(12) United States Patent (10) Patent No.: US 7,838,011 B2 Modi (45) Date of Patent: *Nov

US007838011B2

(12) United States Patent (10) Patent No.: US 7,838,011 B2 Modi (45) Date of Patent: *Nov. 23, 2010

(54) STABILIZED PROTEIN COMPOSITIONS FOR 2004/0247623 A1 12/2004 Cady TOPCAL ADMINISTRATION AND FOREIGN PATENT DOCUMENTS METHODS OF MAKING SAME EP O319638 A1 6, 1989 EP O525167 B1 9, 1995 (76) Inventor: Pankaj Modi, 519 Golf Links Road, EP 0812186 B1 10, 2001 Ancaster, Ontario (CA) L9G 4X6 WO WO9101719 A1 2, 1991 WO WOO1584.72 8, 2001 (*) Notice: Subject to any disclaimer, the term of this WO WOO3O15698 A2 2, 2003 patent is extended or adjusted under 35 WO WO 2004/060384 A2 T 2004 U.S.C. 154(b) by 746 days. WO WO2005046.637 A2 5, 2005 This patent is Subject to a terminal dis OTHER PUBLICATIONS claimer. Carpender et al., Interactions of Stabilizing Additives with Proteins During Freeze-Thawing and Freeze-Drying, International Sympo (21) Appl. No.: 11/057,481 sium on Biological Product Freeze-Drying and Formulation, Oct. 24-26, 1990; Karger (1992), 225-239. (22) Filed: Feb. 14, 2005 Goodnough et al., Stabilization of Botulinum Toxin Type A During Lyophilization, App & Envir. Micro. 58 (10) 3426-3428 (1992). (65) Prior Publication Data Schmidt, et al., Endoproteinase Activity of Type A Botulinum Neurotoxin Substrate Requirements and Activation by Serum Albu US 2006/O182766A1 Aug. 17, 2006 min, J. of Protein Chemistry, 16 (1), 19-26 (1997). (51) Int. Cl. * cited by examiner A6 IK39/08 (2006.01) Primary Examiner Robert Mondesi A6 IK3I/74 (2006.01) Assistant Examiner Khatol Shahnan-Shah A6 IK 47/44 (2006.01) (74) Attorney, Agent, or Firm Gersten Savage LLP A23. I/00 (2006.01) (52) U.S. Cl...... 424/239.1; 424/78.03; 424/284.1; (57) ABSTRACT 5307412 A stabilizing composition that also enhances permeation is (58) Field of Classification Search ...... 424/239.1 provided for the topical or transdermal administration of an See application file for complete search history. active ingredient. The composition comprises collagen, elas (56) References Cited tin, sphingosine and cerebroside. Also provided are pharma ceutical or cosmetic formulations comprising an effective U.S. PATENT DOCUMENTS amount of an active agent and the stabilizing composition as 6,585,993 B2 7/2003 Donovan et al. well as methods of administering active agents topically or 6,638,621 B2 * 10/2003 Anderson ...... 428/402.24 transdermally. 2003/011.8598 A1* 6, 2003 Hunt ...... 424,184.1 2004/0033254 A1* 2/2004 Song et al...... 424/449 10 Claims, 3 Drawing Sheets U.S. Patent Nov. 23, 2010 Sheet 1 of 3 US 7,838,011 B2

U.S. Patent Nov. 23, 2010 Sheet 2 of 3 US 7,838,011 B2 Western Blot Analysis #1

FIG. 2A

Lane-1 Molecular weight marker Lane-3 Stabilized Botulinum Toxin (at time 0 month)

Western Blot Analysis (repeat #2 at 6 months time)

FIG. 2B

Lane-1 Molecular weight marker Lane- 2 Pure Botulinum Toxin (from vial) Lane-3 Stabilized Botulinum Toxin (6 months) U.S. Patent Nov. 23, 2010 Sheet 3 of 3 US 7,838,011 B2

Molecular mass marker ranges from the top represents the molecular weights of 200, 116, 98, 67, 55, 37, 28 and 14 kDa.

Before and After Photographs (Botulinum Toxin Cream)

FIG. 3A

Before

FG.3B

Before After

US 7,838,011 B2 1. 2 STABILIZED PROTEIN COMPOSITIONS FOR ever, these stabilizers are not able to sufficiently stabilize TOPCAL ADMINISTRATION AND botulinum toxin for storage at room temperature. Thus there METHODS OF MAKING SAME is a specific need for enhancement of the stability of botuli num toxin and also to promote its permeation through the FIELD OF INVENTION skin. The process that leads to skin aging and wrinkles is com The present invention relates to methods for stabilizing plex. A primary cause of wrinkling is a build-up of free radical active ingredients in pharmaceutical compositions to provide toxic plaque that binds to collagen and elastin fibers, causing a prolonged shelf-life with enhanced availability of active the skin's supportive structure to become inflexible and ingredients. 10 unhealthy. Laugh lines, Smile lines, crow's feet or facial creases appear in areas where repeated muscle movement BACKGROUND OF THE INVENTION OCCU.S. In 2002 the FDA approved the Botulinum Toxin Type A For storage stability and convenience of handling, pro (BOTOXOR) Cosmetic) to temporarily improve the appearance teinaceous pharmaceutical compositions are often formu 15 of moderate to severe frown lines between the eyebrows lated as a lyophilized (i.e. freeze dried) or vacuum dried (glabellar lines). BOTOXOR) Cosmetic is a purified protein powder and stored at low temperatures between -10C to 4C. produced by the Clostridium botulinum bacterium. The toxin The dry powder is reconstituted with a suitable fluid, such as reduces the activity of the muscles that cause frown lines saline or water, prior to administration to a patient. Alterna between the brows to form over time. While BOTOXOR) Cos tively, a proteinaceous pharmaceutical composition can be metic has been Successful in some cases, there are areas formulated as an aqueous solution or Suspension that includes where it should not be used, such as throat and neck areas, a stabilizer to prevent protein degradation. However, many around the mouth, near the eyes, and the hands. If the muscles proteins are very difficult to stabilize resulting therefore in a in these areas are paralyzed or disabled, it can lead to diffi loss of protein and/or protein activity during the formulation, culty talking, Smiling, eating, Swallowing, moving hands and reconstitution and/or the period of storage. Stability problems 25 fingers, and seeing. can occur as a result of protein denaturation, degradation, Current formulations of BOTOXOR) Cosmetic must be dimerization, and/or polymerization. Various excipients, administered within four hours after reconstitution since the Such as albumin and gelatin have been used with differing toxin molecule is very labile. During this time period, recon degrees of success to try and stabilize a protein active ingre stituted BOTOXOR) Cosmetic is stored in a refrigerator (4.de dient in a pharmaceutical composition. Cryoprotectants such 30 gree. C.). Breakdown of the toxin into toxoid can induce as alcohols have been used to reduce protein denaturation immune responses to the toxoid that can interfere with sub under the freezing conditions of lyophilization. Stabilizers sequent treatments. can work by reducing adhesion of the protein active ingredi Animal derived or donor pool proteins such as gelatin and ent to surfaces, such as the Surfaces of laboratory glassware, serum albumin have been used with Some Success to stabilize vessels, the vial in which the pharmaceutical composition is 35 botulinum toxin. However, there has been a desire for reconstituted or the inside Surface of a syringe used to inject improved methods of stabilizing botulinum toxins. the pharmaceutical composition. Adhesion of a protein active It has been Suggested that a suitable alternative to human ingredient to Surfaces leads to loss of active ingredient and serum albuminas a botulinum toxinstabilizer may be another can also contribute to denaturation of the remaining retained protein or a low molecular weight (non-protein) compound protein active ingredient, both of which reduce the total activ 40 (Carpender et al., Interactions of Stabilizing Additives with ity of the active ingredient present in the pharmaceutical Proteins. During Freeze-Thawing and Freeze-Drying, Inter composition. Stabilizers can also reduce denaturation of the national Symposium on Biological Product Freeze-Drying active ingredient which can occur upon preparation of a low and Formulation, Oct. 24-26 1990; Karger (1992), 225-239. dilution Solution of the active ingredient. As well as being However, many Substances commonly used as carriers and able to stabilize a protein in a composition, an ideal stabiliz 45 bulking agents in pharmaceutical compositions have proven ing agent should have negligible immunogenicity when to be unsuitable as albumin replacements in compositions injected into a human patient. containing Clostridial toxin. For example, the disaccharide Pure botulinum toxin is so labile that it is has limited cellobiose has been found to be unsuitable as a botulinum practical utility to prepare a pharmaceutical composition. toxin stabilizer. The use of cellobiose as an excipient in con Botulinum toxin complexes, such as the toxin type A complex 50 junction with albumin and sodium chloride was reported to are extremely Susceptible to denaturation due to Surface dena result in a much lower level of toxicity/efficacy (10% recov turation, heat, and alkaline conditions. Inactivated toxin ery) after lyophilization of crystalline botulinum toxin type A forms toxoid proteins which may be immunogenic. The with these excipients, as compared to the toxicity after lyo resulting antibodies can render a patient refractory to toxin philization with only human serum albumin (>75% to >90% injection. As with enzymes generally, the biological activities 55 recovery). Goodnough et al., Stabilization of Botulinum of the botulinum toxins is dependent, at least in part, upon Toxin Type A During Lyophilization, App & Envir. Micro. 58 their three-dimensional conformation. Additionally, it is (10) 3426-3428 (1992). Schmidt, et al., Endoproteinase known that dilution of the toxin complex obtained by the Activity of Type A Botulinum Neurotoxin Substrate Require known culturing, fermentation and purification to the much ments and Activation by Serum Albumin, J. of Protein Chem lower toxin concentrations used for pharmaceutical compo 60 istry, 16 (1), 19-26 (1997). sition formulation results in rapid detoxification of the toxin Gelatin has been used in some protein active ingredient unless a suitable stabilizing agent is present. Dilution of the pharmaceutical compositions as an albumin Substitute. How toxin from milligram quantities to a solution containing nano ever, it does not provide for stabilization of botulinum toxin at grams permilliliterpresents significant difficulties because of room temperature. the rapid loss of specific toxicity upon Such great dilution. 65 Several efforts have been made to provide botulinum toxin Current stabilizers that have been used in botulinum toxin formulations that can be stabilized and delivered in alterna formulations are animal derived albumin and gelatin. How tive ways. For example, U.S. Pat. No. 6,585,993 discloses a US 7,838,011 B2 3 4 biocompatible implant for continuous release of a neurotoxin A method for making a stabilized active protein agent over a treatment period extending from one month to five composition, said method comprising the steps of: years. While such an implantable system may be useful for i.admixing sphingosine and cerebroside; certain situations, such as for the treatment of migraine, this ii. dissolving the admixture of step: type of implant system is not feasible for the treatment of 5 1. in alcohol; facial, neck or hand wrinkles. iii. removing the alcohol; United States Patent Application No. 2004/0247623 Sug iv. adding an aqueous solution of the active protein agent to gests a method for the treatment of sensory neuron related form an active agent composition; and distorters through transdermal application of a neurotoxin. V. admixing the active agent composition with a solution of This application is particularly directed to a method of treat 10 Solubilized collagen and low molecular weight elastinto ing migraine. The application suggests that botulinum toxin form a stabilized protein composition. can be administered transdermally through a variety of ways. In a preferred embodiment of the method, the sphingosine For example, the toxin may be incorporated into a transder and cerebroside are admixed in equal amounts. mal patch or it may be administered through electrophoresis. In another preferred embodiment, the alcohol is ethanol The application also suggests that botulinum toxin can be 15 and it is preferably removed by vacuum evaporation. administered using a topical cream. They teach that this In a further preferred embodiment, the active agent com would be achieved by reconstituting botulinum toxin with position comprises micelles. normal saline and then mixing the reconstituted toxin with a In yet another embodiment, the solution of solubilized Suitable cream or base and then massaging it onto the affected collagen and elastin comprises equal amounts of collagen and area. This type of application is unlikely to have much effect elastin. since the reconstituted botulinum toxin will have a very short In a preferred embodiment, the active protein agent is active life. botulinum toxin, preferably botulinum toxin Type A, more International Patent Application WOO158472 describes a preferably botulinum toxin in saline. pharmaceutical composition comprising botulinum toxin and In a further preferred embodiment, a method for preparing a polysaccharide. This application teaches that the polysac 25 cosmetic orpharmaceutical formulation is provided. Accord charide stabilizes the neurotoxin. However, other studies, as ing to the method, the stabilized composition is combined discussed above, have shown that Sacharrides are with other ingredients to form a cream, lotion, gel, ointment poor stabilizers for botulinum toxin. or other formulation that is acceptable for topical administra International Patent Application WO 04/060384 discloses tion. a pharmaceutical botulinum toxin composition which 30 In another aspect of the invention, a composition for trans includes a sequestration agent. The purpose of the sequestra dermal delivery of an active component is provided. The tion agent is to prevent the diffusion of the botulinum toxin composition comprises the active ingredient together with away from the site of injection. This does not address the need collagen, elastin and cerebroside. In one preferred embodi for stable compositions that can be applied to the surface of ment, the active component is botulinum toxin. The botuli the skin. 35 num toxin may be botulinum toxin Type A, Type B, Type C, There is no doubt that BOTOXOR) Cosmetic can smooth out Type D, Type E. Type For Type G or combinations thereof. In fine lines and wrinkles for most users. However, there are a preferred embodiment, botulinum toxin Type A is used. several disadvantages associated with its use. The BOTOXOR) In a preferred embodiment, the composition comprises Cosmetic must be administered in a doctors office. The injec about 1 to 40 wit/wt % of collagen, about 1 to 40 wit/wt % of tions can be painful and there may be bruising. Adverse side 40 elastin, about 0.1 to 15% sphingosine and about 0.1 to 15% effects occur in some injection treated patients. Most com cerebroside. mon side effects for treatment of frown line include droopy In a further preferred embodiment, the composition com eyelids, nausea, flu-like symptoms (fever etc.), headache and prises an agent which enhances the penetration of the botuli respiratory infections. Less frequent reactions may include num toxin through the dermal layer. This enhancing agent is 45 selected from the group consisting of d-limonene, allantoin, facial pain, redness at the injection site, and muscle weakness fulvic acid, myrrh, hydroquinone glycuin, quillaja Saponaria at other sites. Repeated treatments may lead to permanent (QTS), and acanthophyilum squarrusom (ATS). paralysis of facial muscles leaving the face expressionless. The compositions of the present invention are preferably Thus, there was a need for newer methods for stabilization formulated into a cream, lotion, gel, ointment or the like for of botulinum toxin. The present invention addresses that 50 topical administration. need. There was a further need for an alternative method of In a further aspect of the invention, a method of treating administering botulinum toxin that does not involve injec skin is provided. The method comprises applying the com tions. position described above daily to areas of the skin which are wrinkled or damaged. SUMMARY OF THE INVENTION 55 The present invention has many advantages over tradi tional injections with BOTOXR) Cosmetic. The composition The present invention addresses the need for improved of the present invention can be safely used in areas Such as the compositions by providing a method for stabilizing large throat and neck, around the mouth, near the eyes and on the protein active ingredients for storage at room temperature. hands. Another advantage of the present invention is that the The invention also provides a composition prepared using the 60 composition can beformulated as a cream or lotion and it can stabilizing method for the transdermal delivery of the active be stored at room temperature for extended periods of time component. One preferred active component according to the without any loss of activity of the active ingredient. Another present invention is botulinum toxin (MW 150 kD). advantage is that the stabilizing method of the present inven In one aspect of the invention, a method of stabilizing an tion eliminates the need for the use of human serum albumin active ingredient in a topical composition is provided. The 65 as a stabilizing agent. Since human serum albumin is a blood method comprises admixing the active ingredient with sph derived product, the elimination of this from the product ingosine, cerebroside, collagen and elastin. reduces the risk of blood borne diseases being transmitted US 7,838,011 B2 5 6 through an injection. Furthermore, because the stabilizing lagen, or collagen. The primary stabilizer can be a synthetic method keeps the protein stable and reduces the incidence of agent that does not induce an immune response or induces an toxoid formation, there is less likelihood of a patient devel attenuated immune response in a Subject. Additional stabiliz oping antibodies to the botulinum toxin. In addition, the ers may also be included in a pharmaceutical composition. methods and compositions of the present invention are cost These additional or secondary stabilizers may be used alone effective and simple to use. For example, the topical compo or in combination with primary stabilizers, such as proteins sition can be used at home without any costly doctor office and polysaccharides. Exemplary secondary stabilizers visits. It also eliminates the need for painful injections. Rather include, but are not limited to non-oxidizing amino acid than one single large dose of botulinum toxin being delivered derivatives (such as a tryptophan derivate, such as Nacetyl once to a single site, the methods and compositions of the 10 tryptophan (NAT)), caprylate (i.e. sodium caprylate), a present invention allow the botulinum toxin to be adminis polysorbate (i.e. P80), amino acids, and divalent metal cat tered at low dose, daily, to provide an effective treatment with ions such as zinc. A pharmaceutical composition can also enhanced safety and reduced side effects. include preservative agents such as benzyl alcohol, benzoic The methods and compositions of the invention have been acid, phenol, parabens and Sorbic acid or a cresol. Such as an shown to reduce fine lines and wrinkles, increase the moisture 15 M-cresol. level of the skin, increase skin elasticity and resilience, As used herein, the term "enhancing agent” refers to an increase the firmness of the skin, improve skin tone, texture agent that promotes the absorption of an active protein agent and overall radiance, diminish bags under the eyes, rejuve by the skin. Examples of enhancing agents include, but are nate the skin, prevent damage from chemical stress, protect not limited to, alcohols, such as short chain alcohols, long the skin from UV rays and free-radical damage, and remove chain alcohols, or polyalcohols, amines and amides. Such as irregular pigmentation. urea, amino adds or their esters, amides, AZONE(R)), deriva tives of AZONE(R), pyrrolidones, or derivatives of pyrroli BRIEF DESCRIPTION OF THE DRAWINGS dones; terpenes and derivatives of terpenes; fatty acids and their esters; macrocyclic compounds; tensides; or Sulfoxides These and other features of the invention will become more 25 Such as, decylmethylsulfoxide. Liposomes; transfersomes; apparent from the following description in which reference is lecithin vesicles; ethosomes; water, Surfactants, such as made to the appended drawings wherein: anionic, cationic, and nonionic Surfactants; polyols; and FIG. 1 Illustrates an SDS polyacrylamide gel analysis: essential oils can also function as enhancing agents. FIGS. 2A and 2B illustrates representative Western Blot As used herein, the term “topical' is used to refer to admin analyses of the composition of the invention; and 30 istration to the skin. The term “transdermal' is used to refer to FIGS. 3A to 3D are photographs of patients treated with the penetration through the other layer or stratum corneum of the composition of the present invention. skin. The term “transdermal delivery” is meant to refer to delivery of an active agent to the muscles underlying the skin. DETAILED DESCRIPTION It is not meant to refer to systemic delivery to the circulatory 35 system. Large proteins can be difficult to stabilize in formulations In the methods and compositions of the present invention, for storage at room temperatures. Therapeutic proteins are collagen is used as a stabilizer. In addition to stabilizing often stored lyophilized and reconstituted before use. Alter botulinum toxin according to the invention, collagen helps natively, the protein composition may be kept frozen prior to improve the tissue’s underlying foundation and contributes to use. The present invention provides methods for stabilizing 40 hydration. Collagen is able to penetrate the skin without the large proteins, as well as therapeutic or cosmetic composi aid of any penetration enhancers. tions containing proteins as active ingredients. The stabilized In a preferred embodiment of the invention, elastin is also compositions of the invention can be stored for extended included. Elastin is a protein that coil and recoils like a spring periods of time at room temperature. within the elastic fibers of connective tissue and accounts for Botulinum toxin is a relatively large protein for incorpora 45 the elasticity of structures such the skin, blood vessels, heart, tion into a pharmaceutical or cosmetic formulation. The lungs, intestines, tendons, and ligaments. Elastin functions in molecular weight of botulinum toxin type A complex is 150 connective tissue together with collagen. Whereas elastin kD and due to the large size of the molecule it is fairly fragile provides elasticity, collagen provides rigidity to connective and labile. A stabilizer component should be able to interact tissue. Low-molecular weight elastin is the only elastin com with the toxin in a manner which does not denature, fragment 50 plex capable of penetrating the skin without the aid of any or otherwise detoxify the toxin molecule or cause disassocia penetration enhancers. Collagen and low molecular weight tion of the toxin complex. soluble elastin complex in conjunction with Botulinum Toxin The present invention provides novel stabilization methods A help diminish the appearance of wrinkles. and products by using collagen in combination with periph The compositions and methods of the present invention eral low molecular weight soluble elastin. Sphingosine and 55 also include a sphingosine. Sphingolipid or sphingosine-1- cerebroside are additional stabilizing components. Botuli phosphate has been recognized as a bioactive molecule num toxin formulated according to the present invention, involved in the regulation of cell growth, differentiation, sur remained stable and was not denatured or fragmented or vival, and chemotaxis as well as angiogenesis and embryo detoxified in the formulation at room temperature for period genesis. Researchers have identified sphingosine-1-phos of 6 months (as assessed from the stability data of the 6 60 phate as a potent chemoattractant for keratinocytes as well as months and clinical data). an activator of extracellular matrix production by fibroblasts. As used herein, the term “protein stabilizer” or “primary The other species of Ceramides or sphingolipids are (N-acyl stabilizer” refers to an agent that helps to preserve or maintain sphingosine) and dihydroceramide (N-acyl sphinganine). the biological structure (i.e. the three dimensional conforma Galactosylceramide (cerebroside), a metabolite of sphin tion) and/or the biological activity of a protein. Stabilizers can 65 golipids is also included. Cerebroside is a myelin related be proteins or polysaccharides. Examples of protein stabiliz protein that plays an important role in the regulation of cell ers include hydroxyethyl starch (hetastarch), gelatin, col growth, differentiation, survival, and chemotaxis. Cerebro US 7,838,011 B2 7 8 side Sulfates are important membrane constituents. Cerebro stimulates the penetration of collagen. The FDA OTC Panel side is a mild basic or neutral protein. has recognized Allantoin as a skin protectant and a mild The stabilizing agents used in the present invention, col neutral permeation enhancer. lagen in combination with peripheral low molecular weight In another preferred embodiment, fulvic acid is included in soluble elastin (EBP) and a mixture of sphingosine and cere- 5 the preparation. Fulvic acid is a low molecular weight anti broside are non toxic and easy to handle. They provide an oxidant. It enhances the body’s absorption of drugs through effective alternative to albumin and still meet the stringent the transdermal route without any side effects. botulinum toxin containing pharmaceutical composition for Myrrh is a further optional ingredient in the skin cream mulation requirements. preparation of the invention. Myrrh is a gum resin extracted According to one method of the invention, a stabilized 10 from Arabian and Somolian shrubs that helps soothe inflamed protein composition is made by combining collagen and low tissues and rejuvenate skin, hair, and nails. molecular weight elastin in a solvent, such as Saline. In a Eldopaque or HYDROQUINONE Glycuin may also be separate flask, sphingosine and cerebroside are dissolved in included. It is used to lighten the dark colored patches of skin an alcohol, preferably ethanol. The alcohol is then removed, (also called melama, liver spot, age spots, freckles) caused by for example by rotary vacuum evaporation. This results in a 15 birth control pills, hormone medicine, injury to the skin, or coating of sphingosine and cerebroside on the flask. A solu simply aging. It may be included together with Sunscreen to tion containing the protein to be stabilized is added to the help prevent these spots from reoccurring due to Sunlight or flask. This preferably results in the formation of micelles of UV light exposure. sphingosine, cerebroside and protein. This micellar compo Quillaja Saponaria (QTS) and Acanthophyllum squarru sition is added to the mixture of collagen and elastin and 20 Som (ATS) total saponins are two natural enhancers that may stirred. This stabilized composition can then be stored at also be included. They demonstrate moderate activity as skin room temperature. Further details of an exemplary composi penetration enhancers. These compounds may also be tion can be found in Example 1 below. The method of the included in a skin cream or lotion of the present invention. present invention was used to stabilize the protein, botulinum The enhancing agents may be included at various stages of toxin. FIGS. 1 and 2 illustrate SDS-PAGE and Western blots 25 the process. For example, they may be added to the stabilized of compositions of stabilized botulinum toxin. composition at the same time as the micellar composition is Botulinum toxin type A consists of a toxin molecule of added to the collagen and elastin mixture. Preferably, the about 150 kD in noncovalent association with nontoxin pro enhancing compounds are introduced during the formulation teins weighing about 750 kD. The nontoxin proteins are of the stabilized composition into a pharmaceutical or cos believed to preserve or help stabilize the secondary and ter- 30 metic formulation. The preparation of an exemplary formu tiary structures upon which toxicity is dependant. The stabi lation for topical application is described in Example 3 below. lization technique of the present invention based on the use of The present invention provides a composition that is collagen in combination with elastin-binding protein (EBP) capable of delivering a botulinum toxin through a person’s and mixture of sphingosine and cerebroside maintains the skin. The composition contains an enhancing agent that integrity of the complex without denaturing or fragmentation 35 facilitates the permeation of the botulinum toxin through the or detoxification. Thus, the invention provides a novel patient’s skin. The composition is suitable for topical admin method for stabilizing botulinum toxin. istration whereby the composition penetrates the skin and A further advantage is that proteins like collagen, elastin transdermally denervates an underlying muscle. The compo and sphingomyelins are non-immunogenic. sition may be provided on a patch that is adhesively secured to In addition to having enhanced stability, the compositions 40 the skin so that the toxin can pass from the patch through the of the present invention may include components that skin to denervate an, underlying muscle. Compositions con enhance absorption through the skin. Thus, the compositions taining botulinum toxin and an enhancing agent can be used can beformulated into a topical composition Such as a cream, to successfully treat several types of disorders associated with lotion, gel, ointment, spray or mask. As a cream, lotion or gel. neurotransmitter release when applied to a person's skin. and by way of example only, about 0.5 ml to about 20 ml or 45 Examples of disorders amenable to treatment by the topical more may be used on a human Subject's face, depending on administration of the compositions set forth here in include, the area to be treated and at least to some extent, the severity and are not limited to, wrinkles, such as brow furrows, head of the affliction to be treated. The composition may also be aches, such as migraine, headache pain, cervical dystonia, included in make-up formulations such as foundations or lip focal hand dystonia, neurogenic inflammation, hyperhydro balm. 50 sis, blepharospasm, strabismus, hemifacial spasm, eyelid dis The following may be included to enhance absorption/ order, cerebral palsy, focal spasticity, limb spasticity, tics, penetration of botulinum toxin through the thick dermal lay tremors, bruxism, anal fissure, fibromyalgia, dysphagia, lac ers. These agents are approved by the FDA for human use and rimation, and pain from muscle spasms. The compositions are on the GRAS (generally recognized as safe) list of the disclosed herein provide localized relief via delivery of botu FDA. These agents do not interact chemically with botulinum 55 linum toxin. toxin. These additives not only enhance absorption botulinum The compositions and formulations of the invention are toxin through the skin layers but they also aid in the stability useful to reduce the signs of aging. Clinical trials were con preservation because of their neutral nature and antioxidant ducted using a topical cream formulation containing botuli properties. num toxin. Details of the trial can be found in Example 4 In one aspect of the invention d-limonene is included in the 60 below. As can be clearly seen from the before and after pho composition to enhance penetration of the toxin through the tographs in FIG. 3, topical administration of stabilized botu dermal layer. Limonene was found to be an effective enhancer linum toxin cream significantly reduces the signs of aging. at 0.30%, enhancing skin permeation of botulinum toxinType In another aspect of the invention, a pharmaceutical or A approximately fourfold. cosmetic formulation is provided containing an effective In one preferred embodiment of the invention, allantoin is 65 amount of an active agent and an effective amount of the included in the preparation. Allantoin is a Substance which stabilizing enhancing composition, and a pharmaceutically triggers new cell formation, shrinks damaged tissues and acceptable carrier Suitable for topical or transdermal admin US 7,838,011 B2 9 10 istration. The formulation may be in any form suitable for The above disclosure generally describes the present application to the skin. For example, it may take the form of invention. A more complete understanding can be obtained by a cream, a lotion, agel, an ointment, a paste, or a solution. The reference to the following specific Examples. These formulation may include lipoZone, micelles, or microspheres. Examples are described solely for purposes of illustration and The formulation may be: a cosmetic composition that are not intended to limit the scope of the invention. Changes includes in addition to the stabilizers and the active ingredi in form and Substitution of equivalents are contemplated as ents water and other additives that are normally used in cos circumstances may suggest or render expedient. Although metics. For example, it may include thickening agents, pre specific terms have been employed herein, such terms are servatives, emulsifiers, perfumes, dyes or coloring, vegetable intended in a descriptive sense and not for purposes of limi or mineral oil, antiseptic agents, acidifying or alkalizing 10 tation. agents, vitamins, anti-UV agents, Surfactant, Solvents, pH stabilizing agents, and other active ingredients known to be EXAMPLES effective on the skin. The cosmetic composition may be pro vided as a milk, cream, lotion, serum, mask or gel. The cos The examples are described for the purposes of illustration metic composition may also be provided as skin foundation, 15 and are not intended to limit the scope of the invention. lip balm, etc. While the description has focused on transdermal delivery Example 1 of botulinum toxin, it is clearly apparent that the stabilizing and enhancing composition can be used to deliver other active Preparations agents transdermally. Examples of active agents that can be delivered in the way include, but are not limited to androgens, Botulinum toxin (BOTOXOR) Cosmetic) vials were recon androstenediol and androisoxazole (for anabolic disorders), stituted of sterile saline solution (0.9%). The vials were testosterone (hypogonadism, muscle wasting, male impo shaken vigorously to dissolve the botulinum toxin (BOTOXOR) tence, postmenopausal symptoms in women), dihydrotest Cosmetic). The reconstituted vials were kept refrigerated and osterone (hypogonadism, muscle wasting), dehydroepi 25 were utilized within 1 hour of reconstitution. androstenone (muscle wasting, fat reduction, fitness); Inaround bottom flask of 50 mL capacity, 10 mg of soluble estrogens (postmenopausal symptoms, birth control), 17 collagen and 10 mg of Elastin were combined. The mixture betaestradiol, estradiol-3,17-diacetate, estradiol-3-acetate, was solubilized in 10 mL of sterile saline solution (0.9%) with estradiol-17-acetate, estradiol-3,17-valerate, estradiol-3-val continuous stirring. In a separate 50 mL round bottom flask, erate, estradiol-17-valerate, ethinyl estradiol, estrone: 30 5 mg of sphingosine and 5 mg cerebroside were combined. progesterones (prevent endometriosis, prevent endometrial This mixture was dissolved in pure ethanol. The alcohol was cancer, control habitual abortion, suppress or synchronize completely removed by rotary vacuum evaporation to obtain estrus, promote hair growth), progesterone (preg-4-ene-320 a uniform coating of the Sphingosine and cerebroside on the dione), norethindrone, norgestrieone, norgestadienone, norg flask wall. To this flask 800 units of botulinum toxin solution estrel, norgestimate, progestogenic acid, dihydroprogesterol, 35 in 6 ml of (0.9%) saline was added. The flask was swirled and nomagesterol. The testosterone hormone may be used in any then stirred continuously for 5 mins at room temperature to of its usual forms, such as, acetate, propionate, 17-beta-cy uniformly coat the botulinum toxin with the sphingosine and clopentanepropionate, enanthanate, isobutyrate, undeconate, cerebroside micelle coating to prevent degradation. This and the like. Similarly, the estradiols may additionally be used coated and preserved micellar botulinum toxin Solution was in any of the known or newly developed forms, such as, for 40 then added to the flask containing the mixture of collagen and example, pivalate, propionate, cypionate, benzoate and other cross linked low molecular weight elastin. The solution was esters. Other drugs such as insulin, insulin like growth factors, stirred for about 5 mins slowly and then kept at a room vaccines, peptides like GLP, IGF, heparin, hirugen, hirulos, temperature in a brown glass vial. huridine, mumps, measles and rubella Vaccine, typhoid vac cine, hepatitis A vaccine, hepatitis B vaccine, herpes simplex 45 Example 2 virus, bacterial toxoids, cholera toxin B-subunit, influenza vaccine virus, bordetala pertussis, vaccinia virus, adenovirus, SDS-PAGE and Western Blot Analysis canary pox, polio vaccine virus, plasmodium falciparum, bacillus calmette geurin (BCG), klebsiella pneumoniae, HIV The stability of the preserved botulinum toxin (Botox) envelop glycoproteins, bovine Somatropine, estrogens, 50 Solution was analyzed by Standard analytical techniques androgens, insulin growth factors, interleukin-I, interleukin using SDS-PAGE and Western Blot analysis and HPLC II and cytokins, Small molecule drugs such as NSAJD, nar analysis at time Zero (few minutes after the preparation of the cotics, etc. may be delivered using the compositions of the coated stabilized solution) and thereafter every month in present invention. comparison with the uncoated Botox solution. Botulinum Toxin A can be stabilized using the methods of 55 SDS-PAGE was performed in all cases under reducing present invention and the stabilized toxin can be successfully conditions with a BIO-RADR) mini-cell apparatus (Bio-Rad delivered transdermally to achieve similar results to those Laboratories, Calif.) with 10% precast tricine gels. The botu obtained by intramuscular injection of BOTOXR) Cosmetic. linum toxin neat (pure sample diluted with Saline) was loaded The formulation can be applied all over the face and neck and in one lane as the comparator to the stabilized botulinum toxin hands as opposed to a BOTOXR Cosmetic injection which is 60 which was loaded in the second lane. Both columns were targeted primarily to areas around eyes and the forehead to loaded with approximately 100 units of the toxin. The loading reduce the wrinkle. buffer, tank buffer, and the molecular weight markers were The present invention has been described with regard to obtained from Bio-Rad Laboratories. The proteinbands were one or more embodiments. However, it will be apparent to visualized by the Coomassie blue staining technique. Protein persons skilled in the art that a number of variations and 65 concentration (density) from SDS polyacrylamide gels was modifications can be made without departing from the scope measured with a BIO-RAD(R) densitometry system and ana of the invention as defined in the claims. lyzed with the standard imaging software analyzer. The com US 7,838,011 B2 11 12 parison of the Botulinum Toxin Abands, i.e. the neat botuli num toxin without stabilization and the stabilized botulinum -continued toxin revealed no degradation in the stabilized botulinum toxin which was kept at a room temperature for 6 months. The Total Volume of the cream (400 mL) results are shown in FIG. 1. Phase D: Western blot analysis For Western blot analysis, proteins d-limonene O.7% separated on SDS-polyacrylamide gels were transferred to Allantoin O.S90 the nitrocellulose membranes at 25 V for 60 mm with a Fulvic Acid O.S90 BIO-RADR) protein blot module. After the complete transfer Puillaia Saponaria (QTS) O.3% of the protein from the gel, the protein binding sites on the 10 Acanthophyllum squaimson (ATS) O.3% Myrrh Extract O.2% membranes were blocked by incubation at room temperature Hydroquinone Glycuin 4.0% in 5% skim milk-Tris-buffered saline (TBS) for 60 mm. Phase E: Membranes were then incubated at a room temperature with affinity-purified MAb produced against the botulinum toxin Stabilized Botulinum Toxin in Collagen Matrix 800 units (gift from the local University Biotechnology Dept) at 5 15 ug/ml in 5% skim milk-TBS for 4 hrs. After several washes with TBS buffer, the membranes were incubated at room Procedure: temperature with affinity-purified goat anti-mouse IgG Heat Phase A and Phase B separately with agitation to 75 (Sigma-Aldrich, Co.) at 4 mu.g/ml in 5% skim milk-TBS C. Add Phase A to Phase B and mix 30 minutes at 75 C. Cool buffer for 3 hrs. After another four to five washes with the down to 20-22 Cand then add Phase C, D and E and continue TBS buffer, the membranes were incubated with TMB 3.3', to agitate until homogenous and one phase. 5.5'-tetramethylbenzidine membrane substrate (Bio-Rad Laboratories) until color developed. This analysis revealed Example 4 that after 6 months time period the botulinum toxin at room temperature was stable and was not degraded and denatured 25 Clinical Studies in Human Subjects to loose its potency. The results of two exemplary Western Blots are shown in FIGS. 2A and 2B. The effectiveness of the stabilized botulinum toxin (Botox) was tested using a cream formulation. Human Subjects with Example 3 wrinkles applied the cream for a period of month or more. The 30 subjects were photographed before and after one month of the Cream Formulation application of the cream on their face. The clinical guidelines established by Allergan to test their The stabilized botulinum toxin composition was formu Botox injection was followed for the testing of the effective lated into a cream for topical administration as outlined ness of the stabilized formulation in healthy subjects (male below. 35 and female) age range from 40-75 years. Botulinum Toxin Type A was not present in the peripheral blood at measurable levels following the dermal application of the cream at the low doses of 1.7 units per mL of the cream. Total Volume of the cream (400 mL) Each cream vial contained 50 units of Botox in 30 ml of Phase A: 40 cream. The recommended quantities of neurotoxin adminis tered at each treatment (in the morning and at the bed time) De-ionized Water 74.7% Tetra Sodium EDTA O.S.-O.7% did not result in Systemic, adverse clinical effects, i.e. muscle Methyl Paraben O.2% weakness, in patients without other neuromuscular dysfunc Propylene Glycol 3.0%-40% tion. Glycerin 3.0%-40% 45 Phase B: Clinical Studies: Assessment of the Glabellar Lines A randomized, single-center, double blind, placebo-con Cetyl Alcohol (Ado 152NE) 2.0% Cetearyl Alcohol 2.0% trolled (placebo cream contained all the ingredients except Glyceryl Stearate 2.0% the Botox), parallel-group study was conducted to evaluate PEG-1 OO Stearate 1-2% 50 the effectiveness of the stabilized Botox cream of the inven Stearic Acid (Emersol 132) 4.5% tion for use in the temporary improvement of the appearance Sorbitan Palmitate O.S.-O.7% Polysorbate-85 1.0% of moderate to severe Glabellar facial lines. The study Polysorbate 60 O.S.-19. enrolled 40 healthy adult patients (ages 40 to 75 years, female Lanolin Alcohol (Ritachol) 1.0% or male) with Glabellar lines of at least moderate severity at HoHoba Oil O.S.-19. 55 Lanolin 1-2% maximum frown. Patients were excluded if they had an infec Tocopheryl Acetate O.S.-19. tion or skin problems, history of facial nerve palsy, marked Dimethicone 200 O.7-1.0% facial asymmetry, ptosis, excessive dermatochalasis, deep BHA O.1% dermal scarring, thick Sebaceous skin, inability to Substan Propylparaben O.1% tially lessenglabellar lines even by physically spreading them Diazolidinyl UREA O.2% Phase C: 60 apart or had a known history of neuromuscular disorder or other disorder that could interfere with neuromuscular func Fragrance (lilac, jasmine) as needed tion. Subjects were asked to apply the actual stabilized Botox Aloe Vera (powder) 1.5%-2.0% cream twice a day (once in the morning and at the bedtime) CoQ-10 O.S9/o Retinyl. A O.O3-0.05% for a month. The subjects were instructed to wash their face Hyaluronic Acid (pure) 1.0-1.5% 65 with warm water and Soap thoroughly to remove any Sweat, Talcum Powder (TiO2) 1.0-1.5% dirt, and oils from their faces. They were instructed apply the stabilized Botox cream or placebo cream around their eyes, US 7,838,011 B2 13 14 forehead, and neck or on the wrinkled skin areas with the their own appearance (+2 or better). This increased to 93% by Supplied applicator so as to avoid any contamination of the the primary efficacy endpoint day of Day 30, compared to 3% product by hand. All subjects were examined prior to the of placebo-treated patients. Based on resting appearance as beginning of the study by a qualified dermatologist and were judged by the investigator, 62% of subjects achieved a sever declared healthy and fit to enter in the study. They were 5 ity score of none or mild at Day 11, and 94% by the efficacy photographed by an expert photographer prior to the begin endpoint day of Day 30. ning of the study. All subjects were asked to follow their normal daily routine without any restriction and asked to live TABLE I their normal life style. The primary efficacy measurements were the investiga 10 Day Botox-Cream Placebo Difference p-Value tor's rating of glabellar line severity at maximum frown at 7 76% (15/20) 3% (1/20) 7396