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Home , Cyclandelate, Moxisylyte, Tolazoline, Visnadine

III USOO5302172A United States Patent 19 11) Patent Number: 5,302,172 Sage, Jr. et al. (45) Date of Patent: Apr. 12, 1994

54 METHOD AND COMPOSITION FOR 5,068,226 11/1991 Weinshenker et al...... 604/20 ONTOPHORESS 88. M I E. et al...... - 32. www. PPS ...... sessor asses to (75) Inventors: Burton H. Sage, Jr.; Jim E. Riviere, 5,088,977 2/1992 Sibalis ...... 604/20 both of Raleigh, N.C. 5,135,480 8/1992 Bannon et al...... 604/20 (73) Assignees: North Carolina State University, OTHER PUBLICATIONS tpickinson and Lattin, Gary A. "Method to Control Delivery of Un pany, N.J. changed Drugs via Iontophoresis' JRE Nov. 1988. 21 Appl. No.: 653,202 D. I. Abramson, et al., Arch Environ Health 19:103 1a. (1969). (22 Filed: Feb. 8, 1991 D. I. Abramson, et al., American Heart Journal 23;817 Related U.S. Application- Data (1942).H. A. Kontos, et al., Circulation Research 21:679 (1967). 63 Continuation-in-part of Ser. No. 494,062, Mar. 15, N. H. Bellantone, et al., International Journal of Pharma 1990, abandoned. ceutics 30:63 (1986). 51) Int. Cl...... A61N 1/30 Primary Examiner-John D. Yasko (52) U.S. Cl...... 604/20; 604/49 Assistant Examiner-Michael Rafa 58 Field of Search ...... 62. Attorney, Agent, or Firm-Bell, Seltzer, Park & Gibson www. 57 ABSTRACT 56) References Cited 57 The invention discloses methods and compositions for U.S. PATENT DOCUMENTS enhanced iontophoretic delivery of active agents. The 3,991,755 9/1976 Vernon et al...... 604/20 compositions are pharmaceutically acceptable composi 4,406,658 9/1983 Lattin et al...... 604/20 tions for iontophoretic delivery comprising a delivery 4,702,732 10/987 Powers et al...... 9/20 enhancing amount of a vasodilator and active agent. 4,820,2634,752,285 4/19896/1988 SpevakPetelenz et et al. al...... 604/20 Methods comprise adding a delivery enhancings amount 4,950,229 8/1990 Sage, Jr 604/20 of a vasodilator to an active agent and delivering by 5,023,085 6/1991 Francoeur et al...... 60/20 iontophoresis. 5,047,007 10/1990 McNichols et al...... 604/20 5,057,072 10/1991 Phipps ...... 604/20 11 Claims, 3 Drawing Sheets

EFFICIENCYE OF ACTIVE 3O DENT WITH VASODLATOR

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FG. U.S. Patent Apr. 12, 1994 Sheet 2 of 3 5,302,172

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NCY OF ACTIVE ENT WITH VASODLATOR

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O OO3 O1 O3 1 5 %TOLOZOLINE FG 5. U.S. Patent Apr. 12, 1994 Sheet 3 of 3 5,302,172

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O CN O CO O CO o - N n 24 GNWOOOT n l 5,302,172 1. 2 The rate of delivery would still be limited by the stra METHOD AND COMPOSTON FOR tl COrneum. ONTOPHORESS Vasodilators such as , nitrates, , , dipyridamole, , , CROSS REFERENCE TO RELATED 5 mecholyl (metacholine), histamine and nylidrin are APPLICATION known to dilate blood vessels. Their use with iontopho This application is a continuation in part of patent resis, without other agents, has been studied. Studies application Ser. No. 07/494,062, filed Mar. 15, 1990 include, for example, D.I. Abramson et al., American abandoned. Heart Journal, 23:817 (1942) which describes a signifi O cant increase in blood flow when using vasodilators FIELD OF THE INVENTION alone. Iontophoresis of vasodilators as a means of enhancing The invention relates to iontophoretic transdermal delivery of an active agent delivered with it has not delivery. More specifically, the invention relates to been demonstrated. Despite attempts to optimize ionto methods and compositions for enhancing iontophoretic 15 phoretic delivery by such means as varying compound delivery. concentrations and optimizing ionic moieties in the BACKGROUND system, the efficiency of iontophoretic delivery is still During iontophoresis, charged compounds pass from low. a reservoir attached to the skin of a person or animal 20 SUMMARY OF THE INVENTION into the tissue therebeneath. The process is one wherein The invention discloses methods and compositions the rate of delivery is a function of current, active agent for enhanced iontophoretic delivery of active agents. concentration, and presence of other ions. It is a gener The compositions are pharmaceutically acceptable ally held belief that higher concentration of compound, compositions for iontophoretic delivery which com higher levels of current, and lower concentration of 25 prise a delivery enhancing amount of a vasodilator and other ions will result in greater delivery of the com active agent. pound. Other embodiments of the invention include methods L. Brown and R. Langer, Ann. Rev. Med 39:221 for enhancing iontophoretic delivery of active agents (1988) describe the generally held belief that the rate which comprise adding a delivery enhancing amount of limiting barrier for transdermal drug delivery is the 30 a vasodilator to an active agent and delivering by ionto stratum corneum. There continues to be a large re phoresis. search effort to find methods to reduce or eliminate the rate limiting property of the stratum corneum. BRIEF DESCRIPTION OF THE DRAWINGS N.H. Bellantone et al., International Journal of Phar FIG. 1 is a schematic presentation of the skin (10), maceutics 30:63 (1986) describes how iontophoresis can 35 which shows the upper capillary loops (12) of the vas be used in place of other means to enhance drug trans culature of the skin and the deeper blood vessels that port through the epidermal barrier such that the need feed the upper capillary loops and the shunt blood ves for chemical penetration enhancers could be obviated. sels (14) which connect the deeper blood vessels. Alternatively, the article suggests use of penetration FIG. 2 is a plot of data showing that addition of the enhancers could lower drug concentrations or lower vasodilator tolazoline to a concentration of lidocaine energy required for delivery. enhances the iontophoretic rate of delivery of lidocaine. Another technique believed to enhance the delivery FIG. 3 is a plot of data showing the concentration of of certain types of active agents by iontophoresis is a vasodilator wherein delivery of the active agent is disclosed in European patent application 0278 473 Al. enhanced. The application describes the addition of compounds to 45 FIG. 4 is a contour plot showing delivery of an active proteins and other macromolecules to decrease the agent when iontophoresed with a vasodilator. degree of aggregation of the molecules in the active reservoir. The added compounds have the ability to aid DETALED DESCRIPTION OF THE solubility and disassociation of the macromolecules. NVENTION It is also well-known in the iontophoresis art (for 50 While this invention is satisfied by embodiments in example, see "ontophoretic Delivery of Nonpeptide many different forms, there is shown in the drawings Drugs Formulation Optimum for Maximum Skin Per and will herein be described in detail, preferred embodi meability” by J. E. Sanderson et al., J. Pharm Sci 78:361 ments of the invention, with the understanding that the (1989) that the presence of ions other than the desired present disclosure is to be considered as exemplary of compound in the donor reservoir formulation reduces 55 the principles of the invention and is not intended to iontophoretic efficiency. limit the invention to the embodiments illustrated. The In the situation of transdermal delivery where the scope of the invention will be measured by the ap rate limiting barrier is the stratum corneum, the dermal pended claims and their equivalents. vasculature, which acts as the means of compound re The presen invention discloses methods and composi moval from the dermal tissue, has no effect on the deliv tions for enhanced iontophoretic delivery of active ery rate. Regardless of its state of dilation, it is capable agents. of removing all the compound that reaches it. Other Embodiments of the invention include pharmaceuti wise, the vasculature would become the rate limiting cally acceptable compositions for iontophoretic deliv barrier. ery comprising a delivery enhancing amount of vasodi If the stratum corneun is the rate limiting barrier, 65 lator and active agent. placing a vasodilator near the dermal vasculature for In addition, embodiments of the invention provide the purpose of enhancing the blood flow through the methods for enhancing iontophoretic delivery of active dermal vasculature by any means, would have no effect. agents comprising: 5,302,172 3 4. (a) adding a delivery enhancing amount of a vasodi It is understood that most active agents have more lator to an active agent; and than one effect in the body. For example, lidocaine is a (b) delivering of a pharmaceutically acceptable com local anesthetic which also exhibits vasoactive proper position of (a) by iontophoresis. ties (i.e. a vasolidator). Therefore, consideration of The methods and compositions of the present inven these factors for any active agent must be taken into tion are particularly advantageous compared to prior account when determining optimum ranges of each for methods and compositions. Prior methods and composi iontophoretic delivery together. tions typically relied on skin damaging or skin altering The response surface method (RSM) is a known compositions such as permeation enhancers. Unlike skin method that can be used to study the effects of active permeation enhancers that alter the stratum corneum, 10 agent properties and vasodilator properties. Other the compositions and methods of the present invention methods for measuring the effects of the active agent are not directed toward altering the stratum corneum and vasodilator are known. Other methods can be found and yet achieve an increase in active agent delivery. in Chapter II of P. D. Halland, Experimental Design. In Likewise, the benefits obtained by the addition of a Biotechnology (Marcell Dekker Inc., (1989) N.Y.). vasodilator to an active agent is opposite the generally 15 The response surface method was used to determine held belief that lower concentration of other ions en the optimum concentrations of a composition of the hances delivery. active agent lidocaine and the vasodilator tolazoline. The following terms are defined as used in this docu The results are easily determined as set forth in FIG. 4 ment. "Ion' refers to an atom or radical that has lost or and Example 1. A composition for enhanced iontopho gained one or more electrons to acquire an electric 20 retic delivery which comprises lidocaine as an active charge. "Active agent” refers to the entity chosen to be agent and tolazoline as a vasodilator preferable contains delivered by iontophoresis. Thus, active agent refers to lidocaine in an amount of about 2.0% to about 60.0% in the chosen entity and the ionic form of the chosen entity solvent and tolazoline in the amount of about 0.001% to for delivery, such as halide salts of a chosen entity to be about 0.1% in solvent. A more preferred composition delivered (e.g., lidocaine and an ionic form of lidocaine 25 for enhanced iontophoretic delivery of a composition for delivery such as lidocaine hydrochloride). "Patient' which comprises lidocaine and tolazoline contains lido refers to animals, including humans, household animals caine in an amount of about 4.0% to about 25.0% in such as dogs and cats, livestock such as cattle, horses, solvent and tolazoline in an amount of about 0.005% to sheep, pigs, goats and rabbits, laboratory animals such about 0.05% in solvent. Optimum concentrations of as mice and rats, and Zoo animals such as exotic species. 30 other active agents and vasodilators are readily ob The methods and compositions of the invention are tained in substantially the same manner. not limited to practice with any one particular ionto The concentration or amount of vasodilator to active phoretic system. Generally, iontophoretic devices com agent in a formulation or mixture is a function of the prise at least two electrodes, an electrical energy source particular active agent and the vasodilator. More specif (e.g., a battery) and at least one reservoir which con 35 ically, the ease with which the active agent may be tains an active agent to be delivered. Several iontopho delivered by iontophoresis is related to the characteris retic devices are known, such as those disclosed in P. tics of the vasodilator, the active agent, and to some Tyle, Pharmaceutical Biosearch 3:318 (1986). extent the iontophoretic system. Key components of the skin, as shown in FIG. 1, are The data shown in FIG, 2, for the vasodilator tolazo the stratum corneum, epidermis, dermis and more spe line and the active agent lidocaine, demonstrates the cifically, the blood vessels of the dermis. In systemic properties of a preferred composition of the invention. drug delivery, the objective is to get the drug from a FIG. 2, in the upper plot, 25, shows the increase in the donor reservoir adjacent to the stratum corneum into delivery of lidocaine with the addition of an optimum the blood stream. In topical drug delivery, the objective amount of the vasodilator tolazoline. Lower plot, 26, is to get the drug from the donor reservoir adjacent to 45 FIG. 2, shows iontophoresis of a control (-) without a the stratum corneum into the skin below the stratum vasodilator. The improvement in transdermal delivery corneum while avoiding removal by the blood stream. by the iontophoresis of the active agent, lidocaine, with Therefore, the structure of both the stratum corneum a vasodilator, is easily discernable by comparing plots and the vascular is important. 25 and 26. Iontophoretic administration rates are readily When iontophoresis of an active agent is performed, measured by protocols such as those disclosed in J. E. the compound passes through the stratum corneum, Riviere et al., J. Toxicol-Cut & Ocular Toxicol 8:493 through the intervening dermal tissue and into the vas (1990) culature. In a situation wherein the stratum corneum is FIG. 3 shows that there is an optimum concentration the rate-limiting barrier, the blood flow in the vascula of vasodilator when the concentration of the active ture is of little consequence. When the rate of delivery 55 agent is kept constant. With no vasodilator, the effi of the active agent is enhanced over passive delivery, as ciency (e.i. same delivery with lower power) is on the in the case of iontophoresis, to the point where the order of 15%. At the optimum, the efficiency is over ability of the vasculature to remove the compound is 30%. As more vasodilator is added, beyond the opti rate limiting, then the blood flow in the vasculature mum concentration, the efficiency falls to a level lower becomes significant. Iontophoresis of a vasodilator with than the original level. an active agent, therefore, is believed to enhance blood A range of useful concentrations of the vasodilator flow, thus enhancing the rate at which active agent is with respect to the active agent is determined by ana removed by the vasculature. lyzing the amount of active agent iontophoresed. Quan The ability to maintain proper concentration ratios of titation of active agent iontophoresed is obtained by active agent to vasodilator will depend upon the ionto 65 following the procedure set forth in Example 2. phoretic properties of the active agent and vasodilator, The concentration of the vasodilator will effect en and hence the relative proportions of the vasodilator hanced delivery of an active agent in two ways. When combined with the active agent. there is too much vasodilator, it is believed there is a 5,302,172 5 6 change of blood flow at the deeper blood vessels of the dropyridines such as nifedipine, verapamil, , skin, thus opening shunt blood vessels which will divert , , spironolactone, nicardipine hy blood flow from the upper capillary loops, and there drochloride, calcitriol, thiazides such as hydrochloro fore prevent enhancement of active agent delivery. The thiazide, flunarizine, sydononimines such as molsido other effect is the result of the introduction of additional mine, sulfated polysaccharides such as heparin fractions ions that compete with the ions of the active ingredient proteins and peptides such as insulin and analogs during coiontophoresis. The present invention provides thereof, calcitonin and analogs thereof such as elcato a method for determining the optimimum concentration nin, protamine, glucagon, globulins, angiotensin I, angi of vasodilator and active agent, which method accounts otensin II, angiotensin III, lypressin, vasopressin, for competing ions in an iontophoretic system. O somatostatin and its analogs, growth hormones, and The present invention provides a method for obtain oxytocin, and the salts of such compounds with phar ing optimum concentration at which enhancement of maceutically acceptable acids or bases, as may be re delivery is maximized. Iontophoresis of a composition quired. Preferably the active agent is a therapuetic anes of vasodilator and active agent is more efficient than thetic, hormone, protein, analgesic, or other low molec iontophoresing active agent without a vasodilator. The 15 ular weight cations. More preferably the active agent is addition of vasodilator permits the same delivery with a lidocaine, insulin, calcitonin, elcatonin or somatostatin. lower power. Primary requirements of an active agent are that it be The term "active agent' can more narrowly refer to charged or capable of modification to carry a charge. a biologically active compound or mixture of com Appropriate selection of active agents for iontophoretic pounds that have a therapeutic, prophylactic pharmaco 20 applications include selection based on specific conduc logical, physiological, or combinations thereof, effect tivity (i.e., estimates how easily drugs move in solution on the recipient and is sufficiently potent such that it when an electric current is applied). can be delivered through the skin or other membrane to Active agent modification for iontophoretic delivery the recipient in sufficient quantities to produce the de is guided by well-known procedures. For example, to sired result. 25 deliver a positively charged drug, the chloride or hy The active agent for use in the method of the inven drochloride form of the drug can be made and placed in tion can be delivered alone, as a prodrug, or in combina the iontophoretic device reservoir for delivery. General tion with other substances. Other substances can in texts in the field include Remington's Pharmaceutical clude other permeation enhancers, buffers, bacteriostat Sciences, Ed. Arthur Osol, 16th ed., 1980, Mack Publish ics, stabilizers, antioxidants, other active agents and the 30 ing Co., Easton, Pa. Typically the basic (OH or like. amine) or acid (H) form of the active agent is made, In general, active agents include therapeutic agents, depending on whether the anionic (negative charged or combinations thereof, in all of the major therapeutic ion) or cationic (positive charged ion) form of the active areas including, but not limited to, anorexics, anthelmin agent is to be delivered. Common modifications of ac tics, antiasthma agents, anticonvulsants, antidiarrheals, 35 tive agents include modification to a halide salt form. antimigraine preparations, antimotion sickness, antinau For example, to deliver a positively charged active seants, antieoplastics, antiparkinsonism drugs, antipru agent, the chloride or hydrochloride form of the active ritics, antipyretics, anticholinergics, sympathomimetics, agent is made and placed in the iontophoretic device xanthine derivatives, cardiovascular preparations in reservoir for delivery. Likewise, the composition is cluding calcium channel blockers, beta blockers, antiar typically dissolved in a suitable solvent to obtain the rhythmics, antihypertensives, diuretics, vasodilators ionic form for iontophoretic delivery. Suitable solvents including general, coronary, peripheral and cerebral include polar liquids such as water, glycerine, and central nervous system stimulants, cough and cold prep lower alkyl alcohols such as methyl alcohol, ethyl alco arations, decongestants, diagnostics, hormones, hypnot hol, and branched alcohols such as isopropyl alcohol. ics, immunosuppressives, muscle relaxants, parasympa 45 In this invention the effective amount of active agent tholytics, parasympathomimetics, psychostimulants, means that amount needed to produce the intended sedatives and tranguilizers, antiinflammatory agents, result following its iontophoretic administration. The analgesics, antiarthritic agents, antispasmodics, antide effective amount will vary, depending, among other pressants, antipsychotic agents, tranquilizers, antianxi factors, on the physiological effect as determined by the ety agents, narcotic antagonists, cholinergic , 50 serum level of desired active agent, rate of clearance of anticancer agents, antiviral agents, antibiotic agents, active agent, and intradermal metabolism desired. appetite suppressants, antiemetics, anticholinergics anti The term pharmaceutically acceptable composition histamines, antimigraine agents, contraceptive agents, refers to the addition salts, mild complexes, solid and antithrombotic agents, diuretics, antihypertensive liquid carriers, ionic forms, and the like, which do not agents, cardiovascular drugs and the like. Examples of 55 significantly or adversely affect the properties of the specific drugs are steroids such as estradiol, progester active agent or its ability to be iontophoretically deliv one, norgestrel, levonogestrel, norethindrone, medrox ered. Pharmaceutically acceptable compositions can be yprogestrone aceate, and their esters, nitro prepared by reference to general texts in the field, such compounds such as nitroglycerine and isosorbide ni as Remington's Pharmaceutical Sciences, Ed. Arthur trates, nicotine, chlorpheniramine, terfenadine, triproli Osol, 16th ed., 1980, Mack Publishing Co., Easton, Pa. dine, hydrocortisone, oxicam derivatives such as pirox Delivery enhancing amount refers to an amount icam, , mucopolysaccharidases such as thi which enhances delivery of the active agent as com omucase, buprenorphine, fentanyl, naloxone, codeine, pared to the active agent delivered alone, but which lidocaine, , pizotiline, salbutanol, amount does not present serious side effects which out , such as misoprostol and en 65 weigh the advantages of its use. prostil, omeprazole, , benzamides such as The reservoir or similar structure that contains the metoclopramine, scopolamine, peptides such as growth active agent to be delivered can be in the form of any releasing factor and somatostatin, , dihy material suitable for making contact between the ionto 5,302,172 7 8 phoresis unit and the skin. Suitable materials include, modifications are possible and are contemplated within but are not limited to, foams, ion exchange resins, gels the scope of the invention described. and matrices. Iontophoresis gels can be karaya gum, other polysac EXAMPLES charide gels, or similar hydrophilic aqueous gels capa Material Preparation ble of carrying ions. Specific examples of such gels The electrodes used herein have a surface area about include polyvinyl alcohol, polymethyl pyrollidine, 10 cm2 (2.5cm x 4 cm) and are fabricated as a sandwich. methyl cellulose, polyacrylamide, polyhemas, The outer layers of the sandwich consist of about 1/16 polyhema derivatives and the like. The matrix selected inch POREXTM (a thick porous, hydrophilic open-cell should have nonirritating properties to avoid irritating 10 polyethylene foam into which a surfactant has been the person's skin or tissue, suitable viscosity and surfac incorporated during manufacture, obtainable from tant properties to obtain good electrical contact with Porex Technologies, Fairburn, Ga.). The inner layer of the skin or tissue, and the ability to act as a carrier the sandwich electrode consists of about 1.5 cm X5 cm medium for the ions. mesh of silver wire (0.0045 inch silver wire 80x80 Suitable vasodilators for use in the present invention 15 weave, obtainable from Unique Wire Weaving Co., can be selected from the major categories of vasodilat Hillside, N.J.). About 1.5 cm x 1 cm tab of silver mesh is ors generally referred to as cerebral, coronary and pe left protruding from the polyethylene sandwich for ripheral. Specific vasodilators within the cerebral cate purposes of making electrical contact. The sandwich is gory include , cinnarizine, citicoline, cyclan held together by an epoxy type glue (e.g., DEV delate, , diisopropylamine dichloroacetate, CONTM 5-minute epoxy glue, obtainable from Devcon eburnamonine, fenoxedil, flunarizine, ibudilast, ifen Corp., St. Louis, Mo.) along the lateral edges of the prodil, nafronyl, nicametate, , nimodipine, polyethylene. papaverine and penifylline. Using scissors, two rectangular pieces are cut from Specific vasodilators within the coronary category the 1/16 inch thick sheet of POREXTM material. Each include amotriphene, bendazol, benfurodil hemisucci 25 piece is about 2.5cm x4cm. A 1 cm x5 cm piece of the nate, benziodarone, chloracizine, chromonar, cloben silver wire mesh is then cut. The mesh is longitudinally furol, clonitrate, dilazep, dipyridamole, droprenilamine, centered on one piece of the POREXTM, with about 1 efloxate, erythritol, erythrityl tetranitrate, etafenone, cm of material extending out one end. A line of epoxy fendiline, floredil, ganglefene, hexestrol bis(B-die type glue is placed along the inner border of lateral thylaminoethyl ether), hexobendine, itramin tosylate, 30 edges and the end of the POREXTM. Take care not to khellin, lidoflazine, mannitol hexanitrate, medibazine, place the glue on the wire mesh itself. It is both under nicorandil, nitroglycerin, pentaerythritol tetranitrate, stood and desirable that the applied glue will contact pentrinitrol, perhexiline, pimefylline, prenylamine, the edges of the wire mesh and, by so doing, hold the propatyl nitrate, pyridofyline, trapidil, tricromyl, 35 mesh securely in position. However, the amount of that trimetazidine, trolnitrate phosphate, and . contact should be kept at a minimum. Specific vasodilators within the peripheral category Sandwich the wire mesh by placing the second piece include aluminum nicotinate, , bencyclane, of POREXTM over the first. Place the completed unit betahistine, bradykinin, brovincamine, bufeniode, bu in the clamp and allow to dry about 40 minues. Examine flomedil, , , ciclonicate, , the electrode to see that all three glued edges are in cinnarizine, cyclandelate, diisopropylamine dichlor tight contact. Test the integrity of the mesh Porex TM oacetate, eledoisin, isoxsuprine. kallidin, kallikrein, attachment by gently tugging on the protruding edge of , nafronyl, nicametate, nicergoline, nicofura mesh. The mesh should not shift in position. Stre in a nose, , nylidrin, , pentoxi dry area. fylline, , E1, , tolazoline, 45 and niacinate. Preferably the vasodilators are EXAMPLE 1. selected from the peripheral category. The following protocol is referred to as the Response All of the compounds are readily described in The Surface Method (RSM). Drug formulation for each Merck Index, Eleventh Edition (Merck and Co., Inc., experiment is shown in the summary below for the Rahway, N.J. (1989)). 50 active (positive) electrode. Drug formulation for the The treatment regimen for use in the present inven indifferent (negative) electrode is unbuffered normal tion includes the consideration of a variety of factors, saline. including the type, age, weight, sex, medical condition Standard methods for flap surgery and preparation of the patient, severity of the condition and active agent are used herein. Standard methods for flap perfusion to be delivered. An ordinarily skilled physician can 55 and maintenance are used; non-recirculating perfusate is readily determine and prescribe and administer the ef used without exception. Standard flap surgery and fective amount of the agent required to prevent or ar methods for flap perfusion are known by reference to rest the progress of the condition. In so proceeding, the publications such as Monteiro Riviere, N.A., et al., The physician could employ relatively low doses at first and Isolated Perfused Porcine Skin Flap (IPPS) “II Ultra subsequently increase the dose until a maximum re Structural and Histological Characterization of Epider sponse is obtained. Likewise, the decision of where to mal Viability", In Vitro Toxicology, 1:241 (1987). A vol apply the iontophoretic system is a factor, depending on ume of venous exudent is collected, at least 1.0 ML but the area of application, for example, whether the area is not exceeding 3.0 ML at one half hour increments after on the torso or the extremities and whether those areas iontophoresis begins. For control purposes, two venous are hairy, wrinkled, folded or creased. 65 effluent specimens are collected prior to iontophoresis. The following examples illustrate the specific em A total of 18 venous effluent specimens are collected in bodiments of the invention described herein. As would total, two before iontophoresis, and 16 covering 4 hours be apparent to skilled artisans, various changes and of iontophoresis and 4 hours past iontophoresis. 5,302,172 10 Voltage measurements are also taken by measuring tific (Folsom, CA) "megabore" column, Cat. No. output of the constant current generator, for example, 125-1012, DB-1--, 15 m long, 1.5 micron film thick WPI Model A360, available from World Precision in ness; or equivalent, with appropriate injector and struments, New Haven, Conn. detector connections. All venous effluent specimens are analyzed for lido- 5 2. Vortex mixer. caine using the standard lidocaine assay procedure de 3. Beckman Microfuge B, or equivalent. scribed in the following example. Mixing instructions 4. Pipettes for quantitative transfer of 50-250 microliter for the active electrode formulations are listed below: volumes. To Prepare 10 5. Crimper for 12 mm crimp-seal vial caps. Consumables 3.6% lidocaine, add 36.5 mgm lidocaine HCL/ML H2O 1. Microfuge tubes with snap-caps; 1.5 ml, polypropyl 31.6% lidocaine, add 36.5 mgm lidocaine HCL/ML H2O 10% lidocaine, add 115.6 mgm lidocaine HCL/ML H2O ele, 1.45% lidocaine, add 16.8 mgm lidocaine HCL/ML H2O 2. Ammonium hydroxide, 1.5M. 70% lidocaine, add 809 mgm lidocaine HCL/ML H2O 15 3. Solvent mixture: 2%(v/v) 2 - propanol in N-heptane. 0.01% tolazoline, add 122.8 mgm tolazoline HCL/ML H2O 0.1% tolazoline, add 1.228 mgm tolazoline HCL/ML H2O 4. Autosampler vials (12X32mm) and crimp seal (te 0.0316% tolazoline, add 388 mgm tolazoline HCL/ML H2O flon-lined rubber septa) caps. 0.0045% tolazoline, add 55.3 mgm tolazoline HCL/ML H2O 5. Autosampler vial inserts. 0.215% tolazoline, add 2.640 mgm tolazoline HCL/ML H2O FW lidocaine s 234.22 6. Pasteur Pipettes. FW idiocaine HCL is 270,68 7. Lidocaine hydrochloride (minimum purity 99%). FW toazoline = 160.21 8. Plastic (PS) 15 ml centrifuge tubes (Falcon #2095, FW tolazoline HCL = 196.67 Before starting, test mix: 70% lidocaine and 031.6% totazoline obtainable from Falcon Products, Becton Dickinson to insure it goes into solution. Labware, Oxnard, Calif.), or equivalent. Protocol 25 9. Transfer pipette tips. non recirculating perfusate Generally 1.0 ml samples are taken of the venus efflu 4 hours iontophoresis ent (perfusate) of the skin flap. The minimum lidocaine 8 hours sampling, sample every hour level which can be determined by this method is about Before starting, test mix: 70% lidocaine and 0.0316% 10 microgram/ml, in the perfusate. This minimum tolazoline to insure it goes into solution. Protocol-non- 30 could easily be decreased, if desired, by using a larger recirculating perfusate 4hours iontophoresis 8 hours GC injection volume than is indicated. Once step C, sampling, sample every hour below, is completed, the remaining portions of the ex traction, transfer of the organic extract to an autosam % % pler vial and sealing of the vial should be completed as LIDOCAINE TOLAZOLINE 35 soon as possible to avoid errors due to evaporation of RUN CURRENTma (W/V) (W/V) the organic solvent. If the extracted samples are not to 1 1.35 3.16 0.01 be analyzed immediately, they should be stored in a 2 1.35 3.6 0.10 freezer. 3 0.90 0.0 0.036 4 0.45 31.6 0.0 Extraction from the aqueous solution is as follows: 5 0.45 3.16 0.10 40 a. 200 microliter of the sample solution containing the 6 0.45 3.16 0.01 lidocaine is accurately transferred to a 1.5 ml poly 7 0.45 36 0.0 8 1.35 3.16 O.O propylene microfuge tube with snap cap. 9 0.90 10.0 0.036 b. 50 microliter of 1.5 NNH4OH is added to the tube. 10 1.35 31.6 0.01 c. 250 microliter of 2% isopropyl alcohol in normal 11 0.90 1.45 0.036 45 2 0,90 700 0.036 heptane is added and the tube is capped. 13 0.90 10.0 0,004.5 d. The contents are gently swirled utilizing a vortex 14 0.90 10.0 0.0316 mixer for 1 minute. 15 0.90 10,0 0.215 e Separate the organic and aqueous fractions by cen 6 1.65 10.0 0.0316 7 O.90 0.0 0.036 50 trifugation employing a microfuge (about 5-10 18 0.15 0.0 0.036 minutes). 19 0.90 10.0 0.215 f. Place a 100 microliter insert in each GC Autosam 20 0,90 10.0 0.0045 2 1.65 10.0 0.0316 pler vial. 22 0.15 10,0 0.036 g. Using a Pasteur pipette transfer the upper organic 23 0.90 45 0.0316 fraction from the microfuge tube to a GC autosam 24 0.90 700 0.0316 55 pler vial (it must be at least half full) and seal the based on an electrode area of 4.5 cm, and current densities of 100 IA/cm, 200 vial with a crimp seal cap. A/cm, and 300 A/cm A stock solution of lidocaine HCl is prepared which contains 1.0 mg/ml lidocaine as free base (fb.) by dis EXAMPLE 2 60 solving 577.9 mg of the hydrochloride salt in 500 ml Quantitation of lidocaine concentrations in samples deionized water. Quantitative measures are required. generated by perfused skin flap iontophoresis experi ments is performed as follows: Molecular Weights idocaine free base = 234.3 Equipment 65 lidocaine-HCl is 270.8 1. Hewlett-Packard (Palo Alto, Calif.) 58.40a gas chro 500 mg lidocaine fb. = (270.8/234.3) * 500 matograph (GC), or equivalent, with flame ionization 500 mg lidocaine fib. = 577/9 mg lidocaine-HCl detector(FID), auto liquid sampler, J. and W. Scien 5,302,172 11 12 A series of 10 ml standard dilutions which cover the 3. Vortex mix, centrifuge and transfer as described range of sample concentrations is prepared by dilution above. of the stock with the matrix solution. The required 4. Set the GC injection volume to 3 microliters. volume of stock is transferred with a pipette to a volu Although the invention has been described with re 5 spect to specific modifications, the details thereof are metric flask which is filled to the mark and mixed well. not to be construed as limitations, for it will be apparent Two sets of extracted standards are prepared from that various equivalents, changes and modifications the standard dilutions by using the same extraction may be resorted to without departing from the spirit procedure described above for the unknown sample and scope thereof and it is understood that such equiva solutions. 10 lent embodiments are to be included therein. The following program for GC set-up is used with What is claimed is: normal (FID) detection and an injection volume of 1 1. A method for enhancing iontophoretic delivery of microliter. an active agent comprising: (a) providing an iontophoretic delivery system com 15 prising a reservoir and an electrical energy source, TEMP 1: 170 said reservoir containing a pharmaceutically ac TIME 1: O NJ TEMP 250 ceptable composition comprising an active agent Fd TEMP 300 and a delivery enhancing amount of a vasodilator; AUXTEMP (This does not matter) (b) contacting said iontophoretic delivery system to CHT SPD 0.50 the skin of a subject; and ZERO 10.0 (c) delivering said pharmaceutically acceptable com ATTN 2 7 position to the subject by iontophoresis; FID SIG A SLOPE SENSE wherein said delivery enhancing amount of vasodi AREA RE O lator is from about 0.001% to about 0.1% in sol FLOWA 30 25 Vent, FLOWB (This does not matter) 2. The method of claim 1 in which the vasodilator is selected from the group consisting of cerebral vasodilat ors, coronary vasodilators, and peripheral vasodilators. A calibration plot is constructed by plotting the area 3. The method of claim 2 in which the vasodilator is counts for the calibration standards (on the "Y" axis) 30 a cerebral vasodilator. against the concentrations of lidocaine in the standard 4. The method of claim 2 in which the vasodilator is dilutions (on the "X' axis). A linear regression analysis a coronary vasodilator. on the data gives the best straight line fit. The equation 5. The method of claim 2 in which the vasodilator is for the straight line is used to determine the concentra a peripheral vasodilator. tions in the samples from their lidocaine peak area 35 6. The method of claim 3 in which the active agent is counts. The regression analysis and plotting are conve selected from the group consisting of therapeutics, anes niently done with the Statgraphics PC program avail thetics, hormones and proteins. able from STSC, Inc., Rockville, Md. 7. The method of claim 6 in which the active agent is an anesthetic. EXAMPLE 3 8. The method of claim 1 in which the delivery en hancing amount of vasodilator is from about 0.005% to A method similar to that described above was devel about 0.05% in solvent. oped to analyze lower concentration samples (such as 9. The method of claim 1 in which the active agent is found in perfused skin flap experiments). However, a lidocaine and the vasodilator is tolazoline. larger sample volume is required (0.5 ml). This method 45 10. The method of claim 9 in which the lidocaine is gives a detection limit of about 2 microgram/ml. Proce from about 2.0% to 60.0% in solvent and the tolazoline dures outlined in the method above are followed except is from about 0.001% to 0.1% in solvent. for the changes indicated below: 11. The method of claim 10 in which the lidocaine is 1. Pipet 0.5 ml of sample into a 1.5 ml microfuge tube. from about 4.0% to 25.0% in solvent and the tolazoline 2. Add 0.20 ml 1.5 N, NH4OH and 0.20 ml of organic 50 is from about 0.005% to 0.05% in solvent. solvent mixture (2-propanol/heptane). k . . .

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