CA2222629A1 - Dermal patch without a separate absorbent material - Google Patents

Abstract

A membrane (250) comprising an adhesive is secured to the skin (12) of a subject. The membrane (250) retains concentrated analyte in or on the membrane (250) itself, in the adhesive, and in the desquamated keratinized stratum corneum cells of the skin (12).

Description

W O 96~9923 _ PCTrUS96/06915 DERMAL PATCH WITHOUT A SEPARATE ABSOR~E,. I MATE~IAL
Field of the Invention The present invention relates to dermal patches for the detecbon of analytes c ~ -d in a body fluid.
Such patches can detect analytes wlthout . i ~ _ an L L material which is separate from another layer 5of the patch that is exposed to the environment.
B; ~kl 1 ' of the Invention c A Inducing ~"
Early i .. ~ of the components of r _~ - used various means to increase the quantity of r, which they could collect from subjects and i' ~d~l.,. analyze. One such means of inducing 10 1 ,~, - involved placing rubber gloves over the hands of a subject. When r -r- - a- ' ' on the subject's hands, it was collected for analysis (U.S. Patent No. 3,5~ q~ to Fields, et al.).
Chemical means have also been du.. ~(, ' to aLc.,lu.dle the collection r , For example, ' such as, ' r have been ' .,d to increase F :, ~ One way of these r' ' ~ iS to ,' -r~e them into the skin (Gibson, B ' l~ , 23 545r 1959)~
P~ ai r' ' ~', used in ; with abrasion to the skin, have also been employed (see, e.g., U.S. Patent No. 4~756~314 to rr~ , et al.) Heat has been used as well in - : with the detection of analytes under the skin and with the transport of b into a subject's body through the skin. In U.S. Patent No. 4~401~122~ Clark describes a method of allL. " _ the skin of a subject with heat or other means. Clark specifies that a subject's skin can be heated to 3844~C in order to control a chemical reaction beneath the skin or in order to acc~l~.dle diffusion through the skin. Jacques, et al. (in U.S. Patent No. 4~775~361) also describe the use of pulsed laser energy to enhance F~ ..ulaneo..;. transport.
B. re., ' dliUn and Other r7 ~., Media As the result of the collection and analysis of p~. . ). it has been found that r ~ ~ contains a variety of analytes of interest. In order to detect such analytes, a sufficient quantity of r ~ . ' dUUIl must first be collected from a subject so that the F , can be subjected to analysis. Prior art dermal patches were normally -' on the skin of a subject for 24 hours in order to collect sufficient p~., (see, e.g., U.S.
Patent No. 4~706~676 to Peck and U.S. Patent Nos. 4~732~153 and 4r329r999 to Phillips). The result of using this method of collecting an analyte is a long-term L~u,, of the CuuL.e.~ll_ ' of the analyte in the subject's p~ over the wear period. Specific rl I as to when the analyte was in the body or whether the patch was exposed to one large or multiple smaller amounts of the analyte is lost in such long-term wear Other d : media can reveal different ;"O lllai regarding the CD~ ' . ' of an analyte For example, the analysis of a venous blood sample reveals the c~ - aliun of an analyte in the venous L;ll ' I y system at the instant that the sample is taken. A urine sample, on the other hand, contains hl~ Illd6un as t CA 02222629 l997-ll-27 W O 96~9923 2- PCTrUS96/06915 an analyte's - - that is : ~ in between the i i f~ of a blood sample and the _._. " d c~, available from dermal patches. A urine specimen is ", ~ of the ~ _ of an analyte in the body between complete voids, so that the higher the r-l . , of voids is, the closer the urine specimen will represent the situation.
C, ~ ., 'i. Kits for ~ , rL,, A variety of " kits for I _ an analyte in, , _ have been ~ .. ', ' For example, U.S. Patent No. 3,~ -q to Fields, et al. discloses a BAND-AID type test patch suited for determining the chloride ion L ~ in, , ~ as a method of " _ ~ _ cystic fibrosis. The 9~, ail,~ disclosed in Fields r , i._j an ' ~ ~.li._ r collecting pad with an impermeable overlying layer for the purpose of, ~.. ... , When the -' ~: ., pad is saturated, the patch is removed from the skin and exposed to a series of strips _~, ' with inL,. ' q of silver chromate or silver nitrate, the color of which ~ a well known change upon - ._. to the chloride salt.
U.S. Patent No. 4,706,676 to Peck discloses a dermal collection device which: , ises a binder to prevent reverse migration of an analyte, a liquid transfer medium which permits transfer of an analyte from the dermal surface to the binder, and an occlusive cover across the top of the liquid transfer medium and binder.
Peck also discloses the 1" ' of such a dermal collection device to detect various .;. ' ' to which humans are exposed. After the dermal collection device has been worn on a patient's skin for a period of time, the device is removed for analysis, which involves the chemical , of the bound substance of interest from the binding reservoir and i' L~rl~, c ' ~ " 9~_ andlor :1 _ Illec~. of the ---' e of interest by r .~ ' ' ' , y t~ ' -Another dermal collection device, disclosed in U.S. Patent No. 4,756,314 to Cck~,,l,urt, claims to q _l~ collect p~" on a dermal patch. This patch uses a diffusion rate-limited ' - as a means to maintain a constant flow of fluid into the patch. The patch c , i~es an ' ' outer boundary structure, and is therefore an occlusive patch.
However, prior art dermal patches and other means of collecting r ~' " are generally only useful for d~ the presence of analytes which are present in r -~ ' " in relatively high c ~ . such as halide ions. In addition, the occlusive outer layer-type devices of the prior art are , i ' ' to the problem of back diffusion of I ., andlor the analytes contained therein. Occlusive devices also suffer from other problems, including changes in the skin's transport ~.h~.dLI~Ii;,l;..;, (see, Brebner, D.F., J. Ph~rsiol., 175: Z95-302 (1964) and r~ . R.J., Arch Derm~t., 91: 661-666 (1965)), and the ~ of an aqueous state below the patch, which fosters bacterial growth. Prior art dermal patches suffer from a number of other d '~
as well, including being 'o. '' to wear and being subject to losing analytes due to fluid loss.
Summarv of the Invention In one aspect, the present invention - , i~ds a method for d ~, the presence of an analyte in W O 96/39923 3 PCT~US96/06915 a body fluid of a subject. This method e . i__s the steps of:
,l .. ' 1~ securing a ' to the subject in fluid s with a source of the body fluid, wherein the ' . Iacks a separate ' ' material; and after a period of time sufficient to collect enough of an analyte of interest so that the analyte can be detected in an assay, removing the membrane from the subject.
In a preferred embodiment, this method additionally e , ~.~,vs the step of v-A~ _ the analyte from the membrane, which can involve dissolving or degrading the membrane, although the analyte can also be vA~ without h Lv, _ the sl-l dl integrity of the ' _ In an even more preferred; ' ' t, this method also additionally comprises the step of b; '- v the analyte to an assay which can detect the 1 0 analyte.
In another preferred embodiment, the membrane has a first side which includes an adhesive attached thereto, and the ll ._"~ securing step c , i...,j - the adhesive to the skin of a subject such that the adhesive adheres to the skin of the subject. In this 'JL " t, the step of removing the ' ~ from the subject includes the step of removing stratum corneum cells from the skin of the subject. The body fluid which is collected with the patch of this . '-' is r . . and the analyte which is detected is vr~ ***e.
The ' - used in the foregoing methods . ~r,,. ~1~ comprises a material selected from the group c ~ _ of urethane, r ~ vth,: ethylene vinyl acetate, rayon, polyvinyl chloride, and PTFE.
More, ~r~ , the ' is made from urethane, such as T~v ' lll 1625 wound dressing, or is made from F '~eth,; . such as r '~.,lh,' foam or F l~vth~; - tape. The ' ane material is also ".v-fv~l. ' 1 -'- L- t, and can be fluid I . gas r ~ . or occlusive.
The ' should have a surface area of between 3~ ~ OAilllal~l~ 1 cm2 and 120 cm2, and r.Lf~l ' 1y has a surface area of c"". ~ '~ 42 cm2. The ' should also have a thickness of between about 0.005 mm and 3 mm, and more, ufu. ' 1~ a thickness of about 0.02 mm.
In another aspect of the present invention, the invention c . i~bs a dermal patch for retaining analytes expressed from a body fluid of a subject, wherein the patch ~ .,s a fluid 1~ ' ' ' having a first side including an adhesive attached thereto. In this aspect of the invention, the patch lacks a separate . ' b_..l material. r,~r~ , the ' _ i , ivcs Tev ' lll 1625 wound dressing, has a surface area of 3" uAilllalul~ 42 cm2, and is 3" ~ :'~ 0.02 mm thick. In a preferred ' - ' :, the ' a.,a is also ' - L 1. The ' ~ can also be made from a ' ' ' ' or '(,. ' '' material in order to facilitate the a;- of analytes from the Brief DesL.i i of the Fiqures Figure 1 is a r ~ _ view of a dermal patch according to one b- ' of the present I .. :-Figure 1a is a cross-sectional view along the line 1a-1a of the dermal patch of Figure 1.

WO 96/39923 PCT/U',CI'~6915 Figure 2 is a r , '- ~ view of a dermal patch according to a second: ' " of the present invention.
Figure 2a is a cross SJ~.:- ' view along the line 2a 2a of the dermal patch of Figure 2.
Figure 3 is a r . e _ view of a third embodiment of the dermal patch of the present i ..
Figure 3a is a cross s ' view along the line 3a-3a of the patch of Figure 3.
Figure 4 is a r . view of one b~ " of a reagent packet for use in effecting a color change 1., ~ _ to the presence of analyte in the patch of the present invention.Figure 5 is an exploded cross s ' ' view of a fourth ' ' of the present invention.
Figure 6 is a cross ~i ' view of a dermal patch according to a further embodiment of the present 1 0 invention.
Figure 7 is a r , -~ view of a dermal patch according to another ' - " of the present invention.
Figure 8 is an exploded ~I~. ' view of a dermal patch according to yet another r ~ ~ ~' ' of the present invention.
Figure 9 is a plan view of a dermal patch according to a further embodiment of the present invention.
Figure 10 is an exploded el~. ' view according to still another r ' " of the present invention.
Figure 11 is a cross 5 ' view of a dermal patch of the present invention which includes a pooling area.
Figure 12 is a chart " : . _ the results of a test of a dermal patch of the present invention. This chart compares the amount of ***e collected on a patch placed on a subject who ingested ***e versus the amount of BE detected in the urine of that subject over the course of almost 200 hours.
Figure 13 is a graph showing the results of an . : in which a dermal patch was placed on a subject who was ~ d 32 mg of ***e ~ ~ . At the end of each of the time periods shown on the I i ' axis of the graph (ILI~ the amounts of time following the ~ of the ***e~, the patch worn by the subject during such period of time was removed and replaced by a new patch. The amount of ***e found in each of these patches is charted on the vertical axis of the graph over the point on the hl ' axis ll . to the period of time during which the patch was worn.
Figure 14 is a graph similar to that of Figure 13 showing another . ~ involving the same subject.
In this eA~ i t, the subject was - ' ~ ~ - Ld 42 mg of ***e via smoking.
Figure 15A is a cross- ' view of one - b~' of an: ~" a.. ~ tLd dermal patch according to the present invention.
Figure 15B is a view of the top of the patch of Figure 15A when such a patch is present on the skin of a subject.
Figure 15C is a cross sectional view of the chemical ~ . and bag layers shown in Figure 15A.

W O 96/39923 PCTrUS96/06915 Figure 16 is a graph depicting the results of an i in which a subject was _ ~ ~ ' codeine and a dermal patch was then placed on each of the subject's thi~qhs. A heating pad which was heated to 105~F
was then placed over one thigh while the other thigh remained unheated. The patches were replaced each hour for six hours, and a final pair of patches was placed on the subject's thighs for one hour between 24 and 25 hours after the administration of the codeine.
Figure 17 is a top ~ . : view of a dermal patch accordin~q to another embodiment of the present invention placed on a subject's skin.
Detailed Dc...,.i: of the Invention l.'Dermal Patches for Da~ Analytes A.I/on-OcclosiveDermalPatches Referring to Figure 1, there is disclosed a dermal patch 10 according to one embodiment of the present .~ . illustrated as being secured to the surface of the skin 12 of a subject. As will be ~ .;al~d by one of skill in the art, the patch of the present invention may be used for ~..t~..i y purposes as well as on humans.
In addition, the patch can be used in more diverse ~,. ' ~~ such as in agriculture or any other environment where a chemical species is to be detected in a fluid. The preferred use, however, is for ': ~ of .d chemical species or analyte in sweat (PLI,-.- ), and the ensuing ' ~ is I i 'r ~1~ directed to that use.
Moisture expressed from the skin 12 within the perimeter of the test patch 10 first -- ' - in a ~: zone 14 beneath the first side of a gas r I ~ ~ filter or layer 16 which is in fluid withtheskin12. Thec zone14, ~r~. "ycontainsan b~ L material,suchasafluid r '' medium 20 which may be cotton gauze or other L 1~ available fluid p_.l '' material. For example, a layer of any of a variety of known fiber webs such as knitted fabrics, or r . ~ .. rayon or cellulose fibers may be used. Filtration Sciences #39 is a pdli' ' 1~ preferred fi '-~ ' ' medium for use as a z ~
zone in the present invention. In a preferred - '~' t, the ' L material contains binders, such as _ ' ~ for, '; 'I~ binding analytes of interest to the ' i material of the patch. As used herein, the term n~b~ ; : materialn ' _ any fluid r I ~~' material capable of collecting or holding analytes L ' ' in, , dliùn. r~,- r~- ~,, such a material is also able to z ~ dl~ such analytes on the patch.
The term "fluid ~ ~- n iS used herein to describe a material which will permit the passage of the liquid phase of fluids .:A~,,t~sr,d from the skin and which will also allow the passage of the vapor phase of such fluids. A fluid, ' ' filter or layer will thus allow the passaye of water in both the liquid and vapor phases.
"Water" is used herein to denote both the liquid and vapor phases of water unless reference is , - ' 'I~ made to a particular phase.
Moisture from F ~, 2 ' in the interfiber spaces of the medium 20. Under the influence of body heat which is readily z ' ' from the surface of the skin through the liquid phase, the liquid water CA 02222629 l997-ll-27 W O 96/39923 PCT~US96/06915 - , of the F . ~ . will tend to volatilke. Such volatilized water can thereby pass through the gas permeable filter or layer 16, which is located on the side of the medium 20 distal of the skin 12, and leavs the patch 10.
As, ~ ' I, the patch 10 is provided with a gas r I ~ filter 16. The term ~qas r ~ is used to describe a material which permits the passage of gases, including the vapor phase of fluids . GO;;Od from the skin, but ' 'l~ retains the fluid phase within the patch. Any of a variety of suitable available u~ membrane filters may be used for this purpose, such as the Gore-Tex0.45 micron Teflon filter manufactured by W. L. Gore & A ~ . Inc. ~Elkton, Maryland).
Adjacent a second side of the gas, ' ' filter 16is a discharge zone 18. As ~
the gas r ~ ~ filter 16 retains the fluid phase but permits escape of the vapor phase of the fluid -in p . dliu... Thus, the vapor c . t, which primarily consists of vaporked water, ~ ~ escapes through the gas p~. '' filter 16 exitinQ the second side thereof into discharge zone 18, which is in - with the, - .' e. In an .' I.~ ' ~ ' t, not . dl~l~ illustrated, the ~qas I ' ' filter 16 iS replaced by a fluid r ~' ' ~ which permits passage of the liquid phase. In this bc' t, liquid, or a r ' ~- of vapor and liquid, will be permitted to escape from the patch. Any of a variety of fluid ,u '' filters are - . 11~ available which can be used to form a fluid r filter used in this 5c ' - of the present invention. A preferred fluid r -~ filter is c : ' from James River Paper Drape.
A flexible, gas pt:. ' ' outer layer 22is, Lr~ disposed adjacent the second side of filter 16 in the discharge zone 18. This layer serves to protect the filter 16 against physical damage such as abrasion, and can also serve as a barrier for ~ chemical c of the filter material from the outside. Outer layer 22 may comprise any of a variety of ~ available gas, . ' ' tapes and films which are known to one of skill in the art. Outer layer 22 may also be distinct from or integral with tape 26,P ~ below.
Alll..llal;.~ , d, ' v upon the intended 1~,1i( of the patch, outer layer 22 may be deleted ~
where it does not appear that abrasion or external c - ~ will i~ affect the patch 10, or where the ~qas F '' layer 16is made from a material which is itself resistant to abrasion andlor external . thus obviating the need for the outer layer 22.
The patch 10 " ~,dlod in Figure 1 is secured to the surface of the skin by means of a I i, ' dl band of tape 26. Pl~rLl~bl~ the tape 26 will extend around all sides of the patch 10. For example, an annular rinq of tape can be die punched for use with a circular patch, or the center of a ~ ' piece of tape can be removed to expose outer layer 22 or filter 16 of a ,.: ~ ' patch (see Figures 1 and 3, ~"
All~....":.~l~, outer layer 22 and tape 26 can be deleted ' ' and layers 16 and 20 can be secured to the surface of the skin by a bandage or through the use of an adhesive. One such method would be to capture layers 16 and 20 under a bandage or wrapping sl..-. ' ~ the arm or the leg. In this case, the gases andlor -W O 96~9923 7 PCTAUS96/06915 fluids are permitted to escape through layers 16 and 20 and into the bandage, where they may either collect or from which they are dissipated into the environment.
A large variety of h,~ , or other suitable tapes are well known in the art, which may be adapted for use with the patch 10 of the present invention. Different tapes or ~ " ~.. may be desirable depending upon the intended use of the test kit, based upon their ability to adhere to the skin during different - " such as daytime wearing under clothinp, durinp sleep, during profuse sweating for, .'( _ ~ periods or durin~q showers. It has been determined that the most desirable tapes include multiple pel~ which prevent sweat from building up underneath the tape and e.. 'l~ compromising the integrity of the adhesive.
FFef~.. ' '~, a tape, such as ~ ' marketed by Johnson & Johnson, is used. More r af.,.~ , the tape comprises a layer of 3M 1625 Tegaderm wound dressing available from the 3M Company (St. Paul, MN).
Any of a wide variety of means for securing the patch 10 to the skin 12 may be utilized. For example, the tape 26 can be eliminated and gauze layer 20 provided with a lower adhesive layer to perform the same function. One such adhesive ' _ is the MN-100 adhesive ' ~ 'L: ~d by Memtec of r'' ', ''' Jtd. This membrane is fluid, , ' ' so that it passes fluid as would the gauze layer 20, yet has one adhesive side so that it will stick to the skin. All~.l,dti. 1~, outer, ul~Lli.~i Iayer 22 can comprise an annular flange 23, extending radially outwardly beyond the outer edges of filter 16 and gauze 20 (see Figure 2a). The lower surface of the flange 23 is then provided with a suitable adhesive.
The surface temperature of human skin varies regionally. However, it is generally within the range of from about 86~F to about 90~F at rest, and can rise to much higher ~ ~ under - ' of sl exertion. At those i , G.>~ a number of chemical species of interest for the purpose of the present i .~ - . such as creatine kinase or a high or low density 'i~ -r UIL;.-, have a ~urR~ tl) low vapor pressure that ..' " is not a ig ~~; factor in the efi- ~ 1 of the ~ function. At the same time, the . ' - - ' aqueous . , : will have a ;.urriLk,.-tl~ hiyh vapor pressure that it will tend to volatilize, thereby c ~ ~ ~ , g the less volatile r. However, in some ~, " the chemical species of interest will have a high enough vapor pressure, even at the resting , e of human skin or the d~ of another surface to which a patch of the present invention is applied, such that escape of the vapor phase through the gas p.,.. '' filter 16 of the analyte of interest will ;' N. "3~ ~ impair the efficacy of the test patch.
For these analytes, a modified patch must be used.
P. Dermal Patches for ~ : ,7 Volafile Anal~tes Referring to Figures 2 and 2a, there is disclosed a modified patch 11 according to the present invention for use in detecting an analyte having a, . r ~~ to escape through the gas permeable filter 16 as a vapor under ordinary use c ' - The test patch 11 ~ s a c~ dF zone 14 defined on its inner boundary by the skin 12 to which the patch 11 is secured. The outer boundary of the ~l di' zone 14 is defined by the gas F ' ' filter or layer 16, which separates the c t-di' zone 14 from the discharge WO 96~9923 PCT/U~ 915 zone 18. Disposed in the - - zone 14, and adjacent the gas, ' ~ filter 16, is a binder layer 31 for binding and, u -- v the escape of ' ' of the volatile analyte. The binder layer 31 is -, dlLd from the vgauze layer 20 by a porous layer 28, which may comprise any of a variety of films for retaining the binder layer 31 yet F ~ v passage of fluid.
In the ' - ' i" - dt~vd in Figure 2a, 1 . will pool in the interfiber spaces of the ~auze 20, and will percolate through porous layer 28 into the binder layer 31. In that layer, a ' ~ ~I~ active or bl ~ 'l~ active binder material will act to ~ bind the volatile analyte, thereby v.. ~ Q it from escaping as a vapor through ~as permeable filter 16. As discussed in r with the embodiment illustrated in Figure t, it is also possible to replace the gas r ~ filter 16 with a fluid r ~ layer, where the presence of fluid on the outside of the test patch would not be ' ' ' The binder layer 31 may comprise any of a variety of binders d, " ~ upon the nature of the volatile analyte to be d.,tu. ' For example, the binder may ' 'l~ bind with the analyte or adsorb the analyte to be d~ ' Thus, when the analyte being collected is ethanol, the binder layer .. '~ contains activated charcoal. In addition, the binder layer may comprise a specific binding partner of the analyte to be dvtl:l ~ d, such as a F 1~, ' ' or I ' ' antibody or an antigen matched to a specific antibody desired to be vd in the F
The patch 11 is .- ' " 'l~ provided with tape 26 or another means for securinvo the patch to the skin of a subject, as has been detailed in with the - '-" : " t.dlLd in Figure 1. Patch 11 is dlvd~ however, as having a unitary outer layer 22 extending beyond the perimeter of the ' 1~; ~, layers to form an annular flange 23, which is provided with an adhesive on its lower surface. As discussed in Cl - I with the s bc " of Figure 1, outer ~ uluvli.v Iayer 22 permits the escape of water vapor yet protects the filter material from chemical - from the outside. As also discussed above, gas layer 16 can also in some cases function as the outer layer 22.
C. Dermal Patches Having a r laver Referring to Figures 3 and 3a, there is disclosed a further ~ ' ~ " of the test patch of the present invention wherein an inner porous layer 28 and an outer porous layer 30 define a space for c v a ~ t - - ' layer 32. The .L ~d of such a I ' ' layer 32 can desirably have attached thereto a capture reagent, such as ~ - b~ " or other means for binding analytes of interest. The inner layer 28 and outer layer 30 ~ comprise the same material, which may be any suitable material for providing an v~ d flow of fluid through the patch while trapping the _' 3"- in between. One suitable material for porous layers 28, 30 is the fluid I '' and I ~, ~ film known by the name Ultipor (nylon 6) and ' vd by Pall Cl ~JUIdliOII in Glen Cove, New York. ~" ' I '. u~ of suitable nylon filtration ' include Micron Sepal s, lnc. of 1~ vh~ '' ' . and Cuno of Meridan, G~ : : Porous layers 28, 30 may also be i~ed of materials other than nylon, such as F U~vdlL . modified pol~ "l ' and WO 96/39923 PCT/U'_5.'.~915 .9 polysulfone.
The pauze, the inner and outer porous layers and the adhesive taPe in ali - bL '- ' can be cut to size with c . ' dies. The ~auze 20 and the inner porous layer 28 can be fixed to the adhesive ring 26 with c .. ' adhesives, such as those used on the adhesive surface itself. A' . '~, they could be heated or ultrasonically bonded together. The proper amount of m;". ' e~ " can then be placed on top of the inner porous layer, after which the outer porous surface is attached by similar means. Typically, in a one-inch diameter patch, from about 0.05 qrams to about 1 qram of m;~ ' ' will be used, and, ~1... "~ from about 0.1 to about OA prams will be used. The inner and outer porous surfaces may have to be staked or spot-welded together iri some pattern, as will be ~,, I,..;.~t~d by one of skill in the art, to prevent the ' ~ from collecting in one area.
The free adhesive surface is optimally covered by pull-away paper (not illustrated) with adequate space to be qripped with one's fingers. The patch is packaged in a paper or plastic pouch similar to that used in c .. ' band-aid r ~ The ~ '' ' unit could be terminally sterilked or pasteurized prior to sale.
All~ J~, the package could comprise a vapor barrier such as a metallic foil or mylar and even include oxy~en or moisture r' ~ means such as a small packet of any of a variety of known ' such as silica ~el, calcium chloride, calcium c~ ' or others as will be 3" ....;all.d by one of skill in the art.
The total thickness of .5und layer 32 can be varied L '' ~y. However, if a color change is to be used to detect an analyte and the such color change is to be brought about by the patch in r ~~ iall: reagent baths, layer 32 is, I.F~ hl~ no more than about 3 mm thick since color changes occurring at ' " ' sites on thicker layers would not likely be :' v ' ' More, ~le.~bl~, the .' - ' layer is between about 1 mm and about 2 mm thick. If such color change analysis is not, 1l l..~d, the I ' ' layer 32 can -' ,,al;.. l~ be torn open, releasing loose ~ ~' 3.' which can be used to conduct chemical analysis for detecting the presence of an analyte bound to the .' ' by .. - ' means, such as in a cuvette.
0~ , the diameter of the beads in , ' --' layer 32 will be at least about one order of ~ ' larger than the diameter of the pores in inner porous layer 28 and outer porous layer 30. For example, the beads contained in I ~ d layer 32 may have diameters within the range of from about 5 to 50 microns, and within the range of from about 5 to about 10 microns. If 10-micron diameter beads are utilized in the, .l ' layer 32, for example, inner porous layer 28 and outer porous layer 30 will optimally comprise a median pore size of ~" . '~ 1 micron.
The . ' ~Mayer 32 may comprise any of a variety of known materials including F~ 3~ latex, and ~qlass. Beads sized from ~ 0.05 micron to 100 micron which are suitable for the present 3,,'i( : are available from rll~ - of U.IIH ~i r. ,1~.

W O 96~9923 10 PCT~US96/06915 J layer 32 serves as the support for an ' ' ' specific bindin~ partner for the analyte to be determined. Thus, a molecule with a high chemical affinity for a specific e . in the fluid to be analyzed will be ' " ' to the ~ in ~ ~ ' layer 32.
D. Dermal Patches ~/DVing an 1, ' ' Outer Laver Referring to Figure 5, there is disclosed a further - ' ~ of the present _ . pe ' 1~ suited for use under c ' ' in which profuse sweating is not present, such as in passive ! ~--, .- --wherein the test patch is provided with an il . ' ' outer layer 42. In order to minimke any back diffusion of fluid into the skin, an ~' ~ _ layer 44 is provided to form a reservoir for drawing moisture away from the surface of the skin and thrbugh support 46 to which is bound a specific binding partner for at least one analyte to be d ' Layer 44 may include chemical means for binding or collectin~ moisture such as a ' t, as has been, .,. 1~ d d, which is suitable for use in proximity to the skin. The patch may be further provided with an ' I~ ~ porous layer 48 to separate support 46 from the surface of the skin, and the patch is provided with any of the means for - ' to the skin as have been, .,. 1~ ' E, Derma/ Patches which Minimize Lateral Diffusion of Sweat in a Patch Referring to Figure 6, there is disclosed a modified patch 13 according to the present ~ .. n, in which all ~ ~ layers between the skin 12 and the binder layer 31 have been deleted. By disposing the binder layer li.e., the layer having a specific binding partner for an analyte to be ' i' directly adjacent the skin, lateral diffusion of sweat ~ "' the binder layer 31 is minimked. The proximity of the binder layer 31 to the skin 12 allows the output of each duct of the sweat glands to contact or be in fluid: with a relatively small area of the binder layer 31. For a variety of reasons which will be apparent to one of skill in the art, it may also be desired to mount a I ~ o, ~ . . Lr~,. ' 1~ a fluid p~ ' 50 atop the binder layer 31.
The E._. d~ . capacity of the binder layer 31 and the fluid r ~ membrane 50 is ~"Lr~
sufficient relative to the output capacity of the individual sweat ducts, to minimke lateral diffusion of sweat away from the " area of the duct. This ' -' has special . . " for ~ " the chemical r . of insensible r -~ ~ d6U~ andlor non exercise ~ ~ , . . in instances where output from the sweat glands is limited. Due to the ~ ~, ' effect detailed infra, the present b~ is also p~ suited for - ~ low ~ analytes.
By limiting the . , " _ cll.~a~ .ialiLs of moisture or water on the skin, through the use of materiais having a maximal e._, -rdli~- capacity, the instant: ' " allows increase of the i' ~ "', rate of sweat in the patch by ' ~ sweat gland output. Nadel and Stolwijk, J. Applied r,s, ~ , 35(5): 689-694 (1973), disclose that sweat gland activity is ~ ased by water Iying on the skin, finding a d 'r~l. in whole body sweat rate of 40% between wet and dry skin. Mitchell and Hamilton, Bioloaqical t'~ " 178: 345-361 (1948), found that loss of water and solutes in insensible r ~ stops ~ . the surface W O 96~9923 11 PCT~US96/06915 of the skin is covered with a film of water. Brebner and Kerslake, J. r.s, ~o~ 175: 295-302 (1964), postulate that the reason for this . ' is that water in contact with the skin causes the n~ ~ ' , ' cells of the skin to swell and thus block the sweat ducts.
The ability of the present invention to produce a positive response based upon the presence of relatively low ~ ~ , of analyte is particularly adv. _ in view of the fact that, durin~ active exercise, a 114~
diameter area of skin provides approximately 35 microliters of sweat per hour, whereas a similar diameter area of skin produces sweat at a e,.~,...;;.e rate of only about 3.2 ~ . ' a per hour. The present ~
is further l~_ ~ as not requiring the user to exercise, but only to wear the patch for an equal or typically longer period during rest or at normal activity levels.
Thus, homogeneous diffusion of sweat throughout the binder layer is preferably minimized when using the instant invention in conjunction with i ' ' andlor non exercise r ~ - '- andlor a ;' ~ of minute amounts of analyte contained within I .- The minimked lateral diffusion of 1-~ .
i~ ~,' the binder layer 31, according to the present invention, provides a more c~ dled collection of sweat at each sweat duct, thereby providing a greater amount of selected analyte to be determined at that area.
F. Oermal Pat~hes Having Multiple Test Zones Referring to Figure 7, there is shown a modified binder layer 52 for a patch ,' " to the present ~ . . wherein two or more distinct zones are provided on the binder layer 52. The use of a reference zone or of several distinct test zones is contemplated for both the single layer patch " ' in - with Figure 6, as well as the emL-'- discussed in I with Figures 1-3a and 5. The multi-zone binder layer 52 may also be used for certain . ' -' to be discussed ! . 'I~! in c : with Figures 6-10 when specific binding ' - y is used.
One or more of the zones, such as determination zone 60 (Figure 7), is used to test for an analyte of interest within sweat, as detailed I ~,. '~. One or more of the remaining zones, such as reference zone 61, is used as a reference indicator.
Refe.~ .e zone 61 performs a variety of, . ' . " _ upon the desired a,, " r of the test patch. For example, reference zone 61 can be provided with color change -' : y as " '; .~ to provide the wearer with an indication that the patch has been worn for long enough that a sufficient sample volume has traversed the patch to provide a meaningful test for the analyte of choice. For this purpose, reference zone 61 is provided with affinity -' : y for a,b..~ l-,d reference b~ such as IgG, albumin or any other sweat ~ . which is reliably present. r~"cr~ the selected reference ' :- is one which provides a l. ' l~ accurate I of the volume of sweat put through the system.
This use of the reference zone 61 may be r ' by first ~l( ~ " the rough ~ _ ratio of a reference 5' such as albumin to the analyte to be ~ ~ and providing the patch with color change ' n ~ which provides a visual indication of the presence of the reference b : only well after CA 02222629 l997-ll-27 WO 96/39923 -12- PCT/U',C'~C915 the elution of the analyte to be il ~ ' has exceeded ths lower limits of :': n~f~
such as albumin will typically be present in ~ greater ~1 than the analyte. Thus, in order to accomplish the objective of indicating passage of a sufficient sample volume, the ~ :y~ of the patch for the reference ' : is, ~t~ lower than for the analyte. This can be achieved by using a , ., '~ lower amount of a specific binding partner for the reference ' : than for the analyte, other dilutions in the assay, or simply selecting a less abundant reference ' - Selection of a suitable reference ~ and - ' . ~ can be readily made through simple:, ~ by one of skill in the art.
G. Use of Dermal Paf~hes Having Muhfiple Test Zones to Prevent Tampering All~lllaO.~ '~, and pdl ' I) useful in assays for drugs of abuse and their ' 'it . a reference zone 61 (Figure 7) can provide an indication that the skin patch was actually worn by the desired patient, parolee or other subject. One inherent limitation in a test in which a subject desires a negative result is the p ~ , that the subiect will simply remove the patch after ~ : and repl~e it just prior to ,. This , gives rise to the ability of the wearer to ensure false negative results.
However, by provision of a reference zone 61 to detect a known . in sweat, the test results will reveal test patches that have not been worn for the test period. n~f~._ zone 61 thus provides a method of, ".. _ false negative e. ' due to _ or removal of the test patch.
A reference zone 61 to detect a known . , : in sweat may also be provided as a positive control zone to ensure the discovery of false negative test results due to ' ~ _~ of reagents or other of the patch. In non lr~ of-.' screens, the indication produced within the reference zone 61 will l ~r~ be a visible color change by a chemical or L bC'~/ ti~ H~ a~i occurring orbecoming apparent to the wearer when a p l,d~.tL. ' amount of the reference analyte has passed through the h.l~. area.
O,i 'I~, a reference zone 61 may be provided as a negative control zone to enable the discovery of false positive results. A preferred negative control zone will have an i ' ' ' specific binding partner for an analyte known to be absent in human sweat. The analyte's specific binding partner must be known to not cross react with . , present in human sweat. An example of an ,, . ial~ analyte is bd~ " ' _ T4 coat protein.
In yet a further F- '- " ' of the present invention (not " ~al~d) two or more analyte ' zones 60 are provided in a single test patch. The use of multiple test zones is pal ' 1~ useful in 9~, " "
such as a drug of abuse screen where testing for any one or more of a wide variety of analytes may be desired.
For example, a single test patch may be used to screen for any of a plurality of drugs of abuse, such as THC, ri ~."" morphine and ~- :' ' A positive result for any of the drugs on the screen may provide sufficient proof of an offense such as a violation of parole, or can be used to signal the need for more CA 02222629 l997-ll-27 W O 96~9923 -13- PCTrUS96/06915 follow up . ~i~. Used as an inrtial scrrening tool, the present invention offers the ~ " of ~eing r ~ .. . and much less -, _ than s . ' q _ analyses. For these reasons, a screening test patch as disclosed herein is particularly suited for initial screening of larpe populations such as parolees, inmates, military personnel or others where ~ _ is desired.
The analyte determination zone 60 and analyte reference zone 61 may be physically , al.3d on the patch, such as in r i~. circles or discrete zones, as " h.lt~,d in Figure 7, or in the case of only two or three analytes, i"tt.. :.Ju i' . .,' ' In the latter case, positive results of different determinations would be indicated by the 9,, of different colors.
Il; Placement of Dr~rrnal Patches Although a patch of the present invention can be used to collect analytes contained in any of a variety of body fluids, I , is the desired fluid to be collected due to its ', ' ' ' supply and its similarity to blood, albeit with lower analyte - Although ~ found in saliva could also be collected with a patch of the present invention, saliva is often ~ ' with I ' ' not - r~soJ by the body, such as fJrd~ ;. Therefore, in a preferred; bc " t, the patches of the present invention are placed on the skin surface of a subject.
A. G' _ ~ of Sweat alands and Perspir.~tion Sweat glands are classified to be either of two types. Eccrine type sweat ~lands function primarily to regulate body . t: through their relationship to e.. . heat loss. It is the eccrine type sweat gland that provides the sweat . ~ ' with exercise and is therefore the source of r , of interest for many 3~ of the patch of the present invention. Apocrine type sweat glands are larger secreting elements which are localized oniy in relatively isolated areas of the body such as the axilla, pubic and r areas.
Sato and Fusako, American J. r.s~ ~0~,,, 245(2): 203 208 (1983~, estimate that the diameter of the duct of the sweat gland is 3~ 40 microns. A~ , to S~ ' , - and Blank, r/,, '~ eview, 51(4): 702-747 (1971), the average density of sweat glands on the skin surface is ~ LI~ 250 per square c : . Thus, the total surface area of sweat gland ducts of the skin represent 11318 of the total surface area of the patch of the instant invention. The visible result on a test patch of the present invention when, for example, using known ELISA i ' ' ", to 1~ a low r ~ : . analyte, is the , unC6 of a number of tiny color changes on the binder or ' ~ layer - ' with the output of specific ducts. If .i~ ' :
lateral diffusion of sweat is permitted prior to contact with the ' ' ' binding partner, the color change is h~.,.. ~.. ll~ too diffuse to detect with the naked eye.
Although the etiology of F . ali~ is relatively complex, it is known to be caused by both mental states such as mental exercise and emotional stress; thermal stress, as the ' - y body's response to t . dlL.I: control; and exercise stress as the r~ active body's response to i . _: ~ control.

CA 02222629 l997-ll-27 W O 96t39923 14 PCT/U'~G f915 In addition to the foregoing ' ~ can be either insensible or sensible. I '' sweat appears to be caused by water diffusion through dermal and epidermal layers. Its purpose appears to be not related to thermaMI ' at all but to aid in such things as the ., . of ' ~ ' ~
between the skin and surfaces to facilitate grip. Further , ' arise with regard to the spatial : i' of sweat glands and the flow rates of the various types of, . ~ . ~ ~ " ' areas of the palms and soles of the feet sweat - '~, although at a very low rate. The rate of insensible r ,' " iS ~, ' upon the position of the particular area in question relative to the heart. For example, elevating a rmb over the heart ' eaOes the insensible ~ rate in that limb.
At tem, . GO of about 31~C in a resting human adult, insensible, . ~ proceeds at a rate of between about 6-10 grams per square meter per hour from the skin of the arm, leg and trunk, up to about 100 grams per square meter per hour for palmer, planter and facial skin. The latter three areas jointly account for 1p, oAil"à~ 42% of the total water loss from the body due to insensible F .. Such i _,- di- first begins on the dorsal surfaces of the foot and spreads to higher places on the body as the dl...p increases. One reported study ' , ~ ' that the average water loss due to insensible r for a body surface area of 1.75 square meters ranged from 381 ml, 526 ml and 695 ml per day at ambient alr,.~o of 22~C, 27~C and 30~C"., i '~.
In contrast to insensible r ~ which does not appear to be ass ' with a pdli' ' sUrfâCe element of the skin, sensible r-, has been r ' with the eccrine ~qland. The number of actively secreting eccrine glands varies among ~ ': ' ' and depends upon the part of the body observed and the type of sweat response created. Maximum gland density varies from between about 200 per square c on the forearm to over 400 per square : on the thenar:
The , of sensible sweat begins at either when the skin alulr exceeds about 94~F or the rectal i dlU~L exceeds about 0.2~F over normal core i _ L. Maximum rates of sweat volume loss can be as high as 2 liters per hour in average subjects and can be as high as 4 liters per hour for brief periods.
Sensible r- ~ ' dliun begins in the distal parts of the lower eAllu and, .~ LOOOs upward as the .,.,-;-. ' ' i , .' r iS elevated. Thus, the dorsum of the foot begins to sweat long before the chest. The pattern of sensible sweat response also shifts from one region of the body to another as the thermal stress increases. Under mild thermal stress, sweating is present mainly in the lower ~:Aill - As the thermal stress further increases, sweating spreads to the trunk. Due to its large surface area, the trunk becomes the dominant water loss surface. Cl,. 'Iy, ~:Alll 1~ high rates are found in the trunk while rates in the lower ~
may actually decline. The forehead can produce extremely high sweat rates but is amony the last areas to sweat in response to thermal stress.
. r~rL /l of Derm~l Patches Although a patch of the present invention can be worn at any practical location on the body, plrrLI ' ' WO 96/39923 -15- PCT/U~ 915 locations for the patch ioclude the skin on the sole of the foot and areas on the chest, back, and biceps. The patch is able to be worn in I "' in these areas, and these areas are not covered with e _ hair, so that the patch may be secured wlth ' adhesive tapes.
The patch can ~ gr l~ be located on different regions of ths body depending upon a variety of - 5 factors. It is well known that the quantity of r .- '- 9 ' ~ iS a function of both the location on the body, as well as the physical activity during and immediately preceding ~ " This is due to both different densities of sweat glands on different regions of the body, as well as to certain regulatory functions of those glands.
Other desirable ,' Iocations for the patches of the present invention will depend on the : " under which it is desired to detect analytes. Using the parameters described above and other known factors, one of skill in the art will understand how to choose a desirable location on the body of a subject on which to place a patch.
Ill. Chemical Species Dl ' with a Dermal Patch A large variety of chemical species which are ' '' in blood are also present in sweat, although typically in a much lesser I r. Early .. ~i~ into the c . - of, ,, centered ons, including sodium, chloride, calcium and p: Extreme individual variation was found among li- ' Is~ and the el,,.,~ composition also differed ', " " upon whether the sweat was ' ' by thermal, mental or other etiology.
Further research has identified numerous additional ~ , in sweat, includin~q both el~,.,l,l '~ teand more complex biological -' ' - Some " d~ chemical species which have been identified in sweat are identified in Table I below:

CA 02222629 l997-ll-27 TABLE I
Chemical C~ I~ of Sweat di~'' ia antitoxin sulfates ascorbic acid iodine thiamine iron riboflavin fluorine nicotinic acid bromine amino acids bismuth ethanol lactic acid 1 'i, r~ ~ pyruvate glucose c... ~ nitrogen C-14 h~; ~â ammonia C-14 a ~ '( uric acid C-14 urea nicotine thiourea morphine pdl ~ ' ~, uric acid '' ' ~ ' mannitol sucrose atabrin lactate :' ' -sodium chloride l ~
P ~ ~ "
calcium c 'f~v ~f ~-~ .
r i' - ~
parathion androgen steroids 1~1. h~,'ri ' ' ' ~'' insulin phenytoin ~ ~ ' cal L
~l~ldC~i Any of the entries in Table I for which affinity chemistry can be d_..', ~ can be an ..~ , iale subject of a test patch according to the present invention. Since most of the - listed in Table I are r .~ they will be trapped in the - zone 14 of the patch 10 i" ~.dL~d in Figure 1a, or on WO 96/39923 17 Pcr/uss6/0691~
the binder layer 31 of Figure 6. However, some - , most notably ethanol, would volatilke under the influence of body heat, thereby enabling escape in the vapor phase through tha test patch. Where the analyte to be ' d is ethanol or another volatile component, a patch of the present invention may be modified as described in ~ with the embodiment " t~dll,d in Figure 2 to contain specific binding partners for the analyte.
In one preferred embodiment, the analyte to be determined in F . ~ is the enzyme creatine kinase MB (CK-MB) which is G~ G.~sLd from the cardiac muscle durin~ myocardial infarction and other cardiac distress.
A Ir ' ' antibody raised against CK-MB can be immobilized to the m;",~' ' in e~ ,' with any of a'variety of r ._ ' methods, such as the cyanogen bromide technique described in Phd., ~ product literature (F; ~, Inc., Fi c u,, New Jersey).
The antibody which is to be used for the purpose of ~ with CK-MB may be ~ ' ' ' on any of a variety of supports known in the art. For example, anti-CK-MB antibody may be bound to r ~acchd~ polymers using the process described in U.S. Patent No. 3,645,852. Alle" . '~, the antibody may be bound to supports comprising filter paper, or plastic beads made from pn'~Glh~l~,..." F ~ GIIG~
F '~, l,J; or other suitable material as desired. P~Gr.~ , the support will take the form of a "i, ' ~Y
of ~' -' which can r .. '1~ be formed into . ~~~' layer 32, illustrated in Figure 3a.
As an ~ " t; ~ to a .' ' support layer, the specific binding partner could be i ' " ' directly to the inner porous layer 20 or 28 on Fi~ure 3a, to the underside of filter 16 of Figure 1a, or to , . ~r idtG
_' L materials used in any of the emt " 's of the dermal patch. In this manner, the need for ~ . ' layer 32 could be :" ' entirely. Fluid F '' ' ~ which are s~ " ~'I~ designed for binding antibody proteins are . , 'I~ available, such as Zetapor from Cuno, and Protrans, available from ICN in Costa Mesa, C-' ~
The -' ' - bc " useful in the present invention can be produced and isolated by r ~ 9 S
which are well known in the art, such as those discussed by Milstein and Kohler, reported in llature, 256: 495 497 (1975). In particular, Jackson describes a method of producing anti CK-MM (an indicator of the status of skeletal muscles) and anti-CK-MB b~ " in Clin. Chem., 3017: 1157-116Z (1984)).
Alt~,."d6._'~, the - , of a ~ available " Ig : kit can be utilized which ~ dlG
the CK-MM enzyme ~ bound to a bead support. A suitable kit marketed as the Isomune-Ck ~
Kit by Roche of Nutley, New Jersey, is one c 'l~ available - "' This kit includes a goat antisera to human CK-MM and donkey anti goat antibody c~ bound to styrene beads. A mixture would produce an '" ' ; " t having a specific affinity for human CK-MM. A more direct and less , _ . ' G, however, would be to immobilize the anti CK-MM ~ ' ' antibody directly to the microbead support CA 02222629 l997-ll-27 WO 96~9923 -18- PCT/U',.'~915 in as d with methods now well known in the art.
IV. De I ,, Anslytes ~-A. Using Color Change C' ~.), to Detect Anabtes Any of a number of methods known to the art can be used to detect an analyte collected on a patch of the present invention. One such method involves the use of color change ~ to visualke the presence of an analyte on a patch. In this G SL " t, after the test patch has been worn for a suitable period of tirne, it can be removed by the wearer (in non-drug screen tests) and d . ', ~~' to produce a visible indicium of the test result. Such a test patch can be marketed together with a developer packet such as packet 34 shown in Figure 4 which contains known developer reagents for the immunoassay. The reagent packet 34 comprises a container 36 having a, .. ' 1~ secured top 38. A flap 40 on the top 38 of the reagent packet r gripping the top 38 and peeling away from container 36 to reveal the reagent L - ' therein. As an example, a protein el~.,l-.r' ~;,;, stain such as Cc - ~ brilliant blue or amido biack 10b, can be bound to purified analyte contained in the reagent packet 34. When a test patch is immersed in the packet 34, any antibodies on the test patch that are unbound by analyte in the I , will become occupied by stained purified analyte in the packet 34. There will thus be an inverse .1 ' ' ~. between the amount of stain absorbed by the patch and the amount of enzyme passed through the patch. In this f ' - '- t, the user would place the patch in the fluid of the packet 34, wait for some period of time such as 30 seconds or more, rinse the patch under tap water and relate the resultant color of the patch to the presence of the enzyme. A color comparison chart and control zone on the patch having no bound antibody may be provided to aid in this ;,.I~
Allt:ll.dli.elt, the user could support the test patch on an open vessel, such as a small jar or vial, or empty container similar in design to reagent packet 34 securing the adhesive border of the patch to the rim of the vessel, and then pour contents of packet 34 on top of the test patch. Gravity would assist the transport of the contents of packet 34 through the test patch to maximize the efficiency of the 'L- " _ reaction, and to facilitate ~ " Oon of the color change.
The system could readily be designed so that the user performs the i r -' '- of the s of the analyte not in the patch at all but by observing the packet contents once the contents have traversed the patch. This method would be similar to r .~ ' ELISA assay methods where the packet contents contain enzyme s ; ~ which will react to specific enzyme - ': dlt... The enzyme ' t~dlt~ would be added to the packet contents after those contents 1- .~.~ed the test patch.
If the F , a;' ~ contained ' ' of interest, they would bind to the specific ~ ' " ' binding partner on the patch. If this occurred, enzyme s ; ~ - in the packet would pass freely across the test patch and l~ yllldi '1~ modify the enzyme substrate producing a controlled color change in the solution in the packet.
If the pe" d;' contained the desired I ' ' of interest, enzyme r _ would then be bound in transit across the patch and would be . ' '' to cause color change in the substrate solution. Other -WO 96/39923 19 PCT/U'56'C~915 schemes can be readily adapted for use in the present invention by one of skill in the art.
A variety of well known ~ ~ , schemes for visualkin~ the presence of an analyte of interest are well known in the art, and need not be detailed here. However, the optimal immunoassay scheme is qenerally one which is simple and requires the fewest steps. For many types of assays, it will be desirable for the wearer to obtain rapid results such as a color change to d t.~,te a positive or nepative result with as few steps as possible. On the other hand, drug of abuse screens are more likely to be evaluated by slinical staff instead of by the test subject, and there is bss concern for a ~user friendly~ product.
For example, in a patch of the present invention designed for determininq both the presence of CK-MM
arld CK-MB enzyme, the immobilked specific bindin~q partner for each of those enzymes will be S~ , ' to separate regions of the test patch. In this manner, if an en~ ' ' ' i , system is utilized, a common enzyme and a common substrate could be used. Alternatively, a different color can be used to express the presence of different analytes.
P. ~ : _ a ~ of an Anall~te Collected on a Patch qne problem which has been encountered in detecting analytes c ~ ~' in patches, q ~pr ~ 'l~ when such analytes are drugs of abuse, is that many L ._ '- ' systems for ~ drug testing do not test for the analytes which are collected on a patch but rather for the ' ' of such analytes. This is because the analytes i' K are not ., ~,,sud in some body fluids. For example, ***e is present in, ., _ but not in urine. Therefore, urine is not tested for the presence of the ***e molecule itself but rather for the presence of the major urine ' 'i~ of ***e in man, L ,1~ ~, ~ ("BE"), in order to detect ***e use by a subject.
Currently, the primary method for the dia~qnosis of drug abuse is by urine analysis. Many c " _ :- systems, therefore, are desi~qned to screen for drug analytes (or their '-' ' in urine. For example, . pr have d~ .', ' very ~ . ' systems to quantify ***e : bc1it in urine. Such systems are highly sensitive to the presence of the major ***e I l ' ' in human urine, t ,: ~, or BE. However, since the ***e molecule itself is not present in urine, many of these systems, such as the SWA EMIT system (Palo Alto, CA) and Roche RIA system (Nutley, NJ), are virtually blind to the ***e molecule itself.
In order to take e '-_ _ of c .. ' ~' ~ - systems that perform druq abuse testing by urinalysis, it is . l~,.l that the drug contents of a patch of the present invention be - _'' by such !" _ :' systems. U,.rl 1~, most of the kits on the market which test for the presence of analytes such as ***e are designed to detect ' -' of such molecules rather than the analytes i' 1~.,i. In order to utilize such ~ systems to test for a desired analyte, therefore, the contents of a patch must be .' ~ 'l~ modified.
In d -- with another aspect of the present invention, therefore, an analyte c~ ' in a patch which is not :' ' ' by c ._ ' d- _ systems, p~,.i ' l~ systems for r ~1~ ~ _ urinaiysis, is ~ 'I~ modified so that it can be detected by such systems. In this aspect, an analyte passed through the skin of a subject in i .;, is collected on an 9b' ' ' material in the patch. The analyte can then be -' ~ 'I~ modified and detected while still in the _' ~ layer or while bound to a microbead in a ~ ~
layer. All_.. ~ , the analyte can be freed from the ~ ' ' material, such as through chemical elution or by dissolving the ~ material, in order to allow the analyte to be detected by a c . ' diagnostic system. The analyte is then :' ~ 'I~ modified so that it can be detected in such a ~- _ system.
As long as the analyte and the metabolite of that anaiyte which is detected by a diagnostic system are krlown and a means of - ._. _ the analyte into its ' is known, it is within the ! . ' i~ of one of skill in the art to .' 'I~ modify such an analyte so that it can be detected. Thus, any such analyte - ~ u' in a patch of the present invention can be tested using .. ~ " systems. However, an example of how to ' 'I~ modify a particular analyte ~ '~ tested for, ***e, will be detailed below.
Cocaine is l"~i ' ' ' in the body by either pH changes or ~' ' a~e enzymes. Cocaine is unstable at pH valyes higher than 7, and thus can be converted to BE either through exposure to high pH or to ' ' - a~e enzymes. Therefore, in order to t' ~ 'I~ modify the ***e on a patch and convert it to BE
in order to make it ': ' ' by c . ' urinalysis, ***e I ' ' can be extracted from the patch and then exposed to a solution at pH 11 at room l . ~ for 10 minutes or more. Following this ' "
step, the patch extract is returned to a neutral pH and then analyzed with . ' " " systems. As is obvious to one of skill in the art, other methods of h,dr~'~ " the ester linkages of the ***e molecule in order to produce BE, such as through the use of enzymes, can also be r r, .. _d in order to prepare an extract of a patch of the present invention so that it can be detected by .~ - ' " : systems.
C. Eluting Analvtes from Dermal Patches Another difficulty e - ~d in detecting analytes that are contained in r , ~ ~" and collected on a patch is that, unless color change chemistry is used to detect such analytes, these analytes usually have to be removed from the patch in order to detect them. Removin~ the analytes normally involves -' 'l~ eluting them from the patch, which is both labor intensive and time c Therefore, in yet another aspect of the present invention, a patch is provided in which the c ' b_.,t material of the patch on which analytes are collected is ' ' _' ' When such _' ' material is dissolved, the analytes contained therein are made available for detection through further " , ' LS. As in other - ' - " of a patch of the present invention, a patch i ~ _ a ' . ' ' ~ ' h_.. l material is placed in fluid - with the skin of a subject in order to collect analytes contained in, _, Such a patch also, ~ contains a gas pl:~ ' ' layer between the ,' ' material and the outside of the patch in order to allow the fluids . ~:;,.ed through the skin in i~ .., to escape to the outside of the patch in their vapor phase.

CA 02222629 l997-ll-27 W O 96~9923 21 PCTAUS96/06915 The analytes of interest that are collected on the -' ; material are r ~r~ able to h "
the chemical ll.: which results in the dissolution of the ' ' material. Thus, the " nll of the 9' L material will not affect the analysis of the analytes c ~r' in the -' ' material. One of skill in the art will be able to recognke whether a pal: ' analyte will be chemically changed by a particular 5 chemical treatment used to dissolve the ' ~ ' material. If one of skill in the art would be unsure as to whether a particular analyte would ~: ' ' such chemical D t, it is a matter of routine experimentation to treat a sample of the analyte under the conditions of the chemical treatment and then determine whether the analyte has been ' 'I~ altered.
In another embodiment, the chemical treatment of the . ' ' material converts an analyte of interest contained in the '~ L : material into a ~l '' metabolite or into some other d~ -t '' species. For example, the ~ll of ***e with a strong base converts it into BE, a common ***e metabolite found in urine. The same strong base can also be used to dissolve an . ' ~-i disk made from a material sensitive to strong bases. In this . bc " , the dissolving of the . ' ' material does not interfere with the analysis of the analyte contained in the b ' : material, but instead actually allows the analyte to be analyzed.
An -b~ L material for use in this aspect of the present invention can be made from any of a variety of materials which can be ' ~ dissolved. For example, a number of materials are variously . :' ' to chemical attack and ' --' by acids andlor bases. Among these materials are Nylon 616 (sold as Vydyne 909 by M - Co., St. Louis, MO), Phenolic (sold as Polychem 102 by Budd Co.), Polyester (PBT) (sold as Ce!anex 3300-2 by Celanese Plastics), and F '~, ~ ' (TP) (sold as r~ : 2363-55D by The Upjohn Co.).
To dissolve any of these materials, an a,u~ , iall~l~ strong acid or base is added the material, as is known to those of skill in the art.
Al L : materials can also comprise a woven protein web, such as a web made from protein fibers a,l" '~ 0.03 inches thick. Such fibers are disclosed by B ~ , J. Forensic Sciences, 34: 1433 1453 (1989)).
Another ' '~ ' ' material which can be used as the ' ' : material in the patch of the present invention is ~ . In this - ' " :, solvents of ~ ,. ~ can be used to dissolve such ~-b material. Such solvents include chlorinated and aromatic h, h.- L esters, ketones, essential oils of high terpene content and . ~ - Specific examples of such solvents include L1_' ' 9, ' ' ' . :' ~' . and Materials and solvents other than those listed above, of course, can also be used in this aspect of the present ;..~. The foregoing materials and solvents are therefore ' y of this aspect of the present invention and not intended to be limiting.
y. r - t;..~ D.,t~ - of an Analy~te in r~
A. Dermal Patches for the n ~;~ ~, ~ of an Anal)~te CA 02222629 l997-ll-27 W O 96~9923 PcT/u~Gi~9l5 ln another aspect of the present invention, the amount of an analyte that is present in a ~iven volume of a subject's r: . can be ;" ~ d. An - ' - " of this aspect of the present invention is i" dt~d in Figure 11. In this ' - ' t, a fluid, ' ' support layer 120is in fluid c with the skin 12 of a subject mammal such as a human, and is located between the skin 12 of the subject and an ' r" ~
layer 130 made of an a' ~ material.
In the n ~ ~' ' "' ' dtl,d in Figure 11, the support layer 120 also ~ i~ s a rate-limiting structure which limits the passage of, _, from the skin 12 to the -' ~It ._ Iayer 130 to a rate lower than the rate of insensible, , of the subject. The insensible rate of, s, is the rate of F n, _: - of a subject which occurs without regard to the ._~, ' of the temperature of the subject and which is not normally noticed by that subject. For humans, the rate of insensible, ., _: of sweat glands in the arm, leg or trunk is 3" 1 ' ~ 6-10 mllm*m*hr (Randall, W.C., Am. J. PhVs Med, 32: 292(1953)).
Since the rate of I , of the subject will almost always be equal to or greater than the rate of passage of such pdl, d6un through the rate-limited support layer 120, the rate of, , passing into the r ' I _ layer 130 can be kept , " l.,l~ constant.
The rate-limited support layer 120 can be made from any material which can control the rate of diffusion of the c la of, _ For example, diffusion can be c - I " ' by a ' _ The rate of diffusion of any particular ' _ is related to physical chala~.lu.iali~.a of the ' such as its molecular ~ pr i'' ' and, in the case of a porous type of ' its pore ske. One example of a porous type of ' ~ - which can be used as a rate-limited structure in this - h " of the present invention is a polyester- , ILd p ~ ,al ' ~, ~ ' ane, such as that I f~ ~d by Nuclepore ( \Aenlo Park, CA). The pore density, pore size and thickness of the ' can be adjusted to provide the y limited fluid transport rate for this 3~," ' Another example of a porous ' is nylon 6,6, such as that faLlu,~d by Pall Corp. (Glencove, NY).
An _' ~lal;.~ to using a porous type rate-limited ' is to use a ratc ' ~, structure r i ~, a dialysis or osmotic - F ~ ~ ' and. Such ' have the c 1~. " of having ~ ' ' weight i, Ch,;ty, which may increase analyte s~ it;.;ty. For example, if one were ~at~d in collecting a Ih~.
drug or its b~" in the .Jbs~,l,uli. layer 130 and these analytes had a molecular weight of 1000 Dalton, one could choose a dialysis ' - which would pass only molecules which are smaller than 2000 Dalton in size. Larger molecules would be excluded from passing into the ' ~uli.~ Iayer 130. By limiting the '~
which pass into the ' ~JIi.~, Iayer 130, b,l~.r~.. - by other in p~ in the IdbulalOI~
analysis of the analyte in the b~ ,uL._ layer 130 is ! ' ' ' ' Although the support layer 130 of this ' - ' I of the invention has been described as c . ia;u~ a rate-limited structure, one of skill in the art will recognize that the support layer 130 and the rate-limited structure can be two separate ' and5 or all_ La in fluid ~ F - with each other.

CA 02222629 l997-ll-27 WO 96/39923 -23- PCTrUS96/06915 Ths -' ~ _ layer 130 is located distally of the support layer 120 S0 that said support layer 120 is between the subject's skin 12 and the a' ~ layer when the patch is being worn. The ' - yt;._ layer can be made from any number of ~ '-J~ ~ : ' If passive ' I of an analyte is adequate to capture that analyte on the patch, then a layer of medical grade paper such as Filtration Sciences medical grade paper (FS#39) will suffice. If active -' yi- is requiredthen substancessuch as ~ ' ' ' ~ ' specifically tailored for high affinity to the analyte can be chemically coupled to the ' ~ _ layer 130 in order to àle the analyte on the ~' r'- - material as, ..: '~ described.
In this embodiment of the invention, a ~as permeable layer 140, which in a preferred embodiment is also an outer, ule.,ti._ layer, is located distally from the skin 12 of the subject on the side of the .' r'' ~
layer 130 opposite that which borders the support layer 120. The gas, '' . outer, utu~li._ layer 140 can be made, for example, from 1625 Tegaderm wound dressing made by the 3M Company (St. Paul, '' ~t~).
In a preferred - b~ ' t, the gas permeable layer 140 extends beyond the areas of skin 12 covered by the support layer 120 and the aLs.,.yti.~ Iayer 130 when the dermal patch 110 is applied to the skin 12 of a subject. h1 this way, the support layer 120 and s' yli.~ layer 130 are protected from external abrasion and 1 5 wear.
A means for attaching the patch to the skin of a subject is also r ~rtS. ~ ~ applied to a portion of the outer, ul~..l;.~ Iayer 140 which extends beyond the support layer 120 and the ..' ~ yli._ layer 130. Most '~" the means for attaching is an adhesive - pc For example, in a patch 110 in which the outer PIU!~SL~I;.., Iayer 140 (excluding that portion to which an adhesive is applied) is 3~ 14 cm2, an adhesive can be applied to an area of ayy~uAilllnt~l~ 1 cm around the outer perimeter of the outer, ulu~li._ layer 140 on the side of the outer y~ulu~ , Iayer 140 in contact with the subject's skin in order to attach the patch 110 to the skin 12 of a subject.
In a more preferred: bc ' t, a pooling area 150is formed between the outer, uksl.li._ layer 140 and the subject's skin 12 when the patch is worn on the subject's skin 12. Such a pooling area 150 can be formed, for example, by an area 152 of the outer, ul-,.. ti.~ Iayer 140 which extends beyond the support layer 120 and the ' - yli._ Iayer 130 and to which no adhesive is applied. Such a pooling area 150 collects the excess p~., that is not diffused across the support layer 120 and allows it to dissipate into the L..~ ._ I across the outer I Utl..,ti._ layer 140. By providing such a pooling area, the back-diffusion of the r , of p_~:, .: across the skin 12 is d, since excess ~ , di- which is unable to pass into the absulyli._ Iayer 130is shunted into the pooling area 150. Since the rate of flow of P :, into the -'- yL~_ layer 130is~ t~ L~ by the rate-limiting structure of the support layer 120, the material of the ?' Yl;.~ Iayer 130is in fluid : with the pooling area 150 only through the support layer 120.
This pooling area is L ' ' to the subject's skin, and provides a sufficient amount of space to CA 02222629 l997-ll-27 W O 96~9923 24 PCTAUS96/06915 L ' extra 1, _. , which does not pass across the r?~- ! ' structure of the support layer 120.
For example, during times of heavy exercise, the rate of, ,, of the subject might rise well beyond the rate at which, , can be passed into the c ' r ' - layer 130. During such times of heavy r the pooling area 150 acts as a ~shunt" to divert, _, away from the support layer 120. The volatile ~ , of such ~ then c.. , dlethrou~qh the ~qas I '' layer 140. In this way, the back-diffusion of r . and the buildup of bacteria under the re~ ' ' structure of the support layer 120 can be avoided or at least 'i~, ' 6'. Using Oermal Patches to Determine the Amount of an Anahte in Perspiration - In order to ': the lenqth of time a patch has been worn, the amount of a reference analyte ' in a certain volume of r -~ ~ ~- of a subject must first be d ' This analyte must be present in an a" . ~ 1~ constant amount in a given volume of, ,, for the period of time that the patch is worn by a subject. Once such an analyte and its - , in r- -. ~ ~ ' is known, the amount of time a patch is worn can be ': ~ ' because the rate at which r ~ '- passes into the a' ,uL.~ layer is held 1~, u~ -.alal~ constant by the rate-limited structure. Since the rate of passage of r -~ ~ '- iS known and the amount of the reference analyte in a given volume is known, once the total amount of the analyte in _' r~;
layer is known the amount of time the patch has been worn by a subject can be c'The volume of p_., dliUn c ' al~,d on a patch can also be ' ~ ' throu~qh the use of this bc " : of the present invention. The rate-limited structure of the support layer 120 in this embodiment is , Lre,~bl~ designed to allow the passage of, ,, to the ' ,uli._ layer 140 at a rate lower than the minimal rate of passage of p~:" through the skin, thereby assuring a relatively constant rate of flow of p..., ~ di- into the dLau~ i. layer 140. The total volume of, , ~, - al~d on the _' ~.i disk is thus directly related to and can be determined by the duration of wear.
In order to 1 .1~ ': the amount of an analyte contained in a given volume of a subject's F . ~ a patch having a rate limited structure as described above is first placed on the skin of a subject, j trL.aLI~ a mammal. rL;, ,.~ is then passed across this ratelimited structure at a known rate. For example, if the rate at which ~ :. ~, is allowed to pass across the rate-limited structure is equal to or less than the insensible rate of r-, ~ of the subject, F ,, will pass into the C' ' ' material at 1~,, 1~ a constant rate. After a sufficient test period of time has elapsed to allow a ' ' ' amount of the analyte to be tested for to pass into the .' L material, the patch is removed from the skin of the subject. When the patch is removed, the amount of time between the r~ of the patch on the skin of the subject and the removal of the patch is recorded.
In order to then ;' ~LI ~ the total volume of ~ ~., which has passed into the ' ~ layer 140 and c al~d analytes there, the rate of flow of F . , into the ~ b. ~.li.., layer 140 (as drl~
by the rate at which ~ . dliUII passes across the rate-limited structure of the support layer 120) is first Wo 96/39923 ~25- PCT/u~G,!o C91~
multiplied by the amount of time the patch has been worn. This figure indicates the volume of r ~ " which has passed through the support layer 120 and into the ~' ~t;._ layer 130. The total quantity of analyte in the c' ~.i _ layer is then determined. By dividing the total amount of analyte present by the total volume of which has passed into the ' ~ _ layer 130, the average amount of the analyte in a given volume of the subject's I _, ~: can be determined.
The above described aspect of the present invention is thereby suited to be used in many areas of diagnostics where ~ ., information about a particular analyte is ~. For example, this invention can be used to monitor therapeutic drug administration, determine the ' adequacy of a subject's diet, or explore hormonal imbalances in a particular subject.
Vl. ' ~ ~ Analy1e C J~' and C 1 ~ ' ,, E '.11 '' in a Dermal Pabch A, The Problem of Back-Oiffusion An analyte which has passed through the skin in r .,' ~ iS usually removed from the exterior surface of the skin through washing or through various natural I . Thus, such an analyte will not normally e ' on the skin's surface. However, analytes which pass into a dermal patch can become more highly ~ ~,dl-,dthan they normally would on the surface of the skin. If an analyte does become cc dlod on a dermal patch, it becomes possible for that analyte to diffuse back through the skin of the subject wearing the patch, a ,' which has been termed "back-diffusion".
Previous reports in the literature suggest that an analyte will back-diffuse after the r ~ of the analyte on a dermal patch rises above the - of the analyte in the sweat or ~lilidl fluid of a subject. In fact, a ' ' model has even been _ dt~.d to elucidate the 1' ~c~' : of back-diffusion (Peck, Carl C., et al., "Co - Tl. . ' Illal Drug C~" Basis for Use in Acse~ g Drug IntakeandPhal s~kinetics",J. rhal ~kineticsandD p' 'o"~,9:41-58(1981)). Thismodelsuggests that back-diffusion will occur when an analyte is c~ dlod on a dermal patch, and that such back-diffusion must be, ~..,..l~J in order to ~c dll,l~, "' Id the amount of an analyte which passes into a dermal patch.
Thus, many prior art It:f~ll suggest using specific binding; ' : y to prevent back c' 'fL
B. Back-Diffosion and Dermal Patches of the Present Invention It is one of the surprising d .. ;es of the present invention, however, that such specific binding -' : y is not ~ to prevent back d 'fl This discovery was first made during the ~:A,~,_.' ' ~,dled in Figure 12, in which a patch without any specific binding ~ y was placed on the skin of a subject who had ingested ***e. In this test, the L - , ~ of ***e and ***e l ' '- found on the patch were charted for approximately 200 hours following the subject's ingestion of ***e. The results of this test showed that the c ~ _ of ***e on the dermal patch rose " 'y during the first hours of the test, and ' ~aflel stayed at l" ~ '~ the same level for the remaining 200 hours. Thus, the patch was able to 5 dl~ analytes over a period of almost 200 hours without exhibiting , " : back . 'fl WO 96/39923 26 PCT/U',S'~ 6915 r : ~, during that 200 hour time period, the c ~ bf cocame io the subject's system was d ~a~;..g, as shown in Figure 12 by the declining ~ of BE in~the subject's urine. Thus, the dermal patch used in this test was able to maintain a r _ of ***e that was higher than that in the subject's system,again' - that~i, ' back-diffusionwas, .. ' Theseresultswere . - 'inlight of the teachings of the prior art, which would have led one of skill in the art to expect to observe back-diffusion during this test.
It is believed that the surprising results of this test were due to the ionLzation states of the anal'ytz of interest collected on the patch, in this case ***e. In the ~, " dt~d in Figure 12, the ionization states of the ***e molecules collected on the dermal patch used in that: . were affected by the pH
of the dermal patch and the pH of the exterior surface of the skin ' ' the patch relative to the pH of the body fluids beneath the surface of the skin, as well as by the pK, of ***e. It is the _ of these pH and pK, values which affects the ionization state of an analyte.
The D ,t:""e of back-diffusion can be ~ ' ~- 'I~ prevented by L ." ~ the ionization state of an analyte being collected on a dermal patch. For example, the pH of a dermal patch can be ~ , " ' in order to also control the pH of the surface of the skin beneath that patch. Once analytes pass through the area of skin ' ' such a dermal patch having a L ~" ' pH, they will become ionked and thus unable to back-diffuse. Thus, after d~- ~ the pK, of an analyte of interest, standard pharr~
I . - can be used to :': ~ - pH values at which that analyte will become ionized once it passes to the surface of the skin of a subject. In this way, a particular analyte of interest can be collected on a dermal patch without the risk of --' , back-diffusion.
We believe that one reason that the ionized form of various analytes do not back-diffuse is that these ionized analyzed can attach ~ ...s~ to larger molecules that are too large to be capable of back-diffusion.
C. The Effect of Occlusion on Back-Diffusion In order to evaluate this model for r ~" ~, back-diffusion, the pH of skin ~..d~ alll a c ' ., patch such as that used in the - i : i" :al~d in Figure 12 was next ' ~' by ~ , a further test. In this test, patches were ~ ' which had 112" long pieces of litmus paper between the ~
layers and the Tegaderm outer layers of each of the patches, and which further had 112" long pieces of litmus paper between the skin and the a' ~,li.~ Iayers of these patches. Such patches were placed on the chests (below the ' . ' ayll,) and biceps of each of three male .. ' ~ for seven days. The colors of the pieces of litmus paper in each patch were ~.,on;lo,~d while these patches were worn.
The results of this ~ ,i : indicated that in all three ~C ~ the pH of both the ~ ' skin and the . ' ~.li.~ Iayers of each of the patches reached only between about 4.5 and 5Ø Further, this pH was reached and i' Ldlld, d in each case within 24 hours. Human skin normally has a pH of about 4.4.
Thus, the ~, . " of a non occlusive patch does not appear to ';1,~ change the skin's pH.

WO 96~9923 -27- PCT~US96/06915 By s , . occlusion of the skin can bring about a much greater change in the skin's pH. In a study done on the effects of occluding skin, the skin of ten subjects was wrapped with plastic film (Saran brand plastic wrap) for 3" ~ five days. The results of this test showed that the pH of the skin of these subjects shifted gradually over the course of the test from 4.38 before occlusion to 7.05 on the fourth day of occlusion (Aly, Raza, et al., ~Effect of P,~ Occlusion on the Microbial Flora, pH, Ca, :" ' and Trans-Epidermal Water Loss on Human Skin,~ Journa/of I 'i~ ~6. ' 'O"" 71:378-381 (1978)).
As !" e ' in further detail below, a rise in pH values such as that observed in the occlusion tests performed by Aly can significantly affect the amount of back-diffusion from a dermal patch. Prior art patches, which are occlusive in nature, appear to have i , ~ - d problems with back-diffusion due to an ' ' and un " ._.~id shift in the pH of the skin below such patches. By contrast, the e~ ' nature of the dermal patches of the present invention results in only a small change in the pH of the surface of the skin under such patches. Since the skin is naturally slightly acidic, the maintenance of a relatively acid pH will prevent back-diffusion problems. The ~ ~. ' effects of a rise in pH on analyte ~ ; can thus be obviated by using the patches of the present invention.
D. G- ~ " J Back-Diffusion The transport of many substances across the skin, including the back-diffusion of analytes, is believed to occur by means of passive diffusion across the stratum cornium, a structure which has a high lipid content (Orland, 1992). Passive diffusion across a lipid barrier normally occurs only if the ': e in question is non-ionized, because ionized ' ' cannot cross such a barrier (Labaune, J.P., "H " - k of Pha, 1989, pp. 18 25). In order to control back d 'rl . therefore, the pH of the surface of the skin below a dermal patch can be s ~., " d, such as with a buffer, so that analytes which pass through the stratum cornium become ionized once they reach the surface of the skin, thereby losing their ability to pass back through the stratum cornium.
All~.l..,li._l~, back-diffusion can be, ~.. ' by ionization of analytes in other ways known to those having ordinary skill in the art. For example, analytes can be ionized by ul~_lH~.ity, such as by r~
Devices such as the Phoresor lln~ (made by lomed, Inc., Salt Lake City, Utah) can be outfitted to ionize analytes collected on patches. These devices, which were originally designed to deliver drugs by means of Ll~_lll ' attached to the skin, can be adapted to deliver cl~_lli~.;ty to a patch or to the skin adjacent the patch. However, other methods can be used to deliver el~ .ity to the patch or skin, such as by simply attaching a pair of el~_l,,dl c-- : ' to a battery or other source of el~_lliLity.
It is believed that an analyte which has passed into a patch can also be bound onto a patch with an antibody and ' '~ ionized by means of an ionized molecule that is also bound to the antibody.
The degree of ionization of a molecule is easily d: ' if its pK, and the pH of its c...;.. is known. The general 1' ' H~ s " - ' equation for a weak base shows:

W O 96~9923 -28- PCT/u~ 9lS
pH - pK, + log (C~0"1C~o") Where:
Cb,, is the s - of the ionked molecule; and C~ is the c of the ~ ' molecule.
The L ,- of an ionked molecule on either side of a lipid barrier, such as the skin barrier, can be found by extending the above equation for a weak acid molecule:
CB- 1 + 10~
CP _ 1 + 1 O~WP~I
or for a weak base:
CB- 1 + 10~K~PH~

Where:
CB is the total - of the molecule in the i~ ,alilidl fluid of a subject;
CP is the total s~ di' of the molecule in the patch;
pHB is the pH of the ;.~t~:lalilidl fluid;
pHP is the pH of the patch;
By using these, ' ~~' :- l, . for any given analyte and subject, a pH can be selected for a patch which will cause the number of molecules of an analyte in the patch to be larger than the number of ' - of that analyte present in the ~ alilidl fluid of the subject wearing the patch. When using a patch wi~h such a selected pH, ~ ' molecules which pass through the stratum cornium will tend to be ionked when they reach the patch, thereby ~IIL._~Itill9 the back-diffusion of those I
In practice, when collecting an analyte of interest on a dermal patch according to one preferred method of the present invention, a pH value or a range of pH values is first selected according to the e, above.
The a' L material of the patch is then, ~:rL, ' 1~ ~ 3d at the selected pH or range of pH values in order to c~ dle the analyte on the patch. As the patch is worn, the - , ' form of the analyte in the ;"l~la~i~idl fluid of a subject will naturally diffuse across the stratum cornium of a subject's skin in order to try and reach , ' with the ni~n;~Pd form of the analyte on the surface of the subject's skin. Once on the exterior surface of the skin, the - ;~n ~Pd analyte molecule will be ionked due to the selected pH of the patch, thus p,u.~ the analyte molecule from back d-'~_ ~ E In addition, after the ' molecule becomes ionized, the c~ dliUII of , ' analyte molecules on the surface of the skin will be dt ~asc.l and thereby cause more ' analyte molecules on the interior side of the skin barrier to cross to the exterior side in order to try to l~a' '' ' an ~ concL..II: of nn:~Pd analyte ' ' - on each side of the skin barrier.
As discussed above, in prior art occlusive patches, the pH of the patch quickly 3" ' 7.05. This CA 02222629 l997-ll-27 W O 96~9923 29 PCT/U~5~1~G~15 severely limits the ratio of analyte in the patch to analyte in the ~lilidl fluid. It is ~ ,. '' when using both occlusive and ~ ~ patches of this embodiment of the invention, to provide a ratio of analyte in the patch to analyte in the ~ ~lilial fluid of over 10, more, ~f-,. ' 1~ over 100. In one preferred - b~ ' t, as illustrated below, such ratios can be provided by maintaining the pH of the patch below a given level.
An example of this method of collecting analytes on a dermal patch using a selected pH is outlined below. When using a OL ' ~ _ patch, such as is described herein, the pH of the surface of the skin of a subject will remain at about 5Ø Pt. . ~ . as well as plasma and ~ ~titidl fluid all have a pH of about 7.2 (Orland, 1992). Using the equations above, it can be d: ~ ' that when detecting the analyte ***e, which is a weak base and has a p~; of about 8.7, the selected pH of 5.0 for the patch will drive the ratio of ***e 1~ ' on the exterior surface of the skin to that in the ~ ~lilial fluid to over 100. The number of ionized ***e ' ' on the exterior surface of the subject's skin compared to the number of non ionized molecules is also much higher. Thus:
CB = 1 + lo~8 7-7 2) = 1 + 1o1 5 (1) CP 1 + 10 (8.7-5,o) 1 + 103'7 CB = 1 + 31.6 = 32.6 = 0.0065 (2) CP 1 + 5012 5013 CP = CB x 154 (3) ZO Applying the fore~qoing equations to prior art patches, in which the pH would quickly approach 7.05, the result would be CP - CB x 1.4. Thus, by ~ ~ _ a patch pH of 5.0, a 110-fold increase in the ratio of ~on~-,..lldliun of analyte in the patch to c i of analyte in the i..l~ ilidl fluid can be obtained.
It can be seen that for weak base analytes such as ***e, the higher skin pH observed under D~ . type dermal patches will allow back-diffusion to occur. Thus, when it is ~ N ~ to use an D~ . type patch, such as when extra, .~: from the ~.. ;.l : is desired, the pH of the skin under such an occlusive patch should be: I" ' in order to prevent back '~u ~ In these 1" " s, a buffer can be used to control the pH of the surface of the skin below the patch. A buffer of any specified pH can be dled by - ~" " the ratio of acid to base in a mixture ~, an acid and a base, such as a mixture of acetic acid and NaOH. Such a mixture can be made more basic by ~ " the : - of base, in this case NaOH, or can instead be made more acidic by i . ~ ~ the cr- ~ - of acid. A buffer of this kind can maintain a desired pH in the patch and on the surface of the skin under the patch when the patch is worn. Thus, even if an occlusive dermal patch is used to collect an analyte, by controlling the pH of the patch the problem of back diffusion can be virtually eliminated.
- A particuiar a~r~iL of the present invention is the ~ ~.. of the back-diffusion of a drug of abuse which has been collected on a dermal patch of the present invention. Table 2 below lists the pK,'s of the major drugs of abuse, all of which are weak bases (Wilson, J., Abused Drugs, a Ir' dtv,J~Pocket GuiuJe, AACC

W O 96/39923 30 PCT~US96/06915 Press, 1990).

Drua Heroin 7.6 Amphetamine 9.8 Morphine 8.1 r :~ 8.5 Cocaine 8.7 The pK, for most of such analytes of interest is in the range of 7.2 to 10Ø The pK, values of an analyte of interest can be used in - with the ~qeneral 1' ' ~ " ' equation ~qiven above in order to determine the ratio of the ionized form of any ~iven analyte to its ~ ~ ' form in the patch. For analytes in this ran~qe of pK,'s in prior art patches, in which the pH of the patch quickly 3,,l1l' 7.05, the ratio of Cb" to C,~0,~ will vary from 3" UAi~dl.d~ 1.4 to ~ 891. As an example, for ***e, which has a pk, of 8.7, this ratio will be 45 in a prior art occlusive patch. In contrast, using a patch of the present invention in which the pH of the patch is buffered to 5.0, the ratio will be 5012. In preferred Sc " of the present invention for both occlusive and n~ ~ ' ., patches, the ratio of ionized forms of the analyte to forms of the analyte in the patch will be over 1000, and more I ~r~ over 5000.
As in the case of ***e, using the equations described above it can be shown that a pH of about 5.0 on the surface of a subject's skin below a patch will cause the Sc.~ l,,d drugs of abuse to collect on the pafch. Thus, for example, a non-occlusive patch of the present invention will - ~,al~ the above analytes without the problem of back r In addition to solving the back-diffusion problems of prior art dermal patches, the present discovery also makes it possible to improve the ability of a dermal patch to - ale an analyte. This can likewise be " ' ' by adjusting the pH of a patch and the surface of the skin below the patch. By d~ g the pK, of an analyte of interest and using the equations above, an ~, ., i..l. pH for the patch can be selected such that the ~ )r : of the ionized form of the analyte is much greater than the , "' i - of the ' form of the analyte. When ' analytes then pass across the skin and into a patch having a pH selected in this way, they will be ionized, thus driving the further diffusion of non-ionized analyte molecules into the patch. Del~, _ the pK, of an analyte of interest, if it is not already known, is within the I .: 'g of one of skill in the art, and thus requires only routine eA~u~ llldliull.
The present discovery further suggests a method of, _I~ d: _ the amount of an analyte which passes through the body of a subject. Such a method first involves the, ' of a dermal patch on a subject. The back-diffusion of analyte ' ' - collected on this patch is controlled in this method, such as by selecting an diJ~-U~JIidl~ buffer for the patch. After a specified period of time, the patch is removed from the CA 02222629 l997-ll-27 W O 96~9923 31 PCT/U~ 915 subject's skin and the amount of time the patch was worn is recorded. The amount of an analyte which has passed into the patch is then ~-Y~ermined. By . . _ the back-diffusion of an analyte, the amount of analyte collected on the patch over the specified period of time will more closely reflect the amount of the analyte which passed through the subject's system over that period of time.
Vll. F~. _ of Ta, ~ ,. with Dermal Patches In some uses of the present dermal patches, it is -~-. _ to provide a means for indicating whether a wearer has removed a patch during the examination period, particularly in s where a wearer has an incentive to make sure that the patch produces a specific result. For example, if it is desired to determine whether a wearer has ingested a drug of abuse, safeguards are desirably provided to prevent . i _ with the dermal patch.
A. Dermal Patches with Padial Slits One: bc ' of a patch for ",.~: _ tampering is " : dl-,d in Figure 8. In this emb- ' t, the patch 62 is secured to the skin 64 with an adhesive member 65. The adhesive member 65 is, ~ r~ bl, c ~ of a material that is strong enough to hold the patch 62 to the skin 64, but that is relatively easily torn such as during removal of the patch from the skin. A suitable material for use in this preferred i ' - "
is Tegaderm 16Z5, 'c ~.d by '' Mining, and ' h ~ _ Corp. of St. Paul, '' Other ~ s, including Avery and Johnson & Johnson, manufacture similar suitable materials; the Johnson &
Johnson product being sold under the ~. ' k "P ' _." It has been found, however, that with sufficient patience, a wearer could remove an adhesive member of this type and replace it without leaving any visible " - that the adhesive member has been removed. Therefore, in the pa~ preferred be shown, the adhesive member 65 has stress razors 66 in the form of a plurality of radial slits around its outer perimeter. The stress razors 66 can be arranged in any of a wide variety of r- 'ig ~ and densities and accrue the a~a"lage of tearing upon removal, as will be apparent to one of skill in the art.f In the - bc " " t~alud in Figure 8, the radial slits 66 extend 3,, UAi~atUI~ 0.05 inches in length from the outer edge toward the center of the patch 62. The slits 66 may be arranged with any of a variety of regular or irregular spacings ' .b~ . . and, in the preferred '-~ are, Lr~ spaced ~. UAil"al~l~ every 0.10 inches around the perimeter of the patch 62. The adhesive force of the material of the adhesive member 65 is I ~ru.a' 1~ more than the force needed to tear the adhesive member at the stress razors 66, so that if the patch 62 is removed, the material of the adhesive member is torn. Thus, when a patch of this preferred e bc " : is worn, a torn adhesive member serves as an indication that the wearer has likely tampered with the patch. Of course, the . _ ' ~ of the adhesive member 65 may be a .' '-' by providing F ruld6ons rather than slits and the slits or F ru. may be oriented in ~ ~.1i.,..3 other than radially.
During storage prior to use, it is desirable to cover the adhesive member to prevent it from sticking to WO 96/39923 32 Pcr/u~ ,. /C 6915 any surface; o~ the stress razors 66 could become torn prior to use. P~ , in the preferred 'lL '- shown in Figure 8, the patch is provided with an inner cover 69 to protect the adhesive member 65.
The inner cover 69 is removed to expose the adhesive member 65 prior to ," of the patch 62 to a subject's skin. Any of a variety of - r " I - materials known to those of skill in the art may be used for the inner cover 69, such as those: '~ used to cover adhesive ' ' "
The patch 62 is virtually ' ' ' to remove and replace without showing visible signs of , ~ ~
Thus, any analytes in sweat produced from skin under the c ~ 'r. zone 14 during the time the patch is worn should be present in the patch. However,aparticularlyshrewd subject desiring to produce false ne~ative results could obtain additional test patches. This shrewd subject would obtain false negative results by removing the initially applied test patch and replacing the test patch just prior to the time the patch is to be removed for assay. In order to ensure that the patch removed from the subject is the same patch which was initially applied to the subject, an ' ~ " marker which is difficult to .l . ' can be ~ dlGd into the patch. For example, a bar code ' '; strip 67, similar to the bar codes used at , k~l check out stands can be i ~ atud into the patch,, ~r~ just below the adhesive member 65. For best results in, . " against ,.,' of the patch, it is : that the ' - F~; ~, marker not be easily removed and replaced without providing an indication that the patch has been tampered with.
In a preferred ~ ' ~ ' t, the patch 62 has a filter 68 between the outer layer 65 and - 'r.
zone 14, as described above in L :- with Figures 1-3a. In a pal ' 1~ preferred - bc ' t, the filter is a fluid plz~ ' ' filter formed from a James River Paper Drape.
The preferred adhesive members of the b~ " shown in Figure 8, made from adhesive materials, such as Tegaderm, which are relatively weak in strength, have generally been designed for hospital patients who are not expected to perspire at high rates. Therefore, the moisture vapor ll rate (MVTR) of these materials is relatively low. For example, the MVTR of Tegaderm is 1,, uAil"alul~ 810 glm*m*day. However, an active person may perspire at: rates as high as 26000 glm*m*day. C~ 1~, an active person may put out more sweat than these adhesive members can transmit to the - ' ~. If this sweat ~ for any siynificant period of time, channels may be formed between the skin 64 and the adhesive member 65, allowing sweat to exit between the adhesive member and the skin, rather than be absorbed by the patch 62.
P. Dermal Patches with Pinhole r~
In .-cr d -e with a further embodiment of the present invention for p ~.~.. i _ I i _. " ~,dl~d in Figure 9, there is provided a patch 70 having an adhesive member 72 which allows excessive sweat to be freely 1, ' to the outside through pinhole r O di' 73. The pinhole r r d~ may be " ~. but ' lh. ' ; a wide band 75 extending from the outer perimeter of the adhesive member to a narrow band 77 .~.,. " ~, the test region 821 of the patch 70.

W O 96~9923 33 PCT~U~Gi'~915 Sweat produced beneath test region 81, over which there are no pinhole r 5L _ - 73, will be absorbed by the test region and will not be 1, ' to the outside. The test region 81 includes the area of the patch 70 directly under the r : _ zone 14 of the patch as well as the area l~ outside this zone. The narrow band 77 outside the r _ zone 14 of the patch has no pinhole I ~. 73, and . ' 'I~ restricts sweat forming ' th the test region 81 from communicating wrth the wide band 75 where sweat is tl_ ' to the outside.
The width of the narrow band 77, is, ..~ between 0.025 and 0.250 inches, more, efe. ' l~
between 0.05 and 0.125 inches. Narrow band widths less than the preferred width are not expected to keep Gontact with the skin, whereas narrow band widths greater than the preferred width may allow sweat channels to form, creating a path for sweat forming within the test region 81 to communicate with the outside.
C. Use of Soluble Markers to Prevent Tampering A wearer of the patch in screenings for drugs of abuse would be expected to be rather creative in L;l. ..".i ~ the r Ut~._" ~ of the patch. For example, a creative wearer could try to wash out the c~ ~.dl~d sweat r , from the patch while the patch remains on the wearer's skin. Such washing couid be dl , t~d using a needle and syringe, such as those commonly used by L.,._ drug abusers for drug injection. For those patches - ,' ,; " specific binding ~' ~.y, ~ t~.d elution of the L dl~.d r , - IS using water would likely prove e c~ ~ Even for those patches not: , ', " specific binding ~ ' ~, y for the analyte being tested, elution with water alone would be difficult, requiring b~ ' volumes of water without triggering the detection of , " through the removal of the patch from the skin. However, certain analytes could - ' 'I~ be at least partially eluted using other solvents.
Thus, in order to detect i , _ with the patch through elution of the patch's contents using water or other solvents, a known amount of a marker which is readily soluble in either aqueous or ., solvents, can be added to the c - 3t - zone during CL_' t: of the patch. The marker should be easily s, li~ ' ' The marker should also be soluble in either aqueous or r ., solvents :', " ~, on the likely route of elution of the analyte. A~'" 'I~, the marker should be suitable for ~ skin contact and not be readily absorbed by the skin. A variety of dyes used in the, ~' of makeup have these suitable .,ha,dcl~,iali.,a. Oil red N ~ ' " number 29,849-2) sold by Aldrich Chemical Corp. of '"' ' . U'i is a suitable lipid soluble dye. OG01 red and DH60 yellow, both available from Virginia Dare Extract Co. of Brooklyn, N.Y. are suitable water soluble dyes. These water soluble dyes can be easily ., lildl~d by elution from the patch followed by ~ ~ optical density at 6500 nm for the red or 5800 nm for the yellow dye.
The quantity of dye remaining can be compared with the range of the amount of dye found to be remainin~q in patches worn culllil,uoual~ without i , ;..9 for the same length of time.
Non visible markers could also be used to prevent the wearer of the patch from obtaining feedback regarding the extent of marker remaining in the patch. A colorless protein could be used for this purpose. A

W O 96/39923 34 PCT/U~Gt~6915 protein should be chosen that is easily identified in the lab, and also not be expected in human sweat. For example, Bovine gamma globulins, such as those sold by Sigma Chemical Co. of St. Louis, MO, could also be used as a marker. The presence of these markers can be easily a;~LL~ ~d using Bovine IgG RID kit, available from ICN of Costa Mesa, CA.
Thus, when a suitable marker is employed within the patch, when the patch is analyzed for the particular analyte being tested, the patch can also be analyzed for the presence of the marker. For visible markers, such as makeup dyes, the presence of the marker may be analyzed by simply viewing the patch. For - .; ' ' markers, the - . ' ' marker can be assayed along with the analyte. A _ '; decrease in the amount of-marker present would be an indication of . i through elution of the patch with a solvent.
a Use of A~ '- _ to Prevent Tampering A further method of i . i ~ with the patch would be to add an ~ to the patch which r~lGs with the assay -' ~.y. N ~ materials have been used to - ' ' dll~ urine tests for drugs of abuse. The most . '~ used, and generally most effective method of producing a false negative result in a urine test is to dilute the urine by ingestion of excessive amounts of fluids. A~ v ly, this approach would not likely be ~, ' in producing false negative results in the sweat collection patch of the present invention because : :,lilhll _ of drug m. ' ' is less likely to be influenced by ingestion of fluids.
However, the addition of certain ~ ' " to the patch may interfere with the analysis .' ~,y.
For example, acids and bases are known to interfere with assays for many drug I ' -" by altering the ' " ' molecular structure. A~' " '1~, many ' ' ' ' products, such as d~t~,b.,..l~, ammonia, ascorbic ZO acid (Vitamin C), and drain openers have been used to interfere with urine assays. These products produce extremes of pH or changes in other chemical pa~ , and would be expected to result in trauma to the skin if used in s: - with tests using the patch of the present invention. This trauma could be noted by the removing the patch.
However, weak acids and bases, as well as eye drops sold under the ll 1I k "Visine," are also known to interfere with a variety of assays for drug ' -' in urinalysis. However, these materials would not be expected to produce skin trauma. Thus, the use of these materials or other s . ' ~1 v with an assay that do not cause skin trauma might go unnoticed by the i ' removing the patch if the fluid contents of the material have had time to L. . dl~ across the outer layer of the patch. However, "Visine" and most other - ~ ~ tlllL~ would be expected to contain ionic materials.
Thus, in order to detect the use of an - ' ' . t, test strips can be ~ dt~d into the patch which will detect the presence of various ionic materials or of extremes of pH. Litmus paper, such as Hydrion pH test paper, available from Baxter Scientific Products, is well known as an indicator of variances of pH. Accoil" "ly, a short piece, for example 1 cm by 112 cm, of litmus paper could be r ~l~d into the patch to detect the various h~ ' r'd products identified above which are known to be highly acidic or basic.

W O 96~9923 35 PCT~US96/06915 Many test strips are also known for detectin~ the presence of ionic ' For example Baxter Scientific Products suppiies test strips from a variety of ~L ' I,.a for the detection of each of the following ions: aluminum, ammonium, chromate, cobalt, copper, ion, nickel, nitrate, peroxide, sulphite, tin, and calcium.
In addition, test strips sold under the name "Qantab" are available from Baxter Scientific Products which identify the presence of chlorine ions. Other test strips available from the same supplier show ~qlucose, protein, and ketones. Most of these test strips are read by simply ~ , _ the color of the strips with a color chart included with the strips. Thus, the test strips provide a simple method of identKyin~q the ~ t~ of any of a variety of ' "
In order to detect adulterants, such as ~Visine," which contain ionic materials not known to the person F ~ ll " the test, the tester must first assay the ? ' ' . ' usin~ a variety of test strips for ions to ascertain which ions are present in the ' Once the 1~r 1, ial~ ions are detected, the test strips ~
to those ions can be r dlud into the patch in order to provide an indication that the adulterant has been added to the patch.
Curiously, any particular ~' " I might produce false negative results in some assays and false positive results in others. For each assay, the common ~d ' , t~ which could be used to produce false negative results could be identified by testin~q the assays with the addition of small amounts of these known i ' Test strips could then be included which would detect the addition of these adulterants.
In a preferred ~ bL " t, the test strip or strips are placed facing the skin, where the strips are not visible to the wearer. The wearer is thereby not provided any feedback which aids the wearer in dec~"
E. Use of a light A( la~er to Prevent Tampering - Many biological . , ' are known to be affected by various spectral bands of light energy. For example, urine samples for analysis of LSD must be kept from exposure to strong light. Schwartz, Arch Inter.
Med. 148: 2407-12 t1988). Further examples of t ,_ ' which require, ut from light include ***e hrdr~-'' ;dd,r' '' Extra rha, -, - , 29th Ed., p. 1213, and morphine sulphate, Id., p. 1310. It is expected that these and other ~ , ' may be affected by exposure to light while being ~al~d in the collection patch as well.
Many analytes to be determined by a patch of the present invention may require collection and storage in the patch for prolonged periods of time (up to several weeks). These analytes are, therefore, exposed to s L - ', of ~Jh~.lu~ " This quantity of, ': adia; may be ~ greaterthan during a urine assay for the same or similar analyte. Also, many analytes have peculiarly high 3r,asiti.ity to light. Thus, for analytes of peculiarly high 1' tu~ iti.ity or for those requiring prolonged collection and storage, it is pal tiL ' 1~ , Idul to shield phul-.5~....~iL.~ analytes from light during prolonged storage in the patch.
Ar- ~ , in still another ' ' of the present invention, " Lal~d in Fi~ure 10, there is provided a test patch 90 having a light layer 92 between the outer adhesive layer 65 and the W O 96~9923 -36- PCT/U',6/'~6~15 zone 14. In Fisure 10, the adhesive layer 65, is shown having stress razors 66, however, this feature is to be I ' ~ od as being optional in this - ' ' of the invention.
The - layer 92 is provided in order to attenuate the l._ ~ ~ of light into the szone 14 where the biological - . ' of interest is being collected and stored. The layer 92 should be . b~ to the ll of ' : . ' yet should also allow relatively l,stli~.t~d passage of the aqueous ~-, of sweat to the outer adhesive layer 65. The layer 92 should be of sufficient porosity that diffusion of the aqueous components of sweat occurs at least as rapidly as sweat normally accumulates in the patch.
Because light of many . ~... '~ :' is capable of degrading the various biological c . ' which may be of interest, the layer 92 should have optical, a, li.,s which attenuate light i' ~ ,.' a wide ;.,.~.,l-.
can be achieved by either reflection or ' r of incoming li~qht. R~R: may be achieved through, for example, the use of any of a variety of metallic surfaces. When used in --- ,' with certain preferred bL' la of the present invention, the _tl Iayer 92 should allow passage of aqueous r , 1~ of sweat. In order to provide a reflective layer with the suitable, ' "ty, thin metallic foil with small holes can be provided. For example, aluminum foil, commercially available from many sources including Reynolds Aluminum Co., could be r ~I dl~d with a plurality of small holes.
Absorptive, Iayers can be provided throuoh the use of a black surface. R~r.,.di l~, these surfaces would continue to allow F ' "q~ of aqueous - of sweat. It is: Id~l that any dye or, lalion in the d'' '- Iayer 92 not bleed when exposed to the aqueous ~ of sweat and also that it not interfere with any binding ' ~ ~y or in the analysis of the analyte. Any of a variety of thin black papers having these ~.,., I;~s are ~ 1-11~ available and are suitable for use as in the layer. For example, black Deltaware cellulose I ' _ - filters available from Baxter Scientific Products have been found to be especially useful for use as an _ layer. This product is available in a variety of i'~ oS;Ii~, more open pores are preferred. Thus, in the preferred embodiment, 0.6 micron black Deltaware filters are provided.
In an all~",aO.~i to the provision of an _ layer (not shown), the adhesive layer 65 can be made to a - le light, either throuqh _' ~.i or .~,' As an example of an _' ~.L.~ adhesive layer, black colorant, such as fine carbon black powder, could be - ~ al~d into the extrusion of the adhesive sheet.
Vlll. A- ' at-,d Analyte C-" m with Dermal Patches In some ,," li"ns of dermal patch I ' ~' ~, it is :' ~ '' to make long term, integral average ~i d~l~ll liu..s of the -, of an analyte in a subject's p~,., alia~. For example, in order to monitor the - , ' of subjects in a drug abuse program, analytes can be collected from the p~, dLJn of such subjects with dermal patches which are worn for a period of days or weeks. The p,~. 'y described dermal patches of the present invention are eminently suitable for such purposes.

CA 02222629 l997-ll-27 WO 96/3992337 PCT/U~6/~6915 In other '..... . however, rt is desirable to be able to determine the of an analyte in a subject's r in a much shorter period of time. For example, it can be desirable to be able to '~ ~ the rof a ~' - drug in a subject's r at a specific point in time. Also, when ~ i ., for bc abuse at the roadside Isuch as by a law ' ~ officer) or on the job, it is beneficial to be able to obtain results within a very short period of time. This is r 1) the case when the analyte to be detected is one which is rapidly r, by the body, such as alcohol.
It is one of the ~ i"~. of the present invention that a dermal patch can, in a relatively short period of time, collect enough r ~,- _ to allow an analyte carried in such r ~ '- to be detected by - ._ ' assays. While a dermal patch must normally be worn by a subject for at least 4 hours and perhaps for up to 24 hours before a diagnostically ' ' ' amount of an analyte will collect in the patch, this period of time can be ' ; ~ d to less than about two hours, and in a preferred embodiment to less than about 20 minutes, by applying heat to the area of skin where the patch is located.
A. I ~ the Rate of r~ : Increases the F~ate at which an AnalJ~te can be Col/ected The discovery that a desired amount of an analyte can be collected in a short period of time was made during . i designed to track the c . _ ~ of the ***e molecule in the 1,.,., , of a subject. In the i illustrated in Figure 13, a volunteer subject with recent ***e . i who had given his informed consent to pal . in the ! , ~ was _' ~ - ~d 32 mg of ***e HCI i ~ . A
dermal patch was then ~ ': '~ placed on the subject's skin, and after 30 minutes this patch was removed and replaced by a new patch. Dermal patches were replaced at each of the time points shown on the h axis of Figure 13 so that the a~pr :~ of the ***e molecule in the subject's pGI~,-_'- and its r~ Idi- over various time periods could be ' ~ ' The amount of ***e found in each patch is shown on the vertical axis of Fiyure 13. The highest level of ***e was found in the patch which was on the subject's skin from 3~ 2 to 4 hours after the c ' : dliUII of ***e to the subject. After the fourth hour post ~ ' :. . a steady decline in the c di' of ***e in the subject's r ~.- '- occurred.
A similar . i was later c 11 ' on the same subject. In this ! . ~ , 42 mg of ***e was - ' Gd to the subject by having the subject smoke it. The results of this ~ . ~ : are " ~all.d in Figure 14 in the same manner as in Figure 13. As in the t i of Figure 13, a peak in ***e s lldi occurs in the patch which was worn by the subject from 2 to 4 hours post ' ~,.: . followed by a decline in the s ~ . of ***e in the patch which was worn between 4 and 8 hours. The distinct feature of the GAI~_.i ' shown in Figure 14 is the result from patch 5, which was worn from 8 to 24 hours after the ~ ' : di' of the ***e. Rather than following the gradual decrease in ***e s ~,di' seen in the GAIJGI of Figure 13, there is a 'i~ ' : rise in s~ : in the patch worn between 8 and 24 hours post - ~ nd;' in this CA 02222629 l997-ll-27 W O 96/39923 -38- PCTrUS96/0691~
In order to dl ~ the cause of the ' I . 1 between the results of the c . ~ of Figures 13 and 14, we . 8" ~ whether anything different had happened between 8 and 24 hours post ~
in the t:A,~,_.' ' of Figure 14 compared to the experiment of Figure 13. It was discovered that the patch worn between 8 and 24 hours post ?' ~- ,- in the !, ~ of Figure 14 was wet with r . unlike any of the other patches in either - i A, L.~tl~, the subject had actively perspired during the period that he wore this patch.
As a result of the subject's having actively perspired, significantly more analyte diffused into the patch worn between 8 and 24 hours post - ' compared to the patch that was worn between 2 and 4 hours post ~ ' ~ ~ , This was a surprising result because it appeared that any diminution which had occurred in the r - _ of analyte in the subject's, ~ during active, ,, was - - ' for by an increased rate of r This resulted in the collection of a, ~ larger quantity of analyte in the patch worn between 8 and 24 hours post ~ in this compared to the amount collected in the patch worn during the same period of time in the LA,~_.' ' of Figure 13.
Active ,c_.., _: can be caused by an increase in the body's i alu~, such as during exercise.
A further , : was therefore 5~ ' ' ' to see whether an increase in the: L of only one area of the body could cause localked active r In this: ~ t, a subject was ' ~d 60 mg of codeine l "or~ (in the form of Naldecon CX PØ, 30 ml), and a dermal patch was then placed on each of the subject's thighs. A heating pad which was heated to 105~F was then placed over one thigh while the other thigh remained ' - ' The patches were replaced each hour for six hours, and a final pair of patches was placed on the subject's thighs for one hour between 24 and 25 hours after the ' ~ ~.dLI.,, of the codeine.
The results of this experiment are shown in the ~raph of Figure 16. From this graph, it can be seen that the patches applied to the thigh which was heated to 105~C contained more analyte than the patches on the unheated thigh during the first six hours of the , i . Thus, by inducing active p ., in a subject, a desired amount of an analyte present in the subject's, , can be collected from the subject in a shorter period of time.
B. Hedt ~ .
In order to detect analytes contained in, ,, more rapidly, a dermal patch can be used which makes use of a heat ut~ means to raise the i . ~ of a subject's skin in the area where the patch is located. In this aspect of the present invention, the heat ~ , _ means is r.~r~ capable of reaching a l , ~ of between about 100~F and 150~F, and more, ~r~ can be heated to between about 105~F
and 115~F. In one preferred: bc " t, the heat ~ , means reaches a , d of about 115~F.
The . alL.~: of the skin I ', ~ ' the dermal patch should also be _., . - 1~ within the foregoing ~"".~, ~ ranges when collecting r ~- '- according to this aspect of the present invention. Thus, it is to locate the heat g g means such that it is in contact with or is in very close proximity W O 96/39923 39 PCT/U~3C~C915 to the skin of a subject when the dermal patch is on a subject's skin.
In another aspect of the present invention, a subject can be made to actively perspire by raising the ~ ' L of the interior of the subject's body rather than by applying heat to the subject's skin with an external heat Q . v means. By raising the internal ~ ~ of a subject's body, the i . _ ~ of the surface of the subject's skin is also thereby increased. In this embodiment of the invention, a patch is placed on the surface of a subject's skin, after which the subject's core body temperature is raised such as throuy-h heavy excercise or through the a' ~ :_ of 1' _. agents which can raise the subject's body temperature. The I ., ~ which is g .Jt~,d by raising the subject's body temperature is then collected on the patch. Al~ .ali..'~, the internal temperature of only a portion of a subject's body can be raised, and ~ ., a;' can then be collected by a patch placed on the skin of the portion of the subject's body which is being heated. For example, an insulating material can be wrapped around the thigh of a subject in order to increase the i ~ of that subject's thigh, and a patch can be placed on the skin of the subject's thigh in order to collect, ., di' ~ Thus, in this aspect of the present invention, the i , di L of the surface of the skin beneath a patch is raised by using the body's own internal heat, . " ' rather than by eAI~ applying heat to a subject's skin as in other aspects of the present invention.
The i , ~ to which the surface of a subject's skin beneath a dermal patch should be raised in a pdl ' ~ aspect of the present invention is likely to vary, ', " _ largely on the length of time within which an analyte is to be detected. When the skin , d of a human subject is raised to about 115~F, sufficient r (about 0.10 ml) can be collected in a patch of about 10 cm2 within between about 20 minutes and one hour to allow the detection of an analyte. In general, the higher the I di L of the heat ~, , means, the higher the rate at which an analyte will be collected. Thus, heat ~ dlilly means which reach 1 ~ ~ dl~ S higher than 150~F can also be used to collect F However, bS which are high enough to cause burns or other skin damage, should not be produced by the heat j di' ~ means. Not only do such high i , dlu,es cause injury to the subject from whom ~ ,, is being " : d, but they are Z5 also likely to cause tissue damage which may interfere with the transport of r One of skill in the art can perform routine to ' , an a, I, ioldl , dlL.~: for collecting a desired amount of r ~ ' _ ' in a given amount of time. Such routine ~.IJ~l;..._..~L would likely include placing a patch having a, ,' , ~ area on a subject in a particular area of skin, bringing the dl~ of the skin of the subject in that particular area to a specific , ~ with a heat y~
means, the patch on the subject's skin for the desired period of time, removing the patch after the desired period of time, and I - b~y the amount of an analyte contained in the patch. If the amount of analyte in the patch is found to be i 'rh.;....l, the , ~ of the heat ~ di~ means or the area of the patch can be increased in order to increase the amount of r , ' di' which flows into the patch, and thus the amount of an analyte which collects therein. Of course, one could also lengthen the amount of time the patch is in WO 96~9923 40 PCT~US96/0691 contact with the skin in order to increase the amount of analyte in the patch.
C. E- ~"AQs~ dDermalPatches The dermal patches according to this aspect of the present invention can be ~._ ' in much the same manner as other dermal patches disclosed herein. The 'i~ feature of energy . t~ d dermal patches is, however, that they include or are designed to operate with a heat ~ ,- _ means which raises the ,' L of the patch and the surface of the skin near the patch. Therefore, the materials used to make L~1 a~ ~ dermal patches must be able to ~ ;:' ' the increased . t~ to which dermal patches are subjected.
- Either occlusive or - o ' _ materials can be used to construct the outer layers in this aspect of the i .. This is because the d ~, ' effects of using an occlusive patch, such as the tendency of an occlusive patch to foster back ''~u n, are not .~ ' over the short periods of time during which . .: is collected using ~, c~Cistp~l dermal patches.
A rate-limiting structure can be used in this aspect of the present invention if a j i ~;
dt:l~. of the amount of an analyte contained in, . is desired. Such a rate-limiting structure can be placed between the skin of the subject and the ' ~ material of an energy-assisted dermal patch. As long as the rate of F . flow which such a structure allows is slightly less than the expected rate of r ~ . of a subject at the temperature at which a particular energy ~ CPd patch operates, the _., ~
volume of ~ ., aliun which enters the patch can be ~ I -' by recording the amount of time that the patch is worn and then '~ that amount by the rate at which the rate-limited structure allows pe.,, to ZO enter the patch. After then ':, ~ the amount of an analyte in the patch, the 1, r I ' ' - ' i "
of the analyte in a subject's, rM~ can be ~
In a preferred ' - ' t, . ~ sistP~ dermal patches are designed to be used only once. Such single use patches have the ~ of being .. since they can be disposed of after use. S _' patches are also more hygenic because they obviate the possibility that an allergen or hl~ _ agent might be passed onto a ' , user of part or all of an energy a~;,;;,ll,d patch.
1. Electrical Heat 6- di- . Means The heat 9~ ~ ~, means according to this aspect of the invention can take various forms. In one ~ bc ' t, the heat 9~ , means is an electrical device which uses ~ IHL.i~y to generate heat (i.e., an electric heater~. One such electrical heat ~, _ means is an .,I~,IIi 'l~ heated pad, such as those: '~
sold to relieve back or muscle pain. Such a pad can be placed over a dermal patch so that the pad overlies the patch. r~ elal,l~, the pad is large enough in area to also contact the skin ,l ' " the patch. The pad is then heated to an à,u,ull, iale i , al ~:, such as about 1150F, in order to produce sufficient F , ~ to detect an analyte within a desired period of time.
In this ~ bL' t, the dermal patch and heating pad can be made or sold together as a kit.

CA 02222629 l997-ll-27 W O 96/39923 41- PCT/U'~ 915 F~,f.,._"~, the pad is adapted to reversibly secure the dermal patch. After such a patch has been used to collect ,, . it can be removed from the pad for analysis and replaced by a new patch.
In another - bL ' t, a dermal patch can include an ~ b 11~ c~ heating element, such as a metal wire or mesh, which can be reversibly ~ ' to a source of el~ .ity. Such a heatin~ element is , 1:~1,.. '1~ . dlLd from the rest of the patch and from the skin of a subject wearing the patch by a material, such as a plastic material, which does not conduct el~,~tli~.ity but does conduct heat. In this way, the element can be heated without exposing a wearer of the patch to electrical shock.
The electrically-powered heating means described herein can use either alternating or direct current.
In a i' ~, setting, such as a medical office or hospital, the heating means can c .~ use ~
current drawn from a ~ ._ ' electrical outlet by means of an electric cord. In an outdoor or other al r ~, ial~ setting, however, a battery powered electrical heat generating means is likely to be more 2. Chemical Heat 1' Means In another embodiment, the heat generating means of the e y~ a3;~ 1.d dermal patches of the present invention can comprise a chemical - , - which produces heat when it reacts. Such a chemical c , -can be ~v~dlcd into a patch such as the patch shown in Figures 15A and 15B. All~lllali.~, the c , - can be added to a patch after the patch has been placed on the skin of a subject.
One .l - which can be used in this e bL '- ' of the invention is disclosed in U.S. Patent No.

3,976,049 to Yamashita, et al. (reissued as Reissue Patent RE 32,026). This is used in pocket hand warmers for skiing and the like, such as those marketed by John Wagner ~~- ~ s, Inc. (Concord, CA). The r pc comprises iron powder, a metal chloride or sulfate, activated carbon, and water, though the principal heat g di- 9 agent is the iron powder. As disclosed in the Yamashita patent, various iron powders can be used, including cast iron powder, reduced iron powder, and ~1~..l,.1~ li~. iron powder.
The metal chloride or sulfate of the foregoing ~ , can, for example, comprise ferric sulfate, I tar sulfate, sodium sulfate" ~ sulfate, potassium chloride, sodium chloride, calcium chloride, and n ~ chloride. These ~ , ' enhance the reaction of the iron powder when it comes into contact with the oxygen in air. In addition, some of the chloride ~ ' . including calcium and ~l, ~ chloride, can absorb water vapor, and thus help to prevent the escape of water vapor. This ~ the heat produced by the ~ by ".1.._ water vapor from carrying away such heat. These metal sulfates and chlorides are p~ ' 'y present in the , - in an amount of between ,, I 1~ 0.5 30 parts by weight per 100 parts by weight of iron powder.
Another i ' ~ of this heat g _ ~ chemical ~ , is activated carbon. This i ~
serves in part to absorb some of the water vapor released during the LAU" I reaction which takes place when the c ,-- is exposed to air, and thus also helps to retain the heat evolved during the reaction. Other porous materials, such as porous silicates, cotton, and wood powder, can be used in place of the activated W O 96/39923 42 PCTrUS96/06915 carbon, however, if desired. Activated carbon is preferred over other materials though, because rt also serves to absorb any odors released during the reaction of the chemical: - When activated carbon is used in the present r , it should be present in the range of about 2.5400 parts by weight per 100 parts by weight of iron.
5Water is also used in the pf Water should be present in the range of ~" uA;~lla~ 10-250 parts by wei~qht per 100 parts by weight of activated carbon. Other " ." . such as catalysts of the oxidation of iron, can be added as well. For example, it has been found that the addition of small amounts of or copper to the iron powder greatiy increases the oxidation of ths iron. Such catalysts should be added in amounts of a, . I '~ 80-500 parts per million by weight, based on 100 parts by weight 10of iron powder.
In a preferred - Sc " t, the heat E~ of Yamashita is present in a layered bag 220 within the dermal patch of the present invention. As shown in Fi~qure 15C, the chemical composition 223 in this; ' - " is , ' ' by an inner bag layer 221 which contains the o , ~ 223 and allows air to reach it. The inner bag layer 221 can, for example, be made from cloth, such as cotton cloth or a synthetic 15cloth. This layer 221 should have an air p ty in the range of about 9,000-10,000 cubic c - : a of air per square . u; ~ of bag per minute lcclcm2min.).
The outer bag layer 222 also helps to prevent the leakage of the ~ . ~ 223 and in addition helps to prevent the ~" i of moisture from the bag. If vapor, such as water vapor, is allowed to escape from the bag layers 221 and 222 , - ' d, the , - 223 will tend to heat up more slowly, since the vapor .
20will carry heat away from the patch. This outer bag iayer 222 should have a lower air p~ y compared to the inner bag layer 221 in order to help retain such water vapor.
r,~r~ , the outer bag layer 222 has an air, ~ y of about 0.5400 cclcm2-min., and more ,.dLI~ the outer bag layer 222 has an air F ' "'y in the range of about 1-150 cclcm2-min. The outer bag layer 222 can, for example, be made of a plastic film such as F '~lh,; . pol~ "e, nylon, polyester, 25polyvinyl chloride, p 1~ " ' chloride, p~ , or rubber. Such plastic films should contain aeration holes over , . '~ 0.1%-5% of their surface areas so as to provide the requisite air p~. ' "~1,.
The ~ , should also, of course, be mixed while isolated from air or other sources of free oxygen so that it does not b~ oxidke before being ~ dlud into a dermal patch or before being used to raise the i , di ~ of the skin of a subject wearing a dermal patch. Once mixed and formed into a layered bag, the ~ should be sealed from the air until ready for use. If the c is ;"I,C.~ dlad into a dermal patch, the entire patch should be encased in material which resists the influx of air, or at least of oxygen, until it is applied to the skin of a subject. A material such as: ", ' can, for example, be used.
Other heat-producing chemical - can also be used according to the present invention. For example, the ~ ,- - disclosed in U.S. Patent No. 3,301,250 can be used instead of the - ,---described above. Other , known to those of skill in the art can be used as well.
Figures 15A-15C exemplify one possible patch according to the present .. The patch 200 in this n ~ ~ ~- ' . . jjCS an ~' ' material 210, a layered ba~q 220 c ~ - .. a chemical r . and an outer I ~e: _ layer 230. The _' ' material 210 is located ' ' the . L '' _ layer 230 which, when the patch is worn on the skin 240 of a subject, is in contact with such skin. The, .~L :- _ layer 230 , CfL~ ~ has an adhesive r . applied thereto to aid in securing the patch 200 to the skin 240 of a subject.
In the embodiment shown in Figure 15B, the ' L ~ material is circular in shape. However, the L material can be formed into any r .. ~ ~ shape which has a large enough surface area to collect 10 "_._, .:
S ,, " _ the ' ' material of the patch of Figures 15A and 15B is a structure r . i ~ ~, a chemical - , 223 which is capable of ~ .: 9 heat when reacted. The c pc can be the composition described above that comprises iron, water, a metal chloride or sulfate, and activated carbon which dlt~. heat by an e~u ' reaction when exposed to the air. In the ' - " : shown in Figures 15A
15 15C, the layered bag 220 is F -' ' ' the l ol~_l;._ layer 230 and thus is in contact with the skin 240 of a subject wearing the patch. In this embodiment, the - does not directly touch the skin of a subject wearing the patch, but rather is in contact with the skin through the bag so that any potential irritation to the subject's skin due to the chemical ~ pr ~ can be avoided.
Although the layered bag 220 is shown in Figures 15A and 15B as being ' Il, the ~,.ul~ L._ layer 20 230, this is not critical. It is only nec;~ y that the bag 220 be in close enough contact with the skin of a subject when the patch is worn that the heat b dleld by the chemical - . - 223 is sufficient to increase the rate of p_., di' ~ of the subject. In addition, while the layered bag 220 is depicted in Fi~qure 15B as being circular, this also is not critical. Thus, the bag 220 can also be pc -' above the, ul~.li.., layer 230 and can be formed into other shapes.
If the . is one which will not interfere with the detection or collection of an analyte of interest, the . , 223 can allclllali.~ be " ' ; within the -b L material 210. In some bc " : a layered bag may not even be -- y.
One particular P 1~d,,lauc of using chemicai heat ~qenerating means in the energy-assisted dermal patches of the present invention is that patches with chemical heat g .: means can be designed to be single use patches where both the patch and the heat ~c"c, " means can be e 'l~ disposed of after a single use.
The patch " ~dlcd in Figures 15A-15C is an example of such a sin~le use patch. This patch can be stored in an air-tight paGkage until needed. Once removed from the package and exposed to oxygen in the air, the chemical - pc described above which is contained in the layered bag 220 will beging to react and heat up. The patch is then quickly placed on a subject from whom a sample of r . dliu,l iS desired.

W O 96/39923 ~4~ PCT/U~9Gi'~915 After a desired amount of, , ~ has been collected from the subject, the patch is removed from the surface of the subject's skin, and the, , _ in the patch is analyzed. The entire patch, including the chemical heat ~, . means, is then disposed of. The chemical reactants of the chemical heat ~ _ means cannot be easily ,l dlod, and the -' ' material of the patch is . ' with the analytes of the subject from whom F , was collected after use. Therefore, disposal of the used patch and the chemical heat 1, , means is the only practical ', of the used patch -IX. D ~ ~ ~ Allergic S~ with a Dermal Patch A. Dermal Allergic ~
- In a further aspect of the present invention, a patch can be used to determine whether a subject is allergic to a particular allergen. Allergens include various forms of pollen, dust, animal skin and fur, c' such as ~ - ' - or food additives, and foods. The presence of an alleroen on the skin of an individual sensitive to that allergen causes an immune system reaction, known as an allergic reaction, jR that ~ ' Certain c-, of the immune system involved in provoking an allergic reaction, such as IgE, r ' t, and various immune cells, are believed to be able to migrate in the dermis. C . of the immune system also circulate in the blood supplying the skin, and as part of an allergic reaction to an allergen on the skin the permeability of the blood vessels supplying the skin is increased. Immune ~, of the blood are thereby also believed to pal in a dermal allergic reaction. Thus, the presence of an allergen on the skin results in the migration and r ~ ~ of immune I . of the body on the surface of the skin where the allergen is present.
ZO B. Using Dermal Patches to Oetermine A/lergic SL"."t,:;~
A subject, pl~rLI ' 1~ a mammal, can be tested for its ~ iti.::y to an allergen by - _ an allergen to the skin of the subject and then detecting any immune ! . which pass through the skin of the subject and onto a patch of the present invention. In this ' - t, a patch is used which contains an allergen in fluid with the skin of the subject when the patch is worn on the skin of the subject. For example, the allergen can be r : ' in the _' material of the patch.
In a preferred: ' - ' t, an agent is also present in the patch in fluid r with the skin of a wearer of the patch. The agent is one capable of .~ the r ' ty of the - . i_~ in the subject's dermis. Such an agent can thus increase the, ' ' ty of the - . i_~ in the dermis beneath the patch and facilitate the flow of immune ~ . : to the site of the allergen.
To determine whether a subject is allergic to a particular aller~en, a patch of the present invention which ed' ll~ includes an allergen is placed on the surface of the skin of the subject. In this ' - " t, when pe:l, dliun reaches the patch, the allergen is in fluid - with the skin of the subject and contacts the skin so as to cause an allergic reaction in the subject, if the subject is sensitive to the allergen.
The patch will then be able to collect bodily - . on the ~' b_.~t material of the patch which are W O 96~9923 PCT/U~ 915 a ~: I with an allergic reaetion, such as immune system c . . which migrate to the location of the allergen. Once such , have accumulated in the ' ' material, the patch is removed, and the presence of such ~ . is detected. If such allergic ,. ' components are present on the patch, this is indicative that the subject is allergic to the allergen tested.
Allr.llaL.. 1~, the skin of the subject can be exposed to an allergen in any other way, such as simply by placing a sample of the allergen on the skin of the subject. Pe.,, and other ~ , expressed through the skin can then be ' ' in a patch of the present invention located, . ~ to the area of the skin of the subject which was exposed to the allergen. If an analyte indicative of an allergic reaction is then detected in the, , aceumulated on the patch, the subject can be diaynosed as being allergie to the 1 0 allergen.
The patch used in this n ~~~- of the present invention can be any of the types, .,. '~
d~..L,-' ' P~r~ , a specific binding partner capable of binding and c ~ : ~ pal i ' bodily components indicative of an allergic reaction are included in the ~ layer (or ~ , zone) of this aspect of the present invention.
As an example of the present embodiment of the invention, an antigen such as pollen can be placed in the -' ~ layer of the patch so that when r ~~ dte~ the b~ layer and brings moisture to that layer, the allergen can migrate through the ~ li._ layer to the lower surface of the patch in contact with the skin and provoke an allergic reaction, if the subject is prone to develop an allergic reaction to the allergen. Al~llla~ , the allergen can be placed directly on the lower surface of the patch so that it ' l~l~ comes into contact with the skin of a subject wearing the patch.
After an immune response is triggered in a subject who is allergic to the allergen, ~ involved in the response will increase in c~ di- in the vicinity of the patch, since it is the site of the allergen. As sensible and insensible F :, ~ di' pass through the skin and into the patch, the immune c which pass through the skin with such r ~~ ~ _'- _ ' dl~ on the 2~ h_ layer of the patch.
Agents which increase capillary r ~ ~ "'y in the dermis i " '~ beneath the patch are, ~rL.. ' '~
included in the patch. '' ~I '3'C;. ' -., in the " i_~ beneath the skin can thereby be made to diffuse into the i aOIidl space of the skin and from there into F- _, Such r ~- ~ - can then carry these I -' ' into the patch so that they ean be detected.
X. Dermal Pateh Without A S3, ~ A'-; Material In some ~ described bc" . a dermal patch according to the present invention can comprise an _b b_..l material for " analytes that are ~.;,_d in a body fluid of a subject. Such an ' L~"l material is generally separable from an outer layer that protects the ~L i material from .;._.. _.. lal sr " The ' ' material, by itself or in ' with other : of the patch, collects and retains the , ~sed analytes and thus ~ ;v s at least a portion of the c~ Ldi- zone of such a patch.
The use of an _' - L material in a patch according to the present invention may be e e ~ when the analyte sought to be collected from r ~ iS one which also exists in the e...;._ t. For example, when using a patch of the present invention to monitor a subject for drug use, it is : y to prevent drugs in the L.. ;.l t, which might collect on or adhere to the outer layer of the patch, from i " ~ " the results of an assay for drugs collected from the F , ~, - of the subject in the - , zone of the patch. If one were to extract the outer layer and ~' L material together as a unit, drugs deposited on the outer layer, from the environment, would be extracted along with drugs from the ' material, - , zone). The result could be a false diagnosis of drug use by the subject.
In many cases, however, analytes from the skin have a unique signature, and this unique signature can be used to distinguish the origin of the analytes. For example, ***e coming through the skin is accompanied by a ***e : ' Mt EME, that is rarely found outside the body. Thus, the presence of EME together with ***e on a patch of the present invention would indicate that the subject who wore the patch had ingested ***e. Another example is the presence of "" " cross-links in an analyte. r~.i cross links are 1 ' ~ ' markers of bone loss, and have been ' with analytes such as N 1 ' ~, :- ' . ""i ' "
d~cA~"1,i' " and h,d UA~I 1'' F~. ' crosslinks do not appear in the .;.l t, so that the presence of such cross links in an analyte or in a sample ~ ~ a mixture of analytes indicates that the analytes were eA~Ir,~sed from a body fluid. There are therefore 3~," - - where an integral outer layer and e - zone can be analyzed together for the presence of specific analytes known to come from only the body.
In one bc " of the present invention, the r~ : zone can comprise a ' alle 250 that, by itself or in - ' i with an adhesive, collects and retains an analyte of interest. Although an ' - Lrul material can also be used together with the ' 250 in this bL " t, no separate ~ ' S.,..l material is y. The . ' 250 in this - ' - ' is then assayed for the presence of the analyte as in other - bc " Is of the present invention.
It is believed that there are several ways that the patch 10 in this i ' - " : of the present invention functions as a r - ~: zone without a separate -' b.,.~t material. One way that the patch 10 is believed to retain analytes is by retaining them in or against the ' . 250 itself. As analytes dissolved in prl_, _E . reach the underside of the ' . - 250, they become ' " ' The water content of the r--~. aliull then ~. i, dles. As discussed ,.. ~ herein, when the ' 250 is a fluid I '' ' ane or when a patch according to the present invention is worn for only a short time, the analytes cannot "back diffuse" into the body. Thus, these dlud analytes are believed to become passively trapped against or in the ~ alle 250. The analytes are believed to be held there by weak chemical bonds or by ellllalJIll~lll within the structure of the ' ~

W O 96~9923 ~7 PCT/U',-'~6915 Thus, in a preferred embodiment, the membrane 250 is formed from a material which can weakly 'l~ bond or, h~ trap analytes 6A~ C~S~.d from body fluids such as I _, ~ For example, the membrane 250 can be formed from urethane, F 1~. I ' . r~'~dth,l~ foam (such as Foam Medical Tape, Product No. 1772, sold by 3M), ps1~ath,' tape (such as Plastic Medical Tape, Product No. 1526-L, sold by 3M), ethylene vinyl acetate (such as Plastic Medical Tape, Product No. 1527, sold by 3M), ~ .. ,. rayon (such as Medical A' l N .. Product No. 1603, sold by 3M), polyvinyl chloride foam (such as Foam Medical Tape, Product No. 9777-L, sold by 3M), and Gore-texn' (Teflonn~ or PTFE) film. If the ' . 250 is ~' ' t, it can be formed into a layer at least as thick or thickerthan that present in Te~adermTU 1625, which is 0.02 mm in i' ' . in order to increase the ~ b~ ~,t;... qualities of the membrane 250. The ' 250 should also be flexible so as to be able to conform to a body surface to which it is applied.
It is also c N_ " in the present embodiment to use a ' . 250 that does not curl up when it is removed from a subject. If the membrane 250 curls up after collecting analytes, this makes it more difficult to extract any analytes which may be bonded to or trapped in the portions of the membrane 250 which are curled, since such portions will not be in direct contact with the solvent or other agent used to remove the analytes from the ' - 250. ~ materials such as F 1~ ~ ' foams are therefore preferred.
The ' 250 in this: ' - ' of the invention can further include an adhesive on the underside 251 of the membrane 250. In a preferred embodiment, for example, the membrane 250 has a thin layer of an acrylate adhesive on its underside 251. Other r"~ ~,5, such as acrylate c, 1~ , synthetic ' i., cement, hydrogel adhesive (Product No. MSX-1186 from 3M), and Wi I~IICK adhesive (available from Avery ~ ' can aiso be used.
It is believed that analytes are also retained in the adhesive layer on the underside 251 of the ' .
250, either by chemical affinity or through I ' ~ ' ~ - Thus, when choosing _ " . to be used in this bc" , is preferred to choose adhesives from which a given analyte can be ' 11~ andlor :' 'l~ extracted.
It is also believed that analyte is retained in the outer surface of human skin, called the stratum corneum, which is r , s ~ of kl.. ~ ' epithelial cells. These relatively flat cells are ~ d, r ' ' a brick wall pattern in cross section. Epithelial cells :' ' ~d alc and kc._ ~ . forming a new layer of stratum as the outer layer of stratum is slou~qhed off as dry skin. The stratum corneum is typically about 14 cells deep of kcldlilliLcd epithelial cells. One layer is lost per day and one layer replaced per day. It is the stratum corneum that retains water inside the body from ~. ~ and prevents !' " ~ 5 L s from being absorbed across the skin and entering the body.
An analyte can be ~..LI ., d in the i cldi ~!" stratum corneum cells. Resed" ' ~ in the field of I. S' Illdl drug delivery have learned that they must include the fraction of drug deposited in the ~
stratum corneum cells to obtain adequate mass balance studies. (Bucks, Maibach, and Guy, Pha" - LiLal CA 02222629 l997-ll-27 W O 96~9923 PCTrUS96/06915 Research. 515~1988), 313-315). It is also known that hair retains biological analytes, such as drugs of abuse.
Hair is ! , - 1~ of k~ ~ ' cells that are similar at least in chemical ~.hal a~ H;~ to stratum corneum cells (Harkey, M.R., Forensic Science I I, 63(1993L9-18).
If the I ' _ 250 includes an adhesive on its underside 251, when the ' 250is removed from the skin 12 of a subject, the removed adhesive carries with it sections of stratum corneum that in turn carry analyte. When stratum corneum cells are removed with the adhesive and ' of a patch, the stratum corneum cells function as a part of the ~ , zone for the analyte. In a preferred ' ~' t, the adhesive is one which is designed to migrate into the stratum corneum, so that when the patch 10 is removed, many layers of stratum corneum are adhered to the membrane 250. For example, the adhesive can be an ___ . _ adhesive which migrates into the stratum corneum and diffuses into the skin, such as the adhesive which is '~ used to apply the patches used in an EKG test to a patient. In addition, the adhesive used in this: bc ' should be designed to adhere to the I ' ~ 250 and not remain on the skin 12 when the 250is removed frDm a subject.
Thus, the 'alld250iS capable of retaining analytes even if it lacks a separate ~ ' - b_..l material and even if it itself is not .' ' t. This is due to the fact that other L.halaL~t~ of the ' ~.. , can serve to retain analytes, such as the ability of adhesive to retain analytes, the retention by the adhesive of skin cells which ' N~., contain analytes, and the weak chemical bonding of analytes to the surface of the d~e250.
This bc' I of the present invention is shown in Figure 17. As seen in Figure 17, the patch 10 of this ~ bc' ~ d5 a ' or layer 250 which is secured to the skin 12 of a subject. The L~a"d250isp.~r~ fluid p~" '' or at least gas I '' . if the patch 10 is going to be worn for an extended period of time. However, if short term wear is all that is needed to detect a particular analyte, then an occlusive material may also be used for ' 250.
In order to secure ' 250 to the skin 12 of a subject, a first side or underside 251 of the '. 250 can further comprise an adhesive. The adhesive can be any adhesive which can be applied safely to skin, which will secure the ~ u~ 250 to the skin 12 for a sufficient period of time to detect an analyte, and which will not ' t 11~ interfere with the detection of the analyte of interest. D~r~.hl~, the adhesive is one from which the analyte of interest can be extracted, thereby G " _ the detection of the analyte. One of skill in the art will be able to choose suitable adhesives for use in this ~ 'Jc' of the present invention.
In a preferred L- " 1, the III~ LI_ - 250 and adhesive comprise T~ ' ", 1625 wound dressing available from the 3M Company (St. Paul, MN).
In other ~-'- " the ' dne250 can also be secured to the skin 12 of a subject by other means. For example, the ' s 250 can be secured with one or more layers of adhesive tape, such as Tegadermn' 1625 wound dressing or D~ tape (both available from 3M), which overlap the skin 12 of the W O 96/39923 49 PCT/U'-,,''~6915 subject and at least a portion of the I ' ~ 250. A non-adhesive strip could also be used to secure the ' _ 250, such as, for example, by wrapping the strip over the ' . 250 and around a limb of the subject.
The first side 251 of the ' 250 is in fluid andlor physical contact with the skin 12 of a subject wearing the patch 10. A second side 252 of the patch is then in contact with the environment outside the skin 12 of a subject wearing the patch 10.
The ' 250 in this embodiment preferably has a surface area of between approximately 1 and 120 cm2, and more ~ has a surface area of about 42 cm2. The membrane 250 also, ~f~ has a thickness of between about 0.005 mm and 3 mm, and more, .,f~..' 1~ has a thickness of about 0.02 mm.
There are several ~ '~. j to a dermal patch which comprises only a single layer of material. The ~~ ~ of such a patch is greatly simplified, since it is not y to attach a separate piece of bc ' : material to the ' 250 in order to create a dermal patch capable of retaining analytes. The physical profile of the patch is also lower, thus making the patch less visible and less obtrusive when worn under clothing.
In use, a ' 250 of a patch 10 according to this embodiment of the present invention is placed in fluid com ~ with a source of body fluid of a subject mammal. For example, the ' 250 can be placed on the skin 12 of a human to collect analytes , ~..;,_d through the skin 12 in, ., The - 250 is Ib ..'1~ secured to the subject, l ~f~ by means of an adhesive located on the underside 251 of the ' al,e 250. After a period of time sufficient to collect enough of an analyte of interest so that the analyte can be detected in an assay, the patch is removed. The ' alle 250, including the adhesive and any skin cells attached to the ' _ ~ 250 andlor to the adhesive, is treated so as to separate the analyte from the ' _ . adhesive, and cells, or c~ make the analyte available to be tested. The liberated analyte is then subjected to an assay which can detect the analyte.
This bcd of the invention can be used in all the ., " described cl~ herein for the dermal patches of the present .. For example, if the I ' 250 is made from a " ' ' ' material, the ~ ' . can be dissolved after use in order to more easily and, .1~ measure analytes retained in the ' 250. A PVC ' used to collect analytes, for example, can be dissolved in ~.~.' ' in order to free the analytes collected by the ' Al~ , the ' ~ 250 can be made from a d ' '' material such as cellulose, which can be degraded by an enzyme in order to release the collected analytes. The ' ane 250 can also be fitted with a unique code, such as a bar code, as described el~_. ' herein, in order to detect i , i ~ with the patch.
One pal; ' l~ preferred use of the patch 10 in this bc " is in the detection of drugs of abuse.
For example, drugs of abuse such as ***e and codeine can be detected. In this ,' i . the patch 10 , i;,es the ' - 250, which is applied to a subject and then analyzed as described el~ herein.

WO 96/39923 50 ~ PCT/U' ~Gi'cC915 An - to confirm the r 1~ ' ' of this - ' ' of the invention is described below in Example 20.
The following examples describe only specific a~l, ' of the present invention.

F~ of '' - ' ' P~ ' to CK-MB for Use on a Test Patch In -- d with one known process for preparing - ' ' ' " . mice such as Balblc female mice or other mouse strains or even other suitable animals such as rats or rabbits are immunked with an amount of the CK-MB enzyme to initiate an irnmune response. The en~yme dosage and i~ schedule for producing useful g of suitable ~ c~ It.. can be readily J~ d, '- _ on the animal strain used.
The size and spacing of doses of CK-MB or other antigen are of prime importance in the antibody response. r. - ~, a wide range of antigen doses - '~ affords immunity against harmful agents. Thus, a small dose of antigen is usually sufficient to initiate an antibody response, i.e., . g of proteins are ~ -', However, a minimum dosage for initiating an immune response does typically exist, although doses of antigen below the minimum dose --~ y to initiate an antibody response will usually maintain antibody, ~ '~ which is already in process. For example, an initial i ~ with ~ y 50~9 of the enzyme may be followed by a h,~ ~ : series of five ~-When certain - pr ' which are i' ' _s not ~antigenic are mixed with an antigen, enhanced antibody l: ~ ' against the antigen occurs, as u.;' ' by the ~ of large amounts of antibody in the serum, a prolonged period of antibody, I ' : and a response to lower doses of antigen.
Such cgh~ DS are called "adjuvants" and include Freund's ~' and complete adjuvants and alum gels.
Thus, a given dose of antigen is usually more effective when injected ~ with an adjuvant or when injected as repeated small aliquots than when - ' ~d ~.. '~.
Typically, the adjuvants of Freund are preferred. The original "complete" Freund's adjuvant mixture consists of mineral oil, waxes and killed tubercle bacilli. Antigen is added to the adjuvant mixture in an aqueous phase to form a water-in-oil emulsion in which each water droplet is - ,. ' ' by a . oil phase c~: ~, tubercle bacilli. The mixture is r '~ injected 5~ into r,A~,~. ' animals. Injection - ': a marked yl ' reaction with lesions ~ " largely of c " : of ' ~ -rll:;~. . ' " ' cells and l~ L~Iti. The local Iymph node shows a small increase in plasma cells.Following the ; with a primary dose of a soluble protein antigen, specific ' - " normally first appear in the serum after a few days and then increase in number until about the second week. Thereafter, the number of serum - S~ " slowly declines over a period of weeks to months.
The first serum ~ ' - " to appear after il are IgM SL ' - These are usually followed by the alJ,OW~ of IgG : bc' Later, as antibody serum levels increase, IgM ~n ' - " di_ap~.~a;, probably as a result of specific feedback , t.-~;UII of IgG 5c " -CA 02222629 l997-ll-27 W O 96/39923 -51- PCTrUS96/06915 After the "primary ,, n to a protein has passed, a second dose of the same antigen given months or even years later usually elicits an intense and aucl~.all.d "specific Is~ ' y response" in which serum antibody usually be~qins to rise within two or three days of exposure. The serum levels of antibody in a sss ' y response may reach as high as 10 mg per ml.
The animal is subs, 1~ sacrificed and cells taken from its spleen are . , ' ' in an . ~, ial~
medium and fused with myeloma cells, such as those ': ~ ' ' from the murine cell line Sp210-Ag14. The result is hybrid cells, referred to as "hybridomas," which are capable of l_, ~' :- in vitro and which produce a mixture of ' ~ ' ~ specific to each of the various ll ~, ~ '' sites on the CK-MB enzyme.
The myeloma cell line selected should be compatibie with the spleen cells, and optimally should be a cell line of the same species as the spleen cells. Althou~qh the murine cell line Sp210-A~q14 has been found to be effective for use with mouse spleen cells, other myeloma cell lines can . ' llali._~ be used. See, for example, llature, 276: 269-270 (1978).
The myeloma cell line used should, ~f~ be of the so called "drug ~~ Ill" type, so that any unfused myeloma cells will not survive in a selective medium, while hybrid cells will survive. A variety of drug resistant I ,~' are known.
The mixture of unfused spleen cells, unfused myeloma cells and fused cells are diluted and cultured in a selective medium which will not support the growth of the unfused myeloma cells for a time sufficient to allow death of all unfused cells. A drug resistant unfused myeloma cell line will not survive more than a few days in a selective medium such as HAT (h,~ ' ~ ,: and Ih,, " S Hence, the unfused myeloma cells perish. Since the unfused spleen cells are n~ 'ig t, they have only a finite number of " until they fail to ,.i",~ ' - The fused cells, on the other hand, continue to ", ~~ because they possess the 'ig quality ~ ' - ' by the myeloma parent and the enzyme y to survive in the selected medium bull,d by the spleen cell parent.
The from each of a plurality of h,' i' ~ " wells is 6~ ~ t~d for the presence of antibody to a specific site unique to the CK MB enzyme structure. 11,; ' are then selected r I ~ "~
the desired antibody to that specific site. This selection may be, for example, by limiting dilution, in which the volume of diluent is aldt;: -'1~ ' ' ' to isolate a certain number of cells le.g., 1 to 4) in each separate well of a microliter plate. In this way, individual h,, i' -- may be isolated for further cloning.
Once the desired h,' i' has been selected, it can be injected into host animals of the same species as those used to prepare the h,; i' a, ".Lr~lahly sy, or semi~y ~, animals. Injection of the h~b.- ' - will result in the ~., of antibody producing tumors in the host after a suitable b. time, resulting in a very high or ~ of the desired antibody in the blood stream and in the F ' exudate of the host. Although the hosts have normal - ' -" - in their blood and exudate, the cu,,~.,,..l._ of these normal . -'-" is only about 5% of the s s . of the desired ' -' antibody. The - 1I -' W O 96~9923 -52- PCTAUS96/06915 antibody may then be isolated in d~ ..d with h ' s known in the art.

r" ai- of '' .' ' Test Patch One specific l~lr~- ' of the present invention is the dual determination of skeletal muscle and cardiac 5muscle status as a result of exercise. A dermal patch is , ' in a ' with the - ' ~
~ alud at Figures 3 and 3a. The gauze layer is prepared by cutting a circular patch having an a" u..i...~tel~
1-inch diameter from a Johnson & Johnson non-stick gauze pad. The inner and outer porous layers are next prepared by cutting two circular patches of Ultipor (nylon 6~, from Pall G~ I in Glen Cove, New York.
Ultipor ! '_ is both fluid r - ~ and ., ~ . and a membrane is selected having, for example, a 1 micron rating. The . bQ d layer is prepared by t.. ' :~ bonding ' ' antibody raised against CK-MB to a I "i,' ~y of pH~al~l beads having a mean particle size of at least about 10 microns.
The patch is . ' ' ' by f: ' _ a,, . ~ ~ '~ 0.2 gram of I ~ . ' ' across the surface of one of the porous layers. The second porous layer is Ih...~a~l~l disposed adjacent the m;L,.b ~' . and the gauze layer is next placed on top of the second porous layer. At this point, the patch is ' d~,.... The p~. ' dl edges of each of the first and second porous layers and the gauze layers are secured together by -heat sealing i ' , Thereafter, the ' "~ is turned over and an annular torus of adhesive tape having , uAilllat.,l~ a 2-inch outside diameter and slightly less than a 1-inch inside diameter is secured thereto to produce a finished patch.

Cardiac Muscle Status Test The patch of Example 1 is then secured to the chest of a healthy 40-year old male and worn P ~
a 36 mile (130 minute) bicycle ride. Upon removal of the patch following the ride, the test patch is immersed in a first solution &~ an excess of enzyme labeled anti CK MB for 3" ~ 1~ 30 minutes, to permit - u U~.I of labeled antibody with ' " ' analyte. The patch is then rinsed under tap water to remove unbound labeled antibodj and immersed in a second solution - ~ " a substrate for the bound enzyme label, which ' l. es a color change when acted upon by the enzyme. A~, _ of color through the top porous layer indicates the presence of CK-MB, and possible cardiac injury. C~, ; to a color chart permits rough ~;ualiu--.

Test for Use of Mariiuana THC pOl~l,lollàl antibody from sheep (available from ~ v . Bournmouth, England) is diluted 1:100 in PBS (pH 7.5). The a"d~ are bound to Gelman 0.45~ (SU 450) Ultrabind S r~ l~d '' ' a"e, following the protocol in Gelman Original E,, t r~ r ~, a" E P.N. 31,084. The I ' alles are air dried.
Disks, 318 inch in diameter, are cut from the coated Gelman ' a"ea. These 318 inch disks are mounted at CA 02222629 l997-ll-27 W 0 96~9923 53 PCT/u~ 9ls the center of a 114 inch diameter hole cut in the center of a one inch diameter circle of Tegaderm 1625 T. ~..l Dressing (available from ~" : Mining and '' f~ , _ St. Paul M
Three mounted ' _ are secured to the chest of a subject who then smokes a marijuana cigarette.
Three mounted ! ' are also secured to a subject who has never used marijuana in any form and who agrees not to use it for the next seven days. The ' . remain in place until they are removed, seven days later. Each of the removed membranes is flushed five times with 300~/1 of 0.2% Tween 20 in PBS. The ' . are incubated for 30 minutes in 100 ~1 of E-Z Screen C ' ' enzyme conjugate from the E-Z
Screen Test Kit (available from Ec.~ Inc., Burlington, North Carolina).
After ~ ' : each membrane is flushed three times with 300~/1 of 0.2% Tween 20 in PBS, followed by three flushes with PBS alone. The membranes are then incubated in TMB '' ' Peroxide Substrate (available from Kirkegaard & Perry Labs, Gaithersburg, r~ t' I for 10 minutes. A light blue ~ k~ .
appears in all six ' White dots appear over the t k~ ~ ' on the three I ' taken from the subject who smoked a ~ - cigarette, indicating sweat gland output of sweat r : ~ THC ' ;.ali.
No white dots appear on the three ' ~ taken from the subject who has never used marijuana.

Positive Control Patch Mouse anti-human IgG, Fc m ' ' antibody (available from ICN, Costa Mesa, California) is diluted 1:100 in PBS (pH 7.5). The ' - ' - are bound to Gelman OA5,1./(SU450) Ultrabind S ., lcd M( ~
following the protocol in Gelman Original r, ~, ' ~ fa Cl a~, " " P.N. 31,084. The ..._..,h. s are air dried. Disks, 318 inch in diameter, are cut from the coated Gelman membranes. These 318 inch disks are centered and mounted on a 114 inch diameter hole cut in the center of a one inch diameter circle of Tegaderm 1625 Tra,.~alc..l Dressing.
Three mounted ' are secured to the chest of five human subjects. The ' un~. remain in place until they are removed, seven days later. Each of the removed r ' is flushed five times with 300,UI of 0.2% Tween 20 in PBS. The ' are incubated for 30 minutes in 100 ~l of ll.. :._. " ' ~ uAida;~c enzyme c~ to goat - ! IgG, Fc FH~' ' antibody (available from ICN, Costa Mesa, California) diluted 1:1000 in PBS.
After b~i each ' is flushed three times with 300~11 of 0.2% Tween 20 in PBS, followed by three flushes with PBS alone. The ' . s are then incubated in TMB M- ' alld Peroxide S ' ~,alc (available from Kirkegaard & Perry Labs, G- ' ~bu.~, '' y' I) for 10 minutes. Blue dots r ", " ,. to individual sweat ducts appear over the t~ k~ . ' on all of the ' ~nes, indicating that the ! ' ' Iy of the patches is operative by their detection of the IgG expected in the sweat of all subjects.

CA 02222629 l997-ll-27 W o 96~9923 54 PCTAUS96/06915 Chemical r' ' of Cocaine Collected on a Patch A'~IUun disks, 318 inch in diameter, are cut from Gelman I ' (Gelman OA5,u (SU-450) Ultrabind S , ILd '' ' ~' These 318 inch disks are mounted at the center of a 114 inch diameter hole cut in the center of a one inch diameter circle of Tegaderm 1625 Tr. pr Dressin~q (available from Mining and r '~ g, St. Paul, f' ' to form a patch.
Three of such patches are secured to the chest of a subject who then ingests ***e. Three patches are also secured to a subject who has never used ***e in any form and who agrees not to use it for the next seven days. The patches remain in place until they are removed seven days later from each subject.
The ***e ' ' and other - present in the I ' _ ~~ of each patch are then eluted from the ' ~ ~s by soaking each of the I ' in a synthetic urine matrix for 30 to 60 minutes at room c with ' ' agitation to form an analyte solution. Following elution, the analyte solutions derived from each of the patches are brought to a pH of 11 by the addition of NaOH to each of the solutions.
The solutions are reacted for 20 minutes at pH 11 and at room dlUIc, after which the solutions are - 1- ' d with HCI.
Each solution is then subjected to c " : analysis with the Roche RIA system (Nutley, NJ) for detecting the l-,cl '-" of ***e BE. The subject who ingested ***e tests positive for the ***e ' ' BE, while the subject who did not consume ***e over the test period does not test positive for BE.

P,e,-d- and Use of a Di s ' '' AbsorDtion Disk Nylon 616 fibers (Vydyne 909 from '' lu Co.) are formed into an ' ': gauze. Disks 3~ r u~illlalll~ 318 inch in diameter are cut from such gauze and are then mounted at the center of a 114 inch diameter hole cut in the center of a one inch diameter circle of Tegaderm 1625 Tl. ~..l Dressing (available from '' Id Mining and ~ g, St. Paul, ~' l ) to form a patch. Such a patch is then applied to a subject. The subject is directed to ingest ***e, and a quantity of, , is then allowed to ar ': on the patch.
When a sufficient period of time has passed for a ': '' amount of ***e to ' on the patch, the patch is removed from the subject and placed in an insoluble container. A base capable of dissolving the Nylon 616 fibers is then poured over the patch. Once the nylon . b ~: disk is dissolved, the ~ d - of the patch are removed from the container. Since ***e is ~ .c.tud into b ,' ~ (BE) in the presence of a base, the ***e contained in the disk is ,,,ci ' -' ' to BE when the disk is dissolved.
The solution of the dissolved nylon, BE, and the other remaining ls of the used ' s ~.Uu,. disk are next ~f: ' ' This solution is then analyzed using a Roche RIA system (Nutley, NJ). The BE in the solution is detected and the amount of BE o: dle.l in the absu.r disk is dl c~ ' W O 96~9923 55 PCTrUS96/06915 n ti~; . D~t~.l of a C~ of Pe._ To ' ~ how much of an analyte is c ~ ' in a given volume of sweat, a patch is first c t~ ' having a support layer made from a polyester- ,, ILd F 1~ ' ~ I, . membrane, '~ .~ by Nuclepore ~Menlo Park, CA). Over this is placed an ~'~ ' material such as Filtration Sciences medical grade paper (FS#39) for accumulating and r , I~ l _, ~, The surface area of the layer of ~ ' ' ~ material should be the same as or smaller than that of the support layer so that when placed on a subject's skin, only the support layer is in contact with the subject's skin. Over this layer is then placed an outer I UIL..~ layer made of 1625 Tegaderm wound dressing made by the 3M Company (St. Paul, M : ' This outer layer is of a larger surface area than either the support layer or the ahsu.' material and covers both of these layers. The outer layer ., ~le.. the ~' i material from the outside of the patch and helps prevent r , from entering the ' ' layer except through the support layer. The outer perimeter of the outer layer has an adhesive on the side of the outer layer that faces the skin of a subject when the patch is applied to the skin of such a subject in order to secure the patch.Such a patch is next placed on the skin of a subject whose, , is to be tested for the presence of theophylline. The subject wears the patch for 7 days, during which time r ,'~ ' . passes through the support layer at a rate of less than 6 ~ . 1 7 ~ ~ After this the patch is removed and b; : ' to analysis to dl the amount of theophylline . ~ ' in the patch.
To ~ the volume of sweat that has passed into the aLSOI' material of the patch, the rate at which PLI,- passed into the _' ' material is multiplied by the amount of time the patch was worn, i.e., 7 days. The amount of , h~ r ' ~ ~' in the patch is then d(: , -' These numbers are then related in order to determine the amount of analyte contained in a given volume of p~._, by dividing the amount of the analyte in the patch by the volume of r ,- ~' which passed through the support layer into the ~' L~"l material.

Fl~v~.d6~.r. and Use of a Dermal Patch to Determine the Sc..i.iti.;tv of a Subiect to an Allernen In order to determine whether an individual is allergic to cat hair, a ~, ~ c ; " cat hair is first placed on the lower surface of a disk 318 inch in diameter made of Filtration Sciences medical grade paper (FS#39). The upper surface of the disk is mounted at the center of a 114 inch diameter hole cut in the center of a one inch diameter circle of Tegaderm 1625 Tl L.. l Dressing (available from '" Id Mining and M r~ ill9, St. Paul, '' Id). The patch is then placed on the surface of the skin of a human subject for 3~, U~dllldlLI~l 3 days in order to a~ ,~ d0ull on the disk and form a ~ :.dle. The disk is then removed and analyzed to detect IgA against cat hair. The presence of IgA against cat hair indicates that the subject has - . esscd an allergic reaction to the cat hair antigen.

CA 02222629 l997-ll-27 W O 96~9923 -56- PCT/U',G,!~ C915 Although this invention has been described in terms of certain preferred b~' and assay schemes, other ! ' ' ~ and assays that are apparent to those of ordinary skill in the art are also within the scope of this invention. A~ P ~'~, the scope of the invention is intended to be defined only by reference to the appended claims.

C~ ~l a Dermal Patch which Inhibits Back-Diffusion A' ~, disks, 318 inch in diameter, are first cut from 6elman membranes (Gelman 0.45,u (SU-450J
Ultrabind S ., lud '' ' _ ~: These e b~ r'- disks are next soaked in a buffer of 0.1 M acetic acid at a pH of 5.0, and the disk is allowed to dry. The 318 inch disks are then mounted at the center of a 114 inch diameter hole cut in the center of a one inch diameter circle of Tegaderm 1625 Tl_ . L.. l Dressing (available from '' : Mining and r~ c, ~ p, St. Paul, ~' : ' to form a patch. The buffer-soaked ' disk could also be mounted onto the Tegaderm dressing while still wet.

P~G..; the Back-Diffusion of a Druq of Abuse A patch is s ~ d according to Example 10. The buffer is added to the b~ IIL;.. , Iayer (.,b~u-~:
disk) of the patch in order to keep the pH of the patch and the surface of a subject's skin below the patch in the range of 4.5 - 5Ø When the patch is placed on the skin of the subject who has ingested one of the drugs of abuse listed in Table 2 (above), the patch ~ dtes the particular drug ingested, without any c ' back-diffusion.

Qua~ a~ !v D~l~, ~ . the Amount of an Analvte Present in a Subiect A patch is cr ~ ' according to the method of Example 10 with an db;U~ Ji' disk having a surface area of 10 cmZ. This patch is then placed on the biceps of a subject's arm. The subject, weighing 168 pounds, is given 126 mg of ***e, which is ' , '~ nasally ingested. The patch is worn for appluAillldlcly2oo hours, and the subject is not allowed to ingest any more ***e. After 200 hours the patch is removed in order to ': ~ how much ***e has been collected. In this experiment, appro,-i..,al~l~ 500 ng (0.5 mg) of ***e is l~..G.t:d on the patch.

W 096~9923 57 PCT/U~,~/C~915 Pn. di' of a Heat r- ~ . Chemical G~ and a Lavered Baq The following in~qredients were prepared and mixed without substantially exposin~q ehem to the air:
Material Amount Cast Iron Powder 30 mg Ferric Sulfate 5 m~
Active Carbon 30 mg Water 30 mg After mixing, the resultin~q: was placed on a piece of cloth made from cotton that is about 4" long and about 112" wide. The cloth should be about 1 mm thick and allow air to pass through it at a rate of ap~ 500cclcm2min.
This cloth is itself on top of a piece Of r ~y~lh,; film that is sli~qhtly lon~er and wider than the cloth bag. The film is pre-drilled with ho!es of sufficient ske and number to allow air to permeate the film at a rate of p, u~dll~al~ly 4 cclcm2-min.
The pr is spread evenly over the surface of the piece of cloth, and the film and cloth are then folded along their short axes so as to form a relatively long tube that is . I ~ '~ circular in diameter. Any excess amount of the chemical composition that will not fit within this tube is removed and saved for later use.
The exposed edges of the film are then sealed with heat or with an adhesive so as to encase the chemical c within the cloth and film.

P~aldi of an '- ,..-A~si~ted Dermal Patch which Uses Chemical Heat G di' ~. Means A round disk made of an ' ' material is first t ~ I ~ by cutting a portion of a Gelman I,,~ hl. - (Gelman 0.45ju (SU450) Ultrabind S ,, lud 1\1 ' ~ which is 318 inch in diameter. This disk is then placed in the center, on the adhesive side, of a one inch diameter circle of Tegaderm 1625 Tldll~pal~..l Dressing (available from '' la Mining and Manufacturing, St. Paul, '' la). The tube formed from pGl~lh~ film, cloth, and the chemical of Example 13 is then placed around the edges of the disk, generally in the manner shown in Figures 15A and 15B, in order to form a patch. The entire patch is then quickly sealed from the air by .~.. I. ' ~ it with " ,' film.

Usinq an Enerqv Assisted Dermal Patch which Makes Use of Chemical Heatinq Means The patch of Example 14 is removed from the air tight ~ ", ' - package in which it is wrapped and _ W O 96/39923 -58- PCT~US96/06915 is ' - l~ placed on the arm of a subject. The; . _ ~ of the patch and the surface of the skin i ' -' the patch rises to over 1000F, and the patch is ~ ' on the skin of the subject for about one hour. The patch is then removed from the subject's skin and is analyzed in order to detect the presence of an analyte of interest in the patch.

UsinD a Subiect's BodY Heat to Accelerate the Collection of PL.. , with a Dermal Patch A dermal patch is - :, ' by first forming a round disk made of an rb~ ' material which is 318 inch in diameter. Such a disk is cut from a portion of a Gelman ' - (Gelman 0.45~1(SU 450) Ultrabind S,, ~I,d ~' ' ' This disk is then placed in the center, on the adhesive side, of a one inch diameter circle of Tegaderm 1625 T , ~ Dressing (available from '" : Mining and ~' faLIL.i,lg, St. Paul, r" s ta), thereby forming a dermal patch. This patch is then placed in contact with the thigh of a subject, and an insulating material is wrapped around the subject's thigh on top of the patch. The , dlL.~ of the subject's thigh increases, and the subject actively perspires. After about two hours, the patch is removed from ' I :' the insulating material, and the r ~ ' " which has collected on the patch is analyzed in order to detect an analyte of interest.

Usina an Enerav-Assisted Dermal Patch which Makes Use of an Electric Heatina Pad The patch of Example 16 is placed on the skin of a subject's arm. An electric heating pad with a sufficient area to cover the patch and also wrap around the L;~ f~.. of the subject's arm is then placed over the patch and wrapped around the subject's arm. The heating pad is heated to about 120~F and left on the subject's arm for l,, . '~ one hour. The patch is then removed from the subject's arm and is analyzed for the presence of an analyte of interest.

Use of Eneray-Assisted Dermal Patches in Drua of Abuse Testina The patch of Example 14 is removed from its air-tight F ' _ _ and is placed on the surface of the arm of a subject who has caused a fatal traffic accident. The chemical c , -- - heats the skin for about 30 minutes. During this time, eccrine glands and other sweat glands produce, ., dliUII, and this r d6~
flows through the ducts, to the surface of the skin and into the ~' - b~"l material of the patch. After 30 minutes, the patch is removed and 1, , l~d to a lab. The patch is then analyzed to !' ' whether a drug of abuse was present in the subject's I :, W O 96/39923 59 PCTrUS96/0691 Use of Enerav-Assisted Dermal Patches in Medical D- -The patch of Example 14 is removed from its air-ti~ht packa~in~ and is placed on the surface of the arm of a subject who has been takin,a theophylline. The chemical ~ . heats the skin of the subject around the patch, and the patch is left on the subject's arm for about an hour. The patch is then removed and analyzed to P: the r I-_ of theophylline in the subject's r ~ ~ ~- If the ~ of theophylline is hiaher than a desired . . the dose of ' , h~" - which the patient is takina is lowered.

- Five patches ~ only TL~, ' I 1625 wound dressin~ were applied to a subject. These Te~ h lll ~only patches had a collection surface area of 42Cm2. S;X patches comprisina Te~, ' .ll 1625 and another _' ; material were also applied to the subject at the same time. The collection surface area of the ' i material in these patches was 14 cm2. Prior to applyin~ each of the patches, the area of skin under each patch was first swabbed with alcohol in order to remove any - ~ ~ present on the surface of the skin.
Twenty-four hours after the initial l,, " of the patches to the subject, a pre dose urine specimen and one of each of the two patch types were collected. The subject then orally ~ 60 ma of codeine r~ ~ ~ ' (30 ml. Naldecon CX-60). This ,l, ~,..~..l~ a typical codeine dose. At 30 and 48 hours p o ~- ' . urine and one of each type of patch were collected. The results of this . i are shown in Table 3 below:

W O 96~9923 60 PcT/u~6~69l5 Table 3 Day Time Elapsed Event Codeine Level Time (Hours) (n~lml) 11:00 (pre-dose) Apply 5 TeL ' . 1~ patches to stomach of subject Apply 3 patcheswith -' I material to back of subject - Apply 3 patches with ' ' - material to stomach of subject 2 10:45 (pre-dose) Obtain urine sample from subject O
Remove one T ., ' c '~ patch O
Remove one patch with ~' ' material O
11:00 0 ~ ~ codeine to subject 3 17:00 30 Obtain urine sample from subject 620 Remove one T~ patch 40.57 Remove one T g ' :el~ patch 16.30 Remove one patch with ~' ' material 12A0 ~ Remove one patch with c' ' material 23.99 4 11:00 - 48 Obtain urine sample from subject O
Remove one T " ' ,.. s 1~ patch 17.69 Remove one Te" ' orl~ patch 23.19 Remove one patch with ' ' material 14.39 Remove one patch with ' ' material 19.41 Remove one patch with ' ' material 20.17 The pre-dose urine and patch ~ - indicate that no codeine was present in the subject at the beginning of the t:A~ ' At 30 hours post-dose, the urine ~r ' ~ had 620 nglmL of codeine. Urine 5, are c~lla;llt~ d positive by NIDA guidelines when opiate Icodeine) content is over 300 nglmL. The levels of codeine found in the Teg;ll' ", -only patches were similar to those found in the patches havin~ an aL~.L.,,\~ material.
By 48 hours post-dose, the urine codeine level had returned to baseline, as expected. The levels of codeine in the Teg-,d~."lr-only patches were again found to be similar to those found in the patches having an CA 02222629 l997-ll-27 W 0 96~9923 61 PcT/u'iG~cc9l5 c ' ' material. Both patch desi~ns appear to retain the codeine for about 48 hours.
The mean codeine level for all four T v ' -only patches was 24.4 n~lmL, while the mean codeine level for all five patches havinp another ~' ' material was 18.1 nglmL. The collection area of the T~ -only patch, though, was about 40 cm2, over three times that of the collection pad in the patch havinp an absorbent material.
Since the ratio of IL..~ .Ld codeine in the T v ' -only patches was 136% (24.4118.1) compared to the patches with an ' ' material, but the T " ' -only patches had three times the surface area, it appears that in ~eneral the Tepaderm~-only patches do not retain as much codeine as the patches with an c ' ; material. It is believed that this is due to ~ '' ~ ~ ~ in the removal of the codeine from the worn T~ only patches andlor to ~ '' ~ ~ in ' r - of codeine from the skin. ~' ' ' . this data does show that a patch accordin~ to the present invention which lacks a separate ' ~ L material can be used to d , ~ the presence of an analyte in a subject's, .- .
The fore~oin~ examples and specific: ' " : are meant to be . ' y only and do not limit the scope of the present .~: In addition, the .1,~ cited herein are hereby I dll,d by ~

Claims (14)

WHAT IS CLAIMED IS:

1. A dermal patch for retaining analytes in perspiration expressed through the skin of a subject, the dermal patch comprising a fluid or gas permeable membrane, the membrane having a first side, wherein the first side further includes an adhesive attached thereto, wherein the dermal patch lacks a separate absorbent material, and wherein the adhesive and membrane are capable of retaining an analyte by chemical affinity or mechanical entrapment when perspiration evaporates through the membrane.

2. The dermal patch of Claim 1, wherein said membrane comprises urethane, polyurethane, polyethylene ethylene vinyl acetate, rayon, polyvinyl chloride of PTFE.

3. The dermal patch of Claim 1, wherein said membrane comprises a polyurethane film such as Tegaderm TM 1625 wound dressing.

4. The dermal patch of Claim 1, wherein said membrane comprises a polyethylene foam or a polyethylene tape.

5. The dermal patch of Claim 1, wherein said membrane has a surface area of between about 1 cm2 and about 120 cm2.

6. The dermal patch of Claim 1 wherein said membrane has a thickness of between 0.005 mm and about 3 mm.

7. The dermal patch of Claim 1, wherein said membrane has a surface area of about 42 cm2 and a thickness of about 0.02 mm.

8. The dermal patch of Claim 1, wherein said membrane is absorbent.

9. The dermal patch of Claim 1, wherein said membrane is made from a dissolvable or degradable material.

10. A method for determining the presence of an analyte in perspiration expressed through the skin of a subject, comprising the steps of:
removably securing a dermal patch according to Claim 1 to the subject by contacting the adhesive to the subject's skin thereby placing the first side of the membrane in fluid communication with the subject's skin for collecting biological material in perspiration expressed through the subject's skin, the dermal patch lacking a separate absorbent material;
allowing the dermal patch to remain attached to the subject's skin for a period of time sufficient to collect biological material in perspiration expressed through the subject's skin; and then removing the dermal patch from the subject for detection of an analyte of interest in the biological material using an assay for the analyte.

11. The method of Claim 10, wherein the step of removing the dermal patch from the subject includes removing stratum corneum cells from the subject's skin.

12. The method of Claim 10, further comprising the step of releasing said analyte from said membrane by extracting said analyte from said membrane, dissolving said membrane, or degrading said membrane.

13. The method of Claim 10, wherein said analyte is ***e.

14. The method of any one of Claims 10 through 13, further comprising the step of determining the presence of said analyte in said biological material collected by the dermal patch.