Compositions and methods for treating cellulite

Abstract

To provide a method of reducing the severity of cellulite on both buttocks in a human subject.SOLUTION: The present invention provides a method comprising a. step of providing a collagenase composition having at least two of the following twelve characteristics: i. Vmax (min-1) of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay); ii. KM of about 4.1 to 410 nanomolar (SRC assay), or about 0.03 to 3.1 mM (GPA assay); iii. Kcat (sec-1) of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay); and b. step of injecting a therapeutically effective amount of the collagenase composition into dimples in both buttocks, wherein an improvement in the appearance of cellulite is assessed by one or more scales.SELECTED DRAWING: Figure 26

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 733,046, filed Sep. 18, 2018; U.S. Provisional Application No. 62 / 788,916, filed Jan. 6, 2019; U.S. Provisional Application No. 62 / 812,036, filed Feb. 28, 2019; U.S. Provisional Application No. 62 / 823,596, filed Mar. 25, 2019; International Application No. PCT / US2019 / 041494, filed Jul. 11, 2019; and International Application No. PCT / US2019 / 41718, filed Jul. 12, 2019, the entire disclosures of each of which are hereby incorporated by reference in their entireties to the maximum extent permitted by law.

[0002] The present invention relates to the field of evaluation and treatment of cellulite.

Background Art

[0003] Cellulite, also known as edematous fibrosclerotic panniculopathy (EFP), is a cosmetic condition that can be understood as an imbalance between the structural and biomechanical properties of the subcutaneous junction (i.e., delicate containment and extrusion forces) (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). Therefore, the goal of cellulite treatment is to strengthen the subcutaneous interface and / or release the fibrous septa through various types of excision (Rudolph et al., supra). Fibrous septa have been recognized as a contributing underlying cause of cellulite and as a target for cellulite treatment through anatomical and image analysis studies (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 Resance 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ら、, "Cellulite: from standing fat herniation to hypodermal stretch marks," Am J Dermatopath.2000;22(1):34-37; Querleux ら、"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 method is required that destroys, such as by dissolving, the dermal septum (Figure 1) composed of collagen, which is the cause of skin depressions, a source of concern for many women.

[0004] There have been treatments utilized to treat cellulite, but there are no approved pharmacological treatments. Despite the existence 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 cellulite include weight loss, topical agents, massage, liposuction, mesotherapy, radiofrequency, excision, power excision, laser treatment, etc. 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 hold the view that improving the aesthetic condition is not easily achievable. Therefore, a safe and effective non-surgical treatment method for improving the aesthetic results of women with cellulite remains an unmet need.

SUMMARY OF THE INVENTION

[0005] The present disclosure relates to a method for treating cellulite in a human patient by subcutaneous injection of a therapeutically effective amount of collagenase (defined in the detailed description). Such a method relates to evaluating the severity of the patient's cellulite prior to treatment using various scales and evaluation techniques to establish a baseline for the severity of the cellulite in the patient. Thereafter, treatment of the cellulite by subcutaneous injection of collagenase is performed. The dosage of collagenase varies, and the collagenase may be in the form of a pharmaceutical composition comprising collagenase and one or more pharmaceutically acceptable excipients. Such excipients include sterile water for injection, pH adjusters, tonicity adjusters, stabilizers, and the like. The post-treatment evaluation is performed to confirm the effectiveness of the treatment as compared to the baseline. The treatment method of the present disclosure results in a significant reduction in the appearance of cellulite.

[0006] As will be explained in the detailed description, there are four stages to the treatment, but they are optional and the order is not intended to be strictly limited. 1. In the first stage, the clinician selects the cellulite depression to be treated. Next, prior to injection, an evaluation is performed. For example, the clinician and / or patient independently evaluate the severity of the cellulite prior to treatment using one or more of the following scales or other evaluation methods (defined in the detailed description). 〇 Hexcel Cellulite Severity Scale (CSS) (Hexcel CSS) 〇 Hexcel Depression Depth Score 〇 Likert Scale 〇 Dimple Analysis 〇 CR-PCSS (Clinician-Reported Photo Numerical Cellulite Severity Scale) 〇 PR-PCSS (Patient-reported Photonumeric Cellulite Severity Scale) 〇 Investigator Global Aesthetic Improvement Scale (I-GAIS) 〇 Subject Global Aesthetic Improvement Scale (S-GAIS) 〇 Patient-reported Cellulite Impact Scale (PR-CIS) 〇 Abbreviated PR-CIS 〇 Subject Self-rating Scale (SSRS) 〇 Subject Satisfaction with Cellulite Treatment (SSCT) 〇 Clinician Assessment of Cellulite Severity (photographs or other images) 〇 Body-Q 〇 Fitzpatrick Scale 〇 Thigh Cellulite Severity - Patient (TCS-P), Thigh Cellulite Severity - Clinician (TCS-C) 〇 Validated photonumeric or other scales 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 General Meeting 2019))

[0007] Furthermore, pretreatment evaluations by clinicians and patients can be performed by analyzing 1 - 15 photographs, illustrations, drawings, computer images, 3D models, MRI images, thermograms, ultrasounds, and patients' verbal feedback, etc., each having a different cellulite severity assessment or level.

[0008] 2. In the second stage of treatment, the clinician makes dots or other markings over the depression to be treated (Figure 6). This is typically done directly beneath the depression if it is present. Additionally, photographs may be taken and other evaluations may be performed.

[0009] 3. In the third stage of treatment, a therapeutically effective amount of collagenase is subcutaneously injected into the depression in a single or divided dose at one or more treatment sites (as defined in the detailed description). The dosage and injection technique vary. For example, the method may include an injection according to the following procedure. ● Inject the collagenase composition (such as CCH) subcutaneously using a 30-gauge syringe with a 1 / 2-inch needle while the subject is lying face down. As shown in Figure 7 (hereinafter, "Treatment I"), at each injection site, 0.1 mL aliquots are injected into the skin three times at positions A, B, and C for a total of 0.3 mL of the collagenase composition. The depth of injection is 1 / 2 inch corresponding to the length of the treatment needle from the tip to the base, with no downward pressure applied. At each injection site, the needle is inserted with the tip positioned perpendicular (90°) to the skin surface, and the collagenase composition is injected in 0.1 mL aliquots (position A). The needle is slightly withdrawn (but not removed from the skin), repositioned at a 45° deviation from the vertical on the long axis of the depression, and 0.1 mL aliquots of the collagenase composition are injected (position B, in the direction of the head). The needle is again slightly withdrawn and repositioned at a position approximately 45° from the vertical below the long axis of the depression, and 0.1 mL aliquots of the collagenase composition are injected (position C, in the direction of the feet). After injection, the subject remains face down 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 over three treatment sessions, each spaced at least 21 days (+ / - 3-day window) apart. For example, a cumulative dose of 5.04 mg can be administered (i.e., 3 treatment visits × 0.84 mg per treatment area × 2 treatment areas). Other techniques are described in the detailed description.

[0011] 4. In the fourth stage of treatment, post-injection evaluation is performed using the above-mentioned scales and other evaluation methods (e.g., scar analysis). The effectiveness of a particular collagenase treatment may be based on the evaluation of a single clinician or the patient, or on a composite evaluation item consisting of the clinician's evaluation and the patient's evaluation. Here, when improvement is shown on both scales for the same subject, i.e., when improvement at a predetermined level is shown on both the clinician's scale and the patient's scale.

[0012] Collagenase is injected in an amount of about 0.01 mg to about 20 mg as a single dose or divided doses and has one or more of the following characteristics. ● V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 - 410 nanomoles (SRC assay), or about 0.03 - 3.1 mM (GPA assay) M ● k of about 1.1 - 107 (SRC assay), or about 93 - 9,179 (GPA assay) cat (sec -1 ) ● 1 / k of about 376 - 37,222 (SRC assay), or about 4 - 428 (GPA assay), microseconds cat 、microseconds ● k / K of about 5,140 - 508,814 (SRC assay), or about 60 - 5,934 (GPA assay), mM cat / K M 、mM -1 sec -1 ● A molecular weight of about 60 kDa to 130 kDa, or about 70 - about 130 kDa, or about 80 - 120 kDa, or about 90 - 120 kDa, or about 100 - 110 kDa ● The purity of the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 - 30,000 SRC units / mg ● Potency of about 5,000 to about 30,000 f-SRC units / mg ● Potency of about 100,000 to about 400,000 GPA units / mg ● Potency of about 175,000 to about 500,00 f-GPA units / mg ● Potency of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less in area ratio ● Bioburden of 1 cfu / mL or less As used herein, the relevant kinetic parameters can be measured using the cuvette assay or microplate assay described herein (e.g., SRC cuvette assay, SRC microplate assay, GPA cuvette assay, and 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 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0, or 0:1. Each of collagenase I and collagenase II may have a purity by area measured by reverse-phase HPLC 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%.

[0014] In another embodiment, the collagenase composition has a CCH (defined in the detailed description) having 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 by area measured by reverse phase HPLC 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%.

[0015] In other examples, the collagenase composition may be liquid or may be reconstituted from a lyophilized solid form with a diluent. 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 dosage 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 divided doses over about 12 times. 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 in total per treatment visit (as defined in the detailed description). In certain embodiments, the above dosages are for a collagenase composition containing CCH. In another embodiment, the above dosages are for a collagenase composition having one or more of the following characteristics. ● V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● About 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) of K M ● About 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) of K cat (seconds -1 ) ● About 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) of 1 / K cat , microseconds ● About 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) of K 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 of the area ratio measured by reverse-phase HPLC (high-performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● A titer of about 175,000 to about 500,00 f-GPA units / mg ● A titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

[0016] In another embodiment, about 0.84 mg of CCH is injected evenly in about 12 divided doses per treatment site (about 0.07 mg × 12 = about 0.84 mg). In some cases, such treatment with 0.84 mg is done in one treatment visit, or is done every 10 - 40 days in 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 more than 5 treatment visits.

[0017] Furthermore, as described in the detailed description, collagenase injection is effective in the treatment of cellulite. For example, a significant improvement in the appearance of cellulite is demonstrated by the Hexel pit depth score, the Likert scale score, and the depression analysis.

[0018] The most common side effects of CCH injection are injection site reactions such as bruising, pain, nodules, itching, swelling, hardness, discoloration, redness, etc. Bruising at the injection site generally decreases with each additional 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 will be understood from the foregoing 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 combination are not mutually inconsistent. Further, any feature or combination of features can 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 includes at least one drawing drawn in color. Copies of this patent or patent application publication, which include a color drawing, are provided in the country upon payment of the request and the necessary fees.

[0021] The features of the foregoing embodiments will be more readily understood by referring to the following detailed description with reference to the accompanying drawings.

[0022]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

DETAILED DESCRIPTION OF THE INVENTION

[0023] Here, various aspects and embodiments are 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 is thorough and complete and fully conveys 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. Definitions Unless otherwise defined, all terms and phrases used herein shall have the meaning ascribed to them in the art, provided that the contrary is not clearly indicated or is not apparent from the context in which the term or phrase is used. Any methods and materials similar or equivalent to those described herein can be used in the practice or study of the present invention, but specific methods and materials are described herein.

[0025] Unless otherwise specified, the use of individual numerical values is described as approximate values, as if the words "about" or "approximately" were prefixed to those numerical values. Similarly, the numerical values of the various ranges specified in this application are described as approximate values, as if the words "about" or "approximately" were prefixed to both the minimum and maximum values within those ranges, unless otherwise explicitly stated. In this way, fluctuations above and below the described ranges can be used to obtain substantially the same results as the values within the ranges. In this specification, the terms "about" and "approximately" when referring to numerical values shall have their plain and ordinary meanings to those having ordinary knowledge of the art most closely related to the disclosed subject matter, or to the art related to the scope or element in question. How much to broaden from the strict numerical boundaries depends on many factors. For example, the importance of the element, and / or the effect that a given amount of variation has on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art, are taken into account. In this specification, using different numbers of significant figures for different numerical values does not mean restricting the use of the words "about" or "approximately" from serving to broaden a particular numerical value or range. Generally, the words "about" or "approximately" have the effect of broadening the numerical value. Also, the disclosure of a range is intended to include the continuous range of all values between the minimum and maximum values, as well as the broadening of the range obtained by the use of the words "about" or "approximately". Accordingly, the description of numerical ranges in this specification is intended to serve merely as a shorthand reference for individually referring to each individual value within the range, and each individual value is incorporated into this specification as if it were individually described herein.

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

[0027] As used herein, "adverse event" or "AE" means an unfavorable or unintended change in body structure (signs), body function (symptoms), test results (chemical tests, electrocardiograms, X-ray tests, etc.), or exacerbation of a pre-existing condition temporarily associated with the use of the investigational drug, regardless of whether it is 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 shaping their body and losing weight. It measures three areas: appearance, health-related quality of life (HRQL), and patients' medical experience using a scale with 18 independent functions. For the patient-reported outcome instrument, see BODY-Q: User Guide BODY-Q: User Guide, Version 1.0, July 2015, Memorial Sloan Kettering Cancer Center, McMaster University, and Stefan Cano. Body-Q includes a scale for measuring 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 concerned" within a one-week time frame, assuming a Fresh Kincaid reading level. Patients are asked the following question: "Thinking about cellulite, how concerned have you been about it during the past week": [16 questions follow, and patients rank their answers as 1 - very concerned, 2 - moderately concerned, 3 - slightly concerned, 4 - not at all concerned]. The score range is from 16 (very concerned) to 64 (not at all concerned).

[0029] Conventionally, acne clinical examinations have been performed by evaluating the medical, surgical, and concomitant medication histories of the subject in addition to visual examinations of the acne area and its surroundings. The results of this interpretation are subjective and are affected by several irrelevant factors such as the viewing angle, ambient lighting, color of the surrounding skin that is not exposed, the experience and eyesight of the observer, etc. As used herein, "acne analysis" means applying uniformly to all target images the objective image capture and tracking methods disclosed in U.S. Patent Publication No. 2019 / 0035080 to detect visible changes in the skin color evaluated from images of the subject's collagenase-treated area. This objective analysis provides the ability to quantify, distinguish, and evaluate acne at both levels within a subject (within the same subject at different time points) and between subjects (between different subjects), thereby assisting or potentially replacing the visual and clinical examinations of acne by healthcare providers. In this analysis, the L * a * b * color space defined by the International Commission on Illumination (CIE) based on the color opponency theory that "two colors, red and green, or yellow and blue, cannot be represented simultaneously" is used. As shown in the figure below, L * represents lightness, a * represents the red / green coordinate, and b * represents the yellow / blue coordinate. The delta (ΔL * ) of L * , the delta (Δa * ) of a * , and the delta (Δb * ) of b * can be either positive (+) or negative (-). However, the delta E (ΔE * ), which is the sum of the differences, is always positive. ● ΔL * (subtracting L * standard from L * sample) = difference in lightness (+ = lighter, - = darker), small numbers (0 - 50) are dark, and large numbers (51 - 100) are light. ● Δa * (subtracting a * standard from a * sample) = difference between red and green (+ = more red, - = more green) ● Δb* (b * Subtract b from the sample * )(Standard) = Difference between yellow and blue (+ = more yellow, - = more blue) To fully represent the color of an object (here, the mole shown in the image of the subject's treatment area), these three values are required. L * a * b * An objective methodology for image analysis of the collagenase treatment area (pre - and post - treatment images at the time points specified in the protocol), which outputs data in terms of these values, enables the quantification of skin color and its changes quickly, easily, accurately, repeatedly, and without bias. In this method, the inherent variations associated with the conventional subjective visual estimation of images are eliminated. ΔE is calculated as follows. ΔE (Color difference between tissue with mole and normal tissue) = SQRT[(L * B - L * N ) 2 +(A * B - A * N ) 2 +(B * B - B * N ) 2 , where L * B = L of tissue with mole * L * N = L of normal tissue * A * B = A of tissue with mole *. A * N = A of normal tissue * B * B = B of tissue with mole * B * N = B of normal tissue * FIG. 18(B) shows the analysis results of the scars in the treatment area.

[0030] As used herein, "CCH" is a mixture of AUX-I (SEQ ID NO: 5 (FIG. 2)) and AUX-II (SEQ ID NO: 6 (FIG. 3)) collagenases, mixed at an approximate ratio of 1:1 obtained by fermentation of Clostridium histolyticum (also known as Hathewaya histolytica). CCH is commercially available as a lyophilized powder under the trademark XIAFLEX (registered trademark), which is a mixture of AUX-I and AUX-II with specific excipients added, but CCH may also 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) used by physicians / clinicians, and is designed to evaluate the severity of cellulite in five levels.

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

[0033] As used herein, "concavity analysis" means the analysis of one or more selected concavities in which parameters such as the volume, length, width, and surface area of the concavity are measured. The measurements are those described in Eckhouse et al., WO2018 / 116304 and WO2018 / 116305, Cherry Imaging (www.cherryimaging.com) and Canfield Scientific, Inc., as well as Salameh et al., "Novel Stereoscopic Optical System for Objectively Measuring Above-Surface Scar Volume - First-Time Quantification of Responses to Various Treatment Modalities," Dermatol. Surg. 00:1-7 (2017); and U.S. Patent No. 9,996,923. Such measurements of volume, length, width, and surface area may be calculated using digital 3D grayscale images (with X and Y axis rotation capabilities) and digital 3D texture and illuminated images (with X and Y axis rotation capabilities) together with a computer program that analyzes such images. As an example, in the treatment area of the buttocks, images of the left treated buttock and / or the right treated buttock can be taken for each patient before and after treatment. For the treatment site of the thigh, images of the treatment site of each thigh at 0 degrees, 45 degrees, and 90 degrees can be taken before and after treatment. For the treatment area of the thigh, images taken using the method of Canfield Scientific may be taken for each of the treatment sites of the thigh at 0 degrees, 45 degrees, and 90 degrees before and after treatment.

[0034] As used herein, "durability" means the period during which the therapeutic effect persists. 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 may 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 of a subject who has completed the study is regarded as the early termination visit, and the evaluation that is normally conducted on the 71st day for a subject who has completed the study is performed at the early termination visit.

[0036] As used herein, the "Fitzpatrick scale" means a scale used to evaluate the skin type of a subject, as shown in Table 1.

Table 1

[0037] As used herein, the "Hexel cellulite severity scale" or "Hexel CSS" or "Cellulite severity scale (CSS)" means the following photo numerical scale for evaluating five major morphological features of cellulite (Table 2).

Table 2

[0038] As used herein, the "Hexel depression depth score" means evaluating only the depth (B) of the depression from the Hexel CSS (Figure 4). 0 = no depression 1 = superficial depression 2 = moderately deep depression 3 = deep depression

[0039] As used herein, the "image" or "image" means a photograph, illustration, drawing, model, 3D model, computer-generated image, MRI image, etc.

[0040] As used herein, the "Likert scale score" means a score in which an independent blinded evaluator (or patient) compares a photograph of cellulite (2D color, 3D color, 3D grayscale) at baseline on day 1 with a photograph at the time of the visit after treatment at each visit after treatment to identify changes in the treatment area (buttocks or thigh). The score is captured on the following 5-point Likert scale. TIFF2025081344000005.tif72164

[0041] As used herein, "non-target thigh" or "non-target buttock" means a thigh or buttock that has not been selected for evaluation of the primary efficacy assessment item. Such non-target areas can still be treated and used for secondary efficacy assessment.

[0042] "Optional" or "optionally" means that the element, component, or situation described thereafter may or may not occur, and the description includes both the case where the element, component, or situation occurs and the case where it does not occur.

[0043] As used herein, the "Patient-Reported Cellulite Impact Scale (PR-CIS)" is a tool that evaluates the visual and emotional impact of cellulite (satisfied with the appearance of cellulite, concerned, aware, ashamed, looking older, looking overweight, looking out of shape) through six questions, each answered on a numerical scale from 0 (not at all) to 10 (very much). More specifically, the PR-CIS consists of six static questions that assess the visual and emotional impact of cellulite (satisfied with the appearance of cellulite, concerned, aware, ashamed, looking older, looking overweight, looking out of shape). For each question, the subject views a digital image of their buttocks or thighs and answers on an 11-point numerical scale (or interval) from 0 (not at all) to 10 (very much). This evaluation may be done for all thighs and / or buttocks combined, rather than for each area individually. The total score of the PR-CIS is derived from six individual questions. Question 1: Considering the area selected for treatment, how satisfied are you with the appearance of your cellulite? Question 2: Considering the area selected for treatment, how bothered are you by the appearance of your cellulite? Question 3: Considering the area selected for treatment, how aware are you of the appearance of your cellulite? Question 4: Considering the area selected for treatment, how ashamed are you of the appearance of your cellulite? Question 5: Considering the area selected for treatment, how much older do you look because of your cellulite? Question 6: Considering the area selected for treatment, do you look overweight or out of shape because of your cellulite?

[0044] As used herein, the "Patient-Reported Cellulite Impact Scale (abbreviated form)" (PR-CIS abbreviated form) is a tool that evaluates the visual and emotional impact of cellulite (being satisfied with the appearance of cellulite, being concerned about it, being aware of it, being ashamed of it, looking overweight, looking out of shape) using five questionnaire items. Each question is answered on a numerical rating scale from 0 (not at all) to 10 (very much). More specifically, the PR-CIS abbreviated form consists of five static questions that assess the visual and emotional impact of cellulite (being satisfied with the appearance of cellulite, being concerned about it, being aware of it, being ashamed of it, looking overweight, looking out of shape). For each item, the subject views a digital image of the buttocks or thighs and answers on an 11-point numerical rating (or interval) scale from 0 (not at all) to 10 (very much). This evaluation may be done for all thighs and / or buttocks combined, rather than for each individual area separately. As a non-limiting example, the total score of the PR-CIS abbreviated form can be derived from the five individual questions. Question 1: Considering the area selected for treatment, how satisfied are you with the appearance of your cellulite? Question 2: Considering the area selected for treatment, how bothered are you by the appearance of your cellulite? Question 3: Considering the area selected for treatment, how aware are you of the appearance of your cellulite? Question 4: Considering the area selected for treatment, how ashamed are you of the appearance of your cellulite? Question 5: Considering the area selected for treatment, does your cellulite make you look overweight or out of shape? The total score of the PR-CIS abbreviated form can be derived from five other question sets in the full PR-CIS.

[0045] As used herein, the "Patient-reported Photometric Cellulite Severity Scale (PR-PCSS)" is a photometric scale described in PCT patent application PCT / US2018 / 020551 (published as WO2018 / 160905 on September 7, 2018) used by patients, and is designed to evaluate the severity of cellulite in five levels.

[0046] As used herein, "photometric" means using a series of photographs, illustrations, drawings, models, 3D models, computer-generated images, MRI images, images, etc., to each of which different levels of cellulite severity are assigned a scale.

[0047] As used herein, "sequential visits" means two or more clinician visits or times at which changes in cellulite are evaluated by a scale. The time between visits may be about two weeks, three weeks, about one month, about two months, about three months, about four months, about five months, about six months, about one year, about 18 months, about two years, about three years, about four years, or about five years or more.

[0048] As used herein, "severe adverse event" means an adverse event that results in death, an adverse event that immediately threatens life, an adverse event that necessitates or prolongs hospitalization, an adverse event that results in permanent or substantial disability, an adverse event that results in congenital abnormalities / birth defects, or an adverse event that is considered a significant medical event.

[0049] As used herein, "statistically significant" generally means statistical data having a "P" value of less than 0.05. In the context of this disclosure, clinical studies are generally designed to verify the superiority of an intervention (e.g., in this case, a treatment) compared to a control. Since clinical studies involve people who are physiologically different from each other, it is natural for the results to vary. Therefore, statistics are used to determine whether the observed differences are due to chance or due to the treatment method itself. The measurement of statistical significance quantifies the probability that the research results are due to chance. The "P" value, which is commonly used in the measurement of statistical significance, represents the probability that the research results are due to chance rather than an actual treatment effect. Generally, the conventional cutoff value for the "P" value considered to be statistically significant is 0.05 (5%), but it may vary depending on the study design and results. If the "P" value is less than 0.05, it means that the probability that the research results are due to chance is less than 5%. If the "P" value is greater than 0.05 (5%), the difference between the treatment group and the control group is not statistically significant, meaning that the difference is less likely to be due to the treatment and may instead be due to chance.

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

[0051] As used herein, the "Subject Global Aesthetic Improvement Scale (S-GAIS)" and the "Investigator Global Aesthetic Improvement Scale (I-GAIS)" mean the following scales for evaluating the severity and / or degree of improvement of cellulite. The subject is asked the following introductory question: "How would you evaluate the appearance of cellulite after treatment?" The evaluation is carried out in the range from -3 (very deteriorated) to +3 (very improved) according to the subject's answer, as shown in Table 3.

Table 3

[0052] As used herein, "Subject Satisfaction with Cellulite Treatment" (SSCT) means a subject satisfaction evaluation in the range of -2 to +2. As an example, Table 4 below provides such an evaluation for cellulite treatment of the buttocks. The patient is asked "To what extent are you satisfied with the results of the cellulite treatment you received today?", and is asked to select a response / evaluation as shown in Table 4. [Table 4]

[0053] Table 5 shows such an evaluation for cellulite treatment of the thighs. The patient is asked "To what extent are you satisfied with the results of the cellulite treatment of the specific area or areas of the thighs that you received treatment for today?", and is asked to select a response / evaluation as shown in Table 5. [Table 5]

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

[0055] As used herein, "target thigh" or "target buttock" means the thigh or buttock selected to evaluate the main efficacy evaluation item.

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

[0057] As used herein, the term "treatment course" means three treatment sessions (i.e., each time visiting a clinician for treatment).

[0058] As used herein, the term "treatment-emergent adverse event" or "TEAE" means a condition that did not exist before treatment with the investigational drug but appeared after treatment, a condition that existed at the start of treatment but worsened during treatment, or a condition that existed at the start of treatment but resolved during treatment and reappeared (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") mean the photo numerical scale (or a substantially similar scale) shown in Figure 5 used by a patient (TCS-P) or a clinician (TCS-C) to evaluate the severity, improvement, and / or patient satisfaction of thigh cellulite and to assist in the evaluation of the effectiveness of collagenase. The way of using the scale reported by the patient is called TCS-P, and the way of using the scale reported by the clinician is called TCS-C.

[0060] As used herein, the terms "treatment visit" or "treatment" or "treatment session" mean that in one office visit, at least one therapeutically effective amount of at least one active agent useful for the treatment of cellulite is injected or treated on the affected area one or more times.

[0061] As used herein, the terms "validated", "efficacy", "validation" mean the process of demonstrating that a particular scale is accurate and reliable, and also include the reproducibility of visual evaluation to ensure that the same results can be consistently obtained. Furthermore, in validation, the accuracy, correctness, and sensitivity of the scale are verified, and it is confirmed that the measurements made by the scale are excellent in reliability, reproducibility, and robustness.

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

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

[0064] C. Stage 1 - Preliminary Evaluation In the first stage of the method for treating cellulite described herein, the clinician selects the cellulite depressions to be treated based on the following criteria. ● The depression must be one that appears clearly and naturally when the patient takes a relaxed pose (standing position with relaxed gluteal muscles) so that it can be confirmed in a photograph. ● The selected depression must be one that the clinician determines has the highest potential to improve the aesthetics of the entire affected area such as the buttocks or thighs. ● The affected area of the patient is photographed in a relaxed state before treatment. ● An evaluation is performed before injection. That is, the clinician and / or the patient independently evaluate the photograph and score the results using one or more of the following scales or evaluation methods. 〇 Hexcel Cellulite Severity Scale (CSS) (Hexcel CSS) 〇 Hexcel Depression Depth Score 〇 Likert Scale 〇 Depression Analysis 〇 Clinician-Reported Photo Numerical Cellulite Severity Scale (CR-PCSS) 〇 Patient-Reported Photo Numerical Cellulite Severity Scale (PR-PCSS) 〇 Investigator Global Aesthetic Improvement Scale (I-GAIS) 〇 Subject Global Aesthetic Improvement Scale (S-GAIS) 〇 Patient-Reported Cellulite Impact Scale (PR-CIS) 〇 Subject Self-Rating Scale (SSRS) 〇 Subject Satisfaction with Cellulite Treatment (SSCT) 〇 Thigh Cellulite Severity - Patient (TCS-P), Thigh Cellulite Severity - Clinician (TCS-C) 〇 Body-Q 〇 Fitzpatrick Scale 〇 Validated photo numeric or other scales (e.g., Hexel-Merz scale) used by clinicians and / or patients to evaluate cellulite severity, improvement, and / or patient satisfaction

[0065] D. Stage 2 - Marking of Depressions to be Treated In the second stage of the treatment described herein, the clinician can mark the depressions to be treated with dots (plural). Additionally, photos may be taken. See, for example, FIGS. 6 and 15.

[0066] E. Stage 3 - Collagenase Injection In the third stage of treatment, the clinician performs a collagenase injection.

[0067] 1. Types of Collagenase Collagenases useful in the present disclosure include any of the collagenases defined above. As further background, matrix metalloproteinases (MMPs) can be composed of collagenases that fall within the definitions 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. Kinetics of Collagenase Enzyme The collagenase useful in the present disclosure can also be characterized by its enzyme kinetics. Here, the approximate kinetic values of one or more collagenases effective in treating Celite include the following: ● V of about 0.08 - 7.70 (SRC assay) or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 - 410 nanomolar (SRC assay) or about 0.03 - 3.1 mM (GPA assay) M ● kcat of about 1.1 - 107 (SRC assay) or about 93 - 9,179 (GPA assay) cat (sec -1 ) ● 1 / kcat of about 376 - 37,222 (SRC assay) or about 4 - 428 (GPA assay), microseconds cat ● kcat / Km of about 5,140 - 508,814 (SRC assay) or about 60 - 5,934 (GPA assay), mM cat / Km M sec -1 -1 V max = maximum velocity K M = [substrate] at 50% of V max K cat = number of substrate molecules cleaved per second 1 / K cat = microseconds required to cleave one molecule of substrate These values may be determined experimentally by varying the substrate and time using the microplate assay described below. Other assays and parameters may also be employed.

[0069] This value quantitatively represents the behavior of the enzyme based on the Michaelis - Menten equation: TIFF2025081344000010.tif41165

[0070] Here, V 0 is the reaction rate (velocity) at substrate concentration [S], V​​​max is the maximum observable speed, K M is V max is the Michaelis constant correlated with the substrate concentration at which 50% of V is obtained. TIFF2025081344000011.tif24127

[0071] Here, k 1 and k -1 and k 2 are the rate constants of the following steps. TIFF2025081344000012.tif42155

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

[0073] The catalytic constant K cat is 、 the turnover number, that is, the speed at which the ES complex reaches E + P. This reflects the number of catalytic cycles performed by each active site per unit time.

[0074] In certain embodiments, AUX - I and AUX - II have the following characteristics. ● AUXI 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomolar 〇 K cat , sec -1 : about 1.1 - 107 〇 1 / K cat , μs: about 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 - 508,814 ● AUXII 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 - 30.5 〇 K M 、 mM: about 0.03 - 3.1 〇 K cat 、 seconds -1 : about 93 - 9,179 〇 1 / K cat 、 microseconds: about 4 - 428 〇 K cat / K M 、 mM -1 seconds -1 : about 60 - 5,934 Preconditions K cat =V max / [AUX] = (n moles of 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 to a product and reflects both binding and catalytic events. In another embodiment, AUX-I and AUX-II have the following characteristics. TIFF2025081344000013.tif110159

[0075] 3. Regarding the Titer (Specific Activity) of Collagenase Assays have been developed and used to determine the specific activity (titer) of collagenase. Such assays are described in subsections a. through c. and characterize collagenase by its ability to convert a substrate to a product within a predetermined time at a predetermined enzyme concentration. In certain non-limiting embodiments, these assays are used to determine the titer of each of AUX-I and AUX-II, as well as the complex CCH formulation (1:1 ratio of AUX-I and AUX-II). In the SRC assay described below, collagen is used as the substrate for the reaction. The SRC assay uses soluble rat (tail) collagen (SRC) as the 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 the type I digestion of SRC. The reaction is carried out at pH 7.2 and 25 °C for 2.5 hours using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer containing 15 mM divalent calcium ions.

[0076] The bovine tendon collagen (BTC) assay (described below) is based on the procedures of Mandl et al., Arch. Biochem. Biophys. 74: 465-475 (1958), and Arch. Biochem. Biophys. 101: 81-88 (1963), revised by Keller and Mandl. See also Rosen, Arch. Biochem. Biophys. 67: 10-15 (1957). The BTC assay uses insoluble bovine tendon collagen as the substrate and measures the activity of both type I and type II (such as AUX-I and II collagenase). The BTC assay uses a colorimetric method utilizing ninhydrin to detect peptides generated by the type I and type II degradation of BTC. This reaction is also carried out at pH 7.2 but is carried out at 37 °C for 22 hours in tris(hydroxymethyl)aminomethane (TRIS) buffer containing 10 mM divalent calcium ions.

[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 mainly used to measure type II activity, and type I activity is about 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 at pH 7.2 and 25 °C for 10 minutes using HEPES buffer containing 100 mM divalent calcium ions.

[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 of using a low molecular weight hexapeptide clearly defined as a substrate, and two clearly defined tripeptides are produced. In addition, the GPA method emits a fluorescent signal and has high sensitivity. The GPA method is suitable for Michaelis-Menten kinetic analysis because it uses a single substrate and reaction conditions (10-minute incubation) approximating the initial velocity of the enzyme. The SRC method is suitable for collagen-degrading enzymes with a collagen-binding domain, and the GPA method is suitable for collagen-degrading enzymes without a collagen-binding domain called gelatinase.

[0079] a. Assay method and specific activity unit of GPA unit i. Collagenase titer measured by GPA assay (cuvette) The GPA assay is mainly used to measure the activity of class II collagenase. In the first step of the assay, an enzymatic reaction is included that involves the 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). In the second step, the released GPA is subsequently measured 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 the enzymatic cleavage of zGPGGPA (hereinafter, "substrate") by the collagenase. Subsequently, the collagenase-treated sample (containing the released GPA) and the leucine standard sample are each treated with fluorescamine for a certain period of time at room temperature to fluorescently label the free amino groups of the generated GPA and leucine molecules. After excitation at 392 nm, the fluorescence emission of each solution is measured at 480 nm. Using the slopes of the obtained leucine and collagenase sample curves, the activity unit is calculated as follows: Activity (f-GPA units / mg) = (M サンプル / M ロイシン ) × (DF / T) Here, M サンプル = slope of the collagenase sample activity curve M ロイシン = slope of the leucine standard curve DF = dilution rate T = reaction time

[0081] Additional non-limiting details regarding the assay method for GPA are as follows. Buffers and Reagents 1. f-Appell buffer, pH 7.2 (55 mM HEPES, 100 mM calcium acetate) 2.1 mM Leucine Working Stock Solution 3. 200 mM Borate, pH 9.0 4. 0.5 mM Fluorescamine Solution in Acetone 5. 2 mg / mL zGPGGPA Substrate in f-Appel's Buffer

[0082] Solution Preparation Solutions are prepared as follows. f-Appel's Buffer: Dissolve 13.0 g of HEPES and 17.6 g of calcium acetate in approximately 800 mL of water. Adjust the pH to 7.2 with sodium hydroxide and QS to 1 L with water. Store at 2 - 8 °C. 10 mM Leucine Stock Solution: Dissolve 65.5 mg of leucine in 50 mL of water. Leucine needs to be weighed directly into a 100 mL (or equivalent) glass beaker. Weigh approximately 65 mg (target weight) of leucine into the beaker. Based on the weight of the weighed leucine, calculate the amount of water to add to the beaker using the following formula. Add the calculated amount of water to the beaker, confirm that the leucine is completely dissolved, and mix well. Aliquot into 1 mL portions. Store at 20 °C or below. TIFF2025081344000014.tif20123 Here C2 = Mass of weighed leucine (mg) V1 = 50 (mL of water) C1 = 65.5 (mg of leucine) V2 = Amount of water required to produce 10 mM stock solution (mL) 1 mM Leucine Working Stock Solution: Thaw the vial of 10 mM leucine stock solution and dilute to 1 mM by combining 150 μL with 1350 μL of water. Mix well before use. 0.5 N HCl: Dilute HCl with water to 0.5 N and mix well. Store at room temperature. Commercially available 0.5 N hydrochloric acid can also be used. 200 nM Borate, pH 9.0: Dissolve 2.4 g of boric acid in approximately 150 mL of water. Adjust the pH to 9.0 using 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 shake to dissolve. Store protected from light at 2 - 8 °C. Substrate Solution (2 mg / mL zGPGGPA): Prepare 2 mg / mL of the substrate in f - Appel's buffer. Dissolve it with a mechanical shaker / rotator over sufficient time until completely dissolved (about 15 minutes).

[0083] Leucine Standard Curve The standard curve of leucine is prepared according to Table 7. [Table 7] Then, transfer 100 μL of each leucine standard into separate tubes and detect fluorescamine.

[0084] Collagenase Sample Preparation Dilute the collagenase sample to 0.01 mg / mL in two steps with f - Appel's buffer and gently vortex to mix. An example of the dilution method is shown below. 1. 100 μL × 1.0 mg / mL → 1000 μL = 0.1 mg / mL 2. 100 μL × 0.1 mg / mL → 1000 μL = 0.01 mg / mL

[0085] Blank Preparation The blank is 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 the zGPGGPA substrate solution and mix well by vortexing. Transfer 100 μL of each blank into separate tubes to detect impurities that may react with fluorescamine.

[0086] Titer Curve For each collagenase sample, prepare a set of titer curves as follows. Warm the tube containing 53160 matrix and buffer in a 25°C water bath for 15 minutes or more. Label the second set of tubes and add 50 μL of 0.5 N hydrochloric acid to each. Add the diluted collagenase sample preparation (0.01 mg / mL) to the tubes according to Table 8, incubate for 10 minutes, mix, and return to the 25°C water bath. Start the incubation period when the first preparation is added to the pre-warmed matrix.

Table 8

[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 solution). Vortex well to mix. Incubate the tubes at room temperature for at least 10 minutes.

[0088] Fluorometer Settings Set the fluorometer using the following instrument parameters and read the fluorescence of each preparation with 1 hour of derivatization. TIFF2025081344000018.tif77161

[0089] Calculation Plot the concentration (X-axis) of each leucine standard against the fluorescence response (Y-axis) at 480 nm. Slope (m) and coefficient of determination (R 2) is determined. The average fluorescence of the preparations of each titer curve is determined. The titer curves of the collagenase sample and leucine are created by plotting the concentration (X-axis) of each preparation against the average fluorescence response at 480 nm (Y-axis). The slope (m) and coefficient of determination (R 2 ) are determined.

[0090] Measurement of the titer of the collagenase sample 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 the collagenase sample titer curve M ロイシン = slope of the leucine standard curve DF = dilution rate (1100 μL / 50 μL = 22) T = reaction time (10 minutes)

[0091] ii. GPA microplate assay for measuring class II collagenase activity in the collagenase sample This method is the same as the above GPA assay, except that it is performed in a microplate. Similar to the above assay, the microplate assay measures the proteolytic activity of the collagenase sample in the enzymatic cleavage of the substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-prolyl-L-alanine (zGPGGPA) (hereinafter referred to as "substrate"). This assay follows the following methodology, but those skilled in the art will understand that specific modifications (e.g., dilution concentration and time) can be made to achieve the purpose of the assay.

[0092] Reagents 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 18.2 MΩ system or equivalent) 5. 1 M HEPES buffer (Gibco 15630-080 or equivalent) 6. Surfact-Amps20 (trademark) (10% Tween solution) (Pierce Cat.#28320 or equivalent) 7. 1 M 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. 2.5 N NaOH (J.T Baker 5666-02 or equivalent) 10. 0.5 N hydrochloric acid (VWR 101223-134 or equivalent) 11. Acetone (Sigma 270725 or equivalent)

[0093] Preparation of solutions (i) Preparation of assay buffer (50 mM HEPES pH 7.1 / 0.05% Tween20 / 5 mM (Ca(C 2 H 3 O 2 ) 2 ): Pipette 50 mL of 1 M HEPES into 800 mL of pure water. Add 5 mL of 1 M (Ca(C 2 H 3 O 2 ) 2 ) and 5 mL of Surfact-Amps (10% Tween20). Check the pH and adjust to 7.1 ± 0.05 if necessary. Add sufficient water to adjust the volume to 1 L 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) of tripeptide GPA in 10 mL of 0.1 N NaOH, and vortex until completely dissolved. Add a sufficient amount of assay buffer to make 100 mL, aliquot 0.5 mL, and store at -70 °C. The 4 mg / mL tripeptide GPA stock can be stored at -70 °C for 1 year. (iv) Preparation of 4 mg / mL (6.8 mM) Peptide Substrate zGPGGPA: Dissolve 400 mg (+1 mg) of peptide substrate zGPGGPA in 10 mL of 0.1 N NaOH, and vortex until completely dissolved. Add a sufficient amount of assay buffer to make 100 mL. This solution can be stored at 4 °C for 3 months. (v) Preparation of 120 mM Boric Acid pH 9.0: Dissolve 7.4 g (±0.5 g) of boric acid in 800 mL of pure water. Titrate this solution with NaOH to pH 9.0, and add a sufficient amount of pure water to adjust the volume to 1 liter. This solution can be stored at room temperature for a maximum of 3 months. (vi) Preparation of 1 mM Fluorescamine in Acetone: Dissolve 28 ± 2 mg of fluorescamine in 100 mL of acetone. This solution needs to be freshly prepared and protected from light and moisture.

[0094] Preparation and Serial Dilution of Tripeptide GPA Standard The 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 (for example, add 20 μL of 4 mg / mL tripeptide GPA to 980 μL of assay buffer). In column A of the assay plate, pipette 200 μL of the 329 μM tripeptide GPA standard into A1 and A7. Pipette 100 μL of assay buffer into A2 - A6 and A8 - A12. For the serial dilution of the tripeptide GPA standard, transfer 100 μL from A1 to A2 and mix, then transfer 100 μL from A2 to A3, and repeat until A5. Take out 100 μL from the well of A5 to make the final volume 100 μL. Well A6 contains only buffer. For the serial dilution of the second tripeptide GPA standard solution, transfer 100 μL from A7 to A8 and mix, then transfer 100 μL from A8 to A9, and repeat this until well A11. 100 μL is taken out from the well of A11 to make the final volume 100 μL. Well A12 contains only buffer.

[0095] Preparation of Collagenase Sample In the case of a collagenase sample (e.g., a lyophilized collagenase preparation), let the sample return to room temperature for at least 10 minutes and reconstitute it to form a stock solution of 500 ng / mL. Different concentrations may be used. Dilute the stock solution with assay buffer to prepare a test collagenase sample (T1A). Repeat this procedure to prepare triplicate test samples (T1A, T1B, T1C).

[0096] Discussion In this method, in a 96-well plate, mix 50 μL of a collagenase test sample with increasing concentrations and 50 μL of an excess substrate (final concentration 2.0 mg / mL). Add 50 μL of assay buffer to columns C - G of a U-bottom 96-well polypropylene reaction plate. Then, perform a 1 / 1.5 serial dilution using a multi-channel pipette, transfer 100 μL of the collagenase sample from column B to column C, mix, and repeat until reaching column G. Table 9 shows the final collagenase concentrations after adding 50 μL of substrate from column B to column H. The blank is prepared by pipetting 50 μL of assay buffer into column H, and this row does not contain enzyme. 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 at room temperature. Turn on the incubator and temperature probe (the temperature before adding the substrate to the plate is 22 ± 1 °C). Add 50 μL of 4 mg / mL (6.8 mM) zGPGGPA substrate to each column in columns B - H and mix. The reaction start time begins after the substrate is added to the first column. Cover the plate and place it in an incubator at 22 ± 1 °C for a total reaction time of 15 ± 1 minutes. After incubation, add hydrochloric acid to quench the reaction and react the free amino terminus of the peptide with the fluorogenic reagent fluorescamine to quantify the amount of released GPA peptide. To stop the reaction, add 100 μL of 0.5 N hydrochloric acid to each well from row A to row H for each column and mix. The reaction time ends when HCl is added to the first column.

[0098] Detection Add 195 μL of 120 mM borate pH 9.0 to each well of a black reading plate made of polypropylene in a microplate Greiner. Transfer 30 μL of the quenched reaction mixture from the reaction plate to the corresponding well of the reading plate and mix well. Next, add 75 μL of 1 mM fluorescamine to each well of the reading plate (use a polypropylene tray for dispensing fluorescamine / acetone) and mix immediately after each addition. Read the plate using a Molecular Devices M2 fluorescence plate reader with the following settings within 15 minutes after fluorescamine addition. The plate is read using a Molecular Devices M2 fluorescence plate reader within 15 minutes after fluorescamine addition with the following settings: excitation 380 nm, emission 473 nm, cutoff 455 nm, 6 reads / well, PMT medium. The luminescence at 473 nm was plotted against the concentration of GPA (μM), and the luminescence at 473 nm was plotted against the concentration of collagenase (ng / mL). For each plot, linear regression was fitted without using fixed parameters. For the collagenase test samples, the zero-point data were excluded from the linear fit, and plots were created using the entire triple data set for each sample. The slopes of the tripeptide GPA standard samples and the collagenase samples were determined.

[0099] Determination of titer The specific activity of the collagenase sample can be calculated as follows: GPA microplate assay unit = ((slope of the collagenase sample) / (slope of the tripeptide GPA × incubation time)) × 10 6 The specific activity of the collagenase test sample is determined from the slope of the tripeptide GPA standard and calculated using a curve fitting program. Using the microplate method, the enzyme reaction rate according to Michaelis-Menten can be calculated using different substrate concentrations and different times.

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

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

[0102] Treat such a collagenase and leucine standard sample with a reagent for attaching a fluorescent label to the generated GPA. After incubating the leucine standard sample and the collagenase sample at room temperature for 10 minutes, measure the fluorescence of each solution at excitation wavelengths of 392 nm and 480 nm and emission wavelengths, respectively. Next, using the slopes of the obtained leucine and collagenase sample curves, calculate the titer unit as follows: Titer (f-SRC units / mg) = (M サンプル / M ロイシン ) × (DF / T) × CF Here, M サンプル = Slope of the collagenase sample titer curve M ロイシン = Slope of the leucine standard curve DF = Dilution rate (1500 μL / 100 mL = 15) T = Reaction time (2.5 hours × 60 minutes / 1 hour = 150 minutes) CF = Conversion factor (1000 μg / 1 mg = 1000)

[0103] The following shows 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, pH 7.2 3. 200 mM borate, pH 9.0 4. 10 mM leucine stock solution 5. 1 mM leucine working stock solution 6. 1 mM fluorescamine solution in acetone 7. 2 mg / mL rat tail tendon collagen in 0.02 N acetic acid

[0105] Solution Preparation Solutions are prepared as follows:

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

[0107] F-enzyme buffer: Dilute the F-TC assay buffer by combining 4 mL with 16 mL of water. Store at 2 - 8 °C.

[0108] 10 mM leucine stock solution: Dissolve 65.5 mg of leucine in 50 mL of water. Leucine needs to be weighed directly into a 100 mL (or equivalent) glass beaker. Weigh approximately 65 mg (target weight) of leucine into the beaker. Based on the weight of the weighed leucine, calculate the amount of water to add to the beaker using the following formula. Add the calculated amount of water to the beaker, confirm that the leucine is completely dissolved, and mix well. Aliquot into 1 mL. Store at -20 °C or below. TIFF2025081344000020.tif21132 Here, C2 = mass of weighed leucine (mg) V1 = 50 (mL of water) C1 = 65.5 (mg of leucine) V2 = amount of water required to produce 10 mM stock solution (mL)

[0109] 1 mM leucine working stock solution: Thaw the vial of 10 mM leucine stock solution and dilute to 1 mM by combining 150 μL with 1350 μL of water. Mix well before use.

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

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

[0112] 200 mM Borate, pH 9.0: Dissolve 2.4 g of boric acid in approximately 150 mL of water. Adjust the pH to 9.0 using 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, shake to dissolve. Store protected from light at 2 - 8 °C.

[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] The standard curve of leucine is prepared according to Table 10.

Table 10

[0117] Subsequently, 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 to 0.01 mg / mL in two steps with F - enzyme buffer and gently vortexed to mix. An example of the dilution method is shown below. 1. 100 μL × 1.0 mg / mL → 1000 μL = 0.1 mg / mL 2. 100 μL × 0.1 mg / mL → 1000 μL = 0.01 mg / mL The diluted samples are stored at room temperature until use.

[0120] Blanks are prepared according to Table 11. First, combine the sample with 0.5 N hydrochloric acid to inactivate the enzyme, then add buffer and substrate.

[0121] Put the collagenase sample into the tubes labeled according to Table 11. Tubes 1, 2, 4, and 6 were prepared in one preparation, and tubes 3, 5, and 7 were prepared in the second preparation.

Table 11

[0122] Cap the tubes and mix gently by vortexing. Incubate the titer curve in a water bath at 25 °C ± 3 °C for 2.5 hours. After the incubation is complete, remove the tubes for titer measurement from the water bath. Add 750 μL of 0.5 N hydrochloric acid to each preparation and mix well by vortexing. The prepared samples can be stored at 2 - 8 °C for up to 22 hours before detection.

[0123] Settings of the detector and fluorometer

[0124] The leucine standard sample is prepared as described above.

[0125] Set the luminescence spectrometer with the following device parameters and read the fluorescence of each preparation after 1 hour of derivatization. TIFF2025081344000023.tif79163

[0126] Calculation Plot the concentration (X-axis) of each leucine standard substance against the fluorescence reaction (Y-axis) at 480 nm. Determine the slope (m) and the coefficient of determination (R 2 ). Do not force it to zero. Determine the average 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 (X-axis) of the collagenase sample in each preparation against the net fluorescence (Y-axis). Determine the slope (m) and the coefficient of determination (R 2 ). Do not force it to zero. Measurement of the titer of the collagenase sample Titer (f-SRC units / mg) = (M サンプル / M ロイシン ) × (DF / T) × CF Here, M サンプル = Slope of the collagenase sample titer curve M ロイシン = Slope of the leucine standard curve DF = Dilution rate (1500 μL / 100 mL = 15) T = Reaction time (2.5 hours × 60 minutes / 1 hour = 150 minutes) CF = Conversion factor (1000 μg / 1 mg = 1000)

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

[0129] ii. SRC microplate assay for measuring class I collagenase activity in a collagenase sample This method is the same as the above SRC assay, except that it is performed in a microplate. Similar to the above SRC assay, the microplate assay measures collagenase activity against a soluble rat tail tendon collagen (SRC) substrate (hereinafter referred to as the "substrate"). This assay follows the following methodology, but those skilled in the art will understand that specific modifications can be made to achieve the purpose of the assay.

[0130] Reagents 1. Soluble rat collagen substrate (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. 1 M HEPES buffer (Gibco 15630-080 or equivalent) 6. 1 M calcium acetate (Ca(C 2H 3 O 2 ) 2 )(Emerald Biosciences EBS - 100 - CAAC or equivalent) 7. Surfact - Amps 20 (trademark) (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 (J.T Baker 5666 - 02 or equivalent) 12. Acetone (Sigma 270725 or equivalent)

[0131] Preparation of Solutions (i) Preparation of Assay Buffer (50 mM HEPES pH7.1 / 0.05% Tween20 / 5 mM (Ca( 2 H 3 O 2 ) 2 ): Pipette 50 mL of 1 M HEPES into 800 mL of pure water. Add 5 mL of 1 M (Ca( C2 H 3O2 ) 2 ) and 5 mL of Surfact - Amps (10% Tween20). Check the pH and adjust to 7.1 ± 0.1 if necessary. Add sufficient water to adjust the volume to 1 L 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 0.1N NaOH: Add 2 mL of 2.5N 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) of tripeptide GPA in 10 mL of 0.1 N NaOH and vortex until completely dissolved. Add a sufficient amount of assay buffer to make 100 mL, aliquot 0.5 mL, and store at -70 °C. The 4 mg / mL tripeptide GPA stock can be stored at -70 °C for up to 1 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 a sufficient amount of purified water to adjust to 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 of 0.02 N acetic acid directly to the vial containing the substrate (in a non-limiting example, supplied as 100 mg of 3.75 mg / mL SRC). Other concentrations of SRC substrate may be used. The calculation is as follows:

[0132] 100 mg ÷ 3.75 mg / mL = 26.7 mL;

[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] This solution is thoroughly mixed by inverting and can be stored at 2 - 8 °C for up to 3 months. (vi) Preparation of 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, add 1.8 mL of 2 mg / mL SRC substrate stock solution, and mix by inverting. This solution needs to be prepared immediately before adding to the plate. (vii) Preparation of 120 mM boric acid pH 9.0: Dissolve 7.4 g (±0.5 g) of boric acid in 800 mL of pure water. Titrate this solution to pH 9.0 with NaOH and add a sufficient amount of DI water to adjust 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 needed to be freshly prepared and protected from light and moisture.

[0136] Preparation and serial dilution of tripeptide GPA standard A 0.08 mg / mL (329 μM) tripeptide GPA standard is prepared by diluting a 4 mg / mL tripeptide GPA stock 50-fold with assay buffer (e.g., add 20 μL of 4 mg / mL GPA to 980 μL of assay buffer). In column A of the assay plate, pipette 200 μL of the 329 μM tripeptide GPA standard into A1 and A7. Pipette 100 μL of assay buffer into A2 - A6 and A8 - A12.

[0137] For the serial dilution of the tripeptide GPA standard, transfer 100 μL from A1 to A2 and mix, then transfer 100 μL from A2 to A3 and repeat up to A5. Take out 100 μL from the well of A5 to make the final volume 100 μL. Well A6 contains only buffer.

[0138] For the second serial dilution of the tripeptide GPA standard, transfer 100 μL from A7 to A8 and mix, then transfer 100 μL from A8 to A9 and repeat up to A11. Take out 100 μL from the well of A11 to make the final volume 100 μL. Well A12 contains only buffer.

[0139] Preparation of collagenase test samples

[0140] In the case of a collagenase sample (e.g., a lyophilized collagenase preparation), allow the sample to equilibrate to room temperature for at least 10 minutes 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 pieces (T1A). Repeat this procedure to prepare triplicate test samples (T1A, T1B, T1C).

[0141] Discussion

[0142] In this method, in a 96-well plate, 50 μL of a test collagenase sample with increasing concentrations is mixed with 50 μL of an excess substrate (final concentration 0.2 mg / mL). The amount of 50 μL of assay buffer is added to columns C-G of a U-bottom 96-well polypropylene reaction plate. Next, using a multi-channel pipette, serial dilutions of 1 / 1.5 are performed, transferring 100 μL of the collagenase sample from column B to column C, mixing, and repeating until column G is reached. 100 μL is removed from column G and discarded, and a blank is prepared in column H by pipetting 50 μL of assay buffer into it. This column contains no enzyme. Table 12 contains the final collagenase concentrations after adding 50 μL of substrate from column B to column H. [Table 12]

[0143] Collagenase reaction Turn on the incubator and temperature probe (the temperature before adding the substrate to the plate is 22 ± 1 °C). Add 50 μL of 0.6 mg / mL SRC substrate to each well from column B to column H, column by column, and mix. The reaction start time begins after the substrate is added to the first column. Cover the plate and place it in an incubator at 22 ± 1 °C for a total reaction time of 45 ± 5 minutes. To quench the reaction, add 100 μL of 0.5 N HCl to each well of the dilution plate, column by column, and mix. The reaction time ends when HCl is added to the first column.

[0144] Detection

[0145] Add 195 μL of 120 mM borate pH 9.0 to each well of a Greiner polypropylene black reading plate microplate. Transfer 30 μL of the quenched reaction mixture from the reaction plate to the corresponding well of the reading plate and mix well. Next, add 75 μL of 1 mM fluorescamine to each well of the reading plate (using a polypropylene tray for dispensing fluorescamine / acetone) and mix immediately after each addition. Within 15 minutes after fluorescamine addition, read the plate using a Molecular Devices M2 fluorescence plate reader with the following settings. The plate is read with a Molecular Devices M2 fluorescence plate reader within 15 minutes after fluorescamine addition with the following settings: excitation 380 nm, emission 473 nm, cutoff 455 nm, 6 reads / well, PMT medium.

[0146] The emission at 473 nm was plotted against the concentration of GPA (μM), and the emission at 473 nm was plotted against the concentration of collagenase (ng / mL). For each plot, a linear regression was fitted without using fixed parameters. For the collagenase samples, the zero-point data were excluded from the linear fit, and plots were created using the entire triple data set for each sample. Determine the slopes of the tripeptide GPA standard samples and the collagenase samples.

[0147] Determination of specific activity and relative titer The specific activity of the collagenase sample can be calculated as follows: SRC microplate assay unit = ((slope of the collagenase sample) / (slope of the tripeptide GPA × incubation time)) × 10 6

[0148] The specific activity of the collagenase test sample 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 according to Michaelis-Menten can be calculated using different substrate concentrations and different times.

[0149] iii. Collagenase titer measured by SRC assay The collagenase useful in the present disclosure may have a titer of about 500 to about 15,000 SRC units / mg. In certain embodiments, the titer is about 500 to about 12,500 SRC units / mg, or about 700 to about 10,000 SRC units / mg, or about 1,000 to about 7,500 SRC units / mg, or 1,500 to about 6,000 SRC units / mg, or about 2,500 to about 5,000 SRC units / mg. Alternatively, the titer 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. Also, the collagenase may 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 the BTC unit assay The Bovine Tendon Collagen Assay for collagenase is based on the procedure of Mandl et al. (1958) and has been modified by Keller and Mandl (1963). Since the collagen of bovine tendon is an insoluble substrate, it is important to divide it finely. Trypsin is used as a control considering the presence of denatured collagen and other protein impurities. This assay is performed in the presence of calcium ions necessary for collagenase activity. The number of solubilized peptides is determined by reacting the N-terminal amino group of the peptide with ninhydrin and colorimetrically measuring the amount of the formed adduct (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 substrate (collagen) 2. Deionized water (water) 3. Tris assay buffer 4. Trypsin stock solution 5. 0.5 M HCl 6. Leucine standard assay solution (1 mM leucine) 7. Rosen buffer 8. 3% Ninhydrin 9. 50% Isopropanol

[0153] Incubation Set up reaction tubes and label them as follows: 3 tubes for trypsin control, 6 tubes for standard solution, and 6 tubes for each test sample. Label each tube and remove the cap. Weigh 10 ± 1 mg of collagen in the order shown in Table 13 and put the weighed collagen into each reaction tube.

Table 13

[0154] The enzyme amount of 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 higher.

[0155] Set up the reaction tubes according to Table 14 to form a matrix pattern. The following table assumes 2 under test samples. Adjust the number of reaction tubes if the number of samples is large or small, but maintain the pattern. If the volume is constant, it is described in Table 14.

Table 14

[0156] Cap the reaction tube. Gently but thoroughly mix the contents. Place the reaction tube in a 37 °C water bath. Incubate for 22 ± 0.5 hours. Record the actual time (37 °C) when incubation started, the number of the water bath used, the lot number of the collagen Lipid, the collagen correction factor for the lot used, and the lot numbers of all solutions used.

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

[0158] Remove the caps from the reaction tubes and discard the caps. Dispense 2 mL of 0.5 M HCl into each reaction tube to stop the reaction. Thoroughly mix the contents of each tube. Filter the contents of each reaction tube into the appropriate filtrate tube.

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

[0160] Color Development Set up and label the boiling tubes as follows: 6 tubes for water and leucine control (step 1), 2 tubes for each filtrate tube (step 1). Place the following amounts of water and leucine standard assay solution in the 6 leucine control tubes. TIFF2025081344000027.tif36154

[0161] Put 0.8 mL of water into each boiling tube (Step 2). Inject 0.2 mL of the filtrate of each sample into a labeled centrifuge tube. Inject 0.5 mL of Rosen buffer into each centrifuge tube. Under the hood, put 0.5 mL of 3% ninhydrin into each centrifuge tube. Mix the contents of each tube thoroughly with a vortex mixer. Place the boiling tubes in a boiling water bath in the fume hood. Boil for 15 ± 1 minutes. When the boiling time is over, remove the boiling tubes from the water bath. Under the containment hood, dispense 5.0 mL of 50% isopropanol into each boiling tube and mix the contents thoroughly. Before reading the absorbance, let the boiling tubes return to room temperature (at least 10 minutes).

[0162] Reading of absorbance While working under the containment hood, read the absorbance of the tubes. Turn on the spectrophotometer and let it warm up. Set the wavelength of the spectrophotometer to 570 nm. Zero the spectrophotometer with 50% isopropanol. Read the absorbance (A 570 ) of the controls of water, leucine, trypsin and the samples being tested. Record the time when the first sample was read in time units. Record the reading as 1000×A 570 and record the time when the last sample was read in time units. All readings should be taken within 1 hour.

[0163] Calculation principle Calculate the total reading time and the total incubation time in minutes. It is desirable that the total reading time is less than 60 minutes and the total incubation time is 1290 - 1350 minutes. Using the linear least squares method, calculate the slope "b" and the correlation coefficient "r" of the leucine standard sample (x = nmol leucine vs y = A 570 reading). The unit of the "b" value is A 570 / nmol leucine. The b value of leucine should be between 2.88 and 3.33. Calculate the average value of the trypsin control (T). The average reading value of the trypsin control (T) should be 221 - 338. Record this average value with digits after the decimal point (Step A). Duplicate samples A 570 of each reaction tube are averaged. Record this value in units after the decimal point. Average sample A 570 Subtract the average trypsin (Step A) from the reading value of to obtain the net sample reading value.

[0164] Next, calculate the activity per tube (ACT) in nmol leu eq / min as follows. TIFF2025081344000028.tif14143Here, 20 is the dilution factor for the amount of the developed reaction mixture, and "b" is the slope of the leucine standard curve. Record this value to one digit after the decimal point. The activity per tube of the sample being tested should be 1.6 - 5.7 nmol leu eq / min.

[0165] Calculate the activity in BTC units as follows: BTC unit = activity (nmol leu eq / min) × collagen correction factor

[0166] Calculate the activity of the sample in BTC units / mL as follows: TIFF2025081344000029.tif16129

[0167] Calculate the specific activity of the sample in BTC units / mm as follows: TIFF2025081344000030.tif14131

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

[0169] i. BTC units and ABC units A variety of collagenase compositions can be employed, where the collagenase has a specific activity of from about 5,000 BTC units / mg to about 25,000 BTC units / mg, or from 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" means the amount of collagenase(s) present in the composition (different from excipients and other components).

[0170] Furthermore, a variety of collagenase compositions can be employed, where the collagenase has a specific activity of from about 5,000 ABC units / mg to about 25,000 ABC units / mg, or from 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" means the amount of collagenase(s) present in the composition (different from excipients and other components).

[0171] d. Other assays Assay methods 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. Dosage of Collagenase Regarding the dosage of collagenase employed in this specification, in the present disclosure, a therapeutically effective amount of collagenase sufficient to bind and dissolve the septum upon subcutaneous injection is provided, such that the appearance of cellulite is reduced compared to the pre-treatment baseline.

[0173] In one embodiment, collagenase can be injected in an amount of about 0.01 mg to about 20 mg, either as a single injection or in divided doses. In another embodiment, collagenase may be injected in an amount of about 0.05 mg to about 15 mg, either as a single or divided administration. In another embodiment, collagenase may be injected in an amount of about 0.10 mg to about 10 mg, either as a single or divided administration. In another embodiment, collagenase may be injected in an amount of about 0.15 mg to about 5 mg, either as a single or divided administration. In another embodiment, collagenase may be injected in an amount of about 0.20 mg to about 3 mg, either as a single or divided administration. In another embodiment, collagenase may be injected in an amount of about 0.25 mg to about 2 mg, either as a single or divided administration.In yet another embodiment, the collagenase can be injected in an amount of 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 1.55 mg, about 1.60 mg, about 1.65 mg, about 1.70 mg, about 1.75 mg, about 1.80 mg, about 1.85 mg, about 1.90 mg, about 1.95 mg, about 2.00 mg, about 2.05 mg, about 2.10 mg, about 2.15 mg, about 2.20 mg, about 2.25 mg, about 2.30 mg, about 2.35 mg, about 2.40 mg, about 2.45 mg, about 2.50 mg, about 2.55 mg, about 2.60 mg, about 2.65 mg, about 2.70 mg, about 2.75 mg, about 2.80 mg, about 2.85 mg, about 2.90 mg, about 2.95 mg, about 3.00 mg, about 3.05 mg, about 3.10 mg, about 3.15 mg, about 3.20 mg, about 3.25 mg, about 3.30 mg, about 3.35 mg, about 3.40 mg, about 3.45 mg, about 3.50 mg, about 3.55 mg, about 3.60 mg, about 3.65 mg, about 3.70 mg, about 3.75 mg, about 3.80 mg, about 3.85 mg, about 3.90 mg, about 3.95 mg, about 4.00 mg, about 4.05 mg, about 4.10 mg, about 4.15 mg, about 4.20 mg, about 4.25 mg, about 4.30 mg, about 4.35 mg, about 4.40 mg, about 4.45 mg, about 4.50 mg, about 4.55 mg, 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 V of about 2.6 minutes -1 to 5.2 minutes -1 when measured using the SRC assay. In another embodiment, the collagenase has a V of about 3.0 minutes max to 5.0 minutes -1 when measured using the SRC assay. In another embodiment, the collagenase has a V of about 3.4 minutes -1 to 4.8 minutes max when measured using the SRC assay. In yet another embodiment, the collagenase has a V of about 3.5 minutes -1 to 4.5 minutes -1 when measured using the SRC assay. In yet another embodiment, the collagenase has a V that is about 2.0 minutes max when measured using the SRC assay, about 2.1 minutes -1 when measured using the SRC assay, about 2.2 minutes -1 when measured using the SRC assay, about 2.3 minutes max when measured using the SRC assay, about 2.4 minutes max when measured using the SRC assay, about 2.5 minutes -1 when measured using the SRC assay, about 2.6 minutes -1 when measured using the SRC assay, about 2.7 minutes -1 when measured using the SRC assay, about 2.8 minutes -1 when measured using the SRC assay, about 2.9 minutes -1 when measured using the SRC assay, about 3.0 minutes -1 when measured using the SRC assay, about 3.1 minutes -1 when measured using the SRC assay, about 3.2 minutes -1 when measured using the SRC assay, about 3.3 minutes -1 when measured using the SRC assay, about 3.4 minutes -1 when measured using the SRC assay, about 3.5 minutes -1 when measured using the SRC assay, about 3.6 minutes -1 when measured using the SRC assay, about 3.7 minutes -1 when measured using the SRC assay, about 3.8 minutes -1 when measured using the SRC assay, about 3.9 minutes -1 when measured using the SRC assay, about 4.0 minutes -1 when measured using the SRC assay, about 4.1 minutes -1 when measured using the SRC assay, about 4.2 minutes -1 when measured using the SRC assay, about 4.3 minutes -1 when measured using the SRC assay, about 4.4 minutes -1 when measured using the SRC assay, about 4.5 minutes -1 when measured using the SRC assay, about 4.6 minutes -1 when measured using the SRC assay, about 4.7 minutes -1 when measured using the SRC assay, about 4.8 minutes -1 when measured using the SRC assay, about 4.9 minutes -1 when measured using the SRC assay, about 5.0 minutes -1 when measured using the SRC assay, about 5.1 minutes -1 when measured using the SRC assay, about 5.2 minutes -1, about 4.8 minutes -1 , about 4.9 minutes -1 , about 5.0 minutes -1 , about 5.1 minutes -1 , about 5.2 minutes -1 , about 5.3 minutes -1 , about 5.4 minutes -1 , about 5.5 minutes -1 , about 5.6 minutes -1 , about 5.7 minutes -1 , about 5.8 minutes -1 , about 5.9 minutes -1 , or about 6.0 minutes -1 may have. In another embodiment, when measured using the SRC assay, collagenase is about 0.7 minutes -1 ~ 7.6 minutes -1 , or about 1 - 6, or about 2 - 5, or about 3 - 4 minutes -1 of V max can have.

[0175] In one embodiment, when measured using the GPA assay, collagenase is about 135 minutes -1 ~ 268 minutes -1 of V max may have. In another embodiment, when measured using the GPA assay, collagenase is about 150 minutes -1 ~ 250 minutes -1 of V max may have. In another embodiment, when measured using the GPA assay, collagenase is about 175 minutes -1 ~ 225 minutes -1 of V max can have. In yet another embodiment, when measured using the GPA assay, collagenase is about 130 minutes -1 , about 135 minutes -1 , about 140 minutes -1 , about 145 minutes -1 , about 150 minutes -1 , about 155 minutes -1 , about 160 minutes -1 , about 165 minutes -1 , about 170 minutes -1 , about 175 minutes -1 , about 180 minutes -1 , about 185 minutes -1 , about 190 minutes-1 , about 195 minutes -1 , about 200 minutes -1 , about 205 minutes -1 , about 210 minutes -1 , about 215 minutes -1 , about 220 minutes -1 , about 225 minutes -1 , about 230 minutes -1 , about 235 minutes -1 , about 240 minutes -1 , about 245 minutes -1 , about 250 minutes -1 , about 255 minutes -1 , about 260 minutes -1 , about 265 minutes -1 , about 270 minutes -1 , about 275 minutes -1 , or about 280 minutes -1 of V max can have. In another embodiment, when measured using a GPA assay, collagenase is about 4 minutes -1 ~400 minutes -1 , or about 0.3~30.5, or about 10~375, or about 20~350, or about 50~300, or about 100~275 minutes -1 of V max may have.

[0176] In one embodiment, when measured using an SRC assay, collagenase has a K of about 75 mM~147 mM m may have. In another embodiment, when measured using an SRC assay, collagenase has a K of about 80 mM~140 mM m may have. In another embodiment, when measured using an SRC assay, collagenase has a K of about 85 mM~130 mM m may have. In another embodiment, collagenase has a K of about 90 mM~120 mM as measured using an SRC assay mIt may have. In yet another embodiment, when measured using the SRC assay, the collagenase has a K 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 110 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 It may have. In another embodiment, when measured using the SRC assay, the collagenase has 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 nanomoles m It may have.

[0177] In one embodiment, when measured using the GPA assay, the collagenase has a K of about 0.03 mM to 3.1 mM m It may have. In another embodiment, when measured using the GPA assay, the collagenase has a K of about 1.00 mM to 1.60 mM m It may have. In another embodiment, when measured using the GPA assay, the collagenase has a K of about 1.10 mM to 1.50 mM m It may have. In another embodiment, when measured using the GPA assay, the collagenase has a K of about 1.15 mM to 1.40 mM mIt may have. In yet another embodiment, when measured using the GPA assay, collagenase has a K 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 It may have. In another embodiment, when measured using the GPA assay, collagenase has 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 m It may have.

[0178] In one embodiment, collagenase has a K of about 36 seconds -1 ~671 seconds -1 when measured using the SRC assay. cat It may have. In another embodiment, collagenase has a K of about 50 seconds -1 ~600 seconds -1 when measured using the SRC assay. cat It may have. In another embodiment, collagenase has a K of about 60 seconds -1 ~500 seconds -1 when measured using the SRC assay. cat It may have. In another embodiment, collagenase has a K of about 70 seconds -1 ~400 seconds -1 when measured using the SRC assay. cat It may have. In yet another embodiment, when measured using the SRC assay, collagenase has a K of about 100 seconds -1 ~350 seconds -1 when measured using the SRC assay. catmay have. In another embodiment, when measured using the SRC assay, collagenase is about 30 seconds -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 of K cat may have. In another embodiment, the collagenase has a K of about 1 second to 107 seconds -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 of K cat may have.

[0179] In one embodiment, the collagenase has a K of about 90 to 10,000, or about 41,000 seconds -1 ~about 81,000 seconds -1 of K cat may have. In another embodiment, the collagenase has a K of about 45,000 seconds -1 ~about 75,000 seconds -1 of K cat may have. In another embodiment, the collagenase has a K of about 50,000 seconds -1 ~about 70,000 seconds -1 of K catIt may have. In another embodiment, when measured using the GPA assay, collagenase has a K of about 55,000 seconds -1 to about 65,000 seconds -1 of K cat It may have. In yet another embodiment, when measured using the GPA assay, collagenase has a K of about 35,000 seconds -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 of K cat It may have. In another embodiment, when measured using the GPA assay, collagenase has a K of about 1215 seconds -1 to 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 of K cat It may have.

[0180] In one embodiment, when measured using the SRC assay, collagenase has a 1 / K of about 376 to 38,000 μs, or about 14,000 μs to about 28,000 μs catmay have. In another embodiment, when measured using the SRC assay, the collagenase has a 1 / K of about 16,000 μsec to about 26,000 μsec cat may have. In one embodiment, when measured using the SRC assay, the collagenase has a 1 / K of about 18,000 μsec to about 24,000 μsec cat may have. In one embodiment, when measured using the SRC assay, the collagenase has a 1 / K of about 20,000 μsec to about 22,000 μsec cat may have. In yet another embodiment, when measured using the SRC assay, the collagenase has a 1 / K 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, about 16,250 μsec, about 16,500 μsec, about 16,750 μsec, about 17,000 μsec, about 17,250 μsec, about 17,500 μsec, about 17,750 μsec, about 18,000 μsec, about 18,250 μsec, about 18,500 μsec, about 18,750 μsec, about 19,000 μsec, about 19,250 μsec, about 19,500 μsec, about 19,750 μsec, about 20,000 μsec, about 20,250 μsec, about 20,500 μsec, about 20,750 μsec, about 21,000 μsec, about 21,250 μsec, about 21,500 μsec, about 21,750 μsec, about 22,000 μsec, about 22,250 μsec, about 22,500 μsec, about 22,750 μsec, about 23,000 μsec, about 23,250 μsec, about 23,500 μsec, about 23,750 μsec, about 24,000 μsec, about 24,250 μsec, about 24,500 μsec, about 24,750 μsec, about 25,000 μsec, about 25,250 μsec, about 25,500 μsec, about 25,750 μsec, about 26,000 μsec, about 26,250 μsec, about 26,500 μsec, about 26,750 μsec, about 27,000 μsec, about 27,250 μsec, about 27,500 μsec, about 27,750 μsec, about 28,000 μsec, about 28,250 μsec, about 28,500 μsec, about 28,750 μsec, about 29,000 μsec, or about 29,250 μsec of 1 / K catIt may have. In another embodiment, when measured using the SRC assay, the collagenase has 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 cat It may have.

[0181] In one embodiment, when measured using the GPA assay, the collagenase has a 1 / K of about 4 μsec to about 430 μsec cat It may have. In another embodiment, when measured using the GPA assay, the collagenase has a 1 / K of about 14 μsec to about 23 μsec cat It may have. In another embodiment, when measured using the GPA assay, the collagenase has a 1 / K of about 16 μsec to about 21 μsec cat It may have. In yet another embodiment, when measured using the GPA assay 、About 10.0 μs, about 10.2 μs, about 10.4 μs, about 10.6 μs, about 10.8 μs, about 11.0 μs, about 11.2 μs, about 11.4 μs, about 11.6 μs, about 11.8 μs, about 12.0 μs, about 12.2 μs, about 12.4 μs, about 12.6 μs, about 12.8 μs, about 13.0 μs, about 13.2 μs, about 13.4 μs, about 13.6 μs, about 13.8 μs, about 14.0 μs, about 14.2 μs, about 14.4 μs, about 14.6 μs, about 14.8 μs, about 15.0 μs, about 15.2 μs, about 15.4 μs, about 15.6 μs, about 15.8 μs, about 16.0 μs, about 16.2 μs, about 16.4 μs, about 16.6 μs, about 16.8 μs, about 17.0 μs, about 17.2 μs, about 17.4 μs, about 17.6 μs, about 17.8 μs, about 18.0 μs, about 18.2 μs, about 18.4 μs, about 18.6 μs, about 18.8 μs, about 19.0 μs, about 19.2 μs, about 19.4 μs, about 19.6 μs, about 19.8 μs, about 20.0 μs, about 20.2 μs, about 20.4 μs, about 20.6 μs, about 20.8 μs, about 21.0 μs, about 21.2 μs, about 21.4 μs, about 21.6 μs, about 21.8 μs, about 22.0 μs, about 22.2 μs, about 22.4 μs, about 22.6 μs, about 22.8 μs, about 23.0 μs, about 23.2 μs, about 23.4 μs, about 23.6 μs, about 23.8 μs, about 24.0 μs, about 24.2 μs, about 24.4 μs, about 24.6 μs, about 24.8 μs, about 25.0 μs, about 25.2 μs, about 25.4 μs, about 25.6 μs, about 25.8 μs, about 26.0 μs, about 26.2 μs, about 26.4 μs, about 26.8 μs, about 27.0 μs, about 27.2 μs, or about 27.4 μs of 1 / K cat may have. In another embodiment, when measured using a GPA assay, collagenase has about 0.3 μs to about 32 μs, or about 1 to 30, or about 5 to 25, or about 10 to 20, or about 15 μs of 1 / K cat may have.

[0182] In one embodiment, when measured using an SRC assay, collagenase has about 5,140 mM -1 seconds -1 to about 508,814 mM -1 seconds -1 of K cat / Km may have. In another embodiment, when measured using the SRC assay, collagenase has a K of about 0.50 mM -1 seconds -1 ~ about 7.75 mM -1 seconds -1 / K cat / K m may have. In another embodiment, when measured using the SRC assay, collagenase has a K of about 0.75 mM -1 seconds -1 ~ about 7.00 mM -1 seconds -1 / K cat / K m may have. In yet another embodiment, when measured using the SRC assay, collagenase has a K of about 1.00 mM -1 seconds -1 ~ about 6.00 mM -1 seconds -1 / K cat / K m may have. In yet another embodiment, when measured using the SRC assay, collagenase has a K of about 0.10 mM -1 seconds -1 about 0.20 mM -1 seconds -1 about 0.30 mM -1 seconds -1 about 0.40 mM -1 seconds -1 about 0.50 mM -1 seconds -1 about 0.60 mM -1 seconds -1 about 0.70 mM -1 seconds -1 about 0.80 mM -1 seconds -1 about 0.90 mM -1 seconds -1 about 1.00 mM -1 seconds -1 about 1.10 mM -1 seconds -1 about 1.20 mM -1 seconds -1 about 1.30 mM -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 seconds -1 and about 4.50 mM -1 seconds -1 and about 4.60 mM -1 seconds -1 and about 4.70 mM -1 seconds -1 and about 4.80 mM -1 seconds -1 and about 4.90 mM -1 seconds -1 and about 5.00 mM -1 seconds -1 and about 5.10 mM -1 seconds -1 and about 5.20 mM -1 seconds -1 and about 5.30 mM -1 seconds -1 and about 5.40 mM -1 seconds -1 and about 5.50 mM -1 seconds -1 and about 5.60 mM -1 seconds -1 and about 5.70 mM -1 seconds -1 and about 5.80 mM -1 seconds -1 and about 5.90 mM -1 seconds -1 and about 6.00 mM -1 seconds -1 and about 6.10 mM -1 seconds -1 and about 6.20 mM -1 seconds -1 and about 6.30 mM -1 seconds -1 and about 6.40 mM -1 seconds -1 and about 6.50 mM -1 seconds -1 and about 6.60 mM -1 sec -1 and about 6.70 mM -1 seconds -1 and about 6.80 mM -1 seconds -1 and about 6.90 mM -1 seconds -1 and about 7.00 mM -1 seconds -1 and about 7.10 mM -1 seconds -1 and about 7.20 mM -1 seconds-1 , about 7.30 mM -1 seconds -1 , or about 7.40 mM -1 seconds -1 of K cat / K m may have. In another embodiment, when measured using the SRC assay, the collagenase is about 0.0048 mM -1 seconds -1 ~ about 0.47 mM -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 of K cat / K m may have.

[0183] In one embodiment, when measured using the GPA assay, the collagenase is about 60 mM -1 seconds -1 ~ about 6,000 mM -1 seconds -1 of K cat / K m may have. In another embodiment, when measured using the GPA assay, the collagenase is about 30,000 mM -1 seconds -1 ~ about 85,000 mM -1 seconds -1 of K cat / K m may have. In another embodiment, when measured using the GPA assay, the collagenase is about 36,000 mM -1 seconds -1 ~ about 77,000 mM -1 seconds -1 of K cat / K m may have. In yet another embodiment, when measured using the GPA assay, the collagenase is about 40,000 mM -1 seconds -1 ~ about 70,000 mM -1 seconds -1 of K cat / K mmay have. In yet another embodiment, when measured using a GPA assay, the collagenase is about 40,000 mM -1 seconds -1 about 42,000 mM -1 seconds -1 about 44,000 mM -1 seconds -1 about 46,000 mM -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 82,000 mM -1 seconds -1 about 84,000 mM -1 seconds -1 about 76,000 mM -1 seconds -1 about 86,000 mM -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 of K cat / K m may have. In another embodiment, when measured using a GPA assay, collagenase is about 900 mM -1 seconds -1 to about 90,000 mM -1 seconds -1 , or about 2,000 - 80,000, or about 10,000 - 70,000, or about 20,000 - 60,000, or about 30,000 - 50,000, or about 40,000 - 45,000 mM -1 seconds -1 of K cat / K m may have.

[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 may have a molecular weight of about 55 kDa, about 57 kDa, about 60 kDa, about 62 kDa, about 65 kDa, about 67 kDa, about 70 kDa, about 72 kDa, about 75 kDa, about 77 kDa, about 80 kDa, about 82 kDa, about 85 kDa, about 87 kDa, about 90 kDa, about 92 kDa, about 95 kDa, about 97 kDa, about 100 kDa, about 102 kDa, about 105 kDa, about 107 kDa, about 110 kDa, about 112 kDa, about 115 kDa, about 117 kDa, about 120 kDa, about 122 kDa, about 125 kDa, about 127 kDa, about 130 kDa, about 132 kDa, about 135 kDa, or about 137 kDa.

[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 contain less than 1% of clostripain by area ratio. In another embodiment, the collagenase may contain less than 1% of gelatinase by area ratio. In another embodiment, the collagenase may contain less than 1% of leupeptin by area ratio. In yet another embodiment, the collagenase may contain a bioburden of 1 cfu / mL or less.

[0186] In one embodiment, the collagenase may have a titer (i.e., specific activity) of about 500 to about 30,000 SRC units / mg. In another embodiment, the collagenase may have a titer of about 2,500 to about 25,000 SRC units / mg. In another embodiment, the collagenase may have a titer of about 5,000 to about 20,000 SRC units / mg. In yet another embodiment, the collagenase may have a titer of about 500, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, about 25,000, about 25,500, about 26,000, about 26,500, about 27,000, about 27,500, about 28,000, about 28,500, about 29,000, about 29,500, or about 30,000 SRC units / mg.

[0187] In one embodiment, the collagenase may have a titer (i.e., specific activity) of about 5,000 to about 30,000 f-SRC units / mg. In another embodiment, the collagenase may have a titer of about 7,500 to about 25,000 f-SRC units / mg. In another embodiment, the collagenase may have a titer of about 10,000 to about 20,000 f-SRC units / mg. In yet another embodiment, the collagenase may have a titer of about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, about 25,000, about 25,500, about 26,000, about 26,500, about 27,000, about 27,500, about 28,000, about 28,500, about 29,000, about 29,500, or about 30,000 f-SRC units / mg.

[0188] In one embodiment, the collagenase may have an activity of about 100,000 to about 400,000 GPA units / mg. In another embodiment, the collagenase may have an activity of about 150,000 to about 350,000 GPA units / mg. In another embodiment, the collagenase may have an activity of about 200,000 to about 300,000 GPA units / mg. In yet another embodiment, the collagenase may have an activity of 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, 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 an activity titer of about 175,000 to about 500,000 f-GPA units / mg. In another embodiment, the collagenase may have an activity titer of about 250,000 to about 450,000 f-GPA units / mg. In another embodiment, the collagenase may have an activity titer of about 300,000 to about 400,000 GPA units / mg. In yet another embodiment, the collagenase may have an activity titer of about 175,000, about 185,000, about 195,000, about 205,000, about 215,000, about 225,000, about 235,000, about 245,000, about 255,000, about 265,000, about 275,000, about 285,000, about 295,000, about 305,000, about 315,000, about 325,000, about 335,000, about 345,000, about 355,000, about 365,000, about 375,000, about 385,000, about 395,000, about 405,000, about 415,000, about 425,000, about 435,000, about 445,000, about 455,000, about 465,000, about 475,000, about 485,000, or about 495,000 f-GPA units / mg.

[0190] In one embodiment, the collagenase may have an activity of about 5,000 to about 25,000 ABC units / mg. In one embodiment, the collagenase may have an activity of about 7,500 to about 20,000 ABC units / mg. In one embodiment, the collagenase may have an activity of about 10,000 to about 17,500 ABC units / mg. In another embodiment, the collagenase may have an activity of about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, or about 25,000 ABC units / mg.

[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 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0, or 0:1. Each of collagenase I and collagenase II may have a purity by area measured by reverse phase HPLC 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%.

[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 by area measured by reverse phase HPLC 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%.

[0193] In other examples, the collagenase composition may be a liquid or may be reconstituted from a lyophilized solid form with a diluent. The dosage of the mixture is measured by the amount of collagenase present without considering the diluent and may include from about 0.1 mg to about 20 mg in one or more injections. In another embodiment, the dosage 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 is injected in divided doses over about 12 injections. The amount of collagenase composition injected may range from 0.01 mL to 3 mL per injection, or from a total of about 0.2 mL to 150 mL per treatment visit. In certain embodiments, the above dosages are for a collagenase composition having a CCH. In another embodiment, the above dosages are for a collagenase composition having one or more of the following characteristics: ● V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● Approximately 4.1 to 410 nanomoles (SRC assay), or approximately 0.03 to 3.1 mM (GPA assay) of K M ● Approximately 1.1 to 107 (SRC assay), or approximately 93 to 9,179 (GPA assay) of K cat (seconds -1 ) ● Approximately 376 to 37,222 (SRC assay), or approximately 4 to 428 (GPA assay) of 1 / K cat , microseconds ● Approximately 5,140 to 508,814 (SRC assay), or approximately 60 to 5,934 (GPA assay) of K cat / K M , mM -1 seconds -1 ● A molecular weight of approximately 60 kDa to approximately 130 kDa, or approximately 70 to approximately 130 kDa, or approximately 80 to approximately 120 kDa, or approximately 90 to approximately 120 kDa, or approximately 100 to approximately 110 kDa ● The purity of the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of approximately 500 to 30,000 SRC units / mg ● A titer of approximately 5,000 to approximately 30,000 f-SRC units / mg ● A titer of approximately 100,000 to approximately 400,000 GPA units / mg ● A titer of approximately 175,000 to approximately 500,00 f-GPA units / mg ● A titer of approximately 5,000 to approximately 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

[0195] In another embodiment, about 0.84 mg of CCH is injected evenly in about 12 divided doses per treatment site (about 0.07 mg x 12 doses = about 0.84 mg). In some cases, such treatment with 0.84 mg is performed every 10 - 40 days in 2, 3, 4, or 5 treatment visits. In other cases, 0.84 mg is injected in a single treatment visit to multiple treatment areas, or is injected 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 aspect, the amount of collagenase that can be injected into the treatment area is about 0.001 mg to 20 mg of collagenase per treatment visit in one or more injections, and can be administered in equal portions, for example, in about 3 to about 100 injections. The collagenase is in liquid form or reconstituted from a lyophilized solid with a diluent. The dosage of collagenase is measured as the amount of collagenase without the diluent and can 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, injected in one or more divided doses.

[0197] In another embodiment, the dosage is administered at about 0.06 mg, 0.48 mg, 0.84 mg, 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg, or 5.04 mg in 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 divided doses. In other examples, the dosage of collagenase is injected in more than 3 divided doses. The amount of collagenase composition injected may range from 0.01 mL to 3 mL per injection, or from about 1 mL to 150 mL in total per treatment visit.

[0198] In one aspect, the above-described mixture of AUX I and II ("CCH") may be injected with collagenase at about 0.01 mg to 10 mg per treatment visit in one or more injections. For example, the dosage may be divided equally and injected in about 3 to about 50 injections. The collagenase may be in liquid form or reconstituted from a lyophilized form with a diluent. The dosage of the mixture is measured by the amount of collagenase without considering 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, 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, or 10 mg is administered in one or more injections. In another embodiment, the dosage 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 the collagenase composition injected can range from 0.01 mL to 3 mL per injection, or from about 1 mL to 80 mL in total per treatment visit.

[0199] The dosage of collagenase can be expressed in mg per injection (again, without considering the 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 embodiments, the present 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 dosage 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, the dosages based on various specific activities are as follows: TIFF2025081344000031.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. Formulations CCH or other collagenase may be in the form of a pharmaceutical preparation having CCH or collagenase and a pharmaceutically acceptable excipient. Such excipients include sterile water for injection, sodium chloride / calcium chloride, pH adjusters, stabilizers, and the like.

[0204] XIAFLEX® is commercially available from the applicant as a single-use glass vial containing 0.9 mg of CCH as a sterile lyophilized powder for reconstitution. A sterile diluent for reconstitution is also provided in a single-use glass vial. As inactive ingredients, hydrochloric acid, sucrose, and tromethamine are included. The diluent contains calcium chloride dihydrate in 0.9% sodium chloride. XIAFLEX® Prescribing Information (2018).

[0205] In another embodiment, the CCH for cellulite is a sterile lyophilized powder containing 0.92 mg of CCH, sucrose, tris, mannitol, and hydrochloric acid, and is contained in a 5 mL vial. The sterile diluent for reconstitution can be composed of individually filling 5 mL vials with water for injection, normal saline, or a dehydrate of 0.6% sodium chloride and 0.03% calcium chloride in water for injection.

[0206] Collagenase or CCH may be filled in vials of other sizes, such as 10 mL, 15 mL, 20 mL, or 30 mL. For other pH adjusters, saccharides, polyols, and stabilizers, see Rowe et al. Handbook of Pharmaceutical Excipients ( 5th Ed).

[0207] 6. Methods of Treatment: Injection Techniques and Administration Regimens The aforementioned collagenase compositions are useful in methods for treating or reducing the severity of cellulite in human subjects. The present disclosure relates to a method for reducing the severity of cellulite in a human patient, comprising the steps of providing a composition having at least one collagenase, and injecting a therapeutically effective amount of said composition into one or more dimples, wherein the patient exhibits a reduction in the severity of cellulite as compared to a pre-treatment baseline level. As will be described in more detail below, the present composition can be administered by various injection techniques and its effectiveness can be measured by a number of scales and other measurement tools.

[0208] Administration of the collagenase composition described herein may be performed on both sides (two thighs or two buttocks) or all four quadrants (both buttocks and both thighs) in a single subject during a treatment visit. Such treatment visits may be performed 2, 3, 4, 5, or 6 times a year at intervals of 10 to 40 days. Also, even when collagenase was injected into all four quadrants at a high cumulative dose, no quantifiable levels of CCH were present in the systemic circulation (i.e., below the lower limit of quantification of the assay) in the bioanalysis detecting the plasma concentration of AUX-I or AUX-II after subcutaneous administration of up to 3.36 mg of CCH, indicating no systemic absorption and further demonstrating overall safety. For example, when 3.36 mg was administered to humans and 43 times the human equivalent dose (HED) was administered to rats, these amounts were found to be well tolerated. The applicant's studies on the pharmacokinetics of collagenase therapy are 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, U.S. Provisional Application No. 62 / 812,036, filed February 28, 2019, U.S. Provisional Application 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 incorporated herein by reference.

[0209] Table 15 shows various injection parameters of collagenase and a general overview of the techniques related to the treatment of patients.

Table 15

[0210] In certain embodiments, about 0.84 mg of CCH is divided equally about 12 times and injected into the affected area such as four quadrants (i.e., the right or left buttock or the right or left thigh) (about 0.07 mg × 12 injections = about 0.84 mg of CCH). In some cases, such treatment of 0.84 mg is performed every 10 - 40 days for 2, 3, 4, 5, or 6 treatments. In other cases, for 2, 3, 4, 5, or 6 treatments, multiple affected areas or quadrants are injected with 0.84 mg every 10 - 40 days.

[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, the patient is administered collagenase as shown in Table 16 using the procedure according to Treatment I (Figure 7).

Table 16

[0213] In this example, the clinician can select each distinct and treatment - suitable gluteal depression while the subject is standing. Note that there are no restrictions on the selection of the depression to be treated. The treatment consists of 12 injections per buttock (24 injections in total for both buttocks) in one treatment visit. Since the purpose of the treatment is to improve the aesthetics of the entire buttock, the physician is instructed to select the depression that is thought to most improve the aesthetics of the entire buttock. It is possible to treat the same depression in one buttock or a different depression from the previously treated one, but it is desirable to inject within the buttock in 3 treatments (12 injections per buttock). Except when there is no treatable EFP depression in the buttock and the clinician evaluates that buttock as 0 on the CR - PCSS, each buttock receives all 3 treatments. Even if no injection is made in a particular buttock (right or left) at treatment visit 2, the subject is evaluated for the treatment of the contralateral buttock at treatment visit 2 and returns at treatment visit 3, and the subject (PR PCSS) and the principal investigator (CR - PCSS) re - evaluate each buttock. If the principal investigator evaluates either or both buttocks as greater than 0 on the CR - PCSS, an injection is made at treatment visit 3. Additionally, the collagenase treatment can include one or more of the following: ● Treat one or more quadrants; ● Treat regardless of the size of the depression; ● Treat women 45 years of age or older; ● Treat depressions without skin sagging, relaxation, or drooping; ● Treat different depressions at different treatment visits; ● Use a needle of 1 / 2 inch or more; ● Do not limit the distance of the depressions to be injected; ● Do not limit the injection position relying on instruments such as spacers, rulers, paper, etc.; ● Ensure that at least one injection is made directly under the depression; ● Treat depressions with a length of less than about 1 cm or more than about 2 cm; ● Use injections within about 2 cm of each other; ● Use injections within a range of less than about 2 cm of each other, and / or ● Measure the effect using one or more of the metrics and methods described herein. In certain embodiments, when the treatment adheres to the above aspects, the patient experiences a rapid response rate to the treatment. See FIGS. 20 - 23.

[0214] Furthermore, in certain specific embodiments, the parameters of the treated patients are shown in Tables 17 and 18. [Table 17] [Table 18]

[0215] In one embodiment, the osmotic concentration of the reconstituted product is about 50 - about 1,000, about 100 - about 900, about 200 - about 800, about 300 - about 700, about 400 - 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 osmotic concentration 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 for treating or reducing EFP in a subject in need thereof, the method providing at least one of the following advantages over the general procedures and treatments for EFP: a. Easy for physicians to use; b. Shorter treatment time; c. Unexpected effects are obtained, even in light of the general view of physicians that it is difficult to improve aesthetic symptoms; d. No need to use hyaluronidase; e. No need to apply heat; f. No need to use a laser; g. No need for subdermal injections; h. No need for anesthesia (regardless of the bruise); i. No need for compression clothing; j. Do not use vacuum.

[0217] In another embodiment, the method of treating or reducing cellulite does not have an upper limit on the severity of the cellulite to be treated. For example, treatment with collagenase is safe and effective regardless of the prevalence or severity of cellulite.

[0218] F. Stage 4 - Treatment evaluation items and measurement of effectiveness The treatment methods described herein are effective in treating cellulite by several scales described below. Generally, as used herein, the term "day" means the study day measured sequentially from the first day of treatment in the treatment course, except when otherwise specified in Example 5 below, which measures days sequentially from the 71st day of the prior study. For example, as described in Example 5, "day 180" means 180 days after the 71st day reported in Examples 2 and 3. Therefore, 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 (except when "day 180" is used to mean 180 days after the 71st day, or 251 days after the first day of treatment, as in Example 5).

[0219] In a particular embodiment, in a population of patients all having moderate or severe CR-PCSS and / or PR-PCSS evaluations: ● In the CR-PCSS clinically evaluated by the clinician of the treatment area of interest, at least 50% of the patients have an improvement in severity of at least 1 level from the baseline at day 22, day 43, or day 71. ● In the PR-PCSS evaluated by the subject while viewing digital images of the treatment area of interest, at least 50% of the patients have an improvement in severity of at least 1 level from the baseline at day 22, day 43, or day 71. ● In the CR-PCSS evaluated live by clinicians in the target treatment area, at least 5% of the patients have an improvement in severity of at least 2 levels from baseline on day 22, day 43, or day 71. ● In the PR-PCSS evaluated by subjects while viewing digital images of the target treatment area, at least 5% of the patients have an improvement in severity of at least 2 levels from baseline on day 22, day 43, or day 71. ● At least 5% of the patients experience a significant reduction in the size (volume, length, width, depth) of the depression, e.g., at least a 5% reduction, or at least a 10% reduction, or at least a 20% reduction from baseline on day 22, day 43, or day 71. These and other efficacy parameters and benchmarks are detailed below. Further, as shown in the following examples, collagenase injection significantly improved the appearance of cellulite, demonstrated persistence, and generally showed good tolerability.

[0220] 1. Efficacy Measured by CR-PCSS and PR-PCSS Improvement in an individual patient means an improvement of at least 1 level or 1 rating from baseline or the previous score or rating. Improvement in a patient group means that the average score or rating has improved by about 0.1 from baseline or the previous average score or evaluation. A responder is any patient who shows an improvement of at least 25% of the maximum total score or evaluation from baseline. In certain embodiments, the treatment methods detailed herein result in one or more of the following efficacy evaluation items measured by CR-PCSS and / or PR-PCSS: 1. The evaluation of CR-PCSS and / or PR-PCSS has improved by at least 0.1 compared to baseline. 2. The severity in the CR-PCSS evaluated live by clinicians in the treatment area has improved by at least 2 levels from baseline (the "day 1" before treatment) on 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. That, while viewing digital images of the treatment area, the severity in the PR-PCSS evaluated by the subject has improved by at least two levels at 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 from the baseline (day 1). 4. The improvement shown by a two-level composite response at 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 is defined as a subject in whom the severity of the CR-PCSS has improved by at least two levels from the baseline and the severity of the PR-PCSS has improved by at least two levels from the baseline. 5. That, in the CR-PCSS evaluated by the clinician in the treatment area live, the severity has improved by at least one level from the baseline at 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. 6. That, while viewing digital images of the treatment area, the severity of the PR-PCSS evaluated by the subject has improved by at least one level at 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 from the baseline. 7. The improvement shown by a one-level composite response at 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 is defined as a subject in whom an improvement in severity of at least one level from the baseline is seen in the CR-PCSS and an improvement in severity of at least one level from the baseline is seen in the PR-PCSS. 8. In a patient population in which the evaluation of the CR-PCSS and / or PR-PCSS was moderate or severe, the improvement in at least one treatment area is statistically significant compared to placebo, and the improvement is one or more of Nos. 1 to 7 above. 9. After the initial administration, a result was obtained that at least 5% of the patients maintained an improvement level of at least 71 days compared to the pre-treatment baseline. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained such a level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial administration. In other cases, a result was obtained that at least 5% of the patients showed improvement compared to the pre-treatment baseline, and the degree of improvement increased over time. In one treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the initial administration, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and a further increase in improvement was observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was observed 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first treatment, or the second treatment, or the third treatment. 10. On the 180th day, the improvement in the CR-PCSS evaluation compared to the baseline was consistently observed on both the left and right sides of the treatment area. 11. In a patient population where all patients have a moderate or severe CR-PCSS and / or PR-PCSS evaluation, the median time until at least 2-level improvement in CR-PCSS and / or PR-PCSS is obtained 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 population of patients where all patients have a moderate or severe CR-PCSS and / or PR-PCSS evaluation, the median time until at least 1-level improvement in CR-PCSS and / or PR-PCSS is obtained 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 a moderate or severe evaluation of CR-PCSS and / or PR-PCSS, the mean value of the subjects' CR-PCSS and / or PR-PCSS scores was separated from the placebo 21 days after the first treatment and showed continuous and significant improvement in subsequent treatments. 14. In a population of patients all with a moderate or severe evaluation of CR-PCSS and / or PR-PCSS, the percentage of subjects having a two-level composite response measured by CR-PCSS and / or PR-PCSS in at least one treatment area on day 71 was about 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 evaluation of CR-PCSS and / or PR-PCSS, the percentage of subjects having a one-level composite response measured by CR-PCSS and / or PR-PCSS in at least one treatment area on day 71 was about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 16. In a population of patients all with a moderate or severe CR-PCSS and / or PR-PCSS evaluation, more than one-third, or more than one-half, or more than two-thirds, or more than three-fourths of the patients had at least one-level of CR-PCSS and / or at least one-level of PR-PCSS response in at least one treatment area by day 71 after treatment, and the results of CR-PCSS were independent of age, BMI, or skin color. 17. The reduction in the severity of cellulite occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit. 18. In a patient population with a moderate or severe evaluation of CR-PCSS and PR-PCSS, the maximum decrease in the evaluation of CR-PCSS and PR-PCSS from baseline (screening visit) was first observed on day 90. In a patient population with a moderate or severe evaluation of CR-PCSS and / or PR-PCSS, the improvement in cellulite severity (i.e., negative change) at a predetermined time point before 180 days is maintained at the 180-day visit. 20. In a patient population with a moderate or severe CR-PCSS and / or PR-PCSS evaluation, on day 90, the mean (SD) change from baseline in the CR-PCSS and / or PR-PCSS evaluation of the left buttock and left thigh was approximately -0.8 (0.58) and approximately -0.6 (0.62), respectively, and the mean (SD) change from baseline in the CR-PCSS and / or PR-PCSS evaluation of the right buttock and right thigh was approximately -0.7 (0.73) and approximately -0.5 (0.70), respectively. 21. In a patient population with a moderate or severe evaluation of CR-PCSS and / or PR-PCSS, on day 180, the decrease from the baseline (screening visit) in the evaluation of CR-PCSS and / or PR-PCSS is consistent across the left and right sides. 22. In a patient population with a moderate or severe evaluation of CR-PCSS and / or PR-PCSS, an improvement of 2 levels in the evaluation of CR-PCSS and / or PR-PCSS in at least one area was observed in at least 10% of the subjects on day 90 and in at least 15% of the subjects on day 180. 22. In a patient population with a moderate or severe CR-PCSS and / or PR-PCSS evaluation, an improvement of 2 levels in the evaluation of CR-PCSS and / or PR-PCSS in at least one area was observed in at least 10% of the subjects on day 90 and in at least 15% of the subjects on day 180, and the response was similar in the buttock and thigh regions and on the left and right sides. 23. In a patient population with a moderate or severe evaluation of CR-PCSS and / or PR-PCSS, an improvement of 1 level in the evaluation of CR-PCSS and / or PR-PCSS in at least one treatment area was observed in at least 60% of the subjects on day 90 and in at least 65% of the subjects on day 180. 24. In a patient population where all have a moderate or severe CR-PCSS and / or PR-PCSS assessment, an improvement of at least one level in the CR-PCSS and / or PR-PCSS assessment in at least one treatment area was observed in at least 60% of the subjects at day 90 and at least 65% of the subjects at day 180, and the response was similar in the gluteal and thigh regions and on the left and right sides. 25. In a patient population where all have a moderate or severe CR-PCSS and / or PR-PCSS assessment, the median time until at least two levels of CR-PCSS and / or PR-PCSS response were obtained in at least one treatment area was observed to be about 80 days. 26. In a patient population where all have a moderate or severe CR-PCSS and / or PR-PCSS assessment, the median time until at least one level of CR-PCSS and / or PR-PCSS response was obtained in at least one treatment area is about 40 days. 27. In a patient population where all have a moderate or severe CR-PCSS and / or PR-PCSS assessment, at least 50% of the patients are level 1 PR-PCSS and / or PR-PCSS responders at the target gluteal region on day 71. 28. In a patient population where all have a moderate or severe CR-PCSS and / or PR-PCSS assessment, at least 20% of the patients are level 2 PR-PCSS and / or PR-PCSS responders at the target gluteal region on day 71. 29. In a patient population where the CR-PCSS and / or PR-PCSS assessment is moderate or severe, at least 35% of the patients are level 1 CR-PCSS and / or PR-PCSS composite responders at the target gluteal region on day 71. 30. In a patient population where all patients have a moderate or severe CR-PCSS and / or PR-PCSS assessment, at least 5% of the patients are level 2 CR-PCSS and / or PR-PCSS composite responders at the non-target gluteal region on day 71. 31. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS evaluations, the mean change from baseline in PR-PCSS and / or PR-PCSS on day 71 was greater in subjects administered collagenase than in subjects administered placebo at the target hip (approximately 0.9 vs. approximately 0.5, respectively) and the non-target hip (approximately 0.9 vs. approximately 0.5, respectively). 32. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS evaluations, the percentage of subjects with a 1-level PR-PCSS and / or PR-PCSS responder was greater in subjects administered collagenase than in subjects administered placebo at the target hip (approximately 62% vs. approximately 40%, respectively) and the non-target hip (approximately 65% vs. approximately 40%, respectively) on day 71. 33. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS evaluations, the mean change from baseline in CR-PCSS and / or PR-PCSS in subjects administered CCH was greater than in subjects administered placebo at the target hip (approximately 0.7 vs. approximately 0.4 [0.72], respectively) and the non-target hip (approximately 0.8 vs. approximately 0.3, respectively) on day 71. 34. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS evaluations, the percentage of subjects with a 1-level CR-PCSS and / or PR-PCSS responder was greater in subjects treated with collagenase than in subjects treated with placebo at the target hip (approximately 58% vs. approximately 32%, respectively) and the non-target hip (approximately 60% vs. approximately 27%, respectively) on day 71. 35. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS evaluations, the percentage of subjects with a 1-level CR-PCSS and / or PR-PCSS composite responder was greater in subjects treated with collagenase than in subjects treated with placebo at the target hip (approximately 42% vs. approximately 20%, respectively) and the non-target hip (approximately 44% vs. approximately 13%, respectively) on day 71. 36. In a population of patients where all have a moderate or severe CR-PCSS and / or PR-PCSS assessment, 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, two or more levels of composite responders are obtained by the collagenase treatment described herein. 37. In a population of patients where all have a moderate or severe CR-PCSS and / or PR-PCSS assessment, by the collagenase treatment described herein, composite responders at one or more levels 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 a population of patients with a moderate or severe CR-PCSS and PR-PCSS assessment, the maximum decrease from the baseline (screening) of the CR-PCSS and PR-PCSS assessments was first observed on day 22, day 71, or earlier. 39. At baseline (before treatment), in a population of patients with a moderate or severe CR-PCSS and / or PR-PCSS assessment, the decrease in the CR-PCSS and / or PR-PCSS assessment from the baseline (screening visit) is consistent on both sides.

[0221] In another embodiment, during at least one treatment visit, by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area, one or more of the above Results Nos. 1 to 39 are obtained, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay)max (Minute -1 ) ● Approximately 4.1 to 410 nanomoles (SRC assay), or approximately 0.03 to 3.1 mM (GPA assay) of K M ● Approximately 1.1 to 107 (SRC assay), or approximately 93 to 9,179 (GPA assay) of K cat (Second -1 ) ● Approximately 376 to 37,222 (SRC assay), or approximately 4 to 428 (GPA assay) of 1 / K cat , microsecond ● Approximately 5,140 to 508,814 (SRC assay), or approximately 60 to 5,934 (GPA assay) of K cat / K M , mM -1 Second -1 ● Molecular weight of approximately 60 kDa to approximately 130 kDa, or approximately 70 to approximately 130 kDa, or approximately 80 to approximately 120 kDa, or approximately 90 to approximately 120 kDa, or approximately 100 to approximately 110 kDa ● Purity of area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Titer (i.e., specific activity) of approximately 500 to 30,000 SRC units / mg ● Titer of approximately 5,000 to approximately 30,000 f-SRC units / mg ● Titer of approximately 100,000 to approximately 400,000 GPA units / mg ● Titer of approximately 175,000 to approximately 500,00 f-GPA units / mg ● Titer of approximately 5,000 to approximately 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

[0222] In other cases, by injection of approximately 1 mg to approximately 20 mg of collagenase by Treatment I, one or more of the above Results Nos. 1 to 39 can be obtained.

[0223] In another example, injection of about 1 mg to about 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 to 39, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● k of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat 、microseconds ● K / K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / K M 、mM -1 sec -1 ● 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 ● Purity of the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Potency (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● Potency of about 5,000 to about 30,000 f-SRC units / mg ● Potency of about 100,000 to about 400,000 GPA units / mg ● Potency of about 175,000 to about 500,00 f-GPA units / mg ● Potency of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0225] In certain embodiments, a mixture of type I collagenase and type II collagenase in a ratio of about 1:1 at about 1 mg to about 20 mg is injected into at least one treatment area during at least one treatment visit, resulting in one or more of the above Results Nos. 1 to 39, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomolar 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , sec -1 : about 93 to 9,179 〇 1 / K cat , μsec: about 4 to 428 〇 K cat / K M , mM -1 sec -1 : about 60 to 5,934 Other ratios may be adopted (for example, 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0226] In certain embodiments, type I and type II collagenases in a ratio of about 1 mg to about 20 mg of about 1:1 are injected using Treatment I, and one or more of the above Results Nos. 1 to 39 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , sec -1 : about 93 to 9,179 〇 1 / K cat , μsec: about 4 to 428 〇 K cat / K M , mM -1 sec -1 : about 60 to 5,934

[0227] Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0228] In other embodiments, in response to the above treatment, the patient is a two - level CR - PCSS responder showing at least a two - level improvement (-2, -3, or -4 change) in the CR - PCSS assessment from baseline at the time of evaluation. A one - level CR - PCSS responder is a patient showing at least a one - level improvement (change amount of -1, -2, -3, or -4) in the CR - PCSS assessment from baseline at the time of evaluation. A patient showing at least a two - level improvement (-2, -3, or -4 change) in the PR - PCSS assessment from baseline at the time of evaluation is a two - level PR - PCSS responder. A one - level PR - PCSS responder is a patient showing at least a one - level (-1, -2, -3, or -4 change) improvement in the PR - PCSS assessment from baseline at the time of evaluation. In other aspects, a two - level composite responder refers to a patient having both a two - level PR - PCSS responder and a two - level CR - PCSS responder at the time of evaluation. A one - level composite responder is a patient having both a one - level PR - PCSS responder and a one - level CR - PCSS responder at the time of evaluation.

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

[0230] In the case of hexacel CSS, the improvement of an individual patient at any visit refers to an improvement of at least one level or one evaluation from the baseline or the previous score. The improvement of the patient group at the visit is an improvement of about 0.1 in the hexacel CSS score or evaluation from the average from the baseline or the previous average hexacel CSS score or evaluation. A responder is any patient who shows an improvement of at least 25% of the maximum total score or evaluation from the baseline. In certain embodiments, one or more of the following efficacy evaluation items measured by hexacel CSS are obtained by the treatment method detailed above: 1. In a patient population having a hexacel CSS evaluation on the first day of the baseline, by injecting collagenase into at least one treatment area at least once during a treatment visit, a statistically significant number of patients achieved a result that satisfied one or more of the following efficacy evaluation items: ● Transition from severe to moderate (from score 11 - 15 to 6 - 10) ● Transition from severe to mild (from score 11 - 15 to 1 - 5) ● Transition from severe to zero (from score 11 - 15 to 0) ● Transition from moderate to mild (from score 6 - 10 to 1 - 5) ● Transition from moderate to zero (from score 6 - 10 to 0) ● Transition from mild to zero (from score 1 - 5 to 0) 2. In hexacel CSS evaluated live by a clinician in the treatment area, a change in severity of at least two levels 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 from the baseline (the "first day" before treatment). 3. In hexacel CSS evaluated by a clinician while viewing digital images of the treatment area, a change in severity of at least two levels 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 from the baseline (the first day). The change indicated by the level 2 response on the 22nd, 43rd, 71st, 90th, 180th, 251st, 360th, 431st, 720th, 3rd, 4th, or 5th day is defined as a subject in whom the severity of the Heixel CSS has improved by at least 2 levels from the baseline as evaluated by a clinician. 5. The severity of the Heixel CSS evaluated live by a clinician in the treatment area shows a change in severity of at least 1 level on the 22nd, 43rd, 71st, 90th, 180th, 251st, 360th, 431st, 720th, 3rd, 4th, or 5th day from the baseline (day 1). 6. The severity of the Heixel CSS evaluated by a clinician while viewing digital images in the treatment area shows a change in severity of at least 1 level on the 22nd, 43rd, 71st, 90th, 180th, 251st, 360th, 431st, 720th, 3rd, 4th, or 5th day from the baseline (day 1). 7. The change indicated by the level 1 response on the 22nd, 43rd, 71st, 90th, 180th, 251st, 360th, 431st, 720th, 3rd, 4th, or 5th day is defined as a subject in whom the severity of the Heixel CSS has improved by at least 1 level from the baseline as evaluated by a clinician. 8. In a patient population with moderate or severe Heixel CSS evaluation, the improvement in at least one treatment area is statistically significant compared to the placebo, and the change is one or more of the above Nos. 2 to 7. 9. After the initial administration, results were obtained showing that at least 5% of patients maintained an improvement level of at least 71 days relative to the pre-treatment baseline. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained 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 initial administration. In other cases, results were obtained showing that at least 5% of patients showed improvement relative to the pre-treatment baseline and the degree of improvement increased over time. In one treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the initial administration, at least 10%, 20%, 30%, 40%, 50% of patients showed improvement and further increases in improvement were observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of patients showed improvement and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 10. On the 180th day, improvements in the Excel CSS evaluation from the baseline were consistently observed on both the left and right sides of the treatment area. 11. In the population of patients with a moderate or severe Excel CSS evaluation, the median time until at least two levels of Excel CSS improvement were obtained in at least one treatment area was about 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 Excel CSS evaluation, the median time until at least one level of Excel CSS improvement was obtained in at least one treatment area was 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 the population of patients with a moderate or severe Excel CSS evaluation, the average value of the Excel CSS score of the subjects separated from the placebo 21 days after the first treatment and showed continuous significant improvement in subsequent treatments. 14. In a population of patients all having a moderate or severe hexcel CSS assessment, the percentage of subjects having a level 2 response measured by hexcel CSS in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 15. In a population of patients all having a moderate or severe hexcel CSS assessment, the percentage of subjects having a level 1 response measured by hexcel CSS in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 16. In a population of patients having a moderate or severe hexcel CSS assessment, more than one-third, or more than one-half, or two-thirds, or three-fourths of the patients have at least a level 1 hexcel CSS response in at least one treatment area by day 71 after treatment, and the results of hexcel CSS do not depend on age, BMI, or skin color. 17. The reduction in the severity of cellulite occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit.

[0231] In another embodiment, during at least one treatment visit, by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area, one or more of the above Results Nos. 1 - 17 are obtained, where the collagenase has one or more of the following characteristics: ● A V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● A K of about 4.1 - 410 nanomolar (SRC assay), or about 0.03 - 3.1 mM (GPA assay) M ● A K of about 1.1 - 107 (SRC assay), or about 93 - 9,179 (GPA assay) cat (sec -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 ● Molecular weight of approximately 60 kDa to approximately 130 kDa, or approximately 70 to approximately 130 kDa, or approximately 80 to approximately 120 kDa, or approximately 90 to approximately 120 kDa, or approximately 100 to approximately 110 kDa ● Purity of area ratio measured by reverse-phase HPLC (high-performance liquid chromatography) is at least 80% ● Titer (i.e., specific activity) of approximately 500 to 30,000 SRC units / mg ● Titer of approximately 5,000 to approximately 30,000 f-SRC units / mg ● Titer of approximately 100,000 to approximately 400,000 GPA units / mg ● Titer of approximately 175,000 to approximately 500,00 f-GPA units / mg ● Titer of approximately 5,000 to approximately 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0233] In another example, injection of approximately 1 mg to approximately 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 to 17, 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 (min -1 ) ● About 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) of K M ● About 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) of K cat (seconds -1 ) ● About 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) of 1 / K cat , microseconds ● About 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) of K 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 of the area ratio measured by reverse-phase HPLC (high-pressure liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● A titer of about 175,000 to about 500,00 f-GPA units / mg ● A titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

[0234] In another example, by injecting about 1 mg to about 20 mg of CCH by Treatment I, one or more of the above results Nos. 1 to 17 are obtained.

[0235] In certain embodiments, a 1:1 ratio of type I and type II collagenase, from about 1 mg to about 20 mg, is injected into at least one treatment area during at least one treatment visit, and one or more of the above Results Nos. 1-17 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomoles 〇 K cat , sec -1 : about 1.1 - 107 〇 1 / K cat , μsec: about 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 - 30.5 〇 K M , mM: about 0.03 - 3.1 〇 K cat , sec -1 : about 93 - 9,179 〇 1 / K cat , μsec: about 4 - 428 〇 K cat / K M , mM -1 sec -1 : about 60 - 5,934 Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0236] In certain embodiments, type I collagenase and type II collagenase in a ratio of about 1:1 from about 1 mg to about 20 mg are injected using Treatment I, and one or more of the above Results Nos. 1 to 17 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , sec -1 : about 93 to 9,179 〇 1 / K cat , μsec: about 4 to 428 〇 K cat / K M , mM -1 sec -1 : about 60 to 5,934 Other ratios may be employed (e.g., 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0237] 3. Efficacy Measured by the Hexel Depression Depth Score

[0238] In the Hexel depression depth score, improvement in an individual patient at any visit means at least a 1-level or 1-evaluation improvement from the baseline or previous score. Also, improvement in the patient group means an improvement of about 0.1 from the baseline or previous average Hexel depression depth score or evaluation. A responder is any patient showing at least a 25% improvement in the maximum total score or evaluation from baseline. In certain embodiments, one or more of the following efficacy evaluation items measured by the Hexel depression depth score are obtained by the treatment method detailed above: 1. In a patient population where all had a baseline day 1 Hexel depression depth score evaluation, as a result of injecting collagenase into at least one treatment area at at least one treatment visit, a statistically significant number of such patients met one or more of the following efficacy evaluation items: ● Transition from deep depression (3) to moderate depth (2) ● Transition from deep depression (3) to superficial depression (1) ● Transition from deep depression (3) to no depression (0) ● Transition from moderate depth (2) to superficial depression (1) ● Transition from moderate depth (2) to no depression (0) ● Transition from superficial depression (1) to no depression (0) 2. The severity of the Hexel depression depth score evaluated by the clinician in the treatment area in real time changed by at least 2 levels at day 22, 43, 71, 90, 180, 251, 360, 431, 720, 3 years, 4 years, or 5 years from the baseline (the "day 1" before treatment). 3. The change shown by a 2-level response at day 22, 43, 71, 90, 180, 251, 360, 431, 720, 3 years, 4 years, or 5 years is defined as a subject in whom the severity of the Hexel depression depth score improved by at least 2 levels from the baseline as evaluated by the clinician. 4. The severity of the Hexcel depression depth score clinically evaluated by clinicians in the treatment area changed by at least one level 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 from the baseline (day 1). 5. The change shown by a one-level response 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 is defined as a subject in whom the severity of the Hexcel depression depth score has improved by at least one level from the baseline as evaluated by the clinician. 6. In a patient population with moderate or deep depressions in the Hexcel CSS assessment, the improvement in at least one treatment area is statistically significant compared to the placebo, and the change is one or more of No. 2 to No. 7 above. 7. After the first dose, the result was obtained that at least 5% of the patients maintained an improvement level of at least 71 days compared to the pre-treatment baseline. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained such a level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the first dose. In other cases, at least 5% of the patients showed improvement compared to the pre-treatment baseline, and the degree of improvement increased over time. In one treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first dose, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was seen. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was seen 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 8. On day 180, the improvement from the baseline of the Hexcel depression depth score evaluation was consistently seen on both the left and right sides of the treatment area. 9. In a patient population with a moderate or deep hecel pit depth score, the median time until at least one treatment area achieves an improvement of two levels in the hecel pit depth score for the first time is about 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 10. In a patient population with a moderate or deep hecel pit depth score, the median time until at least one treatment area achieves an improvement of one level in the hecel pit depth score for the first time 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 a moderate or deep hecel pit depth score, the mean hecel pit depth score of the subjects separated from the placebo 21 days after the first treatment and showed continuous and significant improvement in subsequent treatments. 12. In a patient population where all have a moderate or deep hecel pit depth score, the percentage of subjects with a two-level response measured by the hecel pit depth score in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 13. In a patient population where all have a moderate or deep hecel pit depth score, the percentage of subjects with a one-level response measured by the hecel pit depth score in at least one treatment area on 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 moderate or deep hecel pit depth score, by day 71 after treatment, more than one-third, or one-half, or two-thirds, or three-fourths of the patients showed a one-level response in the hecel pit depth score in at least one treatment area, and the results of the hecel pit depth score are independent of age, BMI, or skin color. 15. The severity reduction of cellulite occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit. 16. The change in score from baseline to day 71 is in one or more treatment areas of about -0.1 to about -2.0. 17. In a statistically significant patient population, the least squares (LS) mean value is about -0.1 to about -1.5 (95% confidence interval (CI)) in one or more treatment areas.

[0239] In another embodiment, during at least one treatment visit, by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area, one or more of the above results No. 1 to 19 are obtained, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomolar (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● kcat of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / kcat of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat 、microseconds ● kcat / Km of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / Km M 、mM -1 sec -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 of the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Titer (i.e., specific activity) of approximately 500 to 30,000 SRC units / mg ● Titer of approximately 5,000 to approximately 30,000 f-SRC units / mg ● Titer of approximately 100,000 to approximately 400,000 GPA units / mg ● Titer of approximately 175,000 to approximately 500,00 f-GPA units / mg ● Titer of approximately 5,000 to approximately 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

[0240] In other cases, by injecting approximately 1 mg to approximately 20 mg of collagenase by Treatment I, one or more of the above Results No. 1 to 19 are obtained.

[0241] In another example, by injecting approximately 1 mg to approximately 20 mg of collagenase by Treatment I, one or more of the above Results No. 1 to 19 are obtained, 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 (min -1 ) ● K of approximately 4.1 to 410 nanomoles (SRC assay), or approximately 0.03 to 3.1 mM (GPA assay) M ● k of approximately 1.1 to 107 (SRC assay), or approximately 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / k of approximately 376 to 37,222 (SRC assay), or approximately 4 to 428 (GPA assay) cat , μsec ● k / K of approximately 5,140 to 508,814 (SRC assay), or approximately 60 to 5,934 (GPA assay) cat / K M , mM -1 sec -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 of the area ratio measured by reverse-phase HPLC (high-performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● A titer of about 175,000 to about 500,00 f-GPA units / mg ● A titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less in area ratio ● A bioburden of 1 cfu / mL or less

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

[0243] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 are injected into at least one treatment area during at least one treatment visit, resulting in one or more of the above Results Nos. 1 to 19, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M 、mM -1 seconds -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max 、minutes -1 : about 0.3 - 30.5 〇 K M 、mM: about 0.03 - 3.1 〇 K cat 、seconds -1 : about 93 - 9,179 〇 1 / K cat 、microseconds: about 4 - 428 〇 K cat / K M 、mM -1 seconds -1 : about 60 - 5,934 Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, Type I and Type II collagenases may be AUX-I and AUX-II, respectively.

[0244] In certain embodiments, about 1 mg to about 20 mg of a 1:1 ratio of Type I and Type II collagenases are injected using Treatment I and one or more of the above Results Nos. 1 - 19 are obtained, where Collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max 、minutes -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomoles 〇 K cat 、seconds -1 : about 1.1 - 107 〇 1 / K cat 、microseconds: about 376 - 37,222 〇 K cat / KM 、 mM -1 seconds -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max 、 minutes -1 : about 0.3 - 30.5 〇 K M 、 mM: about 0.03 - 3.1 〇 K cat 、 seconds -1 : about 93 - 9,179 〇 1 / K cat 、 microseconds: about 4 - 428 〇 K cat / K M 、 mM -1 seconds -1 : about 60 - 5,934

[0245] Other ratios may be adopted (for example, 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Furthermore, Type I and Type II collagenases may be AUX - I and AUX - II, respectively.

[0246] 4. Efficacy Measured by the Likert Scale for Aesthetic Appearance

[0247] In the Likert scale, the improvement of an individual patient at any visit is an improvement of at least 1 level or 1 assessment from the pre - treatment or previous score. The improvement of a patient group at any visit is an improvement in the average Likert score or assessment by about 0.1 from the pre - treatment or any previous average Likert score or assessment. In certain embodiments, one or more of the following efficacy evaluation items measured by the Likert scale score are obtained by the treatment method detailed above: 1. In a population of patients with cellulite, by injecting collagenase into at least one treatment area during at least one treatment visit, a statistically significant number of such patients were able to meet one or more of the following effectiveness evaluation items: ● The Likert scale score was "improved" (1) ● The Likert scale score was "greatly improved" (2) ● The Likert scale score was "very improved" (3) 2. The appearance of the treatment area on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3-year mark, 4-year mark, or 5-year mark was improved by at least 2 levels on the Likert scale score evaluated by a clinician live compared to before treatment. 3. The appearance of the treatment area on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3-year mark, 4-year mark, or 5-year mark was improved by at least 2 levels on the Likert scale score evaluated by the subject while viewing a digital image of the treatment area compared to before treatment. 4. The appearance of the treatment area on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3-year mark, 4-year mark, or 5-year mark was improved by at least 1 level on the Likert scale score evaluated by a clinician live compared to before treatment. 5. The appearance of the treatment area on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3-year mark, 4-year mark, or 5-year mark was improved by at least 1 level on the Likert scale score evaluated by the subject while viewing a digital image of the treatment area compared to before treatment. 6. In a population of patients with cellulite, the improvement in the Likert scale score in at least one treatment area was statistically significant, and the improvement was one or more of Nos. 2 to 7 above. 7. After the initial administration, results were obtained showing that at least 5% of patients maintained an improvement level of at least 71 days relative to the pre-treatment baseline. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained 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 initial administration. In other cases, results were obtained showing that at least 5% of patients showed improvement relative to the pre-treatment baseline, and the degree of improvement increased over time. In one treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the initial administration, at least 10%, 20%, 30%, 40%, 50% of patients showed improvement, and further increases in improvement were observed. In some treatment methods, at least 10%, 20%, 30%, 40%, or 50% of patients showed improvement, and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months after the first treatment, the second treatment, and the third treatment. 8. On the 180th day, the improvement seen in the Likert scale score evaluation from the baseline was consistent in the right and left treatment areas. 9. In the population of patients with cellulite, the median time to the earliest 2-level improvement in the 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 the population of patients with cellulite, the median time to the earliest 1-level improvement in the 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 the population of patients with cellulite, the mean value of the Likert scale score of the subjects separated from the placebo 21 days after the first treatment and showed continuous and significant improvement in subsequent treatments. 12. In a patient population having cellulite, the percentage of subjects having a two-level response measured by the Likert scale score in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 13. In a patient population having cellulite, the percentage of subjects having a one-level response measured by the Likert scale score in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 14. In a patient population having cellulite, one-third or more, or one-half, or two-thirds, or three-fourths of the patients have a response of at least one level on the Likert scale score in at least one treatment area by day 71 after treatment, and the results of the Likert scale score are independent of age, BMI, or skin color. 15. The reduction in cellulite severity occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit.

[0248] In another embodiment, during at least one treatment visit, by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area, one or more of the above results No. 1 - 15 are obtained, where the collagenase has one or more of the following properties: ● V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 - 410 nanomolar (SRC assay), or about 0.03 - 3.1 mM (GPA assay) M ● K of about 1.1 - 107 (SRC assay), or about 93 - 9,179 (GPA assay) cat (sec -1 ) ● 1 / K of about 376 - 37,222 (SRC assay), or about 4 - 428 (GPA assay) cat, microsecond ● K of about 5,140 - 508,814 (SRC assay), or about 60 - 5,934 (GPA assay) cat / K M , mM -1 second -1 ● Molecular weight of about 60 kDa - about 130 kDa, or about 70 - about 130 kDa, or about 80 - about 120 kDa, or about 90 - about 120 kDa, or about 100 - about 110 kDa ● Purity of area ratio measured by reverse phase HPLC (high - pressure liquid chromatography) is at least 80% ● Titer (i.e., specific activity) of about 500 - 30,000 SRC units / mg ● Titer of about 5,000 - about 30,000 f - SRC units / mg ● Titer of about 100,000 - about 400,000 GPA units / mg ● Titer of about 175,000 - about 500,00 f - GPA units / mg ● Titer of about 5,000 - about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0250] In another example, injection of about 1 mg - about 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 - 15, where the collagenase has one or more of the following characteristics: ● V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (minute -1 ) ● K of about 4.1 - 410 nanomolar (SRC assay), or about 0.03 - 3.1 mM (GPA assay) M ● K of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) cat , microseconds ● K of about 5,140 to 508,814 (SRC assay), or about 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 of the area ratio measured by reverse-phase HPLC (high-performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● A titer of about 175,000 to about 500,00 f-GPA units / mg ● A titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

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

[0252] In certain embodiments, a ratio of approximately 1 mg to approximately 20 mg of type I collagenase and type II collagenase in a ratio of approximately 1:1 is injected into at least one treatment area during at least one treatment visit, and one or more of the above results Nos. 1 to 15 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : approximately 0.08 - 7.70 〇 K M : approximately 4.1 - 410 nanomoles 〇 K cat , sec -1 : approximately 1.1 - 107 〇 1 / K cat , μsec: approximately 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : approximately 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : approximately 0.3 - 30.5 〇 K M , mM: approximately 0.03 - 3.1 〇 K cat , sec -1 : approximately 93 - 9,179 〇 1 / K cat , μsec: approximately 4 - 428 〇 K cat / K M , mM -1 sec -1 : approximately 60 - 5,934 Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0253] In certain embodiments, a 1:1 ratio of type I and type II collagenase, from about 1 mg to about 20 mg, is injected using Treatment I, and one or more of the above Results Nos. 1-15 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomolar 〇 K cat , sec -1 : about 1.1 - 107 〇 1 / K cat , μsec: about 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 - 30.5 〇 K M , mM: about 0.03 - 3.1 〇 K cat , sec -1 : about 93 - 9,179 〇 1 / K cat , μsec: about 4 - 428 〇 K cat / K M , mM -1 sec -1 : about 60 - 5,934 Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0254] 5. Depression Analysis

[0255] In certain embodiments, treatment of the celite with collagenase reduces the parameters of the concavity 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% ● Total 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, as a result of treatment, at least 5% of the patients maintained an improvement level relative to the pre-treatment baseline for at least 71 days after the first administration. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained such a level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the first administration. In other embodiments, treatment resulted in at least 5% of the patients showing improvement relative to the pre-treatment baseline and further showing an increase in improvement over time. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first administration, and a further increase in improvement was observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment.

[0257] In another embodiment, by injecting at least about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit, at least one of the indentation size parameters is reduced 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 about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 - 410 nanomolar (SRC assay), or about 0.03 - 3.1 mM (GPA assay) M ● k of about 1.1 - 107 (SRC assay), or about 93 - 9,179 (GPA assay) cat (sec -1 ) ● 1 / k of about 376 - 37,222 (SRC assay), or about 4 - 428 (GPA assay), microseconds cat 、 ● K of about 5,140 - 508,814 (SRC assay), or about 60 - 5,934 (GPA assay) cat / K M mM -1 seconds -1 ● Molecular weight of about 60 kDa - about 130 kDa, or about 70 - about 130 kDa, or about 80 - about 120 kDa, or about 90 - about 120 kDa, or about 100 - about 110 kDa ● Purity by area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Potency (i.e., specific activity) of about 500 - 30,000 SRC units / mg ● Potency of about 5,000 - about 30,000 f - SRC units / mg ● Potency of about 100,000 - about 400,000 GPA units / mg ● Potency of about 175,000 - about 500,00 f - GPA units / mg ● Potency of about 5,000 - about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0259] In another example, injection of about 1 mg - about 20 mg of collagenase by Treatment I causes at least one of the dimple size parameters to decrease by at least 5%, or at least 10%, or at least 20%, where the collagenase has one or more of the following characteristics: ● V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● About 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) of K M ● About 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) of K cat (seconds -1 ) ● About 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) of 1 / K cat , microseconds ● About 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) of K 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 of the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● A titer of about 175,000 to about 500,00 f-GPA units / mg ● A titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

[0260] In another example, injection of about 1 mg to about 20 mg of CCH by Treatment I causes at least one of the parameters of the dimple size to decrease by at least 5%, or at least 10%, or at least 20%.

[0261] In certain embodiments, at least one treatment visit, inject into at least one treatment area a type I collagenase and a type II collagenase in an approximate 1:1 ratio of about 1 mg to about 20 mg, resulting in at least a 5%, or at least a 10%, or at least a 20% reduction in at least one of the dimple size parameters, and type I collagenase and type II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomoles 〇 K cat , sec -1 : about 1.1 - 107 〇 1 / K cat , μsec: about 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 - 30.5 〇 K M , mM: about 0.03 - 3.1 〇 K cat , sec -1 : about 93 - 9,179 〇 1 / K cat , μsec: about 4 - 428 〇 K cat / K M , mM -1 sec -1 : about 60 - 5,934 Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0262] In certain embodiments, about 1 mg to about 20 mg of type I and type II collagenases in a ratio of about 1:1 are injected using Treatment I, resulting in a reduction of at least 5%, or at least 10%, or at least 20% in at least one of the depression size parameters, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomoles 〇 K cat , sec -1 : about 1.1 - 107 〇 1 / K cat , μsec: about 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 - 30.5 〇 K M , mM: about 0.03 - 3.1 〇 K cat , sec -1 : about 93 - 9,179 〇 1 / K cat , μsec: about 4 - 428 〇 K cat / K M , mM -1 sec-1 : Approximately 60 to 5,934 Other ratios may be adopted (for example, 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0263] 6. Efficacy Measured by the Subject Global Aesthetic Improvement Scale (S-GAIS) and the Investigator Global Aesthetic Improvement Scale (I-GAIS) By the treatment methods detailed above, the responses measured by S-GAIS and I-GAIS are improved. Two-level S-GAIS responders are subjects with an S-GAIS assessment of at least 2 (+2 or +3) at the evaluation time point. One-level S-GAIS responders are subjects with an S-GAIS assessment of at least 1 (+1, +2, +3) at the evaluation time point. Two-level I-GAIS responders are subjects with an I-GAIS assessment of at least 2 (+2 or +3) at the evaluation time point. One-level I-GAIS responders are subjects with an I-GAIS assessment of at least 1 (+1, +2, or +3) at the evaluation time point. The improvement of an individual patient at any visit refers to an improvement of at least one level or one evaluation from the baseline or previous score. The improvement of a patient group refers to an increase in the average score or evaluation by approximately 0.1 from the baseline or previous average score or evaluation.

[0264] In certain embodiments, by the treatment methods detailed above, one or more of the following efficacy evaluation items measured by S-GAIS and / or I-GAIS are obtained: 1. In a patient population with cellulite, by injecting collagenase into at least one treatment area at least once during a treatment visit, a statistically significant number of such patients meet one or more of the following efficacy evaluation items: ● The score of S-GAIS and / or I-GAIS is "improved" (+1) ● The score of S-GAIS and / or I-GAIS is "significantly improved" (+2) ● The score of S-GAIS and / or I-GAIS is "very improved" (+3). 2. In the I-GAIS clinically evaluated by the clinician in the treatment area, at least a two-level improvement was observed on the 22nd, 43rd, 71st, 90th, 180th, 365th, or 730th day. 3. In the S-GAIS evaluated by the subject while viewing the digital image in the treatment area, at least a two-level improvement was observed on the 22nd, 43rd, 71st, 90th, 180th, 365th, or 730th day. 4. The improvement shown by a two-level composite response on the 22nd, 43rd, 71st, 90th, 180th, 365th, or 730th day is defined as a subject in whom the I-GAIS improved by at least two levels as evaluated by the clinician and the S-GAIS improved by at least two levels as evaluated by the patient. 5. In the I-GAIS clinically evaluated by the clinician in the treatment area, at least a one-level improvement was observed on the 22nd, 43rd, 71st, 90th, 180th, 365th, or 730th day. 6. In the S-GAIS evaluated by the subject while viewing the digital image in the treatment area, at least a one-level improvement was observed on the 22nd, 43rd, 71st, 90th, 180th, 365th, or 730th day. 7. The improvement shown by a one-level composite response on the 22nd, 43rd, 71st, 90th, 180th, 365th, or 730th day is defined as a subject in whom the I-GAIS improved by at least one level as evaluated by the clinician and the S-GAIS improved by at least one level as evaluated by the patient. 8. In the patient population with cellulite, the improvement of I-GAIS and / or S-GAIS in at least one treatment area is statistically significant compared to the placebo, and the improvement is one or more of No. 2 to No. 7 above. 9. With this treatment, at least 5% of the patients maintained their level of improvement for 71 days after the first dose. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained 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 cases, at least 5% of the patients showed improvement, and the degree of improvement increased over time. In one treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first dose, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further increases in improvement were observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 10. On the 180th day, the improvements seen in the I-GAIS and S-GAIS evaluations were consistent across the left and right of the treatment area. 11. In the patient population with cellulite, the median time to the earliest 2 levels of improvement in at least one treatment area in I-GAIS and / or S-GAIS is about 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 12. In the patient population with cellulite, the median time to the earliest 1 level of improvement in at least one treatment area in I-GAIS and / or S-GAIS 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 the patient population with cellulite, the mean values of the subjects' I-GAIS and / or S-GAIS separated from the placebo 21 days after the first treatment and showed continuous and significant improvement in subsequent treatments. 14. In a patient population having cellulite, the percentage of subjects having a two-level composite response measured by I-GAIS and / or S-GAIS in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 15. In a patient population having cellulite, the percentage of subjects having a one-level composite response measured by I-GAIS and / or S-GAIS in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 16. In a patient population having cellulite, more than one-third, or more than one-half, or two-thirds, or three-fourths of the patients have at least a one-level I-GAIS and / or S-GAIS response in at least one treatment area by day 71 after treatment, and the GAIS results are independent of age, BMI, or skin color. 17. The reduction in cellulite severity occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit. 18. In a patient population with a moderate or severe CR-PCSS and / or PR-PCSS assessment, at least 60% of the patients showed a one-level S-GAIS response at the target buttocks on day 71. 19. In a patient population with a moderate or severe CR-PCSS and / or PR-PCSS assessment, at least 20% of the patients showed a two-level S-GAIS response at the target buttocks on day 71. 20. In a patient population with a moderate or severe CR-PCSS and / or PR-PCSS assessment, on day 71, the mean S-GAIS was greater in subjects treated with collagenase at the target buttocks compared to subjects treated with placebo (about 1.0 vs. about 0.5, respectively). Similar results were obtained for the non-target buttocks (about 1.0 in subjects treated with collagenase and about 0.5 in subjects treated with placebo). 21. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS evaluations, at day 71, the percentage of level 1 S-GAIS responders was higher in subjects treated with collagenase than in subjects treated with placebo in the target buttocks (approximately 70% vs. approximately 40% respectively) and non-target buttocks (approximately 70% vs. approximately 40% respectively). 22. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS evaluations, at day 71, the mean I-GAIS in subjects administered collagenase to the target buttocks was statistically significantly greater compared to subjects administered placebo (approximately 1.0 vs. 0.3 respectively). Similar results were obtained for the non-target buttocks (approximately 0.6 in subjects receiving collagenase treatment and approximately 0.1 in subjects receiving placebo treatment). 23. In patient populations with moderate or severe CR-PCSS and / or PR-PCSS evaluations, at day 71, the percentage of level 1 I-GAIS responders was higher in subjects treated with collagenase than in subjects treated with placebo in the 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 evaluations, a series of cross-tabulations showed consistency between PR-PCSS and S-GAIS. A level 1 change in PR-PCSS was associated with a similar change in S-GAIS.

[0265] In another embodiment, one or more of the above Results Nos. 1 to 24 are obtained by injecting from 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: ● A V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● A K of about 4.1 to 410 nanomolar (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● K of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (seconds -1 ) ● 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) cat , microseconds ● K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) cat / K M , mM -1 seconds -1 ● 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 ● Purity of area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● Titer of about 5,000 to about 30,000 f-SRC units / mg ● Titer of about 100,000 to about 400,000 GPA units / mg ● Titer of about 175,000 to about 500,00 f-GPA units / mg ● Titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase and leupeptin are 1% or less in area ratio ● Bioburden of 1 cfu / mL or less

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

[0267] In another example, injection of about 1 mg to about 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 to 24, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay) or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomoles (SRC assay) or about 0.03 to 3.1 mM (GPA assay) M ● K of about 1.1 to 107 (SRC assay) or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / K of about 376 to 37,222 (SRC assay) or about 4 to 428 (GPA assay), microseconds cat , microseconds ● K of about 5,140 to 508,814 (SRC assay) or about 60 to 5,934 (GPA assay) cat / K M , mM -1 sec -1 ● 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 ● Purity of the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● Titer of about 5,000 to about 30,000 f-SRC units / mg ● Titer of about 100,000 to about 400,000 GPA units / mg ● Titer of about 175,000 to about 500,00 f-GPA units / mg ● Titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0269] In certain embodiments, a ratio of approximately 1:1 of type I collagenase and type II collagenase, from about 1 mg to about 20 mg, is injected into at least one treatment area during at least one treatment visit, and one or more of the above Results Nos. 1-24 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomolar 〇 K cat , sec -1 : about 1.1 - 107 〇 1 / K cat , μsec: about 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 - 30.5 〇 K M , mM: about 0.03 - 3.1 〇 K cat , sec -1 : about 93 - 9,179 〇 1 / K cat , μsec: about 4 - 428 〇 K cat / K M , mM -1 sec -1 : about 60 - 5,934

[0270] Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0271] In certain embodiments, about 1 mg to about 20 mg of type I and type II collagenases in an approximate 1:1 ratio are injected using Treatment I, and one or more of the results No. 1 - 24 above are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomoles 〇 K cat , sec -1 : about 1.1 - 107 〇 1 / K cat , μsec: about 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 - 30.5 〇 K M , mM: about 0.03 - 3.1 〇 K cat , sec -1 : about 93 - 9,179 〇 1 / K cat , μsec: about 4 - 428 〇 K cat / K M , mM -1 sec -1 : about 60 - 5,934 Other ratios may be employed (e.g., 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

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

[0273] 7. Efficacy Measured by PR-CIS By the treatment method as described above, the response measured by PR-CIS is improved. The total score of PR-CIS is the sum of six items of the scale. The total score of PR-CIS ranges from 0 to 60, indicating that the higher the value, the greater the impact of cellulite. Item 1 of PR-CIS asks about the degree of satisfaction with the appearance of cellulite, and is reversed by subtracting the evaluation reported by the subject from 10. For the PR-CIS total score, subjects whose PR-CIS total score decreased by 12 or more from baseline at the evaluation time point were defined as responders. For each individual PR-CIS impact score, a response was defined as an improvement of at least two score intervals from baseline at each time point. Further, a responder refers to a patient whose maximum total score showed an improvement of at least 20% from baseline. Also, the improvement for each individual patient at each visit means an improvement of at least one level or one evaluation from baseline or the previous score. Also, the improvement for the patient group means that the average score or evaluation increased by about 0.1 from baseline or the previous average score or evaluation. Further, improvement means that the change from baseline is at least one level out of 60.

[0274] In certain embodiments, the treatment methods detailed above provide one or more of the following efficacy evaluation items as measured by PR-CIS: 1. In a patient population with cellulite, by injecting collagenase into at least one treatment area at at least one treatment visit, a statistically significant number of such patients meet one or more of the following efficacy evaluation items: ● PR-CIS shows improvement in at least one area selected from the group consisting of satisfaction with the appearance of cellulite, concern, awareness, embarrassment, looking older, looking overweight / out-of-shape. ● At one or more evaluation time points, the PR-CIS total score decreases by 12 or more from the baseline. ● The PR-CIS impact score shows improvement of at least 2 score intervals from the baseline at one or more evaluation time points. ● Improvement means that the change from the baseline is at least 1 level out of 60. 2. In the PR-CIS of the treatment area, 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, an improvement in severity of at least 12 points from the baseline (the "first day" before treatment) is observed. 3. In a patient population with cellulite, in the PR-CIS of the treatment area, 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 from the baseline (the first day before treatment), the severity improves by at least 12 points statistically significantly compared to the placebo. 4. After the initial administration, results were obtained where at least 5% of patients maintained an improvement level relative to the pre-treatment baseline for 71 days or more. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained 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 initial administration. In other cases, results were obtained where at least 5% of patients showed improvement relative to the pre-treatment baseline, and the degree of improvement increased over time. In one treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the initial administration, at least 10%, 20%, 30%, 40%, 50% of patients showed improvement, and further increases in improvement were observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of patients showed improvement, and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 5. On the 180th day, improvements in the PR-CIS evaluation from the baseline were consistently observed on both the left and right sides of the treatment area. 6. In the patient population with cellulite, for at least one treatment area, the median time until the PR-CIS total score decreased by at least 12 from the baseline at one or more evaluation 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 the patient population with cellulite, the average value of the subjects' PR-CIS scores separated from the placebo 21 days after the first treatment, and showed continuous and significant improvement in subsequent treatments. 8. In the patient population with cellulite, one-third or more, or one-half or more, or two-thirds or more, or three-quarters or more of the patients had a decrease in the PR-CIS total score of at least 12 from the baseline at one or more evaluation time points in at least one treatment area by the 71st day after treatment, and the PR-CIS results were independent of age, BMI, or skin color. 9. The reduction in cellulite severity occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit. 10. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS evaluations, the average change from baseline in the PR-CIS total score on day 71 was approximately -10 in subjects receiving collagenase treatment and approximately -5 in subjects receiving placebo treatment. 11. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS evaluations, the mean change in PR-CIS from baseline on day 71 was statistically significantly better in subjects receiving collagenase treatment than in subjects receiving placebo treatment in the total score (approximately -12 vs -6 respectively) and the shortened score (approximately -10 vs 5 respectively), as well as in the individual impact scores (satisfaction with the appearance of cellulite, bothersomeness, self-consciousness, embarrassment, aged appearance, concern about body shape). 12. In the patient population with moderate or severe CR-PCSS and / or PR-PCSS evaluations on day 71, the proportion of responders for PR-CIS was greater in subjects administered collagenase than in subjects administered placebo in the total score (approximately 45% vs approximately 20% respectively) and the shortened score (approximately 50% vs approximately 25% respectively). Furthermore, for the individual impact scores, the proportion of respondents was also greater in the collagenase-administered group than in the placebo-administered group. These differences were also statistically significant.

[0275] In another embodiment, one or more of the above Results Nos. 1 to 12 are obtained by injecting from 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 characteristics: ● V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● About 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) of K M ● About 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) of K cat (seconds -1 ) ● About 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) of 1 / K cat , microseconds ● About 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) of K 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 of the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● A titer of about 175,000 to about 500,00 f-GPA units / mg ● A titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

[0276] In other cases, by injecting about 1 mg to about 20 mg of collagenase by Treatment I, one or more of the above Results Nos. 1 to 12 are obtained.

[0277] In another example, injection of about 1 mg to about 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 to 12, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● k of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat 、microseconds ● K / K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / K M 、mM -1 sec -1 ● 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 ● Purity of area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● Titer of about 5,000 to about 30,000 f-SRC units / mg ● Titer of about 100,000 to about 400,000 GPA units / mg ● Titer of about 175,000 to about 500,00 f-GPA units / mg ● Titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0279] In certain embodiments, a 1:1 ratio of Type I and Type II collagenase of about 1 mg to about 20 mg is injected into at least one treatment area during at least one treatment visit, resulting in one or more of the above Results Nos. 1 to 12, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , sec -1 : about 93 to 9,179 〇 1 / K cat , μsec: about 4 to 428 〇 K cat / K M , mM -1 sec -1 : about 60 to 5,934

[0280] Other ratios may be employed (e.g., 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0281] In certain embodiments, a ratio of type I collagenase to type II collagenase of about 1 mg to about 20 mg of about 1:1 is injected using Treatment I, and one or more of the results of Results Nos. 1-12 above are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , sec -1 : about 93 to 9,179 〇 1 / K cat , μsec: about 4 to 428 〇 K cat / K M , mM -1 sec -1 : about 60 to 5,934 Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0282] As further described in Examples 2 and 3, the improvement of the patients after treatment was evaluated using the PR-CIS of CCH versus placebo, comparing it to the baseline. As shown in FIG. 11, the treatment with CCH was significantly better than the placebo.

[0283] 8. Efficacy Measured by the Abbreviated PR-CIS By the above treatment method, the response measured in the PR-CIS abbreviated form is improved. The total score of the PR-CIS abbreviated form is the sum of the five items of the scale. The total score of the PR-CIS abbreviated form ranges from 0 to 50, and the higher the numerical value, the greater the adverse effect by cellulite. Item 1 of the PR-CIS asks about how satisfied the subject is with the appearance of cellulite, and it is reversed by subtracting the evaluation reported by the subject from 10. For the PR-CIS abbreviated form total score, a subject whose PR-CIS total score has decreased by 10 or more from the baseline at the evaluation time point is considered a responder. For each individual PR-CIS abbreviated form impact score, a response is considered when there is an improvement at least two score intervals from the baseline at each time point. Further, a responder refers to a patient whose maximum total score shows an improvement of at least 20% from the baseline. Also, for each individual patient, it means that there is an improvement of at least one level or one evaluation from the baseline or the previous score. Also, the improvement of the patient group means that the average score or evaluation has increased by about 0.1 from the baseline or the previous average score or evaluation. Further, improvement means that the change from the baseline is at least one level out of 50.

[0284] In certain embodiments, the treatment method detailed above provides one or more of the following efficacy evaluation items measured in the PR-CIS short form: 1. In a patient population with cellulite, by injecting collagenase into at least one treatment area at at least one treatment visit, a statistically significant number of such patients satisfy one or more of the following efficacy evaluation items: ● In the PR-CIS short form, show improvement in at least one area selected from the group consisting of satisfaction with the appearance of cellulite, concerns, sensations, embarrassment, looking older, looking overweight / looking out of shape. ● At one or more evaluation time points, the total score in the PR-CIS short form decreases by 10 or more from the baseline. ● The PR-CIS short form impact score shows improvement of at least 2 score intervals from the baseline at one or more evaluation time points. ● Further, improvement means that the change from the baseline is at least 1 level out of 50. 2. In the PR-CIS short form of the treatment area, 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, there is an improvement in severity of at least 12 points from the baseline (the "day 1" before treatment). 3. In a patient population with cellulite, in the PR-CIS short form of the treatment area, 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 from the baseline (day 1 before treatment), the severity improves by at least 10 points statistically significantly compared to the placebo. 4. After the initial administration, a result was obtained that at least 5% of the patients maintained an improvement level relative to the pre-treatment baseline for 71 days or more. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained such a level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial administration. In other cases, a result was obtained that at least 5% of the patients showed improvement relative to the pre-treatment baseline, and the degree of improvement increased over time. In a certain treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the initial administration, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further increase in improvement was observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 5. On the 180th day, the improvement from the baseline of the PR-CIS abridged evaluation was consistently observed on both the left and right sides of the treatment area. 6. In the patient population with cellulite, for at least one treatment area, the median time until the total score of the PR-CIS abridged form decreased by at least 10 from the baseline at one or more evaluation 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 the patient population with cellulite, the average value of the PR-CIS abridged score of the subjects separated from the placebo 21 days after the first treatment, and showed continuous and significant improvement in subsequent treatments. 8. In the patient population with cellulite, more than one-third, or more than one-half, or more than two-thirds, or more than three-fourths of the patients had a decrease of at least 10 in the total PR-CIS abridged score from the baseline at one or more evaluation time points in at least one treatment area by the 71st day after treatment, and the results of PR-CIS do not depend on age, BMI or skin color. 9. The reduction in the severity of cellulite occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit. 10. In patient populations with a moderate or severe CR-PCSS and / or PR-PCSS assessment, the average change from baseline in the PR-CIS abbreviated total score on day 71 was approximately -10 in subjects receiving collagenase treatment and approximately -5 in subjects receiving placebo treatment. 11. In patient populations with a moderate or severe CR-PCSS and / or PR-PCSS assessment, the mean change in the PR-CIS abbreviated form from baseline on day 71 was statistically significantly better in subjects receiving collagenase treatment than in subjects receiving placebo treatment, in both the total score (approximately -12 vs. approximately -6, respectively) and the abbreviated score (approximately -10 vs. approximately 5, respectively). Also, statistically significant changes were observed between the collagenase treatment group and the placebo treatment group in the individual impact scores (satisfaction with the appearance of cellulite, bothersomeness, awareness, embarrassment, aged appearance, concern about body shape). 12. In patient populations with a moderate or severe CR-PCSS and / or PR-PCSS assessment on day 71, the proportion of PR-CIS abbreviated responders was greater in subjects administered collagenase than in subjects administered placebo, in both the total score (approximately 45% vs. 20%, respectively) and the abbreviated score (approximately 50% vs. 25%, respectively). Furthermore, for the individual impact scores, the proportion of responders was greater in the collagenase treatment group than in the placebo treatment group. These differences were also statistically significant.

[0285] In another embodiment, one or more of the above Results Nos. 1 to 12 are obtained by injecting from 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 characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min-1 ) ● About 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) of K M ● About 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) of K cat (seconds -1 ) ● About 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) of 1 / K cat , microseconds ● About 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) of K 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 of the area ratio measured by reverse-phase HPLC (high-pressure liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● A titer of about 175,000 to about 500,00 f-GPA units / mg ● A titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

[0286] In other cases, by injection of about 1 mg to about 20 mg of collagenase by Treatment I, one or more of the above Results Nos. 1 to 9 are obtained.

[0287] In another example, injection of about 1 mg to about 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 to 12, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomolar (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● kcat of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / kcat of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat 、 ● kcat / Km of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / Km M 、 -1 sec -1 ● 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 ● Purity of area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Potency (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● Potency of about 5,000 to about 30,000 f-SRC units / mg ● Potency of about 100,000 to about 400,000 GPA units / mg ● Potency of about 175,000 to about 500,00 f-GPA units / mg ● Potency of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0289] In certain embodiments, about 1 mg to about 20 mg of a collagenase type I and collagenase type II in an approximate 1:1 ratio are injected into at least one treatment area during at least one treatment visit, resulting in one or more of the above Results Nos. 1 to 12, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , sec -1 : about 93 to 9,179 〇 1 / K cat , μsec: about 4 to 428 〇 K cat / K M , mM -1 sec -1 : about 60 to 5,934

[0290] Other ratios may be employed (e.g., 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

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

[0292] Other ratios may be adopted (for example, 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0293] 9. Efficacy Measured by the Subject Self-Rating Scale (SSRS) By the treatment method as described above, an improvement effect measured by SSRS was obtained. An SSRS responder is a subject who is at least somewhat satisfied (somewhat satisfied [4], very satisfied [5], or extremely satisfied [6]) with the appearance of cellulite in the affected area at the evaluation time point. Further, a patient with an improvement of 17% or more in the maximum total score from the baseline is defined as a responder. The improvement of an individual patient at any visit means an improvement of at least one level or one evaluation from the baseline or the previous score. The improvement of a group of patients at any visit is an improvement in the average score or evaluation of about 0.1 from the baseline or any previous average score or evaluation. In a specific embodiment, the treatment method results in one or more of the following effectiveness evaluation items measured by the SSRS evaluation: 1. In a population of patients with cellulite, by injecting collagenase into at least one treatment area at least once during a treatment visit, a statistically significant number of such patients can meet one or more of the following effectiveness evaluation items: ● SSRS evaluation of "somewhat satisfied" ● SSRS evaluation of "satisfied" ● SSRS evaluation of "extremely satisfied" 2. The SSRS evaluation of the treatment area improves by at least two levels on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3rd year, 4th year, or 5th year from the baseline (the "first day" before treatment). 3. The SSRS evaluation of the treatment area improves by at least 1 level on the 22nd, 43rd, 71st, 90th, 180th, 251st, 360th, 431st, 720th day, 3rd year, 4th year, or 5th year from the baseline (day 1). 4. In the patient population with cellulite, the improvement in the SSRS evaluation in at least one treatment area is statistically significant compared to the placebo, and the improvement is one or more of the above Nos. 2 to 3. 5. After the first administration, a treatment result was obtained in which at least 5% of the patients maintained an improvement level of at least 71 days compared to the pre-treatment baseline. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained such a level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the first administration. In other cases, at least 5% of the patients showed improvement compared to the pre-treatment baseline, and the degree of improvement increased over time. In one treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first administration, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further increase in improvement was observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 6. On the 180th day after the first injection, the improvement from the baseline of the SSRS evaluation was consistently observed on both the left and right sides of the treatment area. 7. In the patient population with cellulite, the median time to the earliest 2-level improvement in the SSRS evaluation in at least one treatment area is about 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 8. In a patient population having cellulite, the median time to the earliest level 1 improvement in the 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 having cellulite, the mean value of the SSRS assessment of the subjects separates from the placebo 21 days after the first treatment, and shows continuous and significant improvement in subsequent treatments. 10. In a patient population having cellulite, the percentage of subjects having a level 2 composite response measured by the SSRS assessment in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 11. In a patient population having cellulite, the percentage of subjects having a level 1 composite response measured by the SSRS assessment in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 12. In a patient population having cellulite, one-third or more, or one-half or more, or two-thirds or more, or three-fourths or more of the patients have at least a level 1 SSRS assessment response in at least one treatment area by day 71 after treatment, and the SSRS assessment results are independent of age, BMI, or skin color. 13. The reduction in the severity of cellulite occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit. 14. In a patient population in which the CR-PCSS and / or PR-PCSS assessments are moderate or severe, at least 40% of the patients became level 1 SSRS responders on day 71. 15. In a patient population in which the CR-PCSS and / or PR-PCSS assessments are moderate or severe, on day 71, the mean value of the SSRS score is statistically significantly greater for the subjects administered collagenase than for the subjects administered placebo. In a patient population with moderate or severe CR-PCSS and / or PR-PCSS evaluation, at least 50% of the patients are level 1 SSRS responders.

[0294] In another embodiment, during at least one treatment visit, by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area, one or more of the above Results Nos. 1 to 16 are obtained, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● kcat of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / kcat of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat , microseconds ● kcat / K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / K M 、mM -1 sec -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 of the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● Potency of about 175,000 to about 500,000 - GPA units / mg ● Potency of about 5,000 to about 25,000 - ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

[0295] In other cases, by injection of about 1 mg to about 20 mg of collagenase by Treatment I, one or more of the above Results No. 1 to 16 are obtained.

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

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

[0298] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in an approximate 1:1 ratio are injected into at least one treatment area during at least one treatment visit, resulting in one or more of the above Results Nos. 1 to 16, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min-1 : About 0.3 to 30.5 〇 K M , mM: About 0.03 to 3.1 〇 K cat , seconds -1 : About 93 to 9,179 〇 1 / K cat , microseconds: About 4 to 428 〇 K cat / K M , mM -1 seconds -1 : About 60 to 5,934 Other ratios may be adopted (for example, 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, 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 type I and type II collagenases in an approximate 1:1 ratio are injected using Therapy I, and one or more of the above Results Nos. 1 to 16 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , minutes -1 : About 0.08 to 7.70 〇 K M : About 4.1 to 410 nanomoles 〇 K cat , seconds -1 : About 1.1 to 107 〇 1 / K cat , microseconds: About 376 to 37,222 〇 K cat / K M , mM -1 seconds -1 : About 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , minutes-1 : About 0.3 to 30.5 〇 K M , mM: About 0.03 to 3.1 〇 K cat , seconds -1 : About 93 to 9,179 〇 1 / K cat , microseconds: About 4 to 428 〇 K cat / K M , mM -1 seconds -1 : About 60 to 5,934

[0300] Other ratios may be adopted (for example, 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Furthermore, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0301] 10. Efficacy Measured by the Subject Satisfaction with Cellulite Treatment (SSCT) By the treatment method detailed above, improvement measured by SSCT is obtained. Subjects who received a "satisfied" or "very satisfied" response in the SSCT evaluation on the 71st day are considered responders indicating efficacy. Improvement in an individual patient means at least one level or one evaluation improvement from the baseline or previous score or evaluation. Also, improvement in a patient group means that the average score or evaluation has improved by about 0.1 point from the baseline or previous average score or evaluation. Furthermore, in SSCT, there is at least 0.1 improvement compared to placebo. In certain embodiments, the treatment method results in one or more of the following efficacy evaluation items measured by SSCT evaluation: 1. In a population of patients with cellulite, by injecting collagenase into at least one treatment area at at least one treatment visit, a statistically significant number of such patients were able to meet one or more of the following efficacy evaluation items: ● "Satisfied" SSCT evaluation ● "Very satisfied" SSCT evaluation 2. An increase in SSCT assessment (e.g., from 1 to 2, etc.) on the 22nd, 43rd, 71st, 90th, 180th, 251st, 360th, 431st, 720th day, 3rd year, 4th year, or 5th year in the treatment area. 3. In the patient population with cellulite, an increase in SSCT assessment in at least one treatment area was statistically significant compared to placebo, and it was confirmed that the improvement occurred at any of the time points of the 22nd, 43rd, 71st, 90th, 180th, 251st, 360th, 431st, 720th day, 3rd year, 4th year, 5th year. 4. With this treatment method, at least 5% of the patients maintained their improvement level for 71 days after the first administration. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained such a level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the first administration. In other cases, at least 5% of the patients showed improvement, and the degree of improvement further increased over time. In a certain treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first administration, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and a further increase in improvement was observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 5. At the 180th day, the improvement seen in the SSCT assessment is consistent across the left and right of the treatment area. 6. In the patient population with cellulite, the median time to the earliest improvement in SSCT 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. 7. In the patient population with cellulite, the average subject's SSCT assessment separates from placebo 21 days after the first treatment and shows continuous and significant improvement in subsequent treatments. 8. In the patient population with cellulite, the percentage of subjects with improved SSCT evaluation in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 9. In the patient population with cellulite, by day 71 after treatment, more than one-third, or more than one-half, or two-thirds, or three-fourths of the patients have an improved SSCT evaluation in at least one treatment area, and the results of the SSCT evaluation are independent of age, BMI, or skin color. 10. The reduction in the severity of cellulite occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit. 11. In the patient population with a moderate or severe CR-PCSS and / or PR-PCSS evaluation, more than half of the patients are satisfied or very satisfied with the treatment on day 180. 12. In the patient population with a moderate or severe CR-PCSS and PR-PCSS evaluation, the decrease in the sustained CR-PCSS and PR-PCSS evaluation and the score of the subject satisfaction survey on day 180 indicate that three treatment sessions of subcutaneous injection of 0.84 mg CCH 12 times per treatment (×2 treatment areas) into both buttocks or both thighs are effective in reducing cellulite. 13. In the patient population with a moderate or severe CR-PCSS and / or PR-PCSS evaluation, at least 50% of the patients treated with collagenase are satisfied or very satisfied with the cellulite treatment. 14. In the patient population with a moderate or severe CR-PCSS and PR-PCSS evaluation, a statistically significant difference was observed between the collagenase treatment group and the placebo treatment group in the average value of subject satisfaction on day 71.

[0302] In another embodiment, during at least one treatment visit, collagenase in an amount of about 1 mg to about 20 mg is injected into at least one treatment area to obtain one or more of the above results Nos. 1 to 14, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● k of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat 、microseconds ● K / K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / K M 、mM -1 sec -1 ● 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 ● Purity as the area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Potency (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● Potency of about 5,000 to about 30,000 f-SRC units / mg ● Potency of about 100,000 to about 400,000 GPA units / mg ● Potency of about 175,000 to about 500,00 f-GPA units / mg ● Potency of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

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

[0304] In another example, injection of about 1 mg to about 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 to 14, where the collagenase has one or more of the following characteristics: ● A V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● A K of about 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● A k of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● A 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat , microseconds ● A K / K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / K M , mM -1 sec -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 ● A purity with an area ratio measured by reverse-phase HPLC (high performance liquid chromatography) of at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● Potency of about 175,000 to about 500,000 - GPA units / mg ● Potency of about 5,000 to about 25,000 - ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0306] In certain embodiments, about 1 mg to about 20 mg of Type I collagenase and Type II collagenase in an approximate 1:1 ratio are injected into at least one treatment area during at least one treatment visit, and one or more of the above Results Nos. 1 to 14 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomolar 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , sec -1 : about 93 to 9,179 〇 1 / Kcat 、 Microseconds: about 4 - 428 〇 K cat / K M 、 mM -1 Seconds -1 : about 60 - 5,934

[0307] Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, Type I and Type II collagenases may be AUX - I and AUX - II, respectively.

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

[0309] Other ratios may be employed (e.g., 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, 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), Thigh Cellulite Severity - Clinician (TCS-C) Improvement of an individual patient at any visit means at least a one-level or one-grade improvement from the baseline or previous score or evaluation. Improvement of a patient group means that the average score or evaluation has improved by about 0.1 from the baseline or previous average score or evaluation. A responder is any patient who shows at least a 20% improvement in the maximum total score or evaluation from the baseline. In certain embodiments, one or more of the following efficacy evaluation items measured by TCS-C and / or TCS-P are obtained by the treatment method detailed above: 1. The evaluation of TCS-C and / or TCS-P has improved by 0.1 or more compared to the baseline. 2. The severity of TCS-C clinically evaluated by a clinician of the target thigh improves by at least two levels on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3-year mark, 4-year mark, or 5-year mark. 3. The severity of TCS-P evaluated by the subject while viewing digital images of the target thigh improves by at least two levels from the baseline (day 1) on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3-year mark, 4-year mark, or 5-year mark. The improvement shown by a two-level composite response on the 42nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3rd year, 4th year, or 5th year is defined as a subject having at least a two-level severity improvement from the baseline in TCS-C and at least a two-level severity improvement from the baseline in TCS-P. On the 52nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3rd year, 4th year, or 5th year, the severity of TCS-C evaluated by a clinician of the target thigh in a live setting improves by at least one level from the baseline (1st day). On the 62nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3rd year, 4th year, or 5th year, the severity of TCS-P evaluated by the subject while viewing a digital image of the target thigh improves by at least one level from the baseline (1st day). The improvement shown by a one-level composite response on the 72nd day, 43rd day, 71st day, 90th day, 180th day, 251st day, 360th day, 431st day, 720th day, 3rd year, 4th year, or 5th year is defined as a subject having at least a one-level severity improvement from the baseline in TCS-C and at least a one-level severity improvement from the baseline in TCS-P. In a patient population with a moderate or severe TCS-C evaluation, the improvement in at least one treatment area is statistically significant compared to the placebo, and the improvement is one or more of the above Nos. 1 to 7. 9. After the initial administration, results were obtained showing that at least 5% of patients maintained an improvement level of at least 71 days relative to the pre-treatment baseline. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained 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 initial administration. In other cases, results were obtained showing that at least 5% of patients showed improvement relative to the pre-treatment baseline, and the degree of improvement increased over time. In one treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the initial administration, at least 10%, 20%, 30%, 40%, 50% of patients showed improvement, and further increases in improvement were observed. In several treatment methods, at least 10%, 20%, 30%, 40%, 50% of patients showed improvement, and further improvements were observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 10. On the 180th day, improvements in the TCS-C and / or TCS-P evaluations from the baseline were consistently observed in the left and right thigh regions. 11. In a patient population with a moderate or severe TCS-C evaluation, the median time until at least 2 levels of improvement in TCS-C and / or TCS-P are obtained in at least one treatment area is approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days. 12. In a patient population with a moderate or severe TCS-C evaluation, the median time until the earliest 1 level of improvement in TCS-C and / or TCS-P 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. 13. In a patient population with a moderate or severe TCS-C evaluation, the average value of the TCS-C and / or TCS-P scores of the subjects separated from the placebo 21 days after the first treatment, and continuous and significant improvement was shown in subsequent treatments. 14. In a patient population with moderate or severe TCS-C assessment, the percentage of subjects having a two-level composite response measured by TCS-C and / or TCS-P in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 15. In a patient population with moderate or severe TCS-C assessment, the percentage of subjects having a one-level composite response measured by TCS-C and / or TCS-P in at least one treatment area on day 71 is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, or greater than 50%. 16. In a population of patients all having moderate or severe TCS-C assessment, more than one-third, or more than one-half, or more than two-thirds, or more than three-fourths of the patients having at least one level of TCS-C and / or at least one level of TCS-P response in at least one treatment area by day 71 after treatment have TCS-C results that are independent of age, BMI, or skin color. 17. The reduction in cellulite severity occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit.

[0311] In another embodiment, during at least one treatment visit, by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area, one or more of the above results No. 1 - 17 are obtained, where the collagenase has one or more of the following properties: ● V of about 0.08 - 7.70 (SRC assay), or about 0.3 - 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 - 410 nanomolar (SRC assay), or about 0.03 - 3.1 mM (GPA assay) M ● K of about 1.1 - 107 (SRC assay), or about 93 - 9,179 (GPA assay) cat (sec -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 ● Molecular weight of approximately 60 kDa to approximately 130 kDa, or approximately 70 to approximately 130 kDa, or approximately 80 to approximately 120 kDa, or approximately 90 to approximately 120 kDa, or approximately 100 to approximately 110 kDa ● Purity as area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Titer (i.e., specific activity) of approximately 500 to 30,000 SRC units / mg ● Titer of approximately 5,000 to approximately 30,000 f-SRC units / mg ● Titer of approximately 100,000 to approximately 400,000 GPA units / mg ● Titer of approximately 175,000 to approximately 500,00 f-GPA units / mg ● Titer of approximately 5,000 to approximately 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0313] In another example, injection of approximately 1 mg to approximately 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 to 17, 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 (min -1 ) ● About 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) of K M ● About 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) of K cat (seconds -1 ) ● About 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) of 1 / K cat , microseconds ● About 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) of K 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 of the area ratio measured by reverse-phase HPLC (high-performance liquid chromatography) is at least 80% ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● A titer of about 175,000 to about 500,00 f-GPA units / mg ● A titer of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of chymotrypsin, gelatinase, and leupeptin are 1% or less by area ratio ● A bioburden of 1 cfu / mL or less

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

[0315] In certain embodiments, a 1:1 ratio of type I collagenase and type II collagenase, from about 1 mg to about 20 mg, is injected into at least one treatment area during at least one treatment visit, and one or more of the above Results Nos. 1-17 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 - 7.70 〇 K M : about 4.1 - 410 nanomolar 〇 K cat , sec -1 : about 1.1 - 107 〇 1 / K cat , μsec: about 376 - 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 - 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 - 30.5 〇 K M , mM: about 0.03 - 3.1 〇 K cat , sec -1 : about 93 - 9,179 〇 1 / K cat , μsec: about 4 - 428 〇 K cat / K M , mM -1 sec -1 : about 60 - 5,934

[0316] Other ratios may be employed (e.g., 0.1 to 2:1, or 0.25 to 2:1, or 0.5 to 2:1, or 0.75 to 2:1, or 1:0.1 to 2, or 1:0.25 to 2, or 1:0.5 to 2, or 1:0.75 to 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0317] In certain embodiments, a ratio of type I collagenase to type II collagenase of about 1 mg to about 20 mg of about 1:1 is employed in Treatment I and injected, and one or more of the above Results Nos. 1 to 17 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min -1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , sec -1 : about 93 to 9,179 〇 1 / K cat , μsec: about 4 to 428 〇 K cat / K M , mM -1 sec -1 : about 60 to 5,934

[0318] Other ratios may be employed (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be AUX-I and AUX-II, respectively.

[0319] 12. Efficacy Measured by Body-Q With the above-described treatment method, the response measured by Body-Q is improved. For cellulite, there are 16 scale items that patients can answer in the range from "not at all concerned" to "very concerned" over the past week, assuming a reading ability at the Fresh Kincaid level. The score range is from 16 (very concerned) to 64 (not at all concerned). For the Body-Q total score, subjects in whom the Body-Q total score has increased by 16 or more from the baseline at the time of evaluation are considered responders. For each individual Body-Q impact score, a response is that there is an improvement of at least one score interval from the baseline at each time point. In an alternative embodiment, the scale items may be more or less than 16, and a responder is any patient showing an improvement of at least 25% of the maximum total score from the baseline.

[0320] In certain embodiments, with the treatment method detailed above, one or more of the following efficacy evaluation items measured by Body-Q are obtained: 1. In a patient population having cellulite, by injecting collagenase into at least one treatment area at at least one treatment visit, a statistically significant number of such patients satisfy one or more of the following efficacy evaluation items: ● Body-Q shows improvement in at least one field selected from the group consisting of: 〇 At one or more evaluation time points, the total score of Body-Q increases by 16 or more from the baseline 〇 For each individual Body-Q impact score, there is an improvement of at least one score interval from the baseline at each time point, and 〇 The maximum total score improves by at least 25% from the baseline. ● At one or more evaluation time points, the total score of Body-Q increases by 16 or more from the baseline. ● At one or more evaluation time points, the Body-Q impact score shows an improvement of at least one score interval from the baseline. ● Average change from the baseline of the Body-Q evaluation regarding cellulite at day 90 and / or day 180 2. Improvement in severity with an increase of at least 16 points in the Body-Q of the target buttock from the 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. 3. Statistically significant improvement in severity over placebo, as shown by an increase of at least 16 points in the Body-Q of the target buttock from the 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 in a patient population with cellulite. 4. After the initial administration, a result was obtained that at least 5% of the patients maintained an improvement level relative to the pre-treatment baseline for 71 days or more. In certain cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained such a level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years, or 3 years, or 4 years, or 5 years after the initial administration. In other cases, a result was obtained that at least 5% of the patients showed improvement relative to the pre-treatment baseline, and the degree of improvement increased over time. In a certain treatment method, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the initial administration, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further increase in improvement was observed. In some treatment methods, at least 10%, 20%, 30%, 40%, 50% of the patients showed improvement, and further improvement was observed 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months after the first treatment, the second treatment, and the third treatment. 5. On the 180th day after the first injection, the improvement in the Body-Q assessment from the baseline was consistently observed in the left and right buttocks. 6. In the patient population with cellulite, for at least one treatment area, the median time until the Body-Q total score increased by at least 16 from the baseline at one or more evaluation 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 the patient population with cellulite, the average Body-Q score of the subjects separated from the placebo 21 days after the first treatment and continued to show significant improvement in subsequent treatments. 8. In the patient population with cellulite, more than one-third, or more than one-half, or more than two-thirds, or more than three-fourths of the patients had an increase in the Body-Q total score of at least 16 from the baseline at one or more evaluation time points in at least one treatment area by the 71st day after treatment, and the Body-Q results were independent of age, BMI, or skin color. 9. The reduction in the severity of cellulite occurs rapidly within about 7, or 14, or 21, or 30, or 35, or 40, or 45, or 50 days after the first treatment visit.

[0321] In another embodiment, during at least one treatment visit, one or more of the above Results Nos. 1 to 9 are obtained by injecting about 1 mg to about 20 mg of collagenase into at least one treatment area, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● k of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / k of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat , microseconds ● k / K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / K M 、mM -1 sec -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 ● A purity of at least 80% as measured by reverse-phase HPLC (high-performance liquid chromatography) in terms of area ratio ● A titer (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● A titer of about 5,000 to about 30,000 f-SRC units / mg ● A titer of about 100,000 to about 400,000 GPA units / mg ● Potency of about 175,000 to about 500,000 GPA units / mg ● Potency of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0323] In another example, injection of about 1 mg to about 20 mg of collagenase by Treatment I results in one or more of the above Results Nos. 1 to 9, where the collagenase has one or more of the following characteristics: ● V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) max (min -1 ) ● K of about 4.1 to 410 nanomoles (SRC assay), or about 0.03 to 3.1 mM (GPA assay) M ● k of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) cat (sec -1 ) ● 1 / k of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay), microseconds cat 、microseconds ● K / K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay), mM cat / K M 、mM -1 sec -1 ● 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 ● Purity by area ratio measured by reverse phase HPLC (high performance liquid chromatography) is at least 80% ● Potency (i.e., specific activity) of about 500 to 30,000 SRC units / mg ● Potency of about 5,000 to about 30,000 f-SRC units / mg ● Potency of about 100,000 to about 400,000 GPA units / mg ● Potency of about 175,000 to about 500,00 f-GPA units / mg ● Potency of about 5,000 to about 25,000 ABC units / mg ● Impurities selected from the group consisting of clostripain, gelatinase, and leupeptin are 1% or less by area ratio ● Bioburden of 1 cfu / mL or less

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

[0325] In certain embodiments, about 1 mg to about 20 mg of type I collagenase and type II collagenase in an approximate 1:1 ratio are injected into at least one treatment area during at least one treatment visit, and one or more of the above Results Nos. 1 to 9 are obtained, where collagenases I and II have the following characteristics: ● Type I 〇 Assay: SRC microplate 〇 V max , min -1 : about 0.08 to 7.70 〇 K M : about 4.1 to 410 nanomoles 〇 K cat , sec -1 : about 1.1 to 107 〇 1 / K cat , μsec: about 376 to 37,222 〇 K cat / K M , mM -1 sec -1 : about 5,140 to 508,814 ● Type II 〇 Assay: GPA microplate 〇 V max , min-1 : about 0.3 to 30.5 〇 K M , mM: about 0.03 to 3.1 〇 K cat , seconds -1 : about 93 to 9,179 〇 1 / K cat , microseconds: about 4 to 428 〇 K cat / K M , mM -1 seconds -1 : about 60 to 5,934 Other ratios may be adopted (for example, 0.1 to 2:1, or 0.25 to 2:1, or 0....

Claims

1. 1. A method for reducing the severity of cellulite in both buttocks of a human patient, the method comprising: a. The following characteristics: i. A V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) ｍａｘ (minute －１ ). ii. A K of about 4.1 to 410 nanomolar (SRC assay), or about 0.03 to 3.1 mM (GPA assay) Ｍ ; iii. A K of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) ｃａｔ (seconds －１ ). iv. a 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) ｃａｔ , microseconds; v. K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) ｃａｔ / K Ｍ , mM －１ seconds －１ ; vi. 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; vii. A purity of at least 80% by area as measured by reverse phase HPLC (high pressure liquid chromatography); viii. a potency of about 5,000 to about 30,000 f-SRC units / mg; ix. a titer of about 175,000 to about 500,00 f-GPA units / mg; x. a potency of about 5,000 to about 25,000 ABC units / mg; xi. The amount of an impurity selected from the group consisting of clostripain, gelatinase, and leupeptin is 1% or less by area; xii. a bioburden of less than or equal to 1 cfu / mL; providing a collagenase composition having at least two of: b. injecting a therapeutically effective amount of said collagenase composition into the pits of both buttocks, wherein the improvement in the appearance of cellulite is evaluated using a Hexel Cellulite Severity Scale (Hexel CSS), Hexel Dimple Depth Score, Likert Scale, Dimple 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 Severity Scale (P-PCSS), or Hexel Cellulite Severity Scale (Hexel CSS). the cellulite severity 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, a validated photo-numerical or other scale used by a clinician and / or patient to assess cellulite severity, improvement, and / or patient satisfaction; The method comprising:

2. The method of claim 1 , wherein the recesses that are treated are independent of size or distance from one another.

3. 10. The method of claim 1, wherein the depression being treated is free of loose, flaky, or sagging skin.

4. 10. The method of claim 1, wherein the patient undergoes multiple treatment visits and different depressions are treated at different treatment visits.

5. 10. The method of claim 1, wherein the injection is performed with a 1 / 2 inch needle.

6. 10. The method of claim 1, wherein the injection is performed by a clinician without relying on devices such as spacers, rulers, paper, etc. to limit the location of the injection.

7. 10. The method of claim 1, wherein at least one injection is administered directly beneath the recess.

8. 10. The method of claim 1, wherein the multiple injections are administered within 2 cm of each other.

9. 2. The method of claim 1, wherein the length of the depression being treated is less than 1 cm or greater than 2 cm.

10. 10. The method of claim 1, wherein the patient experiences a rapid response rate to treatment.

11. 10. The method of claim 1, wherein the treatment is administered to a patient population all of whom have a baseline CR-PCSS rating of moderate or severe, the treatment is selected from the group consisting of: a. at least 50% of patients have at least one level improvement from baseline in CR-PCSS severity assessed live by the hip clinician at Days 22, 43, or 71; b. at least 50% of patients have at least one level improvement from baseline in subject-assessed PR-PCSS severity while viewing digital images of the buttocks at Days 22, 43, or 71; c. at least 5% of patients have an improvement of at least 2 levels from baseline in CR-PCSS severity as assessed live by the hip clinician at Days 22, 43, or 71; d. at least 5% of patients have an improvement of at least 2 levels from baseline in subject-assessed PR-PCSS severity while viewing digital images of the buttocks at Days 22, 43, or 71; and e. at least 5% of patients experience a decrease in cup size; A method which produces a result selected from the group consisting of:

12. The method of claim 11 , wherein the recess size parameters are selected from the group consisting of volume, length, width, and depth.

13. 12. The method of claim 11, wherein the reduction in pit size is at least a 10% reduction from baseline at days 22, 43, or 71.

14. 10. The method of claim 1, wherein the cumulative collagenase injected is about 5.04 mg.

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

16. 10. The method of claim 1, wherein the collagenase composition has the following characteristics: AUX-I (SRC assay): i. V ｍａｘ ,point －１ : About 3.8 ii. K Ｍ、 mm: about 2.07×10 －４ iii. K ｃａｔ ,Second －１ : About 53 iv. 1 / K ｃａｔ , microseconds: about 18,799 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : About 256,977 b. AUX-II (GPA assay) i. V ｍａｘ ,point －１ : About 15.4 ii. K Ｍ、 mm: about 1.6 iii. K ｃａｔ ,Second －１ : About 4,636 iv. 1 / K ｃａｔ , microseconds: about 216 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : About 2,997 A method comprising the steps of:

17. The method of claim 1 , wherein the composition has at least three of said characteristics.

18. The method of claim 1 , wherein the composition has at least four of said characteristics.

19. 10. The method of claim 1, wherein said composition has at least five of said characteristics.

20. 10. The method of claim 1, wherein the composition comprises about 1 mg to 20 mg of one or more collagenases.

21. 10. The method of claim 1, wherein the composition comprises CCH.

22. 10. The method of claim 1, wherein the composition has a potency of about 10,000 ABC units / 0.58 mg and the therapeutically effective amount is about 1 mg to 20 mg.

23. 10. The method of claim 1, wherein the composition has a potency of about 15,000 ABC units / mg to 20,000 ABC units / mg and the therapeutically effective amount is about 1 mg to 20 mg.

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

25. 10. The method of claim 1, wherein when the treatment is administered to a patient population, the treatment results in at least 5% of patients maintaining a level of improvement relative to a pre-treatment baseline for at least 71 days after the first administration.

26. 26. The method of claim 25, wherein at least 10% of the patients maintain a level of improvement relative to pre-treatment baseline for at least 71 days after the first administration.

27. 26. The method of claim 25, wherein at least 20% of the patients maintain a level of improvement relative to pre-treatment baseline for at least 71 days after the first administration.

28. 10. The method of claim 1, wherein when the treatment is administered to a patient population, the treatment results in at least 5% of patients showing improvement relative to a pre-treatment baseline and showing a further increase in improvement over time.

29. 10. The method of claim 1, wherein the treatment achieves the following efficacy outcomes as measured by CR-PCSS and / or PR-PCSS: a. at least a 2-level improvement from baseline (pre-treatment "Day 1") in live hip clinician-assessed CR-PCSS severity at Days 22, 43, 71, 90, 180, or 365; b. An improvement of at least 2 levels from baseline (Day 1) in PR-PCSS severity as assessed by the patient while viewing digital images of the buttocks at Days 22, 43, 71, 90, 180, or 365; c. improvement as indicated by a 2-level composite response at Days 22, 43, 71, 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 from baseline (Day 1) in CR-PCSS severity assessed live by the hip clinician at Days 22, 43, 71, 90, 180, or 365; e. at least one level improvement from baseline (Day 1) in PR-PCSS severity as assessed by the patient while viewing digital images of the buttocks at Days 22, 43, 71, 90, 180, or 365; f. Improvement as indicated by a one-level composite response at Days 22, 43, 71, 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 scores 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 method produces at least one of the following results:

30. 10. The method of claim 1, wherein the treatment is administered to a subject with one or more of the following efficacy outcomes measured by cupidity analysis: 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 method of claim 1,

31. 1. A method for reducing the severity of cellulite in both buttocks of a human patient, the method comprising: a. The following characteristics: i. A V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) ｍａｘ (minute －１ ). ii. A K of about 4.1 to 410 nanomolar (SRC assay), or about 0.03 to 3.1 mM (GPA assay) Ｍ ; iii. A K of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) ｃａｔ (seconds －１ ). iv. a 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) ｃａｔ , microseconds; v. K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) ｃａｔ / K Ｍ , mM －１ seconds －１ ; vi. 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; vii. A purity of at least 80% by area as measured by reverse phase HPLC (high pressure liquid chromatography); viii. a potency of about 5,000 to about 30,000 f-SRC units / mg; ix. a titer of about 175,000 to about 500,00 f-GPA units / mg; x. a potency of about 5,000 to about 25,000 ABC units / mg; xi. The amount of an impurity selected from the group consisting of clostripain, gelatinase, and leupeptin is 1% or less by area; xii. a bioburden of less than or equal to 1 cfu / mL; providing a collagenase composition having at least two of: b. injecting a therapeutically effective amount of said collagenase composition into both buttock cavities according to Treatment I, whereby bruising is significantly reduced or the darkness is eliminated between about 3 and 20 days after the treatment visit; The method comprising:

32. 32. The method of claim 31, wherein the cumulative collagenase injected is about 5.04 mg.

33. 32. The method of claim 31, wherein the collagenase composition has the following characteristics: AUX-I (SRC assay): i. V ｍａｘ ,point －１ : About 0.08~7.70 ii. K Ｍ : about 4.1 to 410 nanomoles iii. K ｃａｔ ,Second －１ : About 1.1~107 iv. 1 / K ｃａｔ , microseconds: about 376 to 37,222 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : Approximately 5,140 to 508,814 b. AUX-II (GPA assay) i. V ｍａｘ ,point －１ : About 0.3~30.5 ii. K Ｍ、 mM: about 0.03~3.1 iii. K ｃａｔ ,Second －１ : About 93~9,179 iv. 1 / K ｃａｔ , microseconds: about 4 to 428 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : About 60~5,934 A method comprising the steps of:

34. 32. The method of claim 31, wherein the collagenase composition has the following characteristics: AUX-I (SRC assay): i. V ｍａｘ ,point －１ : About 3.8 ii. K Ｍ、 mm: about 2.07×10 －４ iii. K ｃａｔ ,Second －１ : About 53 iv. 1 / K ｃａｔ , microseconds: about 18,799 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : About 256,977 b. AUX-II (GPA assay) i. V ｍａｘ ,point －１ : About 15.4 ii. K Ｍ、 mm: about 1.6 iii. K ｃａｔ ,Second －１ : About 4,636 iv. 1 / K ｃａｔ , microseconds: about 216 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : About 2,997 A method comprising the steps of:

35. 32. The method of claim 31, wherein the composition has at least three of said characteristics.

36. 32. The method of claim 31, wherein the composition has at least four of said characteristics.

37. 32. The method of claim 31, wherein the composition has at least five of said characteristics.

38. 32. The method of claim 31, wherein the composition comprises about 1 mg to 20 mg of one or more collagenases.

39. 32. The method of claim 31 , wherein the composition comprises CCH.

40. 32. The method of claim 31, wherein the composition has a potency of about 10,000 ABC units / 0.58 mg and the therapeutically effective amount is about 1 mg to 20 mg.

41. 32. The method of claim 31, wherein the composition has a potency of about 15,000 ABC units / mg to 20,000 ABC units / mg and the therapeutically effective amount is about 1 mg to 20 mg.

42. 32. The method of claim 31, wherein the therapeutically effective amount is about 1 mg to 10 mg, and the composition has a potency of about 20,000 to about 30,000 f-SRC units / mg, or about 175,000 to about 300,000 f-GPA units / mg.

43. 32. The method of claim 31, wherein when the treatment is administered to a patient population, the treatment results in at least 5% of patients maintaining a level of improvement relative to a pre-treatment baseline for at least 71 days after the first administration.

44. 44. The method of claim 43, wherein at least 10% of the patients maintain a level of improvement relative to pre-treatment baseline for at least 71 days after the first administration.

45. 44. The method of claim 43, wherein at least 20% of the patients maintain a level of improvement relative to pre-treatment baseline for at least 71 days after the first administration.

46. 32. The method of claim 31, wherein when the treatment is administered to a patient population, the treatment results in at least 5% of patients showing improvement relative to a pre-treatment baseline and showing a further increase in improvement over time.

47. 1. A method for reducing the severity of cellulite in both buttocks of a human patient, the method comprising: a. The following characteristics: i. A V of about 0.08 to 7.70 (SRC assay), or about 0.3 to 30.5 (GPA assay) ｍａｘ (minute －１ ). ii. A K of about 4.1 to 410 nanomolar (SRC assay), or about 0.03 to 3.1 mM (GPA assay) Ｍ ; iii. A K of about 1.1 to 107 (SRC assay), or about 93 to 9,179 (GPA assay) ｃａｔ (seconds －１ ). iv. a 1 / K of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay) ｃａｔ , microseconds; v. K of about 5,140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay) ｃａｔ / K Ｍ , mM －１ seconds －１ ; vi. 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; vii. A purity of at least 80% by area as measured by reverse phase HPLC (high pressure liquid chromatography); viii. a potency of about 5,000 to about 30,000 f-SRC units / mg; ix. a titer of about 175,000 to about 500,00 f-GPA units / mg; x. a potency of about 5,000 to about 25,000 ABC units / mg; xi. The amount of an impurity selected from the group consisting of clostripain, gelatinase, and leupeptin is 1% or less by area; xii. a bioburden of less than or equal to 1 cfu / mL; providing a collagenase composition having at least two of: b. injecting a therapeutically effective amount of said collagenase composition into both gluteal cavities according to Treatment I, wherein an improvement in the appearance of said cellulite is established by CR-PCSS; The method comprising:

48. 48. The method of claim 47, wherein the cumulative collagenase injected is about 5.04 mg.

49. 48. The method of claim 47, wherein the collagenase composition has the following characteristics: AUX-I (SRC assay): i. V ｍａｘ ,point －１ : About 0.08~7.70 ii. K Ｍ : about 4.1 to 410 nanomoles iii. K ｃａｔ ,Second －１ : About 1.1~107 iv. 1 / K ｃａｔ , microseconds: about 376 to 37,222 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : Approximately 5,140 to 508,814 b. AUX-II (GPA assay) i. V ｍａｘ ,point －１ : About 0.3~30.5 ii. K Ｍ、 mM: about 0.03~3.1 iii. K ｃａｔ ,Second －１ : About 93~9,179 iv. 1 / K ｃａｔ , microseconds: about 4 to 428 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : About 60~5,934 A method comprising the steps of:

50. 48. The method of claim 47, wherein the collagenase composition has the following characteristics: AUX-I (SRC assay): i. V ｍａｘ ,point －１ : About 3.8 ii. K Ｍ、 mm: about 2.07×10 －４ iii. K ｃａｔ ,Second －１ : About 53 iv. 1 / K ｃａｔ , microseconds: about 18,799 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : About 256,977 b. AUX-II (GPA assay) i. V ｍａｘ ,point －１ : About 15.4 ii. K Ｍ、 mm: about 1.6 iii. K ｃａｔ ,Second －１ : About 4,636 iv. 1 / K ｃａｔ , microseconds: about 216 v. k ｃａｔ / K Ｍ 、mm －１ Second －１ : About 2,997 A method comprising the steps of:

51. 48. The method of claim 47, wherein the composition has at least three of said characteristics.

52. 48. The method of claim 47, wherein the composition has at least four of said characteristics.

53. 48. The method of claim 47, wherein the composition has at least five of said characteristics.

54. 48. The method of claim 47, wherein the composition comprises about 1 mg to 20 mg of one or more collagenases.

55. 48. The method of claim 47, wherein the composition comprises CCH.

56. 48. The method of claim 47, wherein the composition has a potency of about 10,000 ABC units / 0.58 mg and the therapeutically effective amount is about 1 mg to 20 mg.

57. 48. The method of claim 47, wherein the composition has a potency of about 15,000 ABC units / mg to 20,000 ABC units / mg and the therapeutically effective amount is about 1 mg to 20 mg.

58. 48. The method of claim 47, wherein the therapeutically effective amount is about 1 mg to 10 mg, and the composition has a potency of about 20,000 to about 30,000 f-SRC units / mg, or about 175,000 to about 300,000 f-GPA units / mg.

59. 48. The method of claim 47, wherein when the treatment is administered to a patient population, the treatment results in at least 5% of patients maintaining a level of improvement relative to a pre-treatment baseline for at least 71 days after the first administration.

60. 60. The method of claim 59, wherein at least 10% of the patients maintain a level of improvement relative to pre-treatment baseline for at least 71 days after the first administration.

61. 60. The method of claim 59, wherein at least 20% of the patients maintain a level of improvement relative to pre-treatment baseline for at least 71 days after the first administration.

62. 48. The method of claim 47, wherein when the treatment is administered to a patient population, the treatment results in at least 5% of patients showing improvement relative to a pre-treatment baseline and showing a further increase in improvement over time.

63. 48. The method of claim 47, wherein the treatment achieves the following efficacy outcomes as measured by CR-PCSS: a. at least a 2-level improvement from baseline (pre-treatment "Day 1") in live hip clinician-assessed CR-PCSS severity at Days 22, 43, 71, 90, 180, or 365; b. An improvement of at least 2 levels from baseline (Day 1) in PR-PCSS severity as assessed by the patient while viewing digital images of the buttocks at Days 22, 43, 71, 90, 180, or 365; c. improvement as indicated by a 2-level composite response at Days 22, 43, 71, 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 from baseline (Day 1) in CR-PCSS severity assessed live by the hip clinician at Days 22, 43, 71, 90, 180, or 365; e. at least one level improvement from baseline (Day 1) in PR-PCSS severity as assessed by the patient while viewing digital images of the buttocks at Days 22, 43, 71, 90, 180, or 365; f. Improvement as indicated by a one-level composite response at Days 22, 43, 71, 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 method produces at least one of the following results:

Images