CN1195276A - Microporation method of human skin for drug delivery and monitoring applications - Google Patents

Abstract

A method of enhancing the permeability of the skin (120, 274) to an analytic for diagnostic purposes or to a drug for therapeutic purposes is described utilizing micro-pore and optionally sonic energy and a chemical enhancer. If selected, the sonic energy may be modulated by means of frequency modulation, amplitude modulation, phase modulation, and/or combinations thereof. Micro-pore is accomplished by (a) ablating the stratum corneum (274) by localized rapid heating of water such that water is vaporized, thus eroding cells; (b) puncturing the stratum corneum (274) which a micro-lancet calibrated to form a micro-pore of up to about 1000 m in diameter (c) ablating the stratum corneum (274) by focusing a tightly focused beam of sonic energy onto the stratum corneum (274); (d) hydraulically puncturing the stratum corneum (274) with a high-pressure jet of fluid to form a micro-pore of up to about 1000 m in diameter; or (e) puncturing the stratum corneum (274) with short pulses of electricity to form a micro-pore of up to about 1000 m in diameter.

Description

Human skin micro-perforation method for drug delivery and detection

Cross reference to related applications

This application is a continuation-in-part application of application No.08/520,547 filed on 8/29/1995, which in turn is a continuation-in-part application of application No.08/152,442 filed on 11/15/1993 (now U.S. Pat. No.5,458,140) and No.08/152,174 filed on 12/8/1993 (now U.S. Pat. No.5,445,611); the benefit of provisional application No.60/008,043 filed on 30/10/1995 is also claimed.

Background

The present invention relates generally to the field of monitoring analytes in the body and delivering drugs transdermally into the body. More particularly, the present invention relates to minimally invasive to non-invasive methods of increasing skin permeability through stratum corneum microperforation that may be used in conjunction with sonic energy, chemical permeation enhancers, pressure, and the like, to selectively enhance analyte egress from the body for detection, or drug delivery into the body.

The stratum corneum is primarily responsible for the well-known barrier function of the skin. Thus, it provides the greatest barrier to transdermal flux of drugs and other molecules into the body and transdermal flux of analytes out of the body. The stratum corneum (the horny layer outside the skin) is a complex structure of tight keratinocyte remnants separated by lipid functions. Compared to the oral and gastric mucosa, the stratum corneum is more difficult to allow penetration of exogenous or endogenous molecules into the body. The stratum corneum is formed by keratinocytes, which constitute the majority of the epidermal cells, losing their nuclei and becoming corneocytes. These dead cells constitute the stratum corneum, which is only about 10-30 μm thick and, as mentioned above, is a highly resistant, water-tight membrane that protects the body from the invasion of foreign substances and the outward migration of fluids and dissolved molecules. The stratum corneum is constantly renewed by exfoliation of keratinocytes during desquamation and by the formation of new keratinocytes by the keratinization process.

Transdermal flux of drugs or analytes can be increased by varying the resistance (diffusion coefficient) or driving force (diffusion gradient). Flow may be promoted by so-called osmotic or chemical promoters. Chemical promoters are well known in the art and will be described in more detail below.

Another method of increasing the permeability of the skin to drugs is electrophoresis. Electrophoresis involves the application of an external electric field and localized delivery of ionic drugs or non-ionic drugs carried in a stream of water with ion transfer (electro-osmosis). Although it is effective to promote permeation by electrophoresis, controlled drug release and irreversible skin damage are problems that arise with this technique.

Sonic energy has also been used to promote permeation of drugs and other molecules through the skin and synthetic membranes. Ultrasound is defined as a mechanical pressure wave with a frequency exceeding 20kHz, H.Lutz et al, Manual of Ultrasound 3-12 (1984). Sonic energy is generated by passing an alternating current through a piezoelectric crystal or other electromechanical element to cause it to vibrate, r.brucks et al, 6 pharm.res.697 (1089). The use of sonic energy to increase the penetration of drug molecules by the skin has been referred to as sonophoresis (sonophoresis).

Although it is known that skin penetration enhancement theoretically should allow the transfer of molecules from the body through the skin to the body for collection or detection, no viable method has been disclosed. U.S. patent No.5,139,023 to Stanley et al discloses an apparatus and method for non-invasive blood glucose testing. The invention uses chemical penetration enhancers to increase the penetration of glucose by mucosal tissues or skin. The glucose then passively diffuses through the mucosal tissue or the skin, being captured by the receiving medium. The amount of glucose in the receiving medium is measured and the blood glucose level is determined accordingly. However, as described by Stanley et al, the method is more effective when applied to mucosal tissues (e.g., buccal tissues) such that a detectable amount of glucose can be collected in the receiving medium after a delay period of about 10-20 minutes. However, the method taught by Stanley et al results in an extremely long delay period, 2-24 hours depending on the promoter compound used, before detectable amounts of glucose diffusing through human skin (heat-detached epidermis) can be detected in vitro. Such a long delay period may be attributed to the length of time required for the chemical permeation enhancer to passively diffuse transdermally and increase the stratum corneum barrier permeability, aswell as the length of time for glucose to passively diffuse out transdermally. Thus, Stanley et al apparently do not disclose a method for non-invasive transdermal transport of blood glucose and other analytes that would allow for rapid detection of these blood electrolytes, as is required for blood glucose monitoring in diabetic patients and for many other bodily analytes.

It is known to deliver drugs using sonic energy, but the results are largely disappointing because of the little increase in permeability. There has been no consensus on the utility of acoustic energy to increase transdermal flux of drugs. Some studies have reported the success of acoustophoresis, j.davick et al, 68 phys.ther.1672 (1988); griffin et al, 47 phys, ther.594 (1967); griffin&j. touch hstone, 42 am.j. phys.med.77 (1963); griffin et al, 44 am.j.phys.med.20 (1965); levy et al, 83 j.clin.invest.2074; d.bommann et al, 9 pharm. res.559(1992), negative results were obtained from other studies, h.benson et al, 69 phys.therm.113 (1988); j.mcelnay et al, 20 br.j.clin.pharmacol.4221 (1985); h.pratzel et al, 13 j.rheumatol.1122 (1986). Systems employing rodent skin show the most promising results, while systems employing human skin generally show disappointing results. It is well known to those skilled in the art that rodent skin is much more permeable than human skin, and therefore the above results do not teach those skilled in the art how to effectively utilize acoustophoresis, such as applied to transdermal delivery and/or detection in humans.

Copending application No.08/152,442 filed 11/15/1993 (now U.S. patent No.5,458,140) and copending application No.08/152,174 filed 12/8/1993 (now U.S. patent No.5,445,611), both of which are incorporated herein by reference, disclose and claim significant improvements in the use of acoustic energy for the detection of analytes and for the administration of drugs to the body. In these inventions, transdermalsampling of analytes or transdermal drug delivery is accomplished by using sonic energy, whose intensity, phase or frequency or a combination of these parameters is modulated, in combination with a chemical permeation enhancer. Also disclosed are punctures made by needle punching, water jet, laser, electroporation, or other methods that use acoustic energy that can be frequency, intensity, and/or phase modulated to controllably push and/or pump molecules into the stratum corneum.

The formation of micropores (i.e., microperforations) in the stratum corneum to facilitate drug delivery is the subject of various studies, leading to the issuance of patents for these technologies.

Jacques et al, 88 j. invest.dermtol.88-93 (1987) teach a method of administering a drug by ablating the stratum corneum in an area of the skin using a pulsed laser of wavelength, pulse length, pulse energy, pulse number, and pulse repetition rate sufficient to ablate the stratum corneum without significantly damaging the underlying epidermis, and then applying the drug to the ablated area. This work has led to U.S. patent No.4,775,361 to Jacques et al. Ablation of skin by irradiation with ultraviolet laser light was earlier reported by Lane et al, Arch. Jacques et al are limited to using few wavelengths of light and expensive lasers.

U.S. patent No.5,165,418 to Tankovich (hereinafter "Tankovich' 418") discloses a method of obtaining a blood sample by irradiating human or animal skin with one or more laser pulses having energy sufficient to cause vaporization of skin tissue to create a hole in the skin extending through the epidermis and to cut at least one blood vessel, allowing an amount of blood to drain through the hole so that it can be collected. Thus, Tankovich' 418 is not suitable for making stratum corneum permeable non-or minimally invasive so that drugs can be delivered to the body or so that analytes in the body can be analyzed.

U.S. Pat. No.5,423,803 to Tankovich et al (hereinafter "Tankovich' 803") discloses laser ablation of superficial epidermal cells of human skin for cosmetic applications. The method involves applying a light-absorbing "contaminant" to the outer layers of the epidermis, forcing some of this contaminant into the intercellular spaces of the stratum corneum, and irradiating the infiltrated skin with laser pulses of sufficient intensity that the energy absorbed by the contaminant causes the contaminant to explode with sufficient energy to tear some of the epidermal cells. Tankovich' 803 further teaches that the contaminant should have a high energy absorption at the wavelength of the laser beam, so that the laser beam must be a pulsed beam of less than 1 μ s in duration, the contaminant must be pressed into the upper layers of the epidermis, and the contaminant must explode with sufficient energy after absorption of the laser energy to tear open the epidermal cells. The invention also fails to disclose or suggest methods of delivering drugs or collecting analytes.

Raven et al (WO92/00106) describe a method for selectively removing diseased tissue from a body by administering to the selected tissue a compound which is highly absorbing to infrared radiation of wavelength 750-860nm, irradiating the area with corresponding infrared radiation at a power sufficient to cause thermal vaporization of the tissue to which the compound is applied but insufficient to cause thermal vaporization of the tissue to which the compound is not applied. The light-absorbing compound is soluble in water or serum, such as indigo anthocyanin, chlorophyll, porphyrin, heme-containing compound, or polyene structure-containing compound, and has power level of 50-1000W/cm²Or higher.

Konig et al (DD259351) teach a method for the thermal treatment of tumor tissue, comprising depositing a medium which absorbsradiation in the red and/or near infrared spectral region on the tumor tissue, and irradiating the infiltrated tissue with a laser of appropriate wavelength. The absorbing medium may include methylene blue, reduced porphyrins or coacervates thereof, and phthalocyanine blue. This patent lists methylene blue with strong absorption at 600-700nm and krypton lasers emitting at 647 and 676 nm. The power level should be at least 200mW/cm²。

It has been demonstrated that by repeatedly applying and removing scotch tape to the same area of skin, tearing away the stratum corneum from a small area of skin can easily collect any amount of interstitial fluid that can be used to detect many analytes of interest. Similarly, "taped" skin has also proven to be readily permeable, allowing transdermal delivery of the compound into the body. Unfortunately, "tape stripping" leaves an open wound that can heal for weeks, and therefore for other reasons, and is not an acceptable practical method of promoting transdermal transport in a wide range of applications.

As discussed above, it has been demonstrated that pulsed lasers (e.g., excimer lasers operating at 193nm, erbium lasers operating at near 2.9 μm, or CO operating at 10.2 μm₂A laser) may be used to ablate small holes in the human stratum corneum. These laser ablation techniques offer the possibility of selectively and potentially non-invasively opening delivery and/or sampling orifices through the stratum corneum. However, due to the prohibitive costs associated with these light sources, there is currently no commercial product based on this concept. The present invention allows for the use of low cost energy sources to produce the desired micro-ablation of the stratum corneum by direct introduction of thermal energy into the stratum corneum with very well defined spatial and temporal resolution.

In view of the above-described problems and/or deficiencies, it would be a significant advance in the art to devise a method of safely increasing skin permeability for minimally invasive or non-invasive detection of body analytes in a faster time frame. Another significant advance in the art would be to provide a method of enhancing transdermal flux of a drug into a selected area of an individual with minimal or no trauma.

Summary of The Invention

It is an object of the present invention to minimize the barrier properties of the stratum corneum by perforation to allow controlled collection of analytes from the body through the pores of the stratum corneum for detection.

It is a further object of the present invention to provide a method for detecting selected in vivo analytes through micropores in the stratum corneum in combination with sonic energy, penetration enhancers, pressure gradients, and the like.

It is another object of the present invention to provide for controlling the flow rate of a drug or other molecule through the pores of the stratum corneum into the body (and into the blood stream, if desired) transdermally.

It is yet another object of the present invention to provide a method for delivering drugs into the body through the micropores in the stratum corneum in combination with sonic energy, penetration enhancers, pressure gradients, and the like.

These and other objects are achieved by providing a method for detecting the concentration of an analyte in an individual, the method comprising the step of increasing the permeability of stratum corneum of a selected area of the surface of the individual to the analyte by:

(a) perforating the stratum corneum of the selected area to reduce the barrier properties of the stratum corneumto analyte withdrawal by forming micropores in the stratum corneum without causing severe damage to the underlying tissues;

(b) collecting a selected amount of an analyte; and

(c) the collected analytes were quantified.

In a preferred embodiment, the method further comprises applying sonic energy to the perforated selected area at a frequency in the range of about 5KHz to 100MHz, wherein the sonic energy is modulated in a manner selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof. In another preferred embodiment, the method further comprises contacting the selected area of the individual with a chemical enhancing agent, and applying sonic energy to further enhance analyte withdrawal.

The perforation of the stratum corneum is accomplished by a method selected from the group consisting of: (a) removing the stratum corneum from the selected area by contacting the selected area spanning an area of no more than 1000 μm with a heat source to ablate the stratum corneum such that the tissue of the selected area binds the water and other vaporizable materials to an elevated temperature above the vaporization point of the water and other vaporizable materials; (b) piercing the stratum corneum with a calibrated microstome to form micropores having a diameter of less than or equal to about 1000; (c) focusing the tightly focused acoustic energy beam onto the stratum corneum to ablate the stratum corneum; (d) hydraulically puncturing the stratum corneum with a high pressure fluid stream to form micropores having a diameter of less than or equal to about 1000, and (e) puncturing the stratum corneum with a short electrical pulse to form micropores having a diameter of less than or equal to about 1000.

A preferred embodiment for thermally ablating the stratum corneum comprises treating atleast a selected area with an effective amount of a dye that exhibits strong absorption into the emission range of a pulsed light source, and focusing a series of pulses output from the pulsed light source onto the dye to heat the dye sufficiently to conduct heat to the stratum corneum to raise the temperature of tissue associated with the water and other vaporizable materials in the selected area above the vaporization point of the water and other vaporizable materials. Preferably, the pulsed light source emits at a wavelength that is not significantly absorbed by the skin. For example, the pulsed light source may be a laser diode emitting in the range of about 630-. A sensing system may also be provided to determine when the barrier properties of the stratum corneum are eliminated. A preferred sensing system includes a light collecting means for receiving light reflected from a selected area and focusing the reflected light on a photodiode, a photodiode for receiving the focused light and for transmitting a signal to a controller, wherein the signal is indicative of a property of the reflected light, and a controller coupled to the photodiode and to the pulsed light source for receiving the signal and for switching off the light source when a preselected signal is received.

In another preferred embodiment, the method further comprises cooling the selected region of the stratum corneum and adjacent skin tissue with a cooling device such that the selected region and adjacent skin tissue are in a pre-cooled steady state condition prior to piercing.

In yet another preferred embodiment, the method comprises ablating the stratum corneum, draining interstitial fluid from the micropores, collecting the interstitial fluid, and analyzing the collected interstitial fluid for an analyte. After collection of interstitial fluid, an effective amount of energy from a laser diode or other light source may be applied to close thepores, allowing interstitial fluid remaining in the pores to aggregate. It is preferred to apply a vacuum to selected areas of the perforation to facilitate collection of interstitial fluid.

In yet another embodiment, the method comprises irradiating at least the selected area with unfocused light from a pulsed light source prior to perforation of the stratum corneum, and sterilizing the selected area irradiated with light.

Another preferred embodiment of the perforation of the stratum corneum comprises contacting the selected area with a wire to raise the temperature of the selected area from the temperature of the surrounding skin to greater than 100 ℃ in about 10-50ms, and then returning the temperature of the selected area to about the temperature of the surrounding skin in about 30-50ms, wherein this cycle of raising the temperature and returning to about the temperature of the surrounding skin is repeated a number of times sufficient to reduce the barrier properties of the stratum corneum. Preferably, the step of returning to about ambient skin temperature is performed by withdrawing the wire out of contact with the stratum corneum. Preferably there is also provided means for monitoring the electrical impedance between the wire and the subject through selected areas of the stratum corneum and adjacent skin tissue, and means for advancing the position of the wire such that when ablation occurs, the electrical impedance decreases with it, the advancing means comprises an advancing wire which is brought into contact with the stratum corneum when the wire is heated. In addition, it is preferred to provide means for withdrawing the wire from contact with the stratum corneum, wherein the monitoring means is capable of detecting a change in impedance associated with contact with the epidermis layer of the stratum corneum and transmitting a signal to the withdrawal means to withdraw the wire from contact with the stratum corneum. The wire may beheated by an ohmic heating element, there may be a current loop with a high impedance point whose temperature is modulated by a modulated current heated through the current loop, or the wire may be placed in an alternating magnetic field of a modulatable excitation coil (excitation coil) that, when energized with alternating current, generates eddy currents sufficient to heat the wire by internal ohmic losses.

A method of increasing the rate of transdermal flux of an active permeant into a selected area of a subject includes the step of increasing the permeability of the stratum corneum of the selected area of the subject's surface to the active permeant by:

(a) perforating the stratum corneum of the selected area to reduce the barrier properties of the stratum corneum to the flow of active permeants by forming micropores in the stratum corneum without causing severe damage to the underlying tissues; and

(b) contacting the perforated selected area with a composition comprising an effective amount of a permeate to promote permeate flow into the body.

In a preferred embodiment, the method further comprises applying sonic energy to the perforated selected regions for a time and at an intensity and frequency effective to establish a fluid flow effect to enhance the transdermal flux of permeate into the body.

There is also provided a method for applying tattoos (tatoos) to selected areas of the skin on the surface of an individual, comprising the steps of:

(a) perforating the stratum corneum of the selected area to reduce the barrier properties of the stratum corneum to the flow of active permeants by forming micropores in the stratum corneum without causing severe damage to the underlying tissues; and

(b) contacting the perforated selected area with a composition comprising an effective amount of tattoo ink as a permeant to facilitate the flow of said ink into the body.

There is further provided a method for reducing the time delay for an analyte to diffuse from the blood of an individual to interstitial fluid of said individual in a selected area of the skin, the method comprising applying a cooling device to said selected area of the skin.

There is still further provided a method for reducing the vaporization and vapor pressure of interstitial fluid collected from micropores in a selected region of the stratum corneum of the skin of a subject, comprising applying a cooling device to said selected region of the skin.

Brief description of several views of the drawings

Fig. 1 is a schematic diagram of the light delivery of a laser diode and monitoring of the progress of the perforation.

FIG. 2 is a schematic diagram of a closed loop feedback system for monitoring perforation.

Fig. 3A is an optical piercing system including a cooling device.

Fig. 3B is a top view illustrating the cooling device of fig. 3A.

Figure 4 is a schematic diagram of an ohmic heating device with a mechanical actuator.

Fig. 5 is a schematic diagram of a high resistance current loop heating apparatus.

Fig. 6 is a schematic view of an apparatus for regulating heating using induction heating.

FIG. 7 is a schematic diagram of a closed loop impedance detector that uses changes in impedance to determine the degree of perforation.

FIGS. 8A-D show the treatment with copper phthalocyanine and then the respective energy densities of 4000J/ cm ²0, 1,5 and 50 light pulses at 810 nm.

Fig. 9-11 are schematic temperature profiles during simulation of thermal perforation with optical perforation.

Fig. 12 and 13 are schematic diagrams of temperature as a function of time in the stratum corneum and the living epidermis, respectively, during simulated thermal perforation with optical perforation.

Fig. 14-16 illustrate the temperature distribution during thermal perforation simulated with optical perforation (where tissue is cooled prior to perforation), the temperature in the stratum corneum as a function of time, and the temperature in the living epidermis as a function of time, respectively.

Fig. 17-19 illustrate the temperature distribution in a simulated thermal perforation process (where tissue is heated with hot wires), the temperature in the stratum corneum as a function of time, and the temperature in the living epidermis as a function of time, respectively.

Fig. 20-22 illustrate the temperature profile during simulated thermal perforation (where the tissue is heated with a hot wire and the tissue is cooled before perforation), the temperature in the stratum corneum as a function of time, and the temperature in the living epidermis as a function of time, respectively.

Figures 23 and 24 illustrate the temperature profile and temperature in the stratum corneum as a function of time, respectively, during simulated thermal perforation, in which the tissue was optically heated according to the operating parameters of Tankovich' 803.

FIG. 25 is a graphical representation of interstitial fluid (ISF; o) and blood (. lambda.) glucose levels as a function of time.

FIG. 26 is a plot of the distribution of the difference term between the ISF glucoseand blood glucose data of FIG. 25.

FIG. 27 shows a histogram of the relative deviation of the ISF versus blood glucose level obtained in FIG. 25.

FIG. 28 shows a cross-section of an exemplary delivery device for delivering a drug to a selected area on the skin of an individual.

Fig. 29A-C are illustrations of the area of affected skin delivering lidocaine to selected areas of the stratum corneum that are perforated (fig. 29A-B) or not perforated (fig. 29C).

Figure 30 shows a comparative plot of the amount of interstitial fluid collected from the microwells from aspiration (o) alone, in combination with aspiration and ultrasound.

Figures 31, 32 and 33 show an ultrasound transducer/vacuum device for harvesting interstitial fluid, a cross-sectional view of the device and a schematic cross-sectional view of the device, respectively.

Fig. 34A-B show top views of a hand-held ultrasound transducer and a side view of its blade end, respectively.

Detailed Description

Before the present method for facilitating stratum corneum penetration for transdermal drug delivery and analyte sampling is disclosed and described, it is to be understood that this invention is not limited to the particular structures, process steps, and materials disclosed herein as such structures, process steps, and materials may vary somewhat. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

In this specification and the appended claims, it must be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a method of delivering "a drug" includes delivery of a mixture of two or more drugs, reference to "an analyte" includes one or more such analytes, and reference to "a permeation enhancer" includes mixtures of two or more permeation enhancers.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

As used herein, "perforation", "microperforation" or any such similar term means the formation of small holes or pores in the stratum corneum of selected areas of the individual's skin to impair the barrier properties of that layer to analytes passing from beneath the surface of the skin for analysis or to active permeants or drugs entering the body for treatment. Preferably, the holes or pores are no greater than about 1mm in diameter, more preferably no greater than about 100 μm in diameter, and extend into the stratum corneum sufficiently to disrupt the barrier properties of the layer without adversely affecting the underlying tissue.

As used herein, "ablation" means the controlled removal of cells caused by the kinetic energy released when the cell vaporizable component is heated to a temperature at which vaporization occurs, the rapid volume expansion due to this phase change causing the cells and possibly some adjacent cells to be "blown away" from the ablation site.

As used herein, "piercing" or "micro-piercing" means perforating the stratum corneum using mechanical, hydraulic, or electrical means.

To the extent that "ablation" or "piercing" accomplishes the same purpose of perforation (i.e., the formation of a hole or pore in the stratum corneum without significantly damaging underlying tissue), these terms are used interchangeably.

As used herein, "penetration" or "permeation" promotion means an increase in the permeability of the skin to a drug, analyte, dye, colorant, or other chemical molecule (also referred to as a permeant), thereby increasing the rate at which the drug, analyte, or chemical molecule permeates the stratum corneum to facilitate perforation of the stratum corneum, withdrawal of the analyte through the stratum corneum, or transport of the drug through the stratum corneum and into the underlying tissue. The permeation enhancing effect produced by the use of such enhancers can be observed, for example, by observing the diffusion of the dye as a permeant through the skin of an animal or human with a diffusion instrument.

As used herein, "chemical enhancers," "penetration enhancers," and the like include all enhancers that increase the transdermal flux of a permeate, analyte, or other molecule, limited only by function. In other words, all compounds and solvents and other chemical promoters which disturb the cell membrane are included.

As used herein, "dye," "colorant," and the like are used interchangeably to refer to a biologically suitable chromogen that exhibits strong absorption in the emission range of the pulsed light source used to ablate stratum corneum tissue to form micropores.

"transdermal" or "transdermal" as used herein means that the permeate penetrates into and through the skin to a pharmaceutically effective therapeutic blood concentration or deep tissue level, or that molecules ("analytes") present in the body exit through the skin so that the analyte molecules can be collected in vitro.

The term "permeant," "drug," or "pharmacologically active agent" or any other similar term as used herein means any chemical or biological material or compound suitable for transdermal administration by methodspreviously known in the art and/or taught herein that elicits a desired physiological or pharmacological effect which may include, but is not limited to, (1) having a prophylactic effect on the body, preventing an undesired physiological effect such as preventing infection, (2) alleviating the symptoms caused by the disease, such as relieving pain or inflammation produced as a result of the disease, and/or (3) relieving, reducing or completely eliminating the body's disease, which effect may be local, such as providing a local anesthetic effect, or may be systemic the present invention is not directed to new permeants or new types of active agents, but is limited to those existing in the art or to later identified as active agents and suitable for delivery by the present methods of the invention.

As used herein, an "effective" amount of a pharmacologically active agent refers to a sufficient amount of the compound to provide the desired local or systemic effect, at a reasonable benefit/risk ratio, as is the case with any medical treatment. As used herein, an "effective" amount of a penetration or chemical enhancer refers to an amount selected to provide a desired increase in skin permeability and a desired depth of penetration, rate of administration, and amount of drug delivered.

As used herein, "carrier" or "excipient" refers to a carrier material suitable for use with other pharmaceutically active substances in amounts which are not significantly pharmacologically active, and includes any such substance known in the art, such as any liquids, gels, solvents, liquid diluents, solubilizing agents, and the like, which are non-toxic in the amounts employed and which do not interact in a deleterious manner with the intended drug. Examples of suitable carriers for use herein include water, mineral oil, silicones, inorganic gels, aqueous emulsions, liquid sugars, waxes, petrolatum and a wide variety of oils and polymeric materials.

As used herein, "biofilm" refers to a membrane material that exists within a living organism and separates one area of the organism from another, and in many cases from its environment. Thus, skin and mucosa are included.

As used herein, "subject" refers to humans and animals to which the present invention is practiced.

As used herein, "analyte" refers to any chemical or biological material or compound that is suitable for passage through a biological membrane using the techniques taught herein or known in the art, and an individual may want to know its concentration or activity in vivo. Glucose is a specific example of an analyte because it is an analyte that is suitable for passage through the skin, and individuals (e.g., those with diabetes) may want to know their blood glucose concentration. Other examples of analytes include, but are not limited to, compounds such as sodium, potassium, bilirubin, urea, ammonia, calcium, lead, iron, lithium, salicylates, and the like.

As used herein, a "transdermal flow rate" is the rate of efflux of any analyte through the skin of an individual (human or animal) or the rate of entry of any drug, pharmacologically active agent, dye or pigment into and through the skin of an individual (human or animal).

As used herein, the terms "intensity amplitude," "intensity," and "amplitude" are used synonymously to refer to the amount of energy generated by an acoustic energy system.

As used herein, "frequency modulated" or "swept" refers to a continuous, gradual, or stepwise change in amplitude or frequency over a period of time. Frequency modulation is a step or step change in frequency over a period of time, such as from 5.4 to 5.76 MHz in 1 second, or 5-10MHz in 0.1 second, or 10-5MHz in 0.1 second, or any other frequency range or time period suitable for a particular application. Complex modulation may include simultaneous changes in frequency and intensity. For example, FIGS. 4A and 4B of U.S. Pat. No.5,458,140 may represent the case where amplitude and frequency modulation, respectively, is applied to a single acoustic transducer at the same time.

As used herein, "phase modulation" means that the timing relationship of the signals is changed corresponding to the initial stage thereof as shown in fig. 4 of U.S. patent No.5,458,140. The frequency and amplitude of the signal may remain unchanged. Phase modulation may be accomplished with a variable delay, such as by temporarily delaying or advancing a signal selectively with respect to its previous state or with respect to another signal.

As described herein, sonic energy in its various applications, such as frequency, amplitude or phase modulation or combinations thereof, and the use of chemical promoters in combination with the modulated sonic energy, may vary in the frequency range between about 5kHz and 100MHz, with a range between about 20kHz and 30MHz being preferred.

As used herein, "non-invasive" means insertion into the body without the need for a needle, cannula, or other invasive medical instrument.

As used herein, "minimally damaging" means damaging the stratum corneum with a mechanical, hydraulic, or electrical device to create small holes or micropores without causing substantial damage to the underlying tissue.

Method for perforation of the stratum corneum

The formation of micropores in the stratum corneum can be accomplished using various methods known in the art and some modifications thereof as disclosed herein.

The use of laser ablation as described by Jacques et al (in U.S. patent No.4,775,361) and Lane et al (supra) clearly provides a means of ablating the stratum corneum with an excimer laser. It has been found that at a wavelength of 193nm and a pulse width of 14ns, each laser pulse can be used at a wavelength of about 70-480mJ/cm²The irradiation amount of (2) removes approximately 0.24 to 2.8 μm of the horny layer. As the pulse energy increases, more tissue is removed from the stratum corneum, and fewer pulses are required to fully perforate the layer. The lower threshold of the amount of radiation within the thermal relaxation time limit that must be absorbed by the stratum corneum to produce a suitable micro-explosion and cause tissue ablation is about 70mJ/cm in 50 milliseconds (ms)². In other words, a total of 70mJ/cm must be transmitted within a 50 millisecond window². This may be 70mJ/cm in 50 milliseconds²Of 10 individual pulses or of 7mJ/cm²Pulse of 1.4W/cm²Is performed by continuous irradiation. The upper limit of the amount of irradiation is the amount that will ablate the stratum corneum without damaging the underlying tissue, and can be determined empirically from the light source, the wavelength of the light, and other variables within the experience and knowledge of those skilled in the artAnd (4) determining.

The term "transmit" means that the amount of energy is absorbed by the tissue to be ablated. At an excimer laser wavelength of 193nm, the uppermost 1 or 2 micron portion of the stratum corneum tissue absorbs substantially 100% of the energy. Assuming the stratum corneum is about 20 microns thick, at longer wavelengths, such as 670nm, only about 5% of incident light is absorbed within the 20 micron layer. This means that about 95% of the high power beam enters the tissue below the stratum corneum where it is likely to cause significant damage.

The ideal method is to use only the necessary amount of power to perforate the stratum corneum without causing bleeding, heating or other damage to the underlying tissue from which the analyte is to be extracted or to which the drug or other permeant is to be delivered.

It would be advantageous, and therefore advantageous, to use an energy source that is more economical than the energy used from an excimer laser. The costs required for operating and maintaining excimer lasers emitting light at wavelengths in the far ultraviolet region are far greater than, for example, diode lasers emitting light in the visible and infrared regions (600-1800 nm). However, at longer wavelengths, the stratum corneum becomes progressively more transparent and absorption occurs primarily in the underlying tissue.

The present invention provides a method for rapidly and painlessly removing the barrier function of the stratum corneum to facilitate transdermal delivery of therapeutic substances to the body or in vivo selection of analytes for analysis upon topical administration. The method uses a procedure that begins with a small area of heat source in contact with the target area of the stratum corneum.

The heat source must have several important properties as will be discussed below. First, the heat source must be of a suitable size, typically about 1-1000 microns in diameter, to limit contact with the skin to a small range. Second, the heat source must be able to adjust the temperature of the stratum corneum at the point of contact from the surrounding skin surface temperature level (33 ℃) to above 123 ℃ over a cycle time and then fall back to near the surrounding skin surface temperature to minimize damage to living tissue and the sensation produced by the subject. The adjustment can be done electronically, mechanically or chemically.

Furthermore, the depth limiting feature inherent in microperforation is facilitated if the heat source has a sufficiently small thermal mass and a limited energy source to raise its temperature so that when it comes into contact with tissues having a moisture content above 30%, the heat spread in these tissues is sufficient to limit the maximum temperature of the heat source to below 100 ℃. This feature effectively terminates thermal vaporization once the thermal probe has penetrated through the stratum corneum into the underlying layers of the epidermis.

In the case of a heat source in contact with the skin, it goes through a series of one or more temperature regulation cycles from an initial ambient skin temperature to a peak temperature above 123 ℃ to near ambient skin temperature. To minimize or eliminate the perception of microperforation subjects, the duration of these pulses is limited, with the spacing between pulses being long enough to cool the active tissue layers in the skin, especially the weakened dermal tissue, to an average temperature below about 45 ℃. These parameters depend on the thermal time constant (about 30-80 milliseconds) of the viable epidermal tissue between the thermal probe and the weakened tissue in the underlying dermis. As a result of the above-described application of pulsed thermal energy, there is sufficient energy input into the stratum corneum within a very small target site so that the local temperature of the portion of tissue is raised sufficiently above the vaporization temperature of the water bound by the tissue in the stratum corneum. When the temperature rises above 100 c, the moisture of the stratum corneum in this localized area (typically 5-15%) will vaporize and expand away very rapidly, causing the keratinocytes in the stratum corneum in the vicinity of the vaporization zone to be removed by the vapor. Us patent No.4,775,361 states that a stratum corneum temperature of 123 ℃ represents a threshold for flash evaporation of this type to occur. When the pulses of thermal energy are applied sequentially, the stratum corneum is further removed before micro-pores are formed from the stratum corneum to the layer below the epidermis, the stratum lucidum. By limiting the duration of the heat pulse below the thermal time constant of the epidermis and allowing sufficient time for the thermal energy entering the epidermis to dissipate, the temperature rise of the active layer in the epidermis is minimal. This allows the entire microperforation process to be performed without any sensation and damage to the underlying and surrounding tissue of the subject.

The present invention includes a method of painlessly creating micropores in the stratum corneum of a human skin of about 1-1000 microns in width. The key to the successful implementation of this method is the production of a suitable thermal energy source, i.e., a thermal probe, in contact with the stratum corneum. The primary technique required in the manufacture of a suitable thermal probe is to design the device so that it maintains the desired contact with the skin and can be thermally tuned at a sufficiently high frequency.

Suitable thermal probes can be made by topically applying to the stratum corneum a suitable light-absorbing compound, such as a dye or colorant, selected to absorb light of the wavelength emitted by the selected light source. In this case, the selected light source may be a laser diode that emits light at a wavelength that is not generally absorbed by skin tissue. By focusing the light source on a small spot on the local surface of the dye and varying the intensity of the light flux focused thereon, the target area can be temperature modulated. By first applying a suitable light-absorbing compound, such as a dye or colorant or the like, to the stratum corneum locally, the compound being selected to absorb light of the wavelength emitted by the laser source, the energy of the laser source, which emits at a longer wavelength than the excimer laser, can be used. The same concept applies to any wavelength and the skilled person will do only to select a suitable dye or colorant and wavelength of light. The skilled person can find a suitable dye and the maximum absorption wavelength of the dye by simply looking at any reference manual. Such references are The Sigma-Aldrich handbook of Stains, Dyes and Indicators of Green, Aldrich Chemical Company, Inc. Milwaukee, Wisconsin (1991). For example, copper phthalocyanine (dye blue 15; CPC) has an absorption peak near 800 nm; the absorption peak of copper phthalocyanine tetrasulfonic acid (acid blue 249) is near 610 nm; the absorption peak of the indocyanine green is near 775 nm; the peak of the absorbance of the leuco cyanine was around 703 nm. CPC is particularly suitable for this embodiment for the following reasons: it is a very stable and inert compound that has been allowed by the U.S. food and drug administration to be used as a dye in implantable sutures; the material has very strong absorption at the wavelength of 750-950nm, is suitable for various low-cost solid emitters such as laser diodes and LEDs, and in addition, the light bandwidth area of the material cannot be directly and obviously absorbed by skin tissues; the vaporization temperature of CPC is very high (greater than 550 ℃ in vacuum), transferring directly from the solid phase to the gas phase without passing through the liquid phase; the thermal diffusion constant of the CPC is small, so that the optical energy focused thereon selectively heats only the region just at the focal point and rarely diffuses from the "hot spot" to the surrounding CPC, thereby facilitating spatial localization of the contact thermal probe.

The purpose of this disclosure is not to list a detailed list of suitable dyes or colorants, as one skilled in the art can readily determine from readily available data.

The same is true for any particular pulsed light source that is desired. For example, the method mayA mechanically turned on/off and focused incandescent lamp is used as a pulse light source. Various catalogs and sales datashow numerous lasers that can operate in the near ultraviolet, visible, and near infrared regions. A representative laser is a Hamamatsu photonic system of PLP-02 type having an output power of 2X 10 at a wavelength of 415nm^-8J; a PLP-05 type Hamamatsu photonic system having an output power of 15J at a wavelength of 685 nm; SDL-3250 series pulse laser of SDL company, with output power of 2 x 10 at wavelength around 800-810nm⁶J; model SDL-8630 from SDL, whose output power is 500mW at a wavelength around 670 nm; AR-081-15000 type single-phase laser with output power of 15,000mW at wavelength 790-830 nm; model Toshiba American electronic TOLD9150, with an output power of 30mW at a wavelength of 690 nm; diolite 800-50 from LiCONIX, which has an output of 50mW at a wavelength of 780 nm.

In the present invention, the pulsed laser light source can emit radiation over a wide wavelength range of about 100nm to 12,000 nm. The excimer laser has an emission range between about 100 and 400 nm. Commercial excimer lasers currently on the market range in wavelength between about 193 to 350 nm. The emission range of the laser diode is preferably between about 380 and 1550 nm. The frequency doubled laser diode has an emission range between about 190 to 775 nm. Using a laser diode pumped optical parametric oscillator, a longer wavelength region between about 1300 to 3000nm can be utilized. It is expected that these areas will further expand in the future with the intensive development of laser technology research.

The transmitted or absorbed energy need not necessarily be derived from a laser, as any light source can be used, whether from 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 light source. Thus, the particular instrument used to transmit the electromagnetic radiation is less important than the wavelength and energy associated therewith. Any suitable instrument that can transmit the required energy at a suitable wavelength (i.e., between about 100-12,000 nm) is considered to be within the scope of the present invention. The essential feature is that the energy must be absorbed by the light absorbing compound to heat it locally and then conduct sufficient heat to the ablated tissue within the allowed time frame.

In one illustrative embodiment, the thermal probe itself is formed from the following thin layers: it is preferably about 5-1000 microns thick, and is composed of a solid non-bioactive compound, and is applied to a selected area of the skin of the subject that is sufficiently large to cover the area where the micropores are to be made. The selection requirements of the specific formula of the compound are as follows: it has a strong absorption in the spectral range of the light source chosen to provide energy to the light absorbing compound. The probe may be, for example, a small piece of a solid compound, a film treated with a high melting point absorbing compound, or the light absorbing compound is applied directly to the skin as a precipitate or as a suspension in a carrier. Regardless of the structure of the photoabsorption thermal probe, its lateral thermal diffusivity must be sufficiently low to keep any local heating within a defined range, the primary means of thermal loss will be direct conduction into the stratum corneum through the contact point between the skin and the probe.

The required temperature adjustment of the probe can be achieved by focusing the light source on the light absorbing compound and adjusting the intensity of the light source. If the energy absorbed in the irradiated area is sufficiently high, it will cause the light-absorbing compound to heat up rapidly. The energy transmitted, as well as the heating rate and peak temperature of the light absorbing compound at the focal point, can be readily adjusted by varying the pulse width andpeak power of the light source. In this embodiment, only that small portion of the focused, light-absorbing compound that is heated by the incident light energy constitutes the thermal probe, with the addition of another light-absorbing compound that has been applied over a larger area than the actual perforation site. By using a solid phase light absorbing compound with a higher melting point, such as copper phthalocyanine (which remains in the solid phase at temperatures up to 550℃) or the like, the temperature of the thermal probe can be rapidly raised to several hundred degrees Celsius and still remain in contact with the skin, allowing this thermal energy to be conducted into the stratum corneum. Furthermore, the present embodiment includes selecting a light source that: in its emission spectrum, energy is generally rarely absorbed in skin tissue.

Once the target area has the light absorbing compound applied locally thereon, a thermal probe can be formed when the light source is activated and the focused waist of the light beam coincides with the surface of the treated area. The energy density of the focused light waist and the amount of absorption occurring in the light-absorbing compound are set to be sufficient to raise the temperature of the light-absorbing compound in a small spot region defined by the focal point of the light source to 123 ℃ or more in a few milliseconds. When the temperature of the thermal probe rises, energy conducted into the stratum corneum is transferred to these tissues, causing the local temperature of the stratum corneum to rise. When sufficient energy is transferred to the small area of the stratum corneum to cause the local temperature to rise above the boiling point of the water contained in these tissues, the water flashes off, ablating the stratum corneum at that point.

By turning the light source on or off, the temperature of the thermal probe can be rapidly adjusted and selective ablation of thesetissues can be achieved, allowing the fabrication of very precisely sized holes that selectively penetrate only the uppermost 10-30 microns of the skin.

Another feature of this embodiment is that by selecting a light source whose energy is generally less absorbed by the skin or underlying tissue and by designing the focusing and transmission optics to have an aperture of sufficiently high value, the small amount of transmitted light that is not absorbed by the thermal probe itself diverges as it penetrates deep within the body. Since there is little absorption at the transmitted wavelength, substantially no energy is transmitted directly from the light source to the skin. This three-dimensional dilution of the coupling energy in the tissue is due to the beam divergence and the low level of absorption in the untreated tissue, resulting in a completely benign interaction between the beam and the tissue without any damage.

In a preferred embodiment of the present invention, a laser diode is used as a light source emitting light having a wavelength of 800 ± 30 nm. The thermal probe can be formed by topically applying a treated scotch tape having a 0.5cm spot size on the adhesive side formed by the deposition of Copper Phthalocyanine (CPC) fine powder. CPCs have a very large absorption coefficient in the 800nm spectral range, typically absorbing more than 95% of the radiant energy emitted by a laser diode.

Figure 1 shows a system 10 for transmitting light emitted by such laser diodes to selected regions of the skin of a subject and for monitoring the progress of the perforation process. The system includes a controller 18 that controls the intensity, duration and spacing of the light pulses and a laser diode 14 coupled thereto. The laser diode emits a light beam 22 directed to a converging lens 26, which converging lens 26 focuses the light beam on a mirror 30. The beam is then reflected bythe mirror 30 onto the objective lens 34, and the objective lens 34 focuses the beam on a preselected point 38. The preselected point coincides with the plane of the xyz stage and its attachment hole 46 so that a selected region of the subject's skin can be illuminated. The xyz stage is coupled to a controller so that the position of the xyz stage can be controlled. The system also includes a monitoring system including a CCD camera coupled to a monitor 54. The CCD camera is confocal calibrated with the objective lens so that the progress of the perforation process can be monitored visually on a monitor.

In another illustrative embodiment of the invention, a system of photodiodes and concentrators that have been aligned confocally with an ablative light source is provided. Fig. 2 shows a sensor system 60 for this embodiment. The system includes a light source 64 that emits a light beam 68, the light beam 68 being focused through a light delivery system 72 onto a preselected point 76 (e.g., a skin surface 80 of a subject). A part of the light in contact with the skin is reflected and the other light is scattered from the illuminated area. A portion of this reflected and scattered light passes through filter 84 and is then focused by a condenser system 88 onto a photodiode 92. A controller 96 is coupled to the laser diode and the photodiode to control the output of the laser diode and to detect light reaching the photodiode, respectively. Only a selected portion of the spectrum scattered from the skin passes through the filter. By analyzing the deviation of the light reflected and scattered from the target area, the system can detect whether the stratum corneum has ruptured, and then use this feedback to control the light source, removing the light pulse when the microperforation of the stratum corneum is completed. By using this type of active closed loop feedback system, a self-regulating, versatile device is obtained that creates uniformly sized micropores in the stratum corneum, regardless of subject variation, and with minimal power requirements.

In another illustrative embodiment, a cooling device is incorporated at the system-to-skin interface. Fig. 3A is a schematic view thereof. In this system 100, a light source 104 (coupled to a controller 106) emits a light beam 108 that passes through a light delivery system 112 and is focused. The light beam is focused by the light delivery system onto a preselected point 116, such as a selected region 120 of the subject's skin. A cooling device 124, such as a Peltier cooler or other cooler, contacts the skin, cooling the skin surface. In a preferred embodiment of the cooling device 124 (fig. 3B), there is a central aperture 128 through which the focused light beam contacts the skin. Referring again to FIG. 3A, the heat sink 132 is preferably also in contact with the cooling device. By providing a cooling device with a small hole in the centre coinciding with the focal point of the light, the skin device can be pre-cooled to 5-10 ℃ over the whole range to be perforated. This pre-cooling allows a greater safety margin for system operation because its potential for user perception and the possibility of any collateral damage to the epidermis directly beneath the puncture site is significantly reduced compared to non-cooled embodiments. Moreover, pre-cooling minimizes vaporization of interstitial fluid and may also provide beneficial physical properties, such as reduced surface tension of such interstitial fluid, for monitoring purposes. Also, cooling devices are known that can cause a localized increase in blood flow to these cooled tissues, thereby promoting diffusion of analytes from the blood to interstitial fluid.

The method is also applicable to other microsurgical techniques in which a light absorbing compound/thermal probe is applied to the area to be ablated, and then the probe temperature at the selected target site is selectively adjusted using a light source to effect tissue through the resulting vaporization-ablation process.

It is a further feature of the present invention to assist in sealing the microwells after they have been deactivated by a light source. Specifically, in the case of monitoring internal analytes, a microwell is made and several interstitial fluids are extracted through the well. After a sufficient amount of interstitial fluid has been collected, the light source is re-activated under reduced power conditions to facilitate rapid coagulation of interstitial fluid in the microwells. By forcibly causing the liquid to coagulate in the hole, the hole in the body is closed, thereby reducing the risk of infection. Furthermore, the use of a light source both in forming and closing the micro-holes is an automatic sterilization method without any means to actually penetrate into the body. Also, the heat stress induced by the light energy kills any microorganisms that may be present at the ablation site.

The concept of optical sterilization can be extended to add an additional step in the process, namely to apply the light source in an unfocused manner, covering the target area, the illuminated area of which is 100 microns or more larger than the actual pore size of the micropores to be created. By selecting the area over which the unfocused beam will be applied, the light flux density can be correspondingly reduced to an appropriate level below the ablation threshold but sufficient to effectively sterilize the skin surface. The optical microperforation process begins after exposing a large area to a germicidal beam in a continuous step or in a series of pulses for a sufficient time, followed by configuring the system in a sharply focused ablation mode.

Another illustrative embodiment of the present invention is to form the required thermal probe from a metallic solid, such as a small diameter wire. As with the previous embodiments, the contact surface of the heater probe must be capable of being adjusted from ambient skin temperature (33 ℃) to a temperature above 123 ℃ for a desired period of time, with the time allowed at high temperatures (on-time) preferably being between about 1 and 50 milliseconds and the time allowed at low temperatures (off-time) being at least about 10 to 50 milliseconds. In particular, if the temperature can be adjusted to above 150 ℃ for an "on" time of around 5 milliseconds and an off time of 50 milliseconds, very effective thermal ablation can be produced with little or no sensation to the subject.

Several methods of adjusting the temperature of the contact area of the wire probe can be successfully implemented. For example, a short length of wire may be heated to the desired high temperature with an external heating element (e.g., an ohmic heating element used in a soldering iron tip). Figure 4 is an ohmic heating device 140 with a mechanical actuator. The ohmic heating device includes an ohmic heat source 144 coupled to a wire heater probe 148. The ohmic heat source is also coupled to a mechanical adjustment device 156 (e.g., a solenoid) through the insulating fixture 152. In this configuration, a steady state condition may be reached in which the tip of the wire probe will stabilize at some equilibrium temperature determined by the physical parameters of the structure, i.e., the temperature of the ohmic heat source, the wire length and diameter, the temperature of the air surrounding the wire, and the constituent material of the wire. Once the desired temperature is reached, the temperature regulation of the selected area of the subject's skin 160 is performed directly by the mechanical regulation device in the following way: the wire thermal tips were alternately brought into contact with the skin for (preferably) 5 ms on time and then removed and placed in air for (preferably) 50ms off time.

Another illustrative embodiment (fig. 5) is an apparatus 170 that includes a power supply 174 coupled to a controller 178. The power source is associated with a current loop 182 that includes a wire 186, the wire 186 being formed in a configuration having a high resistance point. Preferably, the wires are secured to the fixture 190 with an insulator 194 separating the different portions of the current loop. The required temperature adjustment can then be performed by simply adjusting the current through the wire. If the thermal mass of the wire element is dimensioned appropriately and the heat dissipation provided by the electrodes (via the electrode connections to the power supply) is sufficient, the warm-up time and the cool-down time of the wire element can be controlled within a few milliseconds. Contact of the wire with selected areas of the skin 198 heats the stratum corneum to achieve the selected ablation.

In fig. 6, yet another illustrative embodiment for perforating the stratum corneum with hot wires is shown. In this system 200, the wire 204 may be placed within an adjustable alternating magnetic field formed by the excitation coil 208. Alternating current is applied to the field coil by a controller 212 associated with the field coil to induce eddy currents of sufficient magnitude in the wire heat probe that would generate heat directly through internal ohmic losses. This is essentially a miniaturisation of induction heating systems commonly used for heat treatment of tool heads or degassing electrodes in vacuum tubes or flash tubes. The advantage of induction heating is that the energy delivered to the wire heater probe can be closely controlled and easily adjusted by electronic control of the excitation coil. If the thermal mass of the wire probe itself and the thermal mass of the stratum corneum in contact with the probe tip are known, the temperature of the point 216 of contact with the skin 220 can be very precisely controlled by control of the transmittedinductive energy. Since the skin tissue is substantially non-magnetic at the lower frequencies at which induction heating can take place, the alternating electromagnetic field will have no effect on the skin tissue if a suitably selected frequency is used in the excitation coil.

If mechanically controlled contact regulation is used, another feature of the invention can be achieved by incorporating a simple closed loop control system in which the impedance between the probe tip and the skin of the subject is monitored. In this manner, the probe may be brought into contact with the skin of the subject, contact is shown to have occurred by a stepwise decrease in resistance, the probe is then held on the skin for the desired "on time", and the probe may then be removed. Several types of linear actuators are suitable for this form of closed loop control, such as voice coil mechanisms, simple solenoids, rotary systems with cams or double-armed cranks, and the like. The advantage is that as thermal ablation progresses, the thermal probe can correspondingly penetrate into the skin, always ensuring good contact with the skin, thereby achieving effective transfer of the required thermal energy. Furthermore, the change in the conductivity of the stratum corneum and the epidermis provides an excellent means of confirming whether the perforation process is complete by a closed loop, i.e. the perforation can be terminated when the resistance indicates that the epidermis has been reached.

Fig. 7 is an illustrative embodiment of such a closed loop impedance monitor. In this system 230, there is an ohmic heat source 234 associated with a wire heat probe 238. The heat source is secured to a mechanical regulator 246 by an insulated fastener 242. A controller 250 is associated with the wire and the skin 254, wherein the controller detects changes in impedance of a selected area 258 of the skin and terminates the puncturing process when a predetermined level is reached.

Micro-lancets are the same concept as a hydraulic puncturing device, which is tuned to only penetrate the stratum corneum to administer a permeant (e.g., such as a drug) through formed pores or to draw an analyte through a pore for analysis. Such devices are considered "minimally invasive" as compared to non-invasive devices and/or technologies. It is well known to use a micro-lancet to pierce the underlying stratum corneum to draw blood. These devices are commercially available from manufacturers (e.g., Becton-Dickinson and Lifescan) and can be used in the present invention by controlling the depth of penetration. As an example of a micro-lancet for collecting body fluids, see PCT application WO95/10223 to Erickson et al (published: 20/4.1995). This application shows a device for lancing the dermis of the skin to collect body fluid for examination of blood glucose concentration, etc., which does not penetrate the subcutaneous tissue.

Perforation of the stratum corneum can also be achieved by using ultrasonic devices. Ultrasonic perforation is a variation of the optical device described above, except that instead of using a light source, a very dense focused beam of ultrasonic energy is delivered to the area of the stratum corneum to be ablated. The required energy is of the same level, i.e. a threshold of 70mJ/cm is still necessary²Energy of/50 ms is absorbed. The same pulsed focused ultrasound transducers as described in U.S. patent nos. 08/152,442 and 08/152,174, which are parent applications, may be used to deliver the fluence required for ablation as used in delivering ultrasound energy whose intensity, phase, or frequency, or a combination of these parameters, is adjusted to percutaneously extract an analyte sample or to transdermally administer a drug. This has the advantage that the same transducer used to push the drug through the stratum corneum, or to draw body fluid to the skin surface for analysis, can be used to make the micropores first.

In addition, electroporation can be performed or a short burst of current with sufficient energy can be delivered to the stratum corneum to form micropores. Electroporation can be used to make holes in biological membranes and is therefore known in the art and electroporators are commercially available. Accordingly, the skilled artisan can select the instruments and conditions to be used without undue experimentation in light of the guidelines set forth herein.

The micropores created in the stratum corneum by the method of the present invention allow transdermal delivery of large molecular weight therapeutic compounds at high flow rates. In addition, these non-invasive microorifices into the body may be exposed to various analytes in the body and thus may be used to analytically determine their concentration in the body.

Example 1

In this example, skin samples were prepared as follows. Epidermal membranes were isolated from whole human cadaver skin by heat separation by klingeman and Christopher (88 arch. dermaltol.702 (1963)). The heat separation method includes exposing the whole skin to a temperature of 60 c for 60 seconds, and then gently peeling the stratum corneum and a part of epidermis (epidermal membrane) from the dermis.

Example 2

Samples of the thermally separated horny layer obtained in the manner of example 1 were cut to 1cm²And (6) slicing. Then use theseThe small sample was placed on a glass cover and allowed to stick, and a disc coated on the back with a pressure sensitive adhesive was applied to the skin sample, with a 6mm hole in the center of the disc, and tested against the skin. The samples were ready for experimental testing. In some cases, skin samples arehydrated by soaking them in neutral buffered phosphate solution or purified water for several hours.

As a test on these untreated skin samples, the output of several different infrared laser diodes emitting at wavelengths of about 810, 905, 1480 and 1550nm were applied to these samples. The optical delivery device was designed with a final objective lens with a numerical aperture of 0.4, producing a 25 micron wide optical waist, and the total power delivered to the focus was measured between 50 and 200 milliwatts for 905nm and 1550nm laser diodes operable in a Continuous Wave (CW) mode. The 905nm and 1550nm laser diodes are designed to produce high peak power pulses of about 10-200 nanoseconds in length at a repetition rate of up to 5000 Hz. The peak power level of the pulsed laser was measured at 45 watts at 905nm and 3.5 watts at 1550 nm.

Under these operating conditions, no laser was significantly effective on the skin samples. The target area was irradiated continuously for 60 seconds and then examined under a microscope, and no visible effect was observed. In addition, the conduction from one side of the membrane to the other was measured before and after irradiation with laser light in a modified Franz cell (Franz cell) which was typically used to test transdermal delivery systems based on chemical permeation enhancers, without any change being observed. From these tests on skin samples obtained from 4 different donors, the following conclusions can be drawn: at these wavelengths, the light energy coupled into the skin tissue is so small that no effect is detected.

Example 3

To evaluate the potential perception of live subjects when irradiated with light energy under the conditions of example 2, the output of each laser source was applied to the fingertips, forearms and backs of hands of 6 volunteers. When the lasers are 810, 905 and 1550nm, the subject does not feel when the laser is on or off. In the case of a 1480nm laser, the subject has some perception during irradiation with a 1480nm laser at 70 milliwatts continuous wave, after a short time, a small blister forms under the skin due to absorption of the 1480nm radiation by one of the water absorption bands. It is clear that the energy absorbed is sufficient to induce blister formation, but not sufficient to ablate the stratum corneum. In addition, absorption of 1480nm light occurs primarily in the deeper, fully hydrated (85-90% moisture content) tissues of the epidermis and dermis, but not in the drier (10-15% moisture content) tissues of the stratum corneum.

Example 4

After confirming the lack of effect on the skin in its natural state (example 3), the efficacy of a series of compounds to absorb light energy and then transfer this absorbed energy into the target tissues of the stratum corneum by conduction was evaluated. The compounds tested included ink; black, blue and red markers that "SHARPIE" cards do not rub off; methylene blue; magenta; epolite #67 (a light absorbing compound for molding in polycarbonate lenses for use as laser goggles); iodine tincture; iodine-polypyrrolidone complex ("BETADINE"); copper phthalocyanine; and printing ink.

Using the 2 CW laser diodes described in example 2, positive ablation results were observed in all the thermally separated stratum corneum in vitro specimens prepared according to example 1, but some of them outperformed others. In particular, Copper Phthalocyanine (CPC) and epolite #67 are listed as being the most effective. One possible explanation for the superior performance of CPC is that it has a high boiling point above 500 ℃ and that it is still in the solid phase before this temperature is reached.

Example 5

Since copper phthalocyanine has been approved by the U.S. food anddrug administration for use in implantable sutures and is listed in Merck index as a molecule that is reasonably benign and stable in terms of human biocompatibility, the next step taken is to combine CPC with topical application of a focused light source on the skin of healthy volunteers. A suspension of fine CPC powder in isopropanol was prepared. The application method used was shaking the solution and then applying a small drop at the target site. As the alcohol vaporizes, a fine, uniform coating of solid phase CPC remains on the skin surface.

The device shown in figure 1 was then applied to the skin at the site where the CPC had been topically applied, with the selected area of the subject's skin against the reference plate. The reference plate consists of a thin glass window of approximately 3cm x 3cm with a 4mm sized hole in the center. The area covered by the CPC is then placed within the central bore. The confocal video microscope (fig. 1) was then clearly focused on the skin surface. The skin is placed at a position where the sharpest focus is obtained in the video system, even if the focus of the laser system coincides with the skin surface. The operator then initiates a laser pulse while viewing the effect on the target site with a video microscope. The amount of penetration is visually assessed by the operator as the depth of the microhole increases by measuring the amount of defocus of the laser spot in the microhole, and the operator can dynamically correct as the ablated surface penetrates deeper into the skin tissue by moving the position of the camera/laser source along the "z" axis. The appearance of the bottoms of the pores changes dramatically, becoming wetter and glossier, when the stratum corneum has been removed to the epidermis. Upon observing this change, the operator can turn off the laser. In many cases, interstitial fluid is significantly lost as a result of the removal of the barrier function of the stratum corneum over the small area, depending onthe hydration state of the test substance and other physiological conditions. This visual record of interstitial fluid accessibility to the puncture site is recorded with a video system.

Example 6

The method of example 5 was repeated except that the CPC was applied to a transparent tape and the tape was then adhered to the skin of the subject at the selected site. The results were substantially the same as in example 5.

Example 7

Histological experiments were performed on cadaver skin in accordance with methods well known in the art to determine ablation threshold parameters and associated lesion information for a given dye mixture. The top surface of the skin sample was treated with a solution of Copper Phthalocyanine (CPC) in alcohol. After the alcohol has evaporated, a topical layer of solid phase CPC is distributed on the skin surface with an average thickness of 10-20 microns. Fig. 8A is a cross-section of the full thickness skin prior to laser application, where 270 is the CPC layer, 274 is the stratum corneum layer, and 278 is the underlying epidermis layer. FIG. 8B is a graph of 4000J/cm using a single light pulse at 810nm²And a pulse period of 20 milliseconds is applied to the sample after it is in a circle of 80 microns in diameter. It is noteworthy that even in the center of the ablation crater 282, a significant amount of CPC is present at the surface of the stratum corneum. It should also be noted that laboratory measurements show that only 10% of the light energy incident on the CPC is actually absorbed and the other 90% is reflected or backscattered. Thus, the effective energy flux transmitted to the dye layer, which may cause the required heating, is only about 400J/cm². Fig. 8C is a sample after application of 5 light pulses at 810nm, in which the stratum corneum barrier was removed without any damage tothe underlying tissue. These results are a good indication of the "ideal" optically tuned heat ablation performance. Fig. 8D is the sample after 50 pulses have been applied. Damaged tissue 286 appears in the epidermis layer due to carbonization of non-ablated tissue and thermal denaturation of underlying tissue. Figures 8A-8C show the separation between the stratum corneum and the underlying epidermis layer due to artifacts (artifacts) that occur as a result of dehydration, freezing, and preparation for imaging.

Example 8

To clarify the details of the thermal ablation mechanism, a mathematical model of the skin tissue was developed, in which various embodiments of the thermal ablation method were tried. The model calculates the temperature distribution in a multi-layer semi-infinite medium, locally inputting a given heat flux at the surface, removing heat from the surface at a distance, i.e. carrying out convection between the two. An implicit Alternating Direction (ADI) method is used to solve the axisymmetric time-varying diffusion equation in cylindrical coordinates. (Note: using the constant temperature boundary condition at the lower boundary as z → infinity; making the radial heat flux zero at the maximum radial boundary as r → infinity). Each layer is parallel to the surface of the skin and is respectively (1) dye; (2) the stratum corneum; (3) the lower epidermis; (4) the dermis. For each layer, the depth and thermal properties, density (rho), specific heat (c) and conductivity (k) in the semi-infinite medium must all be determined.

First, the heat transfer coefficient h of the skin is calculated from the "stable", "one-dimensional (1-D)" temperature distribution determined by the ambient air temperature, the skin surface temperature and the dermis temperature. No dye is assumed to be present on the skin surface and "h" is given. The program may allow the technician to use this "h" on the surface of the dye layer or to input another desired "h" for the dye surface. Then, the "stable" temperature profile is calculated for all layers (including the dye layer) using the "h" determined for the dye surface. This temperature profile is the initial condition for time-dependent heating problems. This constitutes the "m-file" initial m. The program then solves for the time-dependent temperature distribution by passage of time, calculation and display of the temperature field of the individual steps.

The embodiments of the methods described herein, for which empirical data has been collected, have been modeled with respect to at least one set of operating parameters, which show that ablation of the stratum corneum can be achieved in an accurate and controlled manner. The analog output is illustrated in two different formats: (1) a cross-sectional view of the skin is shown of different tissue layers, above which is 3 isotherms, which give 3 critical temperature thresholds; (2)2 different temperature versus time curves, one representing a point in the center of the stratum corneum directly below the target site, and the other representing a point on the boundary between the viable cell layer of the epidermis and the bottom surface of the stratum corneum. These curves show how the temperature of each point changes over time when a heat pulse is applied, as if the technician had implanted a micro thermocouple in the tissue. Furthermore, using this model, it is possible to investigate the limits of the parameters within which the method can be used to determine the outer limits of two important aspects of the performance of the method. First, a case is described where a boundary line can be determined within which the method can be used without causing pain or undesirable tissue damage.

As described in several different embodiments of the invention, there is a point at which the effect on the skin tissue of a subject becomes suboptimal for any given heat source, either because the subject feels pain, or because the cells active in the underlying epidermis and/or dermis are subjected to temperature, which if maintained for a sufficiently long duration, will cause damage to these tissues. Thus, experimental simulations were performed with optically heated topical Copper Phthalocyanine (CPC) dye embodiments as a baseline method to establish how the thermal temperature constants of different skin tissue layers substantially define a window within which the method can be used without pain or damage to adjacent tissue layers.

Fig. 9 and 10 are schematic cross-sectional views of a skin layer and a topical dye layer. In each figure, 3 clear isotherms are shown: (1)123 ℃, which is the temperature at which water in the tissue vaporizes resulting in tissue ablation; (2) at 70 ℃, if the temperature lasts for several seconds, the active cells will be damaged; (3)45 ℃, the average temperature at which the subject will feel pain. The pain threshold is described in several basic physiological textbooks, but experience has shown that the threshold is somewhat subjective. Indeed, in repeated experiments on the same subject, different holes formed in the range of only a few millimeters may produce sensations of significantly different degrees, possibly due to the proximity of the puncture site to the nerve endings.

The linearity on the graph shows the different layers of dye and skin, measured in microns, separated by a flat boundary. However, the boundaries of actual skin tissue are far more complex, and in an average sense, this model gives a good approximation of the thermal gradients present in actual tissue, for the lines involved. In this and all subsequent simulations, the linearity of the CPC dye layer and the skin layers were as follows: dye, 10 microns; stratum corneum, 30 microns; lower epidermis, 70 microns; dermis, 100 microns.

Additional conditions for the model used for this particular simulation are shown in the following table:

TABLE 1

Initial conditions of differential thermal model
		Ambient air temperature	Ta＝20℃
Skin surface temperature	Ts＝30℃
		Temperature of dermis	Td＝37℃
Vaporization temperature of dye	Tvap＝550℃
		Vaporization temperature of stratum corneum	Tc1＝123℃
Temperature of tissue damage	Tc2＝70℃
		Temperature of "pain	Tc3＝45℃
Radius of the irradiated area	R			hot30 microns
		Energy density of application	FLUX＝400J/cm2

TABLE 2

Parameter of	Dye material	Stratum corneum	Watch skin	Leather product
					Thermal conductivity	0.00046	0.00123	0.00421	0.00421
Density of	0.67	1.28	1.09	1.09
					Specific heat	0.8	1.88	3.35	3.35

When applying these simulations, the following cautious assumptions are attached:

1. this event is not included in the model when a portion of the stratum corneum may have a temperature that has exceeded the ablation threshold for thermal vaporization of water, and the subsequent loss of thermal energy in the tissue due to this vaporization is also not a factor in the above simulation. This will result in a slight increase in the temperature shown by the underlying tissue from that time on in the simulation.

2. Likewise, when a portion of the Copper Phthalocyanine (CPC) dye layer has reached its vaporization point of 550 ℃, this event is also not included in the model, but only the temperature is rigidly limited to that level. This will also result in a slight subsequent temperature rise of the underlying tissue as the simulation progresses.

Despite the use of these simplified conditions in the model, the correlation between expected performance and empirically observed performance based on clinical studies and histological studies on donor tissue specimens was significant. The key data to note in fig. 9 and 10 are the length of time the heat pulse is applied and the location of the 3 different threshold temperatures shown by the isotherm.

In fig. 9, the pulse length was 21 milliseconds and the 70 ℃ isotherm just crossed the boundary between the stratum corneum and the viable cells in the epidermis. Under these conditions, in vitro experiments on donor skin specimens showed that 50 pulses of thermal energy, 50 milliseconds apart, produced detectable damage to this top layer of living cells (see FIG. 1)8D) In that respect However, in vitro experiments also showed that 5 pulses of thermal energy do not cause significant damage to these tissues under the same these operating parameters. It is believed that even if the nominal damage threshold has been exceeded (at least in the sense of a transient), this temperature must be cumulatively maintained for a certain event to actually cause damage to the cells. However, the basic information obtained by simulation is that if the "on time" of the heat pulse is kept below 20 milliseconds, the flux density is 400J/cm²Even if the ablation threshold isotherm has penetrated deep into the stratum corneum, viable cells in the underlying epidermis are not damaged. In other words, by using a low flux density heat source whose "on time" is adjusted to be suitably short, ablation of the stratum corneum can be achieved without damaging adjacent cells in the epidermis underneath it (see fig. 8C). This may be primarily due to the significantly different thermal diffusivity of the two tissue layers. I.e., the stratum corneum (containing only about 10-20%Moisture) has a much lower thermal conductivity constant of 0.00123J/(S cm K) than the thermal conductivity constant 0.00421J/(S cm K) of the skin. This allows the temperature in the stratum corneum to be gradually raised while remaining within a tight spatial range until ablation occurs.

In fig. 10, the same simulation is further performed starting from the damage threshold critical point operation shown in fig. 9. By heating the dye in a circle with a diameter of 60 microns and a flux density of 400J/cm²The heat pulse was applied for 58 milliseconds and the pain sensation isotherm at 45 ℃ just entered the weakened layer of skin contained by the dermis. Furthermore, the lesion threshold isotherm is significantly deeper into the epidermal layer than it is at the location shown in fig. 9. Linking this simulation to the numerous clinical studies conducted in this way, the accuracy of the model is extremely well documented, as the model shows almost the duration of the "on time" that the thermal probe can be applied to the skin before the subject feels it. In clinical trials, a controllable pulse generator was used to set the "on time" and "off time" of a series of light pulses applied to a localized layer of Copper Phthalocyanine (CPC) on the skin. While maintaining a constant "off-time" of 80 milliseconds, the "on-time" is gradually increased until the subject claims a slight "pain" sensation. Without exception, all subjects in these studies were told "painful" for the first time between "on-times" of 45-60 milliseconds, very close to what the model expected. In addition, the previously described "pain" sensation was noted in these clinical studies as a function of location. Thus, to say "pain" is the point at which a definite sensation can be perceived for the first time. At one site, pain may be declared, while at an adjacent site, the same subject may be declared as merely "detectable".

One element of this clinical study is the insight that, even at the samesite, a non-uniform burst of heat pulses may act together with psychophysiological neural perception of the subject, causing a substantial decrease in sensation. For example, a series of shorter length pulses may be used to saturate nerve cells in a region, temporarily depleting the neural mediators in the synapse, thereby limiting its ability to transmit "pain" information. As a result, this makes longer pulses after these short pulses less noticeable than if they were applied at the beginning of the sequence. Thus, a series of experiments were performed with some arbitrarily manufactured pulse bursts, and the results were consistent with this hypothesis. This similar situation of saturation can be found in the sensation when: when a person just starts to step into a very hot bath, he is initially painful, but as he adapts to the sensation of heat, it quickly becomes tolerable.

Example 9

It is an object of the present invention to achieve painless microperforation of the stratum corneum without significant damage to surrounding viable tissue. As described in the simulations illustrated in example 8 and fig. 9-10, there appears to be a tight boundary within which microperforations can be performed in such a painless, atraumatic manner for a given flux density of thermal energy within the ablation target. Both in vivo and in vitro studies have shown that this allows experimental determination of some of the operating parameters that give good results. The combination of the simulations described below shows the effect of this method when using these specific parameters.

In the first case, a burst of 10 pulses is applied to the skin covered by the CPC, the "on-time" and the "off-time" each being 10 milliseconds. Fig. 11 shows the final temperature distribution in the skin tissue just after the end of the pulse burst. It can be seen that the isotherms representing the three critical temperature thresholds show that stratum corneum ablation has been achieved without any sensation of the nerves of the dermis, and that the damage threshold enters very few active cells in the underlying epidermis. As previously mentioned, in order to actually cause permanent damage to the cells, not only must the epidermal cells be heated to a certain temperature, but they must also be held at this temperature for a period of time, generally considered to be about 5 seconds. Figures 12 and 13 show the temperature (as a function of time) of the stratum corneum and the viable epidermis, respectively, for a total of 10 cycles of heating at "on time" and cooling at "off time". In connection with the in vivo studies carried out, it was noted that in 90% or more of the perforation attempts carried out with the system parameters set in conformity with the above-mentioned simulation, effective perforation of the stratum corneum was achieved without causing pain to the subject, and in the microscopic examination of the perforated site carried out several days later, no significant damage to the tissue was detected. The results of in vitro studies performed on donor skin specimens throughout the thickness are also consistent with model expectations.

Example 10

In the in vivo experimental studies performed and in these simulations, it appears that pre-cooling of the skin helps to reduce the possibility of pain or damage to surrounding tissues, optimizing the microperforation process. In practice, this can be easily achieved by: a simple cooling plate is placed against the skin prior to perforation. For example, applying a Peltier cooling plate (the plate is held at about 5 ℃ for a few seconds) in a circle of 1cm diameter around the perforated target site can significantly reduce the temperature of the tissue. Fig. 3A-B are schematic illustrations of an experimental setup used for this purpose in a laboratory. By comparing fig. 11 with fig. 4, fig. 12 with fig. 15, and fig. 13 with fig. 16 with exactly the same 10-cycle pulse used in the operation described in example 9, it can be seen that much improvement is achieved in controlling the depth of temperature penetration into skin tissue. Furthermore, the lower thermal diffusivity and specific heat of the stratum corneum is advantageous compared to the epidermis and dermis. Once cooled, the highly hydrated tissues of the epidermis and dermis require greater thermal energy input to raise their temperature, while the tissues of the stratum corneum, being drier, can heat up rapidly to the ablation threshold.

Example 11

Once the basic heat-conducting mechanism of energy delivery to skin tissue for effective, painless ablation and micro-perforation of the stratum corneum is understood, several different specific methods of achieving the desired rapid temperature adjustment of the contact points, such as the hot-wire embodiments illustrated in fig. 4-7, can be conceived.

As described herein, one basic embodiment is to use an ohmic heating element (fig. 4), such as the tip of a small cordless soldering iron, around which is wrapped a suitably sized, relatively inactive wire, with the remaining shorter wire protruding from the heater body. When current is applied with a constant current source, the heater will rise to a certain temperature and reach a steady state within a few seconds by releasing heat to the surrounding air. Likewise, the wire that is part of the thermal system will also reach a steady state, so that with this type of component, the very tip of the wire can be raised to almost any arbitrary temperature, up to about 1000 ℃. The tip can be sized to give just the desired pore size.

In the laboratory, a tungsten wire of 80 microns diameter was used, attached to a replaceable tip of a "WAHL" cordless soldering iron, the wire protruding about 2mm from the tip. The temperature of the tip at steady state was measured with a thermocouple, noting that steady state temperatures above 700 ℃ could be easily reached by changing the setting of the constant current. To achieve the desired adjustment, a low-mass, fast-response electromechanical actuator is associated with the tip so that the position of the wire can be translated linearly by more than 2mm at a rate up to 200 Hz. Then, by fixing the whole device on a precision platform, the vibrating tines can be brought into very controlled contact with the skin surface in the following way: the tines are only in contact with the skin for less than 10 milliseconds at each "on time", while an arbitrary length of "off time" can be achieved by setting the pulse generator accordingly. These in vivo studies show that perforation can indeed be achieved before the subject being perforated knows that the wire tip is in contact with the skin.

To compare the performance of this embodiment with the optically heated topical CPC dye embodiment, the following simulation was performed as in example 8. Hot wire implementations can be performed with the same analog code, essentially only by varying the initial conditions. Since contact with the wire is essentially instantaneous, heat does not build up in the CPC layer over time, there is no residual heat on the surface when the wire is physically removed from contact with the skin, and there is residual heat on the heated CPC dye layer, and further, since the wire itself defines the target area for ablation/microperforation, there should be no lateral spread of thermal energy before it is applied to the stratum corneum. Comparative performance of the "hot wire" embodiment is shown in FIGS. 17-19.

Example 12

In this example, the method of example 11 was used, except that the skin was pre-cooled as in example 10. Likewise, in the "hot wire" embodiment, pre-cooling of the target site produces similar beneficial results. The results of the pre-cooling simulation of the "hot wire" method are shown in FIGS. 20-22.

Example 13

As discussed in the background introduction herein, the Tankovich' 803 patent appears similar at first sight to the invention claimed herein. In this example, the simulation model was constructed using the operating parameters specified in the Tankovich' 803 patent, i.e., a pulse width of 1 microsecond and a power level of 40,000,000W/cm². Figures 23 and 24 show that no part of the stratum corneum reaches the threshold for water flash under these conditions, i.e. 123 ℃, and therefore, stratum corneum ablation/microperforation did not occur. In practice, a high peak power, short duration pulse of this type is applied to the topical dye layer, vaporizing only the dye at the skin surface without any effect on the skin. Thus, this example demonstrates that the conditions specified in the Tankovich' 803 patent are not effective in the invention claimed herein.

Example 14

In this example, interstitial fluid obtained after perforation of the skin as in example 6 was collected and analyzed to determine its glucose concentration. Data were obtained from 4 subjects without diabetes and 6 subjects with diabetes who were undergoing the glucose challenge test. Subjects were between the ages of 27-43 years. The aim of this study was to investigate the use of this method in the following areas: sufficient interstitial fluid (ISF) is painlessly collected from the subject to allow an ISF sample to be analyzed for glucose content, and these concentrations are then compared to the glucose concentration in the subject's whole blood.

Glucose analysis was performed on all subjects' blood and ISF using the Miles-Bayer "ELITE" system. All 10 subjects used the same assay, but those with insulin-dependentdiabetes were adjusted for glucose load and insulin administration.

The basic design of this study was to recruit an appropriate number of volunteers (some of which were diabetic and others of which were not), from which a series of pairs of ISF and whole blood were drawn every 3-5 minutes over the entire study period of 3-4 hours. Glucose analysis was performed on both blood and ISF samples to determine the statistical relationship between blood glucose levels and interstitial fluid. To study the postulated temporary lag in ISF glucose levels compared to whole blood glucose levels, subjects were induced to show significant dynamic changes in their glucose levels. This is achieved by the following method: each subject was fasted for 12 hours prior to starting the test and then allowed to glucose load after establishing their baseline glucose levels through a set of 3 fasting blood and ISF glucose levels. After establishment of the baseline level, subjects were subjected to a glucose load in the form of sweet juice according to the following guidelines:

i. for the control group of subjects, the glucose load was calculated according to the standard of 0.75 grams of glucose per pound of body weight.

For a subject with insulin-dependent diabetes mellitus, the glucose load is 50 grams of glucose. In addition, subjects with diabetes will self-inject their normal morning dose of fast acting insulin immediately after the glucose load. In the case of diabetic subjects with fasting glucose levels of 300mg/dl, they are asked to self-inject insulin first, with a glucose load after their blood glucose level has fallen back to 120 mg/dl.

Each subject enrolled was first given a complete description of the study in the "consent by express" file, requiring them to understand and sign prior to formal participation program. After acceptance, they fill in a medical historyquestionnaire. The specific clinical method implemented is as follows:

(a) subjects fasted from 9 pm to the beginning of the study and had only water. During which time coffee, juice or smoke is not allowed.

(b) The subject arrived at the testing facility 9 am the following day.

(c) The subject sits in a chair which is provided to the subject to allow the subject to relax throughout the study.

(d) Upon arrival of the subject, whole blood and ISF samples are initially withdrawn every 3-5 minutes and continued for the next 3-4 hours. The duration of the data collected depends on when the subject's blood glucose levels return to the normal range and stabilize after the glucose load. ISF samples were collected by optical perforation (ISF pumping) as described in more detail below. The volume of each ISF sample was about 5. mu.l to ensure good loading of the ELITE test strip. Each blood sample was taken with a commonly used finger lancet. The ISF and blood samples were immediately subjected to glucose analysis using an ELITE home glucose meter system from Miles-Bayer corporation. To improve the estimation of "true" blood glucose levels, two independent ELITE analyses were performed on each prick finger sample.

(e) To facilitate the ease of collecting ISF continuously from the same site on a given subject throughout the data collection phase, 25 microwells constituting a 5X 5 matrix were punctured on the upper forearm of the subject, each microwell being between 50-80 microns wide and 300 microns apart from each other. A Teflon disc 30mm in diameter and having a 6 mm-sized hole at the center was adhered to the forearm of the subject with a pressure sensitive adhesive so that the 6 mm-sized hole was positioned above the 5X 5 matrix of micropores. Due to this bonding, a simple method can be used: a small pipette is attached and a moderate vacuum (10-12 inches of mercury) is applied to the perforated area to allow the ISF to flow out of the body through the micro-holes. The top of the teflon disc is fitted with a clear glass window to allow the operator to directly view the underlying microperforated skin. When a 5 microliter drop of ISF is formed on the skin surface, it can be easily determined by visually monitoring the microperforation site through the window. This level of vacuum produces a nominal pressure gradient of 5 pounds per inch 2 (PSI). Without the micro-holes, the ISF could not be withdrawn from the subject at all with only a moderate vacuum.

(f) After the first three sample pairs were withdrawn, the subjects were given a glucose load by drinking high-sugar orange juice. For subjects who do not have diabetes, the amount of glucose administered is 0.75 grams per pound of body weight, while for subjects who have diabetes, 50 grams is administered. The diabetic subject also self-injected (periodically) a dose of (daily) fast acting insulin, suitably calculated from the 50 gram glucose level combined with the uptake of the glucose load. For the normal 1.5-2.5 hour lag between the maximal effect of insulin administration to the agent, it is expected that subjects with diabetes will have blood glucose levels that rise by up to 300mg/dl and then fall back to the normal range after insulin action. Subjects who are not diabetic are expected to exhibit standardized glucose tolerance test results, with a peak in blood glucose levels (between 150 and 220 mg/dl) typically occurring 45-90 minutes after glucose loading, and then falling back rapidly to their normal baseline levels around the next hour.

(g) After a glucose load or glucose load plus insulin injection is performed, ISF samples and spiked whole blood samples of the subject are drawn simultaneously over the next 3-4 hours at 5 minute intervals. Sampling was terminated when blood glucose levels in 3 consecutive samples indicated that the subject's glucose had stabilized.

After examining the data, several features were evident. In particular, there was a significant shift in the output shown in mg/dl glucose in the glucose meter compared to the level shown in blood for any particular lot of ELITE test strips. Increased readings are expected due to the absence of red blood cells (hematocrit) in the ISF and due to electrolyte differences between ISF and whole blood. Regardless of the underlying cause of the output shift, it was determined by comparison with a reference analysis that the true ISF glucose level is linearly related to the value obtained by the ELITE system, with a constant proportionality coefficient for the ELITE band of any particular batch. As a result, to compare ISF glucose levels with whole blood measurements, the ISF data is linearly corrected to one level as follows: ISF glucose ═ 0.606^*ISF_ELITE+19.5。

When used to measure ISF glucose levels, the scaling of the ELITE output allows a technician to examine the entire data set for error terms associated with ISF use to assess blood glucose levels. Of course, the correlation between the ISF glucose value and the blood glucose level is the same as that of the conversion type even without any linear conversion.

Based on much published literature on ISF glucose and preliminary data, it would have been expected that a 15-20 minute lag would be observed between ISF glucose levels and glucose levels in whole blood obtained from stingers. This is not the result displayed when analyzing the data. Specifically, when the data set for each subject was analyzed to determine the time shift required to obtain the greatest correlation between ISF glucose and blood glucose levels, the worst case in this group of subjects was found to have a time lag of only 13 minutes, an average time lag of only 6.2 minutes, and nearly immediate (about1 minute) time responses for several subjects.

Based on the minimum amount of hysteresis observed in this data set, the graph shown in FIG. 25 gives all 10 glucose load tests, one after the other on an extended time scale. Given the data without any time shift, showing high level tracking between ISF and blood glucose levels, the entire clinical data set was processed in exactly the same way. If the entire data set is shifted as a whole to find the best time response estimate, the correlation between ISF and blood glucose levels peaks at r 0.97 with a delay time of 2 minutes. This is only an irrelevantly significant improvement by the unbiased correlation of r 0.964. Thus, for the rest of the analyses, their ISF values are processed without adding any time offset. That is, each set of blood and ISF glucose levels is processed as a pair of simultaneously collected data.

After the unbiased erite ISF reading is scaled to reflect the proportion of glucose in the ISF, the error associated with these data can be checked. The simplest approach is to assume that the two ELITE spikes are in fact absolutely correct values for the blood glucose reading, and then only compare the scaled ISF values to these average blood glucose values. These data are specifically as follows: standard deviation of blood-ISF, 13.4 mg/dL; coefficient of variation of ISF, 9.7%; two Elite standard deviations, 8.3 mg/dL; the coefficient of variation of blood (supplied by Miles corporation), 6%.

As shown by these data, the blood-based measurements already contain error terms. In fact, performance data published by the manufacturers indicate that the nominal Coefficient of Variation (CV) of the ELITE system is between 5-7%, depending on the glucose level and the amount of red blood cells in the blood.

Another pattern of differences between ISF glucose and blood glucose is shown in the formof a scatter plot in fig. 26. In this figure, the upper and lower bounds of the 90% confidence interval are also shown for reference. It is noteworthy that all data with blood glucose levels in the range below 100mg/dL fall within these 90% confidence interval error bars with two exceptions. This is important because the lack of a tendency to hypoglycemia is not measured, which can have very serious consequences for a diabetic user. That is, it is better to predict glucose levels between 40-120mg/dL too low than too high.

Basically, if it is assumed that the basic analysis error when using the ELITE system for ISF is comparable to that when using the ELITE system for whole blood, the bias of ISF glucose to blood glucose can be expressed as follows:

ISF_{deviation of}＝〔(ISF_{Practice of})²+ (blood)_{Practice of})²〕^1/2。

Applying this equation to the values given above, the "true" value of the estimate of the ISF error term can be solved:

ISF_{practice of}＝〔(ISF_{Deviation of})²- (blood)_{Practice of})²〕^1/2。

Alternatively, the following equation is used to solve:

ISF_{practice of}＝〔(13.4)²-(8.3)²〕^1/2＝10.5mg/dL。

A histogram of the relative deviation of ISF versus blood glucose levels is shown in fig. 27.

Administration through pores in the stratum corneum

The present invention also includes a method of delivering drugs, including current transdermal delivered drugs, through micropores in the stratum corneum. In one illustrative embodiment, administration is achieved by placing the solution in a reservoir above the perforation site. In another illustrative embodiment, a pressure gradient is used to further facilitate administration. In yet another embodiment, ultrasonic energy is used to further facilitate administration, with or without the use of a pressure gradient. The ultrasonic energy may be manipulated in accordance with conventional transdermal parameters or by using acoustic streaming (as will be described shortly) to cause the delivered solution to pass through the perforated stratum corneum.

Example 15

This example shows the delivery of lidocaine (a local anesthetic) by perforating the stratum corneum. The lidocaine solution also contains a chemical penetration enhancer formulation designed to promote passive diffusion of lidocaine through the stratum corneum. An illustrative delivery device 300 is shown in fig. 28, wherein the device includes a housing 304 surrounding a reservoir 308 for containing a drug-containing solution 312. The top of the housing includes an ultrasonic transducer 316 that provides ultrasonic energy to assist in the administration of the drug-containing solution through the pores 320 in the stratum corneum 324. Pressure may be applied from port 328 in the ultrasound transducer to further assist in the administration of the drug-containing solution through the micropores in the stratum corneum. The delivery device is applied to a selected area of the skin of the subject and the device is placed over at least one, and preferably a plurality of, micropores. An adhesive layer 332 attached to the lower portion of the housing allows the device to be adhered to the skin so that the medicated solution in the reservoir can flow into the pores. As a result of the administration through the micropores, the drug is infused into the underlying epidermis 336 and dermis 340.

The efficacy of the administration using puncture and ultrasound was tested with 5 subjects. The experiment was performedon two sites on the subject's left forearm, spaced approximately 3 inches apart, symmetrically between the thumb and upper arm. The part near the thumb is called part 1, and the part farthest from the thumb is called part 2. Site 1 served as the control site, where lidocaine and the accelerator solution were applied using the same delivery device 300, but without perforation in the stratum corneum or application of ultrasonic energy. 24 holes are pierced in a circle of diameter 1cm in the region 2, these holes being spaced apart by 0.8 mm and being of the grid type. The perforation at site 2 was carried out using the method of example 6. Lidocaine was administered with low levels of ultrasound. The ultrasound was applied using a custom-made Zevex-type ultrasound transducer device set to pulse with a peak-to-peak input of 0.4 volts, producing 1000 short pulses at 10Hz with a fundamental frequency of 65.4KHz, i.e., a pulsed signal was obtained, the transducer was energized for a short 15 millisecond pulse, and then turned off for 85 milliseconds. The measured output of the amplifier to the transducer was 0.090 watts RMS.

After lidocaine application, the feel was measured by rubbing a 30 gauge wire back and forth over the test site. The experiment was performed in two sites, site 1 for 10-20 minutes; two 5 minutes after each other at site 2. The degrees of numbness in the two parts were evaluated on a scale of 10 to 0, with 10 indicating no numbness and 0 indicating complete numbness. The combined results for all 5 subjects are as follows.

The control site, site 1, had little to no tingling for 10-12 minutes (rating 7-10). At about 20 minutes, some numbness was observed at site 1 (level 3) as the solution completely penetrated the stratum corneum. Site 1 was cleaned after lidocaine administration. Site 2 was almost completely numbed in a 1cm diameter circle with micropores (scale 0 to 1). The feel of numbness decreases almost linearly outside the circle of 1cm diameter, is 1 outside the circle of 2.5 cm, and is absent outside the circle of 2.5 cm. The result of the assessment of site 2 after the second application was that the diameter of the complete numbing circle was slightly larger, about 1.2 cm, the numbing effect dropped linearly to 1 in an irregular elliptical pattern with a diameter of 2-2.5 cm of the ellipse perpendicular to the forearm and a diameter of 2-6 cm parallel to the forearm. No numbness was observed outside this area. Graphical representations of illustrative results obtained on representative subjects are shown in figures 29A-C. Fig. 29A and 29B show the results obtained at site 2 (perforated) after 5 and 10 minutes, respectively. Fig. 29C shows the results obtained at site 1 (control site, unperforated).

Ultrasound energy and accelerator for promoting percutaneous flow

The physical action of the ultrasonic energy field produced by the ultrasonic transducer can be utilized in a method by which the acoustic frequency can be modulated to increase the flow rate otherwise achieved. The energy distribution of an ultrasonic transducer can be divided into a near field and a far field as shown in fig. 1 of U.S. patent No.5,445,611, which is incorporated herein by reference. The near field (characterized by length N) is the region from the first energy minimum to the last energy maximum. The region further from the last maximum is the far field. The near (N) field mode includes a large number of closely spaced local pressure peaks and nulls. The length N of the near field is a function of the frequency, the shape and size of the transducer face, and the speed at which the ultrasonic waves travel in the medium. For a single transducer, intensity variations within its normal operating range do not affect the nature of the ultrasonic energy distribution, except in a linear fashion. However, for systems with multiple transducers (all modulatedin frequency and amplitude), the relative intensities of the individual transducers can affect the energy distribution in the acoustic medium, whether the medium is skin or another medium.

By varying the frequency of the ultrasonic energy moderately (e.g., between about 1-20%), the mode of peaks and nulls remains relatively unchanged, but the length N of the near field region varies in proportion to the frequency. Significant changes in frequency (e.g., 2 or more times) will likely produce different sets of resonant or vibrational modes in the transducer, resulting in unpredictable, significantly different near-field energy modes. Thus, for moderate changes in acoustic frequency, the complex pattern of peaks and nulls will compress or expand in an accordion-like manner. By choosing the direction of the frequency modulation, the direction of the shift of these local pressure peaks can be controlled. Selective modulation of the acoustic frequency can control the movement of these localized pressure peaks through the skin toward the interior of the body or toward the surface of the body by applying ultrasonic energy to the skin surface. Modulation from high to low frequencies drives a pressure peak into the body, while modulation from low to high frequencies pushes the pressure peak from the body to the body surface and through the skin to the outside of the body.

Typical parameters for this application, for example, an ultrasound transducer diameter of 1.27 cm, a nominal operating frequency of 10MHz, and an acoustic impedance similar to that of water, then a frequency modulation of 1MHz produces a shift of about 2.5mm of the peak and null of the near-field energy pattern near the stratum corneum. This degree of action provides a good means for accessing the area beneath the stratum corneum, and even the epidermis, dermis or other tissue beneath the dermis, from the standpoint of transdermal and/or transmucosal extraction ofthe analyte. For any given transducer, there may be an optimal frequency range within which frequency modulation is most effective.

The rate of passage of a drug or analyte through the skin can also be increased by altering the resistance (diffusion coefficient) or driving force (diffusion gradient). The rate can be increased by using so-called penetration enhancers, i.e. chemical enhancers.

Chemical promoters consist of two main types of components, namely a binary system consisting of a cell membrane disrupting compound and a solvent or both. Cell membrane disrupting compounds are known in the art and are known to be useful in the preparation of topical medicaments and also to play a role in the extraction of analytes through the skin. These compounds are believed to contribute to skin penetration by disordering the lipid structure of the stratum corneum cell membrane. A broad list of these compounds is described in European patent application No.43,738 (published: 6/13 1982), which is incorporated herein by reference. It is believed that any cell membrane disrupting compound may be used in the present invention.

Suitable solvents include water, glycols (e.g., propylene glycol), and glycerol; monohydric alcohols (e.g., ethanol, propanol) and higher alcohols; DMSO; dimethylformamide; n, N-dimethylacetamide; 2-pyrrolidone; n- (2-hydroxyethyl) pyrrolidone; n-methyl pyrrolidone; 1-dodecylazacycloheptan-2-one and other N-substituted alkylazacycloalkyl-2-ones (azones), and the like.

U.S. patent No.4,537,776, published 27.8.1985, includes an excellent summary of the prior art and background information detailing the use of certain binary systems for promoting penetration. For the sake of the overall disclosure, information and terminology used herein is incorporated by reference.

Likewise, the above-mentioned european patent application No.43,738 teaches how to use selected diols as solvents with a wide variety of cell membrane disrupting compounds to deliver lipophilic pharmacologically active compounds. The disclosure of the european patent application is also incorporated herein by reference, as it discloses in detail cell membrane disrupting compounds and diols.

British patent application GB2,153,223A, published on 21.8.1985, discloses a binary system for promoting penetration of metoclopramide, consisting of monovalent alcohol esters of C8-32 aliphatic monocarboxylic acids (unsaturated and/or branched if the number of carbon atoms is 18-32) or C6-24 aliphatic monohydric alcohols (unsaturated and/or branched if the number of carbon atoms is 14-246) and N-cyclic compounds (e.g. 2-pyrrolidone, N-methylpyrrolidone, etc.).

U.S. patent No.4,973,468 discloses that an enhancer combination of diethylene glycol monoethyl ether or monomethyl ether with propylene glycol monolaurate and methyl laurate can enhance transdermal administration of steroids such as progestins and estrogens. Dual enhancers for transdermal drug delivery consisting of glycerol monolaurate and ethanol are disclosed in U.S. patent No.4,820,720. U.S. patent No.5,006,342 lists a number of enhancers for transdermal delivery,they are prepared from fatty acid esters or C₂To C₄Aliphatic alcohol ethers of alkanediols, wherein each fatty acid/alcohol moiety of the ester/ether contains from 8 to 22 carbon atoms. U.S. patent No.4,863,970 discloses permeation enhancing compositions useful for topical application, which compositions comprise a permeation enhancing vehicle containing an active permeant and a specified amount of one or more cell membrane disrupting compounds (e.g., oleic acid, oleyl alcohol, and glycerol esters of oleic acid; C)₂Or C₃Alkanols and inert diluents such as water, etc.).

Other chemical promoters (not necessarily related to binary systems) include DMSO and aqueous DMSO solutions as taught by Herscher in U.S. Pat. Nos. 3,551,554, 3,711,602 and 3,711,606, and azones (N-substituted alkyl azacycloalkyl-2-ones and compounds such as Cooper in U.S. Pat. No.4,557,943).

Certain chemical enhancers may have undesirable side effects such as toxicity and skin irritation. U.S. patent 4,855,298 discloses a composition containing glycerin in an amount sufficient to produce an anti-irritant effect to reduce skin irritation caused by chemical enhancers containing a skin irritant composition.

Since the combination of stratum corneum microperforation and the application of ultrasonic energy, coupled with the use of chemical enhancers, can enhance the rate of analyte extraction or the rate of permeate transport through the stratum corneum, the specific carrier and specific chemical enhancer used can be selected from a long list of carriers available in the art, some of which have been described above and incorporated herein by reference. It is not necessary to specify or specifically exemplify these compounds as they exist in the art. The present invention is not limited to the use of chemical enhancers themselves, and it is believed that all chemical enhancers that can be used to deliver drugs transdermally will work with dyes in optical microperforations, and with ultrasound energy in withdrawing measurable analytes from beneath the skin and through the skin surface, or delivering permeants or drugs through the skin surface.

Example 16

The modulated sonic energy and chemical enhancers were tested on a human cadaver skin specimen for the ability to control transdermal flux. In these tests, epidermal membranes were isolated from intact human cadaver skin using the thermal isolation method of example 1. The epidermal membrane is cut and placed between the two parts of the permeation cassette, with the stratum corneum facing the upper (donor) layer or the lower (recipient) layer. As shown in fig. 2 of U.S. patent No.5,445,611, the epidermis is supported by a modified Franz box. Each Franz box consists of an upper and a lower layer clamped by one or several clamps. The lower layer has a sampling hole through which material is added or removed. When the upper and lower layers are sandwiched together, the stratum corneum is sandwiched between them. The upper layer of each Franz cell was modified to allow the ultrasound transducer to be placed within 1cm of the corneal layer. Methylene blue was used as an indicator molecule to assess the penetration of the stratum corneum. Visual recordings of the progress and results of each trial were made in time-stamped tape format using video cameras and video tape recorders (not shown). In addition, the sample was removed and the dye that penetrated through the stratum corneum in the test was quantitatively measured with an absorption spectrometer. Suitable chemical promoters may be various solvents as described above and/or compounds that interfere with the coating of the cells. The accelerators used in particular are: the volume ratio of ethanol/glycerin/water/glycerol monooleate/methyl laurate was 50/30/15/2.5/2.5. The system for generating and controlling acoustic energy includes a programmable 0-30MHz arbitrary waveform generator (Stanford resonance Systems Model DS345), a 20 watt 0-30MHz amplifier, and two unfocused ultrasonic immersion vibrators with resonant peaks at 15 and 25MHz, respectively. At the same time, 6 cassettes were prepared for testing samples of stratum corneum from the same donor. After the stratum corneum samples were mounted, they were hydrated with distilled water for at least 6 hours before testing.

Example 17

Action of sonic energy in the absence of chemical promoters

The thermally separated epidermis was placed in aFranz box with the epidermis side up and the stratum corneum side down, unless otherwise indicated, as described in example 16. The lower layer was filled with distilled water and the upper layer with a concentrated solution of methylene blue in distilled water.

Thermally separated skin: the upper layer was filled with methylene blue and immediately sonic energy was applied to one of the cassettes with the transducer fully submerged. This arrangement corresponds to the transducer being placed on the opposite side of the skin fold, or the acoustic energy being reflected off a reflective plate placed at the same location, to "push" the analyte out the other side of the fold into the collection device. The acoustic energy is initially set at a nominal operating frequency of 25MHz, corresponding to the intensity of the 20 volt peak-to-peak (P-P) input waveform. This corresponds approximately to an average input energy of 1 watt to the transducer, and likewise, assuming a nominal conversion efficiency of 1% for this transducer based on manufacturer data, the output acoustic energy is at 0.78cm²About 0.01 watts at the surface of the active area, or 0.13 watts/cm at the acoustic energy intensity². The other 3 control boxes had no sound applied to them. After 5 minutes the sonic energy was turned off. No macroscopic indication of dye flow through the stratum corneum was observed for any of the cells during this interval, indicating that the concentration of dye solution in 2ml of receiving medium was less than about 0.0015% (v/v).

The same test with 3 control cartridges and 1 test cartridge was continued as follows: increasing the intensity of the acoustic energy to the maximum possible output, or about 0.13 watts/cm, from a driver with a p-p input of 70 volts and an average power input of 12 watts²The ultrasound output intensity of (a). The frequency was also set to sweep from 30MHz to 10 MHz. This 20MHz sweep is performed 10 times per second, i.e. at a sweep frequency of 10 Hz. At these input power levels, the acoustic transducer must be monitored to avoid overheating. A contact thermocouple is applied to the body of the vibrator, and the power supply is periodically turned on and off to maintain the maximum temperature of the vibrator below 42 ℃. Penetration of the stratum corneum by methylene blue was still undetectable with the naked eye after applying the maximum power at 50% duty cycle for about 30 minutes with 1 minute on and 1 minute off.

A cold water jacket is then connected to the acoustic transducer to extend the time of excitation at the maximum energy level. The same 3 controls and 1 test cartridge were used and sonic energy was applied to the cartridge at maximum power for 12 hours. At this point, the liquid temperature of the upper layer only rises to 35 ℃ and is only slightly above the normal temperature of the stratum corneum in the body, which is about 31 ℃. Visible evidence of dye flow through the stratum corneum occurred in none of the 4 cells after 12 hours of acoustic energy application as described above.

Example 18

Action of sonic energy in the absence of chemical promoters

Perforated stratum corneum: 6 cartridges were prepared as described in example 16. The clamps that clamped the upper and lower layers of the Franz cartridge were clamped beyond that normally required to seal the upper and lower layers and tightened to the extent that manual perforation was induced, introducing a "pinhole" in the thermally separated skin sample. When the dye solution was added to the upper layer of each box, there was immediately visible evidence of dye leaking into the lower layer through the pores formed in the stratum corneum. Applying sonic energy to a cartridge in which the stratum corneum has pierced such "pinholes" a rapid increase in the transfer of fluid through the pinholes in the stratum corneum was observed. The transfer rate of the indicator dye molecules is directly related to the presence or absence of the application of acoustic energy. That is, the application of acoustic energy causes an immediate pulse (lag time about<0.1 second) of indicator molecules through stratum corneum pinholes. The pulse of indicator molecules immediately stops upon switching off the sonic energy (switching off lags about<0.1 seconds). The pulses may be repeated as described.

Example 19

Action of sonic energy and chemical promoters

Two different chemical promoter formulations were used. Chemical accelerator 1(CE1) is a mixture of ethanol/glycerol/water/glycerol monooleate/methyl laurate (50/30/15/2.5/2.5 by volume). These ingredients are generally recognized as safe, i.e., GRAS, by the FDA for use as pharmaceutical excipients. Chemical enhancer 2(CE2) is an experimental agent that has proven to be extremely effective in enhancing transdermal drug delivery, but is generally considered too irritating for long-term transdermal drug delivery applications. CE2 contained ethanol/glycerol/water/laurone (lauradone)/methyl laurate (volume ratio 50/30/15/2.5/2.5). Laurone is a lauryl (dodecyl) ester of 2-pyrrolidone-5-carboxylic acid ("PCA"), also known as lauroyl PCA.

As described previously (example 16), 6 Franz boxes were positioned, except that the thermally separated epidermis was placed with the epidermis layer down, i.e., the stratum corneum layer side up. Each sample was exposed to distilled water overnight to complete hydration. To start the experiment, the lower layer of distilled water was replaced with methylene blue in all 6 cassettes. The upper layer was filled with distilled water and the boxes were observed for about 30 minutes to confirm no dye passage to ensure that there was no pinhole puncture in any of the boxes. When none was found, the upper layer of distilled water was removed from the 4 boxes. The other 2 boxes served as distilled water controls. The upper layer of two cartridges was then filled with CE1 and the other two cartridges were filled with CE 2.

Sonic energy is applied immediately to one of the two CE2 cartridges. Using 25MHz vibrator, and maximum intensity ≈ 0.13 watt/cm²The frequency was swept from 10MHz to 30MHz every 0.1 seconds. The dye flow was visually detected after applying sonic energy for 10-15 minutes at a 50% duty cycle. No dye flow was detected in the other 5 cassettes.

Sonic energy was then applied to one of two cartridges containing CE1, which were also set. Within 5 minutes, the dye began to appear in the upper layer. Thus, the sonic energy, together with the chemical enhancer, significantly increases the transdermal flux rate of the marker dye through the stratum corneum and reduces lag time.

Example 20

Action of sonic energy and chemical promoters

Formulations of two chemical accelerators, CE1 and CE2, designated CE1MG and CE2MG, were prepared minus glycerin and subjected to the above experiment. The glycerin is replaced by water, so that the proportion of other components is kept unchanged. 3 boxes were prepared with a modified Franz box with the epidermal side of the thermally separated epidermis facing the upper of the two layers. These samples were then hydrated in distilled water for 8 hours. After the hydration step, the distilled water in the lower layer was replaced with either CE1MG or CE2MG and the upper layer was filled with the dye solution. Sonic energy is applied to each of the 3 cartridges sequentially.

A significant increase in the permeability of the stratum corneum samples was observed with the total time of applying pulsed,frequency modulated sonic energy of less than 10 minutes. The change in stratum corneum permeability is fairly uniform throughout the area exposed to chemical promoters and acoustic energy. No "pinhole" penetration of the dye through its crossing stratum corneum was observed. Transcutaneous flow rate can be controlled instantaneously by turning the acoustic energy on or off. Turning off the acoustic energy appears to instantaneously reduce the transdermal flow rate so that active transport of the dye through the skin sample is not seen; the flow rate may be reduced to the extent of passive diffusion. The sonic energy is turned on again and a high level of flow rate is immediately restored. The manner of modulation appears to provide a regular, pulsed increase in transcutaneous flow rate at the rate of modulation. The maximum increase in transcutaneous flow rate for this setting appears to occur at around 27MHz when the acoustic energy is placed at a constant frequency.

After the same results were obtained for all 3 samples, all fluids were drained and both sides of the stratum corneum were rinsed with distilled water. The lower layer was immediately filled with distilled water and the upper layer was refilled with the dye solution. The box was observed for 30 minutes. No pores were observed in the stratum corneum samples, and no significant amount of dye was detected in the lower layer. A small amount of dye is visible in the lower layer, probably due to the dye and promoter captured by the skin sample from its previous exposure. After another 12 hours, the amount of dye detected was still small.

Example 21

Action of sonic energy and chemical promoters

Perforated stratum corneum: 3 cassettes with skin samples thermally separated were prepared with the skin side facing the top side of the cassette from the same supplier as described in example 16. The sample was hydrated for 8hours, and then the lower layer of distilled water was replaced with CE1MG or CE2 MG. The upper layer was filled with a dye solution. Pinholes in the stratum corneum allow the dye to leak through the stratum corneum sample into the underlying cartridge containing the accelerator. Sonic energy is applied. The dye molecules rapidly advance through the pores immediately after the application of sonic energy. As mentioned above, rapid dye flow through the pores is directly and immediately associated with the application of sonic energy.

Example 22

Action of sonic energy and chemical promoters

A low cost acoustic transducer TDK # NB-58S-01(TDK Corp.) was tested for its ability to increase transcutaneous flow. The peak response of the transducer was measured to be about 5.4MHz, with other local peaks appearing at about 7MHz, 9MHz, 12.4MHz, and 16 MHz.

The TDK transducer was then tested for its ability to increase transcutaneous flow with CE1MG at 5.4 MHz. The skin samples were hydrated for 8 hours with the epidermal side facing the lower layer in 3 cassettes. A dye solution was placed in the lower layer. The transducer was placed on top in CE1 MG. A large amount of dye was detected in the collection well of the cartridge by moving through the stratum corneum within 5 minutes using a sweep frequency of 5.3-5.6Mhz as the acoustic energy excitation. Local heating occurs, and the temperature of the oscillator reaches 48 ℃. In the control example where CE1MG was used without acoustic energy, a 24 hour exposure in the collection well resulted in less than 5 minutes exposure using acoustic energy.

This example demonstrates that low cost, low frequency sonic vibrators combined with appropriate chemical promoters can significantly affect the transdermal flow rate. Although high frequency acoustic energy can theoretically concentrate more energy in the stratum corneum, lower frequency modulated acoustic energy can increase transdermal flow rates when combined with chemical enhancers, making this technique useful and practical.

Example 23

Confirmation of molecular migration through human skin

The experiments with TDK transducer and CE1MG as described above were repeated at about 12.4MHz (highest local resonance peak of transducer), frequency sweep from 12.5 to 12.8MHz at a rate of 2Hz, and acoustic energy density below 0.1W/cm². The thermally separated epidermis was facing down the epidermis side with the dye solution in the lower layer and the accelerator solution and sonic energy placed in the upper layer. A large amount of dye within 5 molecules enters the collection well through the stratum corneum. The ohmic heating of the vibrator was significantly lower than the same vibrator operating at 5.4MHz, causing the chemical accelerator temperature to increase to only about 33 ℃.

Even at such low efficiency levels, the results obtained with CE1MG and acoustic energy from the TDK transducer in the detection direction are significant. FIGS. 3A and 3B of U.S. Pat. No.5,445,611 show graphs of data obtained from 3 cassettes measuring the transdermal flow rate in the direction of detection. Even at this point of 5 minutes, there was an easily measurable amount of dye in the chemical accelerator on the outer side of the stratum corneum, indicating that the dye flowed from the epidermis side through the stratum corneum to the "outside" of the skin sample.

In order to optimize the application of sonic energy or sonic energy/chemical facilitator treatment to the method of collecting and detecting analytes from the body, an instrument is required to test the amount of analyte of interest. A test system for taking multiple readings during analyte extraction with sonic or sonic/chemical promoters does not require the unification and normalization of different skin characteristics and flow rates on a sample-by-sample basis. Taking two or more data points with respect to time over the time that the analyte concentration in the collection system increases, a curve fitting method can be used to determine parameters describing the curve of the rate or flow of analyte withdrawal versus the point at which equilibrium is reached, and thereby establish a measure of the concentration in the interval. The general form of this curve is constant from one body to another, only the parameters change. Once these parameters are established, a steady state solution (i.e., equivalent to infinite time) is found for this function, i.e., the solution when equilibrium is established, the concentration of the analyte in vivo is obtained. Thus, this method allows the determination of the required precision to be made in the same length of time for all members of a population (without regard to individual differences in skin penetration).

There are several existing detection techniques that are suitable for use in this application. See D.A. Christensen, in 1648Proceedings of Fiber optics, Medical and Fluorescent Sensors and Applications 223-26 (1992). One approach involves the use of a pair of optical fibers that are placed close together in an almost parallel fashion. One of the fibers is a light source fiber through which light energy is conducted. The other fiber is a sensing fiber attached to a photodiode. As light is transmitted through the light source fiber, a portion of the light energy (the evanescent wave) is present at the fiber surface, which is collected by the detection fiber. The detection fiber conducts the energy of the captured evanescent wave to a photodiode, which measures the energy. The fibers are treated with a binder to attract and bind the analyte to be detected. When an analyte molecule binds to a surface (e.g., analyte glucose bound to an immobilized lectin such as concanavalin a or an immobilized anti-glucose antibody), the amount of evanescent wave coupled between the two fibers changes, as does the amount of energy captured by the fiber being detected and measured by the diode. Several measurements of the evanescent wave are taken in a short time to quickly determine the parameters describing the equilibrium curve, and thus the concentration of the analyte in the body can be calculated. The results of experiments with this system showing measurable flow in 5 minutes (U.S. patent No.5,445,661 fig. 3A and 3B) collected enough data to determine an accurate final reading in 5 minutes.

In its most basic embodiment, the means by which sonic energy is applied and analytes collected comprises an absorbent sheet of natural or synthetic material which serves as a reservoir for the chemical enhancer (if used) and to receive the reservoir to be analyzed from the skin surface. The plate or reservoir is secured in place at selected areas of the skin surface, either passively or by means of suitable fastening means such as tape or adhesive tape.

The acoustic transducer is placed with the plate or reservoir between the skin surface and the transducer and held in place by a suitable tool. The power source is connected to the vibrator and is actuated by a switching device or any other suitable mechanism. After the transducer is activated, sonic energy, modulated in frequency, phase or amplitude, is delivered as needed to deliver the chemical enhancer from the reservoir through the skin surface and then collect the analyte from the skin surface into the reservoir. After a desired fixed or variable time, the vibrator is turned off. The plate or reservoir now containing the analyte of interest can be removed and the analyte quantified using any of the conventional chemical analysis methods used in the laboratory or using a portable instrument. Alternatively, the mechanism for quantifying the analyte may be contained within the device for collecting the analyte, as an integral part of the device or as an accessory. U.S. patent No.5,458,140, which is incorporated herein by reference, describes a device for detecting an analyte.

Example 24

Another method of detecting an analyte (e.g., glucose) after collection of a sample on a perforated skin surface as described above may be achieved by using enzymatic methods. There are a variety of enzymatic methods for measuring glucose in biological samples. One method involves oxidizing glucose in a sample with glucose oxidase to produce gluconolactone and hydrogen peroxide. The hydrogen peroxide is then converted with peroxidase in the presence of a colorless chromogen into water and a colored product.

The intensity of the colored product will be proportional to the amount of glucose in the liquid. The color can be determined by conventional absorption or reflection methods. Calibration with a known glucose concentration allows the amount of color to be used to determine the glucose concentration in the collected analyte. The relationship is determined experimentally, and the glucose concentration in the blood of the subject can be calculated. This knowledge can be used in the same way as the knowledge obtained from the finger stick blood glucose test. Results were obtained in 5-10 minutes.

Example 25

Any system that employs visual display or reading of glucose concentration may indicate to a diagnostician or patient the need for administration of insulin or other suitable medication. In intensive care or other situations where continuous monitoring is required andalmost simultaneous corrective action is required, the display may be connected to an appropriate signaling device that triggers the administration of insulin or other medication. For example, there are insulin pumps implanted in the abdominal or other body cavity, which may be activated by exogenous or endogenous stimuli. Alternatively, using stratum corneum microperforation and other techniques of the present invention that increase the transdermal flow rate, a system for transdermal delivery of insulin can be provided, the flow rate control of which is modulated by signals from the glucose sensing system. In this way, a fully biomedical controlled system is obtained which not only monitors and/or diagnoses medical needs, but at the same time provides corrective measures.

Biomedical control systems of the same nature may also be used in other situations, such as maintaining electrolyte balance or administering analgesics based on measured analyte parameters (e.g., prostaglandins).

Example 26

Like audible sound, ultrasound waves can reflect, refract, and absorb when they encounter another medium with different properties [ d.bommann et al, 9 pharm. res.559(1992)]. Mirrors or lenses may be used to focus or control the distribution of acoustic energy across the tissue under investigation. For many parts of the human body, a cluster of living organisms can be found to support this system. For example, the earlobe is a convenient site and mirrors or lenses may be used to aid in orientation control (e.g., "push" the analyte or permeate through the stratum corneum) which acts similarly to changing acoustic frequencies and intensity.

Example 27

Multiple acoustic vibrators may be used to selectively control the direction of flow through the stratum corneum into or out of the body through the skin. A piece of skin, such as an earlobe, may allow the transducer to be placed on either side of it. The vibrators may be selectively excited or excited in a phased manner to promote percutaneous flow in a desired direction. An array or acoustic loop of transducers may be established, using the concept of phased arrays (similar to that invented for radar and microwave communication systems), to direct and focus acoustic energy to the area of interest.

Example 28

In this example, the procedure of example 19 was followed except that the heat-separated epidermal specimens were first treated with an excimer laser (e.g., Lambda Physik EMG/200 type; 193nm wavelength, 14ns pulse width) to ablate the stratum corneum as described in U.S. Pat. No.4,775,361, which is incorporated herein by reference.

Example 29

In this example, except that the thermally separated skin samples were first treated with 1, 1 '-diethyl-4, 4' -carbocyanine iodide (Aldrich, λ ═ 703nm), then delivered in total 70mJ/cm using a laser diode type TOLD9150 (Toshiba America electronic, 30mW at 690nm) laser diode²A 50ms laser was applied to the dye treated samples to ablate the stratum corneum beyond which it was processed according to the procedure of example 19.

Example 30

In this example, the procedure of example 29 was followed, except that the dye was indigo cyanine green (Sigma cat. No. I-2633; lambda. 775nm) and the laser was Diolite 800-50(LiCoNiX, 50nW at 780 nm).

Example 31

In this example, the procedure of example 29 was followed except that the dye was methylene blue and the laser was SDL-8630(SDL Inc., 500mW at 670 nm).

Example 32

In this example, the procedure of example 29 was followed except that the dye was contained in a solution containing a permeation enhancer (e.g., CE 1).

Example 33

In this example, the procedure of example 29 was followed except that the ultrasound exposure was used to aid in the delivery of the dye and accelerator-containing solution to the stratum corneum.

Example 34

In this example, the procedure of example 31 was followed except that the pulsed light source was a short arc lamp emitting over a wide range of 400-1100nm but with a bandpass filter placed in the system to limit the output to the wavelength region of about 650-700 nm.

Example 35

In this example, the procedure of example 19 was followed except that the heat-separated epidermal sample was first punctured with a calibrated micro-lancet (Becton Dickinson) to create micropores in the stratum corneum without reaching the underlying tissue.

Example 36

In this example, the skin samples were first used at 70-480mJ/cm except for the heat-separated skin²Focused acoustic energy treatment in the/50 ms range to ablate outside the stratum corneum was performed as in example 19.

Example 37

In this example, the procedure of example 19 was followed except that the hydraulic piercing was first performed with a high-pressure liquid jet to form micropores having a diameter of not more than about 100 μm.

Example 38

In this example, the procedure of example 19 was followed except that the stratum corneum was first punctured with short electrical pulses to form micropores no larger than about 100 μm in diameter.

Example 39

Acoustic Streaming (Aconotic Streaming)

The novel mechanism and application of acoustic energy to deliver therapeutic substances into the body and/or to harvest body fluids from the body into an external reservoir via the pores formed in the stratum corneum will now be described. Another aspect of the invention is to use acoustic energy to create an acoustic streaming effect on the fluid surrounding and between intact cells in the epidermis and dermis of human skin. There are many documents that describe that acoustic streaming is a mode of interaction of acoustic energy with liquid media (Nyborg, physical Acoustics Principles and Methods, p.265-331.Vol.II-Part B, academic Press.1965). The first theoretical analysis of the acoustic streaming phenomenon was made by Rayleigh (1884, 1945). In an extensive discussion of this topic, Longuet Higgins (1953-1960) gave results applicable to two-dimensional flow generated near any vibrating cylinder. Nyborg (1958) developed a three-dimensional approximation to an arbitrary surface. As described in Fairbanks et al, 1975 Uitrasonics Symposium Proceedings, IEEE Cat. #75, CHO994-45U, acoustic energy and acoustic streaming phenomena are of great use in facilitating the flow of liquids through porous media, showing measurable flow rates increasing up to 50-fold, which may be passive or simply applying a pressure gradient.

All previous attempts to utilize percutaneous delivery or extraction of ultrasound have focused on methods of interaction of the acoustic energy and the skin tissue intended to facilitate penetration of the stratum corneum. The exact manner in which the interaction is involved is, without exception, assumed to be due to a local increase in the temperature of the stratum corneum and the resulting melting of the lipid domains of the intercellular spaces between the keratinocytes (Srinivasan et al). Other researchers have suggested that formation of micropockets inthe stratum corneum and/or tearing of structures open channels through which fluid can flow more easily. In general, the design of an acoustic system to increase transdermal flow rate is based on the early recognition that the use of existing ultrasound treatment devices designed to produce a "deep heating" effect on a subject, combined with the topical application of a gel-like or liquid formulation containing a drug to be delivered into the body, produces a quantifiable increase in the flow rate of the drug into the body. The use of acoustic energy in the context of the method disclosed in this specification for creating micropores in this barrier layer can be considered as an entirely new and different concept compared to the classical definition of the concept of acoustophoresis.

Based on the experimental findings described in U.S. Pat. Nos. 5,458,140 and 5,445,611, it was found that applying a suitably driven ultrasound transducer to a liquid reservoir on either side of a perforated SC specimen, where liquid can be pumped through the perforated membrane at large flow rates, can produce an "acoustically induced flow" phenomenon when small holes are present or formed in the Stratum Corneum (SC) in Franz boxes used in vitro studies.

The use of the fluid flow regime of the acoustic/fluid interaction for fluid intake or aspiration into the body can now be practically explored using the methods described in this specification to produce controlled micro-pore formation in the stratum corneum of living skin. For example, clinical studies have shown that at 400 μm²A series of 4 micro-holes of 80 μm diameter were formed and a gentle suction (10-12 inches Hg) was applied to this area to aspirate an average of about 1 μ l of interstitial fluid (ISF) leaving the body and collected in a vessel outside the body. The addition of a low power miniature acoustic transducer to this system is configured to rapidly generate inwardly converging concentric pressure waves on 2-6mm of tissue surrounding the puncture site, and has been shown to increase the ISF flow rate by about 50%.

If it is not desired to produce some form of direct absorption of acoustic energy (if heat generation is desired) at the skin tissue, the frequency of the acoustic energy to which the skin tissue is substantially transparent, i.e., in the very low frequency region of 1kHz to 500kHz, can be determined. Using a microscope to observe an in vivo experiment in which the subject's skin is microperforated and ISF is drawn out of the body and pools to the skin surface, a significant acoustic streaming effect is observed even at some of the lowest frequencies tested. The phonon is excited to reveal a macroscopic sign that introduces a dramatic amount of acoustic streaming, since small particulate matter moves with it when the ISF swirls. Typical motion amplitudes can be described as follows: for a circular ISF blend with a skin surface diameter of 3mm, one macroscopic particle is visible that makes approximately 3 complete revolutions per second. This corresponds to a linear velocity of the fluid of more than 2.5 mm/sec. Using less than 100mW/cm²The level of acoustic energy entering the tissue may confirm this overall effect.

One can easily observe the movement of the upper surface of the skin and the liquid above it, but it is rather difficult to assess the dynamic changes occurring inside the layers of skin tissue in response to the coupling of acoustic energy into these tissue interiors. One can assume that if such large linear velocities of fluid (e.g.,>2.5mm/S) can be readily produced on a surface, then an increase in flow of fluid through the intercellular channels of living dermal tissue should also be visible in response to this acoustic energy input. Recently, quantitative measurements were made of the ISF collected through the microwells applying low frequencyacoustic energy to a region within the circumference around the puncture site. In this experiment, the technique of ISF collection based only on light suction (10-12 hours Hg) was replaced with the same instrument but equipped with an acoustic transducer. In a series of 10 two minute collection times, 5 with suction only, 5 with suction plus sonic energy, it was observed that about 50% higher ISF was collected by activating the acoustic source during the same time. These data are shown in FIG. 30. Such an increase in ISF flow rate is achieved without the subject reporting any sensory enhancement due to acoustic energy. The apparatus used in this experiment is illustrated in FIGS. 31-33. The transducer assembly of fig. 31-33 comprises a thick-walled cylinder of piezoelectric material, with an inner diameter of about 8mm and a wall thickness of 4 mm. The cylinder is polarized such that when an electric field is applied between the metalized surfaces of the outer and inner diameters, the thickness of the cylinder wall expands or contracts with the polarity of the electric field. In fact, such a structure allows to obtain a device which rapidly squeezes the tissues sucked into the central hole, generating an inward radial acoustic streaming action of the liquid present in these tissues. This inward acoustic streaming action will bring more ISF to the microperforation site in the center of the hole where it is collected externally away from the body.

Similar devices as shown in fig. 34A-B were constructed and tested, producing similar preliminary results. In the schematic diagrams of fig. 34A-B, an ultrasonic transducer manufactured by Zevex, inc, salt lake, utah was modified to have a blade-shaped extension attached to an ultrasonic sound collector. The 0.5 thick blade end of this extension had a 4mm hole. When activated, the primary motion is longitudinal along the length of the blade, essentially causing a rapid reciprocating motion. The provision of a hole of 4mm causes physicalfluctuation of the metal blade, which at this point results in a very active, but unordered, large displacement. In use, the subject's skin is sucked up into the hole and acoustic energy is introduced into the skin in a manner similar to that shown in figure 33.

The new aspects of this new application of ultrasound lie in the following basic ranges:

1. it is no longer necessary to focus the effect of ultrasound on increasing the permeability of the SC barrier film as proposed by Langer, Kost, bommann et al.

2. A less frequent system can be used which is less absorbed in the skin tissue but still produces the desired fluid induced flow phenomenon in the intercellular channels between the epidermal cells containing interstitial fluid.

3. The mode of interaction of ultrasound with tissue and fluids therein is the so-called "induced flow" mode, which is recognized in the acoustic literature as a unique mode distinct from classical vibrational-type interactions that tear cellular membranes, facilitating passive diffusion processes.

Optimizing the geometry, frequency, power and modulation applied to the ultrasound transducer has been shown to provide a significant increase in flow through the perforated skin site. The optimization of these parameters is designed to examine the non-linear relationship of the flow of the dispensed liquid within the environment at this microscopic scale. With frequencies below 200kHz, large fluidization effects can be observed without any detectable heating or other adverse tissue interactions. The acoustic energy levels required to produce these measurable effects are low, with average power levels typically at 100mW/cm²The following is a description.

Thus, the above embodiments are merely representative of some systems that may be employed to use ultrasound or ultrasound + chemical enhancers for the collection and quantification of analytes for diagnostic and transdermal delivery of permeants. The present invention is directed to the discovery that analytes or permeate delivery can be measured transdermally with little or no damage after keratoporation by the appropriate use of ultrasound, particularly with chemical enhancers. However, the present invention is not limited to these specific descriptions. There are many perforation techniques and promoter systems, some of which may be more effective than others, for detecting or withdrawing certain analytes or delivering permeants through the stratum corneum. However, in light of the basic principles set forth herein, one skilled in the art can readily perform a certain amount of experimentation to obtain the optimum perforation, accelerant, or optimum time, intensity, and frequency of application of the ultrasound waves, and modulation of the frequency, amplitude, and phase of the applied ultrasound waves. Accordingly, the scope of the invention is to be limited only by the scope of the following claims and their functional equivalents.

Claims (61)

1. A method for detecting the concentration of an analyte in an individual, the method characterized by the step of enhancing the permeability of stratum corneum of a selected area of a surface of the individual to the analyte, the steps comprising:

(a) perforating the stratum corneum of the selected area to form micropores in the stratum corneum without causing severe damage to underlying tissue, thereby reducing the barrier properties of the stratum corneum to analyte withdrawal;

(b) collecting a selected amount of an analyte; and

(c) the collected analyte is quantitatively determined.

2. The method of claim 1 further comprising applying sonic energy to the perforated selected areas at a frequency in the range of about 5kHz to 100kHz, wherein the sonic energy is modulated by a means selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof.

3. The method of claim 2 further comprising contacting the selected area of the subject with a chemical enhancer and applying sonic energy to further enhance analyte extraction.

4. The method of claim 1, 2 or 3, wherein said perforating of said stratum corneum is accomplished by a method selected from the group consisting of: (a) will be less than or equal to about 1000 mu m²Contacting the stratum corneum 7 of the selected area with a heat source to ablate the stratum corneum and thereby remove the stratum corneum of the selected area by raising the temperature of the tissue associated with the water and other vaporizable materials in the selected area above the vaporization point of said water and other vaporizable materials; (b) puncturing with a calibrated miniature lancetSaid stratum corneum, to form micropores of diameter less than or equal to about 1000 μm; (c) focusing a tightly focused beam of acoustic energy onto the stratum corneum to ablate the stratum corneum; (d) hydraulically puncturing the stratum corneum with a high pressure fluid stream to form micropores having a diameter of less than or equal to about 1000 μm, and (e) puncturing the stratum corneum with a short electrical pulse to form micropores having a diameter of less than or equal to about 1000 μm.

5. The method of claim 4 wherein said perforations are accomplished by contacting an area of said stratum corneum having awidth no greater than about 1000 μm with a heat source to raise the temperature of the tissue bound water and other vaporizable materials above the vaporization point of said water and other vaporizable materials, thereby removing stratum corneum from the selected area.

6. The method of claim 5, comprising treating the selected area with at least an effective amount of a dye exhibiting strong absorption for the range of pulsed light source emission and focusing a series of pulses output from the pulsed light source on the dye to heat the dye sufficiently to conduct heat to the stratum corneum, raise the temperature of tissue bound water and other vaporizable materials of the selected area above the vaporization point of the water and other vaporizable materials, wherein the dye acts as a heat source.

7. The method of claim 6, wherein the pulsed light source emits at a wavelength that is not significantly absorbed by skin.

8. The method of claim 7 wherein the pulsed light source is a laser diode emitting at about 630 and 1550 nm.

9. The method of claim 7 wherein the pulsed light source is an optical parametric oscillator emitting at about 700 and 3000nm pumped with a laser diode.

10. The method of claim 6, wherein the pulsed light source is selected from the group consisting of an arc lamp, an incandescent lamp, and a light emitting diode.

11. The method of claim 6, further characterized by providing a sensing system that determines when the barrier properties of the stratum corneum have been overcome.

12. The method of claim 11, wherein the sensing system includes a light collecting device that receives light reflected from the selected area and focuses the reflected light onto a photodiode, a photodiode that receives the focused light and sends a signal to a controller, wherein the signal is indicative of a property of the reflected light, and a controller connected to the photodiode and the pulsed light source to receive the signal and to turn off the pulsed light source upon receipt of a preselected signal.

13. The method of claim 6, further comprising cooling the selected area of stratum corneum and adjacent skin tissue with a cooling device to place the selected area and adjacent skin tissue in a selected pre-cooled, steady-state condition prior to perforation.

14. The method of claim 13, wherein the cooling device comprises a Peltier device.

15. The method of claim 6, wherein said ablating results in the outflow of interstitial fluid, said analyte being collected in a selected amount of said interstitial fluid.

16. The method of claim 15, further characterized by further comprising: after collecting the selected amount of interstitial fluid, applying an effective amount of energy from the pulsed light source to close the micropores to cause interstitial fluid retained in the micropores to coagulate.

17. The method of claim 15 further including applying a vacuum to selected areas of said perforated stratum corneum to promote the egress of interstitial fluid.

18. The method of claim 6, further characterized by: irradiating at least the selected area with unfocused light from the pulsed light source prior to perforating the stratum corneum to sterilize the selected area irradiated by the light.

19. The method according to claim 5, comprising contacting the selected area with a wire, wherein the wire acts as a heat source to raise the temperature of the selected area from the temperature of the surrounding skin to greater than 100 ℃ in about 10-50ms, and then to return the temperature of the selected area to about the temperature of the surrounding skin in about 30-50ms, wherein the cycle of raising the temperature and returning to about the temperature of the surrounding skin is repeated a plurality of times sufficient to reduce the barrier properties of the stratum corneum.

20. The method of claim 19 wherein the step of returning to about ambient skin temperature is performed by withdrawing the wire out of contact with the stratum corneum.

21. The method of claim 20 further comprising providing means for monitoring the electrical impedance between said wire and the subject through selected areas of said stratum corneum and adjacent skin tissue and means for advancing the wire such that said advancing means advances the wire as the electrical impedance of ablation decreases to bring the wire into contact with the stratum corneum during heating.

22. The method of claim 21 further comprising means for withdrawing the wire from contact with the stratum corneum, wherein the monitoring means detects a change in impedance associated with contact with the stratum corneum underlying cortex and transmits a signal tothe withdrawal means to withdraw the wire from contact with the stratum corneum.

23. The method of claim 20, wherein the wire is heated with an ohmic heating element.

24. The method of claim 20, wherein the wire is formed into a circuit comprising a high impedance point whose temperature is modulated by passing a modulated current through the circuit.

25. The method of claim 20, wherein the wire is positioned in a modulatable ac magnetic field of the field coil such that energizing the field coil with ac current generates eddy currents sufficient to heat the wire through internal ohmic losses.

26. The method of claim 4 wherein said piercing is accomplished by piercing said stratum corneum with a calibrated micro-lancet to form micro-pores having a diameter of no more than about 1000 μm.

27. The method of claim 4 wherein said perforating is accomplished by ablating the stratum corneum into said stratum corneum by focusing a tightly focused beam of acoustic energy onto said stratum corneum.

28. The method of claim 4 wherein said perforating is accomplished by hydraulically puncturing said stratum corneum with a high pressure fluid stream to form micropores of no more than about 1000 μm in diameter.

29. The method of claim 4 wherein said perforating is accomplished by puncturing said stratum corneum with short electrical pulses to form micropores of no more than about 1000 μm in diameter.

30. The method of claim 4, 6 or 19, wherein the analyte is glucose.

31. The method of claim 30, wherein the quantification of glucose is by a colorimetric assay with glucose oxidase or by a biosensor.

32. A method for enhancing transdermal flux of an active agent into a selected area of a subject, the method characterized by the step of enhancing permeability of the stratum corneum of the selected area of the subject's surface to said active permeant, comprising the steps of:

(a) perforating the stratum corneum of the selected area to form micropores in the stratum corneum without causing severe damage to the underlying tissue, thereby reducing the barrier properties of the stratum corneum to the flow of said active permeants;

(b) contacting the perforated selected area with a composition comprising an effective amount of the permeate to promote flow of the permeate into the body.

33. The method of claim 32, further comprising the step of: applying acoustic energy to the perforated area for a time and at an intensity and frequency effective to produce a fluid-moving effect, thereby promoting transdermal flux of the permeate into the body.

34. The method of claim 33 wherein said sonic energy is applied to said perforated selected areas in a frequency range of about 5kHz to 100MHz, wherein said sonic energy is modulated by a means selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof.

35. The method of claim 34 further comprising contacting the selected area of the subject with a chemical enhancer and applying sonic energy to further enhance the flow of the permeate into the body of the subject.

36. The method of claim 32, 33, 34 or 35, wherein said perforating of said stratum corneum of said selected region is accomplished by a method selected from the group consisting of: (a) contacting the selected area having a width of no greater than about 1000 μm with a heat source to ablate the stratum corneum such that the temperature of the tissue associated with the selected area of water and other vaporizable material is raised above the vaporization point of said water and other vaporizable material, thereby removing the stratum corneum from the selected area; (b) piercing the stratum corneum with a calibrated micro-lancet to form micropores no greater than about 1000 μm in diameter; (c) focusing a tightly focused beam of acoustic energy onto the stratum corneum to ablate the stratum corneum; (d) hydraulically piercing the stratum corneum with a high pressure stream of liquid to form micropores of no greater than about 1000 μm in diameter, and (e) piercing the stratum corneum with a short electrical pulse to form micropores of no greater than about 1000 μm in diameter.

37. The method of claim 36, wherein said perforating is accomplished by: contacting said selected region of said stratum corneum having a width of no greater than about 1000 μm with a heat source to raise the temperature of tissue associated with said water and other vaporizable materials in said selected region above the vaporization point of said water and other vaporizable materials, thereby removing the stratum corneum from said selected region.

38. The method of claim 37, comprising treating the selected area with at least an effective amount of a dye exhibiting strong absorption of the emission range of the pulsed light source, and focusing a series of pulses output from the pulsed light source on the dye to heat the dye sufficiently to conduct heat to the stratum corneum, raise the temperature of tissue associated with the water and other vaporizable materials in the selected area above the vaporization point of the water and other vaporizable materials, wherein the dye acts as a heat source.

39. The method of claim 38, wherein the pulsed light source emits wavelengths that are not significantly absorbed by skin.

40. The method of claim 39 wherein the pulsed light source is a laser diode emitting in the range of about 630 and 1550 nm.

41. The method of claim 39 wherein the pulsed light source is an optical parametric oscillator emitting in the range of about 700-3000nm pumped with a laser diode.

42. The method of claim 39, wherein said pulsed light source is a light source selected from the group consisting of an arc lamp, an incandescent lamp, and a light emitting diode.

43. The method of claim 38, further providing a sensing system to determine when the barrier properties of the stratum corneum are eliminated.

44. The method of claim 43, wherein the sensing system includes a light collecting device that receives light reflected from the selected area and focuses the reflected light on a photodiode, a photodiode that receives the focused light and sends a signal to a controller, wherein the signal is indicative of a property of the reflected light, and a controller coupled to the photodiode and the pulsed light source to receive the signal and to turn off the pulsed light source upon receiving a preselected signal.

45. The method of claim 38, further comprising cooling a selected region of said stratum corneum and adjacent skin tissue with a cooling device such that said region and adjacent skin tissue are in a selected pre-chilled steady state condition prior to piercing.

46. The method of claim 45, wherein the cooling device comprises a Peltier device.

47. The method of claim 38, further comprising irradiating at least the selected area with unfocused light from the pulsed light source prior to the perforation of the stratum corneum, the selected area irradiated with the light being sterilized.

48. The method of claim 37, further comprising contacting the selected area with a wire, wherein the wire acts as a heat source to raise the temperature of the selected area from the temperature of the surrounding skin to greater than 100 ℃ in about 10-50ms, and then to return the temperature of the selected area to about the temperature of the surrounding skin in about 30-50ms, wherein the cycle of raising the temperature and returning to about the temperature of the surrounding skin is repeated a plurality of times sufficient to reduce the barrier properties of the stratum corneum.

49. The method of claim 48 wherein said step of returning to about ambient skin temperature is performed by withdrawing said wire out of contact with the stratum corneum.

50. The method of claim 49 further comprising providing means for monitoring the electrical impedance between said wire and said subject through selected areas of said stratum corneum and adjacent skin tissue and means for advancing the position of said wire such that when ablation occurs with a concomitant decrease in electrical resistance, said advancing means advances the wire to contact the stratum corneum as it heats.

51. The method of claim 50 further comprising means for withdrawing said wire from contact with the stratum corneum, wherein the monitoring means is capable of measuring a change in impedance associated with contact with the stratum corneum sub-epidermis layer and transmitting a signal to said withdrawing means to withdraw said wire from contact with the stratum corneum.

52. The method of claim 49, wherein the wire is heated with an ohmic heating element.

53. The method of claim 49 wherein the wire forms a current loop containing a high impedance point, the temperature of the high impedance point being modulated by a modulated current passing through the current loop.

54. The method of claim 49, wherein the wire is exposed to an alternating current magnetic field of a modulatable exciter coil, such that the exciter coil, when energized with an alternating current, generates eddy currents sufficient to heat the wire via internal ohmic losses.

55. The method of claim 36 wherein said piercing is accomplished by piercing said stratum corneum with a calibrated micro-lancet to form micro-pores having a diameter of no more than about 1000 μm.

56. The method of claim 36 wherein said perforating is accomplished by ablating the stratum corneum into said stratum corneum by focusing a tightly focused beam of acoustic energy onto said stratum corneum.

57. The method of claim 36 wherein said perforating is accomplished by hydraulically puncturing said stratum corneum with a high pressure fluid stream to form micropores of no more than about 1000 μm in diameter.

58. The method of claim 36, wherein said perforating is accomplished by puncturing said stratum corneum with short electrical pulses to form micropores of no more than about 1000 μm in diameter.

59. A method of applying a tattoo to a selected area of the skin on a surface of an individual, comprising the steps of:

(a) perforating the stratum corneum of the selected area to form micropores in the stratum corneum without causing severe damage to the underlying tissue, thereby reducing the barrier properties of the stratum corneum to the flow of active permeants; and

(b) contacting the perforated selected area with a composition comprising an effective amount of tattoo ink to facilitate the flow of said ink into the body.

60. A method for reducing the time delay for an analyte to diffuse from the blood of an individual to interstitial fluid of said individual in a selected area of the skin, characterized in that the method comprises applying cooling means to said selected area of the skin.

61. A method for reducing the vaporization and vapor pressure of interstitial fluid collected from micropores in a selected region of the stratum corneum of the skin of a subject, comprising applying a cooling device to said selected region of the skin.

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