NZ737403B2 - Method of treating or reducing efp - Google Patents

Abstract

Disclosed is the use of collagenase for the preparation of a medicament for treating or reducing edematous fibrosclerotic panniculopathy (EFP) (cellulite) in a patient, wherein said medicament is adapted for administration of a plurality of subdermal injections of collagenase comprising collagenase I and collagenase II to an area affected by EFP, wherein the dose of collagenase per injection is between about 5 to about 200 ABC units, and wherein the collagenase I and collagenase II each have a purity of at least about 95% by area as determined by reverse phase high performance liquid chromatography. A further embodiment also requires the total dose of collagenase for administration to the affected area to be between about 5 to about 5000 ABC units and the collagenase to have a specific activity of about 10,000 ABC units per 0.58mg. I and collagenase II to an area affected by EFP, wherein the dose of collagenase per injection is between about 5 to about 200 ABC units, and wherein the collagenase I and collagenase II each have a purity of at least about 95% by area as determined by reverse phase high performance liquid chromatography. A further embodiment also requires the total dose of collagenase for administration to the affected area to be between about 5 to about 5000 ABC units and the collagenase to have a specific activity of about 10,000 ABC units per 0.58mg.

Description

METHOD OF TREATING OR REDUCING EFP RELATED APPLICATION This application claims the benefit ofUS. Provisional Application No. 61/549,863 filed October 21, 2011. The entire contents of the above-referenced application are incorporated by reference .

BACKGROUND OF THE INVENTION Edematous fibrosclerotic panniculopathy (EFP), more commonly ed to as cellulite, is a condition that presents as a topographical tion in the appearance of the skin and affects about 90% of postpubertal women (Rawlings (2006), Int J Cosmetic Sci 2006; 28: 175-90; Khan et al. (2010), JAm Acad Dermatol 2010; 62: 361-70). EFP appears as dimpled skin and gives the skin what is commonly described as an orange-peel ance. The dermal septae, composed of collagenase, are ed to play a causative role in the dimpling of the skin.

Collagenase, an enzyme that has the specific ability to digest collagen, has been used to treat a variety of collagen-mediated diseases, including, for example, Dupuytren’s cture, Peyronie’s disease, lipoma and adhesive capsulitis. US. Patent No. 4,645,668 and US. Patent App. Pub. No. 20070224184 also disclose certain uses of collagenase. A major source of enase is from the fermentation of the bacterium, Clostrz'dz'um histolyticum. An injectable formulation comprising C. histolytz'cum collagenase I and collagenase II is sold under the trade name XIAFLEX® and is approved by the US. Food and Drug Administration (FDA) for the ent of Dupuytren’s contracture.

W0 2013/059619 PCT/U82012/061063 _ 2 _ The present invention is directed to a method for the treatment or ion of EFP comprising one or a ity of injections of collagenase to an area affected by EFP.

SUMMARY OF THE INVENTION The t invention is based on the discovery that one or more low dose injections of collagenase to an area affected by EFP are effective to reduce or treat such EFP. The amount of collagenase in a single injection may be as low as 5 ABC units, or 0.00029 mg (0.29 ug). The total dose of enase administered depends on the size of the treatment area, and is thus between about 5 to about 5000 ABC units. The concentration of collagenase is n about 50 to about 2000 ABC units/milliliter (ml). 10,000 ABC units is equivalent to 0.58 mg collagenase.

In one embodiment, the invention is directed to a method of treating or reducing EFP in a patient sing administering to said patient one or a ity of subdermal injections of collagenase to an area affected by EFP, n the dose of collagenase per injection is between about 5 to about 200 ABC units, and wherein the plurality of subdermal injections are administered at a plurality of injection sites. In certain aspects, a plurality of subdermal ions are administered at a plurality of injection sites. In additional embodiments, the tration of collagenase stered is between about 50 to about 2000 ABC units per milliliter. In certain additional embodiments, each injection of collagenase is administered in a volume of about 0.5 ml or less. In a further aspect, the total dose of collagenase administered is between about 5 to about 2000 ABC units.

In another embodiment, the invention is directed to a method of treating or reducing EFP in a patient comprising administering to said patient one or a plurality of subdermal injections of collagenase within an area affected by EFP, wherein the total dose of collagenase administered to the affected area is between about 5 to about 5000 ABC units, and wherein the plurality of injections are administered at a plurality of injection sites. In certain aspects, each injection is between about 5 to about 200 ABC units. In some embodiments, the concentration of enase administered is between about 50 to about 2000 ABC units per milliliter and/or each injection of collagenase is administered in a volume of about 0.5 ml or less.

W0 2013/059619 PCT/U82012/061063 _ 3 _ In yet another embodiment, the ion is a method of treating or reducing EFP in a patient comprising administering to said patient one or a plurality of subdermal injections of collagenase to an area affected by EFP, wherein the dose of collagenase administered per ion to the affected area is between about 5 to about 200 ABC units and n the concentration of collagenase administered to the affected area is between about 50 to about 2000 ABC units per milliliter.

In a fiarther aspect, the invention is directed to a method of treating or reducing EFP in a patient sing administering one or a plurality of subdermal injections of enase to an area affected by EFP wherein the concentration of collagenase administered to the affected area is between about 50 to about 2000 ABC units per iter and wherein the volume of each injection of collagenase is about 0.5 ml or less.

In some embodiments, the dose of collagenase administered per injection is between about 5 to about 100 ABC units. In additional embodiments, the dose of enase administered per ion is between about 5 to about 50 ABC units.

The collagenase can be derived from a bacterial source or can be a recombinant form of collagenase. In some embodiments, the collagenase is purified from Clostridium histolytz'cum. In an additional embodiment, the collagenase comprises collagenase I and collagenase II. In yet fiarther embodiments, the collagenase is collagenase I and collagenase II purified from Clostridium histolytz'cum and comprises collagenase I and collagenase II.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other s, es and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts hout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. is a depiction of the ion template where each dot within the octagon represents an injection site. The X within the circle represents the ion site to the “central dimple.” The four comers are labeled as upper right (UR), lower right (LR), upper left (UL), and lower left (LL). is a depiction of buttock and posterior upper thigh with template corner gs. A = Midline of the upper posterior thigh and buttock (from center of the eal fossa); C = Template ; E = Template corner.

DETAILED DESCRIPTION OF THE INVENTION A description of preferred embodiments of the invention follows.

As discussed above, the present invention is based on the discovery that low dose injections of collagenase to an area affected by EFP are effective to lyse collagen and thereby reduce or treat such EFP. The amount of enase in a single injection may be as low as 5 ABC units, or 0.00029 mg. The total dose of collagenase administered depends on the size of the treatment area, and is thus between about 5 to about 5000 ABC units and/or wherein the concentration of collagenase is between about 50 to about 2000 ABC units/milliliter (ml).

The doses and concentrations of collagenase employed according to the method of the t invention are substantially lower than the doses and concentrations of collagenase currently used and approved by the U.S. FDA for the treatment of Dupuytren’s contracture.

The words “a” or “an” are meant to encompass one or more, unless otherwise specified.

“Treating” or “treatment” of EFP includes the administration of the itions or agents described herein to improve the appearance of the skin previously affected by EFP and/or to achieve an improved aesthetic outcome. Treatment of EFP can, for example, encompass a visual reduction in the severity of EFP or a visual reduction in the severity or number of skin dimples.

The invention asses a method of ng or reducing EFP in a patient comprising stering one or a plurality subdermal injections of collagenase to an area affected by EFP, wherein the plurality of injections are injected at a plurality of injections sites within an area ed by EFP, wherein the dose of collagenase administered per injection is about 5 to about 200 ABC units. In additional embodiments, the dose of collagenase per injection is about 5 to about 100 ABC units, or about 5 to about 50 ABC units, or about 10 to about 100 ABC units, or about 10 to about 50 ABC units. In additional embodiments, the total dose of collagenase administered to the affected area is between about 5 to about 5000 ABC units. The invention also asses a method comprising administering collagenase to the area affected by EFP wherein the concentration of collagenase administered to the affected area is about 50 to about 2000 ABC units/ml. The _ 5 _ invention additionally encompasses a method of treating or reducing EFP in a patient sing administering a plurality of injections of enase to an area affected by EFP, wherein the dose of collagenase per ion is between about 5 to about 200 ABC units.

An area affected by EFP (also referred to herein as the “target area” or “treatment area”) is typically an area of the skin on the thighs and/or ks characterized by one or more dimples. In some embodiments, the area of EFP treated with the one or plurality of injections of collagenase is an area of the lateral upper aspect of the thigh or an area of the buttocks and does not involve the gluteal fold. The target area to be treated with collagenase can also be an area having a photonumeric EFP severity scale (CSS) score of Z 10 representing moderate to severe EFP severity within the right or left k or the right or left thigh (Hexsel et al. (2009). A validated photonumeric EFP severity scale. JEADV2009; 23:523-52; the ts of which are expressly incorporated by reference herein). The CSS is a photonumeric scale that looks at five key morphologic features of EFP: (A) number of depressions, (B) depth of depressions, (C) logical appearance of skin surface, (D) laxity, flaccidity or sagging of skin, and (E) the classification scale originally described by Niimberger and Muller, JDermatol Surg Oncol 1978; 4(3): 221-29.) (Hexsel et al., 2009).

Each of these features is ted on a 4-point scale from a low of 0 to a high of 3. The scale ranges from 0 to 15.

The target area treated with the one or plurality of injections of EFP is generally an area of the skin characterized by one or more dimples that are visually evident when the patient is standing. The geometric area of the treatment area s on the size of the area ed by EFP. Thus, for example, if the area affected by EFP includes one dimple, the ric area of the target area can be about 1 cm2 to about 5 cm2. In onal aspects, the area affected by EFP comprises multiple dimples and has a geometric area greater than 1 cm2. In one embodiment, the target area has a geometric area between about 1 cm2 to about 200 cm2. As will be understood, because the target area is generally an area of the thigh or buttocks, the area can, for example, be roughly rectangular in shape and have a length of about 1 to about 15 cm, and a width of about 1 to about 10 cm. In an additional example, the target area has a length of about 6 to about 15 cm and a width of about 4 to about 10 cm. The person of skill in the art will also understand that the target area can be roughly circular or any other geometric shape depending on the desired area for treatment. The target area can optionally be characterized by at least one skin dimple approximately at the center of the area W0 2013/059619 PCT/U82012/061063 _ 6 _ of EFP treated, wherein the area of EFP is treated with the one or a plurality of ions.

The sites for injection of collagenase can be numbered and spaced within the area affected by EFP such as to allow for even distribution and efficacy of the ed collagenase. The number of collagenase injections will generally depend on the size of the area to be treated.

In some embodiments, the number of injection sites is at least 1 or more. In other aspects, the number of injection sites is at least about 3 or more. In other aspects, the number of injection sites is at least about 5 or more. In yet other aspects, the number of injection sites is at least about 7 or more. In a r aspect, the number of injection sites is at least 10 or more.

In one embodiment, in which the area to be treated is a single dimple area or otherwise haVing a geometric area of less than about 5 cm2, there is only a single site of injection, and between about 5 and about 200 ABC units of collagenase is injected.

Optionally, the single injection can be ed into the center of the dimple. In embodiments in which the treatment area is larger, there will be multiple sites of ion and between about 5 to about 200 ABC units of collagenase is administered per injection. Optionally, the plurality of injections include at least one injection administered to the center of one dimple.

The sites of injection can, for example, be about 1 to about 4 cm from one another. In yet other s, the sites of injection can be about 2 to about 3 cm from one another. For example, in certain embodiments, the area affected by EFP has a width about 8 cm and a length of about 10 cm in dimensions and the number of collagenase injections stered is 10 and the distance between injection sites is about 2.5 cm. In certain s, an injection template (for example, made of a thin clear material) which includes markings showing the intended injection sites is placed over the target area and the one or more ions are made at the sites indicated by the template.

The target area is d with one or a plurality of concurrent injections of collagenase. As used herein, concurrent injections are injections administered at the same time or sequentially within the same period of time, i.e., during a single treatment session.

As discussed above, the total dose of collagenase administered to the area affected by EFP is between about 5 to about 5000 ABC units of collagenase. The total dose of collagenase administered depends on the size of the treatment area. The total dose of collagenase is the sum of the doses administered using one or a plurality of ions of collagenase. In certain aspects, each injection of collagenase will comprise equal doses of collagenase. For e, when the number of injections is 10, to be distributed over the treatment area, and each _ 7 _ injection is 200 ABC units of collagenase, the total dose of collagenase to be administered to the target area will be 2000 ABC units. By way of further example, when the number of injections is 10 to be buted over the treatment area, and each injection is 5 ABC units of collagenase, the total dose of collagenase to be administered to the target area will be 50 ABC units. By way of further example, when the number of injections is 5 to be distributed over the treatment area, and, each injection is 5 ABC units of collagenase, the total dose of collagenase to be administered to the target area will be 25 ABC units. When only a single injection is to be performed and the dose selected is 5 ABC units, the total dose of collagenase administered will be 5 ABC units. The concentration of collagenase administered to the area affected by collagenase is between about 50 ABC units/ml to about 2000 ABC units/ml. Collagenase can be administered in a volume per injection of about 0.5 ml or less. In r , collagenase can be administered in a volume per injection of about 0.1 ml to about 0.5 ml. The total volume of collagenase injected can, for example, be between about 0.1 ml (when only a single injection site is used) to about 7 ml (for multiple sites of injections), and higher for larger size treatment areas. In one embodiment where the treatment area is about 80 cm2, the total volume injected is about 1 to about 5 ml, summed from 10 injections of between 0.1 ml and 0.5 ml.

Collagenase is an enzyme that has the ic ability to digest collagen. One commercial source of collagenase is from fermentation by Clostrz'dz'um histolytz'cum. In certain aspects, the collagenase ses a combination of purified Clostrz'dz'um histolytz'cum collagenase I and collagenase II. Preferably, the collagenase I and collagenase II are t in a mass ratio of about 1 to l. Collagenase AUX I has a single polypeptide chain consisting of approximately 1000 amino acids with a lar weight of l 15 kDa. Collagenase AUX II also has a single polypeptide chain consisting of about 1000 amino acids with a molecular weight of 110 kDa. Crude enase obtained from C. histolytz'cum can be purified by a y of methods known to those skilled in the art, including, for example, heparin affinity chromatography, um sulfate precipitation, hydroxylapatite chromatography, size exclusion chromatography, ion exchange chromatography, and metal chelation chromatography. Methods of purification of crude collagenase ed from C. histolytz'cum are also described in US. Pat. No. 7,811,560, the contents of which are sly incorporated herein by reference herein. As discussed above, an inj ectable formulation comprising C. histolytz'cum collagenase I and collagenase II is sold in the US. under the trade W0 2013/059619 PCT/U82012/061063 _ 8 _ name XIAFLEX® and is approved by the US. FDA for the treatment of Dupuytren’s contracture. The enase can, for example, be a parenteral lyophilized product comprised of two collagenases in an approximate 1:1 mass ratio, Collagenase I (AUX-I, Clostridial type I collagenase) and Collagenase II (AUX-II; Clostridial type II collagenase). Preferably, the collagenase comprises collagenase I and collagenase II have a mass ratio of about 1 to 1 and having a purity of at least about 95% by area as determined by reverse phase high performance liquid chromatography (as described, for example, in US. Pat. No. 7,811,560).

Collagenase compositions of the invention can also be prepared by mixing either a specific number of activity units or specific masses of the purified enzymes. Collagenase activity can be measured by the enzyme’s ability to hydrolyze either synthetic peptide or collagen substrate. Those skilled in the art will recognize that enzyme assays other than those disclosed herein may also be used to define and prepare functionally lent enzyme compositions.

It is understood that the terms “Collagenase I”, “ABC I”, “AUX I”, genase AUX I”, and “collagenase ABC I” mean the same and can be used interchangeably.

Similarly, the terms “Collagenase II”, “ABC II”, “AUX II”, “collagenase AUX II”, and “collagenase ABC II” refer to the same enzyme and can also be used interchangeably.

In certain additional embodiments, the collagenase stered according to a method bed herein is a recombinant collagenase.

The collagenase is administered in a pharmaceutical composition sing collagenase and a pharmaceutically acceptable carrier or diluent. In some embodiments, the composition does not include a protease enzyme other than collagenase (other than a small or trace amount of proteolytic enzyme that may be present in the enase purified from a bacterial fermentation). In additional embodiments, the pharmaceutical ition does not include an enzyme other than collagenase (other than a small or trace amount of enzyme that may be present in the collagenase d from a bacterial fermentation). The person of skill in the art will understand that a enase derived from a bacterial fermentation, even after purification, may n small or trace amounts of impurities, including other enzymes, such as other protease enzymes. The small or trace amount of impurity can, for example, be less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of the collagenase ition. In some embodiments, the small or trace amount of impurity can be less than about 1%, 2%, 3%, 4% or 5% by area, as determined by reverse W0 2013/059619 PCT/U82012/061063 _ 9 _ phase high performance liquid chromatography. In another aspect, the pharmaceutical composition consists essentially of collagenase and a pharmaceutically able carrier or diluent. As used herein, a pharmaceutically ition “consisting essentially of enase and a pharmaceutically acceptable carrier or diluent” is intended to exclude from the ceutical compositions other protease enzymes and hyaluronidase other than small or trace s as bed above. In certain additional aspects, a composition “consisting essentially of collagenase” excludes from the compositions an enzyme other than collagenase. In yet another embodiment, the pharmaceutical composition consists of collagenase and a ceutically acceptable salt thereof.

The method of treating or reducing EFP can further include the eatment of the target area with a local anaesthetic agent.

In further aspects, the invention is directed to the treatment or reduction of EFP in a patient consisting essentially of administering one or a plurality injections of collagenase to an area affected by EFP wherein the dose of collagenase administered is between about 5 to about 200 ABC units and optionally, further wherein the concentration of collagenase is about 50 to about 2000 ABC units/ml. In yet an additional aspect, the ion is directed to the treatment or reduction of EFP in a patient consisting of administering a plurality of injections of collagenase wherein the dose of enase stered per ion is between about 5 to about 200 ABC units and optionally, further wherein the concentration of collagenase is about 50 to about 2000 ABC units/ml.

The pharmaceutically acceptable carrier can be one or more liquid carriers or excipients appropriate for injection. As used herein, the term "pharmaceutically acceptable carrier or excipient" means a non-toxic, inert, liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; es such as corn starch and potato starch; cellulose and its tives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; glycols such as propylene ; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; ic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, ing agents, coating agents, perfiaming PCT/U82012/061063 _ 10 _ agents, vatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Injectable preparations, for example, sterile inj e aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending . The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol.

Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium de solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The inj ectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile inj ectable medium prior to use. The sterile solutions can also be lyophilized for later use. In one embodiment, the pharmaceutical composition comprising collagenase is a lyophilized inj ectable composition formulated with lactose. In one embodiment each milligram of injectable collagenase is formulated with 1.9 mg of lactose. In r embodiment, each milligram of injection collagenase preferably has imately 2800 SRC units and 51000 units ed with a potency assay using a tic substrate, szPGGPA.

In another embodiment, the enase composition used according to a method of the invention is a lyophilized inj e ition formulated with sucrose, Tris at a pH level of about 8.0. For example, 1.0 mg of the drug substance of the invention is formulated in 60 mM sucrose, 10 mM Tris, at a pH of about 8.0 (this equates to 20.5 mg/mL of sucrose and 1.21 mg/mL of Tris in the formulation buffer). Generally, a source of calcium is included in the formulation, such as calcium chloride.

The invention will be better understood in connection with the following es, which are ed as an illustration only and not limiting of the scope of the invention.

Various changes and modifications to the disclosed embodiments will be nt to those skilled in the art and such changes and modifications may be made without departing from the spirit of the invention and the scope of the appended claims.

PCT/U82012/061063 _ 11 _ EXAMPLES Example 1: Effectiveness of Collagenase Clostridium Histolfiicum in lysing dermal en in gen minipigs Collagenase idium Histolyticum (Auxilium Product Operation, Malvem, PA) is a parenteral lyophilized product comprised of two collagenases in an approximate l:l mass ratio, Collagenase I (AUX-I, Clostridial type I collagenase) and enase II (AUX-II; Clostridial type II collagenase). These collagenases are isolated and purified from the fermentation of Clostridium histolytz'cum. The vehicle for reconstitution of the lyophilized product was saline (0.9% sodium chloride) with 0.03% (2 mM) calcium chloride. The vehicle for dilution and preparation of the ations described in more detail below was saline (0.9% sodium chloride for ion; Baxter Healthcare Corporation, Deerfield, IL) with 10 mM (013%) TRIZMA ® (Sigma Aldrich, Inc., St. Louis, MO), 60 mM (2.0%) sucrose (Sigma Aldrich, Inc., St. Louis, MO), and 2 mM calcium chloride (pH 8.0) ch Chemical Corporation, Allentown, PA). lN HCl (hydrochloric acid) was used to bring the pH of the e for dilution to 8.0. The e for dilution was mixed throughout preparation, sampling and dose administration procedures. The vehicle for dilution was then sterile-filtered using a 0.22-um PVDF syringe filter into a sterile vial and capped with a septum. The vehicle for dilution was prepared using aseptic technique within a laminar flow hood using sterilized are and utensils.

Dosing formulations were prepared at the test article (Collagenase Clostridium Histolyticum) concentrations indicated in the following table: Table 1 Test Article Concentration a (mg/mL)/ Treatment Number ABC units/ml H 1 0.0015/ 8 .9 2 0.003/ 8 51.2 3 0.005/ 8 _ 12 _ 86.2 4 0.009/ 8 155.2 -7 0.015/ 8 258.6 8-9 0.03/ 8 517.2 0.07/ 8 1206.9 11 0.09/ 8 1551.7 12 0.15/ 8 2586.2 = The test article ations were not adjusted for . = pH measurement using litmus paper.

The dosing formulations were prepared on the day of dosing. The lyophilized product was reconstituted using the vehicle for reconstitution to obtain a 2.5 mg/mL stock test article solution. The vehicle for on was used to dilute the stock test article solution and prepare the formulations to be used for dosing. The formulations were maintained on wet ice prior to, and during dosing. Formulations were prepared using aseptic que within a r flow hood using sterilized glassware and utensils.

Gottingen minipigs were used as the test system on this study. This species and breed of animal is recognized as appropriate for general toxicity studies and has been utilized as a biomedical research model in a wide variety of disciplines. Swine exhibit many similarities with man in cardiovascular anatomy and physiology, digestive physiology, and integumentary structure and function. Because of the similarity of the anatomy and physiology of the skin between swine and man, swine have become standard models for plastic surgery, wound healing, and dermal toxicity studies sen et al, (1998), Scandinavian l ofLaboratory Animal Science 1998, 25 (supplement I):27-30).

Historically, the swine has been used in toxicological assessment of prospective therapeutics.

Gottingen minipigs (4 males and 3 females) were received in good health from Marshall BioResources, North Rose, NY. The animals were approximately 3 to 4 months old at receipt. Each animal received one dose of all 12 treatments, for a total dose per animal of approximately 0.43 mg Collagenase idium Histolyticum. The animals were approximately 4 months old at dose administration. Body weights ranged from 7185 g to 8646 g for the males and 9333 g to 9633 g for the s at dosing. Test article _ 13 _ formulations were administered to animals anesthetized by telazol/xylazine on study day 0 by subcutaneous injection (bolus). The day prior to ion, the designated areas for injection were clipped if necessary. Six injections were stered along the right and left lateral trunk of each animal using an appropriately sized syringe and needle (16-23 gauge). s were not dosed in the same site more than once. Each subcutaneous injection consisted of a different, randomly assigned treatment (combination of formulation concentration and dose volume). Injection sites were spaced sufficiently apart to prevent possible ons from adjacent injection sites. The area immediately surrounding the site of test article deposition was marked using permanent marker and remarked as needed. Individual injection sites were labeled. The selected route of administration for this study was subcutaneous injection.

No drug-related histologic effects were detected in adipocytes, nerves, l structures (hair follicles, sweat glands and sebaceous glands) or the overlying epidermis.

There were no consistent differences in collagen lysis between the sexes or between the animals euthanized at 24 or 48 hours post-dosing in the estimated extent of collagen lysis and the results are combined by gender for the 24 and 48 hour post-dosing sies in the table below. At concentrations less than 0.015 mg/mL, the extent of collagen lysis was generally dose sive. At 2 0.015 mg/mL the extent of collagen lysis was generally proportional to the dose volume injected as d to the total dose or ation concentration (for example, test articles 7 and 8 represent the same dose given at a lower concentration and higher volume for test article 7).

Table 2 Total Dose (mg)/ Concentration Dose ABC Mean Extent of Collagen Lysis Test (mg/mL)/(ABC Volume units )a Article units/ml (mL) Males s Both 1 0.0015/ 0.2 0.0003/ 0.38 2.90 1 .64 .9 5.17 2 0.003/ 0.2 0.0006/ 2.15 1.77 1.96 51.2 10.3 3 0.005/ 0.2 0.001/ 1.81 1.84 1.82 86.2 17.2 0.009/ 0.2 0.0018/ 3.13 2.27 2.70 155.2 31.0 0.015/ 0.05 0.00075/ 1.89 3.04 2.47 258.6 12.9 0.015/ 0.1 0.0015/ 4.15 2.82 3.48 258.6 25.9 0.015/ 0.2 0.003/ 5.84 3.89 4.87 258.6 51.2 0.03/ 0.1 0.003/ 3.93 3.57 3.75 517.2 51.2 0.03/ 0.2 0.006/ 6.05 4.08 5.07 517.2 103.4 0.07/ 0.1 0.007/ 6.37 4.78 5.57 1206.9 120.7 11 0.09/ 0.1 0.009/ 5.50 4.60 5.05 1551.7 155.2 12 0.15/ 0.1 0.015/ 5.72 4.43 5.07 2586.2 258.6 a = For each gender, the mean extent of collagen 1ysis was combined for all three animals at the 24 and 48 hour necropsy als.

Collagenase Clostridium Histolyticum administered as single subcutaneous injections at concentrations ranging from 0.0015 to 0.15 mg/mL resulted in local swelling at sites treated with enase Clostridium Histolyticum concentrations 20.015 mg/mL and collagen 1ysis (the expected pharmacologic effect of C011agenase Clostridium yticum) at all doses, dose volumes, and formulation concentrations in this study. The minimally effective dose in this study was 0.0003 mg protein and the minimally effective concentration W0 2013/059619 PCT/U82012/061063 _ 15 _ was 0.0015 mg/mL. At concentrations less than 0.015 mg/mL, a dose response generally was apparent for the extent of collagen lysis. At concentrations 20.015 mg/mL, the extent of effective collagen lysis reflected the dose volume given more than the total dose or formulation concentration.

Collagen lysis was accompanied consistently by a number of secondary changes.

These changes included the following: hemorrhage and/or acute inflammation seen consistently at all effective dose levels; necrosis of the muscle fibers of the panniculus camosis at 20.003 mg/mL (total doses 2 0.0006 mg protein); perivascular and intramural edema, neovascularization/flbrosis, vascular necrosis, and/or thrombosis seen sporadically at trations 20.009 mg/mL (total doses 20.0018 mg n); and arterial intramural hemorrhage seen sporadically at ation concentrations 20.030 mg/mL (total doses .003 mg protein). There were no Collagenase Clostridium Histolyticum-mediated effects on adipocytes, nerves, l structures or the overlying epidermis.

Results from this study showed some degree of histologically detectable collagen lysis of the dermal septa at all Collagenase Clostridium Histolyticum dose concentrations; however, more areas of te lysis of dermal septa were seen at concentrations 20.015 mg/mL. In this study, there were no Collagenase Clostridium Histolyticum mediated s on adipocytes, nerves, l skin structures or the overlying epidermis. Local effects of Collagenase Clostridium Histolyticum (hemorrhage and/or acute inflammation) at each concentration were ed upon gross and/or microscopic examination at necropsy. There were no consistent ences in collagen lysis between the sexes or between the animals euthanized at 24 or 48 hours post-dosing in the estimated extent of collagen lysis.

At concentrations effective in the complete lysis of the dermal septae, dose and volume-dependent en lysis was noted to occur within a diameter of approximately 2.5 cm in the minipig study. 2012/061063 _ 16 _ Example 2: Phase 1b, open-label= dose-escalation and pharmacokinetic study for the treatment of EFP The objectives of this study are to assess the safety, effectiveness, pharmacokinetics, and immunogenicity of enase idium Histolyticum at increasing total doses in an 80 cm2 area, ranging from 0.0029 mg to 0.116 mg (50 to 2000 ABC units) and sing concentrations ranging from 0.0029 mg/mL to 0.116 mg/mL (50 to 2000 ABC units per mL) in the treatment of adult women with EFP.

Based on the findings discussed above in the minipig study (Example 1), the injection template for the Phase 1 al study is designed to allow for a distance of approximately 2.5 cm between injections. This distance is ed to enable adequate bution and activity of Collagenase Clostridium Histolyticum within the 8 cm x 10 cm area of EFP without significant gaps or overlap within the treatment area. In an t to determine the optimal dose for the treatment of EFP (for example, efficacious with minimal local adverse effects), dose selection was made following consideration of the favorable safety profile of Collagenase Clostridium Histolyticum to date both in clinical development and clinical practice as well as on data from the minipig study. As a result, a starting dose of 0.0029 mg injected as 10 separate 0.00029 mg doses within the 8 cm x 10 cm target EFP area at a concentration of 0.0029 mg/mL is proposed for the Phase 1 study.

This study is a Phase lb, open-label, dose-escalation and pharmacokinetic study.

During the screening visit, while the subject is standing, the investigator or qualified designee will examine the left and right buttock and the left and right thigh and select a quadrant that has a photonumeric EFP severity scale (CSS) score of Z 10 representing moderate to severe EFP severity (Hexsel et al., 2009). The investigator or qualified designee will identify an area of EFP within the selected quadrant that is at least 8 cm x 10 cm (i.e., target EFP area) and is suitable for ent (i.e., that is on the lateral upper aspect of the thigh or within the buttock and does not involve the gluteal fold). The target EFP area must be evident when the subject is standing, t the use of any manipulation such as skin pinching or muscle contraction. Each subject will be screened for study eligibility within 21 days before injection of study drug on Day 1.

W0 59619 PCT/U82012/061063 _ 17 _ In this study, 63 ts will each receive 10 concurrent equal volume injections of Collagenase Clostridium Histolyticum within the target EFP area. Following screening and determination of study eligibility, subjects will be assigned sequentially to Cohorts l, 2, 3, and 4 (Table 3). The first nine subjects will be assigned to Cohort l (1 mL total volume/target EFP area). Within each of following Cohorts 2 to 4, the first nine subjects in each cohort will be assigned to the 5 mL total volume/target EFP area followed by the next nine subjects who will be assigned to the 1 mL total volume/target EFP area. Dosing will start with Cohort l. Dosing in Cohorts 2 and 3 will not begin until the safety of subjects in Cohort 1 has been evaluated by the safety monitoring committee (SMC). Dosing in Cohort 4 will not begin until the safety of subjects in Cohorts 2 and 3 has been evaluated by the SMC.

The doses proposed to treat an 8 cm X 10 cm area of EFP represent between 0.5% (0.0029 mg) and 20% (0. 1 16 mg) of the dose used in a single injection for Dupuytren’s contracture at a concentration that is between 0. 1% (0.0029 mg/mL) and 5% (0. l 16 mg/mL) of the concentration used in the approved product for Dupuytren’s contracture.

Table 3: Study Drug ment by Cohort EFP Dose/ EFP Concentration Target EFP Area as a Percent of the Dose CCH/ Volume/ as a t of the Approved Dupuytren’s Target Target Approved Dupuytren’s tration Cohort EFP Areaa EFP Areaa Concentration Dose (0.58 m_) (2.3 m_/mL) (50 U)b lmL 250U 5 mL (250 U) 1 mL (250 U/mL) 3. _- 750U 5 mL (750 U) 1 mL (750 U/mL) (2000 U) 5 mL (400 U/mL) a A total of 10 single injections at the target injection sites across the 8 cm X 10 cm target EFP area.

CCH is Collagenase idium histolyticum b U is ABC units _ 18 _ The components of Collagenase Clostridium Histolyticum are: mixed collagenase AUX-I and AUX-II, 10 mM tris, 60 mM sucrose. The components of Collagenase Clostridium Histolyticum sterile Diluent A for reconstitution are: 0.03% (2 mM) calcium chloride (CaCL) in 0.9% (154 mM) sodium chloride (NaCL) solution, pH 6.0-7.0, is ed as a ally-sterilized liquid at 3.0 mL per vial. The components of e Collagenase Clostridium Histolyticum buffered sterile Diluent B for filrther dilution are: 0.03% (2 mM) calcium de and 0.12% (10 mM) tromethamine (TRIS) in 0.9% (154 mM) sodium chloride, pH 8.0 de is supplied as a terminally-sterilized liquid at 3.0 mL per vial. After reconstitution with Diluent A and fiarther dilution with Diluent B, the A4500 solution can be kept at room temperature (20° to 25°C/68° to 77°F) for up to one hour or refrigerated at 2° to 8°C (36° to 46°F) for up to 4 hours prior to administration.

An ion template will be made of a thin clear material. The 8 cm x 10 cm injection template will be pre-stamped with the diagram and injection sites shown in Each of the black dots within the octagon represents an injection site. The encircled ‘X’ represents the ion site of the central dimple (i.e., the area of the deepest depression in the target area (. Each black dot ding the site of the central dimple and the four comers) will be cut-out to enable the investigator to mark the target EFP area as needed.

Each dot within the octagon shown in represents an injection site. The X within the circle represents the injection site to the ‘central ’. The four comers are labeled as upper right (UR), lower right (LR), upper left (UL), and lower left (LL).

During the screening visit, while the subject is standing, the investigator or qualified designee will examine the left and right buttock and the left and right posterior upper thigh and select a nt that has moderate to severe EFP (CSS score of 210). The investigator or qualified designee will identify an area of EFP within the selected quadrant that is at least 8 cm x 10 cm (referred to in this example as the target EFP area) and is suitable for treatment (for example, is within posterior upper thigh or within the buttock and does not involve the gluteal fold). The target EFP area must also be evident when the subject is standing, without the use of any manipulation (such as skin pinching or muscle contraction).

WO 59619 PCT/U82012/061063 _ 19 _ The investigator will then place the injection template over the target EFP area (while the subject remains standing) and on the template encircled ‘X’ over the center of the deepest depression (for example, the central dimple) in the target area. The investigator will use a surgical marker and mark the subject’s skin at two comers of the template. The investigator will remove the template and record which two comers of the template were marked (e. g., upper right, lower right, upper left, lower left). The investigator will use a tape measure to measure as follows: 1. Identify the midline of the upper thigh and buttock starting from the center of the popliteal fossa (. 2. Identify the line that is dicular to the e and goes through the first template comer mark (as depicted in [Line BC]). Measure distance between Points B and C (record distance). 3. e the distance from Point A to Point B as depicted in (record distance). 4. Repeat Steps 2 and 3 for measurement of the second template comer mark ( [Points A, D, and E]).

Each recorded measurement will be later used to relocate the 8 cm x 10 cm target EFP area before injection and before each efficacy evaluation time point.

On Day 1, after the injection site has been identified, marked (as described above), photographed, and the pre-inj ection efficacy evaluations have been completed, the subject will be positioned prone on the examination table so that the entire 8 cm x 10 cm target EFP area with the previously marked ion sites is visible and assessable to the investigator.

The injection site area should be prepped with an appropriate antiseptic such as alcohol.

Using a usly prepared 5mL or 1 mL syringe (depending on cohort assignment) with a 30 gauge 14 inch needle, the investigator will administer a dose of either 0.5 mL or 0.1 mL of Collagenase Clostridium Histolyticum into each of the 10 injection sites. At each of the 10 sites, study drug will be injected perpendicular to the subject’s skin to a depth of 14 inch (~7 mm).

The investigator will administer study drug in a sequence that s each of the 10 sites receives a single 0.5 mL or 0.1 mL injection of Collagenase idium yticum.

Following the tenth injection, the m permissible volume of study drug will have been administered (5 mL or 1 mL). Any injectate on the surface of an injection site following injection (suggestive of extravasation) will be noted and the associated injection location will PCT/U82012/061063 be recorded. No local massage or ion will be performed subsequent to injection of study drug.

The following primary endpoints will be evaluated at Day 90: o Investigator global aesthetic improvement scale assessment of the target EFP area; 0 Subject global aesthetic improvement scale assessment of the target EFP area; and o A der analysis based on proportion of subjects with a Z 30% improvement from baseline in surface contour standard deviation of the target EFP area based on 3-D digital photography.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various s in form and details may be made therein Without ing from the scope of the invention encompassed by the ed claims. - 21 –

Claims (33)

CLAIMS What is claimed is:

1. Use of collagenase for the preparation of a medicament for treating or reducing edematous fibrosclerotic panniculopathy (EFP) in a t, wherein said medicament 5 is adapted for administration of a plurality of subdermal ions of collagenase comprising collagenase I and enase II to an area affected by EFP, wherein the dose of collagenase per injection is between about 5 to about 200 ABC units, and wherein the collagenase I and collagenase II each have a purity of at least about 95% by area as determined by e phase high performance liquid tography. 10

2. The use of claim 1, wherein the number of subdermal injections depends on the size of the area to be treated.

3. The use of claim 1, wherein the concentration of collagenase for administration is between about 50 to about 2000 ABC units per milliliter.

4. The use of any one of claims 1 to 3, wherein each injection of collagenase is 15 formulated for administration in a volume of about 0.5 ml or less.

5. The use of any one of claims 1 to 4, wherein the total dose of collagenase for administration is between about 5 to about 2000 ABC units.

6. The use of any one of claims 1 to 5, wherein the dose of collagenase per ion is n about 5 to about 100 ABC units. 20

7. The use of any one of claims 1 to 5, wherein the dose of collagenase per injection is between about 5 to about 50 ABC units.

8. The use of any one of the preceding claims, wherein the collagenase I and collagenase II are present at a 1:1 mass ratio.

9. The use of any one of claims 1 to 5, wherein the collagenase is present in a 25 pharmaceutical composition consisting essentially of collagenase and a pharmaceutically acceptable carrier.

10. The use of any one of claims 1 to 5, wherein the collagenase is present in a pharmaceutical composition consisting of collagenase and a pharmaceutically acceptable r.

11. The use of any one of claims 1 to 5, wherein at least two ions of collagenase 5 are formulated for administration to two injection sites within the affected area.

12. The use of claim 11, wherein at least five injections of collagenase are formulated for administration to five injection sites within the affected area.

13. The use of claim 12, wherein at least ten injections of collagenase are formulated for administration to ten injection sites within the affected area. 10

14. The use of any of the preceding claims, n the affected area of EFP has an area of about 1 cm2 to about 200 cm2.

15. The use of any one of claims 1 to 5, wherein ity of subdermal ions of collagenase are formulated for administration and wherein the distance between injection sites is at least about 1 to about 4 cm. 15

16. The use of claim 15, wherein the distance between injection sites is about 2 to about 3 cm.

17. The use of claim 14, wherein the area affected by EFP has a length of about 10 cm and width of about 8 cm.

18. The use of claim 17, wherein there is a distance of about 2.5 cm between injection 20 sites.

19. The use of any one of claims 1 to 5, wherein each injection has a volume of about 0.5 ml.

20. The use of any one of claims 1 to 5, wherein each injection has a volume of about

0.1 ml. 25 21. Use of collagenase for the ation of a medicament for treating or reducing EFP in a patient, wherein said medicament is adapted for administration of a plurality of subdermal ions of collagenase comprising collagenase I and collagenase II within an area affected by EFP, wherein: (a) the total dose of collagenase for administration to the affected area is between about 5 to about 5000 ABC units, (b) the plurality of injections are formulated for administration at a plurality of injection sites, (c) the enase has a specific activity of about 10,000 ABC units per 0.58 5 mg, and (d) the collagenase I and collagenase II each have a purity of at least about 95% by area as determined by reverse phase high performance liquid tography.

22. The use of claim 21, wherein the number of subdermal injections of collagenase depends on the size of the area to be treated. 10

23. The use of any one of claims 21 and 22, wherein the concentration of collagenase for administration is between about 50 to about 2000 ABC units per milliliter.

24. The use of any one of claims 21 to 23, wherein each injection of enase is formulated for stration in a volume of about 0.5 ml or less.

25. The use of any one of claims 21 to 24, wherein the dose of collagenase per 15 injection is between about 5 to about 100 ABC units.

26. The use of any one of claims 21 to 25, wherein the total dose of collagenase for administration to the ed area is between about 5 to about 2000 ABC units.

27. The use of claim 26, wherein the total dose of collagenase for administration to the affected area is between about 5 to about 1000 ABC units. 20

28. The use of any one of claims 21 to 27, wherein the collagenase I and collagenase II are present at a 1:1 mass ratio.

29. The use of any one of claims 21 to 27, wherein at least two injections of collagenase are formulated for administration to two ion sites within the affected area. 25

30. The use of claim 29, wherein at least five injections of collagenase are formulated for stration to five injection sites within the affected area.

31. The use of claim 30, wherein at least ten injections of collagenase are formulated for administration to ten injection sites within the affected area.

32. The use of claim 1, n the number of subdermal injections of collagenase depends on the size of the area to be treated, n the volume of each injection of 5 collagenase formulated for administration is about 0.5 ml or less.

33. The use of claim 32, wherein each injection has a volume of about 0.2 ml or less.