COMPOSITIONS AND METHODS ENHANCING TRANSDERMAL

DELIVERY OF DRUGS AND BIOLOGICS

Field of the Invention

The present invention generally relates to formulations and methods to deliver therapeutic levels of topically applied drugs to the skin, or to the systemic circulation.

Cross-Reference to Related Applications This application claims priority to U.S. S.N. 60/655,348, entitled "Compositions and Methods Enhancing Transdermal Delivery of Drugs and Biologies", filed on February 23, 2005, by Shikha P. Barman, Hannah Farnham, Lauren K. Roode, and Anna Wan.

Background of the Invention

Drugs are routinely administered orally or by injection. The effectiveness of most drugs relies on achieving a certain concentration in the bloodstream. Many drugs exhibit undesirable behaviors that are specifically related to a particular route of administration. For example, drugs may be degraded in the gastrointestinal (GI) tract by the low gastric pH, local enzymes, or interaction with food or drink in the stomach. The drug or disease itself may forestall or compromise drug absorption because of vomiting or diarrhea. If a drug entity survives its trip through the GI tract, it may face rapid metabolism to pharmacologically inactive forms by the liver, which is known as the first pass effect.

Transdermal drug delivery (TDD) offers several advantages over traditional delivery methods including injections and oral delivery. When compared to oral delivery, TDD avoids gastrointestinal drug metabolism, reduces first-pass effects, and provides sustained release of drugs for up to seven days, as reported by Elias, in Percutaneous Absorption: Mechanisms- Methodology-Drug Delivery, Bronaugh, R. L., Maibach, H. 1. (Ed), pp 1-12, Marcel Dekker, New York, 1989. The skin is a complex structure made up of at least four distinct layers of tissue: the nonviable epidermis or the stratum corneum (SC), the viable epidermis, the viable dermis, and the subcutaneous connective tissue.

Located within these layers are the skin's circulatory system, the arterial plexus, and appendages, including hair follicles, sebaceous glands, and sweat glands. The circulatory system lies in the dermis and tissues below the dermis. The capillaries do not actually enter the epidermal tissue but come within 150 to 200 microns of the outer surface of the skin.

Transport of drugs occurs only across the epidermis where the drug is absorbed by the blood capillaries. In comparison to injections, TDD can reduce or eliminate the pain and possibility of infection associated with injections. Theoretically, the transdermal route of drug administration could be advantageous in the delivery of many therapeutic drugs, including proteins, because many drugs, including proteins, are susceptible to gastrointestinal degradation and exhibit poor gastrointestinal uptake, proteins such as interferons are cleared rapidly from the blood and need to be delivered at a sustained rate in order to maintain their blood concentration at a high value. Transdermal devices are also easier to use than injections.

In spite of these advantages, very few drugs and no proteins or peptides are currently administered transdermally for clinical applications because of the low skin permeability to drugs. This low permeability is attributed to the stratum corneum (SC), the outermost skin layer which consists of flat, dead cells filled with keratin fibers (keratinocytes) surrounded by lipid bilayers. The "brick-and mortar" structure of the SC is comprised of intricate arrangements of lipids, such as phosphatidyl cholines, fatty acids and ceramides into lamellar structures that impart water-barrier properties to the skin. The highly-ordered structure of the lipid bilayers confers an impermeable character to the SC (Flynn, G. L., in Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery.; Bronaugh, R. L., Maibach, H. I. (Ed), pages 27-53, Marcel Dekker, New York, 1989). This property of resistance to absorption of molecules provides the highest order of protection from foreign bodies. This also implies that the skin barrier function has to be disrupted, ideally temporarily, to deliver therapeutics transdermally.

Several methods have been proposed to enhance transdermal drug transport, including the use of chemical enhancers, i.e. the use of chemicals to either modify the skin structure or to increase the drug concentration in a transdermal patch (Burnette, R. R., in Developmental Issues and Research Initiatives; Hadgraft J., G., R. H., Eds., Marcel Dekker: 1989; pp. 247-288; Junginger, et al. in Drug Permeation Enhancement; Hsieh, D.S., Eds., pp. 59-90; Marcel Dekker, Inc. New York 1994). Solvents and skin permeation agents such as alcohol and dimethyl sulfoxide (DMSO) are used to partially dissolve the lipid "brick and mortar" structure to create channels for drug delivery through the skin. However, this approach has shown success only for non-polar hydrophobic drugs; the delivery of macromolecules and hydrophilic small molecules through the skin remains a challenge for transdermal drug delivery. Other methods used to enhance transdermal transport include the use of applications of electric fields to create transient transport pathways [electroporation] or to increase the mobility of charged drugs through the skin [iontophoresis] (Prausnitz Proc. Natl. Acad. Sci. USA 90, 10504-10508 (1993); Walters, K. A., in Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Ed. Hadgraft J., Guy, R.H., Marcel Dekker, 1989). Iontophoresis is an effective and painless method of delivering medication to the skin by applying electrical current to a solution of the medication. Iontophoresis provides an electrical driving force to move compounds. Application of a positive current will drive positively charged drug molecules away from the electrode and into the skin. Similarly, a negative current will drive negatively charge ions into the skin.

Electroporation is believed to work in part by creating transient pores in the lipid bilayers of the SC (Burnette, (Hadgraft and Guy, Eds.), Marcel Dekker, pp. 247-291 (1989)). Electroporation involves application of electric field pulses that create transient aqueous pathways in lipid bilayer membranes, causing a temporary alteration of skin structure. While occurrence of aqueous pores may allow transdermal permeation of neutral

molecules by diffusion, the transport of charged molecules during pulsing occurs predominantly by electrophoresis and electroosmosis.

Another approach that has been explored in transdermal drug delivery is the application of ultrasound, also referred to as sonophoresis. Ultrasound has been shown to enhance transdermal transport of low-molecular weight drugs (molecular weight less than 500) across human skin (Levy, J. Clin. Invest. 1989, 83, 2974-2078; Kost and Langer in "Topical Drug Bioavailability, Bioequivalence, and Penetration"; pp. 91-103, Shah V. P., M.H.I., Eds. (Plenum: New York, 1993); Frideman, R. M., "Interferons: A Primer", Academic Press, New York, 1981). Although a variety of ultrasound conditions have been used for sonophoresis, the most commonly used conditions correspond to therapeutic ultrasound (frequency in the range of between one MHz and three MHz, and intensity in the range of between above zero and two W/cm ² ) (see U.S. Patent No. 4,767,402 to Kost, et al.). SONOPREP® is an ultrasonic skin permeation system and procedure available from Sontra Medical Center Corp. (Franklin, Massachusetts).

However, it is a common observation that the typical enhancement induced by therapeutic ultrasound is less than ten-fold. In many cases, no enhancement of transdermal drug transport has been observed upon ultrasound application.

Therefore, it is an object of the invention to enhance transport of topically applied drugs into and through the skin.

BRIEF SUMMARY OF THE INVENTION Improved methods for transdermal transport of drug formulations are described herein. Formulations designed to enhance transport of therapeutic levels of topically applied drugs into the systemic circulation, methods of making the formulations are also described herein. The formulations contain at least one active agent to be delivered and at least one skin permeation enhancer in a polymeric hydrogel, and optional additional excipients. Methods for enhancing transport of formulations into and through the skin include: (a) pretreatment of the skin with a hydrating solution, (b) physical permeation of the stratum corneum by low frequency ultrasound

(administered by a sono-permeation device, such as SONOPREP ^® , available from Sontra Medical Corporation, Franklin, MA), (c) topical application of a formulation containing the bioactive molecule, and optionally (d) application of an electric potential difference that forces ionized drugs through the skin. Optionally, the formulation contains permeation-enhancing agents. The method may be used with the formulations described herein or with other formulations for topical administration. In a preferred embodiment, the active agent to be delivered is a drug, preferably the drug is a local anesthetic, such as lidocaine. BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA is a graph of average pain scores between 0-5 ("anesthetic effect") (y-axis) as a function of formulation composition, following passive delivery of lidocaine-HCl containing formulations applied for five minutes on SONOPREP ^® -treated skin. Figure IB is a graph of the percentage of subjects with pain scores between 0-1, 1-2, 2-3, 3-4, and 4-5 as a function of formulation composition.

Figure 2 A is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 1 of various lidocaine- hydrochloride containing formulations as a function of onset time (one, two, three, four or five minutes). Figure 2B is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 2 of various lidocaine-hydrochloride containing formulations as a function of onset time.

Figure 3 is a graph depicting the enhanced viscosity of a formulation containing benzyl alcohol and PLURONIC ^® F 127 as compared to a formulation containing PLURONIC ^® F 127 alone.

Figure 4A is a graph of the average pain scores between 0-5 (y-axis) of various lidocaine-hydrochloride containing formulations as a function of the site of delivery (dorsum of hand versus the anticubital (AC) of the forearm) and as a function of application time (one, three, five, and ten minutes). Figure 4B is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 1 of various lidocaine-

hydrochloride containing formulations as a function of the site of delivery and as a function of application time. Figure 4C is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 2 of various lidocaine-hydrochloride containing formulations as a function of the site of delivery and as a function of application time.

Figure 5 A is a graph of average pain scores between 0-5 (y-axis) of various lidocaine-hydrochloride containing formulations delivered iontophoretically by SONOPREP ^® as a function of current strength (0, 0.4, 1.0 niA) and as a function of time of pain insult (0 or 15 minutes). Figure 5B is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 1 of various lidocaine-hydrochloride containing formulations delivered iontophoretically as a function of current strength. Figure 5C is a graph depicting the depth of anesthesia as shown by needle depth (mm) achieved by various lidocaine-hydrochloride containing formulations delivered iontophoretically as a function of current strength.

Figure 6 A is a graph of average pain scores between 0-5 (y-axis) as a function of formulation composition. Figure 6B is a graph of average pain scores between 0-5 (y-axis) as a function of formulation composition. Figure 7A is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 1 of various lidocaine- hydrochloride containing formulations delivered iontophoretically, as a function of length of iontophoresis, as a function of the site of delivery (AC vs. hand), and as a function of time of treatment (passive, 1 or 2 minutes). Figure 7B is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 1 of various lidocaine-hydrochloride containing formulations delivered iontophoretically as a function of the site of delivery (AC or hand) and as a function of conditions of treatment.

Figure 8 is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 1 of various lidocaine-hydrochloride containing formulations delivered iontophoretically as a function of time of (0, 5, 10, 15, and 20 min).

Figure 9 is a graph of pain scores between 0-5 (y-axis) over time (minutes, x-axis) for various lidocaine-hydrochloride containing formulations delivered iontophoretically.

Figure 10 is a graph depicting onset of anesthesia shown by percentage of subjects with pain scores less than 1 of various lidocaine- hydrochloride containing formulations delivered iontophoretically to the dorsum of the hand as a function of time of pain insult.

Figure 11 is a graph of pain scores between 0-5 (y-axis) over time (minutes, x-axis) of various lidocaine-hydrochloride containing formulations delivered iontophoretically to the dorsum of the hand.

DETAILED DESCRIPTION OF THE INVENTION Transport of drugs into and through the skin can be enhanced by the combination of skin pre-treatment, physical and chemical permeation of the stratum corneum, and, optionally, physical transport mechanisms that can "push" the drugs through the skin. The steps that result in enhanced transport are: (a) pretreatment of the skin with a hydrating solution, (b) physical permeation of the stratum corneum by low frequency ultrasound (SONOPREP ^® (Sontra Medical Corporation, Franklin, MA)), (c) application of the bioactive molecule, formulated in a transport vehicle, preferably one that contains permeation-enhancing agents, and optionally (d) application of an electric potential difference that forces ionized drugs through the skin. Application of low-frequency (between approximately 20 and 200 kHz) ultrasound can dramatically enhance transdermal transport of drugs, as described in WO 97/04832 by Massachusetts Institute of Technology. Transdermal transport enhancement induced by low-frequency ultrasound was found to be as much as 1000-fold higher than that induced by therapeutic ultrasound. Another advantage of low-frequency sonophoresis as compared to therapeutic ultrasound is that the former can induce transdermal transport of drugs which do not passively permeate across the skin. Compositions and methods that can deliver efficacious levels of an active agent to the skin that has been pretreated with a sono-permeation device, such as SONOPREP®, are described. Using lidocaine hydrochloride

as a model drag, it was demonstrated in healthy volunteers that rapid-onset of drag activity and adequate duration of the therapeutic effect can be achieved with the compositions described herein. The rapid onset of drag activity by use of sonophoresis has been demonstrated in human clinical trials (Katz, et al., Anesth. Analg. ; 98:371-6 (2004)). In these trials, it was demonstrated that rapid cutaneous anesthesia of the skin was achieved by application of an eutectic mixture of local anesthetics (EMLA) after a brief pre-treatment of the underlying skin with low frequency ultrasound. As described herein, the combined steps of: (a) pre-hydrating the skin site, (b) poration by ultrasound (SONOPREP®), and (c) application of a skin permeating composition that contains an active agent, provide greatly enhanced transdermal delivery of therapeutic levels of active agents to the skin. I. Formulations The formulations for enhanced delivery of therapeutic levels of agents to the skin contain at least one active agent, at least one skin permeation enhancer, a polymeric hydrogel, and optionally one or more other excipient(s). The formulations can be applied to skin on any area of the body, including the scalp and the face. A. Active Agents

A wide variety of active agents, varying in mode of action, polarity, ionizabilibity, molecular weight and solubility, are suitable for administration as described herein.

Suitable active agents include, but are not limited to, proteins and peptides, polysaccharides, nucleic acid molecules, and organic compounds. Examples of drags that may be administered include anesthetics, anti- infectives (antibiotics, antivirals and antifungals), anti-inflammatories (both steroidal and non-steroidal), antigens or vaccines, antibodies, chemotherapeutics, hormones, cardiovascular drags, drags for treatment of disorders such as high cholesterol and diabetes, and other drags that are typically administered either systemically or topically. The active agents can be hydrophilic, hydrophobic, or amphiphilic. Exemplary hydrophilic

molecules include aspirin, heparin or γ-interferons. Exemplary hydrophobic molecules include steroids, such as prednisolone. Exemplary amphiphilic molecules include oligonucleotides and peptides or aptamers, with both hydrophilic and hydrophobic segments. Examples of local anesthetics include amethocaine, amethocaine hydrochloride, benzocaine, butamben, butamben picrate, dibucaine, dimethisoquin hydrochloride, diperodon hydrochloride, ketocaine, lidocaine, lidocaine hydrochloride, pramoxine, pramoxine hydrochloride, prilocaine hydrochloride, procaine hydrochloride, propanocaine hydrochloride, propipocaine, propoxycaine hydrochloride, and pharmaceutically acceptable salts and free-base forms thereof. Examples of antiinflammatories include alclometasone dipropionate, amcinonide, beclamethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol propionate, clobetasone butyrate, desonide, desoxymethasone, diflorasone diacetate, diflucortolone valerate, flumethasone pivalate, fluclorolone acetonide, fluocinolone acetonide, fluocionoide, fluocortin butyl, flucortolones, fluprednidene acetate, flurandrenolone, halcinonide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone acetate, nometasone furoate, triamcinolone acetonide, anti-inflammatory agent is one or more of diclofenac, ibuprofen, acetylsalicylic acid, piroxicam, ketoprofen, felbinac, benzylamine and de-esterified base compounds, esters of base compounds, salts thereof and combinations thereof.

Representative agents for delivery include local anesthetics delivered to an area of the skin that requires a skin biopsy, a venipuncture, or catheterization procedure such as lidocaine and lidocaine hydrochloride; drugs for treating a dermatological condition, such as psoriasis, tretinoin gel or other anti-wrinkle cream; hyaluronate, a polysaccharide that induces tissue regeneration and repair; and immunotherapeutics to elicit a specific immune reaction.

The immunotherapeutic may be targeted to specific immune cells to induce the immunological cascade necessary to generate a desired response.

The depth of pores created by the low frequency ultrasound generated by a device, such as SONOPREP®, renders a direct channel for vaccine delivery to the network of immunomodulatory cells called Langerhans cells (LC). The high percentage of Langerhans cells in the human skin acts as the body's line of defense against foreign antigens. These immunomodulatory cells host the humoral and cellular responses specific to foreign antigens. Delivering vaccines right to the LC network 35 μm into the epidermis allows for needleless vaccine delivery.

Other suitable active agents include adjuvants of biological origin, such as lipopolysaccharides or proteins, or of synthetic origin, such as sodium lauryl sulfate, monophophoryl lipid A, saponins, or highly immunogenic CPG units.

In one embodiment, an adjuvant is delivered to a hydrated, ultrasound pre-treated skin site prior to delivery of the immunotherapeutic to the site. This "primes" or activates the immune cells in the stratum corneum, prior to delivering the vaccine. Specific activation of the immune system by delivery of the adjuvant to a hydrated ultrasound pre-treated skin site includes activation of Langerhans cells and recruitment of monocytes and macrophages to the site of delivery. Delivering the immunotherapeutic or vaccine post activation of the Langerhans cells, presents an effective and efficient way of eliciting an antigen-specific immune response to the vaccine. Iontophoresis may be applied following delivery of the immunotherapeutic to enhance delivery of the adjuvant and immunotherapeutic. In another embodiment, simultaneous delivery of the adjuvant with the immunotherapeutic is performed to a hydrated, ultrasound pre-treated skin site. The adjuvant can be delivered separately at an adjacent site to the vaccine delivery site, or at the same site co-formulated with the vaccine. B. Skin Permeation Enhancers The formulations described herein include at least one skin permeation enhancer. Suitable skin permeation enhancers include, but are not limited to, benzyl alcohol, linoleic acid, alpha-linolenic, oleic acid, cod-

liver-oil, methanol, menthol derivatives, squalene, glycerol derivatives, and sodium taurocholate.

Suitable skin permeation enhancers are well known to one of skill in the art and are also described in U.S. Patent No. 5,947,921 to Johnson, et al. Skin permeation enhancers are generally discussed below. Lipid Bilayer Disrupting Agents

Chemical enhancers are known and commercially available. For example, ethanol has been found to increase the solubility of drugs up to 10,000-fold (Mitragotri, et al. in Encl. of Pharm. Tech.: Swarbrick and Boylan, Eds. Marcel Dekker 1995) and yield a 140-fold flux increase of estradiol, while unsaturated fatty acids have been shown to increase the fluidity of lipid bilayers (Elias, in Percutaneous Absorption: Mechanisms- Methodology— Drug Delivery, Bronaugh and Maibach, Eds., pp 1-12, Marcel Dekker, New York, 1989). Examples of fatty acids which disrupt lipid bilayer include linoleic acid, capric acid, lauric acid, and neodecanoic acid, which can be in a solvent such as ethanol or propylene glycol. The permeation enhancement of three bilayer disrupting compounds, capric acid, lauric acid, and neodecanoic acid, in propylene glycol has been reported by Aungst, et al., Pharm. Res. 7, 712- 718 (1990). The primary mechanism by which unsaturated fatty acids, such as linoleic acid, are thought to enhance skin permeabilities is by disordering the intercellular lipid domain. SC lipid bilayers disordered by unsaturated fatty acids or other bilayer disrupters may be similar in nature to fluid phase lipid bilayers. A separated oil phase should have properties similar to a bulk oil phase. See, for example, Clegg and Vaz in "Progress in Protein-Lipid Interactions" Watts, Ed. (Elsevier, N.Y. 1985) 173-229; Tocanne, et al., FEB 257, 10-16 (1989) and Perry, et al., "Perry's Chemical Engineering Handbook" (McGraw-Hill, NY 1984). A comprehensive list of lipid bilayer disrupting agents is also described in European Patent No. 0 043 738 to Procter & Gamble.

Solubility Enhancers

Suitable solvents include water; diols, such as propylene glycol and glycerol; mono-alcohols, such as ethanol, propanol, and higher alcohols;

DMSO; dimethylformamide; N,N-dimethylacetamide; 2-pyrrolidone; N-(2- hydroxyethyl) pyrrolidone, N-methylpyrrolidone, 1-dodecylazacycloheptan-

2-one and other n-substituted-alkyl-azacycloalkyl-2-ones (azones). U.S. Patent No. 4,537,776 to Cooper contains a summary of exemplary binary systems for permeant enhancement. European Patent No.

0 043 738, also describes the use of selected diols as solvents along with a broad category of cell-envelope disordering compounds for delivery of lipophilic pharmacologically-active compounds. Another binary system for enhancing metaclopramide penetration is disclosed in UK Patent No.

2,153,223 to Nitto Electric Industrial Co. Ltd., consisting of a monovalent alcohol ester of a C8-32 aliphatic monocarboxylic acid (unsaturated and/or branched if C 18-32) or a C6-24 aliphatic monoalcohol (unsaturated and/or branched if C 14-24) and anN-cyclic compound such as 2-pyrrolidone or N- methylpyrrolidone.

Combinations of enhancers consisting of diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate and methyl laurate are disclosed in U.S. Patent No. 4,973,468 to Chiang, et al. for enhancing the transdermal delivery of steroids such as progestogens and estrogens. A dual enhancer consisting of glycerol monolaurate and ethanol for the transdermal delivery of drugs is described in U.S. Patent No.

4,820,720 to Sanders, et al. U.S. Patent No. 5,006,342 to Cleary, et al. lists numerous enhancers for transdermal drug administration consisting of fatty acid esters or fatty alcohol ethers of C2 to C4 alkanediols, where each fatty acid/alcohol portion of the ester/ether is of about 8 to 22 carbon atoms. U.S.

Patent No. 4,863,970 to Patel, et al. discloses penetration-enhancing compositions for topical application including an active permeant contained in a penetration-enhancing vehicle containing specified amounts of one or more cell-envelope disordering compounds such as oleic acid, oleyl alcohol,

and glycerol esters of oleic acid, a C ₂ or C ₃ alkanol and an inert diluent such as water.

Other chemical enhancers, not necessarily associated with binary systems, include dimethylsulfoxide (DMSO) or aqueous solutions of DMSO such as those described in U.S. Patent No. 3,551 ,554 to Herschler; U.S. Patent No. 3,711,602 to Herschler; and U.S. Patent No. 3,711,606 to Herschler, and the azones (n-substituted-alkyl-azacycloalkyl-2-ones) such as noted in U.S. Patent No. 4,557,943 to Cooper.

Some chemical enhancer systems may possess negative side effects such as toxicity and skin irritations. U.S. Patent No. 4,855,298 discloses compositions for reducing skin irritation caused by chemical enhancer- containing compositions having skin irritation properties with an amount of glycerin sufficient to provide an anti-irritating effect.

Combinations of Lipid Bilayer Disrupting Agents and Solvents Ethanol and the unsaturated fatty acid linoleic acid were combined

(LA/ethanol) and studied as described in U.S. Patent No. 5,947,921. Single component enhancer formulations, including polyethylene glycol 200 dilaurate (PEG), isopropyl myristate (IM), glycerol trioleate (GT), ethanol/pH 7.4 phosphate buffered saline in a one-to-one ratio (50% ethanol), and PBS were also examined. Other permeation enhancers include alkyl glucosides, sorbitans and combinations thereof, at concentrations between 0.1-20% w/v.

C. Polymeric Hydrogel

In one embodiment, the drug formulations are preferably administered in a vehicle comprising a polymer that is capable of forming a hydrogel based on changes in temperature. In a preferred embodiment, the hydrogel comprises a block copolymer such as polyethylene oxide- polypropylene oxide-polyethylene oxide (PEO-PPO-PEO), PLURONIC ^® F127 (BASF Corporation, Mount Olive, New Jersey). The term "hydrogel" as used herein refers to a "physically-crosslinked" gel, with the gel state occurring due to interchain interactions. The amphiphilic polymer in aqueous solution forms organized self-assemblies, as in micelles and

micellar aggregates, with the hydrophobic PPO segments arranged in the interior of the micelle ("core") and the hydrophilic PEO segments arranged in the exterior ("corona"). Self-organized assemblies have had applications in drug delivery, such as in liposomal formulations. The hydrophobic cores of the PEO-PPO-PEO micelles have excellent drug partitioning capabilities, providing a thermodynamically favorable environment to maintain solubility of hydrophobic drugs in formulations. PPO segments have limited solubility in water at temperatures greater than 15 ⁰ C. The higher temperatures cause the solutions to gel. The thermosensitivity of the polymer solutions can be utilized to sterile filter drug formulations cold that are otherwise un-filterable in the gelled state. Furthermore, these solutions can be sprayed with ease onto the skin when cold, to achieve uniform drug-incorporated coatings. The solution's gelling temperature varies with the polymer's concentration in solution. The concentration can be adjusted for the formulation to be a gel. The critical point of gelation is a function of the temperature. Therefore, a drug-containing formulation can be filled-and-fϊnished at temperatures less than the gel temperature, and later applied topically at room temperature when the formulation is a gel.

Formulations that are able to form gels due to change in temperature have several advantages over formulations that do not form gels, including:

1. The formulation can be sterile-filtered, cold (at temperatures less than 4 degrees centigrade). This provides an inexpensive method of developing sterile formulations.

2. The formulations can also be autoclaved as another means of sterilization, such as at conditions of 25 psi, 125°C for 15-20 minutes, or other suitable autoclave conditions.

3. The formulations can be sprayed onto the skin, cold to obtain high and uniform coverage of an area to be treated. Once skin contact is established, the formulation forms a gel, thereby preventing run-off. Other applications of this functionality are in delivering uniform formulations to the face, as a mask or as a treatment modality. For example, delivering a

"sprayable gel" containing anesthetics may be an important application for pain management for burn victims.

4. The formulations can be analyzed in the liquid state at temperatures less than 4 ⁰ C. Additional properties of these formulations include:

1. Biocompatibilitv: Polymers based on polyethylene oxide (PEO) and PEO-PPO-PEO segments are biocompatible, due to their low absorption of protein. These polymers have been utilized as vehicles to deliver nucleic acids and small molecule drugs. In addition, these polymers have been demonstrated to have low skin irritation.

2. Hvdrating capability: Polymers based on polyethylene oxide and derivatives thereof, have a capacity to absorb water as "bound" water. The water molecules form "hydration spheres" on the polyether backbone of PEO-PPO-PEO polymers. The high moisture retention ability of the vehicle is important to enable efficient permeation through hydrated skin. It has been demonstrated that permeation of drugs through hydrated skin is higher than through non-hydrated skin.

3. Amphiphilicity: Polymers based on PEO-PPO-PPO segments have both hydrophobic and hydrophilic characteristics, characterized by the hydrophilic/lipophilic balance (HLB). This ratio can be modulated by varying the PEO/PPO ratio of the polymer, with high PEO content resulting in more hydrophilicity. As a result of their hydropliilic/lipophilic balance (amphiphilicity), both hydrophobic and hydrophilic drugs can be dissolved in this polymeric vehicle. For example, hydrophobic drugs such as lidocaine stay in solution due to the formation of hydrophobic micelles by this polymer in aqueous solvents. Furthermore, the hydrophobic units of the polymer (PPO) enable passive permeation through the skin.

4. Neutrality: Polymers based on PEO-PPO-PEO segments are non- ionic. Therefore, they do not interact with ionic drugs nor prevent permeation into the skin, by creation of bulky, macromolecular charged complexes. This functionality of neutrality is especially useful for active transport of drugs by iontophoresis. Iontophoresis requires the active agent

be in a charged state and, preferably, low molecular weight. Thus, a vehicle that is the same charge as the drug would compete with the drug. Alternatively, a vehicle that is of the opposite charge as the drug would interact with the drug to create macromolecular complexes. Transport of macromolecular complexes are inhibited through the skin.

5. Chemically compatible with hvdrophilic and sparingly soluble excipients: Due to its high hydrophilicity, PLURONIC ^® F 127 is chemically compatible with most excipients added to the formulation.

Other hydrogels that can be used include poloxamers such as F68, F108, P105, reverse PLURONICS® (BASF Corp.) of the structure PPO- PEO-PPO, polyethylene oxide based polymers and polysaccharides such as hyaluronic acid, chitosan gluconate, and carboxy methyl cellulose.

D. Excipients

The formulation optionally includes one or more excipients. Suitable excipients include polyethylene glycols or derivatives thereof, cholesterol derivatives, salt forms of fatty acids, and block copolymers comprised of polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO- PEO) segments.

When the active agent is soluble in polar solvents such as water, dimethyl sulfoxide (DMSO), dimethyl formamide, and isopropyl alcohol and in non-polar solvents such as N-methylpyrrolidone (NMP), benzyl alcohol, soybean oil, olive oil, vitamin E and derivatives thereof, one or more of these solvents may be included in the formulation.

Other suitable excipients include lipid desolvation and oil partitioning agents such as dimethyl sulfoxide (DMSO), alcohol, cholesterol, cholesterol derivatives, bile salts, isopropyl myristate, triolein, glycerol, glycerol derivatives, alkyl glucosides, fatty acids such as lauric acid, stearic acid, linoleic acid, and behenic acid, terpenes such as menthol, and camphor, vitamin E, vitamin E acetate, pegylated Vitamin E, ceramides, and sphingolipids and its derivatives such as phosphatidyl choline, lecithin, and phosphatidic acid.

E. Formulation Methods

The formulation may be formulated as an emulsion, dispersion, solution, suspension, or gel. Procedures for making emulsions, dispersions, solutions or suspension are well-known in the art. Procedures for making gels are described above in section LC.

An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in- water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers. The formulation may be an oil-in-water emulsion, or a water-in-oil emulsion, wherein the active agent may be present in only one phase, or in both phases.

The emulsion is prepared by adding the discrete phase (e.g. the oil phase) to the continuous phase (e.g. the aqueous phase), optionally with mixing or agitating to form the emulsion.

As a dispersion, the formulation may contain particles of active agent as the dispersed component. Optionally, the dispersion contains dissolved active agent in the aqueous phase and particulate, crystalline active agent as the dispersed component. This can be achieved by saturating the solution with active agent, resulting in a distribution of dissolved active agent and seed crystalline active agent.

The formulation may contain a mixture of soluble (in an aqueous solvent) and insoluble (in an aqueous solvent) forms of the active agent. In this case, the aqueous continuous phase would contain the active agent (for example, lidocaine hydrochloride) and the dispersed phase would consist of micron-sized or nano-sized particles containing the insoluble form of the active agent (for example, lidocaine). When delivered, this formulation initially releases the soluble form of the active agent. The insoluble nanoparticles then slowly become bioavailable, as they partition into the aqueous phase. In one embodiment, the formulation contains lidocaine as the water-insoluble form of the active agent and lidocaine hydrochloride as the water-soluble form of the active agent.

The formulation may also be formulated into a self-assembling organized system, such as micelles or in micellar aggregates, liquid- crystalline lyotropic solutions, or liposomes. These formulations contain one or more additives that induce self-assembly, such as polyethylene glycols or derivatives thereof, cholesterol derivatives, salt forms of fatty acids, and block copolymers comprised of PEO-PPO-PEO segments. In aqueous solution, these additives all form micelles and micellar aggregates with a hydrophobic "core" and hydrophilic "corona". Optionally, the formulation may be in the form of a cream. The cream may contain microscopic phospholipids spheres which contain the active agent.

In another embodiment, the formulation can be a wax pellet which "melts" when applied to the skin. II. Method of Administration

The method of enhanced transport of drugs into and through the skin of a patient includes the steps of: (a) pretreatment of the skin with a hydrating solution, (b) physical permeation of the stratum corneum by low frequency ultrasound ( such as through the use of a sonophoresis device, such as SONOPREP ^® ), (c) application of the bioactive molecule, formulated in an appropriate transport vehicle, and, optionally, (d) application of an electric potential difference that forces ionized drugs through the skin.

The formulation is preferably administered to the skin at a site selected based on convenience to the patient as well as maximum active agent penetration. For example, the arm, thigh, or stomach represent areas of relatively thin skin and high surface area, while the hands and feet are uneven and calloused.

In one embodiment, the active agent is an immune activating factor, such as a vaccine. A formulation containing the immune activating factor is applied to a low frequency, ultrasonicated site on hydrated skin. The formulation may include permeation enhancers, such as menthol, transcutol, and isopropyl myristate, to enhance delivery into the epidermis. The formulation may also include depot forming agents such as vitamin E, vitamin E α-tocopherol polyethylene glycol succinate (TPGS), and glycerol to prolong the rate at which the therapeutic agent is cleared from the plasma. Additionally, a vasoconstrictor such as epinephrine can be used in the formulation to slow down plasma clearance. Transport of the formulation through channels created by a sonophoresis device, such as SONOPREP ^® , can optionally be enhanced by "pushing" the vaccine or the immunotherapeutic by application of an iontophoretic current, as described in Example 4, below. A. Hydration of the Skin

The skin can be treated with any hydrating solution, for example, a glycerol-containing wipe. The materials used for hydration include glycerol, bile salts, phosphate buffered saline, Tweens, sorbitans and combinations thereof. The hydrating solution can be formulated as a solution, or used as a wipe to hydrate the skin prior to sonication.

B. Application of Low Frequency Ultrasound

Ultrasound is defined as sound at a frequency of higher than about 20 kHz and 10 MHz, with intensities of between greater than zero and three

W/cm2. Ultrasound is preferably administered at frequencies of less than or equal to about 2.5 MHz, more preferably less than about 1 MHz, even more preferably less than 200 kHz, and most preferably less than 50 to 100 kHz, to induce cavitation of the skin to enhance transport. Exposures are typically

for between 20 seconds and 10 minutes, continuously, but may be shorter and/or pulsed. It should be understood that although the normal lower range of ultrasound is 20 kHz, one could achieve comparable results by varying the frequency to less than 20 kHz, that is, into the sound region down to about one kHz. The intensity should not be so high as to raise the skin temperature more than about one to two degrees Centigrade.

As used herein, sonophoresis is the application of ultrasound to the skin. "Low frequency" sonophoresis is ultrasound at a frequency that is less than one MHz, more typically in the range of 20 to 100 kHz, which is applied continuously or, preferably, in pulses, for example, 100 to 500 msec pulses every second at intensities in the range of between above zero and one

W/cm 2 ₅ more typically between 12.5 mW/cm^ and 225 mW/crrA

In the preferred embodiment, a device such as the SONOPREP® Skin Permeation Device is used to apply the low frequency ultrasound. This device is available from Sontra Medical Corporation, Franklin, MA and contains a battery operated power and control unit, a hand piece containing the ultrasonic horn and the disposable coupling medium cartridge, and a return electrode. A clinician performs a skin permeation treatment by applying the ultrasonic hand piece to the patient's skin. The clinician pushes the hand piece down on the patient's skin to activate the ultrasonic horn. The patient holds the return electrode so that the device automatically shuts itself off, based on a drop in skin impedance (as measured by current moving through the return electrode) once the proper level of skin permeation is achieved. The SONOPREP® device applies relatively low frequency

(compared to diagnostic imaging) ultrasonic energy to the skin for 15 seconds (average). The ultrasonic horn contained in the hand piece vibrates at 55,000 times per second (55KHz) and applies the energy to the skin through the liquid coupling medium to create cavitation bubbles that expand and contract in the coupling medium. Ultrasonic cavitation disorganizes the lipid bi-layer of the stratum corneum, creating reversible micro-channels in the skin through which fluids and analytes can be extracted and large

molecules can also be delivered. The SONOPREP ^® device is easy for the health care professional to administer and the treatment can also be self- administered by the patient. The permeability is reversible and the skin goes back to its normal state within 24 hours. Cavitation of a skin permeation fluid by application of low frequency ultrasound via SONOPREP ^® has been demonstrated to create pores or "channels" into the skin, essentially disrupting its barrier properties. The range of diameters created by these pores is 50-75 μm. The depth of the pores is between 18-35 μm into the epidermis. The pores are transient, lasting approximately 24 hours, leaving a window of time for delivery of drug. This modality of permeation of skin by "transient poration" can be utilized effectively not only in the delivery of drugs transdermally, but also to extract serum analytes for continuous diagnostic monitoring, as in blood glucose. C. Application of the Formulation The formulation can be applied as a cream, a gel, in a patch, or in a drug reservoir. The formulations can be applied as a paste or cream to be rubbed into the ultrasonicated site with a gloved hand. The formulations can be applied to the skin as a "cold" spray, which solidifies into a paste-like consistency at skin temperature. This provides the benefit of a "sprayable" formulation that does not "run-off the skin. Wax pellet formulations can be contained within a reservoir patch for application. The formulation can also be imbedded into a matrix on the patch. The formulations may be designed to have a solution-to-gelatin (sol-to-gel) transition temperature (i.e. the gel formation would occur at this temperature) of 32 ⁰ C. This formulation can be applied to the skin as a viscous liquid, to have high molecular mobility while diffusing into the skin. This liquid will then turn into "micro-gel" reservoirs once they become equilibrated at the skin temperature of 32 ⁰ C. This can result in an "in situ" delivery system with prolonged drug delivery functionalities. The formulations may act by lipid desolvation or by partitioning into the lipid layer, by forming nanocrystals in the epidermis, or by forming depots in the lipid lamellae.

The formulation that is applied during the method described herein may be one of the formulations listed above or a topical formulation that is commercially available. Preferably the formulation contains a local anesthetic, such as lidocaine and one or more pharmaceutically acceptable excipients. The formulation can also contain preservatives such as benzyl alcohol and emulsion stabilizers such as carbomer 940. In one preferred embodiment, the formulation is L.M.X.4®, a topical anesthetic cream containing 4% lidocaine available from Ferndale Laboratories Inc. (Ferndale, Michigan). L.M.X.4® contains lidocaine (4%), benzyl alcohol (1.5%), carbomer 940, cholesterol, hydrogenated lecithin, polysorbate 80, propylene glycol, vitamin E acetate, trolamine (triethanolamine), and water. L.M.X.4® contains phospholipids microspheres that help deliver the lidocaine through the skin.

D. Iontopheresis and/or EIectroporation Optionally, iontopheresis and/or electroporation can be used in combination with the methods described herein. Application of electric current may enhance transdermal transport by inducing conductive transport. Iontophoresis involves the application of an electrical current, preferably DC, or AC, at a current density of greater than zero up to about 1 niA/cm ² . Typically, a constant voltage is applied since resistance changes over time, usually in the range of between greater than zero and four volts. Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. Factors that affect iontophoretic delivery are: (a) ionized drug concentration, (b) current strength, (c) ionic competition, (d) molecular size and (e) site of delivery. The formulations have the following functional attributes: (a) chemically compatible with the ionized drug, (b) not susceptible to degradation by current, (c) acts as a drug reservoir, (d) no leakage from the patch, (e) uncharged and neutral, (f) history of human use, (g) skin biocompatible and (h) manufactured USP grade. The excipients that can be added to a formulation that undergo iontophoresis have the following

attributes: (a) compatible with ionized drug and (b) other attributes to enhance the delivery effect, such skin permeation enhancer or depot forming agent. The formulations described in this example, meet all the iontophoretic delivery criteria. The PEO-PPO-PEO-based (PLURONIC ^® F127) formulations described herein, demonstrate chemical compatibility with the drug. Preferably, the formulations do not lose their gel forming capabilities when delivered during iontophoresis.

Application of electric current enhances transdermal transport by different mechanisms. First, application of an electric field provides an additional driving force for the transport of charged molecules across the skin and second, ionic motion due to application of electric fields may induce convective flows across the skin, referred to as electroosmosis. This mechanism is believed to play a dominant role in transdermal transport of neutral molecules during iontophoresis. The advantages of combining sonophoresis with physical enhancers is not restricted to electric current. Effects on transdermal transport may also be observed between ultrasound and pressure (mechanical or osmotic) as well as between ultrasound and magnetic fields since the physical principles underlying the possible enhancement are the same. A pressure gradient can be used to enhance convection (physical movement of liquid) across the skin permeabilized by sonophoresis. Application of magnetic fields to the skin pretreated with ultrasound may also result in a higher transport of magnetically active species across the skin. For example, polymer microspheres loaded with magnetic particles could be transported across the skin using sonophoresis and magnetic fields.

The invention will be further understood from the following non-limiting examples.

Examples These formulations have been optimized for performance for permeation into the skin, depth of penetration and duration of drug effect with skin pre-treatment with SONOPREP ^® . Performance of drug-containing formulations have been demonstrated with and without iontophoresis.

Lidocaine hydrochloride, a common anesthetic, was utilized as the model drug to determine its delivery on ultrasound permeated skin. A standard pain model was utilized to assess the anesthetic effect, in healthy volunteers. Example 1. Delivery of Lidocaine Hydrochloride Formulated in Various Compositions on Low Frequency Ultrasound Pre-Treated Skin.

Lidocaine hydrochloride-containing formulations were applied on a skin target site following (a) hydration with a glycerol-containing wipe and (b) sonication with low frequency ultrasound (SONOPREP ^® ). Following sonication, the formulation was applied for 5 minutes in the sonicated site. All formulations contained 4% w/w lidocaine hydrochloride.

Permeation-enhancing excipients and their respective concentrations were varied in the different formulations. The formulations used were:

LMX: Standard commercial lidocaine HCl formulation, typically applied for a half hour to obtain anesthesia. However, when applied on ultrasound permeated skin, rapid-onset anesthesia with LMX was observed. This was used as the internal control of the formulation screening study.

Formulation 5 contained 18% w/w PLURONIC ^® F127, 2% w/w benzyl alcohol, 76% PBS, in addition to the active drug, pH 5.5.

Formulation 5a contained 18% w/w PLURONIC ^® F 127, 3% w/w benzyl alcohol, 75% PBS, in addition to the active drug, pH 5.5.

Formulation 8 contained 18% w/w PLURONIC ^® F127, 2% w/w benzyl alcohol, 2% w/w taurocholate, 74% PBS, in addition to the active drug, pH 5.5.

Formulation 8a contained 18% w/w PLURONIC ^® F127, 2% w/w benzyl alcohol, 3% w/w taurocholate, 73% w/w PBS, in addition to the active drug, pH 5.5.

Formulation Y contained 18% w/w PLURONIC ^® F127, 2% w/w benzyl alcohol, 2% w/w dodecyl maltoside, 74% w/w PBS, in addition to the active drug, pH 5.5. All formulations were tested for anesthetic effect on the volar forearm of healthy volunteer subjects between the ages of 20-60. The anesthetic effect was caused by delivery of lidocaine HCl to the cutaneous

sensory nerves present in the epidermis. A standard pain model was utilized to assess the anesthetic effect. The pain model was based on a pain scale of 0-5, with 5 being the pain felt by the subject in an untreated area. The pain scale was as follows: O=felt nothing, l=pressure only, 2=much less pain than reference, 3=less pain than reference, 4=little less than reference, 5=same as reference. According to the pain model, the lower the formulation score, the better the anesthetic effect. A 21 G needle was applied vertically in the center of the treated area and a pain score was indicated by the subject. The pain insult was applied to the anticubital of the forearm immediately after 5 minutes of application of 1 OOμl of the formulation. The number of sites treated was 16-20. All pain scores were normalized to the LMX pain score. Scores greater than 1 were better in performance than LMX.

Rapid-onset delivery of lidocaine hydrochloride was observed through ultrasound-permeated skin. These results can be seen in Figure IA. Figure IA is a graph of average pain scores between 0-5 (y-axis), as a function of formulation composition. Formulation 8a demonstrated the best anesthetic effect, due to the presence of 2% w/w sodium taurocholate. Formulation 5a had a better anesthetic effect than formulation 5 and LMX, due to the presence of 3% w/w benzyl alcohol. Formulations 5 and 8 had a lower anesthetic effect than LMX. Formulation Y was equivalent to LMX. Concentrations of benzyl alcohol greater than 3% w/w had issues of skin irritation and did not demonstrate significant enhancement of the anesthetic effect. The percentage of subjects with pain scores between 0-1, 1-2, 2-3, 3- 4, and 4-5 is shown in Figure IB. Formulation 8a containing 2% w/w sodium taurocholate had the highest percentage of subjects with pain scores less than 1.

The onset of anesthesia, as shown by percent of subjects with pain scores less than lor 2 as a function of application time, is shown in Figures 2A and 2B. The formulations were applied for 1, 2, 3, 4 or 5 minutes on the anticubital of the forearm and tested for the anesthetic effect using the pain score model. Formulation 8a had the highest percentage of subjects with pain scores less than 1 in 1 minute.

Figures IA, IB ₅ 2 A, and 2B demonstrate that all formulations tested were generally effective as transdermal delivery formulations. Formulations containing 18% w/w PLURONIC ^® F 127, 4% w/w lidocaine hydrochloride and a combination of other skin permeation enhancing excipients such as sodium taurocholate and benzyl alcohol create an effective gel-based delivery system to deliver drugs transdermally. Example 2. Modulation of the Solution-to-Gelatin Transition Temperature of the Formulations by Benzyl Alcohol.

The inherent temperature sensitive nature of these formulations, which were "liquid" at 4 ⁰ C, enabled facile sterile filtration and fill-and- fϊnish. The solution-to-gelatin (sol-to-gel) transition temperature of the formulations can be modulated by the ratio of benzyl alcohol to PLURONIC ^® F 127. Viscosity measurements were performed with a Brookfield Viscometer as a function of temperature for formulation 18% F 127, formulation 8a, and formulation 8e. Formulation 18% F 127 contained 18% PLURONIC ^® F127, 88% PBS, pH 5.5. Formulation 8e was the same as formulation 8a except that it contained 5% benzyl alcohol. Formulation 8e contained 18% w/w PLURONIC ^® F 127, 5% w/w benzyl alcohol, 3% w/w taurocholate, 70% w/w PBS, in addition to the active drug, pH 5.5. Benzyl alcohol participates in the micellar aggregation phenomenon of a

PLURONIC ^® F 127 based solution, aiding in the creation of hydrophobic micellar aggregates. This is demonstrated by the enhanced viscosity of a PLURONIC ^® F 127 and benzyl alcohol-containing formulations, as compared to 18% w/w PLURONIC ^® F 127, alone. These results are shown in Figure 3. The creation of micellar aggregates due to interaction of

PLURONIC ^® F 127 with benzyl alcohol allows the formulation to dissolve hydrophobic compounds such as menthol. For example, menthol does not dissolve in an aqueous solution of PLURONIC ^® F 127. However, menthol can be dissolved in benzyl alcohol prior to dissolution in an aqueous PLURONIC ^® F127 solution.

Other factors controlling the solution-to-gelation temperature are the respective concentrations of the polymer and the excipient. Micelle-forming

compounds such as surfactants can form "mixed-micelle" systems in aqueous solutions with PLURONIC ^® F 127. Sodium taurocholate and sodium glycocholate form their own micelles in water, thereby "disrupting" micellar aggregates formed by PLURONIC ^® F127 or combinations of PLURONIC ^® F 127 and benzyl alcohol. Micelle-disrupters such as surfactants or salts can be utilized to modulate the sol-to-gel transition temperatures. Example 3: Lidocaine Hydrochloride Containing Compositions Delivered on Hydrated Skin Pre-Treated with Low Frequency Ultrasound. The anesthetic effect on the dorsum of the hand or the anticubital of the forearm of variations in the mixed micelle characteristics of the formulations are shown in Figures 4A, 4B, and 4C. Formulation 8e was the same as formulation 8a, except it had 5% w/w benzyl alcohol. Formulation 8e contained 18% w/w PLURONIC ^® F 127, 5% w/w benzyl alcohol, 3% w/w taurocholate, 70% w/w PBS, in addition to the active drug, pH 5.5.

Formulation 8f was the same as formulation 8a, except it had 5% w/w sodium taurocholate. Formulation 8f contained 18% w/w PLURONIC ^® F 127, 2% w/w benzyl alcohol, 5% w/w taurocholate, 71% w/w PBS ₅ in addition to the active drug. The formulations were of different viscosities due to the interaction of benzyl alcohol and taurocholate with PLURONIC ^® F 127. Therefore, the flow properties of the formulations can be modified by addition of micelle-disrupters or micelles-enhancers. The viscosity of formulation 8e was a stiff gel, the viscosity of formulation 8a was a loose gel with no flow, and the viscosity of formulation 8f was a viscous liquid with some flow. These properties were due to the micelle-enhancing properties of benzyl alcohol and the micelle-disrupting properties of bile salts (sodium taurocholate).

Formulations were applied on the dorsum of the hand or the anticubital of the forearm for 1, 2, 3, 4, or 5 minutes following hydration with a glycerol-containing wipe and sonication with low frequency ultrasound (SONOPREP ^® ). The anesthetic effect was determined using the pain score model. All formulations were effective in delivering lidocaine

hydrochloride on the dorsum of the hand and the anticubital of the forearm. The duration of anesthesia as shown in Figure 4 A was equivalent for all formulations. The onset of anesthesia as shown in Figures 4B, and 4C, was equivalent for all formulations. However, the average pain score was much lower following 1 minute or 3 minute application of the formulation when applied to the anticubital of the forearm (see Figure 4A). In addition, the percentage of patients with pain scores of less than 1 was much higher in patients treated on the anticubital of the forearm (see Figures 4B and 4C). Example 4: Lidocaine Hydrochloride Containing Compositions Delivered on Hydrated Skin Pre-Treated with Low Frequency Ultrasound Followed by Iontophoresis.

The effect of current strength on iontophoretically delivered formulations containing 4% w/w lidocaine hydrochloride on sonicated skin is shown in Figures 5 to 11. The measure of efficacy was determination of effective anesthesia obtained with a standard pain model. Pain scores were determined as described in Example 1, except that formulations were applied at the site for 1 minute followed by iontophoresis for 1 minute. Measurement of perceived pain at the site of treatment was performed with a 21 G needle immediately after iontophoresis and 15 minutes later. Formulations were delivered to the anticubital of the forearm. Pain scores were based on a scale of 0-5, with 5 being the perceived pain in an adjacent untreated area, 0=no feeling, l=pressure only, 2=much less pain than reference, 3=less pain than reference, 4=little less pain than reference, and 5=equivalent to reference. Iontophoresis was delivered at 0 mA, 0.4 mA, or 1.OmA for formulation 8a and delivered at 0 mA for formulation LMX. Enhancement of the analgesic effect occurs when iontophoretic transport of the drug is combined with SONOPREP ^® (see Figure 5A). Pain scores were generally equivalent at current levels of 0.4 mA and 1 mA. A higher number of subjects experienced pain scores between 0 and 1, demonstrating an enhanced analgesic effect, compared to the passive delivery groups (0 mA or no iontophoresis) (see Figure 5B). The depth of anesthesia achieved by iontophoretic delivery on ultrasound-pretreated skin

was higher for both levels of current, compared to passive delivery transport methods (0 mA or no iontophoresis) (see Figure 5C).

The effects of lowering phosphate salts to 0.05 M in the 8a formulation (Formula 8a P), lowering pH of the 8a formulation to 4.5, to ionize the drug completely (Formula 8a 4.5), or adding the permeation enhancer DL Methanol (Formula 8a M) to formulation 8a is shown in Figure 6 A. Treatment was applied for one minute at the dorsum of the hand. Iontophoresis was applied for 2 minutes. Measurement of perceived pain at the site of treatment was performed with a 21G needle immediately after iontophoresis. All formulations contained 4% w/w lidocaine hydrochloride. As shown in Figure 6A, the analgesic effect was enhanced the most, by addition of DL menthol to the 8a formulation (see Formula 8aM).

The effect of eliminating or lowering the phosphate content of the 8a M formulation is shown in Figure 6B. The pH of formulation 8a M was lowered to pH 4.5 (Formula 8a M 4.5). The salt content of formulation 8a M was removed to eliminate ionic competition (Formula 8a MDI). The phosphate content of formulation 8a M was lowered to 0.05M (Formula 8a M P). Treatment was applied for one minute at the dorsum of the hand. Iontophoresis was applied for 2 minutes. Measurement of perceived pain at the site of treatment was performed with a 21 G needle immediately after iontophoresis. Figure 6B shows that eliminating the salt content or lowering the phosphate content significantly enhance the analgesic effect (see Formula 8a MDI and Formula 8a MP).

The effect of iontophoresis on the transport of Formula 8a MDI and LMX on hydrated skin pre-treated with low frequency ultrasound is shown in Figure 7A. Passive delivery of 8MDI and LMX on sonopermeated skin was compared with 8aMDI delivered at 0.4mA for 2 min and 8MDI delivered at 1.0 mA for 1 minute on both the anticubital of the arm and the dorsum of the hand. Formulation LMX and Formulation 8aMDI were applied for 5 minutes. For subjects receiving iontophoresis, 100% of the subjects had pain scores less than 1 on the anticubital of the arm, immediately after application of the drug, and approximately -90% of

subjects had pain scores less than 1 on the dorsum of the hand (see Figure 12). Passive modes of delivery resulted in higher duration of anesthesia for the LMX and 8a MDI formulations (see Figure 7A). Figure 7 A demonstrates rapid anesthetic effect as well as rapid clearance of the anesthetic from the site of application for subjects receiving iontophoretic groups.

The effect of adding epinephrine to extend the anesthetic effect of formulation 8aMDI is shown in Figure 7B. Epinephrine (0.005% w/w) was added to formulation 8aMDI (Formula 8aMDIep). Vitamin E TPGS (1% w/w) was added to formulation 8aMDI (Formula 8aMDI ve). EMLA is a topical anesthetic containing 2.5% lidocaine and 2.5% prilocaine. Treatment was applied for 2 minutes at 0.4mA iontophoresis for formulation 8aMDI ep or formulation 8aMDI ve on the dorsum of the hand or the anticubital of the forearm. Treatment was applied for 5 minutes for formulation LMX. Treatment was applied for one hour for EMLA with no ultrasound. All other formulations were applied after hydration with a glycerol-containing wipe and sonication with low frequency ultrasound (SONOPREP ^® ). Measurement of perceived pain at the site of treatment was performed with a 21 G needle immediately after iontophoresis. Figure 7B demonstrates that adding epinephrine or vitamin E TPGS to the formulation significantly enhanced the duration of the anesthetic effect. Since epinephrine is a vasoconstrictor, it enhanced the duration of the anesthetic effect by lowering the plasma clearance of drug by lowering vascularization at the site of delivery. The effect of increasing vitamin E TPGS concentration in formulation 8aMDI on the duration and level of anesthesia delivered by iontophoretic transport on sonopermeated skin is shown in Figures 8 and 9. The vitamin E TPGS concentration of formulation 8aMDI ve was increased to 3% w/w (8aMDI ve3). Treatment was applied for 2 minutes at 0.4mA iontophoresis for formulation 8aMDI ep, 8aMDI ve, or 8aMDI ve3 on the dorsum of the hand. Treatment was applied for 5 minutes for formulation LMX. Treatment was applied for one hour for EMLA with no ultrasound.

All other formulations were applied after hydration with a glycerol- containing wipe and sonication with low frequency ultrasound (SONOPREP ^® ). Measurement of perceived pain at the site of treatment was performed with a 21G needle at O ₅ 5, 10, 15, 20, 25, or 30 minutes after iontophoresis. Increasing the vitamin E TPGS concentration to 3% w/w enhanced the duration of anesthesia as compared to 1 % w/w vitamin E TPGS in formulation 8aMDI (see Figures 8 and 9).

The effect of combining the depot forming properties of vitamin E TPGS and vasoconstricting properties of epinephrine, in efforts to extend the duration of anesthesia (Formula 8aMDI ve 3 epl) is shown in Figures 10 and 11. Treatment was applied for 2 minutes at 0.4mA iontophoresis for formulation SaMDI ep, 8aMDI ve, 8aMDI ve3, or 9aMDI ve3 epl on the dorsum of the hand. Treatment was applied for 5 minutes for formulation LMX. Treatment was applied for one hour for EMLA with no ultrasound. All other formulations were applied after hydration with a glycerol- containing wipe and sonication with low frequency ultrasound (SONOPREP ^® ). Measurement of perceived pain at the site of treatment was performed with a 21 G needle at 0, 5, 10, 15, 20, 25, or 30 minutes after iontophoresis. Enhanced duration and level of anesthesia was obtained by formulation 8aMDIve3epl . Therefore, this formulation was the best optimized formulation for iontophoretic delivery of lidocaine hydrochloride on ultrasound permeated skin (SONOPREP ^® ).

Example 5: Passive Delivery of Immune Activating Factors into the Epidermis to Induce Antigen-Specific Immune Responses. The presence and extent of delayed-type hypersensitivity (DTH) reactions to recall antigens tetanus toxoid and Candida albicans, when administered by transcutaneous route (SONOPREP ^® ) or intradermal needle injection is shown in Tables 1 and 2.

Groups 1 and 2 consisted of 10 adult healthy volunteers each. 10 ml of whole blood from each volunteer was collected for peripheral blood mononuclear cell (PBMC) isolation. Each volunteer from Group 1 was injected transdermally with two recall antigens tetanus toxoid and Candida

albicans to the volar aspect of the forearm at separate sites using needle administration. For each volunteer from Group 2, the volar aspect of the forearm was first hydrated with a glycerol wipe and subsequently sonicated with SONOPREP ^® . 100 μl of tetanus toxoid was delivered by pipette into a foam reservoir placed over the sonicated site. The reservoir delivery chamber was secured by application of a TEGADERM® (Minnesota Mining and Manufacturing Company, St. Paul, Minnesota) bandage. The delivery reservoir was left on-site between 8-12 hours (overnight). In addition, lOOμl of a solution containing Candida albicans was delivered onto another sonicated site.

The immune response to tetanus toxoid and Candida albicans was quantified by measurement of the presence or absence of induration and the diameter of induration/site.

Tables 1 and 2 demonstrate that transcutaneous administration of tetanus toxoid and Candida albicans recall antigens by SONOPREP ^® produced detectable DTH responses in 9/10 and 10/10 subjects, respectively. The kinetics of appearance and disappearance of DTH responses were similar for the intradermal and SONOPREP ^® groups. As shown in Tables 1 and 2 antigens can be delivered topically, via SONOPREP ^® methods, to elicit antigen-specific immune responses. Table 1. Presence and extent of delayed-type hypersensitivity (DTH) reactions to tetanus toxoid administered via SONOPREP ^® or intradermal needle injection

Abbreviations: standard deviation, S.D., mean represents average of X values (where X= 9 for intradermal and X= 10 for SONOPREP ^® ).

Table 2. Presence and extent of delayed-type hypersensitivity (DTH) reactions to Candida albicans administered via SONOPREP ^® or intradermal needle injection

5 Abbreviations: standard deviation, S.D., mean represents average of X values (where X= 9 for intradermal and X= 10 for SONOPREP ^® ).

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific

10 embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.