Injection technique for treating subcutaneous cellulite

Abstract

The content disclosed herein relates to a method of treating subcutaneous cellulite on the thighs or buttocks of a human subject by administering an effective amount of collagenase and evaluating a reduction in the severity of subcutaneous cellulite through one or more scales.

Description

[0001] This application is a divisional application of application number 201980059156.8 filed on July 12, 2019, and the invention name is “Injection technology for treating subcutaneous fat mass”

[0002] Related applications

[0003] This application claims priority to International Application PCT / US19 / 41494, filed on July 11, 2019, U.S. Provisional Application No. 62 / 697,376, filed on July 12, 2018, and U.S. Provisional Application No. 62 / 733,046, filed on September 18, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present application relates to the field of evaluating and treating subcutaneous cellulite. Background Art

[0005] Cellulite (also known as edematous fibrosclerotic panneuropathy (EFP)) is an aesthetic condition that can be understood as an imbalance between the structural features and biomechanical properties of the subcutaneous junction (i.e., subtle containment and extrusion forces) (Rudolph et al, "Structural Gender Dimorphism and the Biomechanics of the Gluteal Subcutaneous Tissue: Implications for the Pathophysiology of Cellulite," Plast. Reconstr. Surg. 2019; 143(4): 1077-1086). Therefore, the goal of subcutaneous cellulite treatment is to strengthen the subcutaneous interface and / or release fibrous septa through various types of subcutaneous injections (Rudolph et al, supra).Through anatomical and image analysis studies, fibrous septa are considered to be the main cause of subcutaneous fat and the target for treatment of subcutaneous fat (Hexsel et al. "Side-by-side comparison of areas with and without cellulite depressions using magnetic resonance imaging," Dermatol Surg. 2009; 35(10): 1471-1477; Hexsel et al. "Magnetic Resonance Imaging of CelluliteDepressed Lesions Successfully Treated by Subcision," Dermatol Surg. 2016; 42(5): 693-696; Mirrashed F, Sharp JC, Krause V, Morgan J, Tomanek B. "Pilot study of dermal subcutaneous fat structures by MRI in individuals who differ ingender, BMI, and cellulite grading," Skin Res Technol. 2004; 10(3): 161-168; Nürnberger and Müller, "So-called cellulite: an invented disease," J Dermatol Surg Oncol. 1978; 4(3): 221-229; Piérard et al, "Cellulite: from standing fat herniation to hypodermal stretch marks." Am J Dermatopath. 2000; 22(1): 34-37; Querleux etal, "Anatomy and physiology of subcutaneous adipose tissue by in vivo magneticresonance imaging and spectroscopy: relationships with sex and presence of cellulite,” Skin Res Technol. 2002; 8(2): 118-124).To effectively treat subcutaneous cellulite, a treatment is needed that dissolves or destroys the dermal septa, which are composed of collagen. Figure 1 ), and causes dimpling of the skin, which troubles many women.

[0006] Several therapies have been used to treat subcutaneous cellulite; however, there are no approved drug treatments. Despite the availability of various treatment modalities, there is little scientific evidence that any of the current non-drug treatments are beneficial. In fact, much of the evidence is anecdotal, subjective, or based solely on patient self-assessment. Some historical treatments for EFP include weight loss, topical medications, massage, liposuction, medial therapy, radiofrequency, fractionation, dynamic fractionation, and laser therapy. Many of these treatments have adverse side effects (Avram MM, “Cellulite: a review of its physiology and treatment,” J Cosmet Laser Ther. 2004; 6(4): 181-185; Collis et al, “Cellulite treatment: a mythor reality: a prospective randomized, controlled trial of two therapies, endermologie and aminophylline cream,” Plast Reconstr Surg. 1999; 104(4): 1110-1114; Khan MH, Victor F, Rao B, Sadick NS. “Treatment of cellulite: Part I. Pathophysiology.” J Am Acad Dermatol. 2010; 62(3): 361-370; Hexsel DM, Mazzuco R. “Subcision: a treatment for cellulite.” Int J Dermatol. 2000; 39(7): 539-544; Boyce et al. ... al. "Clinical evaluation of a device for the treatment of cellulite: Triactive." Am J Cosmet Surg. 2005; 22: 233-237; DiBernardo BE. "Treatment of cellulite using a 1440-nm pulsed laser with one-year follow-up." Aesthet Surg J. 2011; 31(3): 328-341). Therefore, many doctors believe that improving aesthetic conditions is not easy. Therefore, there is still a need for safe and effective non-surgical treatments to improve the cosmetic results of women with subcutaneous fat. Summary of the Invention

[0007] The disclosure herein meets the above needs and relates to methods for treating subcutaneous cellulite in human patients by subcutaneous injection of a therapeutically effective amount of collagenase (as defined in the specific embodiments). These methods involve a pre-treatment assessment of the severity of the patient's subcutaneous cellulite using various scales and assessment techniques to establish a baseline for the severity of the patient's subcutaneous cellulite. The collagenase is then administered subcutaneously to treat the subcutaneous cellulite. The dosage and administration of the collagenase can vary, and the collagenase can be in the form of a pharmaceutical composition comprising the collagenase and one or more pharmaceutically acceptable excipients. Such excipients may include sterile water for injection, a pH adjuster, and a stabilizer. Post-treatment assessments are performed to confirm the therapeutic effect compared to the baseline. The treatment methods disclosed herein can significantly reduce the appearance of subcutaneous cellulite.

[0008] As described in the specific embodiment, there are four treatment phases, although they are optional and need not be performed in a strict order.

[0009] 1. In the first stage, the clinician selects a cellulite pit for treatment. Next, before injection, an assessment is performed, for example, the clinician and / or the patient independently assess the severity of the cellulite before treatment using one or more of the following scales or other assessment methods (as defined in the specific embodiments):

[0010] Hexsel Cellulite Severity Scale (Hexsel CSS)

[0011] οHexsel Depth Scoring Table

[0012] Likert scale

[0013] οPit analysis

[0014] o Clinician-Reported Photo-Digital Cellulite Severity Scale (CR-PCSS)

[0015] o Patient-Reported Photo-Digital Cellulite Severity Scale (PR-PCSS)

[0016] Investigator-Global Aesthetic Improvement Scale (I-GAIS)

[0017] οSubject Global Aesthetic Improvement Scale (S-GAIS)

[0018] Patient-reported cellulite impact scale (PR-CIS)

[0019] οPR-CIS simplified version

[0020] ο Patient Self-Rating Scale (SSRS)

[0021] οSubjects' Satisfaction Questionnaire for Subcutaneous Cellulite Treatment (SSCT)

[0022] o Clinician's assessment of the severity of subcutaneous cellulite (photographic or other images)

[0023] οBody-Q

[0024] οFitzpatrick Scale

[0025] Thigh Cellulite Response Scale (PR-TCES; CR-TCES)

[0026] o Any validated numerical or other clinically and / or patient-used scale for evaluating cellulite severity, improvement, and / or patient satisfaction (e.g., the Hexel-Merz scale (poster presented at the 2019 American Academy of Dermatology meeting)).

[0027] In addition, pre-treatment assessment by clinicians and patients can be performed by analyzing a series of 3 to 15 photographs, illustrations, drawings, computer images, 3-D models, MRI images, thermographs, ultrasound images, patient verbal feedback, etc., each photograph, illustration, drawing, computer image, 3-D model, MRI image, thermograph, ultrasound image, patient verbal feedback, etc. having a different grade or level of severity of subcutaneous cellulite.

[0028] 2. In the second stage of treatment, the clinician marks the pits to be treated with dots or other markers ( Figure 6 If a low point exists, it is usually placed at the lowest point of the pit. Additional photographs may be taken and additional evaluations performed.

[0029] 3. In the third stage of treatment, a therapeutically effective amount of collagenase is injected subcutaneously into the pit in one or more treatment areas (as defined in the detailed description) in a single dose or multiple doses. The dosage and injection technique vary. For example, the method may include the following procedures (e.g. Figure 10 Deep and superficial injections (as shown in Treatment IV):

[0030] With the needle at an angle of approximately 30° to the skin surface at the injection site and pointing toward the subject's head, push the needle all the way in (1 inch) and inject 0.3 ml of the collagenase component by gently pushing the syringe plunger.

[0031] While maintaining the needle at a 30° angle to the skin surface, gently withdraw approximately half the length of the needle (1 / 2 inch) and reposition the needle toward the subject's side (position B). Inject approximately 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0032] Keeping the needle at a 30° angle to the skin surface, reposition the needle to a position midway between positions A and B (position C, toward the subject's shoulder) and inject approximately 0.3 mL of the collagenase component by gently pushing the syringe plunger.

[0033] Keeping the needle at a 30° angle to the skin surface and the needle pointing completely opposite to position B (position D), gently push the syringe plunger to inject approximately 0.3 mL of the collagenase component.

[0034] • Holding the needle at a 30° angle to the skin surface, reposition the needle to a position midway between positions A and D (position E, toward the subject's other shoulder) and inject approximately 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0035] • A 3 ml syringe with 1.5 ml of collagenase composition is sufficient for one injection site.

[0036] • Use twelve (12) 3 ml syringes per treatment area (each buttock or each thigh) and inject 12 times 1.5 ml at 12 injection sites (a total of 18.0 ml, 5 times 0.3 ml).

[0037] During each visit, a total of 24 injections were administered in 2 treatment areas (2 buttocks or 2 thighs).

[0038] In another embodiment, the needle can be injected into the pit at three angles: about 90 degrees from the skin surface, about 45 degrees from the skin surface and the long axis of the pit, and about 135 degrees from the skin surface and the long axis of the pit. In one non-limiting embodiment, a 1 mL syringe with a 0.1 mL gradient and a 30 gauge syringe are used. 1 The three injections were delivered directly into the well as three 0.1 mL aliquots through a 2 inch needle. Other techniques are described in the Detailed Description section.

[0039] 4. During the fourth treatment phase, post-injection assessments are performed using the above-mentioned scales and other assessment methods (e.g., bruise analysis). The efficacy of a specific collagenase treatment can be based on a single clinician rating or patient rating, or on a composite endpoint consisting of both clinician ratings and patient ratings, where improvement is shown on both scales in the same subject, i.e., both the clinician and patient scales show a pre-specified level of improvement.

[0040] The collagenase is injected in an amount of about 0.01 mg to about 20 mg, which may be injected as a single dose or divided into several divided doses, and has one or more of the following characteristics:

[0041] ·V max (min -1) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0042] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0043] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0044] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0045] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0046] • A molecular weight of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa.

[0047] The purity (by area) is at least 80% as determined by reverse phase HPLC (high pressure liquid chromatography)

[0048] The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[0049] Potency is approximately 5,000 to approximately 30,000 f-SRC units / mg

[0050] Potency is approximately 100,000 to approximately 400,000 GPA units / mg

[0051] Potency is approximately 175,000 to approximately 500,00 f-GPA units / mg

[0052] Potency is approximately 5,000 to approximately 25,000 ABC units / mg

[0053] Less than or equal to 1% (by area) of an impurity selected from clostripain, gelatinase, and leupeptin

[0054] Bioburden less than or equal to 1 cfu / mL

[0055] As used herein, relevant kinetic parameters can be measured using a cuvette assay or a microplate assay as described herein (eg, a SRC cuvette assay, a SRC microplate assay, a GPA cuvette assay, and a GPA microplate assay).

[0056] In some embodiments, the collagenase present in the composition comprises collagenase I and collagenase II present in a ratio of about 1: 1. Other ratios of collagenase I and collagenase II can also be used, such as 0.1-2: 1, or 0.25-2: 1, or 0.5-2: 1, or 0.75-2: 1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0: 1. Each of collagenase I and collagenase II can have an area purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% as measured by reverse phase HPLC.

[0057] In another embodiment, the collagenase composition comprises CCH (as defined in the specific embodiments) containing AUXI and AUXII in a ratio of about 1: 1. Other ratios of AUXI and AUXII can also be used, for example, 0.1-2: 1, or 0.25-2: 1, or 0.5-2: 1, or 0.75-2: 1, or 1: 0.1-2, or 1: 0.25-2, or 1: 0.5-2, or 1: 0.75-2, or 1: 0, or 0: 1. Each of AUXI and AUXII can have an area purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, as measured by reverse phase HPLC.

[0058] In other embodiments, the collagenase composition can be liquid or reconstituted with a lyophilized solid form and a diluent. The dosage of the mixture is measured by the amount of collagenase present, without considering the diluent, and can comprise about 0.1mg to about 20mg of collagenase in one or more injections. In another embodiment, the dosage used in one or more injections is about 0.06mg, 0.48mg, 0.84mg, 1.68mg, 2.52mg, 3.36mg, 4.2mg, 5.04mg, 5.88mg, 6.72mg, 7.56mg or 8.4mg. For example, about 0.06mg, 0.48mg, 0.84mg or 1.68mg is used in about 12 divided injections. The volume of the collagenase composition injected can be between 0.01mL and 3mL at each injection, or the total amount of each treatment follow-up is about 0.2mL to 150mL (as defined in a specific embodiment). In a specific embodiment, the above dosage is for a collagenase composition comprising CCH. In another embodiment, the above dosage is for a collagenase composition having one or more of the following characteristics:

[0059] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0060] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0061] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0062] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0063] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0064] • A molecular weight of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa.

[0065] The purity (by area) is at least 80% as determined by reverse phase HPLC (high pressure liquid chromatography)

[0066] The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[0067] Potency is approximately 5,000 to approximately 30,000 f-SRC units / mg

[0068] Potency is approximately 100,000 to approximately 400,000 GPA units / mg

[0069] Potency is approximately 175,000 to approximately 500,00 f-GPA units / mg

[0070] Potency is approximately 5,000 to approximately 25,000 ABC units / mg

[0071] Less than or equal to 1% (by area) of an impurity selected from clostripain, gelatinase, and leupeptin

[0072] Bioburden less than or equal to 1 cfu / mL

[0073] In another embodiment, about 0.84 mg of CCH is injected in about 12 aliquot injections per treatment area (about 0.07 mg x 12 injections = about 0.84 mg). In some cases, this 0.84 mg treatment occurs in one treatment visit, or 2, 3, 4, or 5 treatment visits every 10-40 days. In other cases, multiple treatment areas are injected with 0.84 mg every 10-40 days for 2, 3, 4, or 5 treatment visits. Such injections can be performed over 5 or more treatment visits.

[0074] Furthermore, as described in the detailed description, collagenase injection is effective for treating subcutaneous cellulite, and significant improvement in the appearance of subcutaneous cellulite can be demonstrated, for example, by Hexsel dimple depth scoring, Likert scale scoring, and dimple analysis.

[0075] Other embodiments of the compositions, methods, and the like of the present invention will become apparent from the following description, drawings, examples, and claims. It will be understood from the foregoing and following descriptions that each and every feature described herein, and each combination of two or more such features, is included within the scope of the present invention, provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment or aspect. Other aspects and embodiments are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The patent or application file contains at least one drawing in color. Copies of this patent or patent application publication in color will be provided by the Office upon request and payment of the necessary fee.

[0077] The above-described features of various embodiments will be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, in which:

[0078] Figure 1 A cross-sectional view of the skin and subdermal tissue depicting collagen septa.

[0079] Figure 2 The amino acid sequence of AUX-I (Seq. ID No. 5) is shown.

[0080] Figure 3 The amino acid sequence of AUX-II (Seq. No. ID 6) is listed.

[0081] Figure 4 The depth of indentation according to the Hexsel Cellulite Severity Scale (CSS) (B) is shown.

[0082] Figure 5 PR- and CR-thigh cellulite efficacy scales are shown.

[0083] Figure 6 An example of the dimples and injection site markings on the subject's buttocks is shown.

[0084] Figure 7 The injection technique used in Treatment I (shaded injection, 3 equal sections) is depicted.

[0085] Figure 8 The injection technique used in Treatment II (shaded injection, 1 aliquot) is depicted.

[0086] Figure 9 The injection technique used in Treatment III (deep injection, 1 aliquot) is described.

[0087] Figure 10 The injection technique used in treatment IV (deep and shaded injections, 5 equal parts) is described.

[0088] Figure 11 The injection technique used in treatment V (shaded injection, 4 equal parts) is depicted.

[0089] Figure 12 An injection technique is described that effectively delivers collagenase or placebo into subcutaneous cellulite pits.

[0090] Figure 13 is the 3D mesh registration (1 day before labeling).

[0091] Figure 14 It is a color distance map used for image registration.

[0092] Figure 15 It is the main pit in the target area.

[0093] Figure 16 It is the main deformable pit in the target area.

[0094] Figure 17 Depicted are the outlines of normal and bruised tissues in the subject's left buttock on days 4, 8, and 15 after injection.

[0095] FIG18(A) depicts the outlines of normal and bruised tissues on days 4, 8, and 15 after injection in the left buttock and provides L*, a*, and b* color measurements of these tissues.

[0096] Figure 18(B) depicts the outlines of normal and bruised tissues in the left buttock after injection on days 4, 8, and 15. The average color and ΔEs of the normal and bruised tissues were calculated based on the L*a*b* color values.

[0097] Figures 19(A)-19(C) Figure 19(A) illustrates the observed changes in pit analysis parameters from day 1, as compared to pre-image markings. Figure 19(B) illustrates the maximum length and maximum width of the pits. Figure 19(C) illustrates the surface area and volume between the pit bottom and the interpolated surface.

[0098] Figure 20 The study flow diagram for the phase 2b randomized, double-blind, placebo-controlled study of CCH in the treatment of edematous fibrosclerotic panneuropathy of the thigh is described. DETAILED DESCRIPTION

[0099] Various aspects and embodiments will be fully described herein. However, these aspects and implementations can be embodied in many different forms and should not be construed as limitations on the invention; rather, the embodiments provided herein will make this disclosure thorough and complete and will fully convey the scope of the subject matter to those skilled in the art. All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0100] A. Definition

[0101] Unless otherwise defined, all terms and phrases used herein have the meanings accorded to such terms and phrases in the art, unless a contrary meaning is clearly indicated or apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are now described.

[0102] Unless otherwise indicated, the use of an individual numerical value refers to the use of an approximation of that value, as if the word "about" or "approximately" were preceding that value. Similarly, unless expressly stated otherwise, the values ​​within the various ranges specified in this application are referred to as approximate values, as if the minimum and maximum values ​​within the stated ranges were preceded by the word "about" or "approximately." Thus, variations above and below the stated ranges can be used to achieve substantially the same results as the values ​​within the stated ranges. As used herein, the terms "about" and "approximately" when referring to a value should have their simple and ordinary meanings to those of ordinary skill in the art most closely associated with the disclosed subject matter, or with the range or element in question. The amount of widening of strict numerical boundaries depends on many factors. For example, some factors that may be considered include the criticality of the element and / or the effect that a change in a given amount would have on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. As used herein, the use of different numbers of significant figures for different values ​​is not intended to limit how the use of the terms "about" or "approximately" will be used to broaden a particular value or range. Therefore, generally speaking, "about" or "approximately" broadens a value. In addition, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values ​​plus the extension of the range provided by use of the term "about" or "approximately", and the recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0103] As used herein, an "affected area" or "treatment area" refers to an area of ​​subcutaneous cellulite in a human patient that is to be treated or has been treated with collagenase (defined below). This may include a quadrant (i.e., left buttock, right buttock, left posterior lateral thigh, right posterior lateral thigh). The affected area or treatment area is not limited to the buttocks or thighs. Rather, any area of ​​the body with subcutaneous cellulite can be used as a treatment area.

[0104] An “adverse event” or “AE” is any unfavorable or unexpected change in body structure (sign), body function (symptom), laboratory result (e.g., chemistry, EKG, X-ray, etc.), or exacerbation of a pre-existing condition that is temporally associated with the use of a study drug, whether or not considered related to the study drug.

[0105] The "Body-Q" is a patient-reported outcome instrument commercially available under license from Memorial Sloan Kettering Cancer Center. It is based on the patient's perception of body contour and / or weight loss. It measures 3 domains: appearance, health-related quality of life (HRQL), and the patient's experience of healthcare via a scale of 18 independent functions. The patient-reported outcome instrument is described in the following: BODY-Q: Instruction Manual BODY-Q: Instruction Manual, Version 1.0, July 2015, Memorial Sloan Kettering Cancer Center, McMaster University, and Stefan Cano. The BODY-Q includes a scale to measure subcutaneous cellulite. See https: / / www.mskcc.org / sites / default / files / node / 174457 / documents / body-q-users- guide.pdf( (July 3, 2019) For cellulite, there are 16 scale items with response options ranging from "not at all" to "extremely bothersome" over the past week, assuming a reading level based on the Flesch-Kincaid scale. Patients are asked, "Thinking about your cellulite, how much has it bothered you in the past week?" [After 16 questions, patients categorize their responses as 1 - Extremely bothersome; 2 - Moderately bothersome; 3 - Somewhat bothersome; 4 - Not at all bothersome]. Scores range from 16 (extremely bothersome) to 64 (not at all).

[0106] Traditionally, clinical examination of bruises involves visual inspection of the bruise and surrounding area, as well as an assessment of the subject's medical, surgical, and concomitant medication history. The results of this interpretation are subjective and are affected by several irrelevant factors, including viewing geometry, ambient lighting, the color of unexposed surrounding skin, and the experience and vision of the observer. "Bruise analysis" as used herein refers to the use of the objective image capture and tracking method disclosed in U.S. Patent Publication No. 2019 / 0035080 to detect visible changes in skin color assessed from images of subjects passing through the collagenase treatment area, which is applied uniformly to all subject images. This objective analysis can assist or even replace visual and clinical inspection of bruises by healthcare providers by providing a quantitative, differentiated way to assess the extent of bruises within subjects (the same subject at different time points) and between subjects (between different subjects). This analysis utilizes the L*a*b* color space defined by the International Commission on Illumination (CIE), which follows the color opposition theory, that is, two colors cannot be red and green at the same time, nor can they be yellow and blue at the same time. As shown below, L* indicates lightness / darkness, a* is the redness / greenness coordinate, and b* is the yellowness / blueness coordinate. DeltaL* (ΔL*), Delta a* (Δa*), and Delta b* (Δb*) can be positive (+) or negative (-). However, the total Delta E (ΔE*) is always positive.

[0107] ΔL*(L*sample-L*standard) = light / dark deviation (+=lighter, -=darker); lower values ​​(0-50) represent dark and higher values ​​(51-100) represent light.

[0108] Δa*(a*sample-a*standard) = red-green deviation (+=more red, -=more green)

[0109] Δb* (b* sample - b* standard) = yellow-blue deviation (+ = more yellow, - = more blue)

[0110] All three values ​​are needed to fully describe the color of an object (in this case, a bruise captured in an image of the treated area of ​​a subject). An objective method for image analysis of collagenase-treated areas (images taken before and at designated time points after treatment), with data output as L*a*b* values, allows for a fast, simple, accurate, reproducible, and unbiased quantification of skin color and any changes therein. This approach eliminates the inherent variability associated with traditional subjective visual estimation of images. ΔE is calculated as follows:

[0111] ΔE (color difference between bruise and normal skin) = SQRT[(L * B -L * N ) 2 +(A * B -A * N ) 2 +(B * B -B * N ) 2 ],in

[0112] L * B = Bruised tissue L *

[0113] L * N =Normal tissue L *

[0114] A * B = Bruised tissue A *

[0115] A * N =Normal tissue A *

[0116] B * B = Bruised tissue B *

[0117] B * N =Normal tissue B *

[0118] Figure 18(B) shows the bruise analysis of the treated area.

[0119] "CCH" refers to AUX-I (Seq. ID No. 5 ( Figure 2 )) and AUX-II (Seq.ID No.6 ( Figure 3 )) Collagenase mixture, in a ratio of approximately 1:1, obtained by fermentation of Clostridium histolyticum (also known as Hathewaya histolyticum). CCH is sold on the market as a lyophilized powder under the trademark It comprises a mixture of AUX-I and AUX-II and specified excipients, although CCH may be used with other suitable excipients.

[0120] The "Clinician-Reported Photo-Digital Cellulite Severity Scale (CR-PCSS)" is a photo-digital scale described in PCT patent application PCT / US2018 / 020551 (published as WO2018 / 160905 on September 7, 2018) that is used by physicians / clinicians to assess the severity of cellulite into 5 levels.

[0121] Unless otherwise specified herein, "collagenase" means any of the following: (a) a collagenase (including mutants) having activity as specified in EC 3.4.24.3 (https: / / www.brenda-enzymes.org / enzyme.php?ecno=3.4.24.3 (accessed on July 3, 2019); (b) a collagenase produced by fermentation of Clostridium histolyticum (also known as Hathewayahistolytica); (c) CCH (as defined above); (d) a collagenase that aligns at least 50% with AUX-I as determined by BLAST. enzymes; (e) collagenases that align at least 50% with AUX-II as determined by BLAST; (f) collagenases produced by fermentation from other organisms (i.e., non-Clostridium histolyticum), such as mammalian, crustacean, fungal, bacterial, or microbial collagenases; (g) collagenases obtained by recombinant techniques; (h) collagenases with a molecular weight between about 65 kDa and about 130 kDa; (i) collagenases designated as class I or class II (also known as collagenase I (or 1), collagenase II (or 2), type I collagenase, type 2 collagenase); (j) mixtures of collagenases I and II; (k) collagenases from strain JCM 1403 (ATCC 19401) or its derivatives; (i) collagenase from strain ATCC 21000 or its derivatives; (m) collagenase from strain ATCC 69334 or its derivatives; (n) collagenase from Clostridium perfringens; (o) collagenase from Vibrio alginolyticus; (p) collagenase from Streptomyces; (q) collagenase from Pseudomonas; (r) collagenase from Achromobacter, (s) collagenases described by Worthington Biochemical (www.Worthington-biochem.com; “Product Highlights”); (t) collagenase from Sigma-Aldrich (www.sigma-aldrich.com); (u) collagenases having one or more of the following characteristics:

[0122] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0123] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0124] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0125] 1 / Kcat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0126] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0127] (v) collagenase described by Nordmark Arzneimittel GmbH & Co. KG.; (w) collagenase from strain 004; or (x) equivalents or mixtures of any of the above. Non-limiting examples of collagenases that can be used in the present disclosure are described in U.S. Patents 7,811,560, 9,757,435, 9,744,138, and WO 2012 / 125948.

[0128] "Pit analysis" refers to an analysis of one or more selected pits, wherein parameters such as the volume, length, width, and surface area of ​​the pits are measured. Measurements can be made by various known methods, such as the methods described in Eckhouse et al. WO 2018 / 116304 and WO 2018 / 116305, and by Cherry imaging (www.cherrymaging.com) and Canfield Scientific. See also Salameh et al., "Novel Stereoscopic Optical System for Objectively Measuring Above-Surface Scar Volume-First-Time Quantification of Responses to Various Treatment Modalities," Dermatol. Surg. 00: 1-7 (2017); and U.S. Patent No. 9,996,923. Such measurements of volume, length, width, and surface area can be calculated using digital 3-D grayscale images (with X and Y axis rotation features) and digital 3-D texture and illumination images (with X and Y rotation features) and computer programs that analyze such images. For example, for a buttocks treatment area, images of the left and / or right buttocks for each patient may be taken before and after treatment. For a thigh treatment area, images of each thigh treatment area may be taken at 0 degrees, 45 degrees, and 90 degrees before and after treatment. For a thigh treatment area, images of each thigh treatment area may be taken at 0 degrees, 45 degrees, and 90 degrees before and after treatment using the Canfield Scientific method.

[0129] "Durability" refers to the period of time over which the effects of the treatment persist. This period can be from 3 months to 20 years, or about 1 to 19 years, or about 2 to 18 years, or about 3 to 17 years, or about 4 to 16 years, or about 5 to 15 years, or about 6 to 14 years, or about 7 to 13 years, or about 8 to 12 years, or about 9 to 11 years. This period can be about 6 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, about 15 years, or about 20 years.

[0130] "Fitzpatrick scale" refers to a scale used to assess a subject's skin type, as shown in Table 1.

[0131] Table 1. Fitzpatrick Scale

[0132] I Light white skin, blue / hazel eyes Often gets sunburned, never tans II Fair skin, blue eyes Burns easily but doesn't tan easily III Yellow skin color Darker skin tone after sunburn IV Light brown skin Rarely, mild sunburn, easily sunburned V bronze skin tone Rarely burns, tans very easily VI Dark brown or black skin tone Never burns, just tans

[0133] The "Hexsel Cellulite Severity Scale" or "Hexsel CSS" or "Cellulite Severity Scale (CSS)" refers to a photographic numerical scale that assesses the five main morphological features of cellulite as follows (Table 2):

[0134] Table 2. Hexsel Cellulite Severity Scale

[0135]

[0136]

[0137] The sum of the scores results in the following classification

[0138] Score Classification of subcutaneous cellulite 1-5 Mild 6-10 Moderate 11-15 severe

[0139] Hexsel et al., J. Eur. Acad., Dermatol. Venereol. 2009; 23(5): 523-528. a. Nürnberger and Müller, J. Dermatol. Surg. Oncol. 1978; 4(3): 221-229. The subjects were assessed in a standing position with their gluteal muscles relaxed. However, if the subjects did not have obvious symptoms of indentation, they were asked to contract their gluteal muscles or perform a squeeze test (by pinching the skin between the thumb and index finger) to differentiate between 0 or 1 points / grade.

[0140] The term "Hexsel Concave Depth Score" is used here to refer to the evaluation of the concave depth of (B) Hexsel CSS only. Figure 4 ):

[0141] 0 = no depression

[0142] 1 = shallow depression

[0143] 2 = Medium deep depression

[0144] 3 = Deep depression

[0145] As used herein, an "image" or "picture" refers to a photograph, diagram, illustration, model, 3D model, computer-generated image, MRI image, or the like.

[0146] "Likert scale score" refers to the evaluation of cellulite by an independent blinded evaluator at each post-treatment visit by comparing the photographs (2D color, 3D color and 3D grayscale) of subcutaneous cellulite before treatment (baseline) on day 1 and the photographs taken at the post-treatment visit. The scores were recorded using the following 5-point Likert scale:

[0147]

[0148] "Optional" or "optional" means that the subsequently described element, component, or condition may or may not occur, so that the description includes instances where the element, component, or condition occurs and instances where it does not.

[0149] The "Patient-Reported Impact of Cellulite Scale (PR-CIS)" as used here refers to a 6-question survey that assesses the visual and emotional impact of cellulite (pleasure, bothersomeness, self-consciousness, embarrassment, looking old or looking overweight or body dysmorphic) using a numerical scale ranging from 0 (not at all) to 10 (extremely noticeable) for each question. More specifically, the PR-CIS is a 6-item static questionnaire that assesses the visual and emotional impact of cellulite (pleasure, bothersomeness, self-consciousness, embarrassment, looking old or looking overweight or body dysmorphic); subjects answer each question using an 11-point numeric rating scale ranging from 0 (not at all) to 10 (extremely noticeable) while viewing digital photographs of their buttocks and / or thighs. This assessment may be for all thighs and / or buttocks together, rather than for each separate area. The total PR-CIS score can be derived from the 6 individual questions:

[0150] Question 1 - Thinking about the area you have chosen for treatment, how satisfied are you with the appearance of your cellulite?

[0151] Question 2 - Thinking about the area you have chosen for treatment, how bothered are you by the appearance of cellulite?

[0152] Question 3 - Thinking about the area you have chosen for treatment, how concerned are you about the appearance of subcutaneous cellulite?

[0153] Question 4 - Thinking about the area you have chosen for treatment, how embarrassed are you about the appearance of subcutaneous cellulite?

[0154] Question 5 - Thinking about the area you chose for treatment, how much older do you look due to the appearance of subcutaneous cellulite?

[0155] Question 6 - Thinking about the area you have chosen for treatment, how fat or out of shape do you appear due to cellulite?

[0156] As used herein, the "Patient-Reported Impact of Cellulite Scale (Reduced Version) (PR-CIS Reduced Version)" refers to a survey using 5 questions to assess the visual and emotional impact of cellulite (happiness, annoyance, self-consciousness, embarrassment, looking old or looking overweight or body dysmorphic), with each question having a rating scale ranging from 0 (not at all) to 10 (extremely noticeable). More specifically, the PR-CIS is a 5-item static questionnaire that assesses the visual and emotional impact of cellulite (happiness, annoyance, self-consciousness, embarrassment, looking old or looking overweight or body dysmorphic); subjects answer each question using an 11-point numerical rating scale while observing digital photographs of their buttocks and / or thighs, with ratings ranging from 0 (not at all) to 10 (extremely). This assessment may be for all thighs and / or buttocks together, rather than for each separate area. In one non-limiting embodiment, the total score of the PR-CIS Reducing Version can be derived from 6 separate questions:

[0157] Question 1 - Thinking about the area you have chosen for treatment, how satisfied are you with the appearance of your cellulite?

[0158] Question 2 - Thinking about the area you have chosen for treatment, how bothered are you by the appearance of cellulite?

[0159] Question 3 - Thinking about the area you have chosen for treatment, how concerned are you about the appearance of subcutaneous cellulite?

[0160] Question 4 - Thinking about the area you have chosen for treatment, how embarrassed are you about the appearance of subcutaneous cellulite?

[0161] Question 5 - Thinking about the area you have chosen for treatment, how fat or out of shape do you appear due to cellulite?

[0162] The total score of the shortened PR-CIS can be derived from the other five questions in the full PR-CIS.

[0163] The “Patient-Reported Photo-Digital Cellulite Severity Scale (PR-PCSS)” as used herein is a patient-reported photo-digital scale, such as PCT International Application PCT / US2018 / 020551 (published on September 7, 2018 as WO2018 / 160905), which is designed to quantify the severity of cellulite into 5 levels.

[0164] As used herein, "photographic digital" refers to the use of a series of photographs, diagrams, illustrations, models, 3D models, computer generated images, MRI images, images, etc., each designating different levels of cellulite severity on a scale.

[0165] As used herein, "consecutive visits" refers to two or more visits by a clinician or a scale to assess changes in subcutaneous fat mass. The interval between visits can be about two weeks, about one month, about two months, about three months, about four months, about five months, about six months, about one year, about two years, about three years, or more than five years.

[0166] As used herein, “serious side effect” means a side effect that results in death, is immediately life-threatening, causes or prolongs hospitalization, causes permanent or substantial disability, congenital anomaly / birth defect, or is considered a major medical event.

[0167] The terms "subject" or "patient" are used interchangeably herein to refer to a human or other mammal.

[0168] The "Subject Global Improvement Scale (S-GAIS)" and "Investigator Global Improvement Scale (I-GAIS)" used here refer to the following scales that assess the severity and / or improvement of subcutaneous cellulite. Subjects were asked the following introductory question: "How would you rate the appearance of your subcutaneous cellulite after treatment?" Scores ranged from -3 (very poor) to +3 (very good), depending on the subject's response, as shown in Table 3.

[0169] Table 3. Subject Global Aesthetic Improvement Scale (S-GAIS) and Investigator Global Aesthetic Improvement Scale (I-GAIS)

[0170]

[0171] As used herein, "subject satisfaction with cellulite treatment" (SSCT) refers to a subject satisfaction rating scale ranging from -2 to +2. For example, Table 4 below provides an evaluation of cellulite treatment on the buttocks. Patients were asked, "Today, are you satisfied with the results of the cellulite treatment you received for the specific area of ​​your buttocks?" They then selected an answer / rating as shown in Table 4.

[0172] Table 4. Subjects' satisfaction with cellulite treatment - buttocks

[0173]

[0174]

[0175] An evaluation of thigh cellulite treatment is provided in Table 5. Patients were asked, “Today, are you satisfied with the results of the treatment for cellulite in this specific area of ​​your thigh?” They then selected an answer / rating as shown in Table 5.

[0176] Table 5. Subjects' Satisfaction Assessment for Cellulite Treatment - Thigh

[0177]

[0178] The "Subject Self-Rating Scale (SSRS)" is a scale used by subjects to rate their satisfaction with their appearance related to subcutaneous cellulite, expressed as a 7-point scale ranging from 0 (extremely dissatisfied) to 6 (extremely satisfied), e.g. Figure 6 shown.

[0179] Table 6. Subject Self-Rating Scale (SSRS)

[0180] score Response Options 6 Very satisfied 5 satisfy 4 Relatively satisfied 3 Neither satisfied nor disappointed 2 Somewhat disappointed 1 disappointment 0 Extremely disappointed

[0181] As used herein, the term "therapeutically effective amount" refers to the amount of collagenase required to reduce the severity of subcutaneous cellulite in a patient or a statistically significant group of patients. The amount of collagenase composition used will be the amount necessary to deliver the amount of collagenase required to achieve the desired result. In practice, this will depend on the collagenase injected, the injection technique, and the enzyme activity in the treatment area.

[0182] As used herein, the term "treatment-emergent adverse event" or "TEAE" refers to any condition that was not present before treatment with the study drug but developed after treatment, or that was present at the start of treatment but worsened during treatment, or that was present at the start of treatment but resolved and then reappeared while the patient was receiving treatment (regardless of the intensity of the AE at the start of treatment).

[0183] The term "Thigh Cellulite Response Scale" or "TCES" as used herein refers to a scale used by clinicians and patients to assess the severity, improvement, and / or patient satisfaction of thigh cellulite and to assist in evaluating the efficacy of collagenase. Figure 5 The patient-reported version is called the PR-TCES; the clinician-reported version is called the CR-TCES.

[0184] As used herein, the term "treatment visit" or "treatment" refers to one or more injections or treatments administered to the affected area to treat cellulite using a therapeutically effective amount of at least one active agent in a single office visit.

[0185] As used in this article, the terms "valid," "validity," or "valid" refer to a process by which a specific scale is demonstrated to be accurate and reliable, including visual assessment of repeatability to ensure that the same results are consistently achieved. Validation further examines a scale's precision, accuracy, and sensitivity to confirm that its measurements are reliable, repeatable, and robust.

[0186] B. Introduction

[0187] The present invention relates to a method of treating subcutaneous cellulite comprising administering a therapeutically effective amount of one or more collagenases to a subject having the appearance of subcutaneous cellulite using certain injection techniques described below.

[0188] Generally, treatment is divided into four phases: (1) the clinician and patient perform a pre-treatment assessment to determine a pre-treatment baseline, and the clinician selects the pit to be treated; (2) if a nadir exists, the clinician marks the pit to be treated at the nadir; (3) the clinician treats the patient with collagenase; and (4) the clinician and patient perform a post-treatment assessment. These phases are described in detail below. Each phase and the steps therein are optional, and the steps do not necessarily need to be performed in a specific order, as the order may vary while achieving comparable results.

[0189] C. Phase 1 - Pre-treatment Assessment

[0190] In the first phase of cellulite treatment described here, clinicians select cellulite pits for treatment based on the following criteria:

[0191] As shown in the photo, when the patient stands in a relaxed position (standing position with the hip muscles relaxed), the dimple should be clearly visible and naturally clear

[0192] The dimples selected should be those that the clinician believes are most likely to improve the aesthetics of the entire buttocks, thigh, or other affected area

[0193] Take a photo of the affected area before treatment while the patient is standing in a relaxed position

[0194] Before injection, an assessment is performed, whereby the clinician and / or the patient independently evaluate the photographs and rate the results using one or more of the following scales or assessments:

[0195] Hexsel Cellulite Severity Scale (Hexsel CSS)

[0196] οHexsel Depth Scoring Table

[0197] Likert scale

[0198] οPit analysis

[0199] o Clinician-Reported Photo-Digital Cellulite Severity Scale (CR-PCSS)

[0200] o Patient-Reported Photo-Digital Cellulite Severity Scale (PR-PCSS)

[0201] Investigator-Global Aesthetic Improvement Scale (I-GAIS)

[0202] ο Subject Global Aesthetic Improvement Scale (S-GAIS)

[0203] Patient-reported cellulite impact scale (PR-CIS)

[0204] ο Patient Self-Rating Scale (SSRS)

[0205] οSubjects' Satisfaction Questionnaire for Subcutaneous Cellulite Treatment (SSCT)

[0206] Thigh Cellulite Response Scale (PR-TCES; CR-TCES)

[0207] οBody-Q

[0208] οFitzpatrick Scale

[0209] o Any valid numerical image or other scale used by clinicians and / or patients to assess cellulite severity, improvement and / or patient satisfaction.

[0210] D. Stage 2 - Marking the pits that need treatment

[0211] In the second phase of treatment described here, the clinician uses dots to mark the pits to be treated. More photographs may be taken.

[0212] E. Stage 3 - Collagenase Injection

[0213] In the third phase of treatment, clinicians treat patients with collagenase.

[0214] 1. Collagenase type

[0215] Collagenases useful in the present invention include any of the collagenases defined above. As further background, matrix metalloproteinases (MMPs) can consist of collagenases that fall within the scope of the present definition. For example, MMP-1 comprises collagenase 1; MMP-8 comprises collagenase 2 / neutrophil collagenase; MMP-13 comprises collagenase 3; and MMP-18 comprises collagenase 4. In addition, cathepsins can be classified as collagenases.

[0216] 2. Collagenase kinetics

[0217] The collagenases useful in the present invention can also be characterized by their enzyme kinetics. Here, the approximate kinetic values ​​of one or more collagenases effective in treating subcutaneous cellulite include:

[0218] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0219] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0220] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0221] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0222] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0223] V max = Maximum speed

[0224] K M = [Substrate] 50% of V max

[0225] K cat = substrate molecules split per second

[0226] 1 / K cat = microseconds required to cleave the substrate molecule.

[0227] These values ​​can be determined experimentally using the microplate assay described below, but using different substrates and times. Other assays and parameters can also be used.

[0228] These values ​​reflect a quantitative expression of the enzyme behavior based on the Michaelis-Menten equation:

[0229]

[0230] Where V0 is the reaction rate (speed) at substrate concentration [S], V maxis the maximum observable rate, K M is the Michaelis constant, which produces 50% V max is related to the substrate concentration.

[0231]

[0232] where k1, k-1 and k2 are the rate constants of the following processes:

[0233]

[0234] where E is the enzyme, S is the substrate, ES is the enzyme-substrate complex, and P is the product.

[0235] Catalytic K cat The turnover number is the rate at which the ES complex is converted to E+P. It reflects the number of catalytic cycles per active site per unit time.

[0236] In certain embodiments, AUX-I and AUX-II have the following characteristics:

[0237] AUX I

[0238] ο Experiment: SRC microplate

[0239] οVmax,min -1 : Approximately 0.08 to 7.70

[0240] οK M : Approximately 4.1 to 410 nanomoles

[0241] οK cat , sec -1 : Approximately 1.1 to 107

[0242] ο1 / K cat , microseconds: approximately 376 to 37,222

[0243] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[0244] AUX II

[0245] ο Experiment: GPA microplate

[0246] οV max , min -1 : Approximately 0.3 to 30.5

[0247] οK M , mM: approximately 0.03 to 3.1

[0248] οK cat , sec -1 : Approximately 93 to 9,179

[0249] ο1 / K cat , microseconds: approximately 4 to 428

[0250] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[0251] Gaga:

[0252] K cat =V max / [AUX] = (nanomoles substrate / nG AUX*min-1) / nG AUX

[0253] Catalytic efficiency (Kcat / KM) generally represents the overall ability of an enzyme to convert a substrate to a product and reflects both binding and catalytic events. In another embodiment, AUX-I and AUX-II include the following features.

[0254]

[0255] ·V max = Maximum speed / degrees

[0256] ·K M = [Substrate] 50% of Vmax

[0257] ·K cat = substrate molecules split per second

[0258] 1 / K cat = the time required to cleave one molecule from the substrate

[0259] ·K cat / K M Often used to represent the catalytic activity of an enzyme

[0260] *Through SRC microplate assay

[0261] + Passed GPA microplate experiment

[0262] 3. Collagenase titer (specific activity)

[0263] A variety of assays have been developed to measure the specific activity (potency) of collagenases. These assays are described in subsections a. to c. and characterize collagenases by their ability to convert substrate to product over a given time period at a predetermined enzyme concentration. In certain non-limiting embodiments, these assays are used to determine the potency of each of AUX-I and AUX-II and the conjugated CCH drug product (a 1:1 ratio of AUX-I and AUX-II). The SRC assay (described below) uses collagen as the reaction substrate. The SRC assay uses soluble rat (tail) collagen (SRC) as a substrate and is used to measure type I collagenase activity, with type II collagenase accounting for approximately 20% of the observed activity of the collagenase mixture. The SRC assay is a fluorometric method that utilizes fluorescamine to detect peptides produced by SRC1-type digestion. The reaction is carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (containing 15 mM divalent calcium ions) at pH 7.2 at 25°C for 2.5 hours.

[0264] The bovine tendon collagen (BTC) assay (described below) is based on the procedure described by Mandl et al., Arch. Biochem. Biophys. 74:465-475 (1958), with modifications as described by Keller and Mandl, Arch. Biochem. Biophys. 101:81-88 (1963). See also Rosen, Arch. Biochem. Biophys. 67:10-15 (1957). The BTC assay uses insoluble bovine tendon collagen as a substrate to measure type I and type II activity (e.g., AUX-I and type II collagenases). The BTC assay is colorimetric and uses ninhydrin to detect peptides produced by BTC type I and type II degradation. The reaction is also performed at pH 7.2, but is carried out at 37°C in tris (hydroxymethyl) aminomethane (tris) buffer containing 10 mM divalent calcium ions for 22 h.

[0265] The third type of collagenase assay, the GPA assay (described below), utilizes a soluble derivatized hexapeptide (carboxy-GPGGPA) as a substrate. The GPA assay primarily measures type II activity, with type I accounting for approximately 10% of the observed activity. Type II collagenase cleaves the hexapeptide into two tripeptides, one of which (GPA) has a free amino terminus that reacts with fluoresceinamine to provide a fluorescent product. The GPA assay is run in HEPES buffer at pH 7.2 containing 100 mM divalent calcium ions at 25°C for 10 minutes.

[0266] Both SRC and BTC assays degrade the natural substrate (collagen), which more closely resembles the therapeutic effect of collagenase injections. The advantage of GPA assays is that they utilize a well-defined, small molecular weight hexapeptide as a substrate and produce two well-defined tripeptides. GPA assays produce a fluorescent signal and are very sensitive. Finally, GPA assays are suitable for Michaelis-Menten kinetic analysis because they use a single substrate and reaction conditions (10 minute incubation) that are close to the initial enzyme velocity. SRC assays are well suited for collagen-degrading enzymes that have a collagen-binding domain, while GPA assays are well suited for collagen-degrading enzymes that do not have a collagen-binding domain, commonly known as gelatinases.

[0267] a. GPA Unit Experimental Method and Specific Activity Unit

[0268] i. Collagenase titer determined by GPA assay (cuvette)

[0269] GPA experiment is mainly used for measuring the effectiveness of type II collagenase.The first step of experiment relates to enzymatic reaction, and described enzymatic reaction comprises that substrate carboxyl-glycyl-L-prolyl-glycyl-prolyl-L-alanine (zGPGPA) is digested into two kinds of peptides by collagenase sample: carboxyl-glycyl-L-prolyl-glycine (zGPG) and glycyl-prolyl-L-alanine (GPA).Second step relates to the GPA that releases is measured with fluorescent derivative fluoresceinamine subsequently.The experiment follows the following method, but those of ordinary skill in the art will appreciate that, under the premise of realizing experimental purpose, some modifications (such as dilution concentration and time) can be carried out.

[0270] The general method is as follows. Prepare a leucine standard. Obtain a collagenase sample and prepare a solution for the first step of the enzymatic cleavage process of zGPGGPA (hereinafter referred to as the "substrate") by collagenase. Following this step, the collagenase-treated sample (containing the released GPA) and the leucine standard are treated with fluorescein for a period of time at room temperature to fluorescently label the generated GPA and the free amino groups in the leucine molecules, respectively. After excitation at 392 nm, the fluorescence emission of each solution is measured at 480 nm. The slopes of the leucine and collagenase sample curves are used to calculate the potency units as follows:

[0271] Potency (f-GPA units / mg) = (M 样品 / M 亮氨酸 )x(DF / T)

[0272] in:

[0273] M 样品 = slope of collagenase titer curve

[0274] M 亮氨酸 =slope of the leucine standard curve

[0275] DF = dilution factor

[0276] T = reaction time

[0277] In addition, non-limiting details about the GPA experimental method are described below:

[0278] Buffers and reagents

[0279] 1. f-Appel buffer, pH 7.2 (55 mM HEPES, 100 mM calcium acetate)

[0280] 2.1 mM leucine working stock solution

[0281] 3.200 mM borate, pH 9.0

[0282] 4. 0.5 mM fluorescamine solution in acetone

[0283] 5. 2 mg / mL zGPGGPA substrate in f-Appel's buffer

[0284] Solution preparation

[0285] Prepare the solution as follows:

[0286] f-Appel buffer: Dissolve 13.0 g HEPES and 17.6 g calcium acetate in approximately 800 mL of water. Adjust pH to 7.2 with sodium hydroxide and adjust QS to 1 L with water. Store at 2-8°C.

[0287] 10 mM Leucine Stock Solution: Dissolve 65.5 mg of leucine in 50 mL of water. The leucine must be weighed directly into a 100 mL (or equivalent) glass beaker. Weigh approximately 65 mg (target weight) of leucine into the beaker. Based on the weight of the weighed leucine, calculate the amount of water to add to the beaker using the following formula. Add the calculated volume of water to the beaker and mix thoroughly to ensure complete dissolution of the leucine. Aliquot into 1 mL portions. Store at or below 20°C.

[0288] V2(mL)=C2(mg)x V1(50mL)

[0289] C1 (65.5 mg)

[0290] in:

[0291] C2 = weighed leucine weight (mg)

[0292] V1=50 (ml water)

[0293] C1=65.5 (mg leucine)

[0294] V2 = Volume of water required to produce a 10 mM stock solution (ml)

[0295] 1 mM Leucine Working Stock Solution: Thaw one vial of 10 mM Leucine stock solution and dilute to 1 mM by combining 150 μL with 1350 μL of water. Stir thoroughly before use.

[0296] 0.5N HCl: Dilute HCl to 0.5N with water and mix thoroughly. Store at room temperature. Alternatively, commercially available 0.5N HCl can be used.

[0297] 200 nM Borate, pH 9.0: Dissolve 2.4 g of boric acid in approximately 150 mL of water. Adjust the pH to 9 with sodium hydroxide. Bring the volume to 200 mL with water and mix thoroughly. Store at 2-8°C.

[0298] 0.5 mM Fluorescein Solution: Mix 15 mg of fluorescein with 100 ml of acetone and stir to dissolve. Store at 2-8 degrees Celsius in the dark.

[0299] Substrate solution (2 mg / mL zGPGGPA): Prepare 2 mg / mL substrate in f-Appel buffer and dissolve on a mechanical shaker / rotator, allowing sufficient time for complete dissolution (approximately 15 minutes).

[0300] Leucine standard curve

[0301] Prepare a leucine standard curve according to Table 7

[0302] Table 7. Preparation of leucine standard curve

[0303]

[0304] "L1" refers to Leucine Standard Sample 1

[0305] Then, 100 μL of leucine standard was transferred to a test tube and the fluorescamine was detected.

[0306] Collagenase sample preparation

[0307] Dilute the collagenase sample to 0.01 mg / mL in f-Appel buffer in two steps and mix gently. An exemplary dilution scheme is described below:

[0308] 1.100μL x 1.0mg / mL-→1000μL=0.1mg / mL

[0309] 2.100μL x 0.1mg / mL-→1000μL=0.01mg / mL

[0310] Blank preparation

[0311] Prepare a blank by mixing 45 μL of the diluted preparation with 500 μL of 0.5 N hydrochloric acid to inactivate the enzyme. Add 455 μL of the zGPGGPA substrate solution and stir thoroughly. Transfer 100 μL of the blank sample to a separate tube to detect impurities that may react with luciferamine.

[0312] potency curve

[0313] Prepare a set of titration curves for each collagenase sample as follows:

[0314] test tube 2 mg / mL substrate solution f-Appel buffer (μL) 1-2 1 45 3-4 1 30 5-6 1 15

[0315] Heat the tubes containing substrate and buffer in a 25°C water bath for at least 15 minutes. Label the second set of tubes and add 50 μL of 0.5N hydrochloric acid to each tube. Add the diluted collagenase sample preparation (0.01 mg / mL) according to Table 8 to the tubes and incubate for 10 minutes, mixing, and returning to the 25°C water bath. Begin the incubation phase by adding the first preparation to the preheated substrate.

[0316] Table 8. Sample preparation

[0317] preparation test tube Sample (μL) potency curve 1-2 55 potency curve 3-4 70 potency curve 5-6 85

[0318] With 1-2 minutes remaining in the 10-minute incubation, remove the preparations from the water bath and gently vortex to mix. Ten minutes after adding the first preparation to the substrate, transfer 50 μL from each tube to a tube containing 50 μL of 0.5 N HCl. These preparations should be added directly to the acid to inhibit digestion. After quenching all preparations, vortex each tube to mix thoroughly.

[0319] Detection

[0320] Add 400 μL of 200 mM borate buffer and 500 μL of 0.5 mM luciferamine solution to all test tubes containing 100 μL of each preparation (blank, collagenase sample titer curve, and leucine standard). Mix well. Incubate the tubes at room temperature for at least 10 minutes.

[0321] Fluorometer setup

[0322] Set up the fluorimeter using the following instrument parameters and read the fluorescence of each preparation after 1 h of derivatization.

[0323] parameter set up Excitation wavelength 392nm Excitation wavelength 480nm Integration 5.0sec. Cracks (Ex&Em) 5.0nm (passband plate) Path length 3mm

[0324] calculate

[0325] Plot the concentration of each leucine standard (X-axis) based on the fluorescence response at 480 nm (Y-axis). Determine the slope (m) and coefficient of determination (R2). Measure the mean fluorescence of each potency curve preparation. Prepare collagenase sample and leucine potency curves by plotting the concentration (X-axis) of each preparation against the mean fluorescence response at 480 nm (Y-axis). Determine the slope (m) and coefficient of determination (R2) of the resulting linear curve.

[0326] Determine the titer of collagenase samples

[0327] Potency (f-GPA units / mg) = (M 样品 / M 亮氨酸 )x(DF / T)

[0328] in:

[0329] M 样品 = Slope of the collagenase sample titer curve

[0330] M 亮氨酸 =slope of the leucine standard curve

[0331] DF = dilution factor (1100 μL / 50 μL = 22)

[0332] T = reaction time (10 minutes)

[0333] ii. GPA Microplate Assay for Determining Type II Collagenase Activity in Collagenase Samples

[0334] This method is similar to the GPA described above, except that it is performed in a microplate format. As with the above assay, the microplate assay measures the proteolytic activity of a collagenase sample in the enzymatic cleavage of the substrate carboxybenzyloxy-glycyl-L-prolyl-glycyl-glycyl-L-propyl-L-alanine (zGPGGPA) (hereinafter referred to as the "substrate"). The assay follows the following protocol, but those skilled in the art will appreciate that certain modifications (e.g., dilution concentration and time) can be made while remaining within the scope of achieving the objectives of this assay.

[0335] Reagents

[0336] 1. Peptide substrate (zGPGGPA) (Bachem M1260 or equivalent)

[0337] 2. Tripeptide GPA (Bachem H3615 or equivalent)

[0338] 3. Fluorescein (Acros 191675000 or equivalent)

[0339] 4. Purified water (Milli-Q-Plus 18.2MΩ system or equivalent)

[0340] 5.1 M HEPES buffer (Gibco 15630-080 or equivalent)

[0341] 6.Surfact-Amps 20 TM (10% Tween solution) (Pierce Cat. #28320 or equivalent)

[0342] 7.1 M calcium acetate (Ca(C2H3O2)2) (Emerald Biosciences, Cat.# EBS-100-CAAC or equivalent)

[0343] 8. Boric acid (Sigma B7660 or equivalent)

[0344] 9. 2.5N NaOH (JT Baker 5666-02 or equivalent)

[0345] 10. 0.5N hydrochloric acid (VWR 101223-134 or equivalent)

[0346] 11. Acetone (Sigma 270725 or equivalent)

[0347] Solution preparation

[0348] (i) Preparation of assay buffer (50 mM HEPES pH 7.1 / 0.05% Tween 20 / 5 mM (Ca(C2H3O2)2): Pipette 50 mL of 1 M HEPES into 800 mL of deionized water. Add 5 mL of 1 M (Ca(C2H3O2)2) and 5 mL of Surfact-Amps (10% Tween 20). Check the pH and adjust to 7.1 ± 0.05 if necessary. Add enough water to bring the volume to 1 L and filter the solution through a 0.22 μm filter. This assay buffer can be stored at room temperature for 3 months.

[0349] (ii) Preparation of 0.1N NaOH: Add 2 mL of 2.5N NaOH to 48 mL of deionized water. This solution can be stored at room temperature for 3 months.

[0350] (iii) Preparation of a 4 mg / mL stock solution of tripeptide GPA: Dissolve 400 mg (± 1 mg) of tripeptide GPA in 10 mL of 0.1 N NaOH and vortex until completely dissolved. Add sufficient assay buffer to bring the volume to 100 mL, divide the solution into 0.5 mL aliquots, and store at -70°C. The 4 mg / mL stock of tripeptide GPA can be stored at -70°C for one year.

[0351] (iv) Prepare 4 mg / mL (6.8 mM) peptide substrate zGPGGPA: Dissolve 400 mg (+1 mg) of the peptide substrate zGPGGPA in 10 mL of 0.1 N sodium hydroxide and vortex until completely dissolved. Add enough assay buffer to bring the volume to 100 mL. This solution can be stored at 4°C for 3 months.

[0352] (v) Prepare 120 mM boric acid at pH 9.0: Dissolve 7.4 g (± 0.5 g) of boric acid in 800 mL of deionized water. Titrate the solution to pH 9.0 with NaOH. Add enough deionized water to bring the volume to 1 L. This solution can be stored at room temperature for 3 months.

[0353] (vi) Prepare 1 mM fluorescamine in acetone: Dissolve 28 ± 2 mg of fluorescamine in 100 mL of acetone. This solution should be prepared fresh and protected from light and moisture.

[0354] Preparation and serial dilution of tripeptide GPA standards

[0355] Prepare a 0.08 mg / mL (329 μM) tripeptide GPA standard by diluting the 4 mg / mL tripeptide GPA stock solution 50-fold in assay buffer (e.g., diluting 20 μL of 4 mg / mL tripeptide GPA in 980 μL of assay buffer). In the assay plate, row A, pipette 200 μL of the 329 μM tripeptide GPA standard into A1 and A7. Pipette 100 μL of assay buffer into A2-A6 and A8-A12.

[0356] For the tripeptide GPA standard serial dilutions, transfer 100 μL from well A1 to well A2, mix, transfer 100 μL from well A2 to well A3, and repeat to well A5. Remove 100 μL from well A5 to bring the final volume to 100 μL. Well A6 contains only buffer.

[0357] For the second serial dilution of the tripeptide GPA standard, transfer 100 μL from A7 to A8, mix, transfer 100 μL from A8 to A9, and repeat until well A11. Remove 100 μL from well A11 to bring the final volume to 100 μL. Well A12 contains only buffer.

[0358] Preparation of collagenase samples

[0359] For collagenase samples (e.g., lyophilized collagenase drug products), cool the sample to room temperature over at least ten minutes and reconstitute into a 500 ng / mL stock solution. Different concentrations can be used. Prepare the collagenase sample to be tested (T1A) by diluting the stock solution with assay buffer. Repeat this procedure to prepare three test samples (T1A, T1B, T1C).

[0360] discuss

[0361] In this method, 50 μL of increasing concentrations of the collagenase sample to be tested are mixed with 50 μL of excess substrate (2.0 mg / mL final concentration) in a 96-well plate. 50 μL of assay buffer is added to rows CG of a U-bottom 96-well polypropylene reaction plate. 150 μL of the collagenase sample is pipetted to row B. Then, a 1 / 1.5 serial dilution is performed using a multichannel pipette, and 100 μL of the collagenase sample is pipetted from row B to row C, mixed, and the process is repeated until row G is reached. A 100 μL amount is removed from row G and discarded. Table 9 contains the final collagenase concentrations after adding 50 μL of substrate to rows B through H.

[0362] Prepare a row H blank by pipetting 50 μL of assay buffer into row H. This row contains no enzyme. Exemplary concentrations are shown in Table 9.

[0363] Table 9. Experimental target concentrations after substrate addition

[0364]

[0365] Collagenase reaction

[0366] The zGPGGPA substrate is cleaved into zGPG and GPA by type II collagenase during a 15-minute incubation at room temperature. Turn on the incubator and temperature probe (22 ± 1°C before adding substrate to the plate). Add 50 μL of 4 mg / mL (6.8 mM) zGPGGPA substrate to columns 1-12 of rows BH, column by column, and mix. The reaction begins after the addition of substrate to the first column. Cover the plate and place it in a 22 ± 1°C incubator for a total reaction time of 15 ± 1 minutes.

[0367] Following incubation, the reaction is quenched by the addition of hydrochloric acid, and the amount of released GPA peptide is quantified after the free amino terminus of the peptide reacts with the fluorescent reagent fluorescamine. To stop the reaction, 100 μL of 0.5 N HCl is added from row A to each well of row H, column by column, followed by mixing. The reaction time ends after the addition of HCl to the first column.

[0368] Detection

[0369] 195 μL 120mM borate (pH 9.0) was added to each well of a microplate Greiner polypropylene black reader. 30 μL of the quenched reaction mixture was transferred from the reaction plate to the corresponding well of the reader plate and thoroughly mixed. 75 μL 1mM fluorescamine was then added to each well of the reader plate (fluorescamine / acetone was dispensed using a polypropylene tray) and mixed immediately after each addition. The plate was read within 15 minutes after fluorescamine addition using a molecular device M2 fluorescence plate reader using the following settings: excitation 380 nm, emission 473 nm, cut-off 455 nm, 6 reads / well, PMT culture medium.

[0370] GPA concentration (μM) versus 473 nm emission, and collagenase concentration (ng / mL) versus 473 nm emission are plotted. For each plot, a linear regression fit with no fixed parameters was performed. For the collagenase test samples, zero-point data was excluded from the linear fit, and the plot was generated using the entire triplicate data set for each sample. The slopes were determined for the tripeptide GPA standard and the collagenase samples.

[0371] Potency determination

[0372] Calculate the specific activity of the collagenase sample as follows:

[0373] GPA microplate experimental unit = ((collagenase sample slope) / (tripeptide GPA slope x incubation time)) x 10 6 .

[0374] The specific activity of the collagenase test sample is determined by the slope of the tripeptide GPA standard and calculated using a curve fitting program. Using the Michaelis-Menton equation, enzyme kinetics can be calculated using different substrate concentrations and time periods using a microplate assay.

[0375] iii. Collagenase titer determined by GPA assay

[0376] Collagenases useful in the context of the present disclosure may have a titer of about 100,000 to about 300,000 GPA units / mg, or about 175,000 to about 300,000 f-GPA units / mg. In other embodiments, the titer may be about 70,000 to about 400,000 GPA units / mg, or about 100,000 to about 375,000 GPA units / mg, or about 125,000 to about 350,000 GPA units / mg, or about 150,000 to about 325,000 GPA units / mg, or about 175,000 to about 300,000 GPA units / mg, or about 200,000 to about 275,000 GPA units / mg. Alternatively, the titer can be from about 70,000 to about 400,000 f-GPA units / mg, or from about 100,000 to about 375,000 f-GPA units / mg, or from about 125,000 to about 350,000 f-GPA units / mg, or from about 150,000 to about 325,000 f-GPA units / mg, or from about 175,000 to about 300,000 f-GPA units / mg, or from about 230,000 to about 430,000 f-GPA units / mg, or from about 200,000 to about 275,000 f-GPA units / mg. The collagenase can also have a potency of from about 30,100 to 87100 or from about 43,000 to 67,000 GPA microplate assay units. The above GPA can be used to analyze the specific activity of any collagenase.

[0377] b. SRC Unit Analysis Method and Specific Activity Units

[0378] i. Collagenase titer by SRC assay (cup)

[0379] The SRC experiment is mainly used to measure the potency of class I collagenase. The general method is as follows. A leucine standard and a collagenase sample solution are prepared. The first step of the experiment involves the enzymatic reaction of collagenase digesting soluble rat tail tendon collagen (SRC). The second step involves the subsequent measurement of the released peptide fragments / amino acids using the fluorescent derivative fluorescamine. The experiment follows the following method, but a person of ordinary skill in the art will understand that certain modifications (such as dilution concentration and time) can be made while achieving the purpose of the experiment.

[0380] This collagenase and leucine standard sample was treated with a reagent to label the resulting GPA with fluorescein. The leucine standard and collagenase sample were incubated at room temperature for 10 minutes, and the fluorescence of each solution was measured at excitation and emission wavelengths of 392 and 480 nm, respectively. The slopes of the leucine and collagenase sample curves were used to calculate the titer units as follows:

[0381] Potency (f-GPA units / mg) = (M 样品 / M 亮氨酸 )x(DF / T)x CF

[0382] in:

[0383] M 样品 = slope of collagenase titer curve

[0384] M 亮氨酸 =slope of the leucine standard curve

[0385] DF = dilution factor (1500 μL / 100 mL = 15)

[0386] T = reaction time (2.5hr x 60min / 1hr = 150min)

[0387] CF = conversion factor (1000 μg / 1 mg = 1000)

[0388] Additionally, non-limiting details regarding the SRC experimental method are described below.

[0389] Buffers and reagents

[0390] 1. F-TC assay buffer, pH 7.2 (22 g HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 4.4 g calcium acetate)

[0391] 2. F-enzyme buffer, pH 7.2

[0392] 3.200 mM borate, pH 9.0

[0393] 4.10 mM Leucine Stock Solution

[0394] 5.1 mM Leucine working stock solution

[0395] 6.1 mM fluorescamine solution in acetone

[0396] 7. Rat tail collagen at a concentration of 2 mg / mL in 0.02N acetic acid

[0397] Solution preparation

[0398] Prepare the solution as follows

[0399] F-TC Assay Buffer: Dissolve 22g HEPES and 4.4g calcium acetate in approximately 900mL of water. Adjust pH to 7.2 with sodium hydroxide and adjust QS to 1L with water. Store at 2-8°C.

[0400] F-Enzyme-Buffer: Dilute F-TC Assay Buffer by combining 4 ml of F-TC Assay Buffer with 16 mL of water. Store at 2-8°C.

[0401] 10 mM Leucine Stock Solution: Dissolve 65.5 mg of leucine in 50 mL of water. The leucine must be weighed directly into a 100 mL (or equivalent) glass beaker. Weigh approximately 65 mg (target weight) of leucine into the beaker. Based on the weight of the weighed leucine, calculate the amount of water to add to the beaker using the following formula. Add the calculated volume of water to the beaker and mix thoroughly to ensure complete dissolution of the leucine. Aliquot into 1 mL portions. Store at or below -20°C.

[0402]

[0403] in

[0404] C2 = weighed mass of leucine (mg)

[0405] V1=50 (mL of water)

[0406] C1=65.5 (mg of leucine)

[0407] V2 = Volume of water (mL) required to produce a 10 mM stock solution

[0408] 1 mM Leucine Working Stock Solution: Thaw one vial of 10 mM Leucine stock solution and dilute 150 μL to 1 mM by mixing with 1350 μL of water. Stir thoroughly before use.

[0409] 0.5N HCl: Dilute HCl to 0.5N with water and mix thoroughly. Store at room temperature. Alternatively, commercially available 0.5N HCl can be used.

[0410] 0.02N Acetic Acid: Combine 1 mL of 1N acetic acid with 49 mL of water and mix thoroughly. Store at room temperature.

[0411] 200 mM Borate, pH 9.0: Dissolve 2.4 g of boric acid in approximately 150 ml of water. Adjust the pH to 9.0 with sodium hydroxide. Add QS to 200 ml with water and mix thoroughly. Store at 2-8°C.

[0412] 1 mM fluorescamine solution: Dissolve 15 mg of fluorescamine in 50 μl of acetone and stir to dissolve. Store at 2-8°C in the dark.

[0413] Substrate Solution (2 mg / mL Rat Tail Collagen): Dilute rat tail collagen to 2 mg / mL with 0.02 N acetic acid. Store at 2-8°C.

[0414] Leucine standard curve

[0415] Prepare a leucine standard curve according to Table 10.

[0416] Table 10. Preparation of leucine standard curve

[0417]

[0418] Then transfer 100 μL of each leucine standard to a separate centrifuge tube for fluorescein detection.

[0419] Collagenase sample and blank preparation

[0420] Dilute the collagenase sample to 0.01 mg / mL in F-enzyme buffer in two steps and vortex gently to mix. An exemplary dilution scheme is described below:

[0421] 1.100μL x 1.0mg / mL→1000μL=0.1mg / mL

[0422] 2.100μL x 0.1mg / mL→1000μL=0.01mg / mL

[0423] Maintain diluted samples at room temperature until used.

[0424] According to Table 11, blanks were prepared by first combining the sample with 0.5 N hydrochloric acid to inactivate the enzyme, followed by the addition of buffer and substrate.

[0425] The individual collagenase sample tubes were labeled according to Table 11. Tubes 1, 2, 4, and 6 were prepared from one preparation and tubes 3, 5, and 7 were prepared from duplicate preparations.

[0426] Table 11. Blank and collagenase sample preparations

[0427]

[0428] Cap the tubes and swirl gently to mix. Incubate the titer curve preparations in a water bath at 25°C ± 3°C for 2.5 hours. After the incubation period, remove the titer curve tubes from the water bath. Add 750 μL of 0.5N HCl to each preparation and swirl thoroughly to mix. Preparations can be stored at 2-8°C for 22 hours prior to testing.

[0429] Detection / Fluorometer Setup

[0430] Leucine standards were prepared as described above.

[0431] Set up the fluorimeter using the following instrument parameters and read the fluorescence of each preparation after 1 h of derivatization.

[0432] parameter set up Excitation wavelength 392nm Excitation wavelength 480nm Integration 5.0sec. Cracks (Ex&Em) 5.0nm (passband plate) Path length 3mm

[0433] calculate

[0434] Plot the concentration of each leucine standard (X-axis) against the fluorescence response at 480 nm (Y-axis). Determine the slope (m) and coefficient of determination (R²). Determine the mean fluorescence of each titer curve preparation. Do not force zero. Determine the mean fluorescence of each replicate preparation. Calculate the net fluorescence of each collagenase sample preparation.

[0435] F(net) = average collagenase sample (EM 480 )-Blank(EM 480 )

[0436] Plot the concentration of collagenase sample in each formulation (X-axis) versus net fluorescence (Y-axis). Determine the slope (m) and factor of determination (R 2 ). Do not force zeroing.

[0437] Determine the titer of collagenase samples

[0438] Potency (f-SRC units / mg) = (M 样品 / M 亮氨酸 )x(DF / T)x CF

[0439] in:

[0440] M 样品 = Slope of the collagenase sample titer curve

[0441] M 亮氨酸 =slope of the leucine standard curve

[0442] DF = dilution factor (1500 μL / 100 mL = 15)

[0443] T = reaction time (2.5 hr x 60 minutes / 1 hr = 150 minutes)

[0444] CF = conversion factor (1000 μg / 1 mg = 1000)

[0445] The SRC assay described above can be used to analyze the specific activity of any collagenase.

[0446] ii. SRC Microplate Assay for Determining Type I Collagenase Activity in Collagenase Samples

[0447] This method is similar to the SRC assay described above, except that it is performed in a microplate format. As with the SRC assay described above, the microplate assay measures collagenase activity in the enzymatic cleavage of a soluble rat-tail collagen (SRC) substrate (hereinafter referred to as the "substrate"). The assay follows the following protocol, but those skilled in the art will appreciate that certain modifications (e.g., dilution concentration and duration) can be made to achieve the objectives of this assay.

[0448] Reagents

[0449] 1. Soluble rat-tail collagen substrate (BD Biosciences 354236)

[0450] 2. Tripeptide GPA (Bachem H3615 or equivalent)

[0451] 3. Fluorescein (Acros 191675000 or equivalent)

[0452] 4. Purified water (Milli-Q-Plus 18.2MΩ system or equivalent)

[0453] 5.1 M HEPES buffer (Gibco 15630-080 or equivalent)

[0454] 6.1 M calcium acetate (Ca(C2H3O2)2) (Emerald Biosciences, Cat.# EBS-100-CAAC or equivalent)

[0455] 7.Surfact-Amps 20 TM (10% Tween solution) (Pierce Cat. #28320 or equivalent)

[0456] 8. 1.0N acetic acid (Sigma 318590 or equivalent)

[0457] 9. 0.5N hydrochloric acid (VWR 101223-134 or equivalent)

[0458] 10. Boric acid (Sigma B7660 or equivalent)

[0459] 11.2.5N sodium hydroxide (JT Baker 5666-02 or equivalent)

[0460] 12. Acetone (Sigma 270725 or equivalent)

[0461] Solution preparation

[0462] (i) Preparation of assay buffer (50 mM HEPES pH 7.1 / 0.05% Tween 20 / 5 mM (Ca(C2H3O2)2): Pipette 50 mL of 1 M HEPES into 800 mL of deionized water. Add 5 mL of 1 M (Ca(C2H3O2)2) and 5 mL of Surfact-Amps (10% Tween 20). Check the pH and adjust to 7.1 ± 0.1 if necessary. Add enough water to bring the volume to 1 L and filter the solution through a 0.22 μm filter. This assay buffer can be stored at room temperature for 3 months.

[0463] (ii) Preparation of 0.1N NaOH: Add 2 mL of 2.5N NaOH to 48 mL of deionized water. This solution can be stored at room temperature for 3 months.

[0464] (iii) Preparation of a 4 mg / mL stock solution of tripeptide GPA: Dissolve 400 mg (± 1 mg) of tripeptide GPA in 10 mL of 0.1 N NaOH and vortex until completely dissolved. Add sufficient assay buffer to bring the volume to 100 mL, divide the solution into 0.5 mL aliquots, and store at -70°C. The 4 mg / mL stock of tripeptide GPA can be stored at -70°C for one year.

[0465] (iv) Preparation of 0.02N Acetic Acid: Add 1 mL of 1.0N acetic acid to 40 mL of purified water. Add enough purified water to adjust the volume to 50 mL. This solution can be stored at room temperature for one year.

[0466] (v) Preparation of 2 mg / mL SRC substrate stock solution: Add 23.3 mL of 0.02 N acetic acid directly to the test tube containing the substrate (in one non-limiting example, 100 mg of SRC is supplied at a concentration of 3.75 mg / mL). Other concentrations of SRC substrate may be used. Calculation is as follows:

[0467] 100 mg ÷ 3.75 mg / mL = 26.7 mL;

[0468] Total volume (mL) = (3.75mg / mL x 26.7mL) / 2mg / mL;

[0469] Total volume (50.0 mL) - 26.7 mL = 23.3 mL

[0470] The solution was mixed thoroughly by inversion and can be stored at 2-8°C for 3 months.

[0471] (vi) Prepare 0.6 mg / mL SRC substrate working solution: Add 4.2 mL of assay buffer to a 15 mL conical tube. Then, add 1.8 mL of 2 mg / mL SRC substrate stock solution and mix by inversion. This solution should be prepared immediately before adding to the plate.

[0472] (vii) Prepare 120 mM boric acid at pH 9.0: Dissolve 7.4 g (± 0.5 g) of boric acid in 800 mL of deionized water. Titrate the solution to pH 9.0 with NaOH and add enough deionized water to adjust the volume to 1 L. This solution can be stored at room temperature for 3 months.

[0473] (viii) Prepare 1 mM fluorescamine in acetone: Dissolve 28 ± 2 mg of fluorescamine in 100 mL of acetone. This solution should be prepared fresh and protected from light and moisture.

[0474] Preparation and serial dilution of tripeptide GPA standards

[0475] Prepare a 0.08 mg / mL (329 μM) tripeptide GPA standard by diluting a 4 mg / mL tripeptide GPA stock solution 50-fold in assay buffer (e.g., diluting 20 μL of 4 mg / mL tripeptide GPA in 980 μL of assay buffer). In the assay plate, row A, pipette 200 μL of 329 μM tripeptide GPA standard into A1 and A7. Pipette 100 μL of assay buffer into A2-A6 and A8-A12.

[0476] For the tripeptide GPA standard serial dilutions, transfer 100 μL from well A1 to well A2, mix, transfer 100 μL from well A2 to well A3, and repeat to well A5. Remove 100 μL from well A5 to bring the final volume to 100 μL. Well A6 contains only buffer.

[0477] For the second serial dilution of the tripeptide GPA standard, transfer 100 μL from A7 to A8, mix, transfer 100 μL from A8 to A9, and repeat until well A11. Remove 100 μL from well A11 to bring the final volume to 100 μL. Well A12 contains only buffer.

[0478] Preparation of collagenase samples to be tested

[0479] For collagenase samples (e.g., lyophilized collagenase drug product), cool the sample to room temperature over at least ten minutes and reconstitute into a 3.0 μg / mL stock solution. Different concentrations can be used. Prepare the collagenase sample to be tested (T1A) by diluting the stock solution with assay buffer. Repeat this procedure to prepare three test samples (T1A, T1B, T1C).

[0480] discuss

[0481] In this method, 50 μL of increasing concentrations of the collagenase sample to be tested are mixed with 50 μL of excess substrate (2.0 mg / mL final concentration) in a 96-well plate. 50 μL of assay buffer is added to rows CG of a U-bottom 96-well polypropylene reaction plate. 150 μL of collagenase sample is pipetted to row B. Then, a 1 / 1.5 serial dilution is performed using a multichannel pipette, and 100 μL of collagenase sample is pipetted from row B to row C, mixed, and the process is repeated until row G is reached. A 100 μL amount is removed from row G and discarded. A row H blank is prepared by pipetting 50 μL of assay buffer into row H. This row does not contain enzyme. Table 12 contains the final collagenase concentrations after adding 50 μL of substrate to rows B through H.

[0482] Table 12. Experimental target concentrations after substrate addition

[0483]

[0484] Collagenase reaction

[0485] Turn on the incubator and temperature probe (22 ± 1°C before adding substrate to the plate). Add 50 μL of 0.6 mg / mL SRC substrate to each well of rows BH, adding column by column and mixing. The reaction starts after the substrate is added to the first column. Cover the plate and place it in the incubator at 22 ± 1°C for a total reaction time of 45 ± 5 minutes. After the incubation, quench the reaction by adding 100 μL of 0.5 N hydrochloric acid to each well of the dilution plate, adding column by column and mixing. The reaction ends after the HCl is added to the first column.

[0486] Detection

[0487] 195 μL 120mM borate (pH 9.0) was added to each well of a microplate Greiner polypropylene black reader. 30 μL of the quenching reaction mixture was transferred from the reaction plate to the corresponding well of the reading plate and mixed thoroughly. 30 microliters of the annealing reaction mixture were transferred from the reaction plate to the corresponding well of the reading plate and mixed thoroughly. Then, 75 μL 1mM fluorescamine was added to each well of the reading plate (fluorescamine / acetone was distributed using a polypropylene tray) and mixed immediately after each addition. The plate was read within 15 minutes after fluorescamine addition using a molecular device M2 fluorescence plate reader using the following settings: excitation 380 nm, emission 473 nm, cut-off 455 nm, 6 reads / well, PMT culture medium.

[0488] GPA concentration (μM) versus 473 nm emission, and collagenase concentration (ng / mL) versus 473 nm emission are plotted. For each plot, a linear regression fit with no fixed parameters was performed. For the collagenase test samples, zero-point data was excluded from the linear fit, and the plot was generated using the entire triplicate data set for each sample. The slopes were determined for the tripeptide GPA standard and the collagenase samples.

[0489] Determination of specific activity and related titers

[0490] Calculate the specific activity of the collagenase sample as follows:

[0491] SRC microplate experimental unit = ((collagenase sample slope) / (tripeptide GPA slope x incubation time)) x 10 6

[0492] The specific activity of the collagenase test sample is determined by the slope of the tripeptide GPA standard and calculated using a curve fitting program. Using the Michaelis-Menton equation, enzyme kinetics can be calculated using different substrate concentrations and time periods using a microplate assay.

[0493] iii. Collagenase titer determined by SRC assay

[0494] The titer of the collagenase effective in the context of the present disclosure is about 500 to 15,000 SRC units / mg. In certain embodiments, the titer is about 500 to about 12,500 SRC units / mg, or about 700 to about 10,000 SRC units / mg, or about 1,000 to about 7,500 SRC units / mg, or 1,500 to about 6,000 SRC units / mg, or about 2,500 to about 5,000 SRC units / mg. Alternatively, the titer can be about 5,000 to about 35,000 f-SRC units / mg, or about 10,000 to about 30,000 f-SRC units / mg, or about 13,000 to about 23,000 f-SRC units / mg, or about 15,000 to about 25,000 f-SRC units / mg. The collagenase may also have a potency of about 980 to 3,510 or about 1,400 to 2,700 SRC microplate assay units.

[0495] c. Collagenase titer in BTC unit experiment

[0496] The bovine tendon collagenase assay is based on the method published by Mandl et al. (1958), modified by Keller and Mandl (1963). Because bovine tendon collagen is an insoluble substrate, it is important to isolate it carefully. Trypsin is run as a control to account for the presence of denatured collagen or other protein impurities. The assay is performed in the presence of calcium ions, which are required for collagenase activity. The amount of soluble peptide is determined by reacting the N-terminal amino group of the peptide with ninhydrin and measuring the amount of the auxiliary formed by colorimetry (Rosen 1957).

[0497] The purpose of this method is to detect the specific activity of collagenase using collagen substrate.

[0498] Reagents and solutions

[0499] 1. Collagen substrate (collagen)

[0500] 2. Deionized water (water)

[0501] 3.Tris assay buffer

[0502] 4. Trypsin Stock Solution

[0503] 5.0.5M HCl

[0504] 6. Leucine standard experimental solution (1 mM leucine)

[0505] 7. Rosen buffer

[0506] 8.3% ninhydrin

[0507] 9.50% isopropyl alcohol

[0508] nourish

[0509] Set up and label the reaction tubes as follows: three tubes for the trypsin control, six tubes for the reference solution, and six tubes for each sample to be tested. Label each tube and remove the cap. Weigh 10 ± 1 mg of collagen according to the sequence in Table 13 and place the weighed amount into each reaction tube.

[0510] Table 13. Weighing order and reaction tube number

[0511] Weighing order Reaction tube# 1 1 2 4 3 6 4 8 5 10 6 12 7 14 8 16 9 18 10 20 11 2 12 3 13 5 14 7 15 9 16 11 17 13 18 15 19 17 20 19 21 21

[0512] For the test samples, the enzyme activity in each reaction tube (ACT) should be between 1.6 and 5.7 nmol leu eq / min. Undissolved samples should be dissolved in Tris assay buffer before use in the assay. The concentration (before addition to the reaction tube) should be at least 0.0065 mg / mL.

[0513] Set up the reaction tubes for matrix mode according to Table 14. The table below assumes two samples are being tested. If running more or fewer samples, adjust the number of reaction tubes but retain the matrix mode. Table 14 lists the scenarios where the volumes remain constant.

[0514] Table 14. Matrix Mode

[0515]

[0516] *The maximum recommended number of samples is 3

[0517] Cap the reaction tube. Gently but thoroughly mix the contents. Place the reaction tube in a 37°C water bath. Incubate for 22 ± 0.5 hours. Record the actual time a, the temperature at which the incubation started, the temperature at 37°C, the number of water baths used, the lot number of collagen lipids, the collagen correction factor for the lot number used, and the lot numbers of all solutions used.

[0518] Quenching and filtering

[0519] Label the filtrate tubes corresponding to each reaction tube in the incubation. Place a funnel filled with folded filter paper over each labeled filtrate tube. At the end of the incubation period, remove the reaction tubes from the water bath. Record the actual time the incubation ended.

[0520] Open the reaction tubes and discard the caps. Quench the reaction by dispensing 2 mL of 0.5 M HCl into each tube. Mix the contents thoroughly. Filter the contents of each tube into an appropriate filter tube.

[0521] The first two steps need to be completed quickly because undigested collagen can be dissolved by hydrochloric acid in a short time. The filtrate can be refrigerated in the capped filtrate tube for up to 95.5 hours before development. Record the refrigeration and storage times.

[0522] Color rendering

[0523] Set up the boiling test tubes and label them as follows: 6 test tubes for the water and leucine controls (Step 1), and 2 test tubes for each filtrate bucket (Step 1). Place the following amounts of water and leucine standard assay solution into the six leucine control test tubes.

[0524] test tube# 1 2 3 4 5 6 Water (μL) 1000 900 850 800 750 700 Leucine (μL) 0 100 150 200 250 300 Leucine (nmol) 0 100 150 200 250 300

[0525] Use a pipette to move 0.8mL of water to each boiled test tube (step 2). Use a pipette to draw 0.2mL of filtrate from each sample and place it into an appropriately labeled boiled test tube. Add 0.5mL of Rosen buffer to each boiled test tube. Under a sealed hood, distribute 0.5mL of 3% ninhydrin to each boiled test tube. Mix the contents of each tube thoroughly on a vortex mixer. Place the boiled test tubes in a boiling water bath in a fume hood. Boil for 15±1 minutes. At the end of the boiling period, remove the boiled test tubes from the water bath. Under a sealed hood, distribute 5.0mL of 50% isopropanol to each boiled test tube and mix the contents thoroughly. Allow the boiled test tubes to reach ambient temperature (at least 10 minutes) before reading the absorbance.

[0526] Absorbance readings

[0527] While working under a sealed hood, read the absorbance of each test tube. Turn on the spectrophotometer and allow it to warm up. Set the spectrophotometer to a wavelength of 570 nm. Zero the spectrophotometer with 50% isopropanol. Read the absorbance (A) of the water, leucine, trypsin control, and test sample. 570 ). Record the time in hours when the first sample is read. Record the reading as 1000X A 570 , and record the time (in hours) when the last sample was read. All readings should be completed within 1 hour intervals

[0528] Calculation principles

[0529] Calculate the total reading time and total incubation time in minutes. The total reading time should be less than 60 minutes and the total incubation time should be between 1290-1350 minutes. Using the linear least squares method, calculate the slope "b" and the correlation coefficient "r" (x = nmol leucine vs y = A) of the leucine standard. 570 The unit of “b” value is A 470 / nmol leucine. Record the "b" value to two decimal places. The b value for leucine should be between 2.88-3.33. Calculate the average reading for the trypsin control (T). The average reading for the trypsin control (T) should be between 221-338. Record this average value to the nearest whole number (step A). ​​Average replicate A for each reaction tube. 570 Read the number. Record this number to the nearest whole number. From the average sample A 570 The average trypsin concentration (step A) was subtracted from the readings to obtain the net sample readings.

[0530] Calculate the activity (ACT) of each tube in nmol leu eq / min as follows:

[0531]

[0532] Where 20 is the dilution factor for the reaction mixture, and "b" is the slope of the leucine standard curve. Write this number to one decimal place. The activity of each test sample tube should be between 1.6 and 5.7 nmol leu eq / min.

[0533] Activity in BTC units is calculated as follows:

[0534] BTC unit = activity in nmol leu eq / min x collagen correlation coefficient.

[0535] Calculate the activity of the sample in BTC units / mL as follows:

[0536]

[0537] The specific activity of the sample in BTC units / mm was calculated as follows:

[0538]

[0539] Conversion of BTC units to ABC units:

[0540] ABC unit = BTC unit x 1.09

[0541] i.BTC units and ABC units

[0542] Various collagenase compositions can be used, wherein the collagenase has a specific activity of about 5,000 BTC units / mg to about 25,000 BTC units / mg, or about 10,000 BTC units / mg to about 25,000 BTC units / mg, or about 15,000 BTC units / mg, or about 17,500 BTC units / mg, or about 20,000 BTC units / mg, or about 22,500 BTC units / mg, or about 9,175 BTC units / 0.58 mg, or 15,817 BTC units / mg, where "mg" refers to the amount of collagenase present in the composition (other than excipients and other ingredients).

[0543] In addition, various collagenase compositions can be used in which the collagenase has a specific activity of about 5,000 ABC units / mg to about 25,000 ABC units / mg, or about 10,000 ABC units / mg to about 25,000 ABC units / mg, or about 15,000 ABC units / mg or about 17,500 ABC units / mg, or about 20,000 ABC units / mg, or about 22,500 ABC units / mg, or about 10,000 ABC units / 0.58 mg, or 17,241 ABC units / mg, where "mg" refers to the amount of collagenase present in the composition (other than excipients and other ingredients).

[0544] d. Other experiments

[0545] Experimental methods for labeling collagen are described in Gisslow et al., Anal. Biochem., 68:70-78 (1975); Robertson et al., Clinica Chimica Acta, 42:43-45 (1972); and Sakamoto et al., A New Method for the Assay of Tissue Collagenase (36297) (1972). Other methods include those from Worthington Biochemical Company (http: / / www.worthington-biochem.com / CLS / assay.html) (accessed July 3, 2019).

[0546] 4. Collagenase dosage

[0547] With respect to the dosage of collagenase employed herein, the present invention provides a therapeutically effective amount of collagenase sufficient to bind to and dissolve the membrane upon subcutaneous injection, thereby reducing the appearance of subcutaneous cellulite compared to a pre-treatment baseline.

[0548] In one embodiment, collagenase can be injected in an amount of about 0.01 mg to about 20 mg in a single or divided injection. In another embodiment, collagenase can be injected in an amount of about 0.05 mg to about 15 mg in a single or divided injection. In another embodiment, collagenase can be injected in an amount of about 0.10 mg to about 10 mg in a single or divided injection. In another embodiment, collagenase can be injected in an amount of about 0.15 mg to about 5 mg in a single or divided injection. In another embodiment, collagenase can be injected in an amount of about 0.20 mg to about 3 mg in a single or divided injection. In another embodiment, collagenase can be injected in an amount of about 0.25 mg to about 2 mg in a single or divided injection. In yet another embodiment, the collagenase may be injected at about 0.05 mg, about 0.10 mg, about 0.15 mg, about 0.20 mg, about 0.25 mg, about 0.30 mg, about 0.35 mg, about 0.40 mg, about 0.45 mg, about 0.50 mg, about 0.55 mg, about 0.60 mg, about 0.65 mg, about 0.70 mg, about 0.75 mg, about 0.80 mg, about 0.85 mg, about 0.90 mg, about 0.95 mg, about 1 .00mg, 1.05mg, about 1.10mg, about 1.15mg, about 1.20mg, about 1.25mg, about 1.30mg, about 1.35mg, about 1.40mg, about 1.45mg, about 1.50mg, about 1.55mg, about 1.60mg, about 1.65mg, about 1.70mg, about 1.75mg, about 1.80mg, about 1.85mg, about 1.90mg, about 1.95mg, about 2.00mg, 2.05mg, about 2.1 0mg, about 2.15mg, about 2.20mg, about 2.25mg, about 2.30mg, about 2.35mg, about 2.40mg, about 2.45mg, about 2.50mg, about 2.55mg, about 2.60mg, about 2.65mg, about 2.70mg, about 2.75mg, about 2.80mg, about 2.85mg, about 2.90mg, about 2.95mg, about 3.00mg, 3.05mg, about 3.10mg, about 3.15mg, about 3. 20mg, about 3.25mg, about 3.30mg, about 3.35mg, about 3.40mg, about 3.45mg, about 3.50mg, about 3.55mg, about 3.60mg, about 3.65mg, about 3.70mg, about 3.75mg, about 3.80mg, about 3.85mg, about 3.90mg, about 3.95mg, about 4.00mg, 4.05mg, about 4.10mg, about 4.15mg, about 4.20mg, about 4.25mg, about 4.30mg, about 4.35mg, about 4.40mg, about 4.45mg, about 4.50mg, about 4.55mg, about 4.60mg, about 4.65mg, about 4.70mg, about 4.75mg, about 4.80mg, about 4.85mg, about 4.90mg, about 4.95mg, about 5.00mg, 5.05mg, about 5.10mg, about 5.15mg, about 5.20mg, about 5.25mg, about 5.30mg, about 5.35mg, about 5.40mg, about 5.45mg, about 5.50mg, about 5.55mg, about 5.60mg, about 5.65mg, about 5.70mg, about 5.75mg, about 5.80mg, about 5.85mg, about 5.90mg, about 5.95mg, or about 6.00mg.

[0549] In one embodiment, the collagenase has a kinetic energy of approximately 2.6 min according to the results of the SRC assay. -1 to 5.2 minutes -1 V max In other embodiments, the collagenase has a reaction time of approximately 3.0 min, as determined by the SRC assay. -1 to 5.0min -1 V max In other embodiments, the collagenase has a reaction time of approximately 3.4 min according to the results of the SRC experiment. -1 to 4.8 minutes -1 V max In still another embodiment, the collagenase has a reaction time of about 3.5 min according to the results of the SRC experiment. -1 to 4.5 minutes -1 V max In still another embodiment, the collagenase has a reaction time of about 2.0 min according to the results of the SRC experiment. -1 , about 2.1 minutes -1 , about 2.2 minutes -1 , about 2.3 minutes -1 , about 2.4 minutes -1 , about 2.5 minutes -1 , about 2.6 minutes -1 , about 2.7 minutes -1 , about 2.8 minutes -1 , about 2.9 minutes -1 , about 3.0 minutes -1 , about 3.1 minutes -1 , about 3.2 minutes -1, about 3.3 minutes -1 , about 3.4 minutes -1 , about 3.5 minutes -1 , about 3.6 minutes -1 , about 3.7 minutes -1 , about 3.8 minutes -1 , about 3.9 minutes -1 , about 4.0 minutes -1 , about 4.1 minutes -1 , about 4.2 minutes -1 , about 4.3 minutes -1 , about 4.4 minutes -1 , about 4.5 minutes -1 , about 4.6 minutes -1 , about 4.7 minutes -1 , about 4.8 minutes -1 , about 4.9 minutes -1 , about 5.0 minutes -1 , about 5.1 minutes -1 , about 5.2 minutes -1 , about 5.3 minutes -1 , about 5.4 minutes -1 , about 5.5 minutes -1 , about 5.6 minutes -1 , about 5.7 minutes -1 , about 5.8 minutes -1 , about 5.9 minutes -1 , or about 6.0 minutes -1 V max In other embodiments, the collagenase has a reaction time of approximately 0.7 min, as determined by the SRC assay. -1 to 7.6 minutes -1 , or about 1 to 6, or about 2 to 5, or about 3 to 4 minutes -1 V max .

[0550] In one embodiment, the collagenase has a bioavailability of approximately 135 min according to the results of the GPA experiment. -1 To 268 minutes -1 V max In other embodiments, the collagenase has a reaction time of about 150 min according to the results of the GPA experiment. -1 to 250 minutes -1 V max In other embodiments, the collagenase has a reaction time of approximately 175 min according to the results of the GPA experiment.-1 to 225 minutes -1 V max In still another embodiment, the collagenase has a bioavailability of approximately 130 min according to the results of the GPA experiment. -1 , about 135 minutes -1 , about 140 minutes -1 , about 145 minutes -1 , about 150 minutes -1 , about 155 minutes -1 , about 160 minutes -1 , about 165 minutes -1 , about 170 minutes -1 , about 175 minutes -1 , about 180 minutes -1 , about 185 minutes -1 , about 190 minutes -1 , about 195 minutes -1 , about 200 minutes -1 , about 205 minutes -1 , about 210 minutes -1 , about 215 minutes -1 , about 220 minutes -1 , about 225 minutes -1 , about 230 minutes -1 , about 235 minutes -1 , about 240 minutes -1 , about 245 minutes -1 , about 250 minutes -1 , about 255 minutes -1 , about 260 minutes -1 , about 265 minutes -1 , about 270 minutes -1 , about 275 minutes -1 , or about 280 minutes -1 V max In other embodiments, the collagenase has a reaction time of about 4 min according to the results of the GPA experiment. -1 to 400 minutes -1 V max , or according to the results of the GPA experiment, the collagenase has a reaction time of about 0.3 to 30.5, or about 10 to 375, or about 20 to 350, or about 50 to 300, or about 100 to 275 min -1 V max .

[0551] In one embodiment, according to the results of the SRC experiment, the K of the collagenase is m In other embodiments, the collagenase K is about 75mM to 147mM, as determined by the SRC assay. m In other embodiments, the collagenase K is about 80mM to 140mM, as determined by the SRC assay. m In other embodiments, the collagenase K is about 85mM to 130mM, as determined by the SRC assay. m In still other embodiments, according to the results of the SRC experiment, the K of the collagenase is about 90mM to 120mM. m It is about 70mM, about 72mM, about 75mM, about 77mM, about 80mM, about 82mM, about 85mM, about 87mM, about 90mM, about 92mM, about 95mM, about 97mM, about 100mM, about 102mM, about 105mM, about 107mM, about 110mM, about 112mM, about 115mM, about 117mM, about 120mM, about 122mM, about 125mM, about 127mM, about 130mM, about 132mM, about 135mM, about 137mM, about 140mM, about 142mM, about 145mM, about 147mM, about 150mM, about 152mM, about 155mM, or about 157mM. In other embodiments, according to the results of the SRC experiment, the K of the collagenase m is about 4.4mM to 437mM, or according to the results of the SRC experiment, the K m It is about 5 to 400, or about 20 to 375, or about 50 to 325, or about 100 to 275, or about 150 to 250 mM, or about 4.1 to 410 nanomolar.

[0552] In one embodiment, according to the results of the GPA experiment, the K m In other embodiments, the collagenase K is about 0.03mM to 3.1mM according to the results of the GPA experiment. m In other embodiments, the collagenase K is about 1.00 mM to 1.60 mM, as determined by the GPA assay. m In other embodiments, the collagenase K is about 1.10mM to 1.50mM, as determined by the GPA assay. mIn still other embodiments, according to the results of the GPA experiment, the K of the collagenase is about 1.15mM to 1.40mM. m is about 0.80mM, about 0.82mM, about 0.85mM, about 0.87mM, about 0.90mM, about 0.92mM, about 0.95mM, about 0.97mM, about 1.00mM, about 1.02mM, about 1.05mM, about 1.07mM, about 1.10mM, about 1.12mM, about 1.15mM, about 1.17mM, about 1.20mM, about 1.22mM, In other embodiments, the collagenase has a K of about 1.25 mM, about 1.27 mM, about 1.30 mM, about 1.32 mM, about 1.35 mM, about 1.37 mM, about 1.40 mM, about 1.42 mM, about 1.45 mM, about 1.47 mM, about 1.50 mM, about 1.52 mM, about 1.55 mM, about 1.57 mM, about 1.60 mM, about 1.62 mM, about 1.65 mM, or about 1.67 mM. ... m is about 0.027mM to 2.7mM, or according to the results of the GPA experiment, the K m It is about 0.1 to 2, or about 0.5 to 1.5 or about 1 to 1.35 mM.

[0553] In one embodiment, according to the results of the SRC experiment, the K of the collagenase is cat In about 36 seconds -1 to 671 seconds -1 In other embodiments, according to the results of the SRC experiment, the K of the collagenase is cat In about 50 seconds -1 to 600 seconds -1 In other embodiments, according to the results of the SRC experiment, the K of the collagenase is cat In about 60 seconds -1 to 500 seconds -1 In other embodiments, according to the results of the SRC experiment, the K of the collagenase is cat In about 70 seconds -1 to 400 seconds -1 In still another embodiment, according to the results of the SRC experiment, the K of the collagenase cat In about 100 seconds -1 to 350 seconds -1In other embodiments, the Kcat of the collagenase is about 30 sec -1 , about 40 seconds -1 , about 50 seconds -1 , about 60 seconds -1 , about 70 seconds -1 , about 80 seconds -1 , about 90 seconds -1 , about 100 seconds -1 , about 110 seconds -1 , about 120 seconds -1 , about 130 seconds -1 , about 140 seconds -1 , about 150 seconds -1 , about 160 seconds -1 , about 170 seconds -1 , about 180 seconds -1 , about 190 seconds -1 , about 200 seconds -1 , about 210 seconds -1 , about 220 seconds -1 , about 230 seconds -1 , about 240 seconds -1 , about 250 seconds -1 , about 260 seconds -1 , about 270 seconds -1 , about 280 seconds -1 , about 290 seconds -1 , about 300 seconds -1 , about 310 seconds -1 , about 320 seconds -1 , about 330 seconds -1 , about 340 seconds -1 , about 350 seconds -1 , about 360 seconds -1 , about 370 seconds -1 , about 380 seconds -1 , about 390 seconds -1 , about 400 seconds -1 , about 410 seconds -1 , about 420 seconds -1 , about 430 seconds -1 , about 440 seconds -1 , about 450 seconds -1 , about 460 seconds -1, about 470 seconds -1 , about 480 seconds -1 , about 490 seconds -1 , about 500 seconds -1 , about 510 seconds -1 , about 520 seconds -1 , about 530 seconds -1 , about 540 seconds -1 , about 550 seconds -1 , about 560 seconds -1 , about 570 seconds -1 , about 580 seconds -1 , about 590 seconds -1 , about 600 seconds -1 , about 610 seconds -1 , about 620 seconds -1 , about 630 seconds -1 , about 640 seconds -1 , about 650 seconds -1 , about 660 seconds -1 , about 670 seconds -1 , about 680 seconds -1 , about 690 seconds -1 , about 700 seconds -1 , about 710 seconds -1 , about 720 seconds -1 , about 730 seconds -1 , about 740 seconds -1 , about 750 seconds -1 or about 760 seconds -1 In other embodiments, according to the results of the SRC experiment, the K of the collagenase cat In about 1 sec -1 to 107 seconds -1 or about 10 to 100, or about 20 to 80, or about 30 to 70, or about 40 to 60 sec, according to the results of the SRC experiment. -1 between.

[0554] In one embodiment, according to the results of the GPA experiment, the K cat In about 90 seconds -1 to 10000 seconds -1 or between 41,000 seconds -1 to 81,000 seconds -1In other embodiments, according to the results of the GPA experiment, the K of the collagenase is cat At approximately 45,000 seconds -1 to 75,000 seconds -1 In other embodiments, according to the results of the GPA experiment, the K of the collagenase is cat In about 50000 seconds -1 to 70000 seconds -1 In other embodiments, according to the results of the GPA experiment, the K of the collagenase is cat At about 55000 seconds -1 to 65000 seconds -1 In still another embodiment, according to the results of the GPA experiment, the K of the collagenase cat It is about 35,000 seconds -1 , approximately 37,500 seconds -1 , about 40,000 seconds -1 , approximately 42,500 seconds -1 , about 45,000 seconds -1 , approximately 47,500 seconds -1 , about 50,000 seconds -1 , approximately 52,500 seconds -1 , about 55,000 seconds -1 , approximately 57,500 seconds -1 , about 60,000 seconds -1 , approximately 62,500 seconds -1 , about 65,000 seconds -1 , approximately 67,500 seconds -1 , about 70,000 seconds -1 , approximately 72,500 seconds -1 , about 75,000 seconds -1 , approximately 77,500 seconds -1 , about 80,000 seconds -1 , approximately 82,500 seconds -1 , or approximately 85,000 seconds -1 In other embodiments, according to the results of the GPA experiment, the K cat At about 1215 seconds -1 To about 120,000 seconds -1or, depending on the results of a GPA experiment, between about 2,000 and 100,000, or about 10,000 and 90,000, or about 20,000 and 80,000, or about 30,000 and 70,000, or about 40,000 and 60,000 seconds. -1 between.

[0555] In one embodiment, according to the results of the SRC experiment, the 1K cat In other embodiments, the collagenase has a K of 100 μs, which is between about 376 μs and 38,000 μs, or between about 14,000 μs and about 28,000 μs. cat In one embodiment, the 1 / K of the collagenase is about 16,000 μsec to about 26,000 μsec. cat In one embodiment, the 1 / K of the collagenase is about 18,000 μsec to about 24,000 μsec. cat In still another embodiment, according to the results of the SRC experiment, the 1 / K catis about 12,500μsec, about 12,750μsec, about 13,000μsec, about 13,250μsec, about 13,500μsec, about 13,750μsec, about 14,000μsec, about 14,250μsec, about 14,750μsec, about 15,000μsec, about 15,250μsec, about 15,500μsec, about 15,750μsec, about 16,000μsec, about 16,250μsec, about 16,500μsec, about 16,750μsec sec, approximately 17,000μsec, approximately 17,250μsec, approximately 17,500μsec, approximately 17,750μsec, approximately 18,000μsec, approximately 18,250μsec, approximately 18,500μsec, approximately 18,750μsec, approximately 19,000μsec, approximately 19,250μsec, approximately 19,500μsec, approximately 19,750μsec, approximately 20,000μsec, approximately 20,250μsec, approximately 20,500μsec, approximately 20,750μsec, approximately 21,00 0μsec, approximately 21,250μsec, approximately 21,500μsec, approximately 21,750μsec, approximately 22,000μsec, approximately 22,250μsec, approximately 22,500μsec, approximately 22,750μsec, approximately 23,000μsec, approximately 23,250μsec, approximately 23,500μsec, approximately 23,750μsec, approximately 24,000μsec, approximately 24,250μsec, approximately 24,500μsec, approximately 24,750μsec, approximately 25,000μsec, approximately 25 , 250 μsec, about 25,500 μsec, about 25,750 μsec, about 26,000 μsec, about 26,250 μsec, about 26,500 μsec, about 26,750 μsec, about 27,000 μsec, about 27,250 μsec, about 27,500 μsec, about 27,750 μsec, about 28,000 μsec, about 28,250 μsec, about 28,500 μsec, about 28,750 μsec, about 29,000 μsec, or about 29,250 μsec. In other embodiments, the 1 / K of the collagenase is determined as determined by the SRC assay. cat Between about 370 μsec and about 36,700 μsec, or as determined by the SRC assay, the 1 / K catAt about 750 to 30,000, or about 2,500 to 25,000, or about 5,000 to 20,000, or about 10,000 to 18,000, or about 15,000 μsec.

[0556] In one embodiment, according to the results of the GPA experiment, the 1 / K cat In other embodiments, according to the results of the GPA experiment, the 1 / K cat In another embodiment, according to the results of the GPA experiment, the 1 / K cat In still another embodiment, according to the results of the GPA experiment, the 1 / K catis about 10.0μsec, about 10.2μsec, about 10.4μsec, about 10.6μsec, about 10.8μsec, about 11.0μsec, about 11.2μsec, about 11.4μsec, about 11.6μsec, about 11.8μsec, about 12.0μsec, about 12.2μsec, about 12.4μsec, about 12.6μsec, about 12.8μsec, about 13.0μsec, about 13.2μsec, about 13.4μsec, about 13.6μsec, about 13.8μsec, about 14.0μsec, about 14.2μ sec, approximately 14.4μsec, approximately 14.6μsec, approximately 14.8μsec, approximately 15.0μsec, approximately 15.2μsec, approximately 15.4μsec, approximately 15.6μsec, approximately 15.8μsec, approximately 16.0μsec, approximately 16.2μsec, approximately 16.4μsec, approximately 16.6μsec, approximately 16.8μsec, approximately 17.0μsec, approximately 17.2μsec, approximately 17.4μsec, approximately 17.6μsec, approximately 17.8μsec, approximately 18.0μsec, approximately 18.2μsec, approximately 18.4μsec, approximately 18. 6μsec, approximately 18.8μsec, approximately 19.0μsec, approximately 19.2μsec, approximately 19.4μsec, approximately 19.6μsec, approximately 19.8μsec, approximately 20.0μsec, approximately 20.2μsec, approximately 20.4μsec, approximately 20.6μsec, approximately 20.8μsec, approximately 21.0μsec, approximately 21.2μsec, approximately 21.4μsec, approximately 21.6μsec, approximately 21.8μsec, approximately 22.0μsec, approximately 22.2μsec, approximately 22.4μsec, approximately 22.6μsec, approximately 22.8μsec, approximately 2 3.0μsec, about 23.2μsec, about 23.4μsec, about 23.6μsec, about 23.8μsec, about 24.0μsec, about 24.2μsec, about 24.4μsec, about 24.6μsec, about 24.8μsec, about 25.0μsec, about 25.2μsec, about 25.4μsec, about 25.6μsec, about 25.8μsec, about 26.0μsec, about 26.2μsec, about 26.4μsec, about 26.8μsec, about 27.0μsec, about 27.2μsec or about 27.4μsec. In other embodiments, the 1 / K of the collagenase is determined according to the results of the GPA experiment. catBetween about 0.3 μsec and about 32 μsec, or according to the results of the GPA experiment, the 1 / K cat At about 1 to 30, or about 5 to 25, or about 10 to 20, or about 15 μsec.

[0557] In one embodiment, according to the results of the SRC experiment, the K of the collagenase is cat / K m At about 5,140mM -1 sec -1 to approximately 508,814 mM -1 sec -1 In another embodiment, according to the results of the SRC experiment, the K of the collagenase is cat / K m At about 0.50mM -1 sec -1 to approximately 7.75 mM -1 sec -1 In another embodiment, according to the results of the SRC experiment, the K of the collagenase is cat / K m At about 0.75mM -1 sec -1 to approximately 7.00 mM -1 sec -1 In still another embodiment, according to the results of the SRC experiment, the K of the collagenase cat / K m At about 1.00 mM -1 sec -1 to about 6.00mM -1 sec -1 In still another embodiment, according to the results of the SRC experiment, the K of the collagenase cat / K m is about 0.10mM -1 sec -1 , about 0.20mM -1 sec -1 , about 0.30mM -1 sec -1 , about 0.40mM -1 sec -1 , about 0.50mM -1 sec -1 , about 0.60mM -1 sec -1 , about 0.70mM -1 sec -1, about 0.80mM - 1 sec -1 , about 0.90mM -1 sec -1 , about 1.00mM -1 sec -1 , about 1.10mM -1 sec -1 , about 1.20mM -1 sec -1 , about 1.30mM -1 sec -1 , about 1.40mM -1 sec -1 , about 1.50mM -1 sec -1 , about 1.60mM -1 sec -1 , about 1.70mM - 1 sec -1 , about 1.80mM -1 sec -1 , about 1.90mM -1 sec -1 , about 2.00mM -1 sec -1 , about 2.10mM -1 sec -1 , about 2.20mM -1 sec -1 , about 2.30mM -1 sec -1 , about 2.40mM -1 sec -1 , about 2.50mM -1 sec -1 , about 2.60mM - 1 sec -1 , about 2.70mM -1 sec -1 , about 2.80mM -1 sec -1 , about 2.90mM -1 sec -1 , about 3.00mM -1 sec -1 , about 3.10mM -1 sec -1 , about 3.20mM -1 sec -1 , about 3.30mM-1 sec -1 , about 3.40mM -1 sec -1 , about 3.50mM - 1 sec -1 , about 3.60mM -1 sec -1 , about 3.70mM -1 sec -1 , about 3.80mM -1 sec -1 , about 3.90mM -1 sec -1 , about 4.00mM -1 sec -1 , about 4.10mM -1 sec -1 , about 4.20mM -1 sec -1 , about 4.30mM -1 sec -1 , about 4.40mM - 1 sec -1 , about 4.50mM -1 sec -1 , about 4.60mM -1 sec -1 , about 4.70mM -1 sec -1 , about 4.80mM -1 sec -1 , about 4.90mM -1 sec -1 , about 5.00mM -1 sec -1 , about 5.10mM -1 sec -1 , about 5.20mM -1 sec -1 , about 5.30mM - 1 sec -1 , about 5.40mM -1 sec -1 , about 5.50mM -1 sec -1 , about 5.60mM -1 sec -1 , about 5.70mM -1 sec -1 , about 5.80mM -1sec -1 , about 5.90mM -1 sec -1 , about 6.00mM -1 sec -1 , about 6.10mM -1 sec -1 , about 6.20mM - 1 sec -1 , about 6.30mM -1 sec -1 , about 6.40mM -1 sec -1 , about 6.50mM -1 sec -1 , about 6.60mM -1 sec -1 , about 6.70mM -1 sec -1 , about 6.80mM -1 sec -1 , about 6.90mM -1 sec -1 , about 7.00mM -1 sec -1 , about 7.10mM - 1 sec -1 , about 7.20mM -1 sec -1 , about 7.30mM -1 sec -1 , or approximately 7.40 mM -1 sec -1 In other embodiments, according to the results of the SRC experiment, the K of the collagenase cat / K m At about 0.0048mM -1 sec -1 to about 0.47mM -1 sec -1 or according to the results of the SRC experiment, the K cat / K m At about 0.009 to about 0.3, or about 0.01 to about 0.25, or about 0.1 to about 0.25 mM -1 sec -1 between.

[0558] In one embodiment, according to the results of the GPA experiment, the K cat / K mAt about 60mM - 1 sec -1 to about 6000mM -1 sec -1 In another embodiment, according to the results of the GPA experiment, the K of the collagenase is cat / K m At approximately 30,000 mM -1 sec -1 to approximately 85,000 mM -1 sec -1 In another embodiment, according to the results of the GPA experiment, the K of the collagenase is cat / K m At approximately 36,000 mM -1 sec -1 to approximately 77,000 mM -1 sec -1 In still another embodiment, according to the results of the GPA experiment, the K of the collagenase cat / K m At approximately 40,000 mM -1 sec -1 to approximately 70,000 mM -1 sec -1 In still another embodiment, according to the results of the GPA experiment, the K of the collagenase cat / K m is about 40,000mM -1 sec -1 , about 42,000mM -1 sec -1 , about 44,000mM -1 sec -1 , about 46,000mM -1 sec -1 , about 48,000mM -1 sec -1 , about 50,000mM -1 sec -1 , about 52,000mM -1 sec -1 , about 54,000mM -1 sec -1 , about 56,000mM -1 sec -1 , about 58,000mM -1 sec -1 , about 60,000mM -1sec -1 , approximately 62,000mM -1 sec -1 , about 64,000mM -1 sec -1 , approximately 66,000mM -1 sec -1 , about 68,000mM -1 sec -1 , about 70,000mM -1 sec -1 , about 72,000mM -1 sec -1 , about 74,000mM -1 sec -1 , about 76,000mM -1 sec -1 , about 78,000mM -1 sec -1 , about 80,000mM -1 sec -1 , about 82,000mM -1 sec -1 , about 84,000mM -1 sec -1 , about 86,000mM -1 sec -1 , about 88,000mM -1 sec -1 , about 90,000mM -1 sec -1 , about 92,000mM -1 sec -1 , approximately 94,000mM -1 sec -1 , or approximately 96,000mM -1 sec -1 In other embodiments, according to the results of the GPA experiment, the K cat / K m At about 900mM -1 sec -1 to approximately 90,000 mM -1 sec -1 or according to the results of the SRC experiment, the K cat / K m At about 2,000 to about 80,000, or about 10,000 to about 70,000, or about 20,000 to about 60,000 mM -1 sec-1 , or about 30,000 to 50,000, or about 40,000 to 45,000 mM -1 sec-1.

[0559] In one embodiment, the molecular weight of the collagenase is between about 60kDa and about 130kDa. In other embodiments, the molecular weight of the collagenase is between about 70kDa and about 130kDa. In other embodiments, the molecular weight of the collagenase is between about 80kDa and about 120kDa. In still another embodiment, the molecular weight of the collagenase is between about 90kDa and about 120kDa. In other embodiments, the molecular weight of the collagenase is between about 100kDa and about 110kDa. In still another embodiment, the collagenase has a molecular weight of about 55 kDa, about 57 kDa, about 60 kDa, about 62 kDa, about 65 kDa, about 67 kDa, about 70 kDa, about 72 kDa, about 75 kDa, about 77 kDa, about 80 kDa, about 82 kDa, about 85 kDa, about 87 kDa, about 90 kDa, about 92 kDa, about 95 kDa, about 97 kDa, about 100 kDa, about 102 kDa, about 105 kDa, about 107 kDa, about 110 kDa, about 112 kDa, about 115 kDa, about 117 kDa, about 120 kDa, about 122 kDa, about 125 kDa, about 127 kDa, about 130 kDa, about 132 kDa, about 135 kDa, or about 137 kDa.

[0560] In one embodiment, the purity of the collagenase measured by reverse HPLC is at least 80%. In other embodiments, the purity of the collagenase measured by reverse HPLC is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In still another embodiment, the collagenase can include less than or equal to 1% clostripain by area. In other embodiments, the collagenase also includes less than or equal to 1% gelatinase by area. In other embodiments, the collagenase can also include less than or equal to 1% leupeptin by area. In still another embodiment, the collagenase includes less than or equal to 1 cfu / mL biological contaminants.

[0561] In one embodiment, the titer (i.e., specific activity) of the collagenase is between about 500 and about 30,000 SRC units / mg. In other embodiments, the titer of the collagenase is between about 2,500 and about 25,000 SRC units / mg. In other embodiments, the titer of the collagenase is between about 5,000 and about 20,000 SRC units / mg. In yet another embodiment, the titer of the collagenase is about 500, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15 ,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, about 25,000, about 25,500, about 26,000, about 26,500, about 27,000, about 27,500, about 28,000, about 28,500, about 29,000, about 29,500 or about 30,000 SRC units / mg.

[0562] In one embodiment, the titer (ie, specific activity) of the collagenase is between about 5,000 and about 30,000 f-SRC units / mg. In other embodiments, the titer of the collagenase is between about 7,500 and about 25,000 f-SRC units / mg. In other embodiments, the titer of the collagenase is between about 10,000 and about 20,000 f-SRC units / mg. In still another embodiment, the titer of the collagenase is about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000 0, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23, 000, about 23,500, about 24,000, about 24,500, about 25,000, about 25,500, about 26,000, about 26,500, about 27,000, about 27,500, about 28,000, about 28,500, about 29,000, about 29,500, or about 30,000 f-SRC units / mg.

[0563] In one embodiment, the titer (i.e., specific activity) of the collagenase is between about 100,000 and about 400,000 GPA units / mg. In other embodiments, the titer of the collagenase is between about 150,000 and about 350,000 GPA units / mg. In other embodiments, the titer of the collagenase is between about 200,000 and about 300,000 GPA units / mg. In yet another embodiment, the titer of the collagenase is about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000. , about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about 300,000, about 310,000, about 320,000, about 330,000, about 340,000, about 350,000, about 360,000, about 370,000, about 380,000, about 390,000 or about 400,000 GPA units / mg.

[0564] In one embodiment, the titer of the collagenase is between about 175,000 and about 500,000 f-GPA units / mg. In other embodiments, the titer of the collagenase is between about 250,000 and about 450,000 f-GPA units / mg. In other embodiments, the titer of the collagenase is between about 300,000 and about 400,000 f-GPA units / mg. In yet another embodiment, the titer of the collagenase is about 175,000, about 185,000, about 195,000, about 205,000, about 215,000, about 225,000, about 235,000, about 245,000, about 255,000, about 265,000, about 275,000, about 285,000, about 295,000, about 305,000, about 315,000, about 325,000, About 335,000, about 345,000, about 355,000, about 365,000, about 375,000, about 385,000, about 395,000, about 405,000, about 415,000, about 425,000, about 435,000, about 445,000, about 455,000, about 465,000, about 475,000, about 485,000, or about 495,000 f-GPA units / mg.

[0565] In one embodiment, the titer of the collagenase is between about 5,000 and about 25,000 ABC units / mg. In one embodiment, the titer of the collagenase is between about 7,500 and about 20,000 ABC units / mg. In one embodiment, the titer of the collagenase is between about 10,000 and about 17,500 ABC units / mg. In other embodiments, the titer of the collagenase is about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15, 000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, or about 25,000 ABC units / mg.

[0566] In some embodiments, the collagenase present in the composition includes collagenase I and collagenase II, and the ratio of collagenase I to collagenase II is about 1: 1. Other ratios of collagenase I and collagenase II can also be used, for example, 0.1-2: 1, or 0.25-2: 1, or 0.5-2: 1, or 0.75-2: 1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0: 1. The purity of each of collagenase I and collagenase II is at least 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% by area, as measured by reverse phase HPLC.

[0567] In other embodiments, the collagenase composition comprises CCH comprising AUXI and AUXII in a ratio of approximately 1: 1. Other ratios of AUXI and AUXII can also be used, for example, 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1. The purity of each of AUXI and AUXII is at least 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% by area as measured using reverse phase HPLC.

[0568] In other embodiments, the collagenase composition can be a liquid or can be reconstituted from a lyophilized form with a diluent. The dosage of the mixture is determined solely by the amount of collagenase present, without reference to a diluent, and can include from about 0.1 mg to about 20 mg in one or more injections. In other embodiments, the administered dose is about 0.06 mg, 0.48 mg, 0.84 mg, 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg, 5.04 mg, 5.88 mg, 6.72 mg, 7.56 mg, or 8.4 mg in a single injection or multiple injections.

[0569] For example, approximately 0.06 mg, 0.48 mg, 0.84 mg, or 1.68 mg are administered in approximately 12 injections. The volume of the injected collagenase composition can range from 0.01 mL to 3 mL per injection, or approximately 0.2 mL to 150 mL for a total treatment visit. In specific embodiments, the above-mentioned dosage is the dosage of a collagenase composition comprising CCH. In other embodiments, the above-mentioned dosage is the dosage of a collagenase composition comprising one or more of the following characteristics:

[0570] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0571] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0572] ·K cat (sec -1) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0573] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0574] ·K cat / K M ,mM -1 sec -1 5,140 to 508,814 (SRC test), or approximately 60 to 5,934 (GPA test)

[0575] • A molecular weight of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa.

[0576] The purity is at least 80% by area as measured by reverse phase HPLC (high pressure liquid chromatography)

[0577] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0578] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0579] Potency of about 100,000 to about 400,000 GPA units / mg

[0580] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0581] Potency of about 5,000 to about 25,000 ABC units / mg

[0582] The impurity content is less than or equal to 1% by area, and the impurities are selected from clostripain, gelatinase and leupeptin

[0583] A bioburden of less than or equal to 1 cfu / mL.

[0584] In other embodiments, approximately 0.84 mg of CCH is injected into each treatment area in approximately 12 aliquot injections (approximately 0.07 mg x 12 injections = approximately 0.84 mg). In some cases, the above treatment of 0.84 mg is administered every 10-40 days over 2, 3, 4, or 5 visits. In other cases, 0.84 mg is injected into more than one treatment area at a single visit or over 2, 3, 4, or 5 visits every 10-40 days. In other embodiments, there are more than five treatment visits (visits).

[0585] In another aspect, the amount of collagenase that can be injected into the treatment area in one or more injections per treatment visit is about 0.001 mg to 20 mg, either in one injection or in multiple injections, for example, divided equally into 3 to about 100 injections. The collagenase is in liquid form or reconstituted from a lyophilized solid with a diluent. The dosage of collagenase is measured by the amount of collagenase without taking into account the diluent and can include about 0.1 mg to 1 mg, or 0.25 mg to 0.75 mg, or 0.1 mg to 2 mg, or 0.25 mg to 1.75 mg, or 0.5 mg to 1 mg, 0.1 mg to 3 mg, or 0.25 mg to 2.75 mg, or 0.5 mg to 2.5 mg, or 0.75 mg to 2.25 mg, or 1 mg to 2 mg, or 0.1 mg to 4 mg, or 0.25 mg to 3.75 mg, or 0.5 mg to 3.5 mg, or 0.75 mg to 3 mg, or 1 mg to 3 mg. In other embodiments, the dose is about 0.001 mg, 0.01 mg, 0.04 mg, 0.05 mg, 0.07 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg per injection or multiple injections. g, 3.75mg, 4.0mg, 4.25mg, 4.5mg, 4.75mg, 5.0mg, 5.25mg, 5.5mg, 5.75mg, 6mg, 6.25mg, 6.5mg, 6.75mg, 7mg, 7.25mg, 7.5mg, 7.75m g, 8 mg, 8.25 mg, 8.5 mg, 8.75 mg, 9 mg, 9.25 mg, 9.5 mg, 9.75 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0586] In other embodiments, the dose administered in one or more injections is about 0.06 mg, 0.48 mg, 0.84 mg, 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg, or 5.04 mg. In another embodiment, 0.06 mg, 0.48 mg, 0.84 mg, 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg, or 5.04 mg is administered to the treatment area in 12 injections. In other embodiments, the dose of collagenase is administered in three or more injections. The volume of each injection of the collagenase composition can be between 0.01 mL and 3 mL, or the total volume per treatment visit can be about 1 mL to 150 mL.

[0587] In one aspect, the above-described AUXI and II mixture ("CCH") can be injected in one or more injections at a dose of about 0.01 mg to 10 mg of collagenase per treatment visit, for example, divided evenly among about 3 to about 50 injections. The collagenase can be in liquid form or can be reconstituted from a lyophilized form with a diluent. The dosage of the mixture is measured by the amount of collagenase without taking into account the diluent and can include about 0.1 mg to 1 mg, or 0.25 mg to 0.75 mg, or 0.1 mg to 2 mg, or 0.25 mg to 1.75 mg, or 0.5 mg to 1 mg, 0.1 mg to 3 mg, or 0.25 mg to 2.75 mg, or 0.5 mg to 2.5 mg, or 0.75 mg to 2.25 mg, or 1 mg to 2 mg, or 0.1 mg to 4 mg, or 0.25 mg to 3.75 mg, or 0.5 mg to 3.5 mg, or 0.75 mg to 3 mg, or 1 mg to 3 mg, or about 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg , 0.6mg, 0.7mg, 0.8mg, 0.9mg, 1mg, 1.1mg, 1.2mg, 1.3mg, 1.4mg, 1.5mg, 1.6mg, 1.7mg, 1.8mg, 1.9mg, 2mg, 2.25mg, 2.5mg, 2.75mg, 3mg, 3.25mg, 3.5mg, 3.75mg, 4.0mg, 4.25mg, 4.5mg, 4.75mg, 5.0mg, 5.25mg, 5.5mg, 5.75mg, 6mg, 6.25mg, 6.5mg, 6.75mg, 7mg, 7.25mg, 7.5mg, 7.75mg, 8mg, 8.25mg, 8.5mg, 8.75mg, 9mg, 9.25mg, 9.5mg, 9.75mg or 10mg. In other embodiments, the dose of CCH administered is approximately 0.06 mg, 0.48 mg, 0.84 mg, or 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg, or 5.04 mg per injection or multiple injections. For example, approximately 0.06 mg, 0.48 mg, 0.84 mg, or 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg, or 5.04 mg is administered distributed over 12 injections. The volume of the collagenase composition injected can range from 0.01 ml to 3 ml per injection, or a total of approximately 1 ml to 80 ml is administered per medical visit.

[0588] The dosage of collagenase can also be expressed in mg per injection (again without taking into account diluent), for example, from about 0.001 mg to 0.5 mg per injection, about 0.01 mg to about 5 mg per injection, or about 0.005 mg to about 0.1 mg per injection, or about 0.005 mg, 0.04 mg or 0.07 mg per injection.

[0589] In certain aspects, the present invention contemplates injecting about 500 ABC units to about 50,000 ABC units per treatment visit, or about 10,000 ABC units to about 25,000 ABC units per treatment visit. In other embodiments, the dose of collagenase per injection is about 50 ABC units to about 2,500 ABC units, or about 85 ABC units to about 2,000 ABC units, or about 1,500 ABC units to about 1,750 ABC units, or about 200 ABC units to about 1,500 ABC units, or about 300 ABC units to 1,500 ABC units, or about 300 ABC units to about 1,250 ABC units, or about 500 ABC units to about 1,000 ABC units.

[0590] In certain embodiments, the dosage is based on the specific activity of:

[0591]

[0592] *To calculate milligrams, multiply the specific activity in SRC units and SRC units / mg by the inverse of the specific activity in SRC units and SRC units / mg. For example, when the dose is 500 SRC units and the specific activity is 500 SRC units / mg, the milligram amount equivalent to a 500 SRC unit dose is (500 SRC units) * (1 / (500 SRC units / mg)) = 1.00 mg

[0593] The milligram amount of SRC units and ABC units / mg specific activity is calculated by multiplying the SRC units by 6.3 ABC units / SRC unit, and then multiplying by the inverse of the ABC units / mg specific activity. For example, when the dose is 500 SRC units and the specific activity is 10,000 ABC units / 0.58 mg, the milligram amount equivalent to 500 SRC units is (500 SRC units) * (6.3 ABC units / SRC unit) * (1 / (10,000 ABC units / 0.58 mg)) = 0.18 mg.

[0594] In certain aspects, the present invention contemplates injecting collagenase in an amount of about 5,000 BTC units to about 25,000 BTC units, or about 10,000 BTC units to about 25,000 BTC units, or about 15,000 BTC units, or about 17,500 BTC units, or about 20,000 BTC units, or about 22,500 BTC units, or about 9,175 BTC units, about 15,817 BTC units.

[0595] 5. preparation

[0596] The CCH or other collagenases may be in the form of a pharmaceutical composition comprising CCH or collagenase and a pharmaceutically acceptable excipient, which may include sterile water for injection, sodium chloride, or calcium chloride, a pH adjuster, and a stabilizer.

[0597] A non-limiting example is The applicant commercially supplies CCH in single-use glass vials containing 0.9 mg of CCH as a sterile lyophilized powder for reconstitution. The single-use glass vials also contain a sterile diluent for reconstitution. Inactive ingredients include hydrochloric acid, sucrose, and tromethamine. The diluent contains calcium chloride dihydrate in 0.9% sodium chloride. Prescribing Information(2018).

[0598] In other embodiments, CCH is provided as a sterile lyophilized powder comprising 0.92 mg of CCH, sucrose, Tris, mannose, and hydrochloric acid in a 5 ml tube. Sterile diluents for reconstitution may include water for injection, normal saline, or 0.6% sodium chloride and 0.03% calcium chloride dihydrate in water for injection, filled into separate 5 ml tubes.

[0599] The collagenase or CCH can also be filled into test tubes of other sizes, for example, 10 ml, 15 ml, 20 ml or 30 ml. Other pH regulators, sugars, polyols and stabilizers can be found in Rowe et al., Handbook of Pharmaceutical Excipients (5 th Ed.)

[0600] 6. Treatment Methods: Injection Techniques and Dosage Regimens

[0601] The collagenase composition described above can be used effectively in a method for treating or reducing the severity of subcutaneous cellulite in a human. The present invention relates to a method for reducing the severity of subcutaneous cellulite in a human patient, comprising: providing a composition comprising at least one collagenase; and injecting a therapeutically effective amount of the composition into one or more pits, wherein the patient exhibits a reduction in the severity of subcutaneous cellulite compared to a baseline severity before treatment. As described in further detail below, the composition can be administered using various injection techniques and its efficacy can be measured using a number of scales and other measurement tools.

[0602] Applicant has previously described related treatment methods and hereby incorporates by reference their patent applications in their entirety, such as PCT patent application PCT / US2018 / 020551 (published on September 7, 2018, with publication number WO2018 / 160905); U.S. Provisional Application No. 62 / 697376, filed on July 12, 2018, entitled “Injection Technology for Treating Edema-Fibrinous Sclerotic Panneuropathy”; U.S. Provisional Application No. 62 / 733046, filed on September 18, 2018, entitled “Compositions and Methods for Treating Edema-Fibrinous Sclerotic Panneuropathy with Four-Quadrant Single-Dose Collagenase”; and U.S. Patent Application Publication No. US20180327731, filed on March 28, 2018, entitled “Methods for Producing Collagenase”.

[0603] Administration of the collagenase compositions described herein can be to a single subject bilaterally (two thighs or two buttocks) or to all four quadrants (two buttocks and two thighs) during a treatment visit. Such treatment visits may be performed every 10-40 days for 2, 3, 4, or 5 treatment visits over a period of one year.

[0604] Table 15 summarizes the various collagenase injection parameters and the associated techniques used to treat patients.

[0605] Table 15. Various injection parameters of collagenase and related techniques used to treat patients

[0606]

[0607]

[0608]

[0609] Table 16 summarizes five non-limiting examples of injection techniques and treatments contemplated by the present invention, corresponding to Figure 7-11 Injection technique shown.

[0610]

[0611] Treatments I to V are described in detail below.

[0612] Treatment I: superficial collagenase injection, divided into 3 equal parts

[0613] like Figure 7 As shown, in this example ("Treatment 1"), collagenase was injected subcutaneously perpendicular to the long axis of the pit while the subject was in a prone position. Each injection consisted of a single subcutaneous injection of collagenase, divided into three 0.1 ml aliquots (for a total injection volume of 0.3 ml). During each treatment visit, 8 syringes (4 syringes per treatment area) were prepared for administration. Each syringe contained 0.9 ml of the collagenase composition (3 injections per syringe). The dose may vary for each subject, with a total dose of approximately 0.5 mg to approximately 5 mg of collagenase per treatment area.

[0614] More specifically, perform the following steps:

[0615] If the depression is an elongated, trough-like depression (position a), hold the needle perpendicular to the skin surface and perpendicular to the long axis of the depression, push the needle all the way in (1 / 2 inch) and inject 0.1 ml of the collagenase composition by gently pushing the syringe plunger. In most cases, the plane containing injection points A, B, and C will be parallel to the long axis of the subject's body.

[0616] Gently withdraw the needle and reposition it so that it is at an angle of approximately 45° to the skin surface, toward the subject's head (position B). Push the needle fully in and inject 0.1 mL of the collagenase composition by gently pushing the syringe plunger. Position B is best directed toward the subject's head.

[0617] Gently withdraw the needle and reposition it so that it is at an angle of approximately 45° to the skin surface, toward the subject's foot (position C). Push the needle fully in and inject 0.1 mL of the collagenase composition by gently pushing the syringe plunger. Position B is best toward the subject's foot.

[0618] • Completely withdraw the needle from the injection site and move to the next injection site.

[0619] • A syringe containing 0.9 ml of collagenase composition is sufficient for three injection sites.

[0620] • Use four (4) 0.9 ml syringes at each treatment site (each buttock or each thigh) for a total of 12 injections of 0.3 ml (3 equal portions of 0.1 ml each) at 12 injection sites.

[0621] At each treatment visit, a total of 24 injections were administered in two treatment areas (2 buttocks or 2 thighs).

[0622] Treatment II: Superficial collagenase injection, 1 aliquot

[0623] like Figure 8 As shown, in this example ("Treatment II"), collagenase was administered subcutaneously while the subject was in the prone position. Each injection consisted of a single subcutaneous injection of collagenase, i.e., a single superficial injection of 0.3 ml aliquots. During each treatment visit, eight syringes (four syringes per treatment area) were prepared for administration. Each syringe contained 0.9 ml of collagenase (three injections per syringe). The total dose of collagenase per subject was approximately 0.5 mg to 5 mg per treatment area.

[0624] During each treatment visit, 8 syringes (4 syringes per treatment area) were prepared for administration. Each syringe contained 0.9 ml of collagenase composition (3 injections per syringe). More specifically, the following procedure was followed:

[0625] Position the needle so that it is at an angle of approximately 30° to the skin surface at the injection site, toward the subject's head, push the needle all the way in (1 / 2 inch), and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0626] Completely withdraw the needle from the injection site and move to the next injection site.

[0627] • A syringe containing 0.9 ml of collagenase composition is sufficient for three injection sites.

[0628] • Use four (4) 0.9 ml syringes at each treatment site (each buttock or each thigh) for a total of 12 injections of 0.3 ml (3 equal portions of 0.1 ml each) at 12 injection sites.

[0629] At each treatment visit, a total of 24 injections were administered in two treatment areas (2 buttocks or 2 thighs).

[0630] Treatment III: Deep injection of collagenase, 1 equal part

[0631] like Figure 9 As shown, in this example ("Treatment III"), collagenase was administered subcutaneously while the subject was in the prone position. Each injection consisted of a single subcutaneous injection of collagenase, i.e., a single deep injection of 0.3 ml aliquots. During each treatment visit, eight syringes (four syringes per treatment area) were prepared for administration. Each syringe contained 0.9 ml of collagenase (three injections per syringe). The total dose of collagenase per subject was approximately 0.5 mg to 5 mg per treatment area.

[0632] More specifically, follow this procedure:

[0633] Position the needle so that it is at an angle of approximately 30° to the skin surface at the injection site, toward the subject's head, push the needle all the way in (1 inch), and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0634] Position the needle so that it is at an angle of approximately 30° to the skin surface at the injection site, toward the subject's head, push the needle all the way in (1 inch), and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0635] • A syringe containing 0.9 ml of collagenase composition is sufficient for three injection sites.

[0636] • Use four (4) 0.9 ml syringes at each treatment site (each buttock or each thigh) for a total of 12 injections of 0.3 ml (3 equal portions of 0.1 ml each) at 12 injection sites.

[0637] Discard used needles and syringes.

[0638] At each treatment visit, a total of 24 injections were administered in two treatment areas (2 buttocks or 2 thighs).

[0639] Treatment IV: Deep and superficial collagenase injections, divided into 5 equal parts

[0640] like Figure 10 As shown, in treatment IV, collagenase is injected subcutaneously while the subject is in a prone position. Each injection consists of a single skin injection of collagenase, with 0.3 ml aliquots administered five times (total injection volume of 1.5 mL). During each treatment visit, 24 syringes (12 syringes per treatment area) are prepared for administration. Each syringe contains 1.5 ml of collagenase (0.3 ml per injection per syringe, 5 injections). The total dose of collagenase per subject in each treatment area ranges from approximately 0.5 mg to 5 mg.

[0641] During each treatment visit, 24 syringes (12 syringes per treatment area) were prepared for administration. Each syringe contained 1.5 mL of collagenase (0.3 mL per syringe per injection, 5 times). More specifically, the following procedure was followed:

[0642] Position the needle so that it is at an angle of approximately 30° to the skin surface at the injection site, toward the subject's head, push the needle all the way in (1 inch), and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0643] Gently withdraw the needle (1 / 2 inch) and, maintaining an angle of approximately 30° to the skin surface, reposition it toward the subject's head (position B). Inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0644] • Maintaining the needle at an angle of approximately 30° to the skin surface, reposition the needle at a position midway between positions A and B (position C, toward the subject's shoulder) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0645] • Maintaining the needle at an angle of approximately 30° to the skin surface, reposition the needle precisely opposite position B (position D) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0646] • Maintaining the needle at an angle of approximately 30° to the skin surface, reposition the needle at a position midway between positions A and D (position E, toward the subject's shoulder) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0647] • A 3 ml syringe containing 1.5 ml of collagenase composition is sufficient for one injection site.

[0648] • Use twelve (12) 3 ml syringes at each treatment site (each buttock or each thigh) and inject a total of 12 times 1.5 ml (5 equal portions of 0.3 ml each, for a total dose of 18 mL) at each of the 12 injection sites.

[0649] At each treatment visit, a total of 24 injections were administered in two treatment areas (2 buttocks or 2 thighs).

[0650] Treatment V: superficial collagenase injection, divided into 4 equal parts

[0651] like Figure 11 As shown, in this example ("Treatment V"), collagenase was administered subcutaneously while the subject was in the prone position. Each injection consisted of a single subcutaneous injection of collagenase, i.e., four deep injections of 0.3 mL aliquots (for a total injection volume of 1.2 mL). During each treatment visit, 24 syringes (12 syringes per treatment area) were prepared for administration. Each syringe contained 1.2 mL of collagenase (4 aliquots of 0.3 mL each). The total dose of collagenase per subject per treatment area was approximately 0.5 mg to approximately 5 mg.

[0652] During each treatment visit, 24 syringes (12 syringes per treatment area) were prepared for administration. Each syringe contained 1.2 mL of collagenase composition (4 equal portions of 0.3 mL each). More specifically, the following procedure was followed:

[0653] Position the needle so that it is at an angle of approximately 30° to the skin surface at the injection site, toward the side of the subject, push the needle all the way in (1 / 2 inch), and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0654] Maintaining the needle at an angle of approximately 30° to the skin surface, reposition the needle at position A to approximately 60° from position A and toward the patient's shoulder (position B) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0655] • Maintaining the needle at approximately 30° to the skin surface, reposition the needle approximately 60° from position B and toward the patient's other shoulder (position C) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0656] • Maintaining the needle at an angle of approximately 30° to the skin surface, reposition the needle at position A and toward the other side of the subject (position D) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0657] • A 3 ml syringe containing 1.2 mL of collagenase composition is sufficient for one injection site.

[0658] Twelve (12) 3 ml syringes (each containing 1.2 mL of collagenase composition) were used at each treatment site (each buttock or each thigh) for a total of 12 injections of 1.2 mL (4 equal portions of 0.3 mL each, for a total dose of 14.4 mL) at each of the 12 injection sites.

[0659] A total of 24 injections were administered in two treatment areas (2 buttocks or 2 thighs).

[0660] In a further embodiment, the collagenase is injected into Figure 12 The injection interval varies in the range of about 0.1 cm to about 15 cm, or about 1 cm to about 10 cm, or about 0.5 cm to about 2 cm. Figure 12 As shown, each injection is divided into three 0.1 ml aliquots (0.3 ml per injection) and used. The first aliquot is administered with the needle perpendicular to the skin surface. For the second and third aliquots, the needle is slightly withdrawn and positioned at an angle of approximately 45° to the left and 45° to the right of the vertical axis.

[0661] like Figure 7-11 Treatments I to V and Figure 12The injection technique shown may be used with varying doses, injection angles, volumes, number of syringes, injection depths, and other parameters, as detailed in Tables 15 and 16. All such variations are intended to be within the scope of the present invention.

[0662] In another embodiment, about 0.84 mg of CCH is divided into 12 equal injections and injected into the affected area, such as a quadrant (i.e., right buttock or left buttock or right thigh or left thigh) (about 0.07 mg x 12 injections = about 0.84 mg CCH). In some cases, a 0.84 mg treatment is administered once every 10-40 days for 2, 3, 4, or 5 treatments. In other cases, 0.84 mg is injected into more than one affected area or quadrant every 10-40 days for 2, 3, 4, or 5 treatments.

[0663] In certain embodiments, as shown in Table 17, the patient is administered collagenase.

[0664] Table 17. Collagenase Dosage and Volume

[0665]

[0666] a Each collagenase injection is 0.3 mL, divided into three equal portions of 0.1 mL each.

[0667] Further, in certain embodiments, the parameters for treating a subject are provided in Tables 18 and 19.

[0668] Table 18. CCH freeze-dried formulation parameters

[0669]

[0670] Table 19. CCH dilution parameters

[0671]

[0672] In one embodiment, the osmotic pressure of the recombinant product is about 50 to about 1,000, about 100 to about 900, about 200 to about 800, about 300 to about 700, about 400 to about 600, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1,000 mOsm / kg. In still another embodiment, the osmotic pressure of the recombinant product is about 512 mOsm / kg, about 275 mOsm / kg, about 281 mOsm / kg, or about 227 mOsm / kg.

[0673] In addition to the above methods, the present invention also provides a method of treating or reducing EFP in a subject in need thereof, wherein the method has at least one of the following advantages over conventional procedures and treatments for EFP:

[0674] a.Easy for doctors to administer;

[0675] b. Shorter treatment time;

[0676] c. unexpected therapeutic effects, considering that doctors generally believe that improvement in aesthetic conditions is difficult to achieve;

[0677] d. No need to administer hyaluronidase;

[0678] e. No heating required;

[0679] f. No need to apply laser;

[0680] g. No segmentation;

[0681] h. No anesthesia required (although bruising may occur);

[0682] i. No need to wear tight clothing; and

[0683] j. Do not use vacuum.

[0684] In other embodiments, the methods of treating or reducing subcutaneous cellulite are not specific to the severity of the subcutaneous cellulite being treated, ie, for example, collagenase treatment is safe and effective regardless of the prevalence or severity of cellulite.

[0685] F. Phase 4 - End of Treatment and Efficacy Measurement

[0686] The treatment methods described herein are effective for treating subcutaneous cellulite through a variety of measures described below. As used herein, "day" refers to the study day; thus, for example, day 22 is 21 days after the first injection, etc. Furthermore, in one embodiment, "day" is + / - 7 days.

[0687] 1. Efficacy of CR-PCSS and PR-PCSS assays

[0688] Improvement for each patient at any visit refers to an improvement of at least 1 level or grade compared to the baseline or any previous score or rating. The average score or rating of a group of patients at any visit is about 0.1 higher than the baseline examination or any previous average score or rating. A responder is a patient whose maximum total score or rating is at least 25% higher than the baseline examination results. In certain embodiments, the treatment methods described in detail above produce one or more of the following efficacy endpoints as determined by CR-PCSS and / or PR-PCSS measurements:

[0689] 1. An improvement of at least 0.1 in the CR-PCSS and / or PR-PCSS scores compared to baseline.

[0690] 2. Improvement of at least 2 grades in the severity of the CR-PCSS on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1"), as assessed by an on-site clinician in the target thigh.

[0691] 3. Improvement of at least 2 levels in the severity of the PR-PCSS from baseline (day 1) on day 22, 43, 71, 90, 180, 365, or 730 as assessed by the subject observing a digital image of the target thigh.

[0692] 4. Improvement demonstrated by a composite Grade 2 response at Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as a subject experiencing at least a 2-grade improvement in severity from baseline in the CR-PCSS or at least a 2-grade improvement in severity from baseline in the PR-PCSS.

[0693] 5. At least 1 grade improvement from baseline in the severity of subcutaneous cellulite on the CR-PCSS as assessed by a clinician in the target thigh on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730.

[0694] 6. At least 1 grade improvement compared to baseline in the severity of subcutaneous cellulite in the PR-PCSS as assessed by the subject observing a digital image of the target thigh on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730.

[0695] 7. Improvement demonstrated by a Grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as a subject experiencing at least a 1-grade improvement in severity compared to baseline in the CR-PCSS or at least a 1-grade improvement in severity compared to baseline in the PR-PCSS.

[0696] 8. In the patient population where all CR-PCSS scores were moderate or severe, there was a statistically significant improvement in at least one treatment area compared to placebo, where the improvement was one or more of 1 to 7 above.

[0697] 9. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, and more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[0698] 10. The improvements observed in the CR-PCSS at Day 180 relative to baseline were consistent in both the left and right thighs.

[0699] 11. In all patient populations with moderate or severe CR-PCSS scores, the median time to earliest grade 2 CR-PCSS and / or PR-PCSS improvement in at least one treatment area was approximately 50 days, 60 days, 70 days, 80 days, or 90 days.

[0700] 12. In all patients with moderate or severe CR-PCSS scores, the median time to earliest grade 1 CR-PCSS and / or PR-PCSS improvement in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0701] 13. In all patient groups with moderate or severe CR-PCSS scores, the mean CR-PCSS and / or PR-PCSS scores of the subjects separated from placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[0702] 14. In the patient population with moderate or severe CR-PCSS scores in all treatment areas, the percentage of subjects with a Grade 2 composite response as measured by CR-PCSS and / or PR-PCSS in at least one treatment area on Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0703] 15. In all patient populations with moderate or severe CR-PCSS scores, the percentage of subjects achieving a Grade 1 composite response as measured by CR-PCSS and / or PR-PCSS in at least one treatment area on Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0704] 16. Among all patients with moderate or severe CR-PCSS scores, more than one-third, one-half, two-thirds, or three-quarters of patients had a CR-PCSS and / or PR-PCSS response of at least grade 1 in at least one treated area at day 71 after treatment, where the CR-PCSS results were not related to age, BMI, or skin color.

[0705] 17. The severity of subcutaneous cellulite is rapidly reduced within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment.

[0706] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 17 above, wherein the collagenase has one or more of the following characteristics:

[0707] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0708] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0709] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0710] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0711] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0712] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0713] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[0714] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0715] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0716] Potency of about 100,000 to about 400,000 GPA units / mg

[0717] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0718] Potency of about 5,000 to about 25,000 ABC units / mg

[0719] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[0720] Bioburden less than or equal to 1 cfu / mL

[0721] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above.

[0722] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above, wherein the collagenase has one or more of the following characteristics:

[0723] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0724] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0725] ·K cat (sec -1) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0726] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0727] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0728] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0729] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[0730] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0731] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0732] Potency of about 100,000 to about 400,000 GPA units / mg

[0733] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0734] Potency of about 5,000 to about 25,000 ABC units / mg

[0735] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[0736] Bioburden less than or equal to 1 cfu / mL

[0737] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above.

[0738] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of collagenase I and collagenase II in a ratio of about 1:1 is injected into at least one treatment area and one or more of the above results 1 to 17 are achieved, wherein the collagenase I and collagenase II have the following characteristics:

[0739] ·Type I

[0740] ο Experiment: SRC microplate

[0741] οVmax,min -1 : Approximately 0.08 to 7.70

[0742] οK M : Approximately 4.1 to 410 nanomoles

[0743] οK cat , sec -1 : Approximately 1.1 to 107

[0744] ο1 / K cat , microseconds: approximately 376 to 37,222

[0745] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[0746] Type II

[0747] ο Experiment: GPA microplate

[0748] οV max , min -1 : Approximately 0.3 to 30.5

[0749] οK M , mM: approximately 0.03 to 3.1

[0750] οK cat , sec -1 : Approximately 93 to 9,179

[0751] ο1 / K cat , microseconds: approximately 4 to 428

[0752] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[0753] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[0754] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of collagenase I and collagenase II in a ratio of about 1:1 is injected into at least one treatment area and one or more of the above results 1 to 17 are achieved, wherein the collagenase I and collagenase II have the following characteristics:

[0755] ·Type I

[0756] ο Experiment: SRC microplate

[0757] οVmax,min -1 : Approximately 0.08 to 7.70

[0758] οK M : Approximately 4.1 to 410 nanomoles

[0759] οK cat , sec -1 : Approximately 1.1 to 107

[0760] ο1 / K cat , microseconds: approximately 376 to 37,222

[0761] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[0762] Type II

[0763] ο Experiment: GPA microplate

[0764] οV max , min -1 : Approximately 0.3 to 30.5

[0765] οK M , mM: approximately 0.03 to 3.1

[0766] οK cat , sec -1 : Approximately 93 to 9,179

[0767] ο1 / K cat , microseconds: approximately 4 to 428

[0768] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[0769] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[0770] In other embodiments, in response to the above treatment, the subject is a grade 2 CR-PCSS responder, which means that the subject shows a CR-PCSS rating of at least 2 grades higher than the baseline at the assessment time point (a change of -2, -3 or -4). A grade 1 CR-PCSS responder means that the subject's CR-PCSS score is at least 1 grade higher than the baseline at the assessment time point (a change of -1, -2, -3 or -4). The subject is a grade 2 PR-PCSS responder, which means that the subject's PR-PCSS score is at least 2 grades higher than the baseline at the assessment time point (a change of -2, -3 or -4). A grade 1 PR-PCSS responder means that at the assessment time point, the subject's PR-PCSS score is at least 1 grade higher than the baseline (a change of -1, -2, -3 or -4). In other aspects, a grade 2 composite responder means that the subject is both a grade 2 PR-PCSS responder and a grade 2 CR-PCSS responder at the assessment time point. A grade 1 composite responder means that the subject is both a grade 1 PR-PCSS responder and a grade 1 CR-PCSS responder at the assessment time point.

[0771] 2. Efficacy measured by the Hexsel Cellulite Severity Scale (Hexsel CSS)

[0772] In Hexsel CSS, improvement for an individual patient at any visit is defined as an improvement of at least 1 level or grade relative to baseline or any previous score. Improvement for a group of patients at any visit is defined as an improvement of approximately 0.1 from baseline or any previous mean Hexsel CSS score or rating. A responder is defined as a patient whose maximum total score or rating improves by at least 25% from baseline. In certain embodiments, the treatment methods detailed above result in one or more of the following efficacy endpoints as measured by Hexsel CSS:

[0773] 1. In the population of all patients with a baseline Day 1 Hexsel CSS score, injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[0774] Severe to moderate (score changes from 11-15 to 6-10)

[0775] Change from severe to mild (score changes from 11-15 to 1-5)

[0776] From severe to zero (score changes from 11-15 to 0)

[0777] From moderate to mild (score changes from 6-10 to 1-5)

[0778] From moderate to zero (a score of 6-10 changes to 0)

[0779] From mild to zero (score changes from 1-5 to 0)

[0780] 2. At least a 2-grade improvement in the severity of Hexsel CSS on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") before treatment, as assessed by a live clinician in the target thigh.

[0781] 3. At least a 2-grade improvement in Hexsel CSS severity on Day 22, 43, 71, 90, 180, 365, or 730 from baseline (Day 1), as assessed by the subject observing a digital image of the target thigh.

[0782] 4. Improvement demonstrated by a composite Grade 2 response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as at least a 2-grade improvement in severity from baseline on the Hexsel CSS as assessed by the subject by the clinician and at least a 2-grade improvement in severity from baseline as assessed by the subject.

[0783] 5. At least 1 grade improvement in the severity of subcutaneous cellulite in the Hexsel CSS compared to baseline on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730 from baseline (Day 1).

[0784] 6. At least 1 grade improvement in the severity of subcutaneous cellulite in the Hexsel CSS compared to baseline on Day 22, 43, 71, 90, 180, 365, or 730 from baseline (Day 1).

[0785] 7. Improvement demonstrated by a Grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as at least a 1-grade improvement in severity from baseline on the Hexsel CSS as assessed by the subject by the clinician and at least a 1-grade improvement in severity from baseline as assessed by the subject.

[0786] 8. In patients with all Hexsel CSS scores being moderate or severe, there is a statistically significant improvement in at least one treatment area compared to placebo, where the improvement is one or more of 2 to 7 above.

[0787] 9. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, and more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[0788] 10. The improvements observed in Hexsel CSS at Day 180 relative to baseline were consistent in both the left and right thighs.

[0789] 11. In all patients with moderate or severe Hexsel CSS scores, the median time to earliest 2-grade Hexsel CSS improvement in at least one treatment area was approximately 50 days, 60 days, 70 days, 80 days, or 90 days.

[0790] 12. In all patients with moderate or severe Hexsel CSS scores, the median time to earliest Grade 1 Hexsel CSS improvement in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0791] 13. In all patient groups with moderate or severe Hexsel CSS scores, the mean Hexsel CSS scores of the subjects separated from placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[0792] 14. In all patient populations with moderate or severe Hexsel CSS scores, the percentage of subjects with a Grade 2 composite reaction as measured by Hexsel CSS in at least one treatment area on Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0793] 15. In all patient populations with moderate or severe Hexsel CSS scores, the percentage of subjects achieving a Grade 1 composite response as measured by Hexsel CSS in at least one treatment area on Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0794] 16. Among all patients with a moderate or severe Hexsel CSS score, more than one-third, one-half, two-thirds, or three-quarters of patients had a Hexsel CSS reaction of at least grade 1 in at least one treated area at day 71 after treatment, with Hexsel CSS results independent of age, BMI, or skin color.

[0795] 17. The severity of subcutaneous cellulite is rapidly reduced within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment.

[0796] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 17 above, wherein the collagenase has one or more of the following characteristics:

[0797] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0798] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0799] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0800] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0801] ·Kcat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0802] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0803] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[0804] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0805] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0806] Potency of about 100,000 to about 400,000 GPA units / mg

[0807] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0808] Potency of about 5,000 to about 25,000 ABC units / mg

[0809] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[0810] Bioburden less than or equal to 1 cfu / mL

[0811] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above.

[0812] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above, wherein the collagenase has one or more of the following characteristics:

[0813] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0814] ·KM , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0815] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0816] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0817] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0818] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0819] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[0820] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0821] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0822] Potency of about 100,000 to about 400,000 GPA units / mg

[0823] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0824] Potency of about 5,000 to about 25,000 ABC units / mg

[0825] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[0826] Bioburden less than or equal to 1 cfu / mL

[0827] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above.

[0828] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 17 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[0829] ·Type I

[0830] ο Experiment: SRC microplate

[0831] οVmax,min -1 : Approximately 0.08 to 7.70

[0832] οK M : Approximately 4.1 to 410 nanomoles

[0833] οK cat , sec -1 : Approximately 1.1 to 107

[0834] ο1 / K cat , microseconds: approximately 376 to 37,222

[0835] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[0836] Type II

[0837] ο Experiment: GPA microplate

[0838] οV max , min -1 : Approximately 0.3 to 30.5

[0839] οK M , mM: approximately 0.03 to 3.1

[0840] οK cat , sec -1 : Approximately 93 to 9,179

[0841] ο1 / K cat , microseconds: approximately 4 to 428

[0842] οK cat / K M ,mM -1 sec-1 : Approximately 60 to 5,934

[0843] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[0844] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 17 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[0845] ·Type I

[0846] ο Experiment: SRC microplate

[0847] οVmax,min -1 : Approximately 0.08 to 7.70

[0848] οK M : Approximately 4.1 to 410 nanomoles

[0849] οK cat , sec -1 : Approximately 1.1 to 107

[0850] ο1 / K cat , microseconds: approximately 376 to 37,222

[0851] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[0852] Type II

[0853] ο Experiment: GPA microplate

[0854] οV max , min -1 : Approximately 0.3 to 30.5

[0855] οK M , mM: approximately 0.03 to 3.1

[0856] οK cat , sec -1 : Approximately 93 to 9,179

[0857] ο1 / K cat , microseconds: approximately 4 to 428

[0858] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[0859] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[0860] 3. Therapeutic effect determined by Hexsel depression depth score

[0861] In the Hexsel Indentation Depth Scale, an improvement in an individual patient at any visit is an improvement of at least 1 level or grade relative to baseline or any previous score. An improvement in a group of patients at any visit is an improvement of about 0.1 in the mean Hexsel Indentation Depth score or grade relative to baseline or any previous mean Hexsel Indentation Depth score or grade. A responder is a patient whose maximum total score or grade improves by at least 25% from the baseline test result. In certain embodiments, the treatment methods detailed above produce one or more of the following efficacy endpoints as measured by the Hexsel Indentation Depth Score:

[0862] 1. In the population of all patients with a baseline Day 1 Hexsel Indentation Depth Score, injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of patients meeting one or more of the following efficacy endpoints:

[0863] Changed from deep depression (3) to moderate depression (2)

[0864] Changed from deep depression (3) to shallow depression (1)

[0865] Changed from Deep Recess (3) to No Recess (0)

[0866] Changed from moderate depression (2) to shallow depression (1)

[0867] Changed from moderately concave (2) to not concave (0)

[0868] Changed from shallow depression (1) to no depression (0)

[0869] 2. An improvement of at least 2 grades in severity of the Hexsel Indentation Depth Score from baseline ("Day 1") on Day 22, 43, 71, 90, 180, 365, or 730, as assessed by an on-site clinician in the target thigh.

[0870] 3. At least a 2-grade improvement in the severity of the Hexsel Indentation Depth Score on Day 22, 43, 71, 90, 180, 365, or 730 from baseline (Day 1), as assessed by the subject observing a digital image of the target thigh.

[0871] 4. Improvement demonstrated by a composite Grade 2 response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as an improvement of at least 2 grades in severity from baseline in the Hexsel Indentation Depth Scale as assessed by the subject and an improvement of at least 2 grades in severity from baseline in the Hexsel Indentation Depth Scale as assessed by the subject.

[0872] 5. On Day 22, 43, 71, 90, 180, 365, or 730 from Baseline (Day 1), the severity of subcutaneous cellulite in the Hexsel dimple depth score, as assessed by the subject on digital images of the target thigh, showed at least a 1-grade improvement compared to baseline.

[0873] 6. At least a 1-grade improvement in the severity of subcutaneous cellulite on the Hexsel dimple depth score as assessed by the subject on digital images of the target thigh on Day 22, 43, 71, 90, 180, 365, or 730 starting from Baseline (Day 1).

[0874] 7. Improvement demonstrated by a Grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as at least a 1-grade improvement in severity from baseline in the Hexsel Indentation Depth Scale as assessed by the subject and at least a 1-grade improvement in severity from baseline in the Hexsel Indentation Depth Scale as assessed by the subject.

[0875] 8. In patients with all Hexsel CSS scores being moderate or severe, there is a statistically significant improvement in at least one treatment area compared to placebo, where the improvement is one or more of 2 to 7 above.

[0876] 9. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, and more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[0877] 10. At Day 180, the improvements observed in the Hexsel Indentation Depth Score relative to baseline were consistent in both the left and right thighs.

[0878] 11. In all patients with moderate or severe Hexsel Indentation Depth Scores, the median time to the earliest 2-grade improvement in Hexsel Indentation Depth Score in at least one treated area was approximately 50 days, 60 days, 70 days, 80 days, or 90 days.

[0879] 12. In all patients with moderate or severe Hexsel Indentation Depth Scores, the median time to earliest Grade 1 improvement in Hexsel Indentation Depth Score in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0880] 13. In all patient groups with moderate or severe Hexsel indentation depth scores, the mean Hexsel indentation depth score of the subjects separated from the placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[0881] 14. In all patient populations with moderate or severe Hexsel Indentation Depth Scores, the percentage of subjects with a Grade 2 composite reaction as measured by the Hexsel Indentation Depth Score in at least one treated area on Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0882] 15. In all patient populations with moderate or severe Hexsel Indentation Depth Scores, the percentage of subjects with a Grade 1 composite reaction as measured by the Hexsel Indentation Depth Score in at least one treated area on Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0883] 16. Among all patients with moderate or severe Hexsel Indentation Depth Scores, more than one-third, one-half, two-thirds, or three-quarters of the patients had a Hexsel Indentation Depth Score response of at least Grade 1 in at least one treated area on Day 71 after treatment, and the Hexsel Indentation Depth Score results were not related to age, BMI, or skin color.

[0884] 17. The severity of subcutaneous cellulite is rapidly reduced within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment.

[0885] 18. The change in score from baseline to Day 71 in one or more treatment areas ranged from about -0.1 to about -2.0.

[0886] 19. The least squares (LS) mean of one or more treatment regions within a statistically significant patient population is approximately -0.1 to approximately -1.5 (95% confidence interval (CI)).

[0887] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 19 above, wherein the collagenase has one or more of the following characteristics:

[0888] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0889] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0890] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0891] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0892] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0893] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0894] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[0895] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0896] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0897] Potency of about 100,000 to about 400,000 GPA units / mg

[0898] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0899] Potency of about 5,000 to about 25,000 ABC units / mg

[0900] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[0901] Bioburden less than or equal to 1 cfu / mL

[0902] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 19 described above.

[0903] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 19 above, wherein the collagenase has one or more of the following characteristics:

[0904] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0905] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0906] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0907] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0908] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0909] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0910] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[0911] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0912] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0913] Potency of about 100,000 to about 400,000 GPA units / mg

[0914] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0915] Potency of about 5,000 to about 25,000 ABC units / mg

[0916] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[0917] Bioburden less than or equal to 1 cfu / mL

[0918] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 19 above.

[0919] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 is injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 19 is achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[0920] ·Type I

[0921] ο Experiment: SRC microplate

[0922] οVmax,min -1 : Approximately 0.08 to 7.70

[0923] οK M : Approximately 4.1 to 410 nanomoles

[0924] οK cat , sec -1 : Approximately 1.1 to 107

[0925] ο1 / K cat , microseconds: approximately 376 to 37,222

[0926] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[0927] Type II

[0928] ο Experiment: GPA microplate

[0929] οV max , min -1 : Approximately 0.3 to 30.5

[0930] οK M , mM: approximately 0.03 to 3.1

[0931] οK cat , sec -1 : Approximately 93 to 9,179

[0932] ο1 / K cat , microseconds: approximately 4 to 428

[0933] οK cat / K M ,mM -1 sec-1 : Approximately 60 to 5,934

[0934] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[0935] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 is injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 19 is achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[0936] ·Type I

[0937] ο Experiment: SRC microplate

[0938] οVmax,min -1 : Approximately 0.08 to 7.70

[0939] οK M : Approximately 4.1 to 410 nanomoles

[0940] οK cat , sec -1 : Approximately 1.1 to 107

[0941] ο1 / K cat , microseconds: approximately 376 to 37,222

[0942] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[0943] Type II

[0944] ο Experiment: GPA microplate

[0945] οV max , min -1 : Approximately 0.3 to 30.5

[0946] οK M , mM: approximately 0.03 to 3.1

[0947] οK cat , sec -1 : Approximately 93 to 9,179

[0948] ο1 / K cat , microseconds: approximately 4 to 428

[0949] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[0950] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Furthermore, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[0951] 4. Measuring therapeutic effect using the Likert Aesthetic Scale

[0952] On the Likert scale, improvement for each patient at any visit is an improvement of at least 1 level or grade compared to the baseline or any previous score or rating. The average score or rating for a group of patients at any visit is about 0.1 higher than the baseline examination or any previous average score or rating. In certain embodiments, the treatment methods described in detail above produce one or more of the following efficacy endpoints, as determined by Likert scale score measurement:

[0953] 1. In the subcutaneous cellulite patient population, collagenase injection into at least one treatment area during at least one treatment visit resulted in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[0954] Likert scale score was “improved” (1)

[0955] Likert scale score was “significantly improved” (2)

[0956] Likert scale rating of “very much improved” (3)

[0957] 2. On-site clinician assessment of the target thigh, with at least a 2-grade improvement in the appearance of the treated area as measured on the Likert scale on Days 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") before treatment.

[0958] 3. The subject observed digital images of the target thigh and the Likert scale score showed at least a 2-level improvement in the appearance of the treated area compared to pre-treatment on days 22, 43, 71, 90, 180, 365, or 730.

[0959] 4. On-site evaluation by a clinician of the target thigh, with the Likert scale score showing at least 1 grade improvement in the appearance of the treated area compared to pre-treatment on days 22, 43, 71, 90, 180, 365, or 730.

[0960] 5. The subject observed digital images of the target thigh and the Likert scale score showed at least 1 level improvement in the appearance of the treated area compared to pre-treatment on days 22, 43, 71, 90, 180, 365, or 730.

[0961] 6. In patients with subcutaneous cellulite, a statistically significant improvement in Likert scale score in at least one treated area, wherein the improvement is one or more of 2 to 7 above.

[0962] 7. The treatment resulted in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment resulted in at least 5% of patients showing improvement compared to their pre-treatment baseline, and more improvement over time. Some treatments resulted in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[0963] 8. The improvements observed from baseline in Likert scale scores at Day 180 were consistent in both the left and right thighs.

[0964] 9. In all patients with subcutaneous cellulite, the median time to earliest improvement of a 2-level Likert scale score in at least one treated area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0965] 10. In the overall patient population with subcutaneous cellulite, the median time to earliest improvement of a 2-grade Likert scale score in at least one treated area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0966] 11. In the entire patient population with subcutaneous cellulite, the mean Likert scale scores of the subjects separated from the placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[0967] 12. The percentage of subjects with subcutaneous cellulite who had a Grade 2 reaction as measured on a Likert scale in at least one treated area at Day 71 was approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0968] 13. The percentage of subjects with subcutaneous cellulite who have a Grade 1 reaction as measured on a Likert scale in at least one treated area at Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0969] 14. Among all patients with subcutaneous cellulite, more than one-third, one-half, two-thirds, or three-quarters of patients had a Likert scale response of at least grade 1 in at least one treated area at day 71 after treatment, with Likert scale results being independent of age, BMI, or skin color.

[0970] 15. The severity of subcutaneous cellulite is rapidly reduced within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment.

[0971] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 15 above, wherein the collagenase has one or more of the following characteristics:

[0972] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0973] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0974] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0975] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0976] ·K cat / K M ,mM -1 sec-1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0977] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0978] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[0979] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0980] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0981] Potency of about 100,000 to about 400,000 GPA units / mg

[0982] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0983] Potency of about 5,000 to about 25,000 ABC units / mg

[0984] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[0985] Bioburden less than or equal to 1 cfu / mL

[0986] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 15 described above.

[0987] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above results 1 to 15, wherein the collagenase has one or more of the following characteristics:

[0988] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0989] ·K M, about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[0990] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0991] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0992] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0993] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0994] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[0995] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0996] Potency of about 5,000 to about 30,000 f-SRC units / mg

[0997] Potency of about 100,000 to about 400,000 GPA units / mg

[0998] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[0999] Potency of about 5,000 to about 25,000 ABC units / mg

[1000] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1001] Bioburden less than or equal to 1 cfu / mL

[1002] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above-described results 1 to 15.

[1003] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 15 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1004] ·Type I

[1005] ο Experiment: SRC microplate

[1006] οVmax,min -1 : Approximately 0.08 to 7.70

[1007] οK M : Approximately 4.1 to 410 nanomoles

[1008] οK cat , sec -1 : Approximately 1.1 to 107

[1009] ο1 / K cat , microseconds: approximately 376 to 37,222

[1010] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1011] Type II

[1012] ο Experiment: GPA microplate

[1013] οV max , min -1 : Approximately 0.3 to 30.5

[1014] οK M , mM: approximately 0.03 to 3.1

[1015] οK cat , see- 1 : Approximately 93 to 9,179

[1016] ο1 / K cat , microseconds: approximately 4 to 428

[1017] οK cat / K M ,mM -1 sec-1 : Approximately 60 to 5,934

[1018] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[1019] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 17 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1020] ·Type I

[1021] ο Experiment: SRC microplate

[1022] οVmax,min -1 : Approximately 0.08 to 7.70

[1023] οK M : Approximately 4.1 to 410 nanomoles

[1024] οK cat , sec -1 : Approximately 1.1 to 107

[1025] ο1 / K cat , microseconds: approximately 376 to 37,222

[1026] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1027] Type II

[1028] ο Experiment: GPA microplate

[1029] οV max , min -1 : Approximately 0.3 to 30.5

[1030] οK M , mM: approximately 0.03 to 3.1

[1031] οK cat , sec -1 : Approximately 93 to 9,179

[1032] ο1 / K cat , microseconds: approximately 4 to 428

[1033] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1034] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[1035] 5. pit analysis

[1036] In certain embodiments, treatment of cellulite with collagenase can reduce dimple size parameters as follows:

[1037] · depth : reduce by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%,

[1038] Or 2.5%, or 2%, or 1%

[1039] · width : reduce by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%,

[1040] Or 2.5%, or 2%, or 1%

[1041] · length : reduce by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%,

[1042] Or 2.5%, or 2%, or 1%

[1043] · Total volume : Reduce by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%, or 2.5%, or 2%, or 1%

[1044] · surface area : Reduce by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%, or 2.5%, or 2%, or 1%

[1045] In certain embodiments, the treatment results in at least 5% of patients maintaining an improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, and more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40% or 50% of patients showing improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with more improvements.

[1046] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit reduces a dimensional parameter of at least one dimple by at least 5%, or at least 10%, or at least 20%, wherein the collagenase has one or more of the following characteristics:

[1047] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1048] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1049] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1050] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1051] ·K cat / K M ,mM -1 sec -1Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1052] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1053] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1054] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1055] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1056] Potency of about 100,000 to about 400,000 GPA units / mg

[1057] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1058] Potency of about 5,000 to about 25,000 ABC units / mg

[1059] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1060] Bioburden less than or equal to 1 cfu / mL

[1061] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V can reduce a dimensional parameter of at least one pit by at least 5%, or at least 10%, or at least 20%.

[1062] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area according to Treatments I to V can reduce the dimensional parameters of at least one pit by at least 5%, or at least 10%, or at least 20%, wherein the collagenase has one or more of the following characteristics:

[1063] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1064] ·K M, about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1065] ·K cat (sec -1 ) of about 1.1 to 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[1066] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1067] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1068] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1069] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1070] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1071] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1072] Potency of about 100,000 to about 400,000 GPA units / mg

[1073] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1074] Potency of about 5,000 to about 25,000 ABC units / mg

[1075] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1076] Bioburden less than or equal to 1 cfu / mL

[1077] In another embodiment, injection of about 1 mg to about 20 mg of CCH according to any of Treatments I to V can reduce a dimensional parameter of at least one pit by at least 5%, or at least 10%, or at least 20%.

[1078] In certain embodiments, in at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 injected into at least one treatment area can reduce a dimensional parameter of at least one dimple by at least 5%, or at least 10%, or at least 20%, wherein the collagenase has one or more of the following characteristics:

[1079] ·Type I

[1080] ο Experiment: SRC microplate

[1081] οVmax,min -1 : Approximately 0.08 to 7.70

[1082] οK M : Approximately 4.1 to 410 nanomoles

[1083] οK cat , sec -1 : Approximately 1.1 to 107

[1084] ο1 / K cat , microseconds: approximately 376 to 37,222

[1085] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1086] Type II

[1087] ο Experiment: GPA microplate

[1088] οV max , min -1 : Approximately 0.3 to 30.5

[1089] οK M , mM: approximately 0.03 to 3.1

[1090] οK cat , sec -1 : Approximately 93 to 9,179

[1091] ο1 / K cat , microseconds: approximately 4 to 428

[1092] οK cat / KM ,mM -1 sec -1 : Approximately 60 to 5,934

[1093] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[1094] In certain embodiments, injection of about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 into at least one treatment area using at least one of treatments I to V can reduce the dimensional parameters of at least one dimple by at least 5%, or at least 10%, or at least 20%, wherein the type I collagenase and type II collagenase have the following characteristics:

[1095] ·Type I

[1096] ο Experiment: SRC microplate

[1097] οVmax,min -1 : Approximately 0.08 to 7.70

[1098] οK M : Approximately 4.1 to 410 nanomoles

[1099] οK cat , see- 1 : Approximately 1.1 to 107

[1100] ο1 / K cat , microseconds: approximately 376 to 37,222

[1101] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1102] Type II

[1103] ο Experiment: GPA microplate

[1104] οV max , min -1 : Approximately 0.3 to 30.5

[1105] οK M , mM: approximately 0.03 to 3.1

[1106] οK cat , sec -1 : Approximately 93 to 9,179

[1107] ο1 / K cat , microseconds: approximately 4 to 428

[1108] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1109] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[1110] 6. The Subject Global Aesthetic Improvement Scale (S-GAIS) and the Investigator Global Aesthetic Improvement Scale (I-GAIS) were used to measure Efficacy

[1111] The above treatment methods can achieve improved therapeutic effects as measured by S-GAIS and I-GAIS. A Grade 2 S-GAIS responder is a subject whose S-GAIS score is at least 2 (+2 or +3) at the assessment time point. A Grade 1 S-GAIS responder is a subject whose S-GAIS score is at least 1 (+1, +2 or +3) at the assessment time point. A Grade 2 I-GAIS responder is a subject whose I-GAIS score is at least 2 (+2 or +3) at the assessment time point. A Grade 1 I-GAIS responder is a subject whose I-GAIS score is at least 1 (+1, +2 or +3) at the assessment time point. Improvement for an individual patient at any visit means an improvement of at least 1 grade or 1 level compared to baseline or any previous score. The average score or score of a group of patients at any visit is approximately 0.1 higher than the average score or score at baseline or any previous visit.

[1112] In certain embodiments, the treatment methods described in detail above produce one or more of the following efficacy endpoints as measured by S-GAIS and / or I-GAIS:

[1113] 1. In a population of patients with subcutaneous cellulite, the injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[1114] S-GAIS and / or I-GAIS score of "improved" (+1)

[1115] S-GAIS and / or I-GAIS score of "much improved" (+2)

[1116] S-GAIS and / or I-GAIS score of "very much improved" (+3)

[1117] 2. At least a 2-grade improvement in I-GAIS as assessed by a clinician in the target thigh on day 22, day 43, day 71, day 90, day 180, day 365, or day 730

[1118] 3. At least a 2-grade improvement in the S-GAIS can be observed on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, as assessed by the subject observing a digital image of the target thigh.

[1119] 4. Improvement demonstrated by a Grade 2 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as at least a 2-grade improvement in severity compared to baseline on the I-GAIS as assessed by the clinician and at least a 2-grade improvement in severity compared to baseline on the S-GAIS as assessed by the subject.

[1120] 5. At least 1-grade improvement in I-GAIS observed on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730 as assessed by a clinician in the target thigh

[1121] 6. At least a 1-grade improvement in the S-GAIS can be observed on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, as assessed by the subject observing a digital image of the target thigh.

[1122] 7. Improvement demonstrated by a Grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as at least a 1-grade improvement in severity compared to baseline on the I-GAIS as assessed by the clinician and at least a 1-grade improvement in severity compared to baseline on the S-GAIS as assessed by the subject.

[1123] 8. In a patient population with subcutaneous cellulite, in which all scores are moderate or severe, statistically significant improvement in I-GAIS and / or S-GAIS in at least one treated area compared to placebo, wherein the improvement is one or more of 2 to 7 above.

[1124] 9. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, and more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[1125] 10. Improvements from baseline observed in I-GAIS and / or S-GAIS at Day 180 were consistent in both the left and right thighs.

[1126] 11. In the overall patient population with subcutaneous cellulite, the median time to earliest grade 2 improvement in I-GAIS and / or S-GAIS score in at least one treated area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1127] 12. In the overall patient population with subcutaneous cellulite, the median time to earliest grade 1 improvement in I-GAIS and / or S-GAIS score in at least one treated area is approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1128] 13. In the overall patient population with subcutaneous cellulite, the mean scores of the subjects on the I-GAIS and / or S-GAIS separated from placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[1129] 14. The percentage of subjects with a Grade 2 reaction as measured by I-GAIS and / or S-GAIS in at least one treated area on Day 71 in the total patient population with subcutaneous cellulite is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1130] 15. The percentage of subjects with a Grade 1 response as measured by I-GAIS and / or S-GAIS in at least one treated area at Day 71 in the total patient population with subcutaneous cellulite is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1131] 16. Among all patients with subcutaneous cellulite and a Hexsel CSS score of moderate or severe, more than one-third, one-half, two-thirds, or three-quarters of patients had an I-GAIS and / or S-GAIS response of at least Grade 1 in at least one treated area on Day 71 after treatment, where the I-GAIS and / or S-GAIS response was not related to age, BMI, or skin color.

[1132] 17. The severity of subcutaneous cellulite is rapidly reduced within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment.

[1133] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 17 above, wherein the collagenase has one or more of the following characteristics:

[1134] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1135] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1136] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1137] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1138] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1139] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1140] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1141] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1142] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1143] Potency of about 100,000 to about 400,000 GPA units / mg

[1144] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1145] Potency of about 5,000 to about 25,000 ABC units / mg

[1146] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1147] Bioburden less than or equal to 1 cfu / mL

[1148] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above.

[1149] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above, wherein the collagenase has one or more of the following characteristics:

[1150] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1151] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1152] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1153] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1154] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1155] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1156] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1157] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1158] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1159] Potency of about 100,000 to about 400,000 GPA units / mg

[1160] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1161] Potency of about 5,000 to about 25,000 ABC units / mg

[1162] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1163] Bioburden less than or equal to 1 cfu / mL

[1164] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above.

[1165] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 17 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1166] ·Type I

[1167] ο Experiment: SRC microplate

[1168] οVmax,min -1 : Approximately 0.08 to 7.70

[1169] οK M : Approximately 4.1 to 410 nanomoles

[1170] οK cat , sec -1 : Approximately 1.1 to 107

[1171] ο1 / K cat , microseconds: approximately 376 to 37,222

[1172] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1173] Type II

[1174] ο Experiment: GPA microplate

[1175] οV max , min -1 : Approximately 0.3 to 30.5

[1176] οK M , mM: approximately 0.03 to 3.1

[1177] οK cat , sec -1 : Approximately 93 to 9,179

[1178] ο1 / K cat , microseconds: approximately 4 to 428

[1179] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1180] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[1181] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 17 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1182] ·Type I

[1183] ο Experiment: SRC microplate

[1184] οVmax,min -1 : Approximately 0.08 to 7.70

[1185] οK M : Approximately 4.1 to 410 nanomoles

[1186] οK cat , sec -1 : Approximately 1.1 to 107

[1187] ο1 / K cat , microseconds: approximately 376 to 37,222

[1188] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1189] Type II

[1190] ο Experiment: GPA microplate

[1191] οV max , min -1 : Approximately 0.3 to 30.5

[1192] οK M , mM: approximately 0.03 to 3.1

[1193] οK cat , sec -1 : Approximately 93 to 9,179

[1194] ο1 / K cat , microseconds: approximately 4 to 428

[1195] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1196] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be expressed as AUX-I and AUX-II, respectively.

[1197] 7. Efficacy measured by PR-CIS

[1198] The treatments detailed above can achieve improved treatment outcomes, as measured by the PR-CIS. The PR-CIS total score is the sum of the six items on the scale. Item 1 of the PR-CIS asks the subject how satisfied they are with the appearance of cellulite, which is inverted by subtracting the subject's reported assessment from 10. The PR-CIS total score can range from 0 to 60, with higher values ​​indicating a greater negative impact of cellulite. For the PR-CIS total score, a responder is defined as a subject whose PR-CIS total score decreases by at least 12 points from baseline at the assessment time point. For the individual PR-CIS impact score, a response is defined as an improvement of at least two score intervals from baseline at each time point. Furthermore, a responder is defined as any patient who demonstrates at least a 20% improvement from the maximum total score at baseline. Improvement for an individual patient at any visit is defined as an improvement of at least one level or grade compared to baseline or any previous score. The mean score or grade for a group of patients at any visit is defined as an improvement of approximately 0.1 from baseline or any previous mean score or grade. Furthermore, improvement is defined as a change of at least one level or grade from baseline out of 60.

[1199] In certain embodiments, the treatment methods described in detail above can achieve one or more of the following efficacy endpoints, as determined by PR-CIS:

[1200] 1. In a patient population with subcutaneous cellulite, the injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of patients meeting one or more of the following efficacy endpoints:

[1201] PR-CIS shows improvement in at least one domain selected from the group consisting of happiness, annoyance, self-consciousness, embarrassment, looking old, and looking overweight / unhealthy PR-CIS total score decreases by at least 12 points from baseline at one or more assessment time points

[1202] PR-CIS impact scores showing improvement of at least 2 score intervals from baseline at one or more assessment time points

[1203] Improvement is defined as a change of at least 1 level from baseline in 60 points

[1204] 2. Improvement of at least 12 points in severity as measured by the PR-CIS on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") before treatment for the target thigh.

[1205] 3. In patients with subcutaneous cellulite, a statistically significant improvement in severity as measured by the PR-CIS on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") compared to placebo was achieved, meaning an improvement of at least 12 points.

[1206] 4. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, with more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[1207] 5. The improvement from baseline in PR-CIS scores observed at Day 180 was consistent in both the left and right thighs.

[1208] 6. In the total patient population with subcutaneous cellulite, the median time to a reduction of at least 12 in the PR-CIS total score compared to baseline, as measured at one or more assessment time points, in at least one treated area is approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1209] 7. In the entire patient population with subcutaneous cellulite, the mean PR-CIS score of the subjects separated from the placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[1210] 8. Among all patients with subcutaneous cellulite, more than one-third, one-half, two-thirds or three-quarters of the patients had a reduction of at least 12 in the PR-CIS total score compared to baseline at one or more assessment time points on day 71 after treatment, and the PR-CIS score results were not related to age, BMI or skin color.

[1211] 9. The severity of subcutaneous cellulite decreases rapidly within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment visit.

[1212] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 9 above, wherein the collagenase has one or more of the following characteristics:

[1213] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1214] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1215] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1216] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1217] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1218] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1219] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1220] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1221] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1222] Potency of about 100,000 to about 400,000 GPA units / mg

[1223] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1224] Potency of about 5,000 to about 25,000 ABC units / mg

[1225] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1226] Bioburden less than or equal to 1 cfu / mL

[1227] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 9 described above.

[1228] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above results 1 to 9, wherein the collagenase has one or more of the following characteristics:

[1229] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1230] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1231] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1232] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1233] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1234] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1235] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1236] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1237] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1238] Potency of about 100,000 to about 400,000 GPA units / mg

[1239] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1240] Potency of about 5,000 to about 25,000 ABC units / mg

[1241] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1242] Bioburden less than or equal to 1 cfu / mL

[1243] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V produces one or more of the above results 1 to 9.

[1244] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 9 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1245] ·Type I

[1246] ο Experiment: SRC microplate

[1247] οVmax,min -1 : Approximately 0.08 to 7.70

[1248] οK M : Approximately 4.1 to 410 nanomoles

[1249] οK cat , sec-1 : Approximately 1.1 to 107

[1250] ο1 / K cat , microseconds: approximately 376 to 37,222

[1251] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1252] Type II

[1253] ο Experiment: GPA microplate

[1254] οV max , min -1 : Approximately 0.3 to 30.5

[1255] οK M , mM: approximately 0.03 to 3.1

[1256] οK cat , sec -1 : Approximately 93 to 9,179

[1257] ο1 / K cat , microseconds: approximately 4 to 428

[1258] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1259] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1260] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 9 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1261] ·Type I

[1262] ο Experiment: SRC microplate

[1263] οVmax,min -1 : Approximately 0.08 to 7.70

[1264] οK M : Approximately 4.1 to 410 nanomoles

[1265] οK cat , sec -1 : Approximately 1.1 to 107

[1266] ο1 / K cat , microseconds: approximately 376 to 37,222

[1267] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1268] Type II

[1269] ο Experiment: GPA microplate

[1270] οV max , min -1 : Approximately 0.3 to 30.5

[1271] οK M , mM: approximately 0.03 to 3.1

[1272] οK cat , sec -1 : Approximately 93 to 9,179

[1273] ο1 / K cat , microseconds: approximately 4 to 428

[1274] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1275] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1276] 8. Efficacy measured by the shortened version of PR-CIS

[1277] The treatments described in detail above demonstrate improved treatment outcomes, as measured by the PR-CIS (Abbreviated Impact Score). The PR-CIS (Abbreviated Impact Score) total score is the sum of the five items on the scale. Item 1 of the PR-CIS asks about the subject's satisfaction with the appearance of cellulite, which is inverted by subtracting the subject's reported assessment from 10. The PR-CIS (Abbreviated Impact Score) total score can range from 0 to 50, with higher numbers indicating a greater negative impact of cellulite. For the PR-CIS (Abbreviated Impact Score), a responder is defined as a subject whose PR-CIS total score decreases by at least 10 points compared to baseline at the assessment time point. For individual PR-CIS (Abbreviated Impact Score), a response is defined as an improvement of at least two score intervals from baseline at each time point. Furthermore, a responder is defined as any patient who demonstrates at least a 20% improvement from the maximum total score at baseline. Improvement for an individual patient at any visit is defined as an improvement of at least one level or grade compared to baseline or any previous score. The mean score or grade for a group of patients at any visit improves by approximately 0.1 compared to the mean score or grade at baseline or any previous visit. Furthermore, improvement was defined as a change of at least 1 point out of 50 from baseline.

[1278] In certain embodiments, the treatment methods described above can achieve one or more of the following effects as measured by the reduced PR-CIS:

[1279] 1. In a patient population with subcutaneous cellulite, injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of patients meeting one or more of the following efficacy endpoints:

[1280] PR-CIS-Abridged version showing improvement in at least one domain selected from the group consisting of happiness, annoyance, self-consciousness, embarrassment, looking old, and looking overweight / unhealthy

[1281] A decrease of at least 10 points from baseline in the PR-CIS abbreviated version total score at one or more assessment time points

[1282] PR-CIS Abbreviated Impact Score showing improvement of at least 2 score intervals from baseline at one or more assessment time points

[1283] Furthermore, improvement is defined as a change of at least 1 level out of 50 from baseline

[1284] 2. Improvement of at least 12 points in severity as measured by the abbreviated PR-CIS on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") before treatment for the target thigh.

[1285] 3. In patients with subcutaneous cellulite, a statistically significant improvement in severity as measured by the PR-CIS-Abridged version on days 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") compared to placebo was achieved, meaning an improvement of at least 10 points.

[1286] 4. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, with more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[1287] 5. The improvement from baseline in the PR-CIS abbreviated version score observed at Day 180 was consistent in both the left and right thighs.

[1288] 6. In the total patient population with subcutaneous cellulite, the median time to a reduction of at least 10 points in the PR-CIS-reduced total score compared to baseline in at least one treated area at one or more assessment time points was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1289] 7. In the entire patient population with subcutaneous cellulite, the mean PR-CIS reduced version score of the subjects separated from the placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[1290] 8. Among all patients with subcutaneous cellulite, more than one-third, one-half, two-thirds, or three-quarters of patients achieved a reduction of at least 10 points in the PR-CIS-reduced total score at one or more assessment time points compared to baseline at day 71 after treatment, with the PR-CIS score being independent of age, BMI, or skin color.

[1291] 9. The severity of subcutaneous cellulite decreases rapidly within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment visit.

[1292] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of the above results 1 to 9, wherein the collagenase has one or more of the following characteristics

[1293] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1294] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1295] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1296] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1297] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1298] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1299] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1300] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1301] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1302] Potency of about 100,000 to about 400,000 GPA units / mg

[1303] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1304] Potency of about 5,000 to about 25,000 ABC units / mg

[1305] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1306] Bioburden less than or equal to 1 cfu / mL

[1307] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 9 described above.

[1308] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above results 1 to 9, wherein the collagenase has one or more of the following characteristics:

[1309] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1310] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1311] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1312] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1313] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1314] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1315] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1316] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1317] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1318] Potency of about 100,000 to about 400,000 GPA units / mg

[1319] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1320] Potency of about 5,000 to about 25,000 ABC units / mg

[1321] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1322] Bioburden less than or equal to 1 cfu / mL

[1323] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above-described results 1 to 9.

[1324] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 9 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1325] ·Type I

[1326] ο Experiment: SRC microplate

[1327] οVmax,min -1 : Approximately 0.08 to 7.70

[1328] οK M : Approximately 4.1 to 410 nanomoles

[1329] οK cat , sec -1 : Approximately 1.1 to 107

[1330] ο1 / K cat , microseconds: approximately 376 to 37,222

[1331] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1332] Type II

[1333] ο Experiment: GPA microplate

[1334] οV max , min -1 : Approximately 0.3 to 30.5

[1335] οK M , mM: approximately 0.03 to 3.1

[1336] οK cat , sec -1 : Approximately 93 to 9,179

[1337] ο1 / K cat , microseconds: approximately 4 to 428

[1338] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1339] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1340] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 9 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1341] ·Type I

[1342] ο Experiment: SRC microplate

[1343] οVmax,min -1 : Approximately 0.08 to 7.70

[1344] οK M : Approximately 4.1 to 410 nanomoles

[1345] οK cat, sec -1 : Approximately 1.1 to 107

[1346] ο1 / K cat , microseconds: approximately 376 to 37,222

[1347] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1348] Type II

[1349] ο Experiment: GPA microplate

[1350] οV max , min -1 : Approximately 0.3 to 30.5

[1351] οK M , mM: approximately 0.03 to 3.1

[1352] οK cat , sec -1 : Approximately 93 to 9,179

[1353] ο1 / K cat , microseconds: approximately 4 to 428

[1354] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1355] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1356] 9. The efficacy of the test was determined by the Subject Self-Rating Scale (SSRS)

[1357] The treatment methods described in detail above produce an improved treatment effect as measured by the SSRS. An SSRS responder is a subject who is at least somewhat satisfied (somewhat satisfied [4], very satisfied [5], or very satisfied [6]) with the appearance of cellulite on the thighs at Day 71. Additionally, a responder is any patient who has improved by at least 17% in the maximum total score compared to baseline. An improvement for an individual patient at any visit is an improvement of at least 1 level or grade compared to baseline or any previous score. The average score or score for a group of patients at any visit is an improvement of about 0.1 over the baseline examination or any previous average score or score. In certain embodiments, the treatment method produces one or more of the following efficacy endpoints as measured by the SSRS rating:

[1358] 1. In a population of patients with subcutaneous cellulite, the injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[1359] SSRS rating is "relatively satisfactory"

[1360] SSRS score is "Satisfactory"

[1361] SSRS rating is "very satisfactory"

[1362] 2. An improvement of at least 2 grades in severity as measured by the SSRS score on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") before treatment for the target thigh.

[1363] 3. Improvement of at least 1 grade in severity as measured by the SSRS score on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") before treatment for the target thigh.

[1364] 4. In a patient population with subcutaneous cellulite, the improvement in SSRS score in at least one treated area is statistically significant compared to placebo, wherein the improvement is one or more of 2 to 3 above.

[1365] 5. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, at least 5% of patients show improvement compared to their pre-treatment baseline, and more improvement is seen over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements seen.

[1366] 6. The improvements observed in SSRS scores at Day 180 relative to baseline were consistent in both the left and right thighs.

[1367] 7. In all patients with subcutaneous cellulite, the median time to the earliest 2-grade improvement in SSRS score in at least one treated area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1368] 8. In the overall patient population with subcutaneous cellulite, the median time to earliest improvement of 1 grade SSRS score in at least one treated area is approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1369] 9. In the entire patient population with subcutaneous cellulite, the mean SSRS score of the subjects separated from the placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[1370] 10. The percentage of subjects with subcutaneous cellulite who have a Grade 2 response as measured by the SSRS score on Day 71 in at least one treated area is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1371] 11. The percentage of subjects with subcutaneous cellulite who have a Grade 1 response as measured by the SSRS score on Day 71 in at least one treated area is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1372] 12. Among all patients with subcutaneous cellulite, more than one-third, one-half, two-thirds, or three-quarters of the patients had an SSRS measurement score of at least 1 in at least one treated area on day 71 after treatment, where the SSRS measurement score results were not related to age, BMI, or skin color.

[1373] 13. The severity of subcutaneous cellulite decreases rapidly within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment visit.

[1374] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 13 above, wherein the collagenase has one or more of the following characteristics:

[1375] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1376] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1377] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1378] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1379] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1380] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1381] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1382] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1383] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1384] Potency of about 100,000 to about 400,000 GPA units / mg

[1385] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1386] Potency of about 5,000 to about 25,000 ABC units / mg

[1387] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1388] Bioburden less than or equal to 1 cfu / mL

[1389] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 13 described above.

[1390] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 13 above, wherein the collagenase has one or more of the following characteristics.

[1391] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1392] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1393] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1394] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1395] ·K cat / K M ,mM-1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1396] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1397] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1398] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1399] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1400] Potency of about 100,000 to about 400,000 GPA units / mg

[1401] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1402] Potency of about 5,000 to about 25,000 ABC units / mg

[1403] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1404] Bioburden less than or equal to 1 cfu / mL

[1405] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 13 above.

[1406] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 13 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1407] ●Type I

[1408] ο Experiment: SRC microplate

[1409] οVmax,min -1 : Approximately 0.08 to 7.70

[1410] οK M : Approximately 4.1 to 410 nanomoles

[1411] οK cat , sec -1 : Approximately 1.1 to 107

[1412] ο1 / K cat , microseconds: approximately 376 to 37,222

[1413] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1414] Type II

[1415] ο Experiment: GPA microplate

[1416] οV max , min -1 : Approximately 0.3 to 30.5

[1417] οK M , mM: approximately 0.03 to 3.1

[1418] οK cat , sec -1 : Approximately 93 to 9,179

[1419] ο1 / K cat , microseconds: approximately 4 to 428

[1420] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1421] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1422] In certain embodiments, any of Treatments I to V is used to inject about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 into at least one treatment area and achieve one or more of the above results 1 to 13, wherein the type I collagenase and type II collagenase have the following characteristics:

[1423] ·Type I

[1424] ο Experiment: SRC microplate

[1425] οVmax,min -1 : Approximately 0.08 to 7.70

[1426] οK M : Approximately 4.1 to 410 nanomoles

[1427] οK cat , sec -1 : Approximately 1.1 to 107

[1428] ο1 / K cat , microseconds: approximately 376 to 37,222

[1429] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1430] Type II

[1431] ο Experiment: GPA microplate

[1432] οV max , min -1 : Approximately 0.3 to 30.5

[1433] οK M , mM: approximately 0.03 to 3.1

[1434] οK cat , sec -1 : Approximately 93 to 9,179

[1435] ο1 / K cat , microseconds: approximately 4 to 428

[1436] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1437] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1438] 10. Measuring efficacy based on subject satisfaction with cellulite treatment (SSCT)

[1439] The treatment methods described in detail above produce an improved therapeutic effect as measured by the SSCT. In the SSCT assessment, subjects whose responses to cellulite on the thighs were "satisfied" or "very satisfied" on day 71 were considered responders showing efficacy. Improvement in an individual patient at any visit refers to an improvement of at least 1 level or grade compared to the baseline or any previous score. The average score or rating of a group of patients at any visit is about 0.1 higher than the baseline examination or any previous average score or rating. Further, for SSCT, improvement is at least 0.1 higher compared to placebo. In certain embodiments, the treatment method produces one or more of the following efficacy endpoints as measured by the SSCT rating:

[1440] 1. In a population of patients with subcutaneous cellulite, the injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[1441] SSCT score is "satisfactory"

[1442] SSCT score was “very satisfactory”

[1443] 2. For the target thigh, the SSCT score increases (e.g., 1 to 2, etc.) on day 22, day 43, day 71, day 90, day 180, day 365, or day 730.

[1444] 3. In a patient population with subcutaneous cellulite, a statistically significant increase in SSCT score in at least one treated area compared to placebo at Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730.

[1445] 4. The treatment results in at least 5% of patients maintaining improved results compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, at least 5% of patients show improvement compared to their pre-treatment baseline, and more improvements are seen over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements seen.

[1446] 5. The improvement from baseline in SSCT scores observed at Day 180 was consistent in both the left and right thighs.

[1447] 6. In the overall patient population with subcutaneous cellulite, the median time to earliest improvement in SSCT score in at least one treated area is approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1448] 7. In the entire patient population with subcutaneous cellulite, the mean SSRS score of the subjects separated from the placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[1449] 8. The percentage of subjects with subcutaneous cellulite who have an improved SSCT score in at least one treated area at Day 71 is between about 1% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, or greater than 50%.

[1450] 9. Among all patients with subcutaneous cellulite, more than one-third, one-half, two-thirds, or three-quarters of patients had an improved SSCT score in at least one treated area at day 71 after treatment, where the SSCT score results were not related to age, BMI, or skin color.

[1451] 10. The severity of subcutaneous cellulite decreases rapidly within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment visit.

[1452] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 10 above, wherein the collagenase has one or more of the following characteristics:

[1453] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1454] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1455] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1456] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1457] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1458] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1459] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1460] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1461] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1462] Potency of about 100,000 to about 400,000 GPA units / mg

[1463] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1464] Potency of about 5,000 to about 25,000 ABC units / mg

[1465] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1466] Bioburden less than or equal to 1 cfu / mL

[1467] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above-described results 1 to 10.

[1468] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above results 1 to 10, wherein the collagenase has one or more of the following characteristics:

[1469] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1470] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1471] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1472] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1473] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1474] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1475] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1476] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1477] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1478] Potency of about 100,000 to about 400,000 GPA units / mg

[1479] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1480] Potency of about 5,000 to about 25,000 ABC units / mg

[1481] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1482] Bioburden less than or equal to 1 cfu / mL

[1483] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above results 1 to 10.

[1484] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 10 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1485] ·Type I

[1486] ο Experiment: SRC microplate

[1487] οVmax,min -1 : Approximately 0.08 to 7.70

[1488] οK M : Approximately 4.1 to 410 nanomoles

[1489] οK cat , see- 1 : Approximately 1.1 to 107

[1490] ο1 / K cat , microseconds: approximately 376 to 37,222

[1491] οK cat / K M ,mM -1 sec -1: Approximately 5,140 to 508,814

[1492] Type II

[1493] ο Experiment: GPA microplate

[1494] οV max , min -1 : Approximately 0.3 to 30.5

[1495] οK M , mM: approximately 0.03 to 3.1

[1496] οK cat , see -1 : Approximately 93 to 9,179

[1497] ο1 / K cat , microseconds: approximately 4 to 428

[1498] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1499] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1500] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 10 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1501] ·Type I

[1502] ο Experiment: SRC microplate

[1503] οVmax,min -1 : Approximately 0.08 to 7.70

[1504] οK M : Approximately 4.1 to 410 nanomoles

[1505] οK cat , sec -1 : Approximately 1.1 to 107

[1506] ο1 / K cat , microseconds: approximately 376 to 37,222

[1507] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1508] Type II

[1509] ο Experiment: GPA microplate

[1510] οV max , min -1 : Approximately 0.3 to 30.5

[1511] οK M , mM: approximately 0.03 to 3.1

[1512] οK cat , see- 1 : Approximately 93 to 9,179

[1513] ο1 / K cat , microseconds: approximately 4 to 428

[1514] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1515] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1516] 11. Thigh Cellulite Treatment Scale (CR-TCES; PR-TCES)

[1517] An improvement in an individual patient at any visit is an improvement of at least 1 level or grade relative to the baseline or any previous score. An improvement in a group of patients at any visit is an improvement of about 0.1 relative to the baseline or any previous mean score. A responder is a patient whose maximum total score or score improves by at least 20% from the baseline test results. In certain embodiments, the treatment methods detailed above produce one or more of the following efficacy endpoints measured by CR-TCES and / or PR-TCES:

[1518] 1. CR-TCES and / or PR-TCES score improved by at least 0.1 compared with baseline.

[1519] 2. For the target thigh, the severity of the disease as measured by the CR-TCES score improved by at least 2 levels on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1"), as assessed by a clinician on-site.

[1520] 3. For the target thigh, the severity of the disease as measured by the PR-TCES score improved by at least 2 levels on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1"), as assessed by a clinician on-site.

[1521] 4. Improvement demonstrated by a grade 2 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as at least a 2-grade improvement in severity from baseline in the CR-TCES or at least a 2-grade improvement in severity from baseline in the PR-TCES.

[1522] 5. At least 1 grade improvement in the severity of subcutaneous cellulite on the CR-TCES as assessed by a clinician in the target thigh on day 22, 43, 71, 90, 180, 365, or 730 from baseline (day 1).

[1523] 6. At least 1 grade improvement in the severity of subcutaneous cellulite on the PR-TCES assessed by a clinician in the target thigh on day 22, 43, 71, 90, 180, 365, or 730 from baseline (day 1).

[1524] 7. Improvement demonstrated by a grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730, defined as at least a 1-grade improvement in severity from baseline in the CR-TCES or at least a 1-grade improvement in severity from baseline in the PR-TCES.

[1525] 8. In the patient population where all CR-TCES scores were moderate or severe, there was a statistically significant improvement in at least one treatment area compared to placebo, wherein the improvement was one or more of 1 to 7 above.

[1526] 9. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, and more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[1527] 10. The improvements observed from baseline in CR-TCES and / or PR-TCES scores at Day 180 were consistent in the left and right thighs.

[1528] 11. In all patient populations with moderate or severe CR-TCES scores, the median time to earliest improvement of grade 2 CR-TCES and / or PR-TCES score in at least one treatment area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1529] 12. In all patient populations with moderate or severe CR-TCES scores, the median time to earliest improvement of a grade 1 CR-TCES and / or PR-TCES score in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1530] 13. In all patient groups with moderate or severe CR-TCES scores, the subjects' average CR-TCES and / or PR-TCES scores separated from placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[1531] 14. In all patient populations with moderate or severe CR-TCES scores, the percentage of subjects achieving a Grade 2 composite response as measured by CR-TCES and / or PR-TCES in at least one treatment area on Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1532] 15. In all patient populations with moderate or severe CR-TCES scores, the percentage of subjects achieving a Grade 1 composite response as measured by CR-TCES and / or PR-TCES in at least one treatment area on Day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1533] 16. In the population of patients with moderate or severe CR-TCES scores, more than one-third, one-half, two-thirds, or three-quarters of the patients had at least 1 CR-TCES and / or at least 1 PR-TCES response in at least one treated area on day 71 after treatment, where the CR-TCES score measurement results were not related to age, BMI, or skin color.

[1534] 17. The severity of subcutaneous cellulite decreases rapidly within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment visit.

[1535] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 17 above, wherein the collagenase has one or more of the following characteristics:

[1536] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1537] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1538] ·K cat (sec -1 ) is from about 1.1 to about 107 (SRC experiment), or,

[1539] About 93 to 9,179 (GPA experiment)

[1540] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1541] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1542] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1543] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1544] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1545] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1546] Potency of about 100,000 to about 400,000 GPA units / mg

[1547] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1548] Potency of about 5,000 to about 25,000 ABC units / mg

[1549] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1550] Bioburden less than or equal to 1 cfu / mL

[1551] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above.

[1552] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above, wherein the collagenase has one or more of the following characteristics:

[1553] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1554] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1555] ·K cat (sec-1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1556] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1557] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1558] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1559] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1560] A potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg A potency of about 5,000 to about 30,000 f-SRC units / mg

[1561] Potency of about 100,000 to about 400,000 GPA units / mg

[1562] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1563] Potency of about 5,000 to about 25,000 ABC units / mg

[1564] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from

[1565] The group consisting of bacterial protease, gelatinase and leupeptin

[1566] Bioburden less than or equal to 1 cfu / mL

[1567] In another embodiment, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 17 above.

[1568] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 10 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1569] ·Type I

[1570] ο Experiment: SRC microplate

[1571] οVmax,min -1 : Approximately 0.08 to 7.70

[1572] οK M : Approximately 4.1 to 410 nanomoles

[1573] οK cat , sec -1 : Approximately 1.1 to 107

[1574] ο1 / K cat , microseconds: approximately 376 to 37,222

[1575] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1576] Type II

[1577] ο Experiment: GPA microplate

[1578] οV max , min -1 : Approximately 0.3 to 30.5

[1579] οK M , mM: approximately 0.03 to 3.1

[1580] οK cat , sec -1 : Approximately 93 to 9,179

[1581] ο1 / K cat , microseconds: approximately 4 to 428

[1582] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1583] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1584] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 17 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1585] ·Type I

[1586] ο Experiment: SRC microplate

[1587] οVmax,min -1 : Approximately 0.08 to 7.70

[1588] οK M : Approximately 4.1 to 410 nanomoles

[1589] οK cat , sec -1 : Approximately 1.1 to 107

[1590] ο1 / K cat , microseconds: approximately 376 to 37,222

[1591] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1592] Type II

[1593] ο Experiment: GPA microplate

[1594] οV max , min -1 : Approximately 0.3 to 30.5

[1595] οK M , mM: approximately 0.03 to 3.1

[1596] οK cat , sec -1 : Approximately 93 to 9,179

[1597] ο1 / K cat, microseconds: approximately 4 to 428

[1598] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1599] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1600] 12. Efficacy measured by Body-Q

[1601] The treatment methods described in detail above produce improved treatment effects as measured by the Body-Q. For subcutaneous cellulite, there are 16 scale items measuring patient response over the past week, with options ranging from "not at all" to "very bothersome" and combined into a Flesch-Kincaid scale reading level. Scores range from 16 (very bothersome) to 64 (not at all). For the Body-Q total score, a responder is a patient whose Body-Q total score increases by at least 16 points compared to baseline at the assessment time point. For each Body-Q impact score, a response is an improvement of at least one score interval compared to baseline at each time point. In an alternative embodiment, the scored items can be greater or less than 16, and the responder is a patient whose maximum total score improves by at least 25% compared to baseline.

[1602] In certain embodiments, the treatment methods detailed above produce one or more of the following efficacy endpoints as measured by Body-Q:

[1603] 1. In a population of patients with subcutaneous cellulite, the injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[1604] Body-Q shows improvement in at least one aspect selected from the group consisting of:

[1605] An increase of at least 16 points in the total score of the Body-Q measure compared to baseline at one or more assessment time points;

[1606] o an improvement of at least 1 point compared to baseline at each time point for each Body-Q impact score; and

[1607] o Improvement from baseline of at least 25% in the maximum total score.

[1608] An increase of at least 16 in the Body-Q total score compared to baseline at one or more assessment time points.

[1609] Body-Q impact scores improved by at least 1 point at each time point compared to baseline

[1610] Mean change from baseline in subcutaneous cellulite identified by Body-Q occurred at Day 90 and / or Day 180

[1611] 2. For the target thigh, improvement in severity is an increase of at least 16 points in the Body-Q score on Day 22, 43, 71, 90, 180, 365, or 730 from baseline ("Day 1") before treatment.

[1612] 3. In patients with subcutaneous cellulite, a statistically significant improvement in severity of at least 16 points compared to placebo was achieved on the Body-Q test on Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730 from baseline (the first day before treatment) in the target thigh.

[1613] 4. The treatment results in at least 5% of patients maintaining their improvement compared to their pre-treatment baseline for 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintain this level of improvement for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial dose. In other cases, treatment results in at least 5% of patients showing improvement compared to their pre-treatment baseline, with more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40%, or 50% of patients showing improvement at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with further improvements.

[1614] 5. At day 180 after the first injection, the improvements observed in Body-Q scores compared to baseline were consistent in both the left and right thighs.

[1615] 6. In the total patient population with subcutaneous cellulite, the median time to an increase of at least 16 from baseline in the Body-Q total score was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1616] 7. In the entire patient population with subcutaneous cellulite, the mean Body-Q score of the subjects separated from the placebo 21 days after the first treatment and showed sustained and significant improvement after subsequent treatments.

[1617] 8. Among all patients with subcutaneous cellulite, more than one-third, one-half, two-thirds or three-quarters of the patients had a Body-Q score of at least 16 points higher than baseline at one or more evaluation time points in at least one treated area on day 71 after treatment, where the Body-Q score was not related to age, BMI or skin color.

[1618] 9. The severity of subcutaneous cellulite decreases rapidly within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment visit.

[1619] In other embodiments, injection of about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of results 1 to 9 above, wherein the collagenase has one or more of the following characteristics:

[1620] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1621] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1622] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1623] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1624] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1625] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1626] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1627] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1628] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1629] Potency of about 100,000 to about 400,000 GPA units / mg

[1630] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1631] Potency of about 5,000 to about 25,000 ABC units / mg

[1632] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1633] Bioburden less than or equal to 1 cfu / mL

[1634] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of Results 1 to 9 described above.

[1635] In other cases, injection of about 1 mg to about 20 mg of collagenase according to any of Treatments I to V produces one or more of the above results 1 to 9, wherein the collagenase has one or more of the following characteristics:

[1636] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1637] ·K M , about 4.1 to 410 nanomolar (SRC experiments), or about 0.03 to 3.1 mM (GPA experiments)

[1638] ·K cat (sec -1) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1639] 1 / K cat , microseconds are approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1640] ·K cat / K M ,mM -1 sec -1 Approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[1641] a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1642] The purity is at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography)

[1643] Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1644] Potency of about 5,000 to about 30,000 f-SRC units / mg

[1645] Potency of about 100,000 to about 400,000 GPA units / mg

[1646] Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg

[1647] Potency of about 5,000 to about 25,000 ABC units / mg

[1648] The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase and leupeptin

[1649] Bioburden less than or equal to 1 cfu / mL

[1650] In another embodiment, injection of about 1 mg to about 20 mg of CCH according to any of Treatments I to V produces one or more of the above-described outcomes 1 to 9.

[1651] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 9 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1652] ·Type I

[1653] ο Experiment: SRC microplate

[1654] οVmax,min -1 : Approximately 0.08 to 7.70

[1655] οK M : Approximately 4.1 to 410 nanomoles

[1656] οK cat , sec -1 : Approximately 1.1 to 107

[1657] ο1 / K cat , microseconds: approximately 376 to 37,222

[1658] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1659] Type II

[1660] ο Experiment: GPA microplate

[1661] οV max , min -1 : Approximately 0.3 to 30.5

[1662] οK M , mM: approximately 0.03 to 3.1

[1663] οK cat , sec -1 : Approximately 93 to 9,179

[1664] ο1 / K cat , microseconds: approximately 4 to 428

[1665] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1666] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1667] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area using any of Treatments I to V and one or more of the above results 1 to 9 are achieved, wherein the type I collagenase and type II collagenase have the following characteristics:

[1668] ·Type I

[1669] ο Experiment: SRC microplate

[1670] οVmax,min -1 : Approximately 0.08 to 7.70

[1671] οK M : Approximately 4.1 to 410 nanomoles

[1672] οK cat , sec -1 : Approximately 1.1 to 107

[1673] ο1 / K cat , microseconds: approximately 376 to 37,222

[1674] οK cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1675] Type II

[1676] ο Experiment: GPA microplate

[1677] οV max , min -1 : Approximately 0.3 to 30.5

[1678] οK M , mM: approximately 0.03 to 3.1

[1679] οK cat , sec -1 : Approximately 93 to 9,179

[1680] ο1 / K cat, microseconds: approximately 4 to 428

[1681] οK cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1682] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenase can be denoted as AUX-I and AUX-II, respectively.

[1683] 13. Evaluation of treatment effects through 3D photography / image measurement

[1684] As described in the following examples, three-dimensional photography or other images can be used for the evaluation of treatment effects, in particular pit analysis. The pit analysis performed using three-dimensional images (as described above) can include calculating pit volume, length, width and area. The calculations can be performed by various known methods, such as the methods described in Eckhouse et al. WO 201...

Claims

1. A cosmetic method for reducing the severity of subcutaneous cellulite on the thigh of a human patient, wherein: A collagenase composition is provided, wherein the collagenase composition has at least two of the following characteristics: iV max , measured by SRC experiments to be approximately 0.08 to 7.70 min -1 , or, as measured by GPA experiments, from about 0.3 to 30.5 min -1 ; ii.K M , measured by SRC experiments to be approximately 4.1 to 410 nanomolar, or, Measured by GPA experiments to be approximately 0.03 to 3.1 mM; iii.K cat , measured by SRC experiments to be approximately 1.1 to approximately 107 sec -1 , Alternatively, as measured by the GPA experiment, it is approximately 93 to 9,179 seconds -1 ; iv.1 / K cat , measured by SRC experiments to be approximately 376 to 37,222 microseconds, Alternatively, it was measured by GPA experiments to be approximately 4 to 428 microseconds; vK cat / K M , measured by SRC experiments to be approximately 5,140 to 508,814 mM -1 sec -1 , or, as measured by GPA experiments, approximately 60 to 5,934 mM -1 sec -1 ; vi. a molecular weight of about 60 kDa to about 130 kDa, or about 70 kDa to about 130 kDa, or about 80 kDa to about 120 kDa, or about 90 kDa to about 120 kDa, or about 100 kDa to about 110 kDa; vii. a purity of at least 80% by area as determined by reverse phase HPLC (high pressure liquid chromatography); viii. a potency of about 5,000 to about 30,000 f-SRC units / mg; ix. a potency of about 175,000 to about 500,000 f-GPA units / mg; x. a potency of about 5,000 to about 25,000 ABC units / mg; xi. less than or equal to 1% by area of ​​impurities selected from the group consisting of clostripain, gelatinase and leupeptin; xii. A bioburden of less than or equal to 1 cfu / mL; and wherein a therapeutically effective amount of the collagenase composition is injected IV or V according to an injection technique; and The cosmetic method is for improving the appearance of subcutaneous fat, wherein the improvement in the appearance of subcutaneous fat is established by a scale or other measurement tool selected from the group consisting of: Hexsel Subcutaneous Fat Severity Grading Scale (Hexsel CSS), Hexsel Dimple Depth Rating Scale, Likert Scale, Dimple Analysis, Clinician-Reported Photo-Digital Cellulite Severity Scale (CR-PCSS), Patient-Reported Photo-Digital Cellulite Severity Scale (PR-PCSS), Investigator Global Aesthetic Improvement Scale (I-GAIS), Subject Global Aesthetic Improvement Scale (S-GAIS), Patient-Reported Cellulite Impact Scale (PR-CIS), PR-CIS Short Form, Patient Self-Rating Scale (SSRS), Subject Satisfaction with Cellulite Treatment (SSCT), Clinician Assessment of Cellulite Severity (Photographic or Other), Body-Q, Thigh Cellulite Treatment Effectiveness Scale (PR-TCES; CR-TCES), and any valid photo-digital or other scale used by clinicians and / or patients to assess cellulite severity, improvement, and / or patient satisfaction; or The color intensity of the bruises significantly decreased or resolved approximately 3 to 20 days after the treatment visit.

2. The cosmetic method according to claim 1, wherein: The collagenase composition was injected IV according to the injection technique.

3. The cosmetic method according to claim 1, wherein: The collagenase composition includes AUX-I and AUX-II and has the following characteristics: a. AUX-I measured by SRC experiment: iV max : Approximately 0.08 to 7.70 minutes -1 ; ii.K M : about 4.1 to 410 nanomoles; iii.K cat : About 1.1 to about 107 seconds -1 ; iv.1 / K cat : approximately 376 to 37,222 microseconds; vK cat / K M : Approximately 5,140 to 508,814 mM -1 sec -1 ; b. AUX-II measured by GPA experiment: iV max : About 0.3 to 30.5 minutes -1 ; ii.K M : about 0.03 to 3.1 mM; iii.K cat : Approximately 93 to 9,179 seconds -1 ; iv.1 / K cat : approximately 4 to 428 microseconds; vK cat / K M : Approximately 60 to 5,934 mM -1 sec -1 .

4. The cosmetic method according to claim 1, wherein the collagenase composition comprises AUX-I and AUX-II, having the following characteristics: a. AUX-I measured by SRC experiment: i.Vmax: about 3.8 minutes -1 ; ii.K M : Approximately 2.07x10 -4 mM; iii.K cat : About 53 seconds -1 ; iv.1 / K cat : approximately 18,799 microseconds; vk cat / K M : Approximately 256,977mM -1 sec -1 ; b. AUX II measured by GPA experiment: i.Vmax: about 15.4 minutes -1 ; ii.K M : about 1.6mM; iii.K cat : Approximately 4,636 seconds -1 ; iv.1 / K cat : about 216 microseconds; vk cat / K M :Approximately 2,997mM -1 sec -1 .

5. The cosmetic method of claim 1, wherein the composition comprises at least three characteristics.

6. The cosmetic method of claim 1, wherein the composition comprises at least four characteristics.

7. The cosmetic method of claim 1, wherein the composition comprises at least 5 characteristics.

8. The cosmetic method of claim 1, wherein the composition comprises about 1 mg to 20 mg of one or more collagenases.

9. The cosmetic method of claim 1, wherein the composition comprises CCH.

10. The cosmetic method of claim 1, wherein the composition has a potency of about 10,000 ABC units / 0.58 mg and the therapeutically effective amount is about 1 mg to 20 mg.

11. The cosmetic method of claim 1 , wherein the composition has a potency of about 15,000 ABC units / mg to 20,000 ABC units / mg and the therapeutically effective amount is about 1 mg to 20 mg.

12. The cosmetic method of claim 1, wherein the therapeutically effective amount is about 1 mg to 10 mg and the composition has a potency of about 20,000 to about 30,000 f-SRC units / mg or about 175,000 to about 300,000 f-GPA units / mg.

13. The cosmetic method of claim 1, wherein the cosmetic method results in at least 5% of patients maintaining the improved level compared to pre-treatment baseline for at least 71 days after administration of the initial dose.

14. The cosmetic method of claim 13, wherein the cosmetic method results in at least 10% of patients maintaining the improved level compared to pre-treatment baseline for at least 71 days after administration of the initial dose.

15. The cosmetic method of claim 13, wherein the cosmetic method results in at least 20% of patients maintaining the improved level compared to pre-treatment baseline for at least 71 days after administration of the initial dose.

16. The cosmetic method of claim 1, wherein the cosmetic method results in an improvement in at least 5% of patients compared to a pre-treatment baseline, and the improvement becomes more pronounced over time.

17. The cosmetic method according to claim 1, wherein the method produces one or more of the following efficacy endpoints as determined by CR-PCSS and / or PR-PCSS measurements: a. At least a 2-grade improvement in CR-PCSS severity as assessed by a clinician in the target thigh on Day 22, 43, 71, 90, 180, or 365 from baseline ("Day 1") before treatment; b. At least a 2-grade improvement in the severity of the PR-PCSS on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365 from baseline (Day 1), as assessed by the subject observing a digital image of the target thigh; c. Improvement demonstrated by a composite Grade 2 response on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365, defined as a subject experiencing at least a 2-grade improvement in severity from baseline in the CR-PCSS or at least a 2-grade improvement in severity from baseline in the PR-PCSS; d. At least 1 grade improvement in the severity of subcutaneous cellulite on the CR-PCSS as assessed by a clinician in the target thigh on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365 compared to baseline (Day 1); e. At least 1 grade improvement compared to baseline in the severity of subcutaneous cellulite on the PR-PCSS as assessed by the subject observing a digital image of the target thigh on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365; f. Improvement demonstrated by a Grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365, defined as the subject experiencing at least a 1-grade improvement in severity from baseline in the CR-PCSS or at least a 1-grade improvement in severity from baseline in the PR-PCSS; and g. In all patients with moderate or severe CR-PCSS scores, statistically significant improvement in at least one treatment domain compared to placebo was achieved, where: The improvement is one or more of the above a to f.

18. The cosmetic method according to claim 1, wherein the method comprises measuring the Hexsel depression depth scale to produce one or more of the following efficacy endpoints: a. An improvement of at least 2 grades in severity in the Hexsel Indentation Depth Score from baseline ("Day 1") on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365, as assessed by a clinician in the target thigh; b. An improvement of at least 2 grades in severity in the Hexsel Indentation Depth Score on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365 from baseline (Day 1), as assessed by the subject observing a digital image of the target thigh; c. Improvement demonstrated by a composite Grade 2 response at Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365, defined as an improvement of at least 2 grades in severity from baseline on the Hexsel Indentation Depth Scale as assessed by the subject, and an improvement of at least 2 grades in severity from baseline on the Hexsel Indentation Depth Scale as assessed by the subject; d. At least a 1-grade improvement in the severity of subcutaneous cellulite on the Hexsel dimple depth score of the target thigh as assessed by a clinician on day 22, 43, 71, 90, 180, or 365 compared to baseline (day 1); e. At least a 1-grade improvement in the severity of subcutaneous cellulite as assessed by the subject on the Hexsel dimple depth score on digital images of the target thigh on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365 compared to baseline; f. Improvement demonstrated by a Grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365, defined as at least a 1-grade improvement in severity compared to baseline on the Hexsel Indentation Depth Scale as assessed by the subject's clinician and at least a 1-grade improvement in severity compared to baseline on the Hexsel Indentation Depth Scale as assessed by the subject's physician; and g. In the group of patients whose HexselCSS scores were moderate or severe, Statistically significant improvement compared to placebo in at least one treatment area, where The improvement is one or more of the above a to f.

19. The cosmetic method of claim 1 , wherein the method produces one or more of the following efficacy endpoints as determined by pit analysis: a. Depth is reduced by at least 5%; b. The width is reduced by at least 5%; c. The length is reduced by at least 5%; d. The total volume is reduced by at least 5%; and e. Surface area is reduced by at least 5%.

20. The cosmetic method of claim 1 for improving the appearance of subcutaneous cellulite established by CR-TCES and / or PR-TCES.

21. The cosmetic method according to claim 20, wherein the method produces one or more of the following efficacy endpoints as determined by CR-TCES and / or PR-TCES: a. An improvement of at least 2 grades in severity on the CR-TCES score from baseline ("Day 1") on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365, as assessed by a clinician in the target thigh; b. An improvement of at least 2 grades in severity on the PR-TCES score from baseline (day 1) on day 22, day 43, day 71, day 90, day 180, or day 365, as assessed by the subject observing a digital image of the target thigh; c. Improvement demonstrated by a Grade 2 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365, defined as a subject experiencing at least a 2-grade improvement in severity compared to baseline on the CR-TCES and at least a 2-grade improvement in severity compared to baseline on the PR-TCES; d. At least 1 grade improvement in the severity of subcutaneous cellulite on the CR-TCES score assessed by a clinician in the target thigh on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365 compared to baseline (Day 1); e. At least 1 grade improvement in the severity of subcutaneous cellulite on the PR-TCES score compared to baseline, as assessed by the subject on digital images of the target thigh on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365; f. Improvement demonstrated by a Grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365, defined as the subject experiencing at least a 1-grade improvement in severity from baseline in the CR-PCSS or at least a 1-grade improvement in severity from baseline in the PR-PCSS; and g. In all patients with moderate or severe CR-TCES scores, statistically significant improvement in at least one treatment domain compared to placebo was achieved, where: The improvement is one or more of the above a to f.

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