Compositions and methods for treating cellulite

Abstract

The present disclosure relates to methods for treating cellulite in the thighs or buttocks of a human subject by administering an effective amount of collagenase, and then assessing the reduction in cellulite severity on one or more scales. [Selection diagram] Figure 26

Description

[Technical field]

[0001] Related Applications This application is a continuation of U.S. Provisional Application Serial No. 62 / 733,046, filed September 18, 2018, U.S. Provisional Application Serial No. 62 / 788,916, filed January 6, 2019, U.S. Provisional Application Serial No. 62 / 812,036, filed February 28, 2019, U.S. Provisional Application Serial No. 62 / 823,596, filed March 25, 2019, International Application No. PCT / US2019 / 041494, filed July 11, 2019, and International Application No. PCT / US2019 / 41718, filed July 12, 2019, which are hereby incorporated by reference in their entireties to the fullest extent permitted by law.

[0002] The present invention relates to the field of cellulite assessment and treatment. [Background technology]

[0003] Cellulite (also known as edematous fibrosclerosing subcutaneous tissue (EFP)) is an aesthetic condition that can be understood as an imbalance between the structural and biomechanical properties (i.e., delicate containment and extrusion forces) of the subcutaneous junction (see 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). Thus, the goal of cellulite treatment is to strengthen the subcutaneous interface and / or release the fibrous septa through various types of ablation (Rudolph et al., supra). Fibrous septa have been recognized by anatomical and imaging studies as a contributing underlying cause of cellulite and as a target for the treatment of cellulite (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 Resistance Imaging of Cellulite"."Magnetic Resonance Imaging of Cellulite Depressed 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 in gender, BMI, and cellulite grading, "Skin Res Technol.2004;10(3):161-168; Nurnberger and Muller, "So-called cellulite: an invented disease, "J Dermatol Surg Oncol.1978;4(3):221-229; Pierard et al., "Cellulite: from standing fat herniation to hypodermal stretch marks," Am J Dermatopath.2000;22(1):34-37; Querleux (Eiichi et al., "Anatomy and physiology of subcutaneous adipose tissue by in vivo magnetic resonance imaging and spectroscopy: relationships with sex and presence of cellulite," Skin Res Technol.2002;8(2):118-124). To effectively treat cellulite, a treatment is required that dissolves or destroys the dermal septum (Figure 1), which is made of collagen and is the cause of the skin dimples that are a source of concern for many women.

[0004] Although there are treatments that have been utilized to treat cellulite, there are no approved pharmacological treatments. Despite the availability of multiple treatments, there is little scientific evidence that current non-pharmacological treatments are beneficial. In fact, much of the evidence is anecdotal, subjective, or based solely on patient self-assessment. Historical treatments for EFP include weight loss, topicals, massage, liposuction, mesotherapy, radiofrequency, excision, powered ablation, and laser treatments. Many of these treatments have undesirable 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 myth or 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., "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).Thus, many physicians are of the opinion that aesthetic improvement is not easily attainable, and therefore there remains an unmet need for safe and effective non-surgical treatments to improve the aesthetic outcome in women with cellulite. The prior art documents relevant to the invention of this application are as follows (including documents cited during the international phase after the international filing date and documents cited when the application entered the national phase in other countries). (Prior art document) (Patent Documents) (Patent Document 1) International Publication No. 2013 / 059619 (Patent Document 2) International Publication No. 2007 / 100675 (Patent Document 3) International Publication No. 2018 / 160905 Summary of the Invention

[0005] The present disclosure satisfies the above needs and relates to a method of treating cellulite in a human patient by subcutaneous injection of a therapeutically effective amount of collagenase (as defined in the detailed description). Such a method involves a pre-treatment assessment of the patient's cellulite severity using various scales and assessment techniques to establish the patient's baseline of cellulite severity. Treatment of cellulite is then performed by subcutaneous injection of collagenase. Dosages of collagenase may vary, and the collagenase may be in the form of a pharmaceutical composition comprising collagenase and one or more pharma- ceutically acceptable excipients. Such excipients include sterile water for injection, pH adjusters, tonicity adjusters, stabilizers, and the like. Post-treatment evaluations are performed to confirm the effectiveness of the treatment compared to the baseline. The disclosed method of treatment results in a significant reduction in the appearance of cellulite.

[0006] As explained in the detailed description, there are four stages in the treatment, which are optional and the order in which they are performed is not intended to be strictly limiting. 1. In the first step, the clinician selects the cellulite depression to be treated. Then, prior to injection, an assessment is made. For example, the clinician and / or patient independently assess the pre-treatment severity of cellulite using one or more of the following scales or other assessment methods (defined in the detailed description): Hexel Cellulite Severity Scale (CSS) (Hexel CSS) Hexel pit depth score Likert scale Depression analysis o CR-PCSS (Clinician-Reported Photo-Numerical Cellulite Severity Scale) 〇 PR-PCSS (Patient-Reported Photo-Numerical Cellulite Severity Scale) Investigator Global Aesthetic Improvement Scale (I-GAIS) Subject Global Aesthetic Improvement Scale (S-GAIS) Patient-Reported Cellulite Impact Scale (PR-CIS) ○ PR-CIS abbreviation Subject Self-Rating Scale (SSRS) Subject satisfaction with cellulite treatment (SSCT) Clinician assessment of cellulite severity (photographs or other images) Body-Q Fitzpatrick scale 〇 Thigh Cellulite Severity - Patient (TCS-P), Thigh Cellulite Severity - Clinician (TCS-C) A validated photonumeric or other scale used by clinicians and / or patients to assess cellulite severity, improvement, and / or patient satisfaction (e.g., the Hexsel-Merz scale (Hexsel et al., "Validated Assessment Scales for Cellulite Dimples on the Buttocks and Thighs in Female Patients," Dermatologic Surgery: August 2019 (Volume 45) Issue p S2-S11 and poster presentation at the American Academy of Dermatology Annual Meeting 2019)

[0007] Additionally, pretreatment evaluation by the clinician and / or patient can be performed by analyzing 1-15 photographs, illustrations, drawings, computer images, 3D models, MRI images, thermograms, ultrasounds, patient verbal feedback, etc., each with a different cellulite severity rating or level.

[0008] 2. In the second stage of treatment, the clinician places a dot or other marking on the depression to be treated (Figure 6), typically directly below the depression if present, and may take photographs and perform other evaluations.

[0009] 3. In the third stage of treatment, a therapeutically effective amount of collagenase is injected subcutaneously into one or more treatment sites (as defined in the detailed description) in a single or divided dose. Dosages and injection techniques vary. For example, the method may include injection according to the following procedure: ● The collagenase composition (e.g., CCH) is injected subcutaneously with the subject in a prone position using a syringe with a 30 gauge ½ inch needle. As shown in FIG. 7 (hereafter, “Treatment I”), each injection site receives one dermal injection of 0.3 mL of collagenase composition, with three aliquots of 0.1 mL at positions A, B, and C. The injection depth is ½ inch, which corresponds to the length of the treatment needle from tip to base, with no downward pressure applied. At each injection site, the needle is inserted and positioned at 90° perpendicular to the skin surface, and the collagenase composition is injected in 0.1 mL aliquots (position A). The needle is then slightly withdrawn (but not removed from the skin) and repositioned over the long axis of the cavity at 45° from perpendicular, and a 0.1 mL aliquot of collagenase composition is injected (position B, toward the head). The needle is again withdrawn slightly and returned to a position approximately 45° from vertical down the long axis of the cavity, and a 0.1 mL aliquot of collagenase composition is injected (position C, toward the paws). After injection, the subject remains prone for 5 minutes.

[0010] As an example, Treatment I may be employed to administer 0.84 mg of the collagenase composition as 12 subcutaneous injections per treatment site during three treatment sessions, each spaced at least 21 days apart (+ / - 3 day window). For example, a cumulative dose of 5.04 mg may be administered (i.e., 3 treatment visits x 0.84 mg per treatment area x 2 treatment areas). Other techniques are described in the detailed description.

[0011] 4. In the fourth phase of treatment, post-injection evaluation is performed using the scales mentioned above or other assessment methods (e.g., bruising analysis). The efficacy of a particular collagenase treatment may be based on a single clinician or patient assessment, or on a composite endpoint consisting of a clinician and a patient assessment, where the same subject may show improvement on both scales, i.e., a pre-specified level of improvement on both the clinician and the patient scales.

[0012] The collagenase is injected in an amount of about 0.01 mg to about 20 mg, in a single dose or in divided doses, and has one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 A molecular weight of about 60 kDa to 130 kDa, or about 70 to about 130 kDa, or about 80 to 120 kDa, or about 90 to 120 kDa, or about 100 to 110 kDa. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL As used herein, the relevant kinetic parameters can be measured using the cuvette or microplate assays described herein (e.g., the SRC cuvette assay, the SRC microplate assay, the GPA cuvette assay, and the GPA microplate assay).

[0013] In some embodiments, the collagenase present in the composition comprises collagenase I and collagenase II in a ratio of about 1:1. Other ratios of collagenase I and collagenase II may be employed 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, or 0:1. Each of collagenase I and collagenase II may have a purity of 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.

[0014] In another embodiment, the collagenase composition has a CCH (as defined in the detailed description) with a ratio of AUX I and AUX II of about 1:1. Other ratios of AUX I and AUX II may be employed 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, or 0:1. Each of AUX I and AUX II may have a purity of 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.

[0015] In other examples, the collagenase composition may be liquid or reconstituted with a diluent from a lyophilized solid form. The dosage of the mixture is measured by the amount of collagenase present without considering the diluent and may have from about 0.1 mg to about 20 mg in one or more injections. In another embodiment, the dose administered 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 one or more injections. For example, about 0.06 mg, 0.48 mg, 0.84 mg, or 1.68 mg is injected in about 12 separate injections. The amount of collagenase composition injected may range from 0.01 mL to 3 mL per injection, or from about 0.2 mL to 150 mL total per treatment visit (as defined in the detailed description). In certain embodiments, the above dosages are for a collagenase composition comprising CCH. In other embodiments, the above dosages are for a collagenase composition having one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0016] In another embodiment, about 0.84 mg of CCH is injected equally into each treatment area in about 12 separate injections (about 0.07 mg x 12 injections = about 0.84 mg). In some cases, such treatment with 0.84 mg is administered in a single treatment visit, or every 10-40 days for 2, 3, 4, or 5 treatment visits. 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 administered in 5 or more treatment visits.

[0017] Additionally, as described in the detailed description, collagenase injections are effective in treating cellulite, e.g., significant improvement in the appearance of cellulite as demonstrated by Hexel pit depth scores, Likert scale scores, and pit analysis.

[0018] The most common side effects of CCH injections are injection site reactions, which include bruising, pain, nodules, itching, swelling, hardness, discoloration, and redness. Injection site bruising generally decreases with each treatment.

[0019] Additional embodiments of the compositions, measures, methods, etc. of the present invention will become apparent from the following description, drawings, examples, and claims. As understood from the above and following description, each and every feature described herein, and each and every combination of two or more of such features, are included within the scope of the present disclosure, provided that the features included in such combinations are not mutually inconsistent. Furthermore, any feature or combination of features may be expressly excluded from any embodiment or aspect. Additional aspects and embodiments are described in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings. [Brief description of the drawings]

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided nationwide upon request and payment of the necessary fee.

[0021] The features of the foregoing embodiments will be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, in which:

[0022] [Figure 1] FIG. 1 is a cross-sectional view of the skin and subcutaneous tissue depicting collagen septa. [Diagram 2] FIG. 2 is an amino acid sequence listing of AUX-I (SEQ ID NO: 5). [Diagram 3] FIG. 3 is an amino acid sequence listing of AUX-II (SEQ ID NO: 6). [Figure 4] FIG. 4 shows the Hexcellulite Severity Scale (CSS) (B) depression depth. [Diagram 5] FIG. 5 shows an example of the Thigh Cellulite Severity-Patient (TCS-P); Thigh Cellulite Severity-Clinician (TCS-C) scale. [Figure 6] Figure 6 shows an example of a subject's buttocks dimple and injection site markings. [Figure 7] FIG. 7 shows the injection technique used in Treatment I. [Figure 8] Figure 8 shows bar graphs of the primary and key secondary endpoints of composite responders in the RELEASE-1 and RELEASE-2 studies. Composite responders were defined as patients who experienced an improvement of ≥2 or ≥1 level from baseline in both CR-PCSS and PR-PCSS assessments of the target hip at Day 71. [Figure 9] Figure 9 is a series of photographs comparing the composite response to CCH 0.84 mg on day 71 with baseline. Figure 9A shows a 2-level improvement in both CR-PCSS and PR-PCSS. Figure 9B shows a 1-level improvement in both CR-PCSS and PR-PCSS. [Figure 10]Figure 10 is a bar graph of the primary and key secondary endpoints of composite responders in the non-target hip (for data analysis) at day 71. Composite responses were defined as patients who achieved an improvement of ≥2 or ≥1 level of severity from baseline in both CR-PCSS and PR-PCSS assessments at day 71. [Figure 11] FIG. 11 is a bar graph showing the frequency of PR-PCSS and S-GAIS responders at day 71 in the intent-to-treat (ITT) population. [Figure 12] 12 is a bar graph showing the mean improvement from baseline in PR-CIS total score in the modified intention to treat (mITT) population at Day 71. Baseline values ​​were used for women who did not have a PR-CIS assessment at Day 71. [Figure 13] Figure 13 shows pre-marking image registration with 3D registration to a grid (Day 1 Pre-marking). The image is centered relative to the grid in 3D space. Using the grid as a reference, the Image Analysis Technologist (IAT) positions the baseline image so that the approximate center of the image is at the origin of the grid. The thighs / buttocks face forward in the +z direction, the top of the thighs / buttocks face in the +y direction, and the bottom of the thighs / buttocks face in the -y direction. [Figure 14] FIG. 14 is a color-by-distance map for image registration. [Figure 15] Figure 15 shows the main depression of the region of interest (1). Using the post-marking image of day 1 as a reference, the technician searches for the target depression on the pre-marking image of day 1. Then, the technician traces the boundary of the main depression on the tracked pre-marking image. [Figure 16] Figure 16 is a series of photographs showing the transposition of the major depression in the area of ​​interest. The tracing of the depression on the tracked pre-marking image is transcribed onto the follow-up images at days 22, 43, and 71 based on each follow-up image's unique surface tracking relationship to the baseline. [Figure 17] FIG. 17 shows contours of normal and bruised tissue on days 4, 8, and 15 after injection into the left buttock of a subject. [Figure 18] Figure 18(A) shows the outlines of normal and bruised tissues on days 4, 8, and 15 after injection in the left buttock, along with the L*, a*, and b* color measurements of those tissues. Figure 18(B) shows the outlines of normal and bruised tissues on days 4, 8, and 15 after injection in the left buttock. The average color and ΔE of normal and bruised tissues were calculated from the L*a*b* color values. [Figure 19] 19(A)-19(C) show an exemplary depression analysis. FIG. 19(A) shows the depression analysis parameters observations and changes from the pre-marking image on day 1. FIG. 19(B) shows the maximum depression length and maximum depression width. FIG. 19(C) shows the surface area and volume between the depression base and the interpolated surface. [Figure 20] 20 is a line graph of the mean PR-PCSS assessment over time for the target hip (mITT population) in Example 3. The bottom line is CCH treatment versus placebo (top line), as described in Examples 2 and 3. [Figure 21] 21 is a line graph of the mean PR-PCSS assessment over time for the non-target hips (mITT population) in Example 3. The bottom line is CCH treatment versus placebo (top line) as described in Examples 2 and 3. [Figure 22] 22 is a line graph of the mean CR-PCSS assessment score over time for the target hip (mITT population) in Example 2. The bottom line is CCH treatment versus placebo (top line) as described in Examples 2 and 3. [Diagram 23] 23 is a line graph of the mean CR-PCSS assessment score over time for the non-target hip (mITT population) in Example 2. The bottom line is CCH treatment versus placebo (top line) as described in Examples 2 and 3. [Figure 24]FIG. 24 depicts the target and non-target hip 2-level combined responders at day 71 (ITT population). [Diagram 25] FIG. 25 depicts the study design for Example 5. [Figure 26] FIG. 26 depicts the mean PR-CIS item scores from days 1 to 71 in Phase 3 (302 / -303) and from days 71 to 180 in Example 5 (Study 304). [Figure 27] FIG. 27 depicts the proportion of SSCTA responders for CCH treatment vs. placebo treatment at Day 180 compared to Day 71 in Phase 3. [Figure 28] Figure 28 depicts the disposition of subjects in Study 202. Note a.: Subjects underwent up to four observation-only visits during a three-month period beginning 90 days after Day 1 of the double-blind study (201) (i.e., within 20 days ± 4 days of the end of the double-blind study) until Study 201 drug blind was broken by the provider. Observation periods were defined within each treatment area and were from Screening A to the first treatment date in Study 202, or to the end date of Study 202 if not treated in Study 202. Note b.: The "other" category included subjects who either failed screening, refused to participate in the treatment phase, subjects whose site was closed on Study Day 272 of subject enrollment, or subjects who did not adhere to the study visit. Note c.: After completing treatment (Day 71 of the treatment phase), subjects were followed through Day 360 with observational assessments at three-month intervals. For subjects who received CCH in Study 201, long-term durability was assessed through Day 720 for the treatment site administered. [Figure 29] FIG. 29 depicts the mean PR-PCSS assessments over time in the CCH treatment area in Study 202. [Diagram 30] FIG. 30 depicts the mean PR-PCSS ratings over time for re-exposed subjects (treated at the buttocks and thighs) in Study 202. [Diagram 31] FIG. 31 depicts the mean PR-PCSS ratings over time for re-exposed buttocks treated subjects during the first and second treatment courses in Study 202. [Diagram 32]FIG. 32 depicts the mean CR-PCSS ratings over time in the CCH treatment area in Study 202. [Diagram 33] FIG. 33 depicts the mean CR-PCSS ratings over time for re-exposed subjects (treated at the buttocks and thighs) in Study 202. [Diagram 34] FIG. 34 depicts the mean CR-PCSS ratings over time for re-exposed buttocks treated subjects during the first and second treatment courses in Study 202. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Various aspects and embodiments are now fully described herein. However, these aspects and embodiments can be embodied in many different forms and should not be construed as limiting. Rather, these embodiments are provided so that the disclosure will be 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.

[0024] A.Definition Unless otherwise defined, all terms and phrases used herein include the meaning that the term and phrase has achieved in the art unless expressly indicated to the contrary 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 study of the present invention, specific methods and materials are described herein.

[0025] Unless otherwise indicated, the use of individual numerical values ​​is described as an approximation, as if the numerical value were preceded by the word "about" or "approximately". Similarly, the numerical values ​​in the various ranges specified in this application are described as approximations, as if both the minimum and maximum values ​​in the range are preceded by the word "about" or "approximately", unless otherwise indicated. In this manner, variations above and below the stated ranges can be used to obtain substantially the same results as the values ​​within the range. In this specification, the terms "about" and "approximately" when referring to numerical values ​​shall have their plain and ordinary meaning to a person of ordinary skill in the art to which the disclosed subject matter most closely relates or to the art related to the range or element in question. The extent to which the boundaries are extended from the exact numerical values ​​will depend on many factors, such as the importance of the elements and / or the effect of a given amount of variation on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. In this specification, the use of different amounts of significant figures for different numerical values ​​is not meant to limit the use of the word "about" or "approximately" to serve to extend a particular numerical value or range. In general, the word "about" or "approximately" has the effect of extending the numerical value. The disclosure of ranges also contemplates continuous ranges including every value between the minimum and maximum values, as well as expansions of that range obtained through the use of the words "about" or "approximately." Accordingly, the recitation of ranges of numerical values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if each separate value were individually set forth herein.

[0026] As used herein, "affected area" or "treatment area" refers to the area of ​​cellulite in a human patient that is or has been treated with Collagenase (defined below). This may include the quadrants (i.e., left buttock, right buttock, left posterolateral thigh, right posterolateral thigh). The affected area or treatment area is not limited to the buttocks or thighs. Rather, any part of the body that has cellulite may be treated as a treatment area.

[0027] As used herein, "adverse event" or "AE" means any unfavorable or unintended change in body structure (signs), body function (symptoms), laboratory results (e.g., chemistry tests, ECG, x-rays), or aggravation of a pre-existing condition temporarily associated with the use of an investigational drug, whether or not considered to be related to the investigational drug.

[0028] "Body-Q" is a patient-reported outcome measure commercially available under license from Memorial Sloan Kettering Cancer Center. Body-Q is based on patients' perceptions of their body contouring and weight loss. It uses scales with 18 independent features to measure three domains: appearance, health-related quality of life (HRQL), and patient experience of health care. The patient-reported outcome instrument is described in BODY-Q: Instructions for UseBODY-Q: Instructions for Use, Version 1.0, July 2015, Memorial Sloan Kettering Cancer Center, McMaster University, and Stefan Cano. BODY-Q includes a scale to measure cellulite. See https: / / www.mskcc.org / sites / default / files / node / 174457 / documents / body-q-users-guide.pdf (accessed July 3, 2019). For cellulite, there are 16 scale items with response options ranging from "not at all" to "very bothersome" in the past week time frame, assuming a Flesch-Kincaid reading level. Patients are asked: "How much have you been bothered by thinking about cellulite in the past week?" [16 questions follow, and patients rank their answers as 1-very bothersome, 2-moderately bothersome, 3-a little bothersome, 4-not bothersome at all]. Scores range from 16 (very bothersome) to 64 (not bothersome at all).

[0029] Traditionally, clinical examination of bruises has been performed by visual inspection of the bruise area and its surroundings, as well as evaluating the subject's medical, surgical, and concomitant medication history. The results of this interpretation are subjective and influenced by several extraneous factors, including the viewing angle, the surrounding lighting, the color of the unexposed surrounding skin, and the observer's experience and visual acuity. As used herein, "bruise analysis" refers to the application of the objective image capture and tracking method disclosed in U.S. Patent Publication No. 2019 / 0035080 uniformly to all subject images to detect visible changes in skin color as assessed from images of the collagenase-treated areas of the subject. This objective analysis may assist or replace the visual and clinical examination of bruises by health care providers by providing the ability to quantify, differentiate, and evaluate bruises at both an intrasubject (within the same subject at different time points) and intersubject (between different subjects) level. This analysis uses the L color scale defined by the International Commission on Illumination (CIE) based on the color refutation theory that "two colors, red and green, and yellow and blue, cannot be represented simultaneously." * a * b * The color space used is L, as shown below. * represents light and dark, a * are the red / green coordinates, b * represents the yellow / blue coordinate. * Delta (ΔL * ), a * Delta (Δa * ), b * Delta (Δb * ) can be positive (+) or negative (-). However, the sum of the differences, Delta E (ΔE * ) is always positive. ● ΔL * (L * Sample to L * Subtract the standard) = difference in brightness (+=bright, -=dark), the smaller the number (0~50), the darker, and the larger the number (51~100), the brighter. ● Δa * (a * From the sample * Subtract standard) = difference between red and green (+=more red, -=more green) ● Δb* (b * Sample from b * subtract standard) = difference between yellow and blue (+=more yellow, -=more blue) These three values ​​are necessary to fully represent the color of an object (here a birthmark in an image of the subject's treatment area). * a * b * The objective methodology of image analysis of collagenase-treated areas (pre- and post-treatment images at protocol-specified time points) that outputs data in values ​​allows for fast, easy, accurate, repeatable, and unbiased quantification of skin color and changes therein. This method eliminates the inherent variability associated with traditional subjective visual estimation of images. ΔE is calculated as follows: ΔE(color difference between bruised and normal tissue)=SQRT[(L * B -L * N ) 2 +(A * B -A * N ) 2 +(B * B -B * N ) 2 ], where L * B = Bruised tissue L * L * N = normal tissue L * A * B = Bruised tissue A *. A * N = normal tissue A * B * B = Bruised tissue B * B * N = normal tissue B * FIG. 18(B) shows the bruising profile of the treated area.

[0030] As used herein, "CCH" refers to a mixture of collagenases AUX-I (SEQ ID NO: 5 (FIG. 2)) and AUX-II (SEQ ID NO: 6 (FIG. 3)) in an approximately 1:1 ratio obtained by fermentation of Clostridium histolyticum (also known as Hathewaya histolytica). CCH is commercially available as a lyophilized powder under the trademark XIAFLEX® and is a mixture of AUX-I and AUX-II plus certain excipients, although CCH may be used in combination with other suitable excipients.

[0031] As used herein, "Clinician-Reported Photo-Numerical Cellulite Severity Scale (CR-PCSS)" is a photo-numerical scale described in PCT patent application PCT / US2018 / 020551 (published as WO2018 / 160905 on September 7, 2018) that is designed for use by physicians / clinicians to rate the severity of cellulite on a 5-point scale.

[0032] Unless otherwise specified herein, "collagenase" means any of the following: a) collagenase (including variants) having activity as defined in EC 3.4.24.3 (https: / / www.brenda-enzymes.org / enzyme.phpecno=3.4.24.3 (accessed July 3, 2019); (b) collagenase derived from Clostridium histolyticum (Hathewaya (c) CCH (as defined above); (d) collagenases having at least 50% sequence identity with AUX-I as determined by BLAST; (e) collagenases having at least 50% sequence identity with AUX-II as determined by BLAST; (f) collagenases produced by fermentation of organisms of other source (i.e., non-Clostridium histolyticum), e.g., mammalian, crustacean, fungal, bacterial or microbial collagenases; (g) collagenases obtained by recombinant techniques; (h) collagenases having a molecular weight of about 65 kDa to about 130 kDa; (i) class I or are collagenases designated as class II (also called collagenase I (or 1), collagenase II (or 2), type I collagenase, and type 2 collagenase);j) a mixture of collagenase I and II;(k) collagenase from JCM1403 strain (ATCC19401) or its derivatives;(l) collagenase from ATCC21000 strain or its derivatives;(m) collagenase from ATCC69334 strain or its derivatives;(n) collagenase from C. perfringens;(o) collagenase from Vibrio alginolyticus;(p) collagenase from Streptomyces;(q) collagenase from Pseudomonas;(r) collagenase from Achromobacter (s) collagenases from Worthington Biochemical Corp. (www.Worthington-biochem.com; "Product Highlights"); (t) collagenases from Sigma-Aldrich (www.sigma-aldrich.com); (u) collagenases having one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 ; (v) collagenase described by Nordmark Arzneimittel GmbH & Co. KG; (w) collagenase from strain 004; or (x) equivalents or mixtures of any of the foregoing. Non-limiting examples of collagenases that can be used in the present disclosure are described in U.S. Patent Nos. 7,811,560, 9,757,435, 9,744,138, and WO2012 / 125948.

[0033] As used herein, "dimple analysis" refers to an analysis of one or more selected depressions, in which parameters such as the volume, length, width, and surface area of ​​the depressions are measured. Measurements are described in Eckhouse et al., WO2018 / 116304 and WO2018 / 116305, Cherry Imaging (www.cherryimaging.com) and Canfield Scientific, Inc., and in 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. Pat. No. 9,996,923. Such measurements of volume, length, width, and surface area may be calculated using digital 3D grayscale images (with X and Y axis rotation capabilities) and digital 3D textured and illuminated images (with X and Y axis rotation capabilities) together with a computer program that analyzes such images. As an example, for buttock treatment areas, images may be taken of the left treated buttock and / or the right treated buttock for each patient before and after treatment. For thigh treatment areas, images may be taken of each thigh treatment area at 0 degrees, 45 degrees, and 90 degrees before and after treatment. For thigh treatment areas, images taken using the Canfield Scientific method may be taken of each thigh treatment area at 0 degrees, 45 degrees, and 90 degrees before and after treatment.

[0034] As used herein, "durability" refers to a period of time during which the therapeutic effect is sustained. This period can range from about 3 months to about 20 years, or about 1 year to 19 years, or about 2 years to 18 years, or about 3 years to 17 years, or about 4 years to 16 years, or about 5 years to 15 years, or about 6 years to 14 years, or about 7 years to 13 years, or about 8 years to 12 years, or about 9 years to 11 years. The 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.

[0035] As used herein, "Early Termination Visit" means that the final visit for subjects who terminate the study is considered the Early Termination Visit, and the assessment normally performed on Day 71 for subjects who terminate the study is performed at the Early Termination Visit.

[0036] As used herein, "Fitzpatrick Scale" refers to the scale used to assess a subject's skin type, as shown in Table 1. [Table 1]

[0037] As used herein, "Hexel Cellulite Severity Scale" or "Hexel CSS" or "Cellulite Severity Scale (CSS)" refers to the following photo-numerical scale that assesses the five major morphological characteristics of cellulite (Table 2): [Table 2] The total points are categorized as follows: TIFF0007628492000003.tif75157

[0038] As used herein, "Hexel pit depth score" refers to the assessment of only pit depth (B) from the Hexel CSS (Figure 4). 0=no depression 1=superficial depression 2 = Medium depth depression 3=deep depression

[0039] As used herein, "picture" or "image" refers to a photograph, illustration, drawing, model, 3D model, computer-generated image, MRI image, and the like.

[0040] As used herein, "Likert scale score" refers to the score given by an independent blinded assessor (or patient) at each post-treatment visit to identify changes in the treated area (buttocks or thighs) by comparing cellulite photographs (2D color, 3D color, 3D grayscale) taken pre-treatment (baseline) on Day 1 with those taken at the post-treatment visit. The score is captured on a 5-point Likert scale as follows: TIFF0007628492000004.tif72164

[0041] As used herein, "non-target thigh" or "non-target buttock" refers to a thigh or buttock that is not selected for evaluation of the primary efficacy endpoint. Such non-target areas may still receive treatment and may be used for secondary efficacy evaluations.

[0042] "Optional" or "optionally" means that the subsequently described element, component, or circumstance may or may not occur, and the description includes instances when the element, component, or circumstance occurs and instances when it does not occur.

[0043] As used herein, the "Patient-Reported Cellulite Impact Scale (PR-CIS)" is a six-item assessment of the visual and emotional impact of cellulite (satisfied with the appearance of cellulite, bothered, aware, embarrassed, looks old, looks fat, looks out of shape) with each question being given on a numerical rating scale ranging from 0 (not at all) to 10 (very much). More specifically, the PR-CIS is a six-item static assessment of the visual and emotional impact of cellulite (satisfied with the appearance of cellulite, bothered, aware, embarrassed, looks old, looks fat, looks out of shape) with each item being answered by subjects using an 11-point numerical rating (or interval) ranging from 0 (not at all) to 10 (very much) while viewing a digital image of their buttocks or thighs. The assessment may also be made across all thighs and / or buttocks together, rather than assessing each site individually. The total PR-CIS score is derived from the six individual questions. Question 1: Considering the areas selected for treatment, how satisfied are you with the appearance of your cellulite? Question 2: Thinking about the area selected for treatment, how bothered are you with the appearance of your cellulite? Question 3: Thinking about the areas selected for treatment, how self-conscious are you about the appearance of your cellulite? Question 4: Thinking about the areas selected for treatment, how embarrassed are you about the appearance of your cellulite? Question 5: Considering the areas selected for treatment, how old does your cellulite make you look? Question 6: Considering the areas selected for treatment, will cellulite make me look fat or out of shape?

[0044] As used herein, the "Patient-Reported Cellulite Impact Scale (abbreviated)" (PR-CIS abbreviation) is a five-item assessment of the visual and emotional impact of cellulite (satisfied with the appearance of cellulite, bothered, aware, embarrassed, looks fat, looks out of shape) using a numerical rating scale ranging from 0 (not at all) to 10 (very much). More specifically, the PR-CIS abbreviation is a five-item static questionnaire that assesses the visual and emotional impact of cellulite (satisfied with the appearance of cellulite, bothered, aware, embarrassed, looks fat, looks out of shape) and each item is answered by subjects using an 11-point numerical rating (or interval) ranging from 0 (not at all) to 10 (very much) while viewing a digital image of the buttocks or thighs. The assessment may be performed for all thighs and / or buttocks together, rather than assessing each site individually. As a non-limiting example, the PR-CIS abbreviation total score may be derived from five individual questions. Question 1: Considering the areas selected for treatment, how satisfied are you with the appearance of your cellulite? Question 2: Thinking about the area selected for treatment, how bothered are you with the appearance of your cellulite? Question 3: Thinking about the areas selected for treatment, how self-conscious are you about the appearance of your cellulite? Question 4: Thinking about the areas selected for treatment, how embarrassed are you about the appearance of your cellulite? Question 5: Considering the areas selected for treatment, will cellulite make me look fat or out of shape? The PR-CIS abbreviated total score can be derived from the other five question sets in the main PR-CIS section.

[0045] As used herein, "Patient-Reported Photo-Numerical Cellulite Severity Scale (PR-PCSS)" is a photo-numerical scale described in PCT patent application PCT / US2018 / 020551 (published as WO2018 / 160905 on September 7, 2018) that is designed to rate the severity of cellulite on a 5-point scale.

[0046] As used herein, "Photo-Numerical" means the use of a series of photographs, illustrations, drawings, models, 3D models, computer generated images, MRI images, images, etc., each of which is assigned a different level of cellulite severity on a scale.

[0047] As used herein, "sequential visits" refers to two or more clinician visits or times during which changes in cellulite are assessed by a scale. The time between visits may be about 2 weeks, 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 18 months, about 2 years, about 3 years, about 4 years, or about 5 years or more.

[0048] As used herein, a "serious adverse event" means an adverse event that results in death, is immediately life-threatening, requires or prolongs hospitalization, results in permanent or substantial disability, results in a congenital anomaly / birth defect, or is otherwise considered a major medical event.

[0049] As used herein, "statistically significant" generally refers to statistical data with a "P" value less than 0.05. In the context of this disclosure, clinical studies are generally designed to test the superiority of an intervention (e.g., in this case, a treatment) compared to a control. Clinical studies involve people who are physiologically different from one another, so it is natural that there will be variability in the results. Statistics are then used to determine whether the observed differences are due to chance or the treatment itself. A measure of statistical significance quantifies the probability that the study results are due to chance. A "P" value, which is often used to measure statistical significance, represents the probability that the study results are due to chance rather than an actual treatment effect. Generally, the conventional cutoff value for a "P" value considered statistically significant is 0.05 (5%), but may vary depending on the study design and results. A "P" value less than 0.05 means that there is less than a 5% chance that the study results are due to chance. A "P" value greater than 0.05 (5%) means that the difference between the treatment and control groups is not statistically significant, meaning that the difference is unlikely to be attributable to the treatment and may instead be attributable to chance.

[0050] The terms "subject" and "patient" are used interchangeably herein and refer to a human or other mammal.

[0051] As used herein, "Subject Global Aesthetic Improvement Scale (S-GAIS)" and "Investigator Global Aesthetic Improvement Scale (I-GAIS)" refer to the following scales for assessing cellulite severity and / or improvement. Subjects are asked the following introductory question: "How would you rate the appearance of your cellulite after treatment?" Ratings are made on a scale ranging from -3 (much worse) to +3 (much improved) depending on the subject's response, as shown in Table 3. [Table 3]

[0052] As used herein, "Subject Satisfaction with Cellulite Treatment" (SSCT) refers to a subject satisfaction rating ranging from -2 to +2. As an example, Table 4 below provides such ratings for a buttock cellulite treatment. Patients are asked, "How satisfied are you with the results of the cellulite treatment you received today?" and are asked to select a response rating as shown in Table 4. [Table 4]

[0053] Such ratings for cellulite treatment of the thighs are shown in Table 5. Patients are asked, "How satisfied are you with the results of the cellulite treatment for the particular area or areas of the thighs that you had treated today?" and asked to select an answer / rating as shown in Table 5. [Table 5]

[0054] As used herein, the "Subject Self-Rating Scale (SSRS)" is a scale for subjects to rate their satisfaction with their cellulite-related appearance on a 7-point integer scale ranging from 0 (extremely dissatisfied) to 6 (extremely satisfied), as shown in Table 6. [Table 6]

[0055] As used herein, "target thigh" or "target hip" refers to the thigh or hip selected for evaluating the primary efficacy endpoint.

[0056] As used herein, the term "therapeutically effective amount" refers to the amount of collagenase required to reduce the severity of cellulite in a patient or a statistically significant population of patients. The amount of collagenase composition employed will be that amount necessary to provide the amount of collagenase necessary to obtain the desired result. In practice, this will vary depending on the collagenase injected, the injection technique, and the enzyme activity in the area to be treated.

[0057] As used herein, a "course of treatment" refers to three treatment sessions (ie, each visit to a clinician to receive treatment).

[0058] As used herein, the term "treatment adverse event" or "TEAE" means a condition that was not present before treatment with an investigational product but appears after treatment, 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 during treatment (regardless of the intensity of the AE at the start of treatment).

[0059] As used herein, the terms "Thigh Cellulite Severity-Patient" ("TCS-P") and "Thigh Cellulite Severity-Clinician" ("TCS-C") refer to the photo-numerical scale shown in FIG. 5 (or a substantially similar scale) used by a patient (TCS-P) or clinician (TCS-C) to assess thigh cellulite severity, improvement, and / or patient satisfaction and to aid in the evaluation of collagenase efficacy. The patient-reported method of use of the scale is referred to as TCS-P and the clinician-reported method of use of the scale is referred to as TCS-C.

[0060] As used herein, the term "treatment visit" or "treatment" or "treatment session" means one or more injections or treatments into the affected area of ​​a therapeutically effective amount of at least one active agent useful in the treatment of cellulite during a single office visit.

[0061] As used herein, the terms "validated," "validated," and "validation" refer to the process of demonstrating that a particular scale is accurate and reliable, including the reproducibility of visual assessment to ensure that the same results are consistently obtained. Validation also examines the precision, accuracy, and sensitivity of the scale to ensure that the measurements made by the scale are reliable, reproducible, and robust.

[0062] B. Introduction The present disclosure relates to a method for treating cellulite comprising administering to a subject having the appearance of cellulite a therapeutically effective amount of one or more collagenases using certain injection techniques described below.

[0063] Generally, there are four stages in the treatment: (1) the clinician and patient perform a pre-treatment assessment to determine a pre-treatment baseline and select the depression to be treated; (2) if there is a fundus, the clinician marks the depression to be treated at the fundus; (3) the clinician treats the patient with collagenase; and (4) the clinician and patient perform a post-treatment assessment. These stages are detailed below. Each stage and the steps within it are optional, and the order of the steps is not intended to be limiting.

[0064] C. Phase 1 – Preliminary Evaluation In the first step of the methods for treating cellulite described herein, the clinician selects the cellulite depression to be treated based on the following criteria: The depression must be naturally evident when the patient is in a relaxed pose (standing with the hip muscles relaxed) as can be seen in a photograph. The depression chosen should be the one that the clinician believes will have the greatest potential to improve the overall aesthetic appearance of the affected area, such as the buttocks or thighs. ● Photograph the affected area while the patient is relaxed before treatment Assessment is performed prior to injection, i.e., the clinician and / or patient independently evaluate the photographs and score the results using one or more of the following scales or assessment methods: Hexel Cellulite Severity Scale (CSS) (Hexel CSS) Hexel pit depth score Likert scale Depression analysis Clinician-reported Photo-Numerical Cellulite Severity Scale (CR-PCSS) Patient-reported Photo-Numerical 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) Subject Self-Rating Scale (SSRS) Subject satisfaction with cellulite treatment (SSCT) 〇 Thigh Cellulite Severity - Patient (TCS-P), Thigh Cellulite Severity - Clinician (TCS-C) Body-Q Fitzpatrick scale A validated photo-numerical or other scale (e.g., the Hexel-Merz scale) used by clinicians and / or patients to assess cellulite severity, improvement, and / or patient satisfaction.

[0065] D. Step 2 - Marking the area to be treated In the second stage of the treatment described herein, the clinician can mark the depression to be treated with a dot(s). A photograph may also be taken. See, for example, Figures 6 and 15.

[0066] E. Stage 3 - Collagenase Injection In the third stage of treatment, clinicians administer collagenase injections.

[0067] 1. Types of collagenase Collagenases useful in the present disclosure include any of the collagenases defined above. For further background, matrix metalloproteases (MMPs) may be comprised of collagenases that fall under the definition herein. For example, MMP-1 has collagenase 1, MMP-8 has collagenase 2 / neutrophil collagenase, MMP-13 has collagenase 3, and MMP-18 has collagenase 4. Additionally, cathepsins can also be classified as collagenases.

[0068] 2. Collagenase Enzyme Kinetics Collagenases useful in the present disclosure may also be characterized by their enzymatic kinetics, where approximate kinetic values ​​for one or more collagenases effective in treating cellulite include the following: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 V max = Maximum speed K M =V max [Substrate] at 50% K cat = number of molecules of substrate cleaved per second 1 / K cat = microseconds required to cleave one molecule of substrate These values ​​may be determined empirically using the microplate assay described below, varying substrates and times, or other assays and parameters may be employed.

[0069] This value quantitatively describes the enzyme's behavior according to the Michaelis-Menten equation: TIFF0007628492000009.tif41165

[0070] Here, V 0 is the reaction rate (velocity) at the substrate concentration [S], Vmax is the maximum observable velocity, K M is V max is the Michaelis constant, which is related to the substrate concentration at which 50% of TIFF0007628492000010.tif24127

[0071] where k 1 , k -1 , k 2 is the rate constant for the next step TIFF0007628492000011.tif42155

[0072] Here, E is the enzyme, S is the substrate, ES is the enzyme-substrate complex, and P is the product.

[0073] Catalytic constant K cat teeth 、 The turnover number, or how quickly the ES complex progresses to E+P, reflects the number of catalytic cycles each active site performs per unit time.

[0074] In certain embodiments, AUX-I and AUX-II have the following characteristics: ● AUXI Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● AUXII Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Prerequisites K cat =V max / [AUX]=(nmol substrate / nG AUX*min-1) / nG AUX Catalytic efficiency (K cat / K M ) generally represents the overall ability of an enzyme to convert a substrate into a product and reflects both binding and catalytic events. In another embodiment, AUX-I and AUX-II have the following characteristics: TIFF0007628492000012.tif110159

[0075] 3. Collagenase titer (specific activity) Assays have been developed and used to determine the specific activity (potency) of collagenases. Such assays are described in subsections a. through c. and characterize collagenases by their ability to convert substrates to products in a given time at a pre-determined enzyme concentration. In certain non-limiting embodiments, these assays are used to determine the potency of each of AUX-I and AUX-II, as well as the combined CCH formulation (1:1 ratio of AUX-I and AUX-II). The SRC assay, described below, uses collagen as a substrate for the reaction. 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 contributing approximately 20% of the observed activity of the collagenase mixture. The SRC assay utilizes fluorescamine to detect peptides generated by type I digestion of SRC. The reaction is carried out in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer containing 15 mM divalent calcium ion at pH 7.2 at 25° C. for 2.5 hours.

[0076] The bovine tendon collagen (BTC) assay (described below) is based on the procedure of Mandl et al., Arch. Biochem. Biophys. 74: 465-475 (1958), revised 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 and measures both type I and type II activity (e.g., AUX-I and II collagenases). The BTC assay is a colorimetric method that utilizes ninhydrin to detect peptides generated by the degradation of types I and II of BTC. The reaction is also performed at pH 7.2, but in tris(hydroxymethyl)aminomethane (TRIS) buffer containing 10 mM divalent calcium ion for 22 hours at 37°C.

[0077] The GPA assay (described below) is a collagenase assay that uses a soluble derivatized hexapeptide (carbobenzoxy-GPGGPA) as a substrate. The GPA assay is primarily used to measure type II activity, with type I activity being 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 fluorescamine to give a fluorescent product. The GPA assay is performed in HEPES buffer containing 100 mM divalent calcium ion at pH 7.2 for 10 min at 25°C.

[0078] Both the SRC and BTC assays degrade the natural substrate (collagen) and are similar to the therapeutic effect of collagenase injection. The GPA assay has the advantage that it uses a well-defined low molecular weight hexapeptide as a substrate, resulting in the production of two well-defined tripeptides. The GPA method also produces a fluorescent signal and is highly sensitive. The GPA method uses a single substrate and reaction conditions (10 min incubation) that approximate the initial rate of the enzyme, making it suitable for Michaelis-Menten kinetic analysis. The SRC method is suitable for collagenases that have a collagen-binding domain, while the GPA method is suitable for collagenases that do not have a collagen-binding domain, known as gelatinases.

[0079] a. GPA unit assay method and specific activity unit i. Collagenase titer measured by GPA assay (cuvette) The GPA assay is mainly used to measure the titer of class II collagenase. The first step of the assay involves an enzymatic reaction involving digestion of the substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-prolyl-L-alanine (zGPGGPA) by the collagenase sample into two peptides: carbobenzoxy-glycyl-L-prolyl-glycine (zGPG) and glycyl-prolyl-L-alanine (GPA). The second step is followed by measurement of the liberated GPA using the fluorescent derivative fluorescamine. The assay follows the method below, but those skilled in the art will understand that certain modifications (e.g., dilution concentration and dilution time) may be made to achieve the purpose of the assay.

[0080] The general method is as follows: Prepare a leucine standard; Obtain a collagenase sample and prepare a solution for use in the first step of enzymatic cleavage of zGPGGPA (hereafter "substrate") by collagenase; Then treat the collagenase-treated sample (containing free GPA) and the leucine standard with fluorescamine for a period of time at room temperature in order to fluorescently label the free amino groups of the resulting GPA and leucine molecules, respectively; Measure the fluorescence emission of each solution at 480 nm after excitation at 392 nm; Use the resulting slope of the leucine and collagenase sample curves to calculate potency units as follows: Titer (f-GPA units / mg) = (M サンプル / M ロイシン )×(DF / T) Where: M サンプル = Slope of collagenase sample titer curve M ロイシン = slope of leucine standard curve DF = dilution factor T = reaction time

[0081] Additional non-limiting details regarding assay methods for GPA are as follows. Buffers and Reagents 1. f-Appel's buffer, pH 7.2 (55 mM HEPES, 100 mM calcium acetate) 2.1mM Leucine working stock solution 3. 200mM borate, pH 9.0 4. 0.5 mM fluorescamine solution in acetone 5. 2mg / mL zGPGGPA substrate in f-Appel's buffer

[0082] Solution preparation The solutions are prepared as follows. f-Appel's buffer: Dissolve 13.0 g HEPES and 17.6 g calcium acetate in approximately 800 mL water. Adjust pH to 7.2 with sodium hydroxide and QS to 1 L with water. Store at 2-8°C. 10mM Leucine Stock Solution: Dissolve 65.5mg of leucine in 50mL of water. Leucine should be weighed directly into a 100mL (or equivalent) glass beaker. Weigh approximately 65mg of leucine (target weight) into the beaker. Based on the weight of leucine weighed out, calculate the amount of water to add to the beaker using the following formula: Add the calculated amount of water to the beaker and mix thoroughly, ensuring the leucine is completely dissolved. Dispense into 1mL aliquots. Store at or below -20°C. TIFF0007628492000013.tif20123 where, C2 = mass of leucine measured (mg) V1 = 50 (mL of water) C1 = 65.5 (mg leucine) V2 = volume of water needed to make a 10 mM stock solution (mL) 1 mM Leucine Working Stock Solution: Thaw a vial of 10 mM Leucine stock solution and dilute to 1 mM by combining 150 µL with 1,350 µL of water. Mix well before use. 0.5N HCl: Dilute HCl to 0.5N with water and mix well. Store at room temperature. You can also use commercially available 0.5N hydrochloric acid. 200 nM Borate: pH 9.0: Dissolve 2.4 g Boric Acid in approximately 150 mL water. Adjust pH to 9.0 with sodium hydroxide. Dilute to 200 mL with water and mix well. Store at 2-8°C. 0.5 mM fluorescamine solution: Mix 15 mg of fluorescamine with 100 mL of acetone and dissolve by shaking. Store at 2-8°C in the dark. Substrate solution (2 mg / mL zGPGGPA): Prepare 2 mg / mL of substrate in f-Appel's buffer. Dissolve on a mechanical shaker / rotator for sufficient time until completely dissolved (approximately 15 min).

[0083] Leucine standard curve A standard curve for leucine is prepared according to Table 7. [Table 7] Then, 100 μL of each leucine standard is transferred to a separate tube and fluorescamine is detected.

[0084] Collagenase sample preparation Dilute the collagenase sample in two steps to 0.01 mg / mL in f-Appel's buffer and mix by gentle vortexing. An example of the dilution method is shown below. 1.100μL×1.0mg / mL-→1000μL=0.1mg / mL 2.100μL×0.1mg / mL-→1000μL=0.01mg / mL

[0085] Blank preparation Blanks are prepared by combining 45 µL of the diluted preparation with 500 µL of 0.5 N hydrochloric acid to inactivate the enzyme. Add 455 µL of zGPGGPA substrate solution and mix thoroughly by vortexing. Transfer 100 µL of each blank to a separate tube to detect impurities that may react with fluorescamine.

[0086] Titer curve For each collagenase sample, a set of titration curves is generated as follows. TIFF0007628492000015.tif53160Warm the tubes containing substrate and buffer in a 25°C water bath for at least 15 minutes. Label a second set of tubes and add 50 μL of 0.5 N HCl to each. Add the diluted collagenase sample preparation (0.01 mg / mL) according to Table 8 to the tubes, incubate for 10 minutes, mix and return to the 25°C water bath. The incubation period begins when the first preparation is added to the pre-warmed substrate. [Table 8] Remove preparations from the water bath with 1-2 minutes remaining of the 10-minute incubation and vortex gently to mix. Ten minutes after adding the first reagent to the substrate, transfer 50 μL from each tube to a tube containing 50 μL of 0.5 N hydrochloric acid. To hasten the digestion, preparations should be added directly to the acid. After all preparations have been quenched, vortex each tube to mix well.

[0087] detection Add 400 μL of 200 mM borate buffer and 500 μL of 0.5 mM fluorescamine solution to all detection tubes containing 100 μL of each preparation (blank, collagenase sample titer curve, leucine standard). Vortex thoroughly to mix. Incubate tubes at room temperature for a minimum of 10 minutes.

[0088] Fluorometer setup Set up the fluorometer using the following instrument parameters to read the fluorescence of each preparation at 1 h of derivatization: TIFF0007628492000017.tif77161

[0089] calculation Plot the concentration of each leucine standard (X-axis) against the fluorescence response at 480 nm (Y-axis). The slope (m) and coefficient of determination (R 2Determine the mean fluorescence of each titration curve preparation. Construct titration curves for collagenase samples and leucine by plotting the concentration of each preparation (X-axis) against the mean fluorescence response at 480 nm (Y-axis). Measure the slope (m) and coefficient of determination (R 2 ) to determine

[0090] Titering collagenase samples Titer (f-GPA units / mg) = (M サンプル / M ロイシン )×(DF / T) Where: M サンプル = Slope of collagenase sample titer curve M ロイシン = slope of leucine standard curve DF = dilution factor (1100 μL / 50 μL = 22) T = reaction time (10 min)

[0091] ii. GPA Microplate Assay for Measuring Class II Collagenase Activity in Collagenase Samples This method is similar to the GPA assay described above, except that it is performed in a microplate. As with the above assay, the microplate assay measures the proteolytic activity of collagenase samples in the enzymatic cleavage of the substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-prolyl-L-alanine (zGPGGPA) (hereinafter referred to as "substrate"). The assay follows the following methodology, although one skilled in the art will understand that certain modifications (e.g., dilution concentration and time) can be made to achieve the objectives of the assay.

[0092] reagent 1. Peptide substrate (zGPGGPA) (Bachem M1260 or equivalent) 2. Tripeptide GPA (Bachem H3615 or equivalent) 3. Fluorescamine (Acros 191675000 or equivalent) 4. Purified water (Milli-Q-Plus 18.2MΩ system or equivalent) 5.1M HEPES buffer (Gibco 15630-080 or equivalent) 6. Surfact-Amps20™ (10% Tween solution) (Pierce Cat. #28320 or equivalent) 7.1M Calcium Acetate (Ca(C 2 H 3 O 2 ) 2 )(Emerald Biosciences Cat.#EBS-100-CAAC or equivalent) 8. Boric Acid (Sigma B7660 or equivalent) 9. 2.5N NaOH (JT Baker 5666-02 or equivalent) 10. 0.5N Hydrochloric Acid (VWR 101223-134 or equivalent) 11. Acetone (Sigma 270725 or equivalent)

[0093] Preparation of solutions (i) Assay buffer (50 mM HEPES pH 7.1 / 0.05% Tween 20 / 5 mM (Ca(C 2 H 3 O 2 ) 2 Preparation of 1M (Ca(C)): Mix 50 mL of 1M HEPES with a pipette into 800 mL of pure water. 2 H 3 O 2 ) 2 ) and 5 mL of Surfact-Amps (10% Tween 20). Check the pH and adjust to 7.1 ± 0.05 if necessary. Adjust the volume to 1 L with enough water and filter the solution through a 0.22 micron filter. This assay buffer can be stored at room temperature for up to 3 months. (ii) Preparation of 0.1 N NaOH: Add 2 mL of 2.5 N NaOH to 48 mL of pure water. This solution can be stored at room temperature for up to 3 months. (iii) Preparation of 4 mg / mL tripeptide GPA stock solution: Dissolve 400 mg (± 1 mg) tripeptide GPA in 10 mL of 0.1 N NaOH and vortex until completely dissolved. Add sufficient assay buffer to make 100 mL and store in 0.5 mL aliquots at -70°C. The 4 mg / mL tripeptide GPA stock can be stored at -70°C for one year. (iv) Preparation of 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 NaOH and vortex until completely dissolved. Add enough Assay Buffer to 100 mL. This solution can be stored at 4°C for up to 3 months. (v) Preparation of 120 mM Boric Acid pH 9.0: Dissolve 7.4 g (± 0.5 g) Boric Acid in 800 mL of DI water. Titrate this solution with NaOH to pH 9.0 and adjust the volume to 1 liter with sufficient DI water. This solution may be stored at room temperature for up to 3 months. (vi) Preparation of 1 mM fluorescamine in acetone: Dissolve 28±2 mg of fluorescamine in 100 mL of acetone. This solution should be freshly prepared and protected from light and moisture.

[0094] Preparation of tripeptide GPA standards and serial dilutions A 0.08 mg / mL (329 μM) tripeptide GPA standard is prepared by diluting the 4 mg / mL tripeptide GPA stock 50-fold with assay buffer (e.g., 20 μL of 4 mg / mL tripeptide GPA into 980 μL of assay buffer). In row A of the assay plate, pipette 200 μL of the 329 μM tripeptide GPA standard into A1 and A7. Pipette 100 μL volumes of assay buffer into A2-A6 and A8-A12. For serial dilutions of the tripeptide GPA standard, transfer 100 μL from A1 to A2, mix, transfer 100 μL from A2 to A3, and repeat through A5. Remove 100 μL from well A5 to bring the final volume to 100 μL. Well A6 contains buffer only. For the second serial dilution of the tripeptide GPA standard, transfer 100 μL from A7 to A8, mix, then transfer 100 μL from A8 to A9, and repeat through well A11. Remove 100 μL from well A11 for a final volume of 100 μL. Well A12 contains buffer only.

[0095] Collagenase sample preparation For collagenase samples (e.g., lyophilized collagenase preparations), allow samples to come to room temperature for at least 10 min and reconstitute to form a 500 ng / mL stock solution. Different concentrations may be used. Dilute the stock solution with assay buffer to prepare the test collagenase sample (T1A). Repeat this procedure to prepare triplicate test samples (T1A, T1B, T1C).

[0096] Discussion In this method, 50 μL of increasing concentrations of collagenase test sample are mixed with 50 μL of excess substrate (final concentration 2.0 mg / mL) in a 96-well plate. A volume of 50 μL of assay buffer is added to row CG of a U-bottom 96-well polypropylene reaction plate. Then, using a multichannel pipette, perform a 1 / 1.5 serial dilution by transferring 100 μL of collagenase sample from row B to row C, mixing, and repeating until row G is reached. Table 9 shows the final collagenase concentrations after adding 50 μL of substrate from row B to row H. A blank is prepared in column H by pipetting 50 μL of assay buffer, containing no enzyme in this row. Exemplary concentrations are shown in Table 9. [Table 9]

[0097] Collagenase reaction The zGPGGPA substrate is cleaved into zGPG and GPA by class II collagenase during a 15 minute incubation period at room temperature. Turn on the incubator and temperature probe (temperature is 22 ± 1 °C before adding substrate to plate). Add 50 μL 4 mg / mL (6.8 mM) zGPGGPA substrate per column to columns 1-12 in row BH and mix. The reaction start time begins after the substrate is added to the first column. Cover the plate and place in a 22 ± 1 °C incubator for a total reaction time of 15 ± 1 minutes. After incubation, the reaction is quenched by adding hydrochloric acid, and the amount of released GPA peptide is quantified by reacting the free amino termini of the peptides with the color-developing reagent fluorescamine. To quench the reaction, 100 μL of 0.5 N hydrochloric acid is added to each well in rows A through H, column by column, and mixed. The reaction time is terminated when HCl is added to the first column.

[0098] detection Add a volume of 195 μL of 120 mM borate pH 9.0 to each well of a microplate Greiner polypropylene black reading plate. Transfer 30 μL of the quenched reaction mixture from the reaction plate to the corresponding well of the reading plate and mix well. Then add 75 μL of 1 mM fluorescamine to each well of the reading plate (using a polypropylene tray to dispense fluorescamine / acetone), mixing immediately after each addition. Read the plate within 15 minutes after adding fluorescamine using a Molecular Devices M2 Fluorescence Plate Reader with the following settings: Excitation 380 nm, Emission 473 nm, Cutoff 455 nm, 6 reads / well, PMT medium. Concentration of GPA (μM) versus emission at 473 nm and concentration of collagenase (ng / mL) versus emission at 473 nm were plotted. For each plot, a linear regression was fitted with no fixed parameters. For collagenase test samples, the zero point data was excluded from the linear fit and plots were generated using the entire triplicate data set for each sample. The slopes for the tripeptide GPA standard samples and collagenase samples were calculated.

[0099] Titer determination The specific activity of a collagenase sample can be calculated as follows: GPA Microplate Assay Units = ((Collagenase Sample Slope) / (Tripeptide GPA Slope x Incubation Time)) x 10 6 The specific activity of collagenase test samples is determined from the slope of the tripeptide GPA standard and calculated with a curve fitting program. Using the microplate method, the enzyme reaction rate can be calculated by Michaelis-Menten method using different concentrations of substrate and different times.

[0100] iii. Collagenase titer measured by GPA assay Collagenases useful in the present disclosure may have a potency 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 potency 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 potency may be about 70,000 to about 400,000 f-GPA units / mg, about 100,000 to about 375,000 f-GPA units / mg, about 125,000 to about 350,000 f-GPA units / mg, about 150,000 to about 325,000 f-GPA units / mg, about 175,000 to about 300,000 f-GPA units / mg, about 230,000 to about 430,000 f-GPA units / mg, or about 200,000 to about 275,000 f-GPA units / mg. The collagenase may also have a potency of about 30,100 to about 87,100, or about 43,000 to about 67,000 GPA microplate assay units. The GPA assay described above may be employed to analyze the specific activity of any collagenase.

[0101] b. SRC unit assay method and specific activity unit Collagenase titer measured by i.SRC assay (cuvette) The SRC assay is mainly used to measure the titer of class I collagenase. The general method is as follows: Leucine standard solution and collagenase sample solution are prepared. The first step of the assay involves an enzymatic reaction involving digestion of soluble rat tail tendon collagen (SRC) with collagenase. In the second step, the released peptide fragments / amino acids are measured with fluorescamine, a fluorogenic derivative. The assay follows the following methodology, but those skilled in the art will understand that certain modifications (e.g., dilution concentration and time) can be made to achieve the purpose of the assay.

[0102] These collagenase and leucine standard samples are treated with a reagent to fluorescently label the resulting GPA. The leucine standard sample and collagenase sample are incubated at room temperature for 10 minutes, after which the fluorescence of each solution is measured at excitation and emission wavelengths of 392 nm and 480 nm, respectively. The slopes of the resulting curves for the leucine and collagenase samples are then used to calculate titer units as follows: Titer (f-SRC units / mg) = (M サンプル / M ロイシン )×(DF / T)×CF Where: M サンプル = Slope of collagenase sample titer curve M ロイシン = slope of leucine standard curve DF = dilution factor (1500 μL / 100 mL = 15) T = reaction time (2.5 hours x 60 minutes / 1 hour = 150 minutes) CF = Conversion factor (1000μg / 1mg = 1000)

[0103] Below are non-limiting details regarding the SRC assay method.

[0104] Buffers and Reagents 1. F-TC assay buffer, pH 7.2 (22 g HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 4.4 g calcium acetate) 2.F-enzyme buffer, pH7.2 3. 200mM borate, pH 9.0 4.10mM Leucine stock solution 5.1mM Leucine working stock solution 6. 1 mM fluorescamine solution in acetone 7. 2mg / mL rat tail tendon collagen in 0.02N acetic acid

[0105] Solution preparation The solutions are prepared as follows:

[0106] F-TC Assay Buffer: Dissolve 22 g HEPES and 4.4 g calcium acetate in approximately 900 mL water. Adjust pH to 7.2 with sodium hydroxide and QS to 1 L with water. Store at 2-8 °C.

[0107] F-Enzyme Buffer: Dilute F-TC Assay Buffer by combining 4 mL with 16 mL of water. Store at 2-8 °C.

[0108] 10mM Leucine Stock Solution: Dissolve 65.5mg leucine in 50mL water. Leucine should be weighed directly into a 100mL (or equivalent) glass beaker. Weigh approximately 65mg leucine (target weight) into the beaker. Based on the weight of leucine weighed out, calculate the amount of water to add to the beaker using the following formula: Add the calculated amount of water to the beaker and mix thoroughly, ensuring the leucine is completely dissolved. Dispense into 1mL aliquots. Store at -20°C or below. TIFF0007628492000019.tif21132, where C2 = mass of leucine measured (mg) V1 = 50 (mL of water) C1 = 65.5 (mg leucine) V2 = volume of water needed to make a 10 mM stock solution (mL)

[0109] 1 mM Leucine Working Stock Solution: Thaw a vial of 10 mM Leucine stock solution and dilute to 1 mM by combining 150 µL with 1,350 µL of water. Mix well before use.

[0110] 0.5N HCl: Dilute HCl to 0.5N with water and mix well. Store at room temperature. You can also use commercially available 0.5N hydrochloric acid.

[0111] 0.02N Acetic Acid: Mix 1mL of 1N Acetic Acid with 49mL of water and mix well. Store at room temperature.

[0112] 200 mM Borate, pH 9.0: Dissolve 2.4 g boric acid in approximately 150 mL water. Adjust pH to 9.0 with sodium hydroxide. Dilute to 200 mL with water and mix well. Store at 2-8 °C.

[0113] 1 mM fluorescamine solution: Dissolve 15 mg of fluorescamine in 50 mL of acetone and shake to dissolve. Store at 2-8°C in the dark.

[0114] Substrate solution (2 mg / mL rat tail tendon collagen): Dilute stock rat tail collagen to 2 mg / mL with 0.02 N acetic acid. Store at 2-8 °C.

[0115] Leucine standard curve

[0116] A standard curve for leucine is prepared according to Table 10. [Table 10]

[0117] Then, 100 μL of each leucine standard is transferred to separate centrifuge tubes and fluorescamine is detected.

[0118] Preparation of collagenase samples and blanks

[0119] Samples are diluted in two steps to 0.01 mg / mL in F-enzyme buffer and mixed by gentle vortexing. An example of the dilution method is shown below. 1.100μL×1.0mg / mL→1000μL=0.1mg / mL 2.100μL×0.1mg / mL→1000μL=0.01mg / mL The diluted samples are stored at room temperature until use.

[0120] Blanks are prepared according to Table 11 by first combining the sample with 0.5N hydrochloric acid to inactivate the enzyme, followed by the addition of buffer and substrate.

[0121] Place collagenase samples in tubes labeled according to Table 11. Tubes 1, 2, 4, and 6 are from the first preparation, and tubes 3, 5, and 7 are from the second preparation. [Table 11]

[0122] Cap the tubes and vortex briefly to mix. Incubate the titer curves in a 25°C ± 3°C water bath for 2.5 hours. After incubation is complete, remove the titer tubes from the water bath. Add 750 μL of 0.5 N hydrochloric acid to each preparation and vortex thoroughly to mix. Preparations can be stored at 2-8°C for up to 22 hours before detection.

[0123] Detector and fluorometer setup

[0124] Leucine standard samples are prepared as described above.

[0125] Set up the luminescence spectrometer with the following instrument parameters to read the fluorescence of each formulation after 1 hour of derivatization: TIFF0007628492000022.tif79163

[0126] calculation Plot the concentration of each leucine standard (X-axis) against the fluorescence response at 480 nm (Y-axis). The slope (m) and coefficient of determination (R 2 ) Do not force to zero. Determine the mean fluorescence of each duplicate preparation. Calculate the net fluorescence of each collagenase sample. F(Net) = Average collagenase sample (EM 480 )-Blank (EM 480 )

[0127] Plot the amount of collagenase sample in each preparation (X-axis) against the net fluorescence (Y-axis). Calculate the slope (m) and coefficient of determination (R 2 ) and must not be forced to zero. Titering collagenase samples Titer (f-SRC units / mg) = (M サンプル / M ロイシン )×(DF / T)×CF Where: M サンプル = Slope of collagenase sample titer curve M ロイシン = slope of leucine standard curve DF = dilution factor (1500 μL / 100 mL = 15) T = reaction time (2.5 hours x 60 minutes / 1 hour = 150 minutes) CF = Conversion factor (1000μg / 1mg = 1000)

[0128] The SRC assay described above can be adapted to analyze the specific activity of any collagenase.

[0129] ii. SRC Microplate Assay for Measuring Class I Collagenase Activity in Collagenase Samples This method is similar to the SRC assay described above, except that it is performed in a microplate. Like the SRC assay described above, the microplate assay measures collagenase activity against a soluble rat tail tendon collagen (SRC) substrate (hereafter referred to as "substrate"). The assay follows the following methodology, although one of skill in the art will recognize that certain modifications may be made to accomplish the objectives of the assay.

[0130] reagent 1. Soluble Rat Collagen Matrix (BD Biosciences 354236) 2. Tripeptide GPA (Bachem H3615 or equivalent) 3. Fluorescamine (Acros 191675000 or equivalent) 4. Purified water (Millipore, Milli-Q-Plus 18.2 MΩ system or equivalent) 5.1M HEPES buffer (Gibco 15630-080 or equivalent) 6.1M Calcium Acetate (Ca(C 2H 3 O 2 ) 2 )(Emerald Biosciences EBS-100-CAAC or equivalent) 7. Surfact-Amps 20™ (10% Tween solution) (Pierce Cat. #28320 or equivalent) 8. 1.0N Acetic Acid (Sigma 318590 or equivalent) 9. 0.5N Hydrochloric Acid (VWR 101223-134 or equivalent) 10. Boric Acid (Sigma B7660 or equivalent) 11. 2.5N Sodium Hydroxide (JT Baker 5666-02 or equivalent) 12. Acetone (Sigma 270725 or equivalent)

[0131] Preparation of solutions (i) Assay buffer (50 mM HEPES pH 7.1 / 0.05% Tween 20 / 5 mM (Ca(C 2 H 3 O 2 ) 2 Preparation of 1M HEPES: Mix 50 mL of 1 M HEPES with 800 mL of pure water using a pipette. Add 5 mL of 1 M (Ca( C2 H 3O2 ) 2 ) and 5 mL of Surfact-Amps (10% Tween 20). Check the pH and adjust to 7.1 ± 0.1 if necessary. Adjust the volume to 1 L with enough water and filter the solution through a 0.22 micron filter. This assay buffer can be stored at room temperature for up to 3 months. (ii) Preparation of 0.1 N NaOH: Add 2 mL of 2.5 N NaOH to 48 mL of pure water. This solution can be stored at room temperature for up to 3 months. (iii) Preparation of 4 mg / mL tripeptide GPA stock solution: Dissolve 400 mg (± 1 mg) tripeptide GPA in 10 mL of 0.1 N NaOH and vortex until completely dissolved. Add sufficient assay buffer to make 100 mL and store in 0.5 mL aliquots at -70°C. The 4 mg / mL tripeptide GPA stock can be stored at -70°C for one year. (iv) Preparation of 0.02 N acetic acid: Add 1 mL of 1.0 N acetic acid to 40 mL of purified water. Add enough purified water to make 50 mL. This solution can be stored at room temperature for up to 1 year. (v) Preparation of 2 mg / mL SRC substrate stock solution: Add 23.3 mL 0.02N acetic acid directly to the vial in which the substrate is supplied (in one non-limiting example, supplied as 100 mg of SRC at 3.75 mg / mL). Other concentrations of SRC substrate may be used. The calculation is as follows:

[0132] 100mg÷3.75mg / mL=26.7mL;

[0133] Total volume (mL) = (3.75 mg / mL × 26.7 mL) / 2 mg / mL;

[0134] Total volume (50.0 mL) - 26.7 mL = 23.3 mL

[0135] The solution is thoroughly mixed by inversion and can be stored at 2-8 °C for up to 3 months. (vi) Prepare 0.6 mg / mL SRC substrate working solution: Add 4.2 mL of Assay Buffer to a 15 mL conical tube. Add 4.2 mL of Assay Buffer to a 15 mL conical tube and 1.8 mL of 2 mg / mL SRC substrate stock solution and mix by inversion. This solution should be prepared immediately prior to adding to the plate. (vii) Preparation of 120 mM Boric Acid pH 9.0: Dissolve 7.4 g (± 0.5 g) Boric Acid in 800 mL of DI water. Titrate this solution to pH 9.0 with NaOH and add enough DI water to make up to 1 L. This solution can be stored at room temperature for up to 3 months. (viii) Preparation of 1 mM fluorescamine in acetone: Dissolve 28±2 mg of fluorescamine in 100 mL of acetone. This solution should be freshly prepared and protected from light and moisture.

[0136] Preparation of tripeptide GPA standards and serial dilutions Tripeptide GPA standards at 0.08 mg / mL (329 μM) are prepared by diluting the 4 mg / mL tripeptide GPA stock 50-fold in assay buffer (e.g., 20 μL of 4 mg / mL GPA into 980 μL of assay buffer). In row A of the assay plate, 200 μL of 329 μM tripeptide GPA standard is pipetted into A1 and A7. In A2-A6 and A8-A12, 100 μL of assay buffer is pipetted.

[0137] For serial dilutions of the tripeptide GPA standard, transfer 100 μL from A1 to A2, mix, then transfer 100 μL from A2 to A3 and repeat through A5. Remove 100 μL from well A5 to bring the final volume to 100 μL. Well A6 contains buffer only.

[0138] For the second tripeptide GPA standard serial dilution, transfer 100 μL from A7 to A8, mix, then transfer 100 μL from A8 to A9 and repeat until All. 100 μL is removed from well A11, resulting in a final dilution of 100 μL. Well A12 contains buffer only.

[0139] Preparation of collagenase test samples

[0140] For collagenase samples (e.g., lyophilized collagenase preparations), allow samples to come to room temperature for at least 10 min and reconstitute to form a 3.0 µg / mL stock solution. Different concentrations may be used. Dilute the stock solution with assay buffer to prepare collagenase test strips (T1A). Repeat this procedure to prepare triplicate test samples (T1A, T1B, T1C).

[0141] Discussion

[0142] In this method, 50 μL of increasing concentrations of test collagenase samples are mixed with 50 μL of excess substrate (final concentration 0.2 mg / mL) in a 96-well plate. A volume of 50 μL of assay buffer is added to row CG of a U-bottom 96-well polypropylene reaction plate. Then, using a multichannel pipette, perform a 1 / 1.5 serial dilution by transferring 100 μL of collagenase sample from row B to row C, mixing, and repeating until row G is reached. A volume of 100 μL is removed and discarded from row G, and a blank is prepared in row H by pipetting 50 μL of assay buffer into row H. This row contains no enzyme. Table 12 contains the final collagenase concentrations after adding 50 μL of substrate from row B to row H. [Table 12]

[0143] Collagenase reaction Turn on the incubator and temperature probe (temperature is 22 ± 1 °C before adding substrate to plate). Add 50 μL 0.6 mg.mL SRC substrate per column to each well in rows B through H and mix. Reaction initiation time begins after substrate is added to the first column. Cover plate and place in 22 ± 1 °C incubator for 45 ± 5 minutes total reaction time. To quench the reaction, add 100 μL 0.5 N HCl per column to each well of the dilution plate and mix. Reaction time ends with addition of HCl to the first column.

[0144] detection

[0145] Add a volume of 195 μL of 120 mM borate pH 9.0 to each well of a microplate Greiner polypropylene black reading plate. Transfer 30 μL of the quenched reaction mixture from the reaction plate to the corresponding well of the reading plate and mix well. Then add 75 μL of 1 mM fluorescamine to each well of the reading plate (using a polypropylene tray to dispense fluorescamine / acetone), mixing immediately after each addition. Read the plate within 15 minutes after adding fluorescamine using a Molecular Devices M2 Fluorescence Plate Reader with the following settings: Excitation 380 nm, Emission 473 nm, Cutoff 455 nm, 6 reads / well, PMT medium.

[0146] Concentration of GPA (μM) versus emission at 473 nm and concentration of collagenase (ng / mL) versus emission at 473 nm were plotted. For each plot, a linear regression was fitted with no fixed parameters. For collagenase samples, the zero point data was excluded from the linear fit, and plots were generated using the entire triplicate data set for each sample. The slopes for the tripeptide GPA standard samples and collagenase samples were calculated.

[0147] Determination of specific activity and relative titer The specific activity of a collagenase sample can be calculated as follows: SRC Microplate Assay Units = ((Collagenase Sample Slope) / (Tripeptide GPA Slope x Incubation Time)) x 10 6

[0148] The specific activity of collagenase test samples is determined from the slope of the tripeptide GPA standard and calculated with a curve fitting program. Using the microplate method, the enzyme reaction rate can be calculated by Michaelis-Menten method using different concentrations of substrate and different times.

[0149] iii. Collagenase titer measured by SRC assay Collagenases useful in the present disclosure may have a potency of about 500 to about 15,000 SRC units / mg. In certain embodiments, the potency 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 about 1,500 to about 6,000 SRC units / mg, or about 2,500 to about 5,000 SRC units / mg. Alternatively, the potency may 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 titer of about 980 to about 3,510, or about 1,400 to about 2,700 SRC microplate assay units.

[0150] c. Collagenase titer in BTC unit assay The Bovine Tendon Collagen Assay for collagenase is based on the procedure of Mandl et al. (1958) and modified by Keller and Mandl (1963). Since bovine tendon collagen is an insoluble substrate, it is important to break it into small pieces. Trypsin is used as a control to allow for the presence of denatured collagen and other protein impurities. The assay is performed in the presence of calcium ions, which are necessary for collagenase activity. The number of solubilized peptides is determined by reacting the N-terminal amino group of the peptide with ninhydrin and measuring the amount of adduct formed colorimetrically (Rosen 1957).

[0151] The purpose of this procedure is to test the specific activity of the collagenase enzyme using a collagen substrate.

[0152] Reagents and solutions 1. Collagen matrix (collagen) 2. Deionized water (water) 3. Tris Assay Buffer 4. Trypsin Stock Solution 5. 0.5M HCl 6. Leucine standard assay solution (1 mM leucine) 7. Rosen's Buffer 8.3% Ninhydrin 9.50% Isopropanol

[0153] incubation Set up and label reaction tubes as follows: 3 tubes for trypsin control, 6 tubes for reference solutions, and 6 tubes for each test sample. Label and remove the caps for each tube. Weigh out 10 ± 1 mg of collagen in the order in Table 13 and place the weighed collagen into each reaction tube. [Table 13]

[0154] The amount of enzyme in the test sample should contain an activity of 1.6-5.7 nmol leu eq / min per reaction tube (ACT). Undissolved samples should first be dissolved in Tris Assay Buffer before use in the assay. The concentration (before adding to the reaction tube) should be 0.0065 mg / mL or greater.

[0155] Set up reaction tubes according to Table 14 so that you have a matrix pattern. The table below assumes 2 under test samples. For more or less samples, adjust the number of reaction tubes but maintain the pattern. For constant volumes, see Table 14. [Table 14]

[0156] Cap the reaction tube. Mix contents gently but thoroughly. Place reaction tube in a 37°C water bath. Incubate for 22 ± 0.5 hours. Record the actual time incubation began (37°C), the water bath number used, the Collagen Lipid lot number, the Collagen Correction Factor for the lot used, and the lot numbers of all solutions used.

[0157] Quenching and Filtration Label the filtrate tubes that correspond to each incubated reaction tube. Place a funnel with filter paper and folded filter paper into each labeled filtrate tube. When the incubation period is over, remove the reaction tubes from the water bath. Record the actual time that the incubation is over.

[0158] Remove the caps from the reaction tubes and discard the caps. Add 2 mL of 0.5 M HCl to each reaction tube to stop the reactions. Mix the contents of each tube thoroughly. Filter the contents of each reaction tube into the appropriate filtrate tube.

[0159] The previous two steps should be completed as quickly as possible, since undigested collagen may dissolve in hydrochloric acid in a short time. The filtrate can be stored in a capped filtrate tube in the refrigerator for up to 95.5 hours before color development. Record the refrigeration and storage time.

[0160] Available colors Set up and label boiling tubes as follows: 6 tubes for water and leucine controls (Step 1), 2 tubes for each filtrate tube (Step 1). Add the following volumes of water and leucine standard assay solution to the 6 leucine control tubes: TIFF0007628492000026.tif36154

[0161] Add 0.8 mL of water to each boiling tube (step 2). Add 0.2 mL of filtrate from each sample to the appropriately labeled bring tube. Add 0.5 mL of Rosen's buffer to each bring tube. Under the hood, add 0.5 mL of 3% ninhydrin to each bring tube. Thoroughly mix the contents of each tube on a vortex mixer. Place the boiling tubes in a boiling water bath in a fume hood. Boil for 15 ± 1 minutes. When the boiling time is over, remove the boiling tubes from the water bath. Under a containment hood, dispense 5.0 mL of 50% isopropanol into each boiling tube and mix the contents thoroughly. Allow the boiling tubes to return to room temperature (at least 10 minutes) before reading the absorbance.

[0162] Absorbance reading While working under the containment hood, read the absorbance of the tubes. Turn on the spectrophotometer and allow it to warm up. Set the spectrophotometer wavelength to 570 nm. Zero the spectrophotometer with 50% isopropanol. Read the absorbance (A) of the water, leucine, trypsin controls and the sample under test. 570 ) and record the time in hours when the first sample was taken. Multiply the reading by 1000 x A. 570 Record the reading as 0.01 and record the time in hours that the last sample was read. All readings should be taken within an hour.

[0163] calculation principle Calculate the total read time and total incubation time in minutes. The total read time should be less than 60 minutes and the total incubation time should be between 1290 and 1350 minutes. Use a linear least squares method to fit the leucine standard samples (x = nmol leucine vs y = A 570 The slope of the reading (b) and the correlation coefficient (r) are calculated. The unit of the b value is A 570 / nmol leucine. The b value for leucine should be between 2.88 and 3.33. Calculate the average of the trypsin control (T). The average reading for the trypsin control (T) should be between 221 and 338. Record this average to the nearest decimal place (Step A). 570 Average the readings. Record this number to the nearest decimal place. Average Sample A 570 Subtract the average trypsin (step A) from the reading to obtain the net sample reading.

[0164] Calculate the activity per tube (ACT) in nmol leu eq / min as follows: TIFF0007628492000027.tif14143where 20 is the dilution factor for the volume of reaction mixture developed and "b" is the slope of the leucine standard curve. Record this number to one decimal place. The activity per tube of the sample being tested should be between 1.6 and 5.7 nmol leu eq / min.

[0165] We calculate activity in BTC as follows: BTC units = activity (nmol leu eq / min) x collagen correction factor

[0166] Calculate the activity in BTC units / mL of the sample as follows: TIFF0007628492000028.tif16129

[0167] Calculate the specific activity of the sample in BTC units / mm as follows: TIFF0007628492000029.tif14131

[0168] The conversion from BTC to ABC is: ABC units = BTC units x 1.09

[0169] i.BTC and ABC units Various collagenase compositions can be employed, where 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(s) present in the composition (as distinct from excipients and other components).

[0170] Additionally, various collagenase compositions can be employed, wherein 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(s) present in the composition (as distinct from excipients and other components).

[0171] d. Other Assays Assays using labeled collagen have been reported by Gisslow et al., Anal.Biochem., 68: 70-78 (1975); Robertson et al., Clinica Chimica Acta, 42:43-45 (1972); Sakamoto et al., A New Method for the Assay of Tissue Collagenase (36297) (1972). Another assay is the Worthington Biochemical Corp. Assay (http: / / www.worthington-biochem.com / CLS / assay.html) (accessed July 3, 2019).

[0172] 4. Collagenase Dosage With regard to the doses of collagenase employed herein, the present disclosure provides a therapeutically effective amount of collagenase sufficient to bind and dissolve septa upon subcutaneous injection, thereby reducing the appearance of cellulite compared to a pre-treatment baseline.

[0173] In one embodiment, collagenase may be injected in an amount of about 0.01 mg to about 20 mg, either in a single dose or in divided doses. In another embodiment, collagenase may be injected in an amount of about 0.05 mg to about 15 mg, either in a single dose or in divided doses. In another embodiment, collagenase may be injected in an amount of about 0.10 mg to about 10 mg, either in a single dose or in divided doses. In another embodiment, collagenase may be injected in an amount of about 0.15 mg to about 5 mg, either in a single dose or in divided doses. In another embodiment, collagenase may be injected in an amount of about 0.20 mg to about 3 mg, either in a single dose or in divided doses. In another embodiment, collagenase may be injected in an amount of about 0.25 mg to about 2 mg, either in a single dose or in divided doses.In yet another embodiment, the collagenase is 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 1.00 mg, about 1.05 mg, about 1.10 mg, about 1.15 mg, about 1.20 mg, about 1.25 mg, about 1.30 mg, about 1.35 mg, about 1.40 mg, about 1.45 mg, about 1.50 mg, about 1.60 mg, about 1.70 mg, about 1.80 mg, about 1.9 ...95 mg, about 1.00 mg, about 1.05 mg, about 1.10 mg, about 1.15 mg, about 1.20 mg, about 1.25 mg, about 1.30 mg, about 1.35 mg, about 1.40 mg, about 1.45 mg, about 1.50 mg, about 1.60 mg, about 1.70 mg, about 1.80 mg, about 1.90 mg, about 1.95 mg, approx. 1.50 mg, approx. 1.55 mg, approx. 1.60 mg, approx. 1.65 mg, approx. 1.70 mg, approx. 1.75 mg, approx. 1.80 mg, approx. 1.85 mg, approx. 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 3.00m g, approx. 3.05 mg, approx. 3.10 mg, approx. 3.15 mg, approx. 3.20 mg, approx. 3.25 mg, approx. 3.30 mg, approx. 3.35 mg, approx. 3.40 mg, approx. .80mg, about 3.85mg, about 3.90mg, about 3.95mg, about 4.00mg, about 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 4.60 mg, about 4.65 mg, about 4.70 mg, about 4.75 mg, about 4.80 mg, about 4.85 mg, about 4.90 mg, about 4.95 mg, about 5.00 mg, about 5.05 mg, about 5.10 mg, about 5.15 mg, about 5.20 mg, about 5.25 mg, about 5.30 mg, about 5.35 mg, about 5.40 mg, about 5.45 mg, about 5.50 mg, about 5.55 mg, about 5.60 mg, about 5.65 mg, about 5.70 mg, about 5.75 mg, about 5.80 mg, about 5.85 mg, about 5.90 mg, about 5.95 mg, or about 6.00 mg.

[0174] In one embodiment, the collagenase has a reactivity of about 2.6 minutes as measured using an SRC assay. -1 ~5.2 minutes -1 V max In another embodiment, the collagenase may have a cleavage time of about 3.0 minutes as measured using an SRC assay. -1 ~5.0 minutes -1 V max In another embodiment, the collagenase may have a cleavage time of about 3.4 minutes as measured using an SRC assay. -1 ~4.8 minutes -1 V max In yet another embodiment, the collagenase may have a cleavage time of about 3.5 minutes as measured using an SRC assay. -1 ~4.5 minutes -1 V max In yet another embodiment, the collagenase may have a V max But about 2.0 minutes -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 , approx. 2.8 minutes -1 , about 2.9 minutes -1 , approx. 3.0 min -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 , approx. 3.7 minutes -1 , approx. 3.8 minutes -1 , about 3.9 minutes -1 , approx. 4.0 min -1 , approx. 4.1 minutes -1 , about 4.2 minutes -1 , approx. 4.3 minutes -1 , approx. 4.4 minutes -1 , about 4.5 minutes -1 , about 4.6 minutes -1 , approx. 4.7 minutes -1, approx. 4.8 minutes -1 , approx. 4.9 minutes -1 , approx. 5.0 min -1 , approx. 5.1 min -1 , approx. 5.2 minutes -1 , approx. 5.3 minutes -1 , approx. 5.4 minutes -1 , about 5.5 minutes -1 , about 5.6 minutes -1 , approx. 5.7 minutes -1 , approx. 5.8 minutes -1 , about 5.9 minutes -1 , or about 6.0 minutes -1 In another embodiment, the collagenase may have a reactivity of about 0.7 min as measured using an SRC assay. -1 ~7.6 minutes -1 , or about 1 to 6, or about 2 to 5, or about 3 to 4 minutes -1 V max may have the following structure:

[0175] In one embodiment, the collagenase has a hydrolysis time of about 135 minutes as measured using a GPA assay. -1 ~268 minutes -1 V max In another embodiment, the collagenase may have a cleavage time of about 150 minutes as measured using a GPA assay. -1 ~250 minutes -1 V max In another embodiment, the collagenase may have a cleavage time of about 175 minutes as measured using a GPA assay. -1 ~225 minutes -1 V max In yet another embodiment, the collagenase may have a cleavage time of about 130 minutes as measured using a GPA assay. -1 , approx. 135 min. -1 , approx. 140 min. -1 , approx. 145 min. -1 , approx. 150 min. -1 , approx. 155 min. -1 , approx. 160 min. -1 , approx. 165 min. -1 , approx. 170 min. -1 , approx. 175 min. -1 , approx. 180 min. -1 , approx. 185 min. -1 , approx. 190 min.-1 , approx. 195 min. -1 , about 200 minutes -1 , about 205 minutes -1 , approx. 210 min. -1 , approx. 215 min. -1 , approx. 220 min. -1 , approx. 225 min. -1 , approx. 230 min. -1 , approx. 235 min. -1 , approx. 240 min. -1 , approx. 245 min. -1 , approx. 250 min. -1 , approx. 255 minutes -1 , approx. 260 minutes -1 , approx. 265 min. -1 , approx. 270 min. -1 , approx. 275 min. -1 , or about 280 minutes -1 V max In another embodiment, the collagenase may have a cleavage time of about 4 minutes as measured using a GPA assay. -1 ~400 minutes -1 , or 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 minutes -1 V max

[0043]

[0176] In one embodiment, the collagenase has a K of about 75 mM to 147 mM as measured using the SRC assay. m In another embodiment, the collagenase may have a K of about 80 mM to 140 mM as measured using an SRC assay. m In another embodiment, the collagenase may have a K of about 85 mM to 130 mM as measured using the SRC assay. m In another embodiment, the collagenase may have a K of about 90 mM to 120 mM as measured using the SRC assay. mIn yet another embodiment, the collagenase may have a concentration of about 70 mM, about 72 mM, about 75 mM, about 77 mM, about 80 mM, about 82 mM, about 85 mM, about 87 mM, about 90 mM, about 92 mM, about 95 mM, about 97 mM, about 100 mM, about 102 mM, about 105 mM, about 107 mM, about 1 K of about 10 mM, about 112 mM, about 115 mM, about 117 mM, about 120 mM, about 122 mM, about 125 mM, about 127 mM, about 130 mM, about 132 mM, about 135 mM, about 137 mM, about 140 mM, about 142 mM, about 145 mM, about 147 mM, about 150 mM, about 152 mM, about 155 mM, or about 157 mM m In another embodiment, the collagenase may have a K of about 4.4 mM to 437 mM, or 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 as measured using the SRC assay. m

[0043]

[0177] In one embodiment, the collagenase has a K of about 0.03 mM to 3.1 mM as measured using a GPA assay. m In another embodiment, the collagenase may have a K of about 1.00 mM to 1.60 mM as measured using a GPA assay. m In another embodiment, the collagenase may have a K of about 1.10 mM to 1.50 mM as measured using a GPA assay. m In another embodiment, the collagenase may have a K of about 1.15 mM to 1.40 mM as measured using a GPA assay. mIn yet another embodiment, the collagenase may have a concentration of about 0.80 mM, about 0.82 mM, about 0.85 mM, about 0.87 mM, about 0.90 mM, about 0.92 mM, about 0.95 mM, about 0.97 mM, about 1.00 mM, about 1.02 mM, about 1.05 mM, about 1.07 mM, about 1.10 mM, about 1.12 mM, about 1.15 mM, about 1.17 mM, , about 1.20 mM, about 1.22 mM, 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 In another embodiment, the collagenase may have a K of about 0.027 mM to 2.7 mM, or about 0.1 to 2, or about 0.5 to 1.5, or about 1 to 1.35 mM, as measured using a GPA assay. m

[0043]

[0178] In one embodiment, collagenase has a cleavage time of about 36 seconds as measured using an SRC assay. -1 ~671 seconds -1 K cat In another embodiment, the collagenase may have a pH of about 50 seconds as measured using an SRC assay. -1 ~600 seconds -1 K cat In another embodiment, the collagenase may have a pH of about 60 seconds as measured using an SRC assay. -1 ~500 seconds -1 K cat In another embodiment, the collagenase may have a pH of about 70 seconds as measured using an SRC assay. -1 ~400 seconds -1 K cat In yet another embodiment, the collagenase may have a cleavage time of about 100 seconds as measured using an SRC assay. -1 ~350 seconds -1 K catIn another embodiment, the collagenase may have a pH of about 30 seconds as measured using an SRC assay. -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 K cat In another embodiment, the collagenase may have a cleavage time of about 1 s as measured using an SRC assay. -1 ~107 seconds -1 , or about 10 to 100, or about 20 to 80, or about 30 to 70, or about 40 to 60 seconds -1 K cat may have the following structure:

[0179] In one embodiment, the collagenase has a cleavage time of about 90 to 10,000, or about 41,000 seconds as measured using a GPA assay. -1 ~about 81,000 seconds -1 K cat In another embodiment, the collagenase may have a cleavage time of about 45,000 sec as measured using a GPA assay. -1 ~about 75,000 seconds -1 K cat In another embodiment, the collagenase may have a protein activity of about 50,000 s as measured using a GPA assay. -1 ~about 70,000 seconds -1 K catIn another embodiment, the collagenase may have a protein synthesis time of about 55,000 s as measured using a GPA assay. -1 ~about 65,000 seconds -1 K cat In yet another embodiment, the collagenase may have a cleavage time of about 35,000 sec as measured using a GPA assay. -1 , about 37,500 seconds -1 , about 40,000 seconds -1 , about 42,500 seconds -1 , about 45,000 seconds -1 , about 47,500 seconds -1 , about 50,000 seconds -1 , about 52,500 seconds -1 , about 55,000 seconds -1 , about 57,500 seconds -1 , about 60,000 seconds -1 , about 62,500 seconds -1 , about 65,000 seconds -1 , about 67,500 seconds -1 , about 70,000 seconds -1 , about 72,500 seconds -1 , about 75,000 seconds -1 , about 77,500 seconds -1 , about 80,000 seconds -1 , about 82,500 seconds -1 , or about 85,000 seconds -1 K cat In another embodiment, the collagenase may have a cleavage time of about 1215 seconds as measured using a GPA assay. -1 ~about 120,000 seconds -1 , or about 2,000 to 100,000, or about 10,000 to 90,000, or about 20,000 to 80,000, or about 30,000 to 70,000, or about 40,000 to 60,000 seconds -1 K cat may have the following structure:

[0180] In one embodiment, the collagenase has a 1 / K of about 376 to 38,000 μsec, or about 14,000 to about 28,000 μsec, as measured using an SRC assay. catIn another embodiment, the collagenase may have a 1 / K of about 16,000 μsec to about 26,000 μsec as measured using an SRC assay. cat In one embodiment, the collagenase may have a 1 / K of about 18,000 μsec to about 24,000 μsec as measured using an SRC assay. cat In one embodiment, the collagenase may have a 1 / K of about 20,000 μsec to about 22,000 μsec as measured using an SRC assay. cat In yet another embodiment, the collagenase may have a kinetic energy of about 12,500 μsec, about 12,750 μsec, about 13,000 μsec, about 13,250 μsec, about 13,500 μsec, about 13,750 μsec, about 14,000 μsec, about 14,250 μsec, about 14,750 μsec, about 15,000 μsec, about 15,250 μsec, about 15,500 μsec, about 15,750 μsec, about 16,000 μsec, Approx. 16,250μs, Approx. 16,500μs, Approx. 16,750μs, Approx. 17,000μs, Approx. 17,250μs, Approx. 17,500μs, Approx. 17,750μs, Approx. 18,000μs, Approx. 18,250μs , approximately 18,500μs, approximately 18,750μs, approximately 19,000μs, approximately 19,250μs, approximately 19,500μs, approximately 19,750μs, approximately 20,000μs, approximately 20,250μs, approximately 20,500μs , approximately 20,750μs, approximately 21,000μs, approximately 21,250μs, approximately 21,500μs, approximately 21,750μs, approximately 22,000μs, approximately 22,250μs, approximately 22,500μs, approximately 22,750μs seconds, approx. 23,000 μs, approx. 23,250 μs, approx. 23,500 μs, approx. 23,750 μs, approx. 24,000 μs, approx. 24,250 μs, approx. 24,500 μs, approx. 24,750 μs, approx. μ seconds, approx. 25,250 μ seconds, approx. 25,500 μ seconds, approx. 25,750 μ seconds, approx. 26,000 μ seconds, approx. 26,250 μ seconds, approx. 26,500 μ seconds, approx. 26,750 μ seconds, approx. 27,000 μ seconds, approx. 1 / K of 0μs, approximately 27,500μs, approximately 27,750μs, approximately 28,000μs, approximately 28,250μs, approximately 28,500μs, approximately 28,750μs, approximately 29,000μs, or approximately 29,250μs catIn another embodiment, the collagenase may have a 1 / K of about 370 μsec to about 36,700 μsec, or 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, as measured using an SRC assay. cat may have the following structure:

[0181] In one embodiment, the collagenase has a 1 / K of about 4 μsec to about 430 μsec as measured using a GPA assay. cat In another embodiment, the collagenase may have a 1 / K of about 14 μsec to about 23 μsec as measured using a GPA assay. cat In another embodiment, the collagenase may have a 1 / K of about 16 μsec to about 21 μsec as measured using a GPA assay. cat In yet another embodiment, the collagenase may have, as measured using a GPA assay, 、Approximately 10.0μ seconds, approximately 10.2 μ seconds, approximately 10.4 μ seconds, approximately 10.6 μ seconds, approximately 10.8 μ seconds, approximately 11.0 μ seconds, approximately 11.2 μ seconds, approximately 11.4 μ seconds, approximately 11.6 μ seconds, approximately 11.8 μ seconds, approximately 12.0 μ seconds , approximately 12.2 μs, approximately 12.4 μs, approximately 12.6 μs, approximately 12.8 μs, approximately 13.0 μs, approximately 13.2 μs, approximately 13.4 μs, approximately 13.6 μs, approximately 13.8 μs, approximately 14.0 μs, approximately 14.2 μs , approximately 14.4 μs, approximately 14.6 μs, approximately 14.8 μs, approximately 15.0 μs, approximately 15.2 μs, approximately 15.4 μs, approximately 15.6 μs, approximately 15.8 μs, approximately 16.0 μs, approximately 16.2 μs, approximately 16.4 μs , approximately 16.6 μs, approximately 16.8 μs, approximately 17.0 μs, approximately 17.2 μs, approximately 17.4 μs, approximately 17.6 μs, approximately 17.8 μs, approximately 18.0 μs, approximately 18.2 μs, approximately 18.4 μs, approximately 18.6 μs , about 18.8μ seconds, about 19.0μ seconds, about 19.2 μ seconds, about 19.4 μ seconds, about 19.6 μ seconds, about 19.8 μ seconds, about 20.0 μ seconds, about 20.2 μ seconds, about 20.4 μ seconds, about 20.6 μ seconds, about 20.8 μ seconds seconds, approx. 21.0μs, approx. 21.2μs, approx. 21.4μs, approx. 21.6μs, approx. 21.8μs, approx. 22.0μs, approx. 22.2μs, approx. 22.4μs, approx. 22.6μs, approx. seconds, approx. 23.2μs, approx. 23.4μs, approx. 23.6μs, approx. 23.8μs, approx. 24.0μs, approx. 24.2μs, approx. 24.4μs, approx. 24.6μs, approx. 24.8μs, approx. 1 / K of approximately 25.4μs, approximately 25.6μs, approximately 25.8μs, approximately 26.0μs, approximately 26.2μs, approximately 26.4μs, approximately 26.8μs, approximately 27.0μs, approximately 27.2μs, or approximately 27.4μs cat In another embodiment, the collagenase may have a 1 / K of about 0.3 μsec to about 32 μsec, or about 1 to 30, or about 5 to 25, or about 10 to 20, or about 15 μsec, as measured using a GPA assay. cat may have the following structure:

[0182] In one embodiment, the collagenase has a concentration of about 5,140 mM as measured using the SRC assay. -1 seconds -1 ~ approx. 508,814 mM -1 seconds -1 K cat / Km In another embodiment, the collagenase may have a concentration of about 0.50 mM as measured using the SRC assay. -1 seconds -1 ~about 7.75mM -1 seconds -1 K cat / K m In another embodiment, the collagenase may have a concentration of about 0.75 mM as measured using the SRC assay. -1 seconds -1 ~ approx. 7.00 mM -1 seconds -1 K cat / K m In yet another embodiment, the collagenase may have a concentration of about 1.00 mM as measured using the SRC assay. -1 seconds -1 ~ approx. 6.00 mM -1 seconds -1 K cat / K m In yet another embodiment, the collagenase may have a concentration of about 0.10 mM as measured using the SRC assay. -1 seconds -1 , about 0.20mM -1 seconds -1 , about 0.30mM -1 seconds -1 , about 0.40 mM -1 seconds -1 , about 0.50 mM -1 seconds -1 , about 0.60mM -1 seconds -1 , about 0.70mM -1 seconds -1 , about 0.80mM -1 seconds -1 , about 0.90mM -1 seconds -1 , about 1.00 mM -1 seconds -1 , about 1.10mM -1 seconds -1 , about 1.20mM -1 seconds -1 , about 1.30mM -1 seconds -1 , about 1.40 mM -1 seconds -1 , about 1.50 mM -1 seconds-1 , about 1.60 mM -1 seconds -1 , about 1.70 mM -1 seconds -1 , about 1.80 mM -1 seconds -1 , about 1.90 mM -1 seconds -1 , about 2.00 mM -1 seconds -1 , about 2.10 mM -1 seconds -1 , about 2.20 mM -1 seconds -1 , about 2.30 mM -1 seconds -1 , about 2.40 mM -1 seconds -1 , about 2.50 mM -1 seconds -1 , about 2.60 mM -1 seconds -1 , about 2.70 mM -1 seconds -1 , about 2.80 mM -1 seconds -1 , about 2.90 mM -1 seconds -1 , about 3.00 mM -1 seconds -1 , about 3.10 mM -1 seconds -1 , about 3.20 mM -1 seconds -1 , about 3.30 mM -1 seconds -1 , about 3.40 mM -1 seconds -1 , about 3.50 mM -1 seconds -1 , about 3.60 mM -1 seconds -1 , about 3.70 mM -1 seconds -1 , about 3.80 mM -1 seconds -1 , about 3.90 mM -1 seconds -1 , about 4.00 mM -1 seconds -1 , about 4.10 mM -1 seconds -1 , about 4.20 mM -1 seconds -1 , about 4.30 mM -1 seconds -1 , about 4.40 mM-1 second -1 、about 4.50 mM -1 second -1 、about 4.60 mM -1 second -1 、about 4.70 mM -1 second -1 、about 4.80 mM -1 second -1 、about 4.90 mM -1 second -1 、about 5.00 mM -1 second -1 、about 5.10 mM -1 second -1 、about 5.20 mM -1 second -1 、about 5.30 mM -1 second -1 、about 5.40 mM -1 second -1 、about 5.50 mM -1 second -1 、about 5.60 mM -1 second -1 、about 5.70 mM -1 second -1 、about 5.80 mM -1 second -1 、about 5.90 mM -1 second -1 、about 6.00 mM -1 second -1 、about 6.10 mM -1 second -1 、about 6.20 mM -1 second -1 、about 6.30 mM -1 second -1 、about 6.40 mM -1 second -1 、about 6.50 mM -1 second -1 、about 6.60 mM -1 sec -1 、about 6.70 mM -1 second -1 、about 6.80 mM -1 second -1 、about 6.90 mM -1 second -1 、about 7.00 mM -1 second -1 、about 7.10 mM -1 second -1 、about 7.20 mM -1 second-1 , about 7.30mM -1 seconds -1 , or about 7.40 mM -1 seconds -1 K cat / K m In another embodiment, the collagenase may have a concentration of about 0.0048 mM as measured using the SRC assay. -1 seconds -1 ~about 0.47mM -1 seconds -1 , or about 0.009 to about 0.3, or about 0.01 to about 0.25, or about 0.1 to 0.25 mM -1 seconds -1 K cat / K m may have the following structure:

[0183] In one embodiment, the collagenase is about 60 mM as measured using the GPA assay. -1 seconds -1 ~ approx. 6,000 mM -1 seconds -1 K cat / K m In another embodiment, the collagenase may have a concentration of about 30,000 mM as measured using a GPA assay. -1 seconds -1 ~ approx. 85,000 mM -1 seconds -1 K cat / K m In another embodiment, the collagenase may have a concentration of about 36,000 mM as measured using a GPA assay. -1 seconds -1 ~ approx. 77,000 mM -1 seconds -1 K cat / K m In yet another embodiment, the collagenase may have a concentration of about 40,000 mM as measured using a GPA assay. -1 seconds -1 ~ approx. 70,000 mM -1 seconds -1 K cat / K mIn yet another embodiment, the collagenase may have a concentration of about 40,000 mM as measured using a GPA assay. -1 seconds -1 , about 42,000 mM -1 seconds -1 , about 44,000 mM -1 seconds -1 , about 46,000mM -1 seconds -1 , about 48,000 mM -1 seconds -1 , about 50,000 mM -1 seconds -1 , about 52,000 mM -1 seconds -1 , about 54,000 mM -1 seconds -1 , about 56,000 mM -1 seconds -1 , about 58,000 mM -1 seconds -1 , about 60,000 mM -1 seconds -1 , about 62,000 mM -1 seconds -1 , about 64,000 mM -1 seconds -1 , about 66,000 mM -1 seconds -1 , about 68,000 mM -1 seconds -1 , about 70,000 mM -1 seconds -1 , about 72,000 mM -1 seconds -1 , about 74,000 mM -1 seconds -1 , about 76,000 mM -1 seconds -1 , about 78,000 mM -1 seconds -1 , about 80,000 mM -1 seconds -1 , about 82,000mM -1 seconds -1 , about 84,000mM -1 seconds -1 , about 76,000 mM -1 seconds -1 , about 86,000mM -1 seconds -1 , about 88,000 mM -1 seconds-1 , about 90,000 mM -1 seconds -1 , about 92,000 mM -1 seconds -1 , about 94,000 mM -1 seconds -1 , or about 96,000 mM -1 seconds -1 K cat / K m In another embodiment, the collagenase may have a concentration of about 900 mM as measured using a GPA assay. -1 seconds -1 ~ approx. 90,000 mM -1 seconds -1 , or about 2,000 to 80,000, or about 10,000 to 70,000, or about 20,000 to 60,000, or about 30,000 to 50,000, or about 40,000 to 45,000 mM -1 seconds -1 K cat / K m may have the following structure:

[0184] In one embodiment, the collagenase may have a molecular weight of about 60 kDa to about 130 kDa. In another embodiment, the collagenase may have a molecular weight of about 70 kDa to about 130 kDa. In another embodiment, the collagenase may have a molecular weight of about 80 kDa to about 120 kDa. In yet another embodiment, the collagenase may have a molecular weight of about 90 kDa to about 120 kDa. In another embodiment, the collagenase may have a molecular weight of about 100 kDa to about 110 kDa. In yet another embodiment, the collagenase is 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 a, 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.

[0185] In one embodiment, the collagenase may have a purity of at least 80% as measured by reverse phase HPLC. In another embodiment, the collagenase may have a purity of 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% as measured by reverse phase HPLC. In yet another embodiment, the collagenase may comprise 1% or less clostripain by area. In another embodiment, the collagenase may comprise 1% or less gelatinase by area. In another embodiment, the collagenase may comprise 1% or less leupeptin by area. In yet another embodiment, the collagenase may comprise a bioburden of 1 cfu / mL or less.

[0186] In one embodiment, the collagenase may have a potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg. In another embodiment, the collagenase may have a potency of about 2,500 to about 25,000 SRC units / mg. In another embodiment, the collagenase may have a potency of about 5,000 to about 20,000 SRC units / mg. In yet another embodiment, 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 In some embodiments, the antibody may have a potency of about 1,000, 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.

[0187] In one embodiment, the collagenase may have a potency (i.e., specific activity) of about 5,000 to about 30,000 f-SRC units / mg. In another embodiment, the collagenase may have a potency of about 7,500 to about 25,000 f-SRC units / mg. In another embodiment, the collagenase may have a potency of about 10,000 to about 20,000 f-SRC units / mg. In yet another embodiment, 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, about 15,500, about 16,000, about 16,500, about The antibody may have a titer of 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.

[0188] In one embodiment, the collagenase may have a potency of about 100,000 to about 400,000 GPA units / mg. In another embodiment, the collagenase may have a potency of about 150,000 to about 350,000 GPA units / mg. In another embodiment, the collagenase may have a potency of about 200,000 to about 300,000 GPA units / mg. In yet another embodiment, the collagenase is at 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 250,000, It may have a potency of 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.

[0189] In one embodiment, the collagenase may have a potency of about 175,000 to about 500,000 f-GPA units / mg. In another embodiment, the collagenase may have a potency of about 250,000 to about 450,000 f-GPA units / mg. In another embodiment, the collagenase may have a potency of about 300,000 to about 400,000 GPA units / mg. In yet another embodiment, the collagenase is at 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 335,0 In some embodiments, the antibody may have a titer of about 300, 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.

[0190] In one embodiment, the collagenase may have a titer of about 5,000 to about 25,000 ABC units / mg. In one embodiment, the collagenase may have a titer of about 7,500 to about 20,000 ABC units / mg. In one embodiment, the collagenase may have a titer of about 10,000 to about 17,500 ABC units / mg. In another embodiment, 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 In some embodiments, the antibody may have a potency of about 1,500, 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.

[0191] In some embodiments, the collagenase present in the composition comprises collagenase I and collagenase II in a ratio of about 1:1. Other ratios of collagenase I and collagenase II may be employed 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, or 0:1. Each of collagenase I and collagenase II may have a purity of 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.

[0192] In another embodiment, the collagenase composition has a CCH with a ratio of AUXI and AUXII of about 1: 1. Other ratios of AUXI and AUXII may be employed 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, or 0:1. Each of AUXI and AUXII may have a purity of 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.

[0193] In other examples, the collagenase composition may be liquid or may be reconstituted with a diluent from a lyophilized solid form. The dosage of the mixture is measured by the amount of collagenase present without considering the diluent and may comprise from about 0.1 mg to about 20 mg in one or more injections. In another embodiment, the dose administered 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 one or more injections.

[0194] For example, about 0.06 mg, 0.48 mg, 0.84 mg, or 1.68 mg are injected in about 12 separate injections. The amount of collagenase composition injected may range from 0.01 mL to 3 mL per injection, or from about 0.2 mL to 150 mL total per treatment visit. In certain embodiments, the above dosages are for a collagenase composition having a CCH. In other embodiments, the above dosages are for a collagenase composition having one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0195] In another embodiment, about 0.84 mg of CCH is injected equally into each treatment area in about 12 separate injections (about 0.07 mg x 12 injections = about 0.84 mg). In some cases, such treatment with 0.84 mg is given every 10-40 days in 2, 3, 4, or 5 treatment visits. In other cases, multiple treatment areas are injected with 0.84 mg in one treatment visit or every 10-40 days in 2, 3, 4, or 5 treatment visits. In other embodiments, there are more than 5 treatment visits.

[0196] In another embodiment, the amount of collagenase that can be injected into the treatment area is from about 0.001 mg to 20 mg of collagenase per treatment visit in one or more injections, which can be administered, for example, in equal portions from about 3 to about 100 injections. The collagenase can be in liquid form or reconstituted with a diluent from a lyophilized solid. The dosage of collagenase is measured in the amount of collagenase without diluent and may have about 0.1 mg to 1 mg, 0.25 mg to 0.75 mg, 0.1 mg to 2 mg, 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 dosage 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, 3.75 mg, 4.0 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5.0 mg, 5.25 mg, 5.5 mg, 5.75 mg, 6 mg, 6.25 mg, 6.5 mg, 6.75 mg, 7 mg, 7.25 mg, 7.5 mg, 7.75 mg, 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 given in single or divided injections.

[0197] In another embodiment, the dosage 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 administered in one or more injections. In another example, 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 is injected into the treatment area in about 12 separate injections. In another example, the collagenase dosage is injected in three or more separate injections. The amount of collagenase composition injected can range from 0.01 mL to 3 mL per injection, or from about 1 mL to 150 mL total per treatment visit.

[0198] In one embodiment, the mixture of AUX I and II described above ("CCH") may be injected in one or more injections of about 0.01 mg to 10 mg of collagenase per treatment visit, e.g., a dose divided equally into about 3 to about 50 injections. The collagenase may be liquid or reconstituted with a diluent from a lyophilized form. The dosage of the mixture is measured in terms of the amount of collagenase, without taking into account the diluent, and is 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 0.1 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, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 10 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 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 administered in one or more injections. In another embodiment, the dose of CCH administered is about 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 in one or more injections. For example, about 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 in 12 injections. The amount of collagenase composition injected can range from 0.01 mL to 3 mL per injection, or from about 1 mL to 80 mL total per treatment visit.

[0199] The dosage of collagenase may also be expressed in mg per injection (again without considering diluent), such as about 0.001 mg to 0.5 mg, about 0.01 mg to about 5 mg, about 0.005 mg to about 0.1 mg, or about 0.005 mg, 0.04 mg, or 0.07 mg per injection.

[0200] In certain aspects, the disclosure 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 another embodiment, 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 150 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 about 1,250 ABC Units, or about 500 ABC Units to about 1,000 ABC Units.

[0201] In certain embodiments, dosages based on various specific activities are as follows: TIFF0007628492000030.tif139164

[0202] In certain embodiments, the present disclosure 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, or about 15,817 BTC units.

[0203] 5. Preparation The CCH or other collagenase may be in the form of a pharmaceutical formulation comprising the CCH or collagenase and pharma- ceutically acceptable excipients, such as sterile water for injection, sodium chloride / calcium chloride, pH adjusters, stabilizers, and the like.

[0204] XIAFLEX® is commercially supplied by the applicant in single-use glass vials containing 0.9 mg of CCH as a sterile lyophilized powder for reconstitution. Sterile diluent for reconstitution is also supplied in a single-use glass vial. Inactive ingredients include hydrochloric acid, sucrose, and tromethamine. Diluent contains calcium chloride dihydrate in 0.9% sodium chloride. XIAFLEX® Prescribing Information (2018).

[0205] In another embodiment, CCH for Cellulite is a sterile lyophilized powder containing 0.92 mg of CCH, sucrose, Tris, mannitol, and hydrochloric acid in a 5 mL vial. Sterile diluents for reconstitution can consist of water for injection, normal saline, or 0.6% sodium chloride and 0.03% calcium chloride dehydrate in water for injection, filled into individual 5 mL vials.

[0206] Collagenase or CCH may be filled into other vial sizes, such as 10 mL, 15 mL, 20 mL, or 30 mL. Other pH adjusters, sugars, polyols, and stabilizers are described in Rowe et al. Handbook of Pharmaceutical Excipients ( 5th Ed).

[0207] 6. Method of treatment: injection technique and dosing regimen The aforementioned collagenase compositions are useful in methods of treating or reducing the severity of cellulite in human subjects. The present disclosure relates to a method of reducing the severity of cellulite in a human patient, comprising providing a composition having at least one collagenase and injecting a therapeutically effective amount of the composition into one or more cavities, wherein the patient exhibits a reduction in the severity of cellulite compared to pre-treatment baseline levels. As described in more detail below, the compositions can be administered by a variety of injection techniques, and efficacy can be measured by a number of scales and other measurement tools.

[0208] Administration of the collagenase compositions described herein may be performed bilaterally (two thighs or two buttocks) or in all four quadrants (both buttocks and both thighs) in a single subject at a treatment visit. Such treatment visits may occur every 10-40 days for two, three, four, five or six treatment visits over a one-year period. Additionally, even when collagenase was injected into all four quadrants at high cumulative doses, bioassays detecting AUX-I or AUX-II concentrations in plasma following subcutaneous administration of CCH up to 3.36 mg demonstrated no quantifiable levels of CCH in the systemic circulation (i.e., below the assay lower limiting dose level), indicating no systemic absorption and further demonstrating overall safety. For example, humans were administered 3.36 mg and rats were administered 43 times the human equivalent dose (HED), and these amounts were found to be well tolerated. Applicants' work on the pharmacokinetics of collagenase therapy is described in U.S. Provisional Application No. 62 / 733,046, filed September 18, 2018, U.S. Provisional Application No. 62 / 788,916, filed January 6, 2019, U.S. Provisional Application No. 62 / 812,036, filed February 28, 2019, U.S. Provisional Application No. 62 / 812,036, filed March 25, 2019, International Application No. PCT / US2019 / 041494, filed July 11, 2019, and International Application No. PCT / US2019 / 41718, filed July 12, 2019, which are incorporated herein by reference.

[0209] A general overview of the various collagenase injection parameters and the techniques involved in treating patients is provided in Table 15. [Table 15] TIFF0007628492000032.tif234155TIFF0007628492000033.tif41150

[0210] In certain embodiments, about 0.84 mg of CCH is injected into affected areas, such as the four quadrants (i.e., right or left buttocks or right or left thighs) in about 12 equal injections (about 0.07 mg x 12 injections = about 0.84 mg of CCH). In some cases, such treatments of 0.84 mg are administered every 10-40 days for 2, 3, 4, 5, or 6 treatments. In other cases, multiple affected areas or quadrants are injected with 0.84 mg every 10-40 days for 2, 3, 4, 5, or 6 treatments.

[0211] In one embodiment, a surgical marker is used to circle each of the depressions selected for treatment. In another embodiment, the circles of the selected treatment areas do not overlap. In yet another embodiment, the circles of the selected treatment areas overlap.

[0212] In certain embodiments, patients are administered collagenase as shown in Table 16 using a protocol according to Treatment I (FIG. 7). [Table 16]

[0213] In this example, the clinician may select a dimple in each buttock that is evident when the subject is standing and suitable for treatment. There is no restriction on the selection of dimples to be treated. Treatment consists of 12 injections per buttock (24 total injections across both buttocks) per treatment visit. Because the goal of treatment is to improve the overall aesthetic appearance of the buttocks, the clinician is instructed to select the dimple that they believe will best improve the overall aesthetic appearance of the buttocks. The same dimples may be treated in one buttock or a different dimple from the one previously treated may be treated, but injections within the buttock are preferred for all three treatments (12 injections per buttock). Each buttock will receive all three treatments unless the buttock does not have a treatable EFP dimple and the clinician rates the buttock as a 0 on the CR-PCSS. If no injection was administered to a particular buttock (right or left) at Treatment Visit 2, the subject will still be evaluated for treatment of the contralateral buttock at Treatment Visit 2 and will return at Treatment Visit 3 to have each buttock assessed again by the subject (PR PCSS) and the investigator (CR-PCSS). If the investigator rates either or both buttocks greater than 0 on the CR-PCSS, an injection will be administered at Treatment Visit 3. Additionally, collagenase treatment may include one or more of the following: ● Treat one or more quadrants; Treat any size of depression; • Treating women over 45 years of age; ● Treating depressions where the skin is loose, lax or sagging; • Treating different recesses at different treatment visits; ● Use needles 1 / 2 inch or larger; • Do not limit the distance of the cavity to be injected; • Do not rely on spacers, rulers, paper, or other devices to restrict injection location; • Ensure that at least one injection is given just below the recess; • Treating depressions less than about 1 cm or more than about 2 cm in length; • Use injections within about 2 cm of each other; Use injections within less than about 2 cm of each other, and / or • Measure the effectiveness using one or more of the scales and methods described herein. In certain embodiments, when the treatment adheres to the above aspects, patients experience rapid response rates to the treatment. See Figures 20-23.

[0214] Additionally, in certain specific embodiments, parameters of treated patients are provided in Tables 17 and 18. [Table 17] [Table 18]

[0215] In one embodiment, the osmolality of the reconstituted 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 yet another embodiment, the osmolality of the reconstituted product is about 512 mOsm / kg, about 275 mOsm / kg, about 281 mOsm / kg, or about 227 mOsm / kg.

[0216] In addition to the above methods, the present disclosure provides a method of treating or reducing EFP in a subject in need thereof, which provides at least one of the following advantages over common procedures and treatments for EFP: a.Easy for physicians to use; b. Reducing time to treatment; c) The results are unexpected, even in light of the general opinion of doctors that cosmetic improvement is difficult; d. no need to use hyaluronidase; e. No need to apply heat; f.No need to use lasers; g.No subcision; h. No need for anesthesia (despite bruising); i.No need for compression garments; j. Do not use a vacuum.

[0217] In another embodiment, the methods of treating or reducing cellulite do not impose an upper limit on the severity of cellulite treated, e.g., treatment with collagenase is safe and effective regardless of the prevalence or severity of cellulite.

[0218] F. Phase 4 - Treatment endpoints and efficacy measurements The treatment methods described herein are effective in treating cellulite by several measures, as described below. Generally, as used herein, the term "day" refers to the study day measured sequentially from the first treatment in a treatment course to the first day of collagenase treatment in a particular treatment area, except as otherwise specified in Example 5 below, where days are measured sequentially from day 71 of the prior study. For example, as described in Example 5, "day 180" refers to 180 days after day 71 as reported in Examples 2 and 3. Thus, outside the context of Example 5, day 1 is the first day of treatment, day 71 is 70 days after day 1, and day 180 is 179 days after day 1 (unless "day 180" is used to mean 180 days after day 71 or 251 days after the first day of treatment, as in Example 5).

[0219] In certain embodiments, in a population of patients all with moderate or severe CR-PCSS and / or PR-PCSS ratings: At least 50% of patients achieve at least one level improvement from baseline in CR-PCSS severity at Day 22, Day 43, or Day 71 as assessed live by a clinician in the therapeutic area of ​​interest. At least 50% of patients achieve at least one level improvement from baseline in PR-PCSS severity at Day 22, Day 43, or Day 71, as assessed by subjects while viewing digital images of the targeted treatment area. At least 5% of patients achieve at least a 2-level improvement from baseline in CR-PCSS severity at Day 22, Day 43, or Day 71 as assessed live by a clinician in the therapeutic area of ​​interest. At least 5% of patients achieve at least a 2-level improvement from baseline in PR-PCSS severity at Day 22, Day 43, or Day 71, as assessed by subjects while viewing digital images of the targeted treatment area. ● At least 5% of patients experience a significant reduction in cavity size (volume, length, width, depth), e.g., at least a 5% reduction, or at least a 10% reduction, or at least a 20% reduction from baseline to day 22, day 43, or day 71. These and other efficacy parameters and benchmarks are detailed below. Moreover, as shown in the Examples below, collagenase injections significantly improved the appearance of cellulite, demonstrated durability, and were generally well tolerated.

[0220] 1. Efficacy measured by CR-PCSS and PR-PCSS Improvement in an individual patient means an improvement of at least one level or rating from baseline or a previous score or rating. Improvement in a patient population means an improvement in the mean score or rating of about 0.1 from baseline or a previous mean score or rating. A responder is any patient who shows at least a 25% improvement in the maximum total score or rating from baseline. In certain embodiments, the treatment methods detailed herein result in one or more of the following efficacy endpoints as measured by CR-PCSS and / or PR-PCSS: 1. CR-PCSS and / or PR-PCSS improvement of at least 0.1 compared to baseline. 2. Improvement of at least 2 levels on the CR-PCSS scale as assessed live by a therapeutic area clinician from baseline (pre-treatment "Day 1") to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Years 3, 4, or 5. 3. Improvement of at least 2 levels in PR-PCSS severity as assessed by the subject while viewing digital images of the treated area from baseline (Day 1) to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5. 4. Improvement as demonstrated by a 2-level composite response at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5 is defined as subjects achieving at least a 2-level improvement from baseline in CR-PCSS severity and at least a 2-level improvement from baseline in PR-PCSS severity. 5. Improvement of at least one level from baseline on the CR-PCSS scale as assessed live by a therapeutic area clinician at days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5. 6. Improvement in PR-PCSS severity, assessed by the subject while viewing digital images of the treated area, of at least one level from baseline to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5. 7. Improvement as demonstrated by a 1-level composite response is defined as subjects who experience at least one level of improvement from baseline on the CR-PCSS and at least one level of improvement from baseline on the PR-PCSS at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5. 8. In patients with moderate or severe CR-PCSS and / or PR-PCSS scores, improvement in at least one treatment domain is statistically significant compared to placebo, and the improvement is one or more of the above No. 1 to No. 7. 9. Results show that at least 5% of patients maintain their improvement level relative to pre-treatment baseline for at least 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such level for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, results show that at least 5% of patients show improvement relative to pre-treatment baseline, and the improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the improvement increases over time, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, 50% of patients showed improvement, with further improvement seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 10. At day 180, improvements in CR-PCSS assessments versus baseline were consistent across the left and right treatment regions. 11. In a patient population in which all patients have moderate or severe CR-PCSS and / or PR-PCSS scores, the median time to a first 2-level CR-PCSS and / or PR-PCSS improvement in at least one therapeutic area is approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 12. In a population of patients all with moderate or severe CR-PCSS and / or PR-PCSS ratings, the median time to an earliest 1-level CR-PCSS and / or PR-PCSS improvement in at least one therapeutic 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. 13. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS scores, subjects' mean CR-PCSS and / or PR-PCSS scores separated from placebo 21 days after the first treatment and showed sustained and significant improvement with subsequent treatments. 14. In a population of patients all with moderate or severe CR-PCSS and / or PR-PCSS ratings, the proportion of subjects having a 2-level composite response as measured by CR-PCSS and / or PR-PCSS in at least one therapeutic area at Day 71 is approximately 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 15. In a population of patients all with moderate or severe CR-PCSS and / or PR-PCSS ratings, the proportion of subjects having a 1-level composite response as measured by CR-PCSS and / or PR-PCSS in at least one therapeutic area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 16. In a population of patients who all have a moderate or severe CR-PCSS and / or PR-PCSS rating, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of patients have a CR-PCSS response of at least 1 level and / or a PR-PCSS response of at least 1 level in at least one treatment domain by Day 71 after treatment, and the CR-PCSS result is not dependent on age, BMI, or skin color. 17. Reduction in cellulite severity occurs rapidly within approximately 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit. 18. In the patient population with moderate or severe CR-PCSS and PR-PCSS scores, the greatest decline in CR-PCSS and PR-PCSS scores from baseline (screening visit) was first observed at day 90. 19. In a patient population with moderate or severe CR-PCSS and / or PR-PCSS scores, improvements (i.e., negative changes) in cellulite severity at predefined time points prior to Day 180 are maintained at the Day 180 visit. 20. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS assessments, at day 90, the mean (SD) changes from baseline in the CR-PCSS and / or PR-PCSS assessments of the left buttock and left thigh were approximately -0.8 (0.58) and approximately -0.6 (0.62), respectively, and the mean (SD) changes from baseline in the CR-PCSS and / or PR-PCSS assessments of the right buttock and right thigh were approximately -0.7 (0.73) and approximately -0.5 (0.70), respectively. 21. In patients with moderate or severe CR-PCSS and / or PR-PCSS scores, there is a consistent reduction from baseline (screening visit) in CR-PCSS and / or PR-PCSS scores on both sides at Day 180. 22.In patients with moderate or severe CR-PCSS and / or PR-PCSS scores, an improvement of 2 levels in at least one domain in CR-PCSS and / or PR-PCSS scores was observed in at least 10% of subjects at Day 90 and in at least 15% of subjects at Day 180. 22. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS scores, an improvement of 2 levels in the CR-PCSS and / or PR-PCSS scores in at least one area was observed in at least 10% of subjects at day 90 and at least 15% of subjects at day 180, with responses similar in the buttocks and thigh areas and in the left and right lateral sides. 23.In patient populations with moderate or severe CR-PCSS and / or PR-PCSS scores, an improvement of 1 level in CR-PCSS and / or PR-PCSS scores in at least one therapeutic domain was observed in at least 60% of subjects at Day 90 and at least 65% of subjects at Day 180. 24. In a patient population, all of whom have moderate or severe CR-PCSS and / or PR-PCSS scores, an improvement of 1 level in the CR-PCSS and / or PR-PCSS scores in at least one treatment area is observed in at least 60% of subjects at day 90 and at least 65% of subjects at day 180, with responses similar in the buttocks and thigh areas and on the left and right sides. 25. In a patient population in which all patients had moderate or severe CR-PCSS and / or PR-PCSS assessments, the median time to an earliest 2-level CR-PCSS and / or PR-PCSS response in at least one therapeutic area was observed to be approximately 80 days. 26. In a patient population in which all patients have moderate or severe CR-PCSS and / or PR-PCSS assessments, the median time to an earliest 1-level CR-PCSS and / or PR-PCSS response in at least one therapeutic area is approximately 40 days. 27. In a patient population in which all patients have moderate or severe CR-PCSS and / or PR-PCSS assessments, at least 50% of patients are PR-PCSS and / or PR-PCSS responders at 1 level in the target hip at Day 71. 28. In a patient population in which all patients have moderate or severe CR-PCSS and / or PR-PCSS assessments, at least 20% of patients are 2-level PR-PCSS and / or PR-PCSS responders in the target hip at day 71. 29. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS assessments, at least 35% of patients are 1-level CR-PCSS and / or PR-PCSS combined responders at the target hip at Day 71. 30. In a patient population in which all patients have moderate or severe CR-PCSS and / or PR-PCSS assessments, at least 5% of patients are 2-level CR-PCSS and / or PR-PCSS combined responders in the non-target hip at day 71. 31.In patient populations with moderate or severe CR-PCSS and / or PR-PCSS scores, the mean change from baseline in PR-PCSS and / or PR-PCSS to day 71 was greater in subjects receiving collagenase than in subjects receiving placebo in the target buttocks (approximately 0.9 vs. approximately 0.5, respectively) and non-target buttocks (approximately 0.9 vs. approximately 0.5, respectively). 32.In patient populations with moderate or severe CR-PCSS and / or PR-PCSS assessments, the proportion of 1-level PR-PCSS and / or PR-PCSS responders was greater in collagenase-treated subjects than in placebo-treated subjects in the target buttocks (approximately 62% vs. approximately 40%, respectively) and non-target buttocks (approximately 65% ​​vs. approximately 40%, respectively) at day 71. 33. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS scores, the mean changes from baseline in CR-PCSS and / or PR-PCSS scores in subjects receiving CCH were greater than those in subjects receiving placebo in the target buttocks (approximately 0.7 vs. approximately 0.4 [0.72], respectively) and non-target buttocks (approximately 0.8 vs. approximately 0.3, respectively) at Day 71. 34. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS assessments, the proportion of 1-level CR-PCSS and / or PR-PCSS responders was greater in collagenase-treated subjects than in placebo-treated subjects in the target buttocks (approximately 58% vs. approximately 32%, respectively) and non-target buttocks (approximately 60% vs. approximately 27%, respectively) at day 71. 35. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS assessments, the proportion of 1-level CR-PCSS and / or PR-PCSS combined responders was greater in collagenase-treated subjects than in placebo-treated subjects in the target buttocks (approximately 42% vs. approximately 20%, respectively) and non-target buttocks (approximately 44% vs. approximately 13%, respectively) at day 71. 36. In a population of patients all with moderate or severe CR-PCSS and / or PR-PCSS ratings, collagenase treatment as described herein results in a combined responder of at least 2 levels that is at least 5% higher than placebo, or at least 7.5% higher than placebo, or at least 10% higher than placebo, or at least 12.5% ​​higher than placebo, or at least 15% higher than placebo, or at least 20% higher than placebo. 37. In a population of patients all with moderate or severe CR-PCSS and / or PR-PCSS ratings, collagenase treatment as described herein results in 1 or more levels of composite responders that are at least 5% higher than placebo, or at least 7.5% higher than placebo, or at least 10% higher than placebo, or at least 12.5% ​​higher than placebo, or at least 15% higher than placebo, or at least 20% higher than placebo, or at least 25% higher than placebo, or at least 30% higher than placebo, or at least 35% higher than placebo, or at least 40% higher than placebo. 38. In patient populations with moderate or severe CR-PCSS and PR-PCSS scores, the greatest reductions from baseline (screening) in CR-PCSS and PR-PCSS scores were first observed on Day 22, Day 71, or earlier. 39. In patients with moderate or severe CR-PCSS and / or PR-PCSS scores at baseline (before treatment), there is a consistent decline in CR-PCSS and / or PR-PCSS scores from baseline (screening visit) in both sides.

[0221] In another embodiment, one or more of Results Nos. 1-39 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay)max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0222] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-39 above.

[0223] In another example, injection of about 1 mg to about 20 mg of collagenase according to Treatment I produces one or more of Results No. 1-39 above, wherein the collagenase has one or more of the following characteristics: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0224] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1 to 39 above.

[0225] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-39 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0226] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-39 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0227] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0228] In another embodiment, in response to the above treatment, the patient is a 2-level CR-PCSS responder, in which the CR-PCSS assessment shows an improvement of at least 2 levels (change of -2, -3, or -4) from baseline at the time of evaluation. A 1-level CR-PCSS responder is a patient who shows an improvement of at least 1 level (change of -1, -2, -3, or -4) from baseline at the time of evaluation. A 2-level PR-PCSS responder is a patient who shows an improvement of at least 2 levels (change of -2, -3, or -4) from baseline at the time of evaluation in the PR-PCSS assessment. A 1-level PR-PCSS responder is a patient who shows an improvement of at least 1 level (change of -1, -2, -3, or -4) from baseline at the time of evaluation in the PR-PCSS assessment. In another embodiment, a 2-level combined responder refers to a patient who has both a 2-level PR-PCSS responder and a 2-level CR-PCSS responder at the time of evaluation. A 1-level combined responder is a patient who has both a 1-level PR-PCSS responder and a 1-level CR-PCSS responder at the time of evaluation.

[0229] 2. Efficacy as measured by the Hexel Cellulite Severity Scale (Hexel CSS)

[0230] In Hexel CSS, improvement for an individual patient at any visit refers to an improvement of at least one level or rating from baseline or previous score. Improvement for a patient population at a visit is an improvement of about 0.1 of the mean Hexel CSS score or rating from baseline or the previous mean Hexel CSS score or rating. A responder is any patient who shows at least a 25% improvement in the maximum total score or rating from baseline. In certain embodiments, the treatment methods detailed above result in one or more of the following efficacy endpoints as measured by Hexel CSS: 1. In the patient population with a baseline Day 1 Hexel CSS assessment, collagenase injection 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: ● Moving from severe to moderate (score 11-15 to 6-10) ● Shift from severe to mild (score 11-15 to 1-5) ● Transition from severe to zero (score 11-15 to 0) ● Transition from moderate to mild (score 6-10 to 1-5) ● Moving from moderate to zero (from a score of 6-10 to 0) ● Transition from mild to zero (score 1-5 to 0) 2. A change in severity of at least 2 levels from baseline (pre-treatment "Day 1") to Day 22, 43, 71, 90, 180, 251, 360, 431, 720, Year 3, Year 4, or Year 5 on the Hexel CSS assessed live by a clinician in the therapeutic area. 3. A change in severity of at least 2 levels from baseline (Day 1) to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3 years, 4 years, or 5 years in the Hexel CSS assessed by a clinician viewing digital images of the treated area. 4. A change demonstrated by a 2-level response at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Years 3, 4, or 5 is defined as subjects experiencing at least a 2-level improvement from baseline in Hexel CSS severity as assessed by a clinician. 5. A change in severity of at least 1 level from baseline (Day 1) to Day 22, Day 43, Day 71, Day 90, Day 180, Day 251, Day 360, Day 431, Day 720, Year 3, Year 4, or Year 5 as assessed live by a therapeutic area clinician. 6. A change in severity of at least one Hexel CSS level from baseline (Day 1) to either Day 22, Day 43, Day 71, Day 90, Day 180, Day 251, Day 360, Day 431, Day 720, Year 3, Year 4, or Year 5 as assessed by a clinician viewing digital images of the treated area. 7. A change in response of at least one level at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Years 3, 4, and 5 is defined as subjects achieving at least one improvement from baseline in Hexel CSS severity as assessed by a clinician. 8. In patients with a moderate or severe Hexel CSS score, improvement in at least one treatment domain is statistically significant compared to placebo, and the change is one or more of the above No. 2 to No. 7. 9. Results show that at least 5% of patients maintain their improvement level relative to pre-treatment baseline for at least 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such level for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, results show that at least 5% of patients show improvement relative to pre-treatment baseline, and the improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the improvement increases over time, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 10. At day 180, improvement from baseline in Hexel CSS assessment was consistent across both sides of the treatment area. 11. In the population of patients with a moderate or severe Hexel CSS rating, the median time to the earliest 2-level Hexel CSS improvement in at least one therapeutic area is approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 12. In the population of patients with a moderate or severe Hexel CSS rating, the median time to an earliest 1-level Hexel CSS improvement in at least one therapeutic 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. 13. In the patient population with moderate or severe Hexel CSS scores, subjects' mean Hexel CSS scores separated from placebo 21 days after the first treatment and continued to show significant improvement with subsequent treatments. 14. In a population of patients all with a moderate or severe Hexel CSS assessment, the proportion of subjects having a 2-level response as measured by Hexel CSS in at least one therapeutic area at Day 71 is approximately 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 15. In a population of patients all with a moderate or severe Hexel CSS assessment, the proportion of subjects having a 1-level response as measured by Hexel CSS in at least one therapeutic area at Day 71 is approximately 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 16. In the population of patients with a moderate or severe Hexel CSS assessment, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of patients have a Hexel CSS response of at least one level in at least one treatment area by Day 71 after treatment, independent of age, BMI, or skin color. 17. Reduction in cellulite severity occurs rapidly within approximately 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit.

[0231] In another embodiment, one or more of Results No. 1-17 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following properties: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0232] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-17 above.

[0233] In another example, injection of about 1 mg to about 20 mg of collagenase from Treatment I produces one or more of Results No. 1-17 above, wherein the collagenase has one or more of the following characteristics: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0234] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-17 above.

[0235] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-17 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0236] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I to obtain one or more of Results No. 1-17 above, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0237] 3. Efficacy measured by Hexel Pit Depth Score

[0238] An individual patient improvement in Hexel Dimple Depth Score at any visit means an improvement of at least one level or rating from baseline or previous score. Also, improvement in a patient population means an improvement of about 0.1 from baseline or previous mean Hexel Dimple Depth score or rating. A responder is any patient who shows at least a 25% improvement in the maximum total score or rating from baseline. In certain embodiments, the treatment methods detailed above result in one or more of the following efficacy endpoints as measured by Hexel Dimple Depth Score: 1. In a patient population that all had a baseline Day 1 Hexel Pit Depth Score assessment, a statistically significant number of patients who received collagenase injection into at least one treatment area during at least one treatment visit met one or more of the following efficacy endpoints: The transition from deep depression (3) to medium depth (2) ●Transition from deep depression (3) to superficial depression (1) ●Transition from deep depression (3) to no depression (0) ●Transition from medium depth (2) to superficial depression (1) ●Transition from medium depth (2) to no depression (0) ● Transition from superficial depression (1) to no depression (0) 2. A change in severity of Hexel pit depth score assessed live by a clinician in the treatment area of ​​at least 2 levels from baseline (pre-treatment "Day 1") to Day 22, Day 43, Day 71, Day 90, Day 180, Day 251, Day 360, Day 431, Day 720, Year 3, Year 4, or Year 5. 3. A change demonstrated by a 2-level response at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Year 3, 4, or 5 is defined as subjects experiencing at least a 2-level improvement from baseline in severity of Hexel Pit Depth Score as assessed by a clinician. 4. A change in severity of at least one level in the Hexel Dimple Depth Score as assessed live by a treatment area clinician from baseline (Day 1) to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Years 3, 4, or 5. 5. A change demonstrated by a 1-level response at Day 22, 43, 71, 90, 180, 251, 360, 431, 720, Year 3, Year 4, or Year 5 is defined as subjects achieving at least a 1-level improvement from baseline in severity of Hexel Pit Depth Score as assessed by a clinician. 6. In patients with moderate or deep depressions as assessed by the Hexel CSS, improvement in at least one treatment area is statistically significant compared to placebo, and the change is one or more of the above items 2 to 7. 7. Results show that at least 5% of patients maintain their improvement level relative to pre-treatment baseline for at least 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such level for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, results show that at least 5% of patients show improvement relative to pre-treatment baseline, and the improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the improvement increases over time, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 8. At day 180, improvements from baseline in Hexel pit depth score assessments were consistently seen in both the left and right treated areas. 9. In patients with moderate or deep Hexel Dimple Depth Score assessments, the median time to the earliest 2-level improvement in Hexel Dimple Depth Score in at least one treatment area is approximately 50, or 60, or 70, or 80, or 90 days. 10. In a patient population with a Hexel Dimple Depth Score rating of moderate or deep, the median time to the earliest 1-level improvement in Hexel Dimple Depth Score in at least one treatment 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. 11. In patient populations with moderate or deep Hexel pit depth scores, subjects' mean Hexel pit depth scores separated from placebo 21 days after the first treatment and showed continued and significant improvement with subsequent treatments. 12. In a patient population in which all patients had a Hexel Dimple Depth Score rating of intermediate or deep, the proportion of subjects who had a 2-level response as measured by the Hexel Dimple Depth Score in at least one treatment area at Day 71 was approximately 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 13. In a patient population all of whom have a Hexel Dimple Depth Score rating of intermediate or deep, the proportion of subjects having a level 1 response as measured by the Hexel Dimple Depth Score in at least one treatment area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 14. In a patient population with a Hexel Dimple Depth Score rating of moderate or deep, by day 71 after treatment, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of patients demonstrate a Hexel Dimple Depth Score response of 1 level in at least one treatment area, and Hexel Dimple Depth Score results are independent of age, BMI, or skin color. 15. Reduction in cellulite severity occurs rapidly within approximately 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days following the initial treatment visit. 16. Change in score from baseline to Day 71 of approximately -0.1 to approximately -2.0 in one or more treatment domains. 17. A least squares (LS) mean of approximately -0.1 to approximately -1.5 (95% confidence interval (CI)) in one or more therapeutic areas in a statistically significant patient population.

[0239] In another embodiment, one or more of Results Nos. 1-19 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following properties: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0240] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-19 above.

[0241] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-19 above, wherein the collagenase has one or more of the following characteristics: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0242] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-19 above.

[0243] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-19 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0244] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-19 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / KM , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0245] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0246] 4. Effectiveness measured by Likert scale for aesthetic appearance

[0247] On the Likert scale, an individual patient's improvement at any visit is an improvement of at least one level or rating from pre-treatment or previous score. A patient group's improvement at any visit is an improvement in the mean Likert score or rating of about 0.1 from pre-treatment or any previous mean Likert score or rating. In certain embodiments, the treatment methods detailed above result in one or more of the following efficacy endpoints as measured by Likert scale scores: 1. In a population of patients with cellulite, injecting 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: ● Likert scale score "improved" (1) ● The Likert scale score was “significantly improved” (2) - Likert scale score of "very improved" (3) 2. Improvement in the appearance of the treated areas at 22 days, 43 days, 71 days, 90 days, 180 days, 251 days, 360 days, 431 days, 720 days, 3 years, 4 years, or 5 years compared to pretreatment on a live clinician-assessed Likert scale score of at least 2 levels. 3. The appearance of the treated areas at 22 days, 43 days, 71 days, 90 days, 180 days, 251 days, 360 days, 431 days, 720 days, 3 years, 4 years, or 5 years has improved by at least 2 levels from pre-treatment on a Likert scale rated by the subject while viewing a digital image of the treated areas. 4. Improvement of at least one level in the appearance of the treated areas compared to pretreatment on a live clinician-assessed Likert scale score at 22 days, 43 days, 71 days, 90 days, 180 days, 251 days, 360 days, 431 days, 720 days, 3 years, 4 years, or 5 years. 5. The appearance of the treated areas at 22 days, 43 days, 71 days, 90 days, 180 days, 251 days, 360 days, 431 days, 720 days, 3 years, 4 years, or 5 years has improved by at least 1 level on a Likert scale score rated by the subject while viewing a digital image of the treated areas compared to pre-treatment. 6. In a population of patients with cellulite, there is a statistically significant improvement in Likert scale score in at least one treatment area, and that improvement is one or more of items 2 through 7 above. 7. Results show that at least 5% of patients maintain their improvement level relative to pre-treatment baseline for at least 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such level for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, results show that at least 5% of patients show improvement relative to pre-treatment baseline, and the improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the improvement increases over time, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 8. At day 180, improvements from baseline in Likert scale scores were consistent between the right and left treatment areas. 9. In a population of patients with cellulite, the median time to improvement in the earliest 2-level Likert scale score in at least one treatment area is about 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 10. In a population of patients with cellulite, the median time to improvement in the earliest 1-level Likert scale score in at least one treatment area is about 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. 11. In a patient population with cellulite, subjects' mean Likert scale scores separated from placebo 21 days after the first treatment and showed continued significant improvement with subsequent treatments. 12. In a patient population with cellulite, the proportion of subjects having a 2-level response as measured by Likert scale scores in at least one treatment area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 13. In a patient population with cellulite, the percentage of subjects having a 1-level response as measured by a Likert scale score in at least one treatment area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 14. In a patient population with cellulite, at least one-third, one-half, two-thirds, or three-quarters of patients have a Likert scale score response of at least one level in at least one treatment area by 71 days after treatment, and the Likert scale score outcome is independent of age, BMI, or skin color. 15. Reduction in cellulite severity occurs rapidly within approximately 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days following the initial treatment visit.

[0248] In another embodiment, one or more of Results No. 1-15 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following properties: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat, microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0249] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-15 above.

[0250] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-15 above, wherein the collagenase has one or more of the following characteristics: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0251] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-15 above.

[0252] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-15 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0253] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-15 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0254] 5. Depression Analysis

[0255] In certain embodiments, treatment of cellulite with collagenase reduces parameters of crater size as follows: ● Depth : 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% ● width : 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% ● length : 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% ● Overall volume : 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% ● surface area : 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%

[0256] In some embodiments, treatment results in at least 5% of patients maintaining a level of improvement relative to pre-treatment baseline for at least 71 days after the first dose. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintaining such levels 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 first dose. In other embodiments, treatment results in at least 5% of patients showing improvement relative to pre-treatment baseline, and even increasing improvement over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showing improvement, and even increasing improvement, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment.

[0257] In another embodiment, at least one of the crater size parameters is reduced by at least 5%, or at least 10%, or at least 20%, by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, where the collagenase has one or more of the following properties: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0258] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I reduces at least one of the pit size parameters by at least 5%, or at least 10%, or at least 20%.

[0259] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I reduces at least one of the pit size parameters by at least 5%, or at least 10%, or at least 20%, where the collagenase has one or more of the following properties: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0260] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I reduces at least one parameter of the pit size by at least 5%, or at least 10%, or at least 20%.

[0261] In certain embodiments, during at least one treatment visit, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area, resulting in a decrease in at least one of the depression size parameters by at least 5%, or at least 10%, or at least 20%, and wherein type I collagenase and type II collagenase have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0262] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, resulting in a decrease in at least one of the pit size parameters by at least 5%, or at least 10%, or at least 20%, where collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds-1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0263] 6. Efficacy as measured by the Subject Global Aesthetic Improvement Scale (S-GAIS) and the Investigator Global Aesthetic Improvement Scale (I-GAIS) The treatment methods detailed above improve responses as measured by the S-GAIS and I-GAIS. A 2-level S-GAIS responder is a subject with an S-GAIS rating of at least 2 (+2 or +3) at the time of evaluation. A 1-level S-GAIS responder is a subject with an S-GAIS rating of at least 1 (+1, +2, +3) at the time of evaluation. A 2-level I-GAIS responder is a subject with an I-GAIS rating of at least 2 (+2 or +3) at the time of evaluation. A 1-level I-GAIS responder is a subject with an I-GAIS rating of at least 1 (+1, +2, or +3) at the time of evaluation. An individual patient improvement at any visit refers to an improvement of at least one level or rating from baseline or a previous score. A patient group improvement refers to an improvement in the mean score or rating of about 0.1 from baseline or the previous mean score or rating.

[0264] In certain embodiments, the methods of treatment detailed above result in one or more of the following efficacy endpoints as measured by S-GAIS and / or I-GAIS: 1. In a patient population with cellulite, a statistically significant number of such patients will meet one or more of the following efficacy endpoints by injecting collagenase into at least one treatment area during at least one treatment visit: ● S-GAIS and / or I-GAIS scores were “improved” (+1) - S-GAIS and / or I-GAIS scores were "significantly improved" (+2) - S-GAIS and / or I-GAIS scores were "very improved" (+3) 2. At least a 2-level improvement on the I-GAIS assessed live by a therapeutic area clinician at Days 22, 43, 71, 90, 180, 365, or 730. 3. At least a 2-level improvement on the S-GAIS, assessed by subjects while viewing digital images of the treated area, at Days 22, 43, 71, 90, 180, 365, or 730. 4. Improvement as demonstrated by a 2-level composite response at Days 22, 43, 71, 90, 180, 365, or 730 is defined as subjects achieving at least a 2-level improvement on the I-GAIS as assessed by a clinician and at least a 2-level improvement on the S-GAIS as assessed by a patient. 5. At least one level of improvement on the I-GAIS assessed live by a therapeutic area clinician at Days 22, 43, 71, 90, 180, 365, or 730. 6. At least one level of improvement on the S-GAIS, as assessed by the subject while viewing digital images of the treated area, at Days 22, 43, 71, 90, 180, 365, or 730. 7. Improvement as demonstrated by a 1-level composite response at Days 22, 43, 71, 90, 180, 365, or 730 is defined as subjects achieving at least 1-level improvement on the I-GAIS as assessed by a clinician and at least 1-level improvement on the S-GAIS as assessed by a patient. 8. In a patient population with cellulite, there is a statistically significant improvement in I-GAIS and / or S-GAIS in at least one treatment area compared to placebo, and the improvement is one or more of No. 2 to No. 7 above. 9. With this treatment, at least 5% of patients maintained their level of improvement for 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintained such levels for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, at least 5% of patients showed improvement and the degree of improvement further increased over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement and further increased improvement was seen 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 10. At day 180, improvements in I-GAIS and S-GAIS assessments were consistent between the left and right treatment areas. 11. In a patient population with cellulite, the median time to improvement in the earliest 2 levels of I-GAIS and / or S-GAIS in at least one treatment area is about 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 12. In a patient population with cellulite, the median time to improvement in the earliest 1 level I-GAIS and / or S-GAIS in at least one treatment area is about 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. 13. In a patient population with cellulite, subjects' mean I-GAIS and / or S-GAIS scores separated from placebo 21 days after the first treatment and showed continued and significant improvement with subsequent treatments. 14. In a patient population with cellulite, the proportion of subjects having a 2-level composite response as measured by I-GAIS and / or S-GAIS in at least one treatment area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 15. In a patient population with cellulite, the proportion of subjects having a composite response of 1 level as measured by I-GAIS and / or S-GAIS in at least one treatment area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 16. In a patient population with cellulite, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of patients have an I-GAIS and / or S-GAIS response of at least 1 level in at least one treatment area by 71 days after treatment, and GAIS results are independent of age, BMI, or skin color. 17. Reduction in cellulite severity occurs rapidly within approximately 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit. 18.In the patient population with moderate or severe CR-PCSS and / or PR-PCSS assessments, at least 60% of patients demonstrated an S-GAIS response of 1 level in the target hip at day 71. 19. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS assessments, at least 20% of patients demonstrated a 2-level S-GAIS response in the target hip at day 71. 20. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS assessments, mean S-GAIS was greater in subjects treated with collagenase in the target buttock compared with placebo-treated subjects (approximately 1.0 vs. approximately 0.5, respectively) at day 71. Similar results were obtained for non-target buttocks (approximately 1.0 in collagenase-treated subjects and approximately 0.5 in placebo-treated subjects). 21. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS assessments, at day 71, the proportion of 1-level S-GAIS responders was greater in collagenase-treated subjects than in placebo-treated subjects in target buttocks (approximately 70% vs. approximately 40%, respectively) and non-target buttocks (approximately 70% vs. approximately 40%, respectively). 22. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS scores, the mean I-GAIS was statistically significantly greater in subjects who received collagenase in the target buttock compared with subjects who received placebo (approximately 1.0 vs. 0.3, respectively) at day 71. Similar results were obtained for non-target buttocks (approximately 0.6 in collagenase-treated subjects and approximately 0.1 in placebo-treated subjects). 23. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS assessments, at day 71, the proportion of I-GAIS level 1 responders was greater in collagenase-treated subjects than in placebo-treated subjects in target buttocks (approximately 70% vs. 25%, respectively) and non-target buttocks (70% vs. 25%, respectively). 24. In a population of patients with moderate or severe CR-PCSS and / or PR-PCSS scores, a series of cross-tabulations showed consistency between PR-PCSS and S-GAIS: a change of one level in PR-PCSS was associated with a similar change in S-GAIS.

[0265] In another embodiment, one or more of Results No. 1-24 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following properties: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0266] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-24 above.

[0267] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-24 above, where the collagenase has one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0268] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-24 above.

[0269] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-24 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0270] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0271] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-24 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0272] Patients assessed improvement after treatment compared to baseline using the S-GAIS in the target and non-target buttocks, as further described in Examples 2 and 3. As shown in Figure 11, treatment with CCH was significantly better than placebo, as indicated by responses of ≧2 and ≧1 levels measured by the S-GAIS.

[0273] 7. Efficacy measured by PR-CIS The above treatment methods improve the response as measured by the PR-CIS. The PR-CIS total score is the sum of the six items on the scale. The PR-CIS total score ranges from 0 to 60, with higher values ​​indicating greater cellulite impact. Item 1 of the PR-CIS asks how satisfied the subject is with the appearance of cellulite, and is reversed by subtracting the subject's reported rating from 10. For the PR-CIS total score, responders were subjects who had a PR-CIS total score reduction of 12 or more from baseline at the time of assessment. For individual PR-CIS impact scores, responses were defined as improvements of at least two score intervals from baseline at each time point. Furthermore, responders are patients whose maximum total score showed at least a 20% improvement from baseline. Furthermore, improvement for an individual patient at each visit means an improvement of at least one level or rating from baseline or previous score. Furthermore, improvement for a patient group means an improvement of about 0.1 in the mean score or rating from baseline or previous mean score or rating. Furthermore, improvement means a change from baseline of at least one level out of 60.

[0274] In certain embodiments, the methods of treatment detailed above result in one or more of the following efficacy endpoints as measured by PR-CIS: 1. In a patient population with cellulite, a statistically significant number of such patients will meet one or more of the following efficacy endpoints by injecting collagenase into at least one treatment area during at least one treatment visit: -PR-CIS will demonstrate improvement in at least one domain selected from the group consisting of satisfaction with the appearance of cellulite, bother, awareness, embarrassment, looking older, and looking fat / out of shape. A reduction in PR-CIS total score of 12 or more from baseline at at least one assessment time point. ●PR-CIS impact score demonstrating an improvement of at least 2 score intervals from baseline at one or more assessment time points. ●Improvement is a change from baseline of at least 1 level out of 60. 2. At least a 12-point improvement in severity from baseline (pre-treatment "Day 1") in the treatment-area PR-CIS at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5. 3. In patients with cellulite, a statistically significant improvement in severity versus placebo of at least 12 points on the PR-CIS in the treatment area from baseline (Day 1 before treatment) to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Years 3, 4, or 5. 4. Results show that at least 5% of patients maintain improvement levels over pre-treatment baseline for at least 71 days after first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such levels for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after first dose. In other cases, results show that at least 5% of patients show improvement over pre-treatment baseline, and the degree of improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the degree of improvement increases, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 5. At day 180, improvement from baseline in PR-CIS assessment was seen consistently in both the left and right treatment regions. 6. In a patient population with cellulite, the median time to a reduction in PR-CIS total score of at least 12 from baseline for at least one treatment area at one or more assessment time points is about 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. 7. In a patient population with cellulite, subjects' mean PR-CIS scores separate from placebo 21 days after the first treatment and show sustained and significant improvement with subsequent treatments. 8. In a patient population with cellulite, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of patients have a reduction in PR-CIS total score of at least 12 from baseline at one or more assessment points in at least one treatment area by 71 days after treatment, and PR-CIS results are not dependent on age, BMI, or skin color. 9. Reduction in cellulite severity occurs rapidly within about 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit. 10. In patients with moderate or severe CR-PCSS and / or PR-PCSS scores, the mean change from baseline in PR-CIS total score at Day 71 was approximately -10 in collagenase-treated subjects and approximately -5 in placebo-treated subjects. 11. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS ratings, mean changes in PR-CIS from baseline to day 71 were statistically significantly more favorable in collagenase-treated subjects than in placebo-treated subjects for total score (approximately -12 vs. -6, respectively) and shortened score (approximately -10 vs. 5, respectively), as well as personal impact scores (satisfaction with the appearance of cellulite, bother, self-consciousness, embarrassment, aged appearance, and body shape concerns). In the patient population with moderate or severe CR-PCSS and / or PR-PCSS scores at day 71, a greater proportion of PR-CIS responders were found in subjects receiving collagenase than in those receiving placebo for the total score (approximately 45% vs. 20%, respectively) and the abbreviated score (approximately 50% vs. 25%, respectively). In addition, a greater proportion of respondents in the collagenase group than in the placebo group achieved individual impact scores. These differences were also statistically significant.

[0275] In another embodiment, one or more of Results No. 1-12 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following properties: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0276] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-12 above.

[0277] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-12 above, wherein the collagenase has one or more of the following characteristics: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0278] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-12 above.

[0279] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-12 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0280] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0281] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-12 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0282] Patient improvement after treatment was assessed compared to baseline using the PR-CIS for CCH vs. placebo, as further described in Examples 2 and 3. As shown in Figure 11, treatment with CCH was significantly better than placebo.

[0283] 8. Efficacy measured with the PR-CIS abbreviation The above treatment methods improve response as measured by the PR-CIS abbreviation. The PR-CIS abbreviation total score is the sum of the five items of the scale. The PR-CIS abbreviation total score ranges from 0 to 50, with higher values ​​indicating greater negative effects from cellulite. Item 1 of the PR-CIS asks how satisfied the subject is with the appearance of cellulite and is reversed by subtracting the subject's reported rating from 10. For the PR-CIS abbreviation total score, a responder is a subject whose PR-CIS total score decreases by 10 or more from baseline at the time of assessment. For individual PR-CIS abbreviation impact scores, a responder is an improvement of at least two score intervals from baseline at each time point. In addition, a responder is a patient whose maximum total score shows at least a 20% improvement from baseline. In addition, for individual patients, an improvement of at least one level or rating from baseline or previous score is defined as an improvement of approximately 0.1 in the mean score or rating from baseline or previous mean score or rating. In addition, an improvement in a patient population is defined as an improvement of approximately 0.1 in the mean score or rating from baseline or previous mean score or rating. Furthermore, improvement refers to a change from baseline of at least 1 level out of 50.

[0284] In certain embodiments, the methods of treatment detailed above result in one or more of the following efficacy endpoints as measured by the PR-CIS abbreviation: 1. In a patient population with cellulite, a statistically significant number of such patients will meet one or more of the following efficacy endpoints by injecting collagenase into at least one treatment area during at least one treatment visit: The PR-CIS abbreviation is improvement in at least one area selected from the group consisting of satisfaction with the appearance of cellulite, bother, awareness, embarrassment, looking older, and looking fat / out of shape. • A decrease of ≥10 from baseline in the PR-CIS abbreviated total score at at least one assessment time point. PR-CIS abbreviated impact score demonstrating an improvement of at least 2 score intervals from baseline at one or more assessment time points. ●Furthermore, improvement is defined as a change from baseline of at least 1 level out of 50. 2. At least a 12-point improvement in severity from baseline (pre-treatment, "Day 1") on the treatment-area PR-CIS abbreviation at days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3 years, 4 years, or 5 years. 3. In the patient population with cellulite, a statistically significant improvement in severity versus placebo of at least 10 points on the PR-CIS abbreviation for the treatment area from baseline (Day 1 before treatment) to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Year 3, Year 4, or Year 5. 4. Results show that at least 5% of patients maintain improvement levels over pre-treatment baseline for at least 71 days after first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such levels for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after first dose. In other cases, results show that at least 5% of patients show improvement over pre-treatment baseline, and the degree of improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the degree of improvement increases, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after first dose. With some treatments, at least 10%, 20%, 30%, 40%, and 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, and 24 months after the first, second, and third treatments. 5. At day 180, improvements from baseline in PR-CIS abbreviated assessment were consistent across the left and right treatment regions. 6. In a patient population with cellulite, the median time to a reduction of at least 10 from baseline in PR-CIS abbreviated total score for at least one treatment area at one or more assessment time points is about 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. 7. In a patient population with cellulite, subjects' mean PR-CIS abbreviated scores separate from placebo 21 days after the first treatment and demonstrate sustained and significant improvement with subsequent treatments. 8. In a patient population with cellulite, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of patients have a reduction in PR-CIS Abbreviated Total Score of at least 10 from baseline at one or more assessment time points in at least one treatment area by 71 days post-treatment, and PR-CIS results are not dependent on age, BMI, or skin color. 9. Reduction in cellulite severity occurs rapidly within about 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit. 10. In patients with moderate or severe CR-PCSS and / or PR-PCSS scores, the mean change from baseline in PR-CIS abbreviated total score at Day 71 was approximately -10 in collagenase-treated subjects and approximately -5 in placebo-treated subjects. 11. In patients with moderate or severe CR-PCSS and / or PR-PCSS scores, the mean change from baseline in the PR-CIS abbreviated form at day 71 was statistically significantly more favorable in collagenase-treated subjects than in placebo-treated subjects for total scores (approximately -12 vs. -6, respectively) and abbreviated scores (approximately -10 vs. 5, respectively). There were also statistically significant changes in the individual impact scores (satisfaction with the appearance of cellulite, bother, awareness, embarrassment, aged appearance, and body shape concerns) between the collagenase-treated and placebo-treated groups. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS scores at day 71, a greater proportion of subjects receiving collagenase were PR-CIS abbreviated responders than subjects receiving placebo for both total score (approximately 45% vs. 20%, respectively) and abbreviated score (approximately 50% vs. 25%, respectively). In addition, a greater proportion of subjects receiving collagenase than placebo responded to the individual impact scores. These differences were also statistically significant.

[0285] In another embodiment, one or more of Results No. 1-12 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following properties: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute-1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0286] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-9 above.

[0287] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-12 above, wherein the collagenase has one or more of the following characteristics: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0288] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-12 above.

[0289] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-12 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0290] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0291] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-12 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0292] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0293] 9. Efficacy measured with the Subject Self-Rating Scale (SSRS) The above treatment methods have resulted in improvement as measured by the SSRS. SSRS responders are subjects who are at least somewhat satisfied (somewhat satisfied [4], very satisfied [5], or extremely satisfied [6]) with the appearance of the cellulite in the affected area at the time of evaluation. In addition, responders are patients who achieve a maximum total score improvement of 17% or more from baseline. Improvement of an individual patient at any visit means an improvement of at least one level or rating from baseline or previous score. Improvement of a patient group at any visit is an improvement of about 0.1 of the mean score or rating from baseline or any previous mean score or rating. In certain embodiments, the treatment methods result in one or more of the following efficacy endpoints as measured by SSRS evaluation: 1. In a population of patients with cellulite, injecting collagenase into at least one treatment area during at least one treatment visit will result in a statistically significant number of such patients meeting one or more of the following efficacy endpoints: ● SSRS rating of "somewhat satisfied" ● SSRS rating of "Satisfied" ● SSRS rating of "extremely satisfactory" 2. At least a 2-level improvement in the SSRS rating in the treatment area from baseline (pre-treatment "Day 1") to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Years 3, 4, or 5. 3. At least one improvement in the SSRS in the treatment area from baseline (Day 1) to Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Years 3, 4, or 5. 4. In a patient population with cellulite, improvement in SSRS assessment in at least one treatment area is statistically significant compared to placebo, and the improvement is one or more of the above No. 2 to No. 3. 5. Treatment results show that at least 5% of patients maintain their improvement level relative to pre-treatment baseline for at least 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such level for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, at least 5% of patients show improvement relative to pre-treatment baseline, and the improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the improvement increases over time, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 6. At 180 days after the first injection, improvements from baseline in SSRS ratings were seen consistently on both sides of the treatment area. 7. In the patient population with cellulite, the median time to improvement in the earliest 2-level SSRS assessment in at least one treatment area is approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 8. In a patient population with cellulite, the median time to improvement in the earliest 1-level SSRS assessment in at least one treatment area is about 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. 9. In a patient population with cellulite, subjects' mean SSRS ratings separate from placebo 21 days after the first treatment and demonstrate sustained significant improvement with subsequent treatments. 10. In a patient population with cellulite, the proportion of subjects having a 2-level composite response as measured by SSRS assessment in at least one treatment area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 11. In a patient population with cellulite, the proportion of subjects having a 1-level composite response as measured by SSRS assessment in at least one treatment area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 12. In a patient population with cellulite, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of patients have an SSRS response of at least one level in at least one treatment area by 71 days after treatment, and the SSRS response is independent of age, BMI, or skin color. 13. Reduction in cellulite severity occurs rapidly within about 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit. 14. In patients with moderate or severe CR-PCSS and / or PR-PCSS scores, at least 40% of patients achieved SSRS level 1 responder at Day 71. 15. In patients with moderate or severe CR-PCSS and / or PR-PCSS scores, at Day 71, the mean SSRS score is statistically significantly greater in subjects receiving collagenase than in subjects receiving placebo. 16. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS scores, at least 50% of patients are SSRS level 1 responders.

[0294] In another embodiment, one or more of Results No. 1-16 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0295] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-16 above.

[0296] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-16 above, where the collagenase has one or more of the following characteristics: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0297] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-16 above.

[0298] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-16 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes-1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0299] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I to obtain one or more of Results No. 1-16 above, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes-1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0300] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0301] 10. Efficacy measured by subject satisfaction with cellulite treatment (SSCT) The treatment methods detailed above result in improvements as measured by SSCT. Subjects who respond to the SSCT assessment at day 71 with a "satisfied" or "very satisfied" response are considered efficacy responders. An individual patient improvement means an improvement of at least one level or rating from baseline or previous score or rating. Additionally, a patient group improvement means an improvement in mean score or rating of about 0.1 points from baseline or previous mean score or rating. Additionally, there is at least a 0.1 improvement in SSCT compared to placebo. In certain embodiments, the treatment methods result in one or more of the following efficacy endpoints as measured by SSCT assessment: 1. In a population of patients with cellulite, injecting 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: ● SSCT rating of "Satisfied" ● SSCT evaluation of "very satisfied" 2. Increase in SSCT assessment (e.g., from 1 to 2) in the treatment area at days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3 years, 4 years, or 5 years. 3. In patients with cellulite, there was a statistically significant increase in SSCT assessment in at least one treatment area compared to placebo, with the improvement confirmed at days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3 years, 4 years, and 5 years. 4. The treatment resulted in at least 5% of patients maintaining their level of improvement for 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintained such levels for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, at least 5% of patients showed improvement and the degree of improvement further increased over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement and further increased improvement was seen 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, and 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, and 24 months after the first, second, and third treatments. 5. At day 180, improvements seen in SSCT assessments were consistent between the left and right treated areas. 6. In the patient population with cellulite, the median time to earliest SSCT-assessed improvement in at least one treatment area is about 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. 7. In a patient population with cellulite, average subject SSCT ratings separate from placebo 21 days after the first treatment and show continued and significant improvement with subsequent treatments. 8. In the patient population with cellulite, the percentage of subjects with improvement in SSCT assessment in at least one treatment area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 9. In a patient population with cellulite, by 71 days after treatment, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of the patients have an improved SSCT assessment in at least one treatment area, and the outcome of the SSCT assessment is independent of age, BMI, or skin color. 10. Reduction in cellulite severity occurs rapidly within about 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit. 11. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS scores, more than half of patients are satisfied or very satisfied with treatment at 180 days. 12. In a patient population with moderate or severe CR-PCSS and PR-PCSS scores, sustained reductions in CR-PCSS and PR-PCSS scores and subject satisfaction survey scores at 180 days indicate that three treatment sessions of 0.84 mg CCH administered subcutaneously in both buttocks or both thighs, 12 times per treatment (× 2 treatment areas), is effective in reducing cellulite. 13. In a patient population with moderate or severe CR-PCSS and / or PR-PCSS scores, at least 50% of patients treated with collagenase are satisfied or very satisfied with their cellulite treatment. 14. In patients with moderate or severe CR-PCSS and PR-PCSS scores, a statistically significant difference was observed in mean subject satisfaction at 71 days between the collagenase and placebo treatment groups.

[0302] In another embodiment, one or more of Results No. 1-14 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following characteristics: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0303] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-14 above.

[0304] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-14 above, where the collagenase has one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0305] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-14 above.

[0306] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-14 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / Kcat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0307] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0308] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-14 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1:About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0309] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0310] 11. Efficacy measured by Thigh Cellulite Severity-Patient (TCS-P) and Thigh Cellulite Severity-Clinician (TCS-C) Improvement for an individual patient at any visit means an improvement of at least one level or grade from baseline or previous score or assessment. Improvement for a patient population means an improvement in the mean score or assessment of about 0.1 from baseline or previous mean score or assessment. A responder is any patient who shows at least a 20% improvement in the maximum total score or assessment from baseline. In certain embodiments, the treatment methods detailed above result in one or more of the following efficacy endpoints as measured by TCS-C and / or TCS-P: 1.TCS-C and / or TCS-P scores improved by 0.1 or more compared to baseline. 2. Improvement of at least 2 levels in live clinician-assessed TCS-C severity in the target thigh at days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5. 3. Improvement of at least 2 levels from baseline (Day 1) in TCS-P severity as assessed by the subject while viewing digital images of the target thigh at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5. 4. Improvement as demonstrated by a 2-level composite response at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Year 3, Year 4, or Year 5 is defined as subjects who have at least a 2-level improvement in severity from baseline on the TCS-C and at least a 2-level improvement in severity from baseline on the TCS-P. 5. Improvement of at least one level from baseline (Day 1) in live clinician-assessed TCS-C severity in the target thigh at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Years 3, 4, or 5. 6. Improvement of at least one level from baseline (Day 1) in TCS-P severity as assessed by the subject while viewing digital images of the target thigh at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, 3, 4, or 5. 7. Improvement as demonstrated by a 1-level composite response at Days 22, 43, 71, 90, 180, 251, 360, 431, 720, Year 3, Year 4, or Year 5 is defined as subjects with at least 1 level of improvement from baseline in severity on the TCS-C and at least 1 level of improvement from baseline in severity on the TCS-P. 8. In patients with moderate or severe TCS-C scores, improvement in at least one treatment domain is statistically significant compared to placebo, and the improvement is in at least one of the above items 1 to 7. 9. Results show that at least 5% of patients maintain their improvement level relative to pre-treatment baseline for at least 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such level for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, results show that at least 5% of patients show improvement relative to pre-treatment baseline, and the improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the improvement increases over time, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 10. At day 180, improvements from baseline in TCS-C and / or TCS-P ratings were consistently seen in both left and right thighs. 11. In patients with moderate or severe TCS-C scores, the median time to the earliest 2-level TCS-C and / or TCS-P improvement in at least one therapeutic area is approximately 50, 60, 70, 80, or 90 days. 12.In patient populations with moderate or severe TCS-C scores, the median time to earliest 1-level improvement in TCS-C and / or TCS-P in at least one therapeutic 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. 13. In patient populations with moderate or severe TCS-C scores, subjects' mean TCS-C and / or TCS-P scores separated compared to placebo 21 days after initial treatment and showed sustained and significant improvement with subsequent treatments. 14.In the patient population with moderate or severe TCS-C scores, the proportion of subjects with a 2-level composite response as measured by TCS-C and / or TCS-P in at least one therapeutic area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 15.In the patient population with moderate or severe TCS-C scores, the proportion of subjects with a 1-level composite response as measured by TCS-C and / or TCS-P in at least one therapeutic area at Day 71 is about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or greater than 50%. 16. In a population of patients who all have a moderate or severe TCS-C rating, more than one-third, or more than one-half, or more than two-thirds, or more than three-quarters of patients have a TCS-C of at least 1 level and / or a TCS-P response of at least 1 level in at least one treatment area by Day 71 after treatment, and the TCS-C result is independent of age, BMI, or skin color. 17. Reduction in cellulite severity occurs rapidly within approximately 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit.

[0311] In another embodiment, one or more of Results No. 1-17 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following properties: V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0312] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-17 above.

[0313] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-17 above, wherein the collagenase has one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376-37,222 (SRC assay) or approximately 4-428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0314] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-17 above.

[0315] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-17 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0316] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0317] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-17 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes -1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934

[0318] Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0319] 12. Efficacy measured by Body-Q The above treatments improve the response as measured by Body-Q. For cellulite, there are 16 scale items that patients can answer ranging from "not at all bothered" to "very bothered" during the past week, assuming Flesch-Kincaid reading comprehension. Scores range from 16 (very bothered) to 64 (not bothered at all). For the Body-Q total score, responders are subjects who have an increase in Body-Q total score of 16 or more from baseline at the time of assessment. For the individual Body-Q impact scores, a response is an improvement of at least one score interval from baseline at each time point. In alternative embodiments, there may be more or less than 16 scale items, and a responder is any patient who shows at least a 25% improvement in maximum total score from baseline.

[0320] In certain embodiments, the methods of treatment detailed above result in one or more of the following efficacy endpoints as measured by Body-Q: 1. In a patient population with cellulite, a statistically significant number of such patients will meet one or more of the following efficacy endpoints by injecting collagenase into at least one treatment area during at least one treatment visit: ● Body-Q demonstrates improvement in at least one area selected from the group consisting of: 〇 An increase in Body-Q total score of ≥ 16 from baseline at at least one assessment time point o An improvement of at least one score interval from baseline for each individual Body-Q impact score at each time point; and o Improve maximum total score by at least 25% from baseline. ● An increase in Body-Q total score of 16 or more from baseline at one or more assessment time points. ● Demonstrate an improvement of at least one score interval from baseline in Body-Q impact score at one or more assessment time points. - Mean change from baseline in Body-Q assessment of cellulite at 90 and / or 180 days 2. Improvement in severity with an increase of at least 16 points in Body-Q in the target hip from baseline (pre-treatment "Day 1") to Day 22, 43, 71, 90, 180, 251, 360, 431, 720, Year 3, Year 4, or Year 5. 3. In a patient population with cellulite, a statistically significant improvement in severity versus placebo as demonstrated by an increase of at least 16 points in Body-Q in the target buttock from baseline (Day 1 before treatment) to Day 22, Day 43, Day 71, Day 90, Day 180, Day 251, Day 360, Day 431, Day 720, Year 3, Year 4, or Year 5. 4. Results show that at least 5% of patients maintain improvement levels relative to pre-treatment baseline for at least 71 days after the first dose. In certain cases, at least 10%, 20%, 30%, 40%, or 50% of patients maintain such levels for at least 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after the first dose. In other cases, results show that at least 5% of patients show improvement relative to pre-treatment baseline, and the degree of improvement increases over time. In some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients show improvement, and the degree of improvement increases, at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first dose. With some treatments, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, with further improvements seen 1, 3, 6, 9, 12, 18, or 24 months after the first, second, or third treatment. 5. At 180 days after the first injection, improvements from baseline in Body-Q ratings were consistently seen in both hips. 6. In a patient population with cellulite, the median time to an increase in Body-Q total score of at least 16 from baseline at one or more assessment time points for at least one treatment area is about 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. 7. In a patient population with cellulite, subjects' mean Body-Q scores separate from placebo 21 days after the first treatment and continue to show significant improvement with subsequent treatments. 8. In a patient population with cellulite, at least one-third, or at least one-half, or at least two-thirds, or at least three-quarters of patients have an increase in Body-Q total score from baseline of at least 16 in at least one treatment area at one or more assessment time points by 71 days after treatment, and Body-Q results are independent of age, BMI, or skin color. 9. Reduction in cellulite severity occurs rapidly within about 7, 14, 21, 30, 35, 40, 45, or 50 days following the initial treatment visit.

[0321] In another embodiment, one or more of Results No. 1-9 above are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, wherein the collagenase has one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0322] In other cases, injection of about 1 mg to about 20 mg of collagenase with Treatment I results in one or more of Results Nos. 1-9 above.

[0323] In another example, injection of about 1 mg to about 20 mg of collagenase with Treatment I produces one or more of Results No. 1-9 above, where the collagenase has one or more of the following characteristics: ● V of approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay) max (minute -1 ) K of approximately 4.1–410 nanomolar (SRC assay) or approximately 0.03–3.1 mM (GPA assay) M K of approximately 1.1 to 107 (SRC assay) or approximately 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of approximately 376 to 37,222 (SRC assay) or approximately 4 to 428 (GPA assay) cat , microseconds ● K of approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 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. ● The purity is at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography). ● Potency (i.e., specific activity) of approximately 500-30,000 SRC units / mg ● Potency of approximately 5,000 to 30,000 f-SRC units / mg ● Potency of approximately 100,000 to 400,000 GPA units / mg ● Potency of approximately 175,000 to 500,00 f-GPA units / mg ● Potency of approximately 5,000 to 25,000 ABC units / mg The impurities selected from the group consisting of clostripain, gelatinase and leupeptin are less than 1% by area ratio. ● Bioburden of less than 1cfu / mL

[0324] In another example, injection of about 1 mg to about 20 mg of CCH with Treatment I results in one or more of Results Nos. 1-9 above.

[0325] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area during at least one treatment visit, and one or more of Results No. 1-9 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes-1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0326] In certain embodiments, about 1 mg to about 20 mg of about a 1:1 ratio of type I collagenase and type II collagenase is injected employing Treatment I, and one or more of Results No. 1-9 above are obtained, wherein collagenase I and II have the following characteristics: ● I-type Assay: SRC microplate 〇 V max , minutes -1 :About 0.08~7.70 〇 K M : Approximately 4.1 to 410 nanomoles 〇 K cat , seconds -1 :About 1.1~107 〇 1 / K cat , microseconds: approx. 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 :About 5,140~508,814 ● Type II Assay: GPA microplate 〇 V max , minutes-1 :About 0.3~30.5 〇 K M , mM: approx. 0.03 to 3.1 〇 K cat , seconds -1 :About 93~9,179 〇 1 / K cat , microseconds: approx. 4 to 428 〇 K cat / K M , mM -1 seconds -1 :About 60~5,934 Other ratios may 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). Additionally, the type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0327] 13. Evaluation of treatment outcomes measured with 3D photographs / images Introduction 3D photography and other images can be used to assess the effectiveness of treatment, particularly for pit analysis and bruising analysis. Pit analysis (as defined above) using 3D images can include calculation of pit volume, length, width, and area. Calculations can be performed by a variety of known methods, such as those described in WO2018 / 116304 and WO2018 / 116305 by Eckhouse et al., Cherry Imaging, and those available from Canfield Scientific, Inc. Such measurements of volume, length, width, and area may be calculated using digital 3D grayscale images (with X and Y axis rotation capabilities) and digital 3D textured and illuminated images (with X and Y axis rotation capabilities) with a computer program that analyzes such images. In one embodiment, for the buttocks treatment area, images may be taken of the left treated buttock and / or the right treated buttock for each patient before and after treatment. In another embodiment, images may be taken of the thigh treatment area at, for example, 0 degrees, 45 degrees, and 90 degrees on the subject's posterior, lateral, and lateral thigh treatment areas, respectively, before and after treatment.

[0328] Clinicians and patients may use photographic and imaging tools to assess cellulite severity, with or without the use of the scales and other tools described herein. In one embodiment, the clinician / investigator or a qualified designee photographs each treatment area (e.g., each buttock or each thigh) in a standardized manner using a provided standardized 3D camera. The subject stands for each imaging session and wears standardized imaging clothing. The clinician / investigator or a qualified designee photographs each of the two treatment areas (e.g., the two buttocks or the two thighs) while the subject stands in a consistent, standardized, relaxed pose. These photographs and the images described above can be used for the efficacy analysis described below. Additional non-limiting and optional details regarding the methodology are described below.

[0329] 3D Photographic Imaging Analysis 3D Photographic Imaging Analysis. Evaluation of treatment efficacy will be performed by 3D photography. The investigator or qualified designee will use a standardized digital camera to photograph each treatment area (both buttocks or both thighs) in a standardized manner before injection, and before and after marking the depression and injection site on treatment days 1, 22, and 43. One set of photographs of each treatment area will be taken at screening and other non-dosing times. Photographs taken on days 4, 8, 15, 22, 43, and 71 (end of study / ET) will be reviewed by a central assessor blinded to treatment group and study visit dates.

[0330] Standard image analysis (IA) procedures used to assess bruising, volume, surface area, and maximum length / width are described below. The camera system may be an IntelliStudio available from Canfield Scientific with custom lighting, a Vectra M1 camera, and Canfield Capture software (v.3.5). Image types, attributes, views, and visit windows are defined and entered directly into the Digital Monitoring System (DMS). Study content set up in the DMS includes: TIFF0007628492000037.tif61156 Images are captured and staged in the DMS and available for review. Photo visits include: Screening, Day 1 / Premarking, Day 1 / Postmarking, Day 4, Day 8, Day 15, Day 22 / Premarking, Day 22 / Postmarking, Day 43 / Premarking, Day 43 Postmarking, Day 71 / ET.

[0331] Depression Analysis: Review the image and ensure that the major depressions are marked with an "X" as indicated in each quadrant. If the major depressions are not marked, perform the following steps: 1. Export the images and layout images to PowerPoint. 2. Contact the site and ask the site surveyor to mark the major depressions with an X on the image in PowerPoint. 3. Correspondence will be stored and shared with the Image Analysis (IA) team.

[0332] Dimple analysis is performed on days 1 (pre-marking), 22, 43, and 71. On days 22, 43, and 71, the observed and change from the day 1 pre-marking images for the dimple analysis parameters, maximum length, maximum width, surface area, and volume between the dimple base and the interpolated surface, are summarized by treatment area and injection type using descriptive statistics (an exemplary dimple analysis is depicted in Figures 19(A)-19(C)). Volume is also analyzed using a linear model.

[0333] An Image Analysis Technologist (IAT) determines the location of the target depression on the Day 1 premarking image using the Day 1 postmarking image as a reference. The boundary of the depression depression is traced on the Day 1 premarking image. This depression tracing is transferred to the Day 22 premarking, Day 43 premarking, and Day 71 ET images. This tracing is used to measure (i) the maximum length (i.e., the longest linear distance across the depression), (ii) the maximum width (i.e., the longest linear distance perpendicular to the maximum length measurement), (iii) the surface area of ​​the depression, and (iv) the volume between the base of the depression and the interpolated surface.

[0334] Birthmark Analysis : Review images to ensure the white reference label is present on each image and outside the bruising area. If the reference label is not present or obstructs the bruising area, a retake will be requested if applicable at that visit. Bruising analysis will be performed on days 1 (pre-marking), 4, 8, and 15.

[0335] Site Marking Analysis : Check with imaging to see if the pit is marked at the visit after marking on Day 1. If the pit is not marked, request rephotography at that visit if necessary.

[0336] Image Analysis (IA) Workflow: Every image selected for analysis is assigned a random tracking number that allows the Image Analysis Technologist (IAT) to know information such as study site, secondary subject identifier, and treatment group. The analysis procedures to be performed at a particular visit are pre-determined based on the name of the visit. Pit analysis is performed on pre-identified major pits and measures volume, surface area, and maximum length / width. Bruise analysis includes the L of the suspected bruise. * a * b * These include color measurements and measurements of unaffected skin outside the suspected birthmark. Site marking analysis involves surface area measurements of the trace as well as measurements of maximum length / width.

[0337] Image Registration : After the IAT undergoes the Day 1 pre-marking (baseline) examination, they open the pre-marking image in the Vectra Analysis Module (VAM) software and center the image on a grid in 3D space. The IAT uses the grid to position the baseline image so that the approximate center of the image is at the origin of the grid. The thighs / buttocks face forward in the +z direction, the upper thighs / buttocks face forward in the +y direction, and the lower thighs / buttocks face forward in the -y direction (Figure 13). All subsequent time points, including the Day 1 post-marking image, are registered to the baseline image's location within the grid using an algorithmic registration function.

[0338] The VAM creates a distance color map that the IAT uses to check the registration. The distance color map represents the distance between the two image models on a color scale of +5 to -5 mm (Figure 14). The IAT verifi...

Claims

1. 1. A collagenase composition for reducing the severity of cellulite in the buttocks of a human patient, said collagenase composition comprising: Collagenase Clostridium histolyticum (CCH), sucrose, Tris, and Mannitol having the collagenase composition, when injected into the buttocks of the patient in a therapeutically effective amount, improves cellulite severity by at least 2 levels as measured by a Clinician-Reported Photo-Numerical Cellulite Severity Scale (CR-PCSS) and a Patient-Reported Photo-Numerical Cellulite Severity Scale (PR-PCSS) 71 days after injection of the composition; Collagenase Compositions.

2. 10. The collagenase composition of claim 1, wherein the composition improves the appearance of the recesses regardless of the size of the recesses or the distance between the recesses.

3. 10. The collagenase composition of claim 1, wherein the composition improves the appearance of depressions in the skin without sagging, loose, or sagging skin.

4. 10. The collagenase composition of claim 1, wherein the patient undergoes multiple treatment visits.

5. 10. The collagenase composition of claim 1, which is injected with a 1 / 2 inch needle.

6. 10. The collagenase composition of claim 1, which is injected by a clinician without relying on devices such as spacers, rulers, paper, etc. to limit the location of the injection.

7. 10. The collagenase composition of claim 1, which is injected just below the recess.

8. 10. The collagenase composition of claim 1, which is injected at multiple sites within 2 cm of each other.

9. 2. The collagenase composition of claim 1, wherein the length of the recess is less than 1 cm or greater than 2 cm.

10. 10. The collagenase composition of claim 1, wherein the patient experiences an improvement in the appearance of cellulite within about 7, about 14, about 21, about 30, about 35, about 40, about 45, or about 50 days after injection of the collagenase composition.

11. 2. The collagenase composition of claim 1, wherein the collagenase composition, when injected into a patient population all having a CR-PCSS baseline rating of moderate or severe, the injection results in a reduction in the severity of inflammatory bowel disease, comprising: a. at least 50% of patients demonstrate an improvement of at least one level of severity from baseline on Day 1 on the hip clinician-assessed live CR-PCSS at Day 22 or Day 43; b. at least 50% of patients demonstrate an improvement of at least one level of severity from baseline on Day 1 on Day 22 or Day 43 in the PR-PCSS assessed by the patient while viewing digital images of the buttocks; c. at least 5% of patients demonstrate an improvement in severity of at least 2 levels from baseline on Day 1 on the hip clinician-assessed live CR-PCSS at Day 22 or Day 43; d. at least 5% of patients demonstrate an improvement in severity of at least 2 levels from baseline on Day 1 on the PR-PCSS assessed by the subject while viewing digital images of the buttocks at Day 22 or Day 43; and e. at least 5% of patients experience a decrease in cup size; A collagenase composition which produces a result selected from the group consisting of:

12. 12. The collagenase composition of claim 11, wherein the depression size is measured by a parameter selected from the group consisting of volume, length, width, and depth.

13. 12. The collagenase composition of claim 11, wherein the injection results in at least a 10% reduction in crater size at 22, 43, or 71 days, measured from baseline.

14. 10. The collagenase composition of claim 1, wherein the collagenase composition is injected to provide a cumulative collagenase dose of about 5.04 mg.

15. 2. The collagenase composition of claim 1, a. The following characteristics measured by the SRC assay: i. V ｍａｘ : About 0.08 to about 7.70 minutes －１ ii. K Ｍ : about 4.1 to about 410 nanomoles iii. K ｃａｔ : About 1.1 to about 107 seconds －１ iv. 1 / K ｃａｔ : about 376 to about 37,222 microseconds v. k ｃａｔ / K Ｍ : About 5,140~about 508,814mm －１ Second －１ AUX-I, b. The following characteristics measured by the GPA assay: i. V ｍａｘ : About 0.3 to about 30.5 minutes －１ ii. K Ｍ :About 0.03~about 3.1mM iii. K ｃａｔ : About 93 to about 9,179 seconds －１ iv. 1 / K ｃａｔ : About 4 to about 428 microseconds v. k ｃａｔ / K Ｍ :About 60~about 5,934mm －１ Second －１ AUX-II, A collagenase composition comprising:

16. 2. The collagenase composition of claim 1, comprising about 1 mg to about 20 mg of CCH.

17. 2. The collagenase composition of claim 1, having a potency of about 10,000 ABC units / 0.58 mg, and said therapeutically effective amount is from about 1 mg to about 20 mg.

18. 2. The collagenase composition of claim 1, having a potency of about 15,000 ABC units / mg to about 20,000 ABC units / mg, and said therapeutically effective amount is about 1 mg to about 20 mg.

19. 2. The collagenase composition of claim 1, wherein the therapeutically effective amount is from about 1 mg to about 10 mg, and the composition has a potency of from about 20,000 to about 30,000 f-SRC units / mg, or from about 175,000 to about 300,000 f-GPA units / mg.

20. 2. The collagenase composition of claim 1, wherein when the collagenase composition is injected into a patient population, the injection results in at least 5% of patients maintaining a level of improvement in the CR-PCSS and / or the PR-PCSS for at least 251 days following the injection.

21. 21. The collagenase composition of claim 20, wherein at least 10% of said patients maintain a level of improvement in said CR-PCSS and / or said PR-PCSS for at least 251 days following said injection.

22. 21. The collagenase composition of claim 20, wherein at least 20% of said patients maintain a level of improvement in said CR-PCSS and / or said PR-PCSS for at least 251 days following said injection.

23. 10. The collagenase composition of claim 1, wherein when said collagenase composition is injected into a patient population, at least 5% of patients show an increasing degree of improvement over time as a result of said injection.

24. 2. The collagenase composition of claim 1, wherein injection of the collagenase composition improves or reduces the following efficacy outcomes as measured by CR-PCSS and / or PR-PCSS: a. at least a 2-level improvement in severity measured from baseline on Day 1 on the hip clinician-assessed live CR-PCSS on Days 22, 43, 90, 180, or 365; b. An improvement of at least 2 levels in severity measured from baseline on Day 1 on the PR-PCSS assessed by the patient while viewing digital images of the buttocks on Days 22, 43, 90, 180, or 365; c. improvement as indicated by a 2-level composite response at Days 22, 43, 90, 180, or 365, defined as patients with at least a 2-level improvement from baseline in the CR-PCSS and at least a 2-level improvement from baseline in the PR-PCSS; d. at least one level improvement in severity measured from baseline on Day 1 on the hip clinician-assessed live CR-PCSS on Days 22, 43, 90, 180, or 365; e. at least one level improvement in severity measured from baseline on Day 1 on the PR-PCSS assessed by the patient while viewing digital images of the buttocks on Days 22, 43, 90, 180, or 365; f. Improvement as indicated by a one-level composite response at Days 22, 43, 90, 180, or 365, defined as patients with at least one level of improvement from baseline in the CR-PCSS and at least one level of improvement from baseline in the PR-PCSS; and g. In a patient population in which all patients had moderate or severe CR-PCSS or PR-PCSS ratings at baseline, there is a statistically significant improvement compared to placebo in at least one treatment domain, and the improvement is one or more of a. through f. above; The collagenase composition according to claim 1, wherein the collagenase composition produces at least one of the following results:

25. 10. The collagenase composition of claim 1, wherein injection of the collagenase composition achieves the following efficacy outcomes as measured by a depression assay: a. At least a 5% reduction in depth; b. A decrease in width of at least 5%; c. At least a 5% decrease in length; d. The overall volume is reduced by at least 5%; e. A reduction in surface area of ​​at least 5%; The collagenase composition provides at least one of the following:

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