destruction occurs.

In Fig. 10, the same modulation process started from the cycle of the critical point of the damage threshold shown in Fig. 9 is carried out further in time. By leaving a heat pulse for 59 ms at the same flux density of 400 W / cm ² within a circle with a diameter of 60 μm of a heated dye, the pain sensitivity isotherm at 45 ^o C just enters the innervated skin layer made up by the dermis. In addition, the damage threshold isotherm moves much further into the epidermal layer than where its location was shown in Fig. 9. The correlation of this simulation with numerous clinical studies using this method provides excellent confirmation of the accuracy of this model in that the model almost accurately shows the duration of the “on”, during which the heat probe can be applied to the skin before the individual feels it. In clinical trials, an adjustable pulsed generator was used to set the “on time” and “off time” of a series of light pulses applied to a layer of copper phthalocyanine dye (FCM) that was locally applied to the skin. While maintaining a constant “off time” of 80 ms, the “on” time gradually increased until a person reported a moderate “pain” sensation. Without exception, all the people who participated in these trials reported the first “pain” with an “on time” of 45 to 60 ms, very close to the time predicted by the model. In addition, in the clinical studies, the previously mentioned variability was noted with respect to the sensation of “pain” from site to site. Accordingly, what is defined as “pain” is the point at which the first unequivocal sensation is noted. In one area, this can be regarded as pain, while in the adjacent area, the same person can describe this sensation simply as “noticeable”.

 One element of this clinical study is the realization that even in the same area, successive uneven thermal pulses interact with the psychophysiological nervous perception of a person, causing a true decrease in perceived sensation. For example, to saturate neurons in this area, a series of shorter thermal pulses can be used that instantly deplete the available supply of neurotransmitters in this syntactic contact and therefore limit the ability to send a “pain” stimulus. This then provides the possibility that longer pulses after these short pulses are less noticeable than if they were applied at the beginning of the sequence of actions. Accordingly, a series of experiments was conducted with some randomly generated series of pulses, and the results were consistent with this hypothesis. An analogy with this situation can be found in the perception when a person enters a very hot bath, which at first is painful, but quickly becomes tolerant as a person gets used to the feeling of warmth.

Example 9
The objective of this invention is to achieve the painless formation of micropores of the stratum corneum, without causing any significant damage to adjacent viable tissues. As described in the simulation illustrated in Example 8 and FIGS. 9-10, it appears that within the destruction target, within which micropore formation can be achieved in such a painless and non-traumatic way, there is a boundary for any given thermal energy flux density. Both in vivo and in vitro studies have shown that this is indeed the case and it has allowed, through empirical methods, to develop some functional parameters that, as it turns out, work very well. The following set of simulations shows how the method works when using these special parameters.

 In the first case, a series of ten pulses with an “on time” of 10 ms, separated by an “off time” of 10 ms, is applied to the skin coated with the CPM. 11 shows the final temperature distribution in the skin tissue immediately after the end of this series of pulses. As is seen, isotherms representing three critical temperature thresholds show that the destruction of the stratum corneum was achieved in the absence of sensation in the nerves of the dermal layer and a very small crossing of the threshold of viable cells of the underlying epidermis. As mentioned earlier, it turns out that for real permanent damage to the cells, the epidermal cells should not only heat up to a certain point, but their temperature should also be maintained at this level for some period of time, which is believed to generally last about 5 seconds. 12 and 13 respectively show the temperature of the stratum corneum and viable epidermis as a function of time, illustrating heating during the “on time” and cooling during the “off time” for all ten cycles. Regarding the relationship of this simulation to in vivo studies, it should be noted that 90% was formed using system parameters established in accordance with the simulation, effective formation of pores without pain for the subject was achieved, and with subsequent microscopic examination of the site of pore formation after several days of visible there was no tissue damage. In vitro studies conducted on samples of donor skin of full thickness were also consistent with the prediction of behavior on the model.

Example 10
When conducting in vivo empirical studies and these simulations, it appears that pre-cooling the skin helps in optimizing the formation of micropores in terms of reducing the likelihood of pain or damage to adjacent tissues. In practice, this can be easily achieved using a simple cold plate against the skin before the pore formation process. For example, the application of a cooled plate of a Peltier device to a circle with a diameter of 1 cm surrounding the target area of pore formation, while holding the plate cooled to about 5 ^{° C.} for about 5 seconds, significantly reduces the temperature of the tissues. A schematic illustration of an experimental device used for this purpose in a laboratory is shown in FIGS. 3A-B. By acting on the exact same series of pulses of ten cycles that was used in the procedure illustrated in Example 9, when comparing FIG. 11 of FIG. 14, FIG. 12 of FIG. 15, and FIG. 13 of FIG. 16 show how much better the regulation of thermal penetration into skin tissue can be improved. It should be emphasized once again that relatively low thermal diffusivity and specific heat capacity of the stratum corneum have advantages in comparison with the epidermis and dermis. After cooling, the highly hydrated tissues of the epidermis and dermis require much greater thermal energy to raise their temperature, while the stratum corneum with its relatively dry structure can quickly heat up to the threshold of destruction.

Example 11
After finding out the fundamental mechanism of thermal conductivity during energy delivery to the skin tissue, which underlies effective painless destruction and the formation of micropores in the keratinized layer, we can understand several different special ways to achieve the required fast modulations of the temperature of the contact point, such as the implementation using a hot wire illustrated on figure 4-7.

The primary embodiment described here uses an active resistance heating element (FIG. 4), such as the tip of a small soldering iron without a wire with a relatively inert wire of a suitable size wound around it with a short length of wire left protruding to the side of the heater body. When electricity is supplied from a direct current source, the heater within a few seconds heats up to a certain temperature and reaches a stable state due to convection losses in the surrounding air. Similarly, the wire, which is part of this thermal system, will reach a steady state so that, using components of this type, the temperature of the tip of the wire itself can rise to almost any arbitrary temperature, roughly up to 1000 ^o C. The size of the tip can be selected to obtain the exact desired size micropores.

The laboratory used tungsten wires with a diameter of 80 μm attached to a removable tip of a soldering iron without a WAHL wire with approximately 2 mm of wire protruding from the tip. Using a thermocouple, the temperature of the tip was measured at its steady state, and it was noted that by changing the direct current adjustment it is easy to achieve stable temperatures in excess of 700 ^o C. To achieve the desired modulation, a fast-response electromechanical starter with a low mass was connected to the tip so that that the position of the wire could linearly move more than 2 mm at a speed of up to 200 Hz. Then, by installing the entire device on a precision platform, this vibrating tip can be brought into contact with the skin surface with a high degree of controllability so that it contacts in one cycle only during the “on time” of less than 10 ms, while the “off time” is arbitrary long periods can be achieved by the corresponding setting of the pulse generator. These in vivo studies have shown that pore formation can actually be achieved even before the subject in whom the pore is formed learns that the end of the wire has been placed in contact with the skin.

 In order to compare the operation of this embodiment with the embodiment using a locally applied CPM dye with optical heating, the following simulations were carried out in accordance with the procedure of Example 8. In essence, by changing only the initial conditions, the implementation using hot wire can be carried out with the same simulation code . Due to the fact that the contact with the wire occurs almost instantaneously, there is no time-dependent heat generation in the CPM dye layer and, when the wire is physically removed from the skin contact, there is no residual heat remaining on the surface, as occurs with the heated CPM dye layer. Along with this, since the wire itself determines the area intended for the destruction / formation of micropores, there should be no lateral diffusion of thermal energy before it acts on the stratum corneum. The comparative functional characteristics of the hot wire embodiment are shown in FIGS. 17-19.

Example 12
In this example, the procedure of example 11 was performed, except that the skin was pre-cooled in accordance with the procedure of example 10. Similarly, pre-cooling of the target area gives positive results, similar to the implementation of the implementation of the "hot wire". The results of the approach in the form of modeling with pre-cooling are shown in Fig.20-22.

Example 13
As discussed earlier in this description, at first glance it appears that the Tankovich '803 patent is similar to the claimed invention. In this example, a simulation model was installed with the functional parameters defined in Tankovich '803, i.e. the pulse width is 1 μs, and the power level is 40,000,000 W / cm ² . On Fig and 24 it is shown that under these conditions, one part of the stratum corneum does not reach the threshold of instantaneous evaporation of water (123 ^o C) and thus, there is no destruction / formation of micropores in the stratum corneum. In practice, applying a pulse with a high peak power and short duration to a locally applied dye layer simply evaporates the dye from the skin surface without affecting the skin. Thus, this example demonstrates that the conditions defined by Tankovich '803 do not work in the present claimed invention.

Example 14
In this example, the interstitial fluid obtained after pore formation in accordance with the procedure of Example 6 was collected and analyzed to determine glucose concentration therein. Data were obtained from four non-diabetic people and six patients with type 1 diabetes who underwent a glucose load test. The age of people was from 27 to 43 years. The aim of the study was to study the possibility of using the method for painlessly taking a sufficient amount of interstitial fluid (LI) in people to enable the analysis of LI for glucose, and then comparing these concentration values with the level of glucose in human whole blood.

 In all people, quantitative determinations of glucose in both blood and ILI were carried out using the Miles-Bayer ELITE system. All ten subjects underwent the same determination procedures, adjusted for glucose load and insulin injection in patients with insulin-dependent diabetes.

The main objective of the study design was to use the smallest number of volunteers, some of whom are diabetic and some who do not have diabetes, who took a series of paired samples of ILI and whole blood every 3-5 minutes during the 3-4 hours of the study period. Quantitative determination of glucose was performed in both blood and ILI, and a statistical relationship was determined between blood glucose levels and interstitial fluid. To study the hypothesis of a temporary delay in glucose levels in the CSI, compared with glucose levels in whole blood, significant and dynamic changes in glucose levels were induced in the subjects. This was achieved by fasting each subject for 12 hours before the start of the test, and then after establishing their initial glucose levels on the basis of a set of three fasting glucose levels in the blood and in the ICF, glucose loading in people. After establishing baseline levels, the subjects were given a glucose load in the form of sweet juice based on the following settings:
i. In control individuals, glucose load was calculated based on 0.75 g glucose per 1 pound of body weight.

 ii. For patients with insulin-dependent diabetes, the glucose load was 50 g of glucose. In addition, immediately after glucose loading, patients with diabetes will inject themselves with their usual morning dose of fast-acting insulin. If a patient with diabetes had fasting glucose levels exceeding 300 mg / dl, they were first asked to inject insulin on their own, and glucose loading was performed after their blood glucose levels fell below 120 mg / dl.

Each involved subject was first given a full description of the study in the “Conscious Consent” document and was asked to understand and sign it before being officially included in the program. After admission, they filled out a medical history questionnaire. The clinical procedure used in detail was as follows:
(a) A person was starving from 9:00 p.m. the night before visiting for research, drinking only water. During this period, he was not allowed to consume caffeine, cigarettes, fruit juice.

 (b) The person was admitted to the facility where the test was conducted by 9:00 the next morning.

 (c) The person was seated in a reclining chair provided for him to relax throughout the study procedure.

 (d) From the moment a person arrives, they take samples and whole blood in the LIS at intervals of three to five minutes and continue for the next three to four hours. The length of the period for which the data were collected was based on when the blood glucose levels in the person returned to their normal range and stabilized after glucose loading. LIS samples were collected using optical pore formation, the LIS pumping method described in more detail below. Each LIS sample was roughly 5 μl by volume to ensure good filling of the ELITE test strip. Blood samples were obtained using a conventional finger puncture lancet. Immediate analysis of samples was performed in the ICF and blood for glucose using the Mikes-Bayer ELITE home blood glucose system. To improve the assessment of “true” blood glucose levels, two separate ELITE quantitative determinations were performed in each finger sample collected into the capillary.

 (e) To facilitate the continuous collection of LIS from the same site throughout the data collection phase for this individual, a matrix of 5 by 5 out of twenty-five micropores was created on the upper forearm of the individual, each micropore ranging from 50 to 80 microns, and the gaps between them were 300 μm. Using pressure-sensitive adhesive on the forearm of an individual, a Teflon disk 30 mm in diameter with a 6 mm hole in the center was attached and positioned so that the 6 mm central hole was placed above the 5 by 5 micropore matrix. This attachment provided a convenient method by which it could be connected small suction hose that delivers a slight vacuum [10 to 12 inches RT. Art. (254-304.8 mm Hg. Art.)] To the area with pores in order to cause the outflow of ILI from the body through micropores. A window made of transparent glass was installed on the upper surface of the Teflon disk, allowing the operator to directly see the skin with the formed micropores directly under it. When a 5-μL CJL bead was formed on the skin surface, this could be easily verified by visual inspection of the area through this window. This vacuum level created a nominal pressure gradient of about 5 psi [0.0344738 Pa]. Without micropores, using only a small vacuum, it was impossible to extract any amount of coolant from the individual's body.

 (f) After drawing the first three pairs of samples, the person was given a glucose load in the form of a very sweet orange juice. The amount of glucose administered was 0.75 g per 1 pound (1 pound = 0.45359 kg) of body weight for non-diabetic people and 50 g for patients with diabetes. Diabetic patients also injected themselves with fast-acting insulin (regular) and an appropriately calculated dose, based on this glucose level of 50 g, which coincides with the ingestion of glucose load. With a typical delay of 1.5 to 2.5 hours between insulin injection and the maximum effect of the injection, it was expected that patients with diabetes would have a shift upwards of their blood glucose levels in the range of up to 300 mg / dl, and then fall rapidly, returning to normal range as the insulin effect develops. Non-diabetic individuals were expected to show standard glucose resistance profiles, usually exhibiting a maximum blood glucose level of 150 to 220 mg / dl 45-90 min after glucose loading, and then rapidly dropping back to their normal initial levels for the next hour or so.

 (g) After administration of a glucose load or glucose load and an injection of insulin, the subjects were pulled over the next three to four hours at the same time as five-minute intervals and samples of whole blood were taken from their puncture in the finger. Sampling was stopped when blood glucose levels in three consecutive samples indicated that the subject's glucose level had stabilized.

After examining the data, several signs became apparent. In particular, for each particular batch of ELITE test strips, there is a distinct shift in the output shown by the meter in mg / dl glucose compared to the level indicated in the blood. Due to the absence of hematocrit in the LIH and normal differences in the concentration of electrolytes in the LIH and whole blood, overestimated readings should be expected. Regardless of the reasons underlying this shift in the output, it was determined by comparison with a reference quantification that the true glucose levels in the LIS are linearly related to the values obtained using the ELITE system, with scale factors constant for each particular batch of ELITE strips . Therefore, to compare the data on measuring glucose levels in the CSF and in whole blood, linear correlation of the first order was applied to the data on the body of the body as follows:
ICL glucose = 0.606 • ECL _ELITE +19.5.

 This change in the scale of the output data of the ELITE glucometer when used to measure glucose levels in the LIS makes it possible to study error vectors in the entire data set associated with the use of the LIS to estimate blood glucose levels. Of course, even in the absence of a linear change in scale, the correlation between glucose values in the CSF and blood glucose levels are the same as in the scaled version.

 Based on most published literature on the subject of glucose in the BCI, as well as preliminary data, it was initially expected that a delay of 15 to 20 minutes will be observed between the levels of glucose in the BCI and the levels of whole blood from a puncture in the finger. This is not supported by data analysis. In particular, when analyzing the data obtained from each individual to determine the time shift required to achieve the maximum correlation between glucose levels in the LVI and blood glucose levels, it was found that the largest time delay in this set of subjects was only 13 min , and the average time delay was only 6.2 minutes, with several subjects having an almost instantaneous determination of glucose levels (with a delay of about 1 minute).

 Based on the minimum delay observed in this data set, the graph shown in FIG. 25 represents all ten glucose load tests carried out one after another over a long period of time. The data are presented without a time shift and show a high level of correlation between glucose levels in the CSF and blood, which was equally observed in the entire set of clinical data. If you shift the entire data set as a whole to identify the best correlation estimate, then these will be the peaks of glucose levels in the CSF and blood with a delay of 2 min at a value of r = 0.97. This is only a slight improvement over the unbiased correlation r = 0.964. Therefore, for the rest of the analysis, the values of ILI are processed without superimposing a time shift on them. That is, each set of data on the level of glucose in the blood in the ILI is considered as simultaneously received data pairs.

 After reducing the time-independent values of the glucose level in the LIS obtained using Elite to a certain scale to reflect the proportional level of glucose available in the LIS, it became possible to study the error associated with this data. The easiest way to do this is to assume that the average value of two values of blood glucose from a puncture in a finger using the ELITE method is actually an absolutely correct value, and then simply compare the values reduced to a certain scale in the ILI with the average values of glucose in the blood. These data are as follows: standard deviation of blood glucose level - ILI 13.4 mg / dl; dispersion coefficient of ILI 9.7%; the standard deviation of the two Elite definitions is 8.3 mg / dl and the dispersion coefficient in the blood (mile) is 6%.

 As these data show, a blood-based measurement already contains an erroneous component. Indeed, work data published by the manufacturer indicate that the ELITE system has a nominal dispersion coefficient (CD) of 5 to 7%, depending on glucose levels and hematocrit in the blood.

 An additional look at the element of difference between the glucose level in the CSF and the level of glucose in the blood is shown in the form of a scatter plot in FIG. This reference also depicts the upper and lower boundaries of the 90% confidence interval. It is interesting to note that with only two exceptions, all data in the range of glucose levels below 100 mg / dl fall under these "whiskers" of the 90% confidence interval. This is important because the consequences of missing the tendency to hypoglycemia would be very significant for a user with diabetes. That is, it would be much better not to predict glucose levels in the range from 40 to 120 mg / dl, rather than excessively predict them.

Essentially, if we assume that the main error of quantification when using the ELITE system for the study of ILI is comparable to the error of the quantification associated with the use of ELITE on whole blood, then the deviation of the glucose level in the ILI from the level of glucose in the blood can be described as:
COLI _{bosom of TCI} = [(COLI _true) ² + _(true COLI) ^{2] 1/2.}

Applying this equation to the above values, you can determine the estimated "true" value of the erroneous component of determining the glucose level in the CSF:
COLI = _{true [(TCI bosom of} COLI) ² - (COLI _true) ^{2] 1/2.}

Or by solving the equation
_True LIS = [(13.4) ² - (8/3) ² ] ^1/2 = 10.5 mg / dl.

 A histogram of the relative deviation of the glucose level in the CSF to the level of glucose in the blood is shown in Fig. 27.

Delivery of drugs through the pores in the stratum corneum
The present invention also includes a method for delivering drugs, including drugs currently administered through the skin, through micropores in the stratum corneum. In one illustrative embodiment, delivery is achieved by placing the solution in a reservoir above the pore forming portion. In another illustrative embodiment, a pressure gradient is used to further enhance delivery. In yet another illustrative embodiment, acoustic energy is used with or without a pressure gradient to further enhance delivery. Acoustic energy can be supplied in accordance with traditional transdermal parameters or by using the effects of entrainment by acoustic waves, which will be described below, to push the feed solution through the stratum corneum with pores formed therein.

Example 15
This example shows the use of pore formation in the stratum corneum for the delivery of lidocaine, a local analgesic. The lidocaine solution also contained a chemical penetration enhancer composition designed to enhance its passive diffusion through the stratum corneum. An illustration of a delivery device 300 is shown in FIG. The device includes a housing 304, including a reservoir 308 for containing the drug-containing solution 312. The upper part of the housing includes an ultrasonic transducer 316 for generating acoustic energy to aid in transporting the drug-containing solution through micropores 320 in the stratum corneum 324. A hole 328 in the ultrasonic transducer allows feeding pressure is applied to it for additional assistance in moving the drug-containing solution through micropores in the stratum corneum. A dispenser is applied to a selected area of an individual's skin so that it is located at least over one, and preferably over a plurality of micropores. An adhesive layer 332 attached to the bottom of the housing allows the device to be attached to the skin so that the drug-containing solution in the reservoir is in communication through the micropore fluid. The delivery of the drug through micropores leads to a transfer to the underlying epidermis 336 and dermis 340.

 The efficacy of drug delivery using pore formation in conjunction with ultrasound was tested in five people. The experiment used two sections on the left forearm of people located at a distance of approximately three inches (7.62 cm) from each other, located at an equal distance between the thumb and the upper part of the forearm. The area near the thumb will be designated as section 1, and the section as far as possible from the thumb will be designated as section 2. Section 1 was used as a control on which lidocaine and an amplifier solution were applied using an identical delivery device 300, but without any formation of micropores of the stratum corneum or acoustic energy. In section 2, 24 pore openings were formed at a distance of 0.8 mm from each other in the form of a lattice enclosed in a circle with a diameter of 1 cm. The micropores in section 2 were created in accordance with the procedure of Example 6. Lidocaine and low-level ultrasound were applied. Ultrasound was supplied using a custom Zevex ultrasonic transducer device kit in high-frequency pulsation mode with a voltage between 0.4 V peaks of a linear amplifier ENI # 2100L with bursts of 1000 single pulses occurring at 10 Hz with a fundamental frequency of 65.4 kHz , i.e. pulse-modulated signal with power being supplied to the converter by bursts of pulses for 15 ms with switching off then for the next 85 ms. The measured rms output power of the amplifier supplied to the converter was 0.090 watts.

 After applying lidocaine, sensations were determined using friction over the test area with a 30 gauge wire. The experiments were carried out in both sections with serial feeding to the same section with the duration of the feeding intervals for section 1 from 10 to 12 minutes and section 2 for 5 minutes. Both sites were evaluated for insensitivity using a scale from 10 to 0, in which 10 indicated a lack of numbness, and 0 indicated a complete lack of sensitivity according to the subjects. The following summary of the results is presented for all 5 subjects.

 In the control plot, plot 1, after 10-12 min, a slight or complete absence of numbness was observed (from 7 to 10 points on a scale). After approximately 20 minutes, as the solution completely penetrated, a numbness was observed in section 1 (3 points on a scale). Upon completion of lidocaine application, section 1 was purified. In section 2, in a circle 1 cm in size containing pores, almost complete numbness was observed (from 0 to 1 point on a scale). Outside of a circle with a diameter of 1 cm, numbness decreased almost linearly to 1 point in a circle with a diameter of 2.5 cm with a complete absence of numbness outside of a circle with a diameter of 2.5 cm. Assessment of the 2nd area after the second application led to the formation of a slightly larger circle of complete numbness with a diameter approximately 1.2 cm, which linearly dropped to 1 point in the form of an uneven oval region with a diameter of 2 to 2.5 cm perpendicular to the forearm and a diameter of 2 to 6 cm parallel to the forearm. Outside of this area, numbness was not observed. A schematic representation of illustrative results obtained from a typical individual is shown in FIGS. 29A-C. On figa and 29B shows the results obtained in section 2 (with pores), respectively, after 5 and 10 minutes On figs shows the results obtained in section 1 (control without pores).

Acoustic energy and transdermal amplifiers
The physics of acoustic energy fields generated by acoustic transducers can be used in a method by which the acoustic frequency can be simulated to improve the flow rate achieved by other methods. As shown in FIG. 1 of US Pat. No. 5,445,611, incorporated herein by reference, the energy distribution of an acoustic transducer can be divided into a field in the near field and a field in the far zone. The field in the near zone, characterized by a length N, represents a zone from the first minimum of energy to the last maximum of energy. The zone distal to the last maximum is a field in the far zone. A field of the type of near (N) zone is suppressed by a large number of peaks located at small intervals and zero values of local pressure. The near field length, N, is a function of the frequency, size and shape of the transducer surface and the speed of sound in the medium through which the ultrasound passes. For one transducer, changes in intensity within its normal functional range do not affect the nature of the distribution of acoustic energy that is different from the linear type. However, for a system with many transducers, all of which are modulated in frequency and amplitude, the relative intensity of the individual transducers affects the distribution of energy in the acoustic medium, regardless of whether it is skin or another medium.

By changing the frequency of acoustic energy in a small amount, for example in the range of about 1 to 20%, the type of peak and zero values remains relatively constant, but the length N of the field in the near zone changes in direct proportion to the frequency. Large frequency changes, say, 2 or more times, will most likely cause a different set of resonances or types of vibration in the transducer, causing a substantially and unpredictably different type of field energy in the near field. Thus, with a small change in acoustic frequency, the complex type of peak and zero values is compressed or expanded like an accordion. By selecting the direction of the frequency modulation, the shift direction of these local pressure peaks can be adjusted. By applying acoustic energy to the skin surface, the selective modulation of the acoustic frequency regulates the movement of these local pressure peaks through the skin either towards the body or towards the surface of the body. High to low frequency modulation drives
pressure peaks into the body, while low to high frequency modulation “pulls” pressure peaks from the body toward the surface and through the skin out of the body.

 If we assume that typical parameters are used for this application, for example, an acoustic transducer with a diameter of 1.27 cm, a nominal functional frequency of 10 MHz and an acoustic impedance similar to that of water, then a frequency modulation of 1 MHz will cause the movement of peak and zero values such as field energy in the near field approximately 2.5 mm close to the stratum corneum. From the point of view of the prospect of removing analytes through the skin and / or through the mucous membranes, this degree of action provides access to the area located significantly below the stratum corneum and even the epidermis, dermis and other tissues underneath. For each given converter, there may be an optimal frequency range within which frequency modulation is most effective.

 The flow of a drug or analyte through the skin can also be increased by a change in either resistance (diffusion coefficient) or driving force (diffusion gradient). The flow can be amplified by the use of so-called penetration enhancers or chemical amplifiers.

 Chemical enhancers include two primary categories of components, i.e. compounds that violate the integrity of the cell membrane, and solvents or binary systems containing both compounds that violate the integrity of the cell membrane, and solvents.

 Compounds that interfere with the integrity of the cell wall are known in the art as being used in topical pharmaceutical preparations and also work when removing analytes through the skin. It is believed that these compounds help in penetrating the skin by disorganizing the lipid structure of the cell walls of the stratum corneum. A complete list of these compounds is described in European Patent Application 43738, published June 13, 1982, which is incorporated herein by reference. It is believed that for the purposes of this invention, any compound that violates the ordered structure of the cell wall can be used.

 Suitable solvents include water, diols such as propylene glycol and glycerol, mono alcohols such as ethanol, propanol and higher alcohols, DMSO, dimethylformamide, N, N-dimethylacetamide, 2-pyrrolidone, N- (2-hydroxy-ethyl) pyrrolidone, N methyl pyrrolidone, 1-dodecylazacycloheptan-2-one and other n-substituted-alkyl-azacycloalkyl-2-ones (azones) and the like.

 US Patent 4,537,776, issued August 27, 1985 to Cooper, provides an excellent generalization of the prior art and background information detailing the use of certain binary systems to enhance penetration. Due to the completeness of this description, the information and terminology used herein is incorporated herein by reference.

 Similarly, European Patent Application 43738, referenced above, describes the use of selected diols as solvents with a wide range of compounds that disrupt the ordered structure of the cell wall for the delivery of lipophilic pharmacologically active compounds. In view of the detailed description of compounds that violate the ordered structure of the cell wall and diols, this description of European patent application 43738 is also included here for reference.

 The binary system for enhancing the penetration of metoclopramide is disclosed in GB Patent Application GB 2153223, published August 21, 1985, and consists of a monovalent alcohol ester of C8-32 aliphatic monocarboxylic acid (unsaturated and / or branched if CIS-32) or C6-24 an aliphatic monoalcohol (unsaturated and / or branched if C14-24) and an N-cyclic compound such as 2-pyrrolidone, N-methylpyrrolidone and the like.

Amplifier combinations consisting of diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate and methyl laurate are disclosed in US Pat. No. 4,973,468 as enhancing transdermal delivery of steroids such as progestogens and estrogens. Dual amplifier consisting of glycerol monolaurate and ethanol for the transdermal delivery of drugs is shown in U.S. Patent 4820720. In U.S. Patent 5,006,342 lists numerous enhancers for transdermal drug administration consisting of fatty acid esters or alcoholic simple fatty acid esters of C ₂ -C alkanediols ₄ , where each fatty acid / alcohol portion of the ester / ether consists of about 8-22 carbon atoms. US Pat. No. 4,863,970 discloses a penetration enhancing composition for topical application comprising an active penetrating agent contained in a penetration enhancing carrier containing certain amounts of one or more disordered cellular compounds such as oleic acid, oleic alcohol and oleic acid glycerol esters; C ₂ or C ₃ alkanol and an inert diluent such as water.

 Other chemical enhancers optionally associated with binary systems include DMSO or aqueous DMSO solutions, such as those disclosed in US Pat. No. 3,551/554 to Herschler; US Pat. No. 3,711,602 to Herschler; in US patent 3,711,606 to Herschler, azones (n-substituted-alkyl-azacycloalkyl-2-ones), such as noted in US Pat. No. 4,557,943 to Cooper.

 Some chemical enhancer systems may have negative side effects, such as toxicity and skin irritation. US Pat. No. 4,855,298 discloses compositions for reducing skin irritation caused by a chemical enhancer containing compositions having skin irritation properties with an amount of glycerol sufficient to provide an anti-irritant effect.

 Because the combination of micropore formation of the stratum corneum and exposure to acoustic energy accompanied by the use of chemical enhancers can increase the rate of removal of the analyte or constant delivery through the stratum corneum, the particular carrier used and, in particular, the chemical enhancer can be selected from a long list of carriers of the prior art , some of which are mentioned above and are incorporated herein by reference. There is no need for a specific detailed description or listing of what is readily available in this area. The invention is not dedicated to the use of chemical enhancers as such, and it is believed that all chemical enhancers that can be used to deliver drugs through the skin will work with dyes in the optical formation of pores, as well as acoustic energy to achieve a measurable removal of analytes from under the skin and through its surface or the delivery of penetrating substances or drugs through the surface of the skin.

Example 16
Modulated acoustic energy and chemical amplifiers were tested for their ability to regulate transdermal flow on skin samples of human corpses. In these tests, the epidermal membrane was separated from the whole skin of human corpses using the thermal separation method of Example 1. The epidermal membrane was dissected and placed between the cell halves for penetration studies with the stratum corneum facing either the upper (donor) compartment or the lower (receiving) compartment . Modified Franz cells were used to fix the epidermis, as shown in FIG. 2 of US Pat. No. 5,445,611. Each Franz cell consists of an upper and a lower chamber, fastened together with one or more clamps. In the lower chamber there is a sampling hole through which materials can be added or removed. A sample of the stratum corneum is fixed between the upper and lower chambers when they are held together by clamps. The upper chamber of each Franz cell is modified to allow the location of the ultrasound transducer within 1 cm of the stratum corneum membrane. Methylene blue was used as an indicator of the molecule to assess penetration through the stratum corneum. Visual registration of the process and the results of each experiment were obtained in magnetic tape format with time stamping using a video camera and video recorder (not shown). In addition, samples were removed for measurement using an absorption spectrometer to quantify the dye that passed through the membrane of the stratum corneum during the experiment. Suitable chemical enhancers can vary over a wide range of solvents and / or compounds that disrupt the ordering of the cell wall structure, as noted above. The particular amplifier used was: ethanol / glycerol / water / glycerol monooleate / methyl laurate in volume ratios of 50/30/15 / 2.5 / 2.5. The device for generating and adjusting acoustic energy included a programmable arbitrary waveform generator (Stanford Research Systems Model DS345) from 0 to 30 MHz and for unfocused ultrasonic immersion transformations having peak resonances at 15 and 30 MHz, respectively. To test samples of the stratum corneum from the same donor, six cells were simultaneously prepared. After inserting samples of the stratum corneum before conducting any tests, they were allowed to soak in distilled water for at least 6 hours.

Example 17
The effect of acoustic energy without chemical amplifiers
As described in Example 16 above, the thermally separated epidermis was placed in a Franz cell with the epidermal side facing up and the side of the stratum corneum facing down, unless otherwise indicated. The lower chambers were filled with distilled water, while the upper chambers were filled with a concentrated solution of methylene blue in distilled water.

Thermally separated epidermis immediately after filling the upper chambers with methylene blue solution acoustic energy was supplied to one of the cells using a completely immersed transducer. This orientation will correspond, for example, to the presence of a transducer on the opposite side of the skin fold, or to a situation in which acoustic energy is reflected from a similarly placed reflector plate and used to “push” the analyte from the outside of the fold into the collection device. The acoustic energy setting was initially set to a nominal operating frequency of 25 MHz with an intensity equivalent set to an inter-peak (R-P) waveform of incoming waves of 20 V. This corresponds roughly to 1 W of the average input power to the transducer and, similarly assuming that the manufacturer's nominal the conversion efficiency for this particular transducer is 1%, acoustic energy is supplied with an output power of about 0.01 W over the surface of 0/78 cm ^{2 of the} active region or acoustics The average intensity is 0.13 W / cm ² . Acoustic energy was not supplied to three other control cells. After 5 minutes, the acoustic energy was turned off. During this interval, in none of the cells was a visual indication of the flow of dye through the stratum corneum, indicating levels that are less than approximately 0.0015% (vol./about.) Of the dye solution in 2 ml of the receiving medium.

The test of the same 3 control cells and 1 experimental cell was carried out as follows. The intensity of acoustic energy was increased to the maximum possible output power level created by the generating equipment at an input voltage between peaks of 70 V and an average input power of 12 W or (≈0.13 W / cm ² ) of the intensity of the output acoustic signal. Frequency setting for modulation or sweep from 30 to 10 MHz was also performed. This 20 MHz sweep was performed ten times per second, i.e. with a swing frequency of 10 Hz. At these input power levels, it was necessary to control the acoustic energy converter to avoid overheating. A contact thermocouple was applied to the transducer case and cycles of power on and off were carried out to maintain the maximum temperature of the transducer below 42 ^o C. After approximately 30 minutes of repeating the supply cycles of maximum power at approximately 50% of the operating cycles lasting 1 min 1 shutdown for 1 min, still not there was a visually detectable penetration of dye methylene blue through the stratum corneum.

Then, a casing of water cooling was attached to the acoustic energy converter to enable longer excitation at the maximum energy level. Using the same 3 control and 1 experimental cell, acoustic energy was supplied to the experimental cell at maximum power for 12 hours. During this time, the temperature of the liquid in the upper chamber rose only to 35 ^° C, only slightly higher than approximately 31 ^° C, normal temperature stratum corneum in vivo. In none of the four cells after 12 hours of acoustic energy supply, as described above, there was no visual evidence of an apparent flow of dye through the stratum corneum.

Example 18
The effect of acoustic energy without chemical amplifiers
Perforated stratum corneum: six cells were prepared as described in Example 16. The clamps securing the upper and lower chambers of the Franz cells were occupied more than the degree required for normal sealing of the upper compartment from the lower compartment, and with a degree for artificial formation of perforations and micropuncture "in thermally separated epidermal samples. When a dye solution was added to the upper chamber of each cell, visual indicators of leakage of dye into the lower chambers through the perforations formed in the stratum corneum immediately appeared. After applying acoustic energy to the cells in which the stratum corneum was thus perforated with small “micropunches”, a rapid increase in fluid transport through the micropuncture in the stratum corneum was observed. The transport speed of the indicator dye molecules was directly related to whether acoustic energy was supplied or not. That is, the supply of acoustic energy caused an immediate (delay time of approximately <0.1 s) impulse of the indicator molecules through micro-punctures in the stratum corneum. This momentum of the indicator molecules stopped immediately after turning off the supply of acoustic energy (turn-off delay of approximately <0.1 s). The pulse may be repeated as described.

Example 19
The influence of acoustic energy with chemical amplifiers
Two different compositions of chemical enhancers were used. Chemical Amplifier One or XI1 was a mixture of ethanol / glycerol / water / glycerol monooleate / methyl laurate in a volume ratio of 50/30/15 / 2.5 / 2.5. These components are generally regarded as safe, i.e. FDA approved GRAS for use as pharmaceutical excipients. Chemical Amplifier Two or XU2 is an experimental composition that has been shown to be very effective at enhancing transdermal drug delivery, but is generally considered too annoying for long-term transdermal delivery. XY2 contained ethanol / glycerin / water / lauradone / methyl laurate in a volume ratio of 50/30/15 / 2.5 / 2.5. Lauradon is a 2-pyrrolidone-5-carboxylic acid lauryl (dodecyl) ester ("PCA") and is also referred to as lauryl PCA.

 Six Franz cells were installed as above (Example 16), except that the thermally separated epidermis was inserted with the downward layer of the epidermis, i.e. side of the stratum corneum facing up. Hydration was carried out by immersing each sample at night in distilled water. To begin the experiment, distilled water in the lower chambers was replaced with a solution of methylene blue dye in all six cells. The upper chambers were filled with distilled water, and the cells were observed for approximately 30 minutes, confirming the absence of passage of the dye in order to verify that there were no micro-punctures in any cells. When they were not found, distilled water in the upper chambers was removed from four of the cells. The other two cells served as controls for distilled water. Then, the upper chambers of two of the experimental cells filled XY1, and the other two experimental cells filled XY2.

Acoustic energy was immediately applied to one of the two cells ХУ2. We used a 25 MHz converter with a frequency sweep every 0.1 s from 10 to 30 MHz with a maximum intensity of ≈0.13 W / cm ² . After 10-15 minutes of supplying acoustic energy at a 50% duty cycle, a dye flow was visually detected. In the other five cells, dye flux was not detected.

 Then, acoustic energy was supplied to one of two cells containing ХУ1 at the same settings. Within 5 minutes, dye began to appear in the upper chambers. Thus, acoustic energy together with a chemical amplifier significantly increased the speed of the transdermal flow of marker dye through the stratum corneum, and also reduced the delay time.

Example 20
The influence of acoustic energy and chemical amplifiers
Compositions of two chemical enhancers XU1 and XU2 were prepared with the exception of glycerol, and these new compositions designated XU1MG and XU2MG were tested as before. Water was replaced with glycerin so that the proportions of the other components remained unchanged. Three cells were prepared in modified Franz cells with the epidermal side of the samples of the thermally separated epidermis facing the upper side of the chambers. These samples were then hydrated in distilled water for 8 h. After the hydration step, distilled water in the lower chambers was replaced by ХУ1МГ or ХУ2МГ and the upper chamber was filled with a dye solution. Acoustic energy was successively applied to each of the three cells.

 After applying pulsed, frequency-modulated acoustic energy with a total duration of less than 10 minutes, a significant increase in the permeability of the stratum corneum samples was observed. The permeability of the stratum corneum samples varied relatively uniformly over the entire area exposed to both the chemical amplifier and acoustic energy. Perforations in the form of "microholes" through which the dye could penetrate through the stratum corneum were not observed. The transdermal flow rate was instantly adjusted by turning acoustic energy on or off. As it turned out, turning off the acoustic energy instantly reduced the transdermal flow rate so that there was no visible active dye transport through the skin sample; apparently, the rate decreased to the rate of passive diffusion. The inclusion of acoustic energy again instantly resumed a high level of flow rate. As it turned out, the modulated type provided a regular pulsating increase in the transdermal flow velocity with a modulated speed. When acoustic energy was tuned to a constant frequency, the maximum increase in the transdermal flow rate for this configuration, as it turns out, occurs at approximately 27 MHz.

 Having obtained the same results with all three samples, the cells were then freed from all liquids and rinsed with distilled water on both sides of the stratum corneum. Then the lower chambers were immediately filled with distilled water, and the upper chambers were filled with a dye solution. Cells were observed for 30 minutes. No holes were observed in the samples of the stratum corneum, and a large amount of dye was not detected in the lower chambers. A small amount of the dye became visible in the lower chambers, probably due to capture of the dye and enhancer by skin samples during their previous contacts. After an additional 12 hours, the amount of dye detected was still very small.

Example 21
The influence of acoustic energy and chemical amplifiers
Perforated stratum corneum: three cells were prepared with samples of a thermally separated epidermis with the epidermal side facing the upper side of the chamber from the same donor as in Example 16. The samples were hydrated for 8 hours, and then distilled water in the lower chambers was replaced with XU1MG or HU2MG. Then the upper chambers were filled with a dye solution. Perforations in the form of micro holes in the samples of the stratum corneum allowed the dye to seep through the samples of the stratum corneum into the chambers containing an amplifier lying below them. Acoustic energy was supplied. Immediately after applying acoustic energy, the dye molecules quickly pushed through the pores. As shown above, the fast flow of dye through the pores directly and directly corrected with the supply of acoustic energy.

Example 22
The influence of acoustic energy and chemical amplifiers
A cheap acoustic energy transformer, TDK # NB-58S-01 (TDK Corp.), was tested for its ability to increase transdermal flow rate. It was determined that the maximum response was approximately 5.4 MHz with other local peaks occurring at approximately 7, 9, 12.4 and 16 MHz.

Then, this TDK transducer was tested at 5.4 MHz for its ability to enhance the transdermal flow rate in combination with HU1MG. Three cells were installed with the epidermal side facing the lower chamber, then skin samples were hydrated for 8 hours. The dye solution was placed in the lower chamber. The converter was placed in the upper chamber immersed in KhU1MG. Using frequency swings from 5.3 to 5.6 MHz as an excitation of acoustic energy, significant amounts of dye were transported through the stratum corneum and were detected in the cell receiver after 5 minutes. Local heating occurred, and the converter reached a temperature of 48 ^o C. In the control using ХУ1МГ without acoustic energy, contact within 24 hours gave a less dye output to the receiver than 5-minute exposure to acoustic energy.

 This example demonstrates that a cheap, low-frequency acoustic energy transducer can dramatically affect the transdermal flow rate when used in combination with an appropriate chemical amplifier. Although higher frequency acoustic energy theoretically will concentrate more energy in the stratum corneum, lower frequency modulated acoustic energy when used with a chemical amplifier can accelerate the transdermal flow rate, making this method applicable and practical.

Example 23
Demonstration of molecular migration through human skin
The tests described above using the TDK transducer and HU1MSG were repeated at approximately 12.4 MHz, one of the highest local maximum responses of the transducer with a frequency sweep at a speed of 2 Hz from 12.5 to 12.8 MHz and an acoustic energy density of less than 0.1 W / cm ² . The epidermal side of the thermally separated epidermis was turned down, the dye solution was in the lower chamber, and the amplifier solution and acoustic energy were placed in the upper chamber. Within 5 minutes, a significant amount of dye was transported through the stratum corneum to the receiver. Heating due to active resistance in the converter was significantly less than in the same converter activated at 5.4 MHz, causing the temperature of the chemical amplifier to increase only to 33 ^o C.

 Even at these low levels of efficiency, the results obtained using the HU1MG and acoustic energy from the TDK transducer were remarkable in the monitoring direction. FIGS. 3A and 3B of US Pat. No. 5,445,611 show plots of data obtained in three separate cells measuring transdermal flow velocity in the monitoring direction. Even at a time point of 5 minutes, easily measured amounts of dye were present in a chemical enhancer on the outer surface of the stratum corneum, indicating transport from the epidermis through the stratum corneum to the “outer” region of the skin sample.

 To optimize the use of acoustic energy or the approach using acoustic energy / chemical amplifier to collect and monitor the analytes from the body, means are needed to quantify the analyte of interest. A quantitative determination device that performs multiple measurements while the unit removes analytes using acoustic energy with or without chemical amplifiers eliminates the need for standardization in a wide population spectrum and normalization for various skin characteristics and flow rates. By plotting two or more data values at a time when the concentration of the analyte in the collection system increases, a curve fitting algorithm can be used to determine the parameters that describe the curve of the ratio of the removal rate of the analyte or the flow rate to the point at which equilibrium is reached, establishing thus a measure of concentration in the range. The general form of this curve does not change from one individual to another, only the parameters change. Once these parameters are established, the solution to the problem of stable equilibrium (i.e., time equal to infinity) of this function, i.e. when complete equilibrium is established, gives the concentration of the analyte in the body. Thus, this approach allows measurements with the desired level of accuracy for the same period of time for all members of the population, regardless of individual differences in skin permeability.

 Currently, there are several detection methods that can be adapted for this application. See D. A. Christensen, 1648 Proceedings of Fiber Optic, Medical and Fluoresceent Sensors and Applications 223-26 (1992). One method involves the use of a pair of optical fibers that are located close to each other in approximately parallel ways. One of the fibers is a supply fiber through which light energy is transmitted. The other fiber is a detector fiber coupled to a photosensitive diode. When light is transmitted through the supply fiber, a part of the light energy, not propagating wave, is present on the surface of the fiber, and part of this light energy is collected by the detection fiber. A detector fiber conducts the captured energy of a non-propagating wave to a photosensitive diode that measures it. The fiber is treated with a binder to attract and bind the analyte, the content of which is to be determined. As the analyte molecules bind to the surface (such as glucose analyte binds to immobilized pectins, such as concanavalin A, or immobilized antibodies to glucose), the quantitative characterization of the non-propagating wave adhesion between the two fibers changes and the amount of energy captured by the detector fiber also changes. and measured using a diode. Several measurements of the detected energy of a non-propagating wave over short periods of time contribute to the rapid determination of parameters describing the equilibrium curve, thus providing the possibility of calculating the concentration of the analyte inside the body. Experimental results showing a measured flow within 5 minutes (FIGS. 3A and 3B of US Pat. No. 5,445,611) with this device provide enough data to collect accurate final readings within 5 minutes.

 In its most basic embodiment, a device that can be used to supply acoustic energy and collect the analyte includes an absorbent pad of either natural or synthetic material, which serves as a reservoir for the chemical amplifier, if used, and for receiving the analyte with skin surface. The pad or reservoir is held in place either passively or by appropriate fastening means, such as a strip of masking tape, on a selected area of the skin surface.

 The acoustic energy transducer is positioned so that the pad or reservoir is between the skin surface and the transducer and is held in place by appropriate means. The power source is connected to the converter and triggered by a switch or any other suitable mechanism. The transducer is turned on to supply acoustic energy, modulated, depending on desire, in frequency, phase or intensity, to deliver a chemical amplifier, if used, from the reservoir through the skin surface, followed by collection of the analyte from the skin surface into the reservoir. After a desired fixed or changing period of time, the inverter switches off. The pad or reservoir, now containing the analyte of interest, can be removed for quantitative analysis of the analyte, for example, in a laboratory using any number of conventional chemical analyzes or using a portable device. Alternatively, a device for quantitative analysis of an analyte can be integrated into the device used to collect the analyte, either as an integral part of the device, or as a nozzle. Devices for monitoring an analyte are described in US Pat. No. 5,458/140, which is incorporated herein by reference.

Example 24
An alternative method for detecting an analyte, such as glucose, after collecting a sample through the surface of the skin with pores formed therein, as described above, can be achieved by using an enzymatic agent. There are several enzymatic methods for determining the glucose content in biological samples. One method involves oxidizing glucose in a sample using glucose oxidase to produce gluconolactone and hydrogen peroxide. In the presence of a colorless chromogen, hydrogen peroxide is then converted, using peroxidase, into water and a colored product
Glucose oxidase
Glucose ------------------> Gluconolactone + H ₂ O ₂ .

2Н ₂ О ₂ + chromogen -----------------> Н ₂ О + colored product
The color intensity of the product will be proportional to the amount of glucose in the liquid. This color can be determined using conventional methods for determining optical density or reflectance. By calibrating a known glucose concentration, a color quantitative characteristic can be used to determine the glucose concentration in the collected analyte. Using tests to determine the relationship, you can calculate the concentration of glucose in the blood of an individual. Then this information can be used in the same way as using the information obtained after conducting a blood glucose test from a puncture in the finger. Results can be obtained in the range of 5 to 10 minutes.

Example 25
Any device using a visual display or a glucose reading device will indicate to the diagnostic or patient the need for insulin or other appropriate medication. In intensive care or in other situations where constant monitoring is desired and almost simultaneous corrective actions are required, the display can be connected to an appropriate signaling device that triggers the administration of insulin or another drug accordingly. For example, there are insulin pumps that are implanted in the abdominal cavity or other body cavity, which can be activated in response to external or internal stimuli. Alternatively, when using the increased transdermal flow rates possible with the formation of micropores in the stratum corneum and other methods described in this invention, insulin delivery can be carried out transdermally with the regulation of flow rates modulated by a signal from a device for determining glucose concentration, i.e. quantitative determination of glucose is carried out by calorimetric quantitative determination using glucose oxidase or electrochemical biosensor. Thus, a complete biomedical regulation device may be available that not only monitors and / or diagnoses a medical need, but also provides a corrective effect.

 A similar type of biomedical control device could be provided in other situations, such as maintaining proper electrolyte balance or administering analgesics in response to a measured parameter of an analyte, such as prostaglandins.

Example 26
Like an audible sound, acoustic waves can undergo reflection, refraction and absorption when they encounter another medium with dissimilar properties [D. Bommannan et al., 9 Pharm. Res. 559 (1992)]. Reflectors or lenses can be used to focus or otherwise regulate the distribution of acoustic energy in the tissue of interest. In many areas of the human body, a fold of tissue can be found to fix this device. For example, an earlobe is a convenient location, which allows the use of a reflector or lens to facilitate directional regulation (for example, “pushing” analytes or penetrating compounds through the stratum corneum with pores formed in it) similar to that implemented by changing the acoustic frequency and intensity.

Example 27
To selectively control the direction of the transdermal flow through the stratum corneum with pores formed in it or into the body, or from the body, many transducers of acoustic energy can be used. A fold of skin, such as an earlobe, allows transducers to be placed on each side of the fold. The transducers can be activated selectively or in a phase manner to enhance the transdermal flow in the desired direction. A chain of transducers or an acoustic circuit can be constructed to use phase diffraction grating concepts similar to those developed for radar and microwave communication systems to direct and focus acoustic energy into a region of interest.

Example 28
In this example, the procedure of Example 19 is performed, except that samples of the thermally separated epidermis are first treated with an excimer laser (for example, the EMG / 200 Lambda Physik; with a wavelength of 193 nm and a pulse width of 14 ns) for destruction of the stratum corneum in accordance with the procedure described in US patent 4775361 included here for reference.

Example 29
In this example, the procedure of Example 19 is carried out, except that samples of the heat-separated epidermis are first treated with 1,1'-4,4'-carbocyaniniodide (Aldrich, λ _max = 703 nm), and then acoustic energy is applied to the treated sample at the overall density of 70 mJ / cm ^2/50 ms by a model TOLD9150 diode laser (Toshiba America Electronics, 30 mW at 690 nm) to ablate the stratum corneum.

Example 30
In this example, the procedure of example 29 is performed, except that the dye is indocyanine green (Sigma cat. 1-2633; λ _max = 775 nm), and the laser is a model Diolite 800-50 (LiCONiX, 50 mW at 780 nm).

Example 31
In this example, the procedure of example 29 is performed, except that the dye is methylene blue and the laser is model SDL-8630 (SDL Inc .; 500 mW at 670 nm).

Example 32
In this example, the procedure of example 29 is performed, except that the dye is contained in a solution including a penetration enhancer, for example XI1.

Example 33
In this example, the procedure of example 29 is performed, except that the dye and the solution containing the enhancer are supplied to the stratum corneum using ultrasound.

Example 34
In this example, the procedure of example 29 is performed, except that the pulsed light source is a short arc lamp emitting light in a wide range from 400 to 1100 nm, but having a passband filter placed in the device to limit the output power to a wavelength region of approximately from 650 to 700 nm.

Example 35
In this example, the procedure of example 19 is carried out, except that the samples of the epidermis separated by heat are first pierced with a micro-scalpel (Becton Dickinson) with a precisely selected size to form micropores in the stratum corneum without reaching the underlying tissue.

Example 36
This example procedure of Example 19, except that the epidermis samples were separated thermally initially processed focused acoustic energy in a range from 70 to 480 mJ / cm ^2/50 ms to ablate the stratum corneum.

Example 37
In this example, the procedure of example 19 is performed, except that the stratum corneum is first punctured hydraulically with a high pressure liquid stream to form micropores with a diameter of up to about 100 microns.

Example 38
In this example, the procedure of example 19 is performed, except that the stratum corneum is first pierced by short pulses of electricity to form micropores with a diameter of up to about 100 microns.

Example 39
Acoustic fluid entrainment
Now a new mechanism and application of acoustic energy will be described in delivering therapeutic substances to the body and / or collecting fluids from the body’s internal environment to the external reservoir through micropores formed in the stratum corneum. An additional issue of this invention is the use of acoustic energy to create the effect of entrainment by acoustic waves acting on fluids flowing around and between intact cells in the epidermis in the dermis of human skin. Acoustic wave entrainment is a well-proven method by which acoustic energy can interact with a liquid medium (Nyborg, Physical Acoustics Principles and Methods, p. 265-531, Vol. II-Part B. Academic Press, 1965). The first theoretical analysis of the phenomenon of drag by acoustic waves was carried out by Rayleigh (1884, 1945). With the combined treatment of an individual, Longuet-Higgins (1953-1960) obtained a result that is applicable to a two-dimensional flow arising directly near any vibrating cylindrical surface. Nyborg (1958) developed a three-dimensional approximation for an arbitrary surface. As described by Fairbanks et al., 1975. Ultrasonics Symposium Proceedings, IEEE Cat. # 75, CHO 994-4SU, acoustic energy and the phenomenon of drag by acoustic waves can be widely used in accelerating the flow of fluid through a porous medium, leading to a detectable increase in flow velocity up to 50 times, which is possible passively or only when creating pressure gradients.

 All previous efforts for transdermal delivery or traction using ultrasound have focused on the methods of interaction between acoustic energy and skin tissues designed to increase the permeability of the stratum corneum. It has been hypothesized that the exact method of the interaction is associated exclusively with a local increase in the temperature of the stratum corneum of the skin and the resulting melting of lipid fragments and intercellular spaces between corneocytes (Srinivasan et al.). Other researchers have suggested that the formation of microcavities and / or the displacement of structures in the stratum corneum opens channels through which fluids can flow more easily. In general, the design of acoustic devices for enhancing the transdermal flow rate was based on an early realization that the use of an existing therapeutic ultrasound device designed to expose an individual to “deep heating” when used in combination with local application of a preparation in the form of a gel or liquid containing the preparation , the introduction of which into the body is supposed, can cause a quantifiable increase in the flow rate of the drug into the body. In the context of the method described here for creating micropores in this barrier layer, the use of acoustic energy can now be characterized in a completely new and different sense than the classically defined concepts of sonophoresis.

 Based on the experimental discovery mentioned in US patents 5458140 and 5445611 that when a small hole in the Franz cells used in in vitro studies existed or created in the stratum corneum (OS), that an appropriately activated ultrasound transducer was applied to the fluid reservoir on either side of the sample An OS with pores, a process of “entrainment by acoustic waves” can be created in which high fluid flow rates were able to pump through this porous membrane.

 When using the method described here for creating controlled micropores in the stratum corneum of living individuals, it is now possible to practically use the method of interaction of acoustic energy / fluid in the form of entrainment by acoustic waves to introduce fluid into and out of the body. For example, clinical studies have shown that by forming a series of four micropores with a diameter of 80 microns in a square of 400 microns and then creating a small [10 to 12 inches RT. Art. (254-304.8 mmHg)] rarefaction in this area, it is possible to cause an average of 1 μl of interstitial fluid for external collection to exit the body from the body to the external chamber. By adding a small, low-power acoustic transducer to this device, which is designed to actively generate inwardly converging concentric circular pressure waves in the tissue area of 2 to 6 mm in size surrounding the porous area, it has been shown that the ICF flow rate can increase by 50 %

Freeing yourself from the desire to create some form of direct absorption of acoustic energy in the skin tissues (as required to create heat), frequencies of acoustic energy for which skin tissues are substantially transparent, i.e. in the region of very low frequencies from 1 to 500 kHz, can be determined. Even at some of the lowest frequencies studied, it was possible to observe significant effects of drag by acoustic waves using a microscope to visually control an in vivo test in which micropores are formed in the skin of an individual and the release of LI from the body is caused with accumulation on the surface of the skin. The power supply to the acoustic transducer was accompanied by pronounced visual signs of the amount of entrainment by the acoustic waves in the form of small pieces of material in the form of particles carried away in the current with turbulent ICL. A typical magnitude of the manifested movement can be described as follows: in a circular accumulation of ILI with a diameter of 3 mm on the surface of the skin, one particle was visible, making roughly 3 full revolutions per second. This is equal to a linear fluid velocity of more than 2.5 mm / s. All this action has been demonstrated at acoustic power levels supplied to the tissue of less than 100 mV / cm ² .

Although it is easy to examine the upper surface of the skin and the fluid activity on it, it is much more difficult to evaluate what is happening in the dynamics within the layers of skin tissue in response to the interaction between these tissues and acoustic energy. It can be assumed that if such high speeds (for example,> 2.5 mm / s) are so easily created on the surface, then in response to the input of this acoustic energy, a noticeable increase in the fluid flow in the intercellular channels present in viable dermal tissues can also be realized . Quantification has now been determined.
the characteristic of the increase in the accumulated ILI collected through this set of micropores when applying low-frequency acoustic energy to the circular region surrounding the areas with formed pores. In this experiment, a collection of LIH based only on a minor dilution [10 to 12 inches Hg. Art. (254-304.8 mmHg)] was alternated using the exact same device, but with an attached acoustic transducer. Over a series of 10 two-minute collection periods, five with simple rarefaction and five with both rarefaction and acoustic energy supply, it was observed that by activating the acoustic energy source over the same time period, it was possible to collect roughly 50% more ILI. These data are shown in FIG. This increase in the LI flow rate was achieved without an increase in sensation caused by acoustic energy noted by the test subject. The device used for this experiment is shown in FIGS. 31-33. The complete assembly of FIG. 31-33 consisted of a thick-walled cylinder of piezoelectric material with an inner diameter of roughly 8 mm and a wall thickness of 4 mm. The cylinder was polarized so that when an electric field is applied through the metallized surfaces of the outer diameter and inner diameter, the wall thickness of the cylinder expands or narrows in response to the polarity of the field. In practice, this design provides a device that quickly squeezes tissue that has been pulled into the central hole, causing an inwardly directed radial effect of entrainment by acoustic waves onto those liquids that are present in these tissues. This inward fascination with acoustic waves is responsible for transporting to the micropore location in the center of the hole a larger amount of LIS, where it can leave the body for external collection.

 The similar device shown in FIG. 34A-B, which gave the same initial results. In the embodiment depicted in FIG. 34A-B, ultrasonic transducer manufactured by Zevex, linc. Salt Lake City, Utah, has been modified to create an additional spatula-shaped extension on the acoustic protrusion. A 4 mm hole was made in the putty-shaped end with a thickness of 0.5 mm of this extension. When activated, the main movement occurs in the longitudinal direction along the length of the spatula, which leads essentially to a rapid movement back and forth. A physical violation of the metal spatula caused by the creation of a 4 mm hole results in a very active, but chaotic, large displacement at this point. In use, the skin of an individual was absorbed into this opening, and then acoustic energy was supplied to the skin by a method similar to that shown in FIG. 33.

A new aspect of this new application of ultrasound is in the following main areas:
1. The concentration of ultrasound function on increasing the permeability of the subcutaneous barrier membrane is no longer required, as described by Langer, Kost, Bommannan and others.

 2. A device with a much lower frequency can be used, which has a very small absorption in the skin tissue, but can nevertheless create the desired phenomenon of fluid entrainment by acoustic waves inside the intracellular channels between epidermal cells that contain interstitial fluid.

 3. The type of interaction in tissues and fluids in it is the so-called type of “entrainment”, recognized in the acoustic literature as a unique and different way, in comparison with vibrational interactions that can shift cell membranes and accelerate the process of passive diffusion.

By optimizing the geometric configuration, frequency, power, and modulations for an acoustic transducer, it has been shown that significant increases in fluid flow through skin areas with pores can be achieved. The optimization of these parameters is intended to use nonlinearities that control the relationships of the fluid flow in this microscopically scaled medium. When frequencies below 200 kHz are used, large fluid effects can be observed without any detectable heating or other negative tissue interactions. The acoustic power levels required to cause these measurable effects are very low, with average power levels that are typically below 100 mW / cm ² .

 Therefore, the above examples only represent devices that can be used using ultrasound or ultrasound and chemical amplifiers in the collection and quantitative analysis of analytes for diagnostic purposes and for transdermal delivery of penetrating substances. The invention is directed to the discovery that the formation of pores in the stratum corneum followed by the correct use of ultrasound, in particular accompanied by the use of chemical enhancers, provides the possibility of non-invasive or minimally invasive transdermal determination of the analyzed substances or the delivery of penetrating substances. However, the invention is not limited to only certain illustrations. There are numerous ways to form pores and enhancer systems, some of which may function better than others, to detect and remove certain analytes or to deliver penetrating substances through the stratum corneum. However, within the framework of the recommendations presented here, specialists in this field can easily carry out a certain number of experiments to obtain the optimal method for the formation of pores, optimal amplifiers or the optimal time, intensity and frequency of the ultrasound used, as well as modulation of the frequency, amplitude and phase of the applied ultrasound. Therefore, the invention is limited in the range of claims only by the following claims and their functional equivalents.

Claims (60)

 1. A method for controlling the concentration of an analyte in an individual’s body, in which the permeability of the stratum corneum of a selected region of the body surface of an individual is increased for the analyte by the following steps: (a) pores are formed in the stratum corneum of the selected region by means of micropores in said stratum corneum, avoiding serious damage to underlying tissues, thus reducing the barrier properties of the specified stratum corneum to remove the specified analyte , (b) collect the selected amount of analyte, and (c) quantify the collected analyte.  2. The method according to claim 1, in which after the formation of pores in the stratum corneum, acoustic energy is applied to the above selected region with pores at a frequency in the range from 5 kHz to 100 MHz.  3. The method according to claim 2, in which the acoustic energy is modulated using an element selected from the groups consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof.  4. The method according to claim 2, further comprising contacting a selected area of the body of the individual with a chemical amplifier with acoustic energy to further enhance the removal of the analyte.  5. The method according to any one of claims 1 to 4, in which the specified formation of pores in the specified stratum corneum is achieved using means selected from the group consisting of: (a) destruction of the stratum corneum by contacting a selected area of the specified stratum corneum with a transverse size up to about 1000 microns with a heat source so that the temperature of the water associated with the tissue and other evaporated substances in the specified selected area rises above the evaporation point of the specified water and other evaporated substances, thus removing the stratum corneum of the selected region, (b) puncture of the specified stratum corneum with a fine-sized microskalpel to form micropores with a diameter of up to about 1000 μm, (c) destruction of the stratum corneum by focusing a rigidly focused beam of acoustic energy on the specified stratum corneum, (d) hydraulic puncture of the specified the stratum corneum with a high-pressure liquid jet to form micropores with a diameter of up to about 1000 microns, and (e) puncture the stratum corneum with short pulses of electricity to form anija micropores of diameter up to about 1000 microns.  6. The method according to claim 5, in which the specified formation of pores is carried out by contacting the specified selected area of the specified stratum corneum with a transverse size of up to 1000 μm with a heat source so that the temperature associated with the tissue of water and other evaporated substances in the specified selected area rises above evaporation points of the indicated water and other evaporated substances, thus removing the stratum corneum in the specified selected area.  7. The method according to claim 6, comprising processing at least said selected region with an effective amount of a dye that exhibits strong absorption in the radiation range of the pulsed light source and focuses the generated series of pulses from said pulsed light source on said dye so that said dye Heats up enough to conduct heat transfer to the specified stratum corneum to raise the temperature of the water associated with the tissue and other evaporated substances in the specified selected region STI above the vaporization point of said water and other substances evaporated, wherein said dye functions as a heat source.  8. The method according to claim 7, in which the specified source of pulsed light radiation emits at a wavelength that is significantly absorbed by the skin.  9. The method of claim 8, in which the specified source of pulsed light radiation is a laser diode emitting in the range from approximately 630 to 1550 nm.  10. The method of claim 8, in which the specified source of pulsed light radiation is a laser diode optical parametric pump generator emitting in the range from about 700 to 3000 nm.  11. The method according to claim 7, in which the specified source of pulsed light radiation is an element selected from the group consisting of arc lamps, incandescent lamps and light emission diodes.  12. The method according to claim 7, further comprising providing a control system for determining when the barrier properties of the stratum corneum are overcome.  13. The method of claim 12, wherein said monitoring system includes means for collecting light for receiving light reflected from said selected area and focusing said reflected light on a photodiode, a photodiode for receiving said focused light and for sending a signal to the control unit, wherein said signal indicates the quality of said reflected light, and a control unit connected to said photodiode and said pulsed light source for receiving said signal and for turning off said and a pulsed light source when a preselected signal is received.  14. The method according to claim 7, further comprising cooling said specified region of the stratum corneum and adjacent skin tissues with a cooling agent such that said selected region and adjacent skin tissues are in a selected, previously cooled, stable state before pore formation.  15. The method according to 14, in which the specified cooling means includes a Peltier device.  16. The method according to claim 7, in which the specified destruction leads to the exudation of the interstitial fluid and the specified analyte is collected in a selected amount of the specified interstitial fluid.  17. The method according to clause 16, further comprising, after collecting the specified selected amount of interstitial fluid, sealing said micropore by applying an effective amount of energy from said laser pulsed light source so that coagulation of the interstitial fluid remaining in said micropore is caused.  18. The method according to clause 16, further comprising creating a vacuum in the specified selected region of the stratum corneum with pores formed to enhance the exudation of interstitial fluid.  19. The method according to claim 7, further comprising, prior to the formation of pores in said stratum corneum, illumination of at least said selected area with unfocused light from said pulsed light source so that the selected area illuminated with said light is sterilized. 20. The method according to claim 6, comprising contacting said selected region with a metal wire, wherein said metal wire functions as a heat source so that the temperature of said selected region rises from ambient skin temperature to a level above 100 ^° C in the range from 10 to 50 ms and then returning the temperature of the indicated selected area to approximately the temperature of the surrounding skin in the range of about 30 to 50 ms, and in which the cycle of raising the temperature and returning to the ambient temperature boiling skin repeated several times, that is effective for reducing the barrier properties of the stratum corneum.  21. The method according to claim 20, in which the specified return to approximately the temperature of the surrounding skin is carried out by removing the specified wire from contact with the stratum corneum.  22. The method according to item 21, further comprising providing means for monitoring the total electrical resistance between said wire and said individual body through said selected region of the stratum corneum and adjacent skin tissues and means for advancing the position of said wire so that as how this destruction occurs with a concomitant decrease in resistance, in which the said means for advancing advances the wire so that the wire is in contact with the stratum corneum during Revan wire.  23. The method according to item 22, further comprising a means for removing said wire from contact with the stratum corneum, in which said control means is capable of detecting a change in impedance associated with contacting the epidermis layer lying under the stratum corneum, and supplying a signal to said means for removing said wire from contact with the stratum corneum.  24. The method according to item 21, in which the specified metal wire is heated using a heating element with active resistance.  25. The method according to item 21, in which the specified metal wire is manufactured so that it contains a current loop having a high resistance point, and the temperature of the specified high resistance point is modulated by passing a modulated electric current through the specified current loop.  26. The method according to item 21, in which the specified metal wire is located in a modulated, alternating magnetic field of the excitation coil so that the excitation of the excitation coil by alternating current causes the formation of eddy currents sufficient to heat the wire using internal active losses.  27. The method according to claim 5, in which the specified pore formation is carried out by puncture of the specified stratum corneum with a microscalpel with a precisely selected size for the formation of micropores with a diameter of up to about 1000 microns.  28. The method according to claim 5, in which the specified formation of the pores is carried out using the destruction of the stratum corneum by focusing a rigidly focused beam of acoustic energy on the specified stratum corneum.  29. The method according to claim 5, wherein said pore formation is carried out by hydraulic puncture of the specified stratum corneum with a high pressure liquid stream to form micropores with a diameter of up to about 1000 microns.  30. The method according to claim 5, in which the specified formation of the pores is carried out using a puncture of the specified stratum corneum with short pulses of electricity to form micropores with a diameter of up to about 1000 microns.  31. The method according to any one of claims 5, 7 or 20, in which the analyte is glucose.  32. The method according to p, in which the quantitative determination of glucose is carried out by means of a calorimetric quantitative determination using glucose oxidase or an electrochemical biosensor.  33. A method of increasing the transdermal flow rate of an active penetrating substance to a selected area of an individual's body, wherein the permeability of the stratum corneum of said selected area of the body surface of the individual is increased for said active penetrating substance by the following steps: (a) pores are formed in the stratum corneum of said selected using means that form micropores in the specified stratum corneum, without serious damage to the underlying tissues, and thus reduce -barrier properties of the stratum corneum for the flow of said active permeant; and (b) contacting the selected area having the created pore with a composition comprising an effective amount of said penetrating agent such that the flow of said penetrating agent into the body is enhanced.  34. The method according to clause 33, in which, after the formation of pores in the stratum corneum, acoustic energy is applied to the aforementioned selected region with pores over time and at an intensity and frequency effective to create the effect of a captivating fluid flow, and thus increase the transdermal velocity the flow of the specified penetrating substance into the body.  35. The method according to clause 34, wherein said acoustic energy is supplied to said selected region with a pore formed therein at a frequency in the range of about 5 kHz to 100 MHz, wherein said acoustic energy is modulated by an element selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof.  36. The method according to p. 35, further comprising contacting a selected area of the body of the individual with a chemical amplifier with acoustic energy to further enhance the flow of the specified penetrating substance into the body of the specified individual.  37. The method according to any one of claims 33-35 or 36, wherein said pore formation in said stratum corneum is achieved by means selected from the group consisting of: (a) destruction of the stratum corneum by contacting a selected region of said stratum corneum with transverse size up to approximately 1000 microns with a heat source so that the temperature of the water associated with the tissue and other evaporated substances in the specified selected area rises above the evaporation point of the specified water and other evaporated substances, thus removing the horns the th layer in the indicated selected region, (b) puncture of the specified stratum corneum with a fine-sized microscalpel to form micropores with a diameter of up to about 1000 microns, (c) destruction of the stratum corneum by focusing a rigidly focused beam of acoustic energy on the specified stratum corneum, (d) hydraulic puncture of the specified stratum corneum with a high pressure liquid stream to form micropores with a diameter of up to about 1000 microns, and (e) puncture of the stratum corneum with short pulses of electricity I formation micropore diameter of about 1000 microns.  38. The method according to clause 37, in which the specified formation of pores is achieved by contacting the specified selected area of the specified stratum corneum with a transverse size of approximately 1000 microns with a heat source so that the temperature associated with the tissue of water and other evaporated substances in the specified selected area rises above the evaporation point of the specified water and other evaporated substances, thus removing the stratum corneum in the specified selected area.  39. The method of claim 38, comprising treating at least said selected region with an effective amount of a dye that exhibits strong absorption in the emission range of the pulsed light source and focuses the generated series of pulses from said pulsed light source on said dye so that said dye is heated sufficiently for conductive heat transfer to the specified stratum corneum for raising the temperature associated with tissue water and other evaporated substances in the specified selected Asti above the vaporization of said water is evaporated, and other substances, wherein said dye functions as a heat source.  40. The method according to § 39, wherein said source of pulsed light radiation emits at a wavelength that is not substantially absorbed by the skin.  41. The method according to p, in which the specified source of pulsed light radiation is a laser diode emitting in the range from approximately 630 to 1550 nm.  42. The method according to p, in which the specified source of pulsed light radiation is a laser diode optical parametric pump generator emitting in the range from about 700 to 3000 nm.  43. The method according to p, in which the specified source of pulsed light radiation is an element selected from the group consisting of arc lamps, incandescent lamps and light emission diodes.  44. The method according to § 39, further comprising providing a control system for determining when the barrier properties of the stratum corneum are overcome.  45. The method according to clause 44, wherein said monitoring system includes a means of collecting light for receiving light reflected from said selected area and focusing said reflected light on a photodiode, a photodiode for receiving said focused light and for sending a signal to the control unit, wherein said signal shows the quality of said reflected light, and a control unit connected to said photodiode and said pulsed light source for receiving said signal and for turning off said mpulsnogo light source when a preselected signal is received.  46. The method according to claim 39, further comprising cooling said indicated selected region of the stratum corneum and adjacent skin tissues with a cooling agent such that said selected region and adjacent skin tissues are in a selected, pre-cooled, stable state before pore formation.  47. The method according to item 46, in which the specified cooling means includes a Peltier device.  48. The method of claim 39, further comprising, prior to the formation of pores in said stratum corneum, illumination of at least said selected area with unfocused light from said pulsed light source so that the selected area illuminated with said light is sterilized. 49. The method of claim 38, comprising contacting said selected region with a metal wire, wherein said metal wire functions as a heat source such that the temperature of said selected region rises from ambient skin temperature to a level above 100 ^{° C.} in the range of 10 to 50 ms and then returning the temperature of the indicated selected area to approximately the temperature of the surrounding skin in the range of about 30 to 50 ms, and in which the cycle of raising the temperature and returning to the ambient temperature skin is repeated several times, which is effective to reduce the barrier properties of the stratum corneum.  50. The method according to 49, in which the specified return to approximately the temperature of the surrounding skin is carried out by removing the specified wire from contact with the stratum corneum.  51. The method according to item 50, further comprising providing means for monitoring the total electrical resistance between said wire and said individual body through said selected region of the stratum corneum and adjacent skin tissue and means for advancing the position of said wire so that as how this destruction occurs with a concomitant decrease in resistance, in which the said means for advancing advances the wire so that the wire is in contact with the stratum corneum during Revan wire.  52. The method of claim 51, further comprising a means for removing said wire from contact with the stratum corneum, wherein said control means is capable of detecting a change in impedance associated with contacting a layer of the epidermis lying under the stratum corneum, and supplying a signal to said means for removing said wire from contact with the stratum corneum.  53. The method according to item 50, wherein said metal wire is heated using a heating element with active resistance.  54. The method according to item 50, in which the specified metal wire is manufactured so that it contains a current loop having a high resistance point, and the temperature of the specified high resistance point is modulated by passing a modulated electric current through the specified current loop.  55. The method according to item 50, in which the specified metal wire is placed in a modulated, alternating magnetic field of the excitation coil so that the excitation of the excitation coil by alternating current causes the formation of eddy currents sufficient to heat the wire using internal active losses.  56. The method according to clause 37, in which the specified formation of the pores is carried out by puncture of the specified stratum corneum with a microscalpel with a precisely selected size for the formation of micropores with a diameter of up to about 1000 microns.  57. The method according to clause 37, in which the specified formation of the pores is carried out using the destruction of the stratum corneum by focusing a rigidly focused beam of acoustic energy on the specified stratum corneum.  58. The method according to clause 37, wherein said pore formation is carried out by hydraulic puncture of the specified stratum corneum with a high pressure liquid stream to form micropores with a diameter of up to about 1000 microns.  59. The method according to clause 37, in which the specified formation of the pores is carried out using a puncture of the specified stratum corneum with short pulses of electricity to form micropores with a diameter of up to 1000 microns.  60. A method of applying a tattoo to a selected area of the skin on the surface of an individual’s body, in which: (a) forming a pore in the stratum corneum of the selected area by means of forming micropores in the stratum corneum without seriously damaging the underlying tissues, and thus lowering the barrier the properties of the stratum corneum for the flow of the specified active penetrating substance, and (b) contacting the selected area having the created pore, with a composition comprising an effective amount of ink for tattoo so that the flow of the indicated ink into the body is enhanced.

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