| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | September 7, 1999 |
| PATENT TITLE |
Chemical and physical enhancers and ultrasound for transdermal drug delivery |
| PATENT ABSTRACT |
Transdermal transport of molecules during sonophoresis (delivery or extraction) can be further enhanced by providing chemical enhancers which increase the solubility of the compound to be transported and/or lipid bilayer solubility, and/or additional driving forces for transport, such as mechanical or osmotic pressure, magnetic fields, electroporation or iontophoresis. In a preferred embodiment the ultrasound is low frequency ultrasound which induces cavitation of the lipid layers of the stratum corneum (SC). This method provides higher drug transdermal fluxes, allows rapid control of transdermal fluxes, and allows drug delivery or analyte extraction at lower ultrasound intensities and other forces or concentrations than that required if each means of enhancing transport is used individually. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | December 18, 1995 |
| PATENT REFERENCES CITED |
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L., "Mechanism of Percutaneous Absorption from Physiochemical Evidence," Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery (Bronaugh, R.L., Maibach, H., Editors, Marcel Dekker, New York) 27-51 (1989). Friedman, R. M., `Interferons: A Primer`, (Academic Press, New York, 1981). Gaertner, W., "Frequency Dependence of Ultrasonice Cavitation," J. Acoust. Soc. Am. 26:977-980 (1954). Ghanem et al., "The effects of ethanol on the transport of lipophilic and polar permeants across hairless mouse skin: Methods/validation of a novel approach," Int. J. Pharm. 78:137-156 (1992). Grups and Frohmuller, "Cyclic Interferon Gamma Treatment of Patients with Metastatic Renal Carcinoma," J. Med. 64(3):218-220 (1989). Hansch and Leo, "Substitutent Constants for Correlation Analysis in Chemistry and Biology" (1979). Junginger, et al., "Visualization of Drug Transport Across Human Skin and the Influence of Penetration Enhancers," "Drug Permeation Enhancement" (Hsieh, D.S., Editors, Marcel Dekker, Inc. New York) 59-89 (1994). Kasting, et al., "Prodrugs for Dermal Delivery: Solubility, Molecular Size, and Functional Group Effects," "Prodrugs: Topical and Ocular Delivery" Sloan, ed. (Marcel Dekker, NY 1992) 117-161. Kost and Langer, "Ultrasound-Mediated Transdermal Drug Delivery," Topical Drug Bioavailability Bioequivalence and Penetration (Maibach, H. I., Shah, V. P., Editors, Plenum Press, New York) 91-104 (1993). Kost, et al., "Ultrasound Effect on Transdermal Drug Delivery," (Ben Gurion University Dept. of Chem. Engineering, Beer Sheva Israel) (MIT, Dept. of Appliced Biological Sciences, Cambridge, MA) CRS Aug. 1986. Krall, L.P., `World Book of Diabetes in Practice` (Editors, Elsvier, 1988). Lee, V. H. L., et al., "Protease Inhibition as an Additional Mechanism for the Nasal Absorption Enhancement Effect of Sodim Taurodihydrofusidate," Proceed. Intern. Symp. Control. Rel. Bioact. Mater 14:55-56 (1987). Lee, V. H. L., et al., "Nasal Peptide and Protein Absorption Promotors: Aminopeptidase Inhibition as a Predictor of Absorption Enhancement Potency of Bile Salts," Proceed. Intern. Symp. Control. Rel. Bioact. Mater 14:53-54 (1987). Levy, et al., "Effect of Ultrasound on Transdermal Drug Delivery to Rats and Guinea Pigs," J. Clin. Invest. 83:2074-2078 (1989). Liu, et al., "Contransport of Estradiol and Ethanol Through Human Skin In Vitro: Understanding the Permeant/Enhancer Flux Relationship," Pharmaceutical Research 8:938-944 (1991). Liu, et al., "Experimental Approach To Elucidate the Mechanism of Ultrasound-Enhanced Polymer Erosion and Release of Incorporated Substances," Macromolecules 25:123-128 (1992). Machluf and Kost, "Ultrasonically enhanced transdermal drug delivery. Experimental approaches to elucidate the mechanism," J. Biomater. Sci. Polymer Edn. 5:147-156 (1993). Mak, et al., "Oleic Acid Concentration and Effect in Human Stratum Corneum: Non-Invasive Determination by Attenuated Total Reflectance Infared Spectroscopy In Vivo," J. Controlled Rel. 12:67-75 (1990). Mitragotri, et al., "Ultrasound-Mediated Transdermal Protein Delivery," Science 269:850-853 (1995). Mitragotri, et al., "A Mechanistic Study of Ultrasonically-Enhanced Transdermal Drug Delivery," J. Pharm. Sci. 84:697-706 (1995). Morimoto, Y., et al., "Prediction of Skin Permeability of Drugs: Comparison of Human and Hairless Rat Skin," J. Pharm. Pharmacol. 44:634-639 (1991). Nagai and Konishi, "Buccaal/Gingival Drug Delivery Systems," Journal of Controlled Release (Elsevier Science Publishers B.V., Amsterdam) 6:353-360 (1987). Newman, J., et al., "Hydrocortisone Phonophoresis," J. Am. Pod. Med. Assoc. 82:432-435 (1992). Olanoff and Gibson, "Method to Enhance Intranasal Peptide Delivery," Controlled Release Technology Pharmaceutical Application (Lee, et al. Editors, American Chemical Society) 301-309 (1987). Ongpipattanankul, et al., "Evidence that Oleic Acid Exists in a Separate Phase Within Stratum Corneum Lipids," Pharm. Res. 8:350-354 (1991). Parkin, et al., "Atopic manifestations in the acquired immune deficiency syndrome: response to recombinant interferon gamma," Br. Med. J., 294:1185-1186 (1987). Perry, et al., "Perry's Chemical Engineering Handbook" (McGraw-Hill, NY 1984). Pishko, et al., "Amperometric Glucose Microelectrodes Prepared through Immobilization of Glucose Oxidase in Redox Hydrogels," Anal. Chem. 63:2268-2272 (1991). Potts and Guy, "Predicting Skin Permeability," Pharm. Res. 9:663-669 (1992). Prausnitz, et al., "Electroporation of mammalian skin:A mechanism to enhance transdermal drug delivery," Proc. Natl. Acad. Sci. USA 90:10504-10508 (1993). Quillen, W.S., "Phonophoresis: A Review of the Literature and Technique," Athl. Train. 15:109-110 (1980). Robinson & Lee, "Influence of Drug Properties on Design," Controlled Drug Delivery 42-43. Rosell, J., et al., "Skin Impedance From 1 Hz to 1 MHz," IEEE Trans. Biomed. Eng. 35:649-651 (1988). Skauen, et al., "Phonophoresis," Int. J. Pharm. 20:235-245 (1984). Stringfellow, Clinical Applications of interferons and their inducers, (Editors, Marcel Dekker, New York, 1986). Tamada, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 22, 129-130 (1995). Tocanne, et al., "Lipid lateral diffusion and membrane organization," FEB 257:10-16 (1989). Tyle and Agrawala, "Drug Delivery by Phonophoresis," Pharm. Res. 6:355-361 (1989). Walker and Hadgraft, "Oleic acid--a membrane `fluidiser` or fluid within the membrane," Int. J. Pharm. 71:R1-R4 (1991). Walmsley, "Applications of Ultrasound in Dentistry," Ultrasound in Med. and Biol. 14:7-14 (1988). Walters, K. A., "Penetration Enhancers and Their Use in Transdermal Therapeutic Systems," Transdermal Drug Delivery: Developmental Issues and Research Initiatives, 197-246 (Hadgraft J., Guy, R.H., Editors, Marcel Dekker, 1989). Wester and Mailbach, "Animal Models for Percutaneous Absorption," Topical Drug Bioavailability Bioequivalence and Penetration (Shah and Maibach, Editors, Plenum Press, New York) 333-349, (1993). Wheatley, et al., "Use of Ussing Chamber for Investigation of Drug Delivery Across Viable Nasal Tissue Membranes," Proceed. Intern. Symp. Control. Rel. Bioact. Mater. (Controlled Release Society, Inc. 14:26-27 (1987). Williams, et al., "On the non-Gaussian distribution of human skin permeabilities," Int. J. Pharm. 86:69-77 (1992). Wilschut, et al., "Estimating Skin Permeation, The Validation of Five Mathematical Skin Permeation Models," Chemosphere 30:1275-1296 (1995). |
| PATENT CLAIMS |
We claim: 1. A method for enhancing transdermal transport of compounds comprising administering to the skin an effective amount of ultrasound in combination with linoleic acid in an ethanol solution. 2. A method for enhancing transdermal transport of compounds comprising administering to the skin an effective amount of ultrasound in combination with a magnetic force field to transport the compound through the skin. 3. The method claim 2 comprising administering to the skin an effective amount of ultrasound and magnetic force in combination with an agent enhancing solubility of the compounds to be transported and an agent enhancing the fluidity of lipid bilayers. 4. The method of claim 1 wherein the combination is linoleic acid in an ethanol solution. 5. The method of claim 2 wherein the ultrasound is administered at a frequency of between 20 kHz and 40 kHz. 6. The method of claim 2 wherein the ultrasound is administered at a frequency of 1 MHz or less. 7. The method of claim 2 wherein the intensity of the ultrasound is less than 2.5 W/cm.sup.2. 8. The method of claim 2 wherein the intensity of the ultrasound is less than 1.5 W/cm.sup.2. 9. The method of claim 2 wherein the ultrasound is administered in combination with an electric force field selected from the group consisting of electroporation and iontophoresis. 10. The method of claim 2 wherein the ultrasound is administered in combination with a mechanical or osmotic force field. 11. The method of claim 2 wherein the ultrasound is administered in combination with iontophoresis. 12. The method of claim 2 wherein the compound to be transported is a drug the patient is in need of. 13. The method of claim 2 wherein the compound to be transported is an analyte to be measured |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION The present invention generally relates to improved methods for drug delivery and measurement of analyte using ultrasound in combination with chemical and/or physical enhancers of transport. The United States government has rights in this invention by virtue of NIH grant GM44884 to R. Langer. Transdermal drug delivery (TDD) offers several advantages over traditional delivery methods including injections and oral delivery. When compared to oral delivery, TDD avoids gastrointestinal drug metabolism, reduces first-pass effects, and provides sustained release of drugs for up to seven days, as reported by Elias, In Percutaneous Absorption: Mechanisms--Methodoloqy-Drag Delivery, Pronaugh, R. L., Maibach, H. 1. (Ed), pp 1-12, Marcel Dekker, New York, 1989. The word "transdermal" is used herein as a generic term. However, in actuality, transport of drugs occurs only across the epidermis where the drug is absorbed in the blood capillaries. When compared to injections, TDD eliminates the associated pain and the possibility of infection. Theoretically, the transdermal route of drug administration could be advantageous in the delivery of many therapeutic proteins, because proteins are susceptible to gastrointestinal degradation and exhibit poor gastrointestinal uptake, proteins such as interferons are cleared rapidly from the blood and need to be delivered at a sustained rate in order to maintain their blood concentration at a high value, and transdermal devices are easier to use than injections. In spite of these advantages, very few drugs and no proteins or peptides are currently administered transdermally for clinical applications because of the low skin permeability to drugs. This low permeability is attributed to the stratum corneum (SC), the outermost skin layer which consists of flat, dead cells filled with keratin fibers (keratinocytes) surrounded by lipid bilayers. The highly-ordered structure of the lipid bilayers confers an impermeable character to the SC (Flynn, G. L., In Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery.; Bronaugh, R. L., Maibach, H. I. (Ed), pages 27-53, Marcel Dekker, New York, 1989). A variety of approaches have been suggested to enhance transdermal transport of drugs. These include: i) use of chemicals to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al. In "Drug Permeation Enhancement"; Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994; Burnette, R. R. In Developmental Issues and Research Initiatives; Hadgraft J., G., R. H., Eds., Marcel Dekker: 1989; pp. 247-288); ii) applications of electric fields to create transient transport pathways ›electroporation! (Prausnitz Proc. Natl. Acad. Sci.USA 90, 10504-10508 (1993); Walters, K. A., in Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Ed. Hadgraft J., Guy, R. H., Marcel Dekker, 1989) or to increase the mobility of charged drugs through the skin ›iontophoresis!, and iii) application of ultrasound ›sonophoresis!. Electroporation is believed to work in part by creating transient pores in the lipid bilayers of the SC (Burnett (1989)). Iontophoresis provides an electrical driving force to move compounds. Chemical enhancers have been found to increase transdermal drug transport via several different mechanisms, including increased solubility of the drug in the donor formulation, increased partitioning into the SC, fluidization of the lipid bilayers, and disruption of the intracellular proteins (Kost and Langer, In Topical Drug Bioavailability, Bioequivalence, and Penetration; Shah and Maibech, ed. (Plennum, N.Y. 1993) pp. 91-103 (1993)). Ultrasound has been shown to enhance transdermal transport of low-molecular weight drugs (molecular weight less than 500) across human skin, a phenomenon referred to as sonophoresis (Levy, J. Clin Invest. 1989, 83, 2974-2078; Langer, R., In "Topical Drug Bioavailability, Bioequivalence, and Penetration"; pp. 91-103, Shah V. P., M.H.I., Eds. (Plenum: New York, 1993); Frideman, R. M., `Interferons: A Primer`, Academic Press, New York, 1981)). Ultrasound has been shown to create cavitation within the SC, which disorders the lipid bilayers and increases drug transport (Walters, In Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadraft, ed. (Marcel Dekker, 1989) pp. 197-233). U.S. Pat. Nos. 4,309,989 to Fahim and 4,767,402 to Kost, et al., disclose various ways in which ultrasound has been used to achieve transdermal drug delivery. Sonophoresis has been shown to enhance transdermal transport of various drugs. Although a variety of ultrasound conditions have been used for sonophoresis, the most commonly used conditions correspond to the therapeutic ultrasound (frequency in the range of 1 MHz-3 MHz, and intensity in the range of 0-2 W/cm.sup.2) (Kost, In Topical Drug Bioavailability Bioequivalence and Penetration, pp. 91-103, Maibach, H. I., Shah, V. P. (Ed) Plenum Press, New York, 1993; U.S. Pat. No. 4,767,402 to Kost, et al.). U.S. Pat. No. 5,445,611 to Eppstein, et al., describes enhancement of ultrasound using the combination of chemical enhancers with modulation of the frequency, intensity, and/or phase of the ultrasound to induce a type of pumping action. However, the intensity and frequencies used in the examples are quite high, which generates heat and decreasing transport over time. In a recent study of sonophoresis, it has been shown that application of ultrasound at therapeutic frequencies (1 MHz) induces growth and oscillations of air pockets present in the keratinocytes of the SC (a phenomenon known as cavitation). These oscillations disorganize the SC lipid bilayers thereby enhancing transdermal transport. However, application of therapeutic ultrasound does not induce transdermal transport of high-molecular weight proteins. It is a common observation that the typical enhancement induced by therapeutic ultrasound is less than ten-fold. In many cases, no enhancement of transdermal drug transport has been observed upon ultrasound application. Accordingly, a better selection of ultrasound parameters is needed to induce a higher enhancement of transdermal drug transport by sonophoresis. Moreover, although efficacy to some degree has been observed using ultrasound for transport of other compounds, the efficiency of transport under conditions acceptable to patients has not been achieved. It is therefore an object of the present invention to provide a method and means for enhancing transdermal transport. It is a further object of the present invention to provide methods for using ultrasound in combination with other means of enhancement for drug delivery and collection of analyte in an efficient, practical manner. SUMMARY OF THE INVENTION Transdermal transport of molecules during sonophoresis (delivery or extraction) can be further enhanced by providing chemical enhancers which increase the solubility of the compound to be transported and/or lipid bilayer solubility, or additional driving forces for transport, such as, mechanical force fields, magnetic fields or iontophoresis. In a preferred embodiment, the ultrasound is low frequency ultrasound which induces cavitation of the lipid layers of the stratum corneum (SC). This method i) provides higher transdermal fluxes, ii) allows rapid control of transdermal fluxes, and iii) allows drug delivery or analyte extraction at lower ultrasound intensities and other forces or concentrations than that required if each means of enhancing transport is used individually. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph of solubility (mg/ml) for corticosterone, dexamethasone, and testosterone in PBS (dark bar), 50% ethanol (hatched .backslash..backslash..backslash.) and linoleic acid in 50% ethanol (striped .vertline..vertline..vertline..vertline.). FIG. 2 is graph of the permeability enhancement for testosterone (288.4 Da), corticosterone (346.5 Da), dexamethasone (392.5 Da) in combination with linoleic acid (dark bars) and ultrasound in combination with linoleic acid enhancement (.backslash..backslash..backslash.) FIG. 3a is a graph of enhancement (log scale) versus molecular weight (Da). FIG. 3b is a graph of permeability enhancement versus molecular weight (Da). FIG. 4a is a graph of fraction of corticosterone transported (%) versus time (hours). FIG. 4b is a graph of the fraction of corticosterone transported (%) versus time (hours). FIG. 5 is a graph of the fraction of corticosterone transported (%) versus time (hours). FIG. 6 is a graph of the amount of calcein transported in one hour (fraction of the amount in the donor.times.10.sup.5) for sonophoresis alone, iontophoresis alone, and sonophoresis in combination with iontophoresis. FIG. 7 is a graph of the glucose concentration in the donor (fraction of receiver concentration). DETAILED DESCRIPTION OF THE INVENTION Sonophoresis: As used herein, sonophoresis is the application of ultrasound to the skin, alone or in combination with chemical enhancers, iontophoresis, electroporation, magnetic force fields, mechanical pressure fields or electrical fields, to facilitate transport of a compound through the skin. In one embodiment, a drug, alone or in combination with a carrier, penetration enhancer, lubricant, or other pharmaceutically acceptable agent for application to the skin, is applied to the skin. In another embodiment, the compound is an analyte such as glucose which is present in a body fluid and extracted by application of the ultrasound, alone or in combination with other forces and/or chemical enhancers. Ultrasound is defined as sound at a frequency of between 20 kHz and 10 MHz, with intensities of between greater than 0 and 3 W/cm.sup.2. Ultrasound B preferably administered at frequencies of less than or equal to about 2.5 MHz to induce cavitation of the skin to enhance transport. As used herein, "low frequency" sonophoresis is ultrasound at a frequency that is less than 1 MHz, more typically in the range of 20 to 40 KHz, which can be applied continuously or in pulses, for example, 100 msec pulses every second, at intensities in the range of between zero and 1 W/cm.sup.2, more typically between 12.5 mW/cm.sup.2 and 225 mW/cm.sup.2. Exposures are typically for between 1 and 10 minutes, but may be shorter and/or pulsed. It should be understood that although the normal range of ultrasound is 20 kHz, one could achieve comparable results by varying the frequency to slightly more or less than 20 kHz. The intensity should not be so high as to raise the skin temperature more than about one to two degrees Centigrade. Application of low-frequency (20 kHz) ultrasound dramatically enhances transdermal transport of drugs. Transdermal transport enhancement induced by low-frequency ultrasound was found to be as much as 1000-fold higher than that induced by therapeutic ultrasound (frequency in the range of 1 MHz-3 MHz, and intensity in the range of 0-2 W/cm.sup.2). Another advantage of low-frequency sonophoresis as compared to therapeutic ultrasound is that the former can induce transdermal transport of drugs which do not passively permeate across the skin. Application of low-frequency ultrasound appears to induce cavitation inside as well as outside the skin. Cavitation occurring at either location may cause disordering of the SC lipids. In addition, oscillations of cavitation bubbles may result in significant water penetration into the disordered lipid regions. This may cause the formation of aqueous channels through the intercellular lipids of the SC. This allows permeants to transport across the disordered lipid domains, then across keratinocytes and the entire SC. This transport pathway may result in an enhanced transdermal transport as compared to passive transport because the diffusion coefficients of permeants through water, which is likely to primarily occupy the channels generated by ultrasound, are up to 1000-fold higher than those through the ordered lipid bilayers, and the transport path length of these aqueous channels may be much shorter (by a factor of up to 25) than that through the tortuous intercellular lipids in the case of passive transport. Many ultrasound devices are available commercially which can be used in the method described herein. For example, the ultrasonic devices used by dentists to clean teeth have a frequency of between about 25 and 40 KHz. Commercially available portable ultrasound tooth-brushes make use of a small sonicator contained within the tooth-brush (Sonex International Corporation). This sonicator is portable and operates on rechargeable batteries. Small pocket-size sonicators carried by patients and used to "inject" drugs whenever required could be readily adapted from these devices. In addition, these devices could be combined with sensors that can monitor drug concentrations in the blood to formulate a self-controlled drug (insulin, for example) delivery method that can decrease the attention required by the patient. Devices typically used for therapeutic or diagnostic ultrasound operate at a frequency of between 1.6 and 10 MHz. These devices can also be modified for use at lower frequencies. Lipid Bilayer Disrupting Agents Chemical enhancers have been found to increase drug transport by different mechanisms. In the preferred embodiment described herein, chemicals which enhance the solubility of compounds to be delivered or measured are used in combination with chemicals which enhance permeability through lipids. Many chemicals having these properties are known and commercially available. For example, ethanol has been found to increase the solubility of drugs up to 10,000-fold (Mitragotri, et al. In Encl. of Pharm. Tech.: Swarbrick and Boylan, eds. Marcel Dekker 1995) and yield a 140-fold flux increase of estradiol, while unsaturated fatty acids have been shown to increase the fluidity of lipid bilayers (Bronaugh and Maiback, editors (Marcel Dekker 1989) pp. 1-12). Examples of fatty acids which disrupt lipid bilayer include linoleic acid, capric acid, lauric acid, and neodecanoic acid, which can be in a solvent such as ethanol or propylene glycol. Evaluation of published permeation data utilizing lipid bilayer disrupting agents agrees very well with the observation of a size dependence of permeation enhancement for lipophilic compound, as discussed below. The permeation enhancement of three bilayer disrupting compounds, capric acid, lauric acid, and neodecanoic acid, in propylene glycol has been reported by Aungst, et al. Pharm. Res. 7, 712-718 (1990). They examined the permeability of four lipophilic compounds, benzoic acid (122 Da), testosterone (288 Da), naloxone (328 Da), and indomethacin (359 Da) through human skin. The permeability enhancement of each enhancer for each drug was calculated according to .epsilon..sub.c/pg =P.sub.e/pg /P.sub.pg, where P.sub.e/pg is the drug permeability from the enhancer/propylene glycol formulation and P.sub.pg is the permeability from propylene glycol alone. The primary mechanism by which unsaturated fatty acids, such as linoleic acid, are thought to enhance skin permeabilities is by disordering the intercellular lipid domain. For example, detailed structural studies of unsaturated fatty acids, such as oleic acid, have been performed utilizing differential scanning calorimetry Barry J. Controlled Release 6, 85-97 (1987) and infrared spectroscopy ongpipattanankul, et al., Pharm. Res. 8, 350-354 (1991); Mark, et al., J. Control. Rel. 12, 67-75 (1990). Oleic acid was found to disorder the highly ordered SC lipid bilayers, and to possibly form a separate, oil-like phase in the intercellular domain. SC lipid bilayers disordered by unsaturated fatty acids or other bilayer disrupters may be similar in nature to fluid phase lipid bilayers. A separated oil phase should have properties similar to a bulk oil phase. Much is known about transport in fluid bilayers and bulk oil phases. Specifically, diffusion coefficients in fluid phase, for example, dimyristoylphosphatidylcholine (DMPC) bilayers Clegg and Vaz In "Progress in Protein-Lipid Interactions" Watts, ed. (Elsvier, N.Y. 1985) 173-229; Tocanne, et al., FEB 257, 10-16 (1989) and in bulk oil phase Perry, et al., "Perry's Chemical Engineering Handbook" (McGraw-Hill, NY 1984) are greater than those in the SC, and more importantly, they exhibit size dependencies which are considerably weaker than that of SC transport Kasting, et al., In: "Prodrugs: Topical and Ocular Delivery" Sloan, ed. (Marcel Dekker, NY 1992) 117-161; Potts and Guy, Pharm. Res. 9, 663-339 (1992); Willschut, et al., Chemosphere 30, 1275-1296 (1995). As a result, the diffusion coefficient of a given solute will be greater in a fluid bilayer, such as DMPC, or a bulk oil phase than in the SC. Due to the strong size dependence of SC transport, diffusion in SC lipids is considerably slower for larger compounds, while transport in fluid DMPC bilayers and bulk oil phases is only moderately lower for larger compounds. The difference between the diffusion coefficient in the SC and those in fluid DMPC bilayers or bulk oil phases will be greater for larger solutes, and less for smaller compounds. Therefore, the enhancement ability of a bilayer disordering compound which can transform the SC lipids bilayers into a fluid bilayer phase or add a separate bulk oil phase should exhibit a size dependence, with smaller permeability enhancements for small compounds and larger enhancements for larger compounds. A comprehensive list of lipid bilayer disrupting agents is described in European Patent Application 43,738 (1982), which is incorporated herein by reference. Exemplary of these compounds are those represented by the formula: R--X wherein R is a straight-chain alkyl of about 7 to 16 carbon atoms, a non-terminal alkenyl of about 7 to 22 carbon atoms, or a branched-chain alkyl of from about 13 to 22 carbon atoms, and X is --OH, --COOCH.sub.3, --COOC.sub.2 H.sub.5, --OCOCH.sub.3, --SOCH.sub.3, --P(CH.sub.3).sub.2 O, COOC.sub.2 H.sub.4 OC.sub.2 H.sub.4 OH, --COOCH(CHOH).sub.4 CH.sub.2 OH, --COOCH.sub.2 CHOHCH.sub.3, COOCH.sub.2 CH(OR")CH.sub.2 OR", --(OCH.sub.2 CH.sub.2).sub.m OH, --COOR', or --CONR'.sub.2 where R' is --H, --CH.sub.3, --C.sub.2 H.sub.5, --C.sub.2 H.sub.7 or --C.sub.2 H.sub.4 OH; R" is --H, or a non-terminal alkenyl of about 7 to 22 carbon atoms; and m is 2-6; provided that when R" is an alkenyl and X is --OH or --COOH, at least one double bond is in the cis-configuration. Solubility Enhancers Suitable solvents include water; diols, such as propylene glycol and glycerol; mono-alcohols, such as ethanol, propanol, and higher alcohols; DMSO; dimethylformamide; N,N-dimethylacetamide; 2-pyrrolidone; N-(2-hydroxyethyl) pyrrolidone, N-methylpyrrolidone, 1-dodecylazacycloheptan-2-one and other n-substituted-alkyl-azacycloalkyl-2-ones and other n-substituted-alkyl-azacycloalkyl-2-ones (azones). U.S. Pat. No. 4,537,776 to Cooper, incorporated herein by reference contains a summary of prior art and background information detailing the use of certain binary systems for permeant enhancement. European Patent Application 43,738, also describes the use of selected diols as solvents along with a broad category of cell-envelope disordering compounds for delivery of lipophilic pharmacologically-active compounds. A binary system for enhancing metaclopramide penetration is disclosed in UK Patent Application GB 2,153,223 A, consisting of a monovalent alcohol ester of a C8-32 aliphatic monocarboxylic acid (unsaturated and/or branched if C18-32) or a C6-24 aliphatic monoalcohol (unsaturated and/or branched if C14-24) and an N-cyclic compound such as 2-pyrrolidone or N-methylpyrrolidone. Combinations of enhancers consisting of diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate and methyl laurate are disclosed in U.S. Pat. No. 4, 973,468 for enhancing the transdermal delivery of steroids such as progestogens and estrogens. A dual enhancer consisting of glycerol monolaurate and ethanol for the transdermal delivery of drugs is described in U.S. Pat. No. 4,820,720. U.S. Pat. No. 5,006,342 lists numerous enhancers for transdermal drug administration consisting of fatty acid esters or fatty alcohol ethers of C.sub.2 to C.sub.4 alkanediols, where each fatty acid/alcohol portion of the ester/ether is of about 8 to 22 carbon atoms. U.S. Pat. No. 4,863,970 discloses penetration-enhancing compositions for topical application including an active permeant contained in a penetration-enhancing vehicle containing specified amounts of one or more cell-envelope disordering compounds such as oleic acid, oleyl alcohol, and glycerol esters of oleic acid; a C.sub.2 or C.sub.3 alkanol and an inert diluent such as water. Other chemical enhancers, not necessarily associated with binary systems, include dimethylsulfoxide (DMSO) or aqueous solutions of DMSO such as those described in U.S. Pat. No. 3,551,554 to Herschler; U.S. Pat. No. 3,711,602 to Herschler; and U.S. Pat. No. 3,711,606 to Herschler, and the azones (n-substituted-alkyl-azacycloalkyl-2-ones) such as noted in U.S. Pat. No. 4,557,943 to Cooper. Some chemical enhancer systems may possess negative side effects such as toxicity and skin irritations. U.S. Pat. No. 4,855,298 discloses compositions for reducing skin irritation caused by chemical enhancer-containing compositions having skin irritation properties with an amount of glycerin sufficient to provide an anti-irritating effect. Combinations of Lipid Bilayer Disrupting Agents and Solvents Ethanol and the unsaturated fatty acid linoleic acid were combined (LA/ethanol) and studied as described in the following examples. Single component enhancer formulations, including polyethylene glycol 200 dilaurate (PEG), isopropyl myristate (IM), glycerol trioleate (GT), ethanol/pH 7.4 phosphate buffered saline in a one-to-one ratio (50% ethanol), and PBS were also examined. The examples compare the effects and mechanisms of (i) a series of chemical enhancers, and (ii) the combination of these enhancers and therapeutic ultrasound (1 MHz, 1.4 W/cm2) on transdermal drug transport. Initial/comprehensive experiments were performed with a model drug, corticosterone, and a series of chemical enhancer formulations, including polyethylene glycol 200 dilaurate (PEG), isopropyl myristate (IM), glycerol trioleate (GT), ethanol/pH 7.4 phosphate buffered saline in a one-to-one ratio (50% ethanol), 50% ethanol saturated with linoleic acid (LA/ethanol), and phosphate buffered saline (PBS). Passive experiments without ultrasound) with PEG, IM, and GT resulted in corticosterone flux enhancement values of only 2, 5, and 0.8, relative the to the passive flux from PBS alone. However, 50% ethanol and LA/ethanol significantly increased corticosterone passive fluxes by factors of 46 and 900. These passive flux enhancements were due to (1) the increased corticosterone solubility in the enhancers, and (2) interactions of linoleic acid with the skin. Specifically, linoleic acid increased the corticosterone permeability by nearly 20-fold over that from 50% ethanol alone. Therapeutic ultrasound (1 MHz, 1.4 W/cm.sup.2) and the chemical enhancers utilized together produced corticosterone fluxes from PBS, PEG, IM, and GT that were greater than the passive fluxes from the same enhancers by factors of between 1.3 and 5.0, indicating that the beneficial effects of chemical enhancers and therapeutic ultrasound can be effectively combined. Ultrasound combined with 50% ethanol produced a 2-fold increase in corticosterone transport above the passive case, but increased by 14-fold the transport from LA/Ethanol. The combination of increased corticosterone solubility in and permeability enhancement from LA/ethanol and ultrasound yields a flux of 0.16 mg/cm.sup.2 /hr, 13,000-fold greater than that from PBS alone. In order to assess the generality of enhancement ability of LA/ethanol and ultrasound, further experiments were performed with two additional model drugs, dexamethasone and testosterone. As with corticosterone, the solubilities in and passive permeabilities from LA/ethanol were much larger than those from PBS alone for dexamethasone and testosterone. The sonophoretic permeabilities from LA/ethanol were also greater for these two drugs than the passive permeabilities. Moreover, the permeability enhancements of the three drugs resulting from the addition of linoleic acid to 50% 3thanol exhibited a clear size dependence, with the degree of enhancement increasing with the size of the drug. The degree of permeation enhancement achieved by adding linoleic acid to 50% ethanol and applying ultrasound exhibits a similar size dependence. These results suggest that linoleic acid and therapeutic ultrasound, which are both lipid bilayer disordering agents, shift the transport of lipophilic molecules from the passive regime to a regime with a very weak size dependence. Physical Enhancers Although principally described herein as the combination of ultrasound with chemical enhancers, physical enhancers can also be used in combination with ultrasound, alone or in combination with chemical enhancers. Physical enhancers, as used herein, include inotophoresis, elect5roporation, magnetic fields, and mechanical pressure. Ultrasound is used to permeabilize the skin followed by the application of various force fields to provide additional driving force for transdermal transport of molecules. Electric Fields (Iontophoresis or Electroporation) Application of ultrasound or electric current alone has been shown to enhance transdermal drug transport and blood analyte extraction. As discussed above, ultrasound-induced cavitation occurring inside or outside the skin causes disordering of the SC lipids. Oscillations of cavitation bubbles may also result in significant water penetration into the disordered lipid regions. This may cause the formation of aqueous channels through the intercellular lipids of the SC, thus allowing permeants to transport across the disordered lipid domains. Once able to diffuse across the lipid domains, molecules may diffuse across keratinocytes and hence across the entire SC. Application of electric current enhances transdermal transport by different mechanisms. First, application of an electric field provides an additional driving force for the transport of charged molecules across the skin and second, ionic motion due to application of electric fields may induce convective flows across the skin, referred to as electroosmosis. This mechanism is believed to play a dominant role in transdermal transport of neutral molecules during iontophoresis. Iontophoresis involves the application of an electrical current, preferably DC, or AC, at a current density of greater than zero up to about 1 mA/cm.sup.2. Typically, a constant voltage is applied since resistance changes over time, usually in the range of between greater than zero and four volts. Attempts have been made to enhance the skin permeability using electric current to achieve transdermal extraction of glucose Tamada, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 22, 129-130 (1995). Although these attempts have been successful to a certain extent, the amounts of glucose extracted by these methods are several orders of magnitude lower than those which could be detected by the currently existing biosensors. The mechanism of sonophoretic transdermal glucose extraction is believed to be similar to that of sonophoretic transdermal drug delivery. Specifically, application of low-frequency ultrasound increases the skin permeability by disordering its lipid bilayers which leads to the formation of aqueous channels through the intercellular lipids of the SC. This allows faster diffusion of glucose present in the interstitial fluids across the permeabilized skin. As used herein, ultrasound is used in combination with application of an electric current. As shown by Example 2, the results obtained using the combination are significantly better than either alone. Mechanical or Osmotic Pressure The advantages of combining sonophoresis with physical enhancers is not restricted to electric current. Effects on transdermal transport may also be observed between ultrasound and pressure (mechanical or osmotic) as well as between ultrasound and magnetic fields since the physical principles underlying the possible enhancement are the same. A pressure gradient can be used to enhance convection (physical movement of liquid) across the skin permeabilized by sonophoresis. This can be particularly useful in transdermal extraction of blood analytes. Application of pressure, for example, a vacuum or mechanical pressure, to the skin pretreated by sonophoresis can result in transdermal extraction of interstitial fluid which can be analyzed to measure concentration of various blood analytes. Magnetic Fields Application of magnetic fields to the skin pretreated with ultrasound may also result in a higher transport of magnetically active species across the skin. For example, polymer microspheres loaded with magnetic particles could be transported across the skin using sonophoresis and magnetic fields. The combination of sonophoresis with any of these additional physical mechanisms for enhanced transport provides the following advantages over sonophoresis or the physical enhancers alone: i) It allows lowering application times to deliver a given drug dose or extract a certain amount of analytes compared to the required times in the presence of ultrasound or one of the other enhancers alone; ii) It reduces the magnitude of the required ultrasound intensity and electric current or pressure to achieve a given transdermal flux compared to that required if, the enhancers were used alone; and iii) It can be used to provide a better control over transdermal transport of molecules compared to that obtained using an enhancer alone. Drug Delivery Drugs to be Administered Drugs to be administered include a variety of bioactive agents, but are preferably proteins or peptides. Specific examples include insulin, erythropoietin, and interferon. Other materials, including nucleic acid molecules such as antisense and genes encoding therapeutic proteins, synthetic organic and inorganic molecules including antiinflammatories, antivirals, antifungals, antibiotics, local anesthetics, and saccharides, can also be administered. The drug will typically be administered in an appropriate pharmaceutically acceptable carrier having an absorption coefficient similar to water, such as an aqueous gel. Alternatively, a transdermal patch such as the one described in the examples can be used as a carrier. Drug can be administered in a gel, ointment, lotion, suspension or patch, which can incorporate anyone of the foregoing. Drug can also be encapsulated in a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated). A number of suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof. The microparticles can have diameters of between 0.001 and 100 microns, although a diameter of less than 10 microns is preferred. The microparticles can be coated or formed of materials enhancing penetration, such as lipophilic materials or hydrophilic molecules, for example, polyalkylene oxide polymers and conjugates, such as polyethylene glycol. Liposome are also commercially available. Administration of Drug The drug is preferably administered to the skin at a site selected based on convenience to the patient as well as maximum drug penetration. For example, the arm, thigh, or stomach represent areas of relatively thin skin and high surface area, while the hands and feet are uneven and calloused. In the preferred embodiment, drug is applied to the site and ultrasound applied immediately thereafter. Chemical and physical enhancers can be applied before, during or immediately after the ultrasound. Chemical enhancers are preferable administered during or before ultrasound. Based on these calculations and the data in the following examples, one can calculate the required dosage and application regime for treatment of a patient, as follows. A typical diabetic patient (70 Kg weight) takes about 12 Units of insulin three times a day (total dose of about 36 Units per day: cited in `World Book of Diabetes in Practice` Krall, L. P. (Ed), Elsvier, 1988). If each insulin dose was to be delivered by sonophoresis in 1 hour, the required transdermal flux would be 12 U/hour. Note that 1 unit (1 U) of insulin corresponds approximately to 40 mg of insulin. The transdermal patch area used in these calculations is 40 cm.sup.2 (the area of a transdermal FENTANYL.TM. patch ›ALZA Corporation!). The donor concentrations used in these calculations are 100 U/ml in the case of insulin (commercially available insulin solution ›Humulin!), 3.times.10.sup.7 in the case of .gamma.-interferon (typical concentration of interferon solution recommended by Genzyme Corporation), and 3.times.10.sup.5 U/ml in the case of erythropoeitin ›Davis, et al., Biochemistry, 2633-2638, 1987!. A typical .gamma.-interferon dose given each time to patients suffering from cancer or viral infections is about 5.times.10.sup.6 U ›(i) Grups, et al., Br. J. Med., 1989, 64 (3): 218-220, (ii) Parkin, et al., Br. Med. J., 1987, 294: 1185-1186!. Similar doses of .alpha.-interferon and .beta.-interferon have also been shown to enhance the immune response of patients suffering from viral infections and cancer (cited in `Clinical Applications of interferons and their inducers`, Ed. Stringfellow D., Marcel Dekker, New York, 1986). If this interferon dose was to be given by sonophoresis in 1 hour, the required transdermal flux would be 5.times.10.sup.6 U/hour. Note that 1 unit of .gamma.-interferon corresponds approximately to 1 pg of .gamma.-interferon. A typical daily erythropoeitin dose given subcutaneously to anemic patients is about 400 U (cited in `Subcutaneous Erythropoeitin, Bommer J., Ritz E., Weinreich T., Bommer G., Ziegler T., Lancet, 406, 1988). If this dose was to be delivered in three steps, each involving sonophoresis for 1 hour, the transdermal flux required would be about 140 U/hour. Note that 1 unit of erythropoeitin corresponds approximately to 7.6 nanograms of erythropoeitin. Optimal selection of ultrasound parameters, such as frequency, pulse length, intensity, as well as of non-ultrasonic parameters, such as ultrasound coupling medium, can be conducted to ensure a safe and efficacious application using the guidelines disclosed herein as applied by one of ordinary skill in the art. Measurement of Analytes Analytes to be Measured A variety of analytes are routinely measured in the blood, lymph or other body fluids. Measurements usually require making a puncture in order to withdraw sample. Examples of typical analytes that can be measured include blood sugar (glucose), cholesterol, bilirubin, creatine, various metabolic enzymes, hemoglobin, heparin, vitamin K or other clotting factors, uric acid, carcinoembryonic antigen or other tumor antigens, and various reproductive hormones such as those associated with ovulation or pregnancy. Transdermal drug delivery, in combination with the non-invasive blood analyte measurements, may be used to formulate self-regulated drug delivery methods which provide: a close control of the blood concentrations, minimal pain, and better patient compliance. Non-invasive blood analysis method includes extraction of various analytes from the skin's interstitial fluids (where the analytes are present at a concentration proportional to the blood concentration) across the skin into a patch, solution or gel, where their concentration can be measured using biosensors. This method of blood analyte measurements should be particularly useful in the case of diabetic patients who require multiple daily blood glucose measurements. Measurement of Analytes The ultrasound is applied to the skin at the site where the sample is to be collected. A reservoir or collecting container is applied to the site for collection of the sample, which is then measured using standard techniques. The ultrasound conditions are optimized as in the case for drug delivery, to maximize analyte recovery, while maintaining the relative levels of the analyte to other components of the sample. Chemical and/or physical enhancers are applied to the site before, during and after the ultrasound, preferably during or before the ultrasound. |
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