WO2007100590A2

WO2007100590A2 - Methods for treating cellulite

Info

Publication number: WO2007100590A2
Application number: PCT/US2007/004549
Authority: WO
Inventors: Marie A. Badalamente
Original assignee: Auxilium Pharmaceuticals, Inc.
Priority date: 2006-02-22
Filing date: 2007-02-20
Publication date: 2007-09-07
Also published as: WO2007100590A3; ZA200807221B; CN101405023A

Abstract

The invention relates to the discovery that collagenase injections are effective in dissolving and lysing the collagenous septa network in the skin which comprises cellulite. As such, the invention relates to methods of treating cellulite in a patient in need of such treatment comprising injecting or otherwise delivering the effective amount of purified collagenase to the collagenous septa network of cellulite in the skin. The invention also relates to the use of collagenase in the manufacture of a medicament to treat cetlulite of the skin.

Description

METHODS FOR TREATING CELLULITE

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/775,690, filed on February 22, 2006. The entire teachings of the above application are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in part, by a grant MO1RR10710 from the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION Dimpling of the skin or the "mattress phenomenon" of the thighs and buttocks is commonly referred to as cellulite. This condition is common and appears in otherwise healthy individuals afflicting women much more frequently than men. Over the counter topical therapies abound for the elimination of cellulite. These products and other over the counter topical applications have proved to be useless, costly, and in fact, have never undergone proper placebo controlled clinical trials. Recent randomized, placebo controlled trials of topical retinol and retinol containing caffeine and ruscogenine have also failed to show merit for the elimination of cellulite.

If the treatment of cellulite is to be successful, then the basic pathophysiology of the condition requires clear definition. It was only in 1999 that Rosenbaum, et al. undertook an investigation of the morphology and biochemistry of cellulite (Rosenbaum, M. Prieto, V., Hellmer, J., Boschmann, M., Krueger, J., Leibel, R.L., Ship, A.G., An Exploratory Investigation of the Morphology and Biochemistry of Cellulite. Plastic & Reconst Surg 101 (7):1934-9, 1998). Seven healthy adult subjects, five women and two men, four affected, three unaffected, underwent sonography of the thigh, measurement of regional in vivo subcutaneous adipose tissue metabolism and full thickness wedge biopsy of the thigh under local anesthesia. The presence of cellulite was defined as evidence of dimpling of the skin of the posterolateral thigh. Any continuous area of skin at least 3cm in diameter in which no dimpling was evident was designated as unaffected. In all affected individuals studies were performed to include both affected and unaffected areas of the thigh. Microscopic examination of the wedge biopsies and in vivo sonographic examination of the thigh both showed a diffuse pattern of extrusion of underlying adipose tissue and to the retinacular dermis in affected, but not unaffected, subjects. The study also demonstrated that women had a diffuse pattern of irregular and discontinuous connective tissue immediately below the dermis but the same layer of connective tissue was smooth and continuous in men. This connective tissue layer was more irregular and discontinuous in affected vs. unaffected individuals. No significant differences were noted in subcutaneous adipose tissue morphology, lipolytic responsiveness, or regional blood flow between affected and unaffected sites within individuals. This study demonstrated that there is a sexual dimorphism in the structural characteristics of the dermal connective tissue that pre-disposes women to develop the irregular extrusion of adipose tissue into the dermis which characterizes cellulite. This study concluded that there was no evidence of any primary role for adipose tissue physiology, blood flow or adipose tissue biochemistry in the etiology of cellulite but that the connective tissue of the female thigh and buttocks is structured to accentuate differences in small sub-dermal adipose tissue deposits.

This conclusion was substantiated by the work of Pierard, et al. who examined 39 autopsy specimens microscopically (Pierard-Franchimont, C, Pierard G.E., Henry, F., Vroome, V. & Cauwenbergh, G. A Randomized, Placebo-Controlled Trial of Topical Retinol in the Treatment of Cellulite. Amer. J. Clin. Dermatology, 1 (6):369-74, 2000). Their control group consisted of four adult women and eleven adult men showing no evidence of cellulite. They state that the lumpy aspect of the dermal hypodermal interface appeared to represent a gender linked (female) characteristic of the thighs and buttocks. Cellulite was identified by this mattress phenomenon microscopically and presented as locally enlarged fϊbrosclerotic strands partitioning the subcutis. They speculated that these structures might represent a reactive process to sustained hypodermal pressure caused by fat accumulation.

In a more recent study by Querleux, et al. the anatomy and physiology of subcutaneous adipose tissue by in vivo magnetic resonance imaging and spectroscopy was studied in relation to sex and the presence of cellulite (Querleux, B., Cornillon, C, Jolivet, O., Bittoun, J., Anatomy and Physiology of Subcutaneous Adipose Tissue by in vivo Magnetic Resonance Imaging and Spectroscopy: Relationships with Sex and Presence of Cellulite. Skin Research And Tech 8(2):118-124, May 2002). These authors concluded that 3D reconstruction of the fibrous septae network showed a higher percentage of septae in the direction perpendicular to the skin surface in women with cellulite.

There remains no effective treatment of cellulite up to date. It is the object of invention to provide such methods for treatment of cellulite.

SUMMARY OF THE INVENTION

The invention relates to the discovery that collagenase injections are effective in lysing the collagen septae network of cellulite in humans to treat cellulite and restore a smooth skin appearance. The invention relates to methods of treating cellulite in a subject in need of such treatment, which involves injecting an effective amount of purified collagenase to the thigh and/or buttocks. The invention also relates to the use of collagenase in the manufacture of a medicament to treat cellulite. The collagenase is preferably purified and substantially free of other enzymes, such as proteases and/or hyaluronidase.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to the discovery that collagenase injections are effective in lysing the collagen septae network of cellulite in humans to treat cellulite and restore a smooth skin appearance. The invention relates to methods of treating cellulite in a subject in need of such treatment, which involves injecting an effective amount of collagenase to the thigh and/or buttocks. The invention also relates to the use of collagenase in the manufacture of a medicament to treat cellulite.

Collagenase injections have been proposed for the treatment of diseases such as Dupuytren's disease, adhesive capsulitis and Peyronie's disease. These diseases are all associated with collagen cords or plaques. (Wegman, Thomas L. US Pat. No 5,589,171 Dec.31, 1996, US Pat. No. 6,086,872 July 11, 2000, US Pat. No. 6,022,539, Feb. 8, 2000, and PCT Application No. PCT/US06/01980, filed on January 19, 2006 entitled "Methods for Treating Adhesive Capsulitis," all of which are incorporated herein by reference in their entirety).

Collagenase injections have also been proposed for the treatment of cellulite when combined with hyaluronidase, a soluble enzyme product prepared from mammalian testes (see Pinelle, Sheldon R. US Pat. No. 4,645,668 Mar. 27, 1985). The patent disclosed one working example for cellulite with a low dose of collagenase (100 units) in combination with hyaluronidase (150 units) for only one female patient. No further details on the improvement of cellulite after the injections were presented. The use of intralesional injection of purified Clostridial collagenase, has been shown to be clinically safe and effective in clinical trials in Dupuytren's disease, in correcting the flexion contracture deformity of the hand(s). Additionally, the use of extracapsular injection of purified Clostridial collagenase has been shown to be clinically safe'and effective in the treatment of adhesive capsulitis (frozen shoulder) in clinical trials in restoring pain free and normal motion of the shoulder. Purified Clostridial collagenase injection has also been used by others in clinical trials in Peyronie's disease, a contracture deformity of the penis.

The published work of the inventor, Dr. Badalamente, in Dupuytren's disease forms the rationale for the proposed invention (Starkweather, K., Lattuga, S., Hurst, L.C., Badalamente, M.A., Guilak, F., Sampson, S.P., Dowd, A., Wisch, D.

Collagenase in the Treatment of Dupuytren's Disease: An in vitro Study. J. Hand Surg. 2lA:490-95, 1996; Badalamente, M.A., Hurst, L.C., Enzyme Injection as a Nonoperative Treatment for Dupuytren's Disease. J. Drug Delivery 3(l):35-40, 1996; Hurst, L.C., Badalamente, M.A. (invited authorship) Non-operative Treatment of Dupuytren's Disease. Hand Clinics, G.M. Rayan (ed). W.B. Saunders 15(1), 97- 107, 1999; Hurst, L.C., Badalamente, M.A. (invited editors & authorship), Dupuytren's Disease, R. Tubinana, R. Tubiana, C. Leclercq, L.C. Hurst, M.A. Badalamente (eds), Martin Dunitz Publishers, London (2000); Badalamente, M.A., Hurst, L.C. Enzyme Injection as a Nonoperative Treatment of Dupuytren's Disease. J Hand Surg 25A(4):629-36, 2000; Badalamente, M.A., Hurst, L.C, Hentz, V.R. Collagen as a Clinical Target: Nonoperative Treatment of Dupuytren's Disease. J Hand Surg 27A(5):788-98, 2002) In Dupuytren's disease, the pathognomonic fibrous cord is often interspersed with a septa-like arrangement of adipose tissue. These present clinically as mattress-type "lumps" of varying sizes and in Dupuytren's disease, are termed nodules. It has been a consistent clinical finding in both Phase 2 and Phase 3 trials for Dupuytren's disease that after purified Clostridial collagenase injection, not only does the collagenous cord dissolve and rupture when subjected to pressure in extension, but the fibro-fatty nodules also resolve, and harmlessly resorb. Therefore, collagenase injected subcutaneously into an area of cellulite was postulated to be a safe and effective treatment for this condition in restoring a smooth appearance of the skin of the thighs and/or buttocks. Collagenase is an enzyme that has specific ability to digest collagen. A preferred form of collagenase is derived from fermentation by Clostridium histoliticum and is purified by a chromatographic technique, such as that disclosed in US Application Serial No. 60/763,470 filed on January 30, 2006 and PCT Application No. PCT/US07/02654 filed on January 29, 2007, incorporated herein by reference.

Recombinant collagenases such as those having the amino acid sequence of the Clostridium histoliticum enzymes can also be used. See USSN 60/889,666 filed February 13, 2007, which is incorporated herein by reference.

The sterilized lyophilized collagenase powder preferably has a minimum assay activity of 50 units per mg.

The collagenase is applied in a liquid carrier that is pharmaceutically acceptable, and preferably inert towards the collagenase. Examples are normal saline, aqueous NaCl/CaC12 buffer, aqueous dextran solution, aqueous hetastarch solution. A preferred collagenase composition comprises a mixture of collagenase I and collagenase II in a mass ratio of about 1 to 1 and having specific activity of at least 700 SRC units/mg, such as at least 1000 SRC units/mg, more preferably at least about 1500 SRC units/mg. One SRC unit will solubilize rat tail collagen into ninhydrin reaction material equivalent to 1 nanomole of leucine per minute, at 25 degrees C, pH 7.4. Collagenase has been described in ABC units as well. The potency assay of collagenase is based on the digestion of undenatured collagen (from bovine tendon) at pH 7.2 and 37 degrees C for 20-24 hours. The numbers of peptide bonds cleaved are measured by reaction with ninhydrin. Amino groups released by a trypsin digestion control are subtracted. One net ABC unit of collagenase will solubilize ninhydrin reactive material equivalent to 1.09 nanomoles of leucine per minute. One SRC unit equals approximately 6.3 ABC units. The collagenase is preferably administered via injection in a liquid carrier that is pharmaceutically acceptable. Preferably, the carrier does not interact or deactivate the collagenase. Examples are normal saline, aqueous NaCl/CaCb buffer (containing 0.9% NaCl and 2mM CaCh). For example, the lyophilized formulation can contain 0.1 mg lactose monohydrate per 1,000 ABC units. Each glass vial used below contained 5,150 ABC units collagenase.

In accordance with the invention, collagenase in a liquid carrier is injected into an area of cellulite on the subject's posterolateral thigh. The amount and concentration of collagenase used is effective to lyse and dissolve the collagen septa network of the cellulite. The injection is preferably sterile and does not exceed 1.0 ml. The total dosage is injected at one, two, three, four or five or more different points into the posterolateral thigh where the cellulite dimples of the thigh are most apparent. The preferred objective is to assure good distribution of the collagenase. Patients preferably rest on the contralateral thigh, in bed, for about one, preferably two hours or more.

In other embodiments, the collagenase can be administered locally or topically, such as, a transdermal patch or topical cream or topical ointment to the area of cellulite or can be administered via an implant, such as, microcapsules or microspheres which release collagenase over time. In one embodiment, the patient is characterized as having an area of at least 10 x 10 cm of cellulite on the posterolateral thigh. The invention can achieve improvement in restoring normal and smooth skin appearance in the 10 x 10 cm area of cellulite on the posterolateral thigh.

In cases where results of a single treatment are considered inadequate, the same procedures, total amount of collagenase and concentration may be repeated as needed, for example, at 4-6 weeks intervals. Areas of cellulite, other than the posterolateral thigh may also require treatment, or repeated treatment at 4-6 week intervals. For example, the front of the thigh and the buttocks may contain areas of cellulite.

EXPERIMENTAL Methods

Ten patients entered the study protocol, all female, mean age 41 ± 10 years. The mean body mass index (BMI) was 28.

The minimum area of cellulite of the posterolateral thigh needed for inclusion was 10 x 10 cm. All patients had areas of cellulite of the posterolateral thigh which exceeded the minimum 10 x 10 cm area. Baseline digital photographs were taken of the target thigh(s). A lO x IO cm circle was drawn on the target thigh to define the treatment area. In a sterile fashion, 10,000 ABC units (0.58mg) were injected at five points in the 10 x 10 cm target cellulite area. The total fluid volume of the injection was 1.0 ml. The buffer used was sterile 0.9% NaCl and 2mM CaCl₂. All patients are followed post injection, at one day, one week, one, three and six months. Post treatment photographs are taken serially.

Patients had the option of choosing to have a similar collagenase injection on the opposite side, for cosmetic symmetry, when they reached the time interval of 4-6 weeks post the first collagenase injection. Quantification of the reduction/elimination of cellulite in the target area of the thigh was by visual inspection and photographic documentation.

The target area of cellulite treated was divided into four equal quadrants in the 10 x 10 cm target treatment area. Reduction/elimination of cellulite in the target treatment area was quantified by visual inspection by quadrant, e.g., 4/4= no quadrants responded to treatment, 3/4= three quadrants responded to treatment, 2/4= two quadrants responded to treatment, 1/4= one quadrant responded to treatment, 0/4= all quadrants responded to treatment. The actual area in cm of any remaining cellulite of the posterolateral target area of cellulite was also measured. Photographs were also used for documentation. Results

All patients experienced a reduction in cellulite of the target thigh after collagenase injection. Table 1 shows the results of the reduction in cellulite in the quadrants of the thighs in the patients treated. There was significant reduction in cellulite appearance of the injected area. Cellulite area was reduced by 77% by day 1 in comparison to baseline. This result was sustained in the longer term. In comparison to baseline, cellulite area was reduced by 74% at 1 week, by 89% at 1 month, by 86% at 3 months and by 76% at 6 months.

Adverse events included tenderness in the injection area, ecchymosis and mild edema which resolved well in a mean of 10, 18 and 6 days respectively.

Table 1

Patient 1 day post 1 week 1 mo 3 mo 6 mo

# Age Sex Thigh Parameter Baseline 1st post 1 st post 1st post 1st post 1st

LY C-

009 37 F Right BMI 33 33 34 34 34 36

Circumference

(cm) 68 68 68 68 68 69

Area (cm) 12x14 12x14 5x5 5x5 5x5 8x9

Quadrants 4 4 2 2 2 3

Patient 1 day post 1 week 1 mo 3 mo 6 mo

# Age Sex Thigh Parameter Baseline 1st post 1 st post 1st post 1st post 1st

NW C-

001 52 F Right BMI 31 31 31 31 32 32

Circumference

(cm) 66 66 64 64 64 64

Area (cm) 16x12 2x2 5x5 . 0 5x1 5x1

Quadrants 4 1 2 0 1 1

3 mo 6 mo

Patient 1 day post 1 week 1 mo post post

# Age Sex Thigh Parameter Baseline 2nd post 2nd post 2nd 2nd 2nd

NW C-

001 52 F Left BMI 32 32 32 32 31 30

Circumference

(cm) 68 68 69 66 63 63

Area (cm) 19x19 19x19 5x5 0 4x0 0

Quadrants 4 4 2 0 1 0

Patient 1 day post 1 week 1 mo 3 mo 6 mo

# Age Sex Thigh Parameter Baseline 1st post 1st post 1st post 1st post 1st

PD C-

004 44 F Left BMi 24 24 25 24 25 25

Circumference

(cm) 57 65 66 63 56 56

Area (cm) 10x10 0 0 4x3 4x3 4x3

Quadrants 4 0 0 1 1 1

3 mo 6 mo

Patient 1 day post 1 week 1 mo post post

# Age Sex Thigh Parameter Baseline 2nd post 2nd post 2nd 2nd 2nd

PD C-

004 44 F Right BMI 25 25 25 25 25

Circumference

(cm) 60 62 62 62 62

Area (cm) 10x10 0 1x4 0 0

Quadrants 4 0 1 0 0

Patient 1 day post 1 week 1 mo 3 mo 6 mo

# Age Sex Thigh Parameter Baseline 1st post 1 st post 1st post 1st post 1st

AP C-

012 54 F Right BMI 23 23 23 23 Lost to

Circumference

(cm) 56 56 56 52 followup

Area (cm) 10x11 3x9 3x5 5x4

Quadrants 4 2 1 1 Patient 1 day post 1 week 1 mo 3 mo 6 mo

# Age Sex Thigh Parameter Baseline 1st post 1st post 1st post 1st post 1st

ER C-

015 44 F Left BMI 22 22 22 22 21

Circumference

(cm) 51 54 52 53 53

Area (cm) 10x10 5x9 4x10 0 0

Quadrants 4 2 3 0 0

Patient 1 day post 1 week 1 mo 3 mo 6 mo

# Age Sex Thigh Parameter Baseline 1st post 1st post 1st post 1st post 1st

AE C-

016 40 F Right BMI 31 31 31 31 31 30

Circumference

(cm) 67 79 72 69 68 64

Area (cm) 12x12 0 9x5 2x3 2x7 2x7

Quadrants 4 0 3 1 2 2

3 mo 6 mo

Patient 1 day post 1 week 1 mo post post

# Age Sex Thigh Parameter Baseline 2nd post 2nd post 2nd 2nd 2nd

AE C-

016 40 F Left BMI 31 31 31 31 32

Circumference

(cm) 69 71 68 68 68

Area (cm) 12x14 0 2x4 1x2 0

Quadrants 4 0 1 1 0

Patient 1 day post 1 week 1 mo 3 mo 6 mo

# Age Sex Thigh Parameter Baseline 1st post 1st post 1st post 1st post 1st

MM C-

017 27 F Right BMI 37 37 37 37 Lost to

Circumference follow

(cm) 74 74 74 74 up

Area (cm) 14x14 0 1x10 6x3

Quadrants 4 0 2 1

Significant improvements in the reduction of cellulite of the posterolateral thigh were seen in patients who received collagenase injection(s). This study has shown that collagenase injection of areas of cellulite is a safe and effective method.

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 changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating cellulite in a subject in need of such treatment comprising delivering an effective amount of purified collagenase to the collagenous septa network of cellulite.

2. The method according to claim 1, wherein the collagenase is derived from the bacterium Clostridium hisolyticum.

3. The method according to claim 1, wherein the purified collagenase is administered alone.

4. The method according to claim 1, wherein the purified collagenase is administered in the absence of triamcinolone or other corticosteroids.

5. The method according to claim 1, wherein the purified collagenase is injected in a dose comprising at least about 700 SRC units, applied in one or more injections.

6. The method according to claim 1, wherein the purified collagenase is injected in a dose comprising at least about 1000 SRC units, applied in one or more injections.

7. The method according to claim 1, wherein the purified collagenase is injected in a dose comprising at least about 1500 SRC units, applied in one or more injections.

8. The method according to claim 1, wherein the purified collagenase is injected in a dose comprising at least about 10000 ABC units, applied in one or more injections.

9. The method according to claim 1, wherein the purified collagenase is injected in a volume of about 1.0 ml.

10. The method according to claim 1, wherein the purified collagenase is injected at multiple sites.

11. The method according to claim I₅ wherein the purified collagenase comprises collagenase I and collagenase II.

12. The method, according to claim 9, wherein the injection is delivered in the area of cellulite, characterized by skin dimpling.

13. The method according to claim 1, wherein the subject is a human patient.

14. The method according to claim 1, wherein the treatment is repeated after about four to six weeks.

15. The method according to claim 1, wherein after one month of receiving at least one administration of collagenase, the patient achieves a significant visual reduction in the appearance of cellulite.