Injection technique for treating subcutaneous cellulite

Abstract

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

Description

[0001] This application is a divisional application of the application filed on July 12, 2019, with the application number 201980059156.8 and the invention title "Injection Technique for Treating Subcutaneous Cellulite".

[0002] Related Applications

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

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

[0005] Cellulite (also known as edematous fibrosclerotic panniculopathy (EFP)) is an aesthetic disease that can be understood as an imbalance between the structural characteristics and biomechanical properties (i.e., subtle containment and extrusion forces) of the subcutaneous junctions (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 aim of cellulite treatment is to strengthen the subcutaneous interface and / or release the fibrous septa through various types of subcutaneous injections (Rudolph et al, ibid).Through anatomical and image analysis studies, fibrous septa are considered to be the main cause of subcutaneous cellulite and are also the targets for treating subcutaneous cellulite (Hexsel et al, “Side-by-side comparison of areas with and without cellulite depressions using magnetic resonance imaging,” Dermatol Surg. 2009;35(10):1471-1477; Hexsel et al. “Magnetic 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; Nürnberger and Müller, “So-called cellulite: an invented disease,” J Dermatol Surg Oncol. 1978;4(3):221-229; Piérard et al, “Cellulite: from standing fat herniation to hypodermal stretch marks,” Am J Dermatopath. 2000;22(1):34-37; Querleux et al, “Anatomy and physiology of subcutaneous adipose tissue by in vivo magnetic resonance imaging and spectroscopy: relationships with sex and presence of cellulite,” Skin Res Technol. 2002;8(2):118-124).To effectively treat subcutaneous cellulite, a treatment method is needed to dissolve or disrupt the dermal septum, which is composed of collagen ( Figure 1 ), and causes skin depressions that trouble many women.

[0006] Some therapies have been used to treat subcutaneous cellulite; however, there are no approved pharmaceutical treatments. Despite the variety of treatment modalities, there is little scientific evidence that any of the current non-pharmaceutical treatments are beneficial. In fact, much of the evidence is anecdotal, subjective, or based solely on patient self-assessment. Some of the historical treatments for EFP have included weight loss, topical medications, massage, liposuction, mesotherapy, radiofrequency, subcision, power-assisted subcision, and laser therapy. Many of these treatments have adverse side effects (Avram MM, “Cellulite: a review of its physiology and treatment,” J Cosmet Laser Ther. 2004;6(4):181-185; Collis et al, “Cellulite treatment: a 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 believe that improving the aesthetic condition is not easy. Accordingly, there remains a need for safe and effective non-surgical therapies to improve the cosmetic appearance of women with subcutaneous cellulite. Summary of the Invention

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

[0008] As described in the specific embodiments, there are a total of four treatment phases, although they are optional and the order of implementation does not need to be strictly restricted.

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

[0010] ο Hexsel Cellulite Severity Scale (Hexsel CSS)

[0011] ο Hexsel Depression Depth Scale

[0012] ο Likert Scale

[0013] ο Pit Analysis

[0014] ο Clinician-Reported Photographic Digital Cellulite Severity Scale (CR-PCSS)

[0015] ο Patient-Reported Photographic Digital Cellulite Severity Scale (PR-PCSS)

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

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

[0018] ο Patient-Reported Cellulite Impact Scale (PR-CIS)

[0019] ο Simplified PR-CIS

[0020] ο Patient Self-Assessment Form (SSRS)

[0021] οSubject Satisfaction Questionnaire for Cellulite Treatment (SSCT)

[0022] οClinician's Assessment of Cellulite Severity (Photographs or Other Images)

[0023] οBody-Q

[0024] οFitzpatrick Scale

[0025] οThigh Cellulite Efficacy Scale (PR-TCES; CR-TCES)

[0026] οAny Valid Image Numbers or Other Clinical Use and / or Patient-Used Scales for Evaluating Cellulite Severity, Improvement, and / or Patient Satisfaction (e.g., Hexsel-Mer z Scale (Poster Published at the American Academy of Dermatology Meeting in 2019).

[0027] In addition, pre-treatment assessments of clinicians and patients can be performed by analyzing a series of 3 to 15 photos, illustrations, drawings, computer images, 3-D models, MRI images, thermograms, sonograms, patient oral feedback, etc., each with different cellulite severity grades or levels.

[0028] 2. In the second stage of treatment, the clinician marks the depressions to be treated with dots or other markers ( Figure 6 ). If there is a lowest point, it is usually placed at the lowest point of the depression. Additional photos can be taken and other assessments can be made.

[0029] 3. In the third stage of treatment, an effective amount of collagenase is subcutaneously injected into the depressions in one or more treatment areas (as defined in the detailed implementation section) in a single dose or multiple doses. The dose and injection technique vary. For example, the method may include deep and shallow injections according to the following procedure (as shown in Figure 10 (Treatment IV)):

[0030] · When the needle is at an angle of about 30° to the skin surface of the injection site and facing the subject's head, push the needle all the way in (1 inch) and inject 0.3 mL of the collagenase component by gently pushing the syringe plunger.

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

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

[0033] · Hold the needle at a 30° angle to the skin surface. The needle is exactly opposite to position B (position D), and gently push the syringe plunger to inject approximately 0.3 mL of the collagenase composition.

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

[0035] · A 3 - mL syringe with 1.5 mL of the collagenase composition is sufficient for one injection site.

[0036] · Use twelve (12) 3 - mL syringes in each treatment area (each buttock or each thigh) and inject 12 needles of 1.5 mL at 12 injection sites (a total of 18.0 mL, 5 portions of 0.3 mL).

[0037] · At each visit, a total of 24 injections are made in 2 treatment areas (2 buttocks or 2 thighs).

[0038] In another embodiment, the needle can be injected into the pit at three angles: at an angle of approximately 90 degrees to the skin surface, at an angle of approximately 45 degrees to the skin surface and the long axis of the pit, and at an angle of approximately 135 degrees to the skin surface and the long axis of the pit. In a non - limiting embodiment, using a 1 - mL syringe with a 0.1 - mL gradient and a 30 - gauge 1 / 2 - inch needle, the above three injections are delivered directly into the pit as three 0.1 - mL aliquots. Other techniques are described in the detailed description section.

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

[0040] The collagenase is injected in an amount of from about 0.01 mg to about 20 mg. The single - dose injection can be divided into several sub - doses and has one or more of the following characteristics:

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

[0042] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

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

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

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

[0046] · Molecular weight is about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa.

[0047] · Purity (by area) determined by reverse-phase HPLC (high-pressure liquid chromatography) is at least 80%

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

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

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

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

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

[0053] · Impurities less than or equal to 1% (by area), the impurities are selected from clostripain, gelatinase and leupeptin

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

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

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

[0057] In another embodiment, the collagenase composition comprises CCH (as defined in the detailed description), and the ratio of AUX I and AUX II contained in CCH is about 1:1. Other ratios of AUX I and AUX II can also 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 1:0.75 - 2, or 1:0, or 0:1. Each of AUX I and AUX II can have an area purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as measured by reverse-phase HPLC.

[0058] In other embodiments, the collagenase composition can be a liquid or reconstituted from a lyophilized solid form with a diluent. The dose of the mixture is measured by the amount of collagenase present, regardless of the diluent, and can contain from about 0.1 mg to about 20 mg of collagenase in one or more injections. In another embodiment, the dose administered in one or more injections is about 0.06 mg, 0.48 mg, 0.84 mg, 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg, 5.04 mg, 5.88 mg, 6.72 mg, 7.56 mg, or 8.4 mg. For example, about 0.06 mg, 0.48 mg, 0.84 mg, or 1.68 mg is administered in about 12 divided injections. The volume of the injected collagenase composition can be between 0.01 mL and 3 mL per injection, or the total volume per treatment follow-up is about 0.2 mL to 150 mL (as defined in the detailed description). In certain embodiments, the above doses are for collagenase compositions containing CCH. In another embodiment, the above doses are used for collagenase compositions having one or more of the following characteristics:

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

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

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

[0062] ·1 / K cat in microseconds is about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

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

[0064] · The molecular weight is about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa.

[0065] · Purity (by area) determined by reverse-phase HPLC (high performance liquid chromatography) of at least 80%

[0066] · Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[0067] · Potency of about 5,000 to about 30,000 f-SRC units / mg

[0068] · Potency of about 100,000 to about 400,000 GPA units / mg

[0069] · Potency of about 175,000 to about 500,00 f-GPA units / mg

[0070] · Potency of about 5,000 to about 25,000 ABC units / mg

[0071] · Impurities of less than or equal to 1% (by area), the impurities being selected from clostripain, gelatinase and leupeptin

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

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

[0074] In addition, as described in the detailed description section, collagenase injection is effective in treating subcutaneous cellulite. For example, through Hexsel depression depth scoring, Likert scale scoring and pit analysis, a significant improvement in the appearance of subcutaneous cellulite can be demonstrated.

[0075] From the following description, drawings, examples and claims, other embodiments of the compositions, scales, methods, etc. of the present invention become apparent. It will be understood from the foregoing and following description that each feature described herein, and each combination of two or more such features, is included within the scope of the present invention, provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features can be specifically excluded from any embodiment or aspect. Other aspects and embodiments are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings. Brief Description of the Drawings

[0077] The patent or application document contains at least one color drawing. After request and payment of the necessary fees, the Patent Office will provide a copy of the color drawing of this patent or patent application publication.

[0078] By referring to the following detailed description and in combination with the accompanying drawings, the above features of each embodiment will be more easily understood, where:

[0079] Figure 1 is a cross-sectional view of the skin and subcutaneous tissue depicting the collagen membrane.

[0080] Figure 2 is a list of the amino acid sequence of AUX-I (Seq.ID No.5).

[0081] Figure 3 is a list of the amino acid sequence of AUX-II (Seq.No.ID 6).

[0082] Figure 4 shows the depth of depression of the Hexsel cellulite severity scale (CSS) (B).

[0083] Figure 5 shows the PR- and CR-thigh cellulite efficacy scale.

[0084] Figure 6 illustrates the pits and injection site markings on the buttocks of the subject.

[0085] Figure 7 describes the injection technique used in Treatment I (injection into the shaded area, divided into 3 equal parts).

[0086] Figure 8 describes the injection technique used in Treatment II (injection into the shaded area, divided into 1 equal part).

[0087] Figure 9 describes the injection technique used in Treatment III (deep injection, divided into 1 equal part).

[0088] Figure 10 describes the injection technique used in Treatment IV (deep and shaded area injection, divided into 5 equal parts).

[0089] Figure 11 describes the injection technique used in Treatment V (injection into the shaded area, divided into 4 equal parts).

[0090] Figure 12 describes an injection technique that can effectively administer collagenase or placebo into the pits of cellulite.

[0091] Figure 13 is a 3D mesh registration (1 day before marking).

[0092] Figure 14 is a color distance map for image registration.

[0093] Figure 15 is the main pit of the target area.

[0094] Figure 16 is the main deformable pit of the target area.

[0095] Figure 17 Describes the contours of normal and bruised tissues at days 4, 8, and 15 after injection into the left buttock of a subject.

[0096] Figure 18(A) depicts the contours of normal and bruised tissues at days 4, 8, and 15 after injection into the left buttock and provides L*, a*, and b* color measurements of these tissues.

[0097] Figure 18(B) depicts the contours of normal and bruised tissues at days 4, 8, and 15 after injection into the left buttock. The average colors and ΔEs of normal and bruised tissues are calculated based on L*a*b* color values.

[0098] Figures 19(A)-19(C) Describes pit analysis. Figure 19(A) illustrates the observed and pre - marked image changes in pit analysis parameters starting from day 1. Figure 19(B) illustrates the maximum length and maximum width of the pit. Figure 19(C) illustrates the surface area and volume between the bottom of the pit and the interpolated surface.

[0099] Figure 20 Describes the study flow chart of a phase 2b randomized, double - blind, placebo - controlled study of CCH for the treatment of thigh edematous fibrosclerotic polyneuropathy. Detailed implementation mode

[0100] All aspects and implementation modes will be described comprehensively here. However, these aspects and implementation aspects can be embodied in many different forms and should not be construed as a limitation of the present invention; on the contrary, the implementation modes provided here will make this disclosure thorough and complete and will fully convey the scope of this subject matter to those skilled in the art. All publications, patents, and patent applications cited herein, whether above or below, are hereby incorporated by reference in their entirety.

[0101] A. Definitions

[0102] Unless otherwise defined, all terms and phrases used herein have the meaning ascribed to such term and phrase in the art, unless the contrary is clearly indicated or apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, particular methods and materials are now described.

[0103] Unless otherwise indicated, the use of an individual numerical value means the use of an approximation of that numerical value, as if the word "about" or "approximate" preceded the numerical value. Similarly, unless otherwise expressly stated, numerical values recited in ranges herein are referred to as approximations, as if the word "about" or "approximate" preceded both the minimum and maximum values of said range. Thus, variations above and below the recited range can be used to obtain substantially the same result as the values within the recited range. As used herein, the terms "about" and "approximate" when referring to a numerical value shall have their plain and ordinary meaning to a person of ordinary skill in the art most closely related to the disclosed subject matter or to a person of ordinary skill in the art related to the range or element being discussed. The amount by which the strict numerical boundaries are widened depends on many factors. For example, some factors that can be considered include the criticality of the element and / or the effect that a change in the given quantity will have on the performance of the claimed subject matter, as well as other considerations known to persons of skill in the art. As used herein, the use of different numbers of significant digits for different numerical values does not mean a limitation on how the words "about" or "approximate" will be used to widen a particular numerical value or range. Thus, generally speaking, "about" or "approximate" widens a numerical value. Additionally, the disclosure of a range is intended as a continuous range, including every value between the minimum and maximum values plus the expansion of the range provided by the use of the terms "about" or "approximately", and the recitation of a range of values herein is only intended as a shorthand method for separately referring to each individual value within that range, and each individual value is incorporated into the specification as if it were recited individually herein.

[0104] As used herein, "affected area" or "treatment area" means the area of cellulite in a human patient to be treated or that has been treated with collagenase (defined below). This may include a quadrant (i.e., left buttock, right buttock, left posterolateral thigh, right posterolateral thigh). The affected area or treatment area is not limited to the buttocks or thighs. On the contrary, any area of the body with cellulite can be a treatment site.

[0105] "Side effect" or "AE" means any adverse or unexpected change in body structure (signs), body function (symptoms), laboratory results (such as chemistry, electrocardiogram, X-ray, etc.), or a worsening of a pre-existing condition that is temporarily associated with the use of the investigational drug, whether or not it is considered related to the investigational drug.

[0106] "Body-Q" is a patient-reported outcome instrument that is commercially available under license from Memorial Sloan Kettering Cancer Center. It is based on patients' perception of body contour and / or weight loss. It measures three domains through an 18-item scale with independent functions: appearance, health-related quality of life (HRQL), and patients' medical experience. The patient-reported outcome tool is as described below: BODY-Q: User Manual BODY-Q: User Manual, Version 1.0, July 2015, Memorial Sloan Kettering Cancer Center, McMaster University, and Stefan Cano. BODY-Q includes scales for measuring cellulite. See https: / / www.mskcc.org / sites / default / files / node / 174457 / documents / body-q-users- guide.pdf(2 (July 3, 2019). For cellulite, there are 16 scale items with response options ranging from "not at all" to "extremely bothered" over a time frame of the past week and assuming a Flesch-Kincaid grade reading level. Patients are asked: "Considering your cellulite, how bothered have you been in the past week?" [After 16 questions, patients will rate their responses as 1 - extremely bothered; 2 - moderately bothered; 3 - somewhat bothered; 4 - not at all bothered]. Scores range from 16 (extremely bothered) to 64 (not at all).

[0107] Traditionally, the clinical examination of bruising has included visual inspection of the bruise and the surrounding area, as well as an assessment of the subject's medical, surgical, and concomitant medication history. The results of such an interpretation are subjective and are influenced by several unrelated factors, including observation geometry, ambient lighting, the color of the non-exposed surrounding skin, and the experience and eyesight of the observer. As used herein, "bruise analysis" refers to the detection of visible changes in skin color evaluated from images of a subject's collagenase-treated area using the objective image capture and tracking method disclosed in U.S. Patent Publication No. 2019 / 0035080, which is uniformly applied to all subject images. This objective analysis can assist and even replace the healthcare provider's visual inspection and clinical examination of bruising by providing a quantified and differentiated way to evaluate the degree of bruising within a subject (over different time points of the same subject) and between subjects (between different subjects). This analysis utilizes the L*a*b* color space defined by the International Commission on Illumination (CIE), which follows the color opponency theory, i.e., two colors cannot be both red and green, nor can they be both yellow and blue at the same time. As shown below, L* indicates light / dark, a* is the red / green coordinate, and b* is the yellow / blue coordinate. DeltaL*(ΔL*), Delta a*(Δa*), and Delta b*(Δb*) can be positive deviations (+) or negative deviations (-). However, the total deviation Delta E(ΔE*) is always positive.

[0108] · ΔL * (L* sample - L* standard) = light - dark deviation (+ = lighter, - = darker); Lower values (0 - 50) indicate dark and higher values (51 - 100) indicate light.

[0109] · Δa * (a* sample - a* standard) = red - green deviation (+ = redder, - = greener)

[0110] · Δb * (b* sample - b* standard) = yellow - blue deviation (+ = yellower, - = bluer)

[0111] All three of these values are needed to fully describe the color of an object (in this case, a bruise captured in an image of the treatment area of a subject). An objective method for image analysis of the collagenase treatment area (images at specified time points before and after treatment), with data output as L*a*b* values, allows for rapid, simple, accurate, repeatable, and unbiased quantification of skin color and any changes to it. This method eliminates the inherent variability of images with traditional subjective visual estimates. ΔE is calculated as follows:

[0112] AE (color difference between bruise and normal skin) = SQRT[(L * B - L * N ) 2 + (A * B - A * N ) 2 + (B * B - B * N ) 2 , where

[0113] L * B = L of bruise tissue *

[0114] L * N = L of normal tissue *

[0115] A * B = A of bruise tissue *

[0116] A * N = A of normal tissue *

[0117] B * B = B of bruise tissue *

[0118] B * N = Normal tissue B *

[0119] Figure 18(B) shows the bruise analysis of the treatment area.

[0120] “CCH” refers to the AUX-I (Seq.ID No.5( Figure 2 )) and AUX-II (Seq.ID No.6( Figure 3 )) collagenase mixture, at a ratio of approximately 1:1, obtained by fermentation of Clostridium histolyticum (also known as Hathewaya histolytica). CCH is sold on the market as a freeze-dried powder under the trademark It includes a mixture of AUX-I and AUX-II and specific excipients, although CCH can be used with other suitable excipients.

[0121] The “Clinician Reported Photographic Digital Cellulite Severity Scale (CR-PCSS)” is a photographic digital scale described in PCT patent application PCT / US2018 / 020551 (WO2018 / 160905, published on September 7, 2018), used by doctors / clinicians to assess the severity of cellulite into 5 levels.

[0122] Unless otherwise specified herein, "collagenase" means any of the following: (a) collagenase having the activity specified in EC 3.4.24.3 (including mutants) (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 alignment with AUX-I as determined by BLAST; (e) collagenase having at least 50% sequence alignment with AUX-II as determined by BLAST; (f) collagenase produced by fermentation of other source organisms (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 between about 65 kDa and about 130 kDa; (i) collagenase designated as Class I or Class II (also known as collagenase I (or 1), collagenase II (or 2), type I collagenase, type 2 collagenase); (j) a mixture of collagenase I and II; (k) collagenase from strain JCM1403 (ATCC 19401) or its derivatives; (l) collagenase from strain ATCC 21000 or its derivatives; (m) collagenase from strain ATCC 69334 or its derivatives; (n) collagenase derived from Clostridium perfringens; (o) collagenase from Vibrio alginolyticus; (p) collagenase from Streptomyces; (q) collagenase from Pseudomonas; (r) collagenase from Achromobacter; (s) collagenase described by Worthington Biochemical Corporation (www.Worthington-biochem.com; "Product Highlights"); (t) collagenase from Sigma-Aldrich Corporation (www.sigma-aldrich.com); (u) collagenase having one or more of the following characteristics:

[0123] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[0124] ·K M is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[0125] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[0126] ·1 / Kcat , in microseconds is approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[0127] ·K cat / K M , mM -1 sec -1 is approximately 5,140 to 508,814 (SRC experiment), or approximately 60 to 5,934 (GPA experiment)

[0128] (v) Collagenase described by Nordmark Arzneimittel GmbH & Co. KG; (w) Collagenase from strain 004; or (x) An equivalent or mixture of any of the above. Non-limiting examples of collagenases that can be used in the present disclosure are described in U.S. Patent Nos. 7,811,560, 9,757,435, 9,744,138, and WO2012 / 125948.

[0129] "Pit analysis" refers to the analysis of one or more selected pits, where parameters such as the volume, length, width, and surface area of the pits are measured. The measurement can be carried out by various known methods, such as the methods described in Eckhouse et al. WO 2018 / 116304 and WO 2018 / 116305, and by Cherry imaging (www.cherrymaging.com) and Canfield Scientific, Inc. See also Salameh et al., "Novel Stereoscopic Optical System for Objectively Measuring Above-Surface Scar Volume - First-Time Quantification of Responses to Various Treatment Modalities," Dermatol. Surg. 00:1 - 7 (2017); and U.S. Patent No. 9,996,923. Such measurements of volume, length, width, and surface area can be calculated using digital 3-D grayscale images (with X and Y axis rotation features) and digital 3-D texture and illumination images (with X and Y rotation features) and a computer program for analyzing such images. For example, for the treatment area of the buttocks, images of the left and / or right treated buttocks of each patient can be taken before and after treatment. For the treatment area of the thigh, images of each thigh treatment area can be taken at 0 degrees, 45 degrees, and 90 degrees before and after treatment. For the treatment area of the thigh, images of each thigh treatment area can be taken at 0 degrees, 45 degrees, and 90 degrees using the Canfield Scientific method before and after treatment.

[0130] "Persistence" refers to the period during which the treatment effect persists. This period can be from 3 months to 20 years, or approximately 1 to 19 years, or approximately 2 to 18 years, or approximately 3 to 17 years, or approximately 4 to 16 years, or approximately 5 to 15 years, or approximately 6 to 14 years, or approximately 7 to 13 years, or approximately 8 to 12 years, or approximately 9 to 11 years. The period can be about 6 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, about 15 years, about 20 years.

[0131] "Fitzpatrick scale" refers to the scale used to evaluate the skin type of a subject, as shown in Table 1.

[0132] Table 1. Fitzpatrick scale

[0133] I Light white skin, blue / hazel eyes Often sunburned, does not tan II Fair skin, blue eyes Prone to sunburn, not easily tanned III Yellowish skin color Skin color darkens after sunburn IV Light brown skin color Rarely, mildly sunburned, easy to V Bronze skin color Rarely sunburned, very easy to tan VI Dark brown or black skin color Never sunburned, only tans

[0134] "Hexsel cellulite severity scale" or "Hexsel CSS" or "Cellulite Severity Scale (CSS)" refers to the photographic digital scale (Table 2) that evaluates 5 main morphological features of cellulite as follows:

[0135] Table 2. Hexsel cellulite severity scale

[0136]

[0137]

[0138] The sum of the scores results in the following classification

[0139] Score Cellulite classification 1-5 Mild 6-10 Moderate 11-15 Severe

[0140] Hexsel et al., J. Eur. Acad., Dermatol. Venereol. 2009; 23(5): 523 - 528. a. Nürnberger and Müller, J. Dermatol. Surg. Oncol. 1978; 4(3): 221 - 229. The subject is evaluated in a standing position with the gluteal muscles relaxed. However, if the subject does not have obvious dimpling symptoms, they are asked to contract the gluteal muscles or perform a pinch test (by pinching the skin between the thumb and index finger) to distinguish between a score of 0 or 1 / grade.

[0141] Here, the use of "Hexsel dimple depth score" refers to the evaluation of only the dimple depth of (B) Hexsel CSS ( Figure 4 ):

[0142] 0 = No depression

[0143] 1 = shallow depression

[0144] 2 = Medium depth of depression

[0145] 3 = Deep depression

[0146] As used herein, an "image" or "picture" refers to a photograph, diagram, illustration, model, 3D model, computer-generated image, MRI image, etc.

[0147] "Likert scale score" refers to the evaluation of subcutaneous cellulite by an independent blinded reviewer at each post-treatment visit by comparing the pre-treatment (baseline) photographs of subcutaneous cellulite on Day 1 (2-D color, 3-D color, and 3-D grayscale) and the photographs at the post-treatment visit. The scores were recorded using the following 5-point Likert scale:

[0148]

[0149] "Optional" or "optional" means that the subsequently described element, component, or condition may or may not occur, so that the description includes instances where the element, component, or condition occurs and instances where it does not occur.

[0150] As used herein, the "Patient-Reported Impact of Cellulite Scale (PR-CIS)" refers to an assessment of the visual and emotional impact of cellulite (pleasure, bothersomeness, self-consciousness, embarrassment, looking old or looking overweight or body dysmorphic) using a 6-question survey, with each question having a rating scale ranging from 0 (not at all) to 10 (extremely noticeable). More specifically, the PR-CIS is a 6-item static questionnaire that assesses the visual and emotional impact of cellulite (pleasure, bothersomeness, self-consciousness, embarrassment, looking old or looking overweight or body dysmorphic); subjects answer each question using an 11-point numeric rating scale ranging from 0 (not at all) to 10 (extremely noticeable) while viewing digital photographs of their buttocks and / or thighs. This assessment may be for all thighs and / or buttocks together, rather than for each separate area. The total PR-CIS score is derived from the 6 individual questions:

[0151] Question 1 - Thinking about the area you chose to treat, how satisfied are you with the appearance of your cellulite?

[0152] Question 2 - Thinking about the area you have chosen to be treated, how bothered are you by the appearance of subcutaneous cellulite?

[0153] Question 3 - Thinking about the area you have chosen to treat, how concerned are you about the appearance of subcutaneous cellulite?

[0154] Question 4 - Thinking about the area you have chosen to be treated, how embarrassed are you about the appearance of subcutaneous cellulite?

[0155] Question 5 - Think about the treatment area you selected. How much older do you look due to the appearance of cellulite?

[0156] Question 6 - Think about the treatment area you selected. How much fatter or out of shape do you look due to cellulite?

[0157] As used herein, the "Patient-Reported Cellulite Impact Scale (abbreviated) (PR-CIS abbreviated)" refers to the use of a 5-question survey to evaluate the visual and emotional impact of cellulite (happiness, annoyance, self-consciousness, embarrassment, looking older or looking overweight or out of shape), with the numerical range of the rating scale for each question being from 0 (not at all) to 10 (extremely obvious). More specifically, the PR-CIS is a 5-item static questionnaire that evaluates the visual and emotional impact of cellulite (happiness, annoyance, self-consciousness, embarrassment, looking older or looking overweight or out of shape); when observing digital photographs of their buttocks and / or thighs, the subjects answer each question using an 11-point numerical rating scale, with the rating range from 0 (not at all) to 10 (extremely). This assessment may be of all the thighs and / or buttocks together, rather than each individual area. In a non-limiting embodiment, the total score of the PR-CIS abbreviated can be obtained from 6 individual questions:

[0158] Question 1 - Think about the treatment area you selected. How satisfied are you with the appearance of cellulite?

[0159] Question 2 - Think about the treatment area you selected. How bothered are you by the appearance of cellulite?

[0160] Question 3 - Think about the treatment area you selected. How concerned are you about the appearance of cellulite?

[0161] Question 4 - Think about the treatment area you selected. How embarrassed are you by the appearance of cellulite?

[0162] Question 5 - Think about the treatment area you selected. How much fatter or out of shape do you look due to cellulite?

[0163] The total score of the PR-CIS abbreviated can be obtained from the other five questions of the full version of the PR-CIS.

[0164] As used herein, the "Patient-Reported Photographic Digital Cellulite Severity Scale (PR-PCSS)" is a patient's photographic digital scale, as in PCT International Application PCT / US2018 / 020551 (published on September 7, 2018 with the publication number WO2018 / 160905), which is designed to quantify the severity of cellulite into 5 levels.

[0165] As used herein, "photographic digital" refers to a series of photographs, diagrams, illustrations, models, 3D models, computer-generated images, MRI images, images, etc., each of which designates different levels of cellulite severity in a scale.

[0166] As used herein, "serial visits" refers to two or more clinician visits or the number of times of evaluating cellulite changes through a scale. The interval between each visit can be about two weeks, about one month, about two months, about three months, about four months, about five months, about six months, about one year, about two years, about three years, or more than five years.

[0167] As used herein, "serious side effects" refers to side effects that result in death, immediately endanger life, cause or prolong the hospitalization of inpatients, cause permanent or substantial disability, congenital anomalies / birth defects, or are regarded as important medical events.

[0168] The terms "subject" or "patient" are used interchangeably herein and refer to humans or other mammals.

[0169] As used herein, the "Subject Global Aesthetic Improvement Scale (S-GAIS)" and the "Investigator Global Aesthetic Improvement Scale (I-GAIS)" refer to the following scales for evaluating cellulite severity and / or improvement. The following introductory question is asked of the subject: "How would you rate the appearance of cellulite after treatment?" The scoring ranges from -3 (very poor) to +3 (very good), depending on the subject's response, as shown in Table 3.

[0170] Table 3. Subject Global Aesthetic Improvement Scale (S-GAIS) and Investigator Global Aesthetic Improvement Scale (I-GAIS)

[0171]

[0172] As used herein, "Subject Satisfaction with Cellulite Treatment" (SSCT) refers to a subject satisfaction rating from -2 to +2. For example, Table 4 below provides an evaluation of cellulite treatment on the buttocks. The patient is asked: "Today, are you satisfied with the result of the cellulite treatment on the specific part of your buttocks that you received?" Then they choose an answer / score, as shown in Table 4.

[0173] Table 4. Evaluation of Subject Satisfaction with Cellulite Treatment - Buttocks

[0174]

[0175]

[0176] Table 5 provides an evaluation of the treatment of cellulite on the thighs. Patients were asked: "Today, are you satisfied with the results of the treatment of cellulite in specific areas of your thighs?" Then they selected an answer / score as shown in Table 5.

[0177] Table 5. Evaluation of Subject Satisfaction with Cellulite Treatment - Thighs

[0178]

[0179] The "Subject Self-Rating Scale (SSRS)" is the scale used by the subject to evaluate his / her appearance satisfaction related to cellulite, expressed as a 7-point scale ranging from 0 (extremely dissatisfied) to 6 (extremely satisfied), as Figure 6 shown.

[0180] Table 6. Subject Self-Rating Scale (SSRS)

[0181] Score Response options 6 Very satisfied 5 Satisfied 4 Somewhat satisfied 3 Neither satisfied nor disappointed 2 Somewhat disappointed 1 Disappointed 0 Extremely disappointed

[0182] The term "therapeutically effective amount" as used herein refers to the amount of collagenase required to reduce the severity of cellulite in a patient or a statistically significant patient population. The amount of the collagenase composition used will be the amount necessary to deliver the amount of collagenase required to achieve the desired result. In practice, this depends on the collagenase injected, the injection technique, and the enzymatic activity in the treatment area.

[0183] The term "treatment-emergent adverse event" or "TEAE" as used herein refers to any condition that was not present before treatment with the study drug but occurred after treatment, or that was present at the start of treatment but worsened during treatment, or that was present at the start of treatment but disappeared while the patient was receiving treatment and then reappeared (regardless of the intensity of the AE at the start of treatment).

[0184] The term "Thigh Cellulite Efficacy Scale" or "TCES" as used herein refers to the photographic digital scale (or substantially similar scale) shown, which is used by clinicians and patients to evaluate the severity, improvement, and / or patient satisfaction of thigh cellulite and to assist in evaluating the efficacy of collagenase. The patient-reported version is called PR-TCES; the clinician-reported version is called CR-TCES. Figure 5 shown.

[0185] The term "treatment visit" or "treatment" as used herein refers to one or more injections or treatments of the affected area, treating cellulite in one outpatient visit with a therapeutically effective amount of at least one active agent.

[0186] As used herein, the terms "valid", "validity", or "validate" refer to a process by which a particular scale is proven to be accurate and reliable, which process includes the reproducibility of visual assessment to ensure that the same results can be consistently obtained. Validation further examines the precision, accuracy, and sensitivity of the scale to confirm that its measurements are reliable, reproducible, and robust.

[0187] B. Introduction

[0188] The present invention relates to a method for treating subcutaneous cellulite, comprising administering a therapeutically effective amount of one or more collagenases to a subject having an appearance of subcutaneous cellulite by using certain injection techniques described below.

[0189] Generally, the treatment is divided into four stages: (1) The clinician and the patient perform a pre-treatment assessment to determine the pre-treatment baseline, and the clinician selects the pits to be treated; (2) If there is a lowest point, the clinician marks the pits to be treated at the lowest point; (3) The clinician treats the patient with collagenase; and (4) The clinician and the patient perform a post-treatment assessment. These stages are described in detail below. Each stage and the steps therein are optional, and the steps do not need to limit the order of implementation because the order may vary, but comparable results can be obtained. _

[0190] C. Stage 1 - Pre-treatment assessment

[0191] In the first stage of treating subcutaneous cellulite described herein, the clinician selects the pits of subcutaneous cellulite to be treated according to the following criteria:

[0192] · As shown in the photograph, when the patient stands in a relaxed position (standing position with relaxed gluteal muscles), the pits should be clearly visible and distinct naturally

[0193] · The selected pits should be those that the clinician believes are most likely to improve the aesthetics of the entire buttocks, thighs, or other affected areas

[0194] · When the patient stands in a relaxed position, photographs of the affected area are taken before treatment

[0195] · Before injection, an assessment is performed, i.e., the clinician and / or the patient independently assess the photographs and score the results using one or more of the following scales or assessment methods:

[0196] ο Hexsel Cellulite Severity Scale (Hexsel CSS)

[0197] ο Hexsel Depression Depth Scale

[0198] ο Likert Scale

[0199] ο Pit Analysis

[0200] ο Clinician-reported Photographic Digital Cellulite Severity Scale (CR-PCSS)

[0201] ο Patient-reported Photographic Digital Cellulite Severity Scale (PR-PCSS)

[0202] ο Investigator's Global Aesthetic Improvement Scale (I-GAIS)

[0203] ο Subject's Global Aesthetic Improvement Scale (S-GAIS)

[0204] ο Patient-reported Cellulite Impact Scale (PR-CIS)

[0205] ο Patient Self-Report Scale (SSRS)

[0206] ο Subject Satisfaction with Cellulite Treatment Questionnaire (SSCT)

[0207] ο Patient-reported Thigh Cellulite Efficacy Scale (PR-TCES; CR-TCES)

[0208] ο Body-Q

[0209] ο Fitzpatrick Scale

[0210] ο Any valid imaging digital or other clinical use and / or patient-administered scale for assessing cellulite severity, improvement, and / or patient satisfaction.

[0211] D. Phase 2 - Marking Dimples Needing Treatment

[0212] In the second phase of the treatment described herein, the clinician dots the dimples to be treated. Additional photographs may be taken.

[0213] E. Phase 3 - Collagenase Injection

[0214] In the third phase of the treatment, the clinician treats the patient with collagenase.

[0215] 1. Collagenase type

[0216] The collagenases useful in the present invention include any of the collagenases defined above. As further background, matrix metalloproteinases (MMPs) can be composed of collagenases within the scope defined herein. For example, MMP-1 contains collagenase 1; MMP-8 contains collagenase 2 / neutrophil collagenase; MMP-13 contains collagenase 3; and MMP-18 contains collagenase 4. Additionally, cathepsins can be classified as collagenases.

[0217] 2. Enzyme kinetics of collagenase

[0218] The collagenases useful in the present invention can also be characterized by their enzyme kinetics. Here, the approximate kinetic values of one or more collagenases that are effective in treating subcutaneous cellulite include:

[0219] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0220] ·K M is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[0221] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0222] ·1 / K cat in microseconds is about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment)

[0223] ·K cat / K M mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or about 60 to 5,934 (GPA experiment)

[0224] V max = maximum velocity

[0225] K M = V at 50% of [substrate] max

[0226] K cat = substrate molecules cleaved per second

[0227] 1 / K cat = microseconds required to cleave substrate molecules.

[0228] These values can be experimentally determined using the microplate assay described below, but with different substrates and times. Other assays and parameters can also be employed.

[0229] These values reflect a quantitative expression of enzyme behavior based on the Michaelis-Menten equation:

[0230]

[0231] where V0 is the reaction rate (velocity) at substrate concentration [S], V maxis the maximum observable rate, K M is the Michaelis constant and is related to the substrate concentration that produces 50% V max .

[0232]

[0233] where k1, k -1 and k2 are the rate constants for the following processes:

[0234]

[0235] where E is the enzyme, S is the substrate, ES is the enzyme - substrate complex, and P is the product.

[0236] Catalytic K cat refers to the turnover number, i.e., how fast the ES complex can be converted into E + P. It reflects the number of catalytic cycles experienced by each active site per unit time.

[0237] In certain embodiments, AUX - I and AUX - II have the following characteristics:

[0238] ·AUX I

[0239] ο Experiment: SRC microplate

[0240] ο Vmax, min -1 : approximately 0.08 to 7.70

[0241] ο K M : approximately 4.1 to 410 nanomoles

[0242] ο K cat , sec -1 : approximately 1.1 to 107

[0243] ο 1 / K cat , microseconds: approximately 376 to 37,222

[0244] ο K cat / K M , mM -1 sec -1 : approximately 5,140 to 508,814

[0245] ·AUX II

[0246] ο Experiment: GPA microplate

[0247] ο V max , min -1 : approximately 0.3 to 30.5

[0248] ο K M, mM: approximately 0.03 to 3.1

[0249] οK cat , sec -1 : approximately 93 to 9,179

[0250] ο1 / K cat , microseconds: approximately 4 to 428

[0251] οK cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[0252] Sum:

[0253] K cat = V max / [AUX] = (nmol substrate / nG AUX*min-1)

[0254] / nG AUX

[0255] The catalytic efficiency (Kcat / KM) generally represents the overall ability of an enzyme to convert a substrate into a product and reflects the binding and catalytic events. In another embodiment, AUX-I and AUX-II include the following characteristics.

[0256]

[0257] ·V max = maximum velocity

[0258] ·K M = Vmax at 50% of [substrate]

[0259] ·K cat = substrate molecules cleaved per second

[0260] ·1 / K cat = time required to cleave one molecule from the substrate

[0261] ·K cat / K M is often used to represent the catalytic activity of an enzyme

[0262] *By SRC microplate experiment

[0263] +By GPA microplate experiment

[0264] 3. Collagenase titer (specific activity)

[0265] A variety of assay methods have been developed to determine the specific activity (titer) of collagenase. Such assay methods are described in subsections a. through c. and characterize collagenase by its ability to convert a substrate to a product at a predetermined enzyme concentration over a given period of time. In certain non-limiting embodiments, these assays are used to determine the titer of each of AUX-I and AUX-II and the combined CCH pharmaceutical product (1:1 ratio of AUX-I and AUX-II). The SRC assay (described below) uses collagen as the reaction substrate. The SRC assay uses soluble rat (tail) collagen (SRC) as the substrate and is used to measure type I collagenase activity, with type II collagenase accounting for approximately 20% of the observed activity of the collagenase mixture. The SRC assay is a fluorescence assay that utilizes fluorescamine to detect the peptides generated from the digestion of SRC type I. The reaction is carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 7.2 containing 15 mM divalent calcium ions at 25 °C for 2.5 h.

[0266] The bovine tendon collagen (BTC) assay experiment (described below) is based on the procedure described by Mandl et al., Arch. Biochem. Biophys. 74:465-475 (1958) and modified according to the description of Keller and Mandl, Arch. Biochem. Biophys. 101:81-88 (1963). See also Rosen, Arch. Biochem. Biophys. 67:10-15 (1957). The BTC assay uses insoluble bovine tendon collagen as the substrate and measures type I and type II activities (such as AUX-I and type II collagenase). The BTC assay method is a colorimetric assay that utilizes ninhydrin to detect the peptides generated from the degradation of BTC type I and type II. This reaction is also carried out at pH 7.2 but at 37 °C in tris(hydroxymethyl)aminomethane (tris) buffer containing 10 mM divalent calcium ions for 22 h.

[0267] The assay for the third type of collagenase, the GPA experiment (described below), utilizes a soluble-derived hexapeptide (carboxy-GPGGPA) as the substrate. The GPA experiment is mainly used to measure type II activity, with type I accounting for approximately 10% of the observed activity. Type II collagenase cleaves the hexapeptide into two tripeptides, one of which (GPA) has a free amino terminus that reacts with fluorescamine to provide a fluorescent product. The GPA runs at pH 7.2 in HEPES buffer containing 100 mM divalent calcium ions at 25 °C for 10 minutes.

[0268] Both the SRC and BTC assays degrade a natural substrate (collagen), which is more closely related to the therapeutic action of collagenase injection. The advantage of the GPA assay is that it uses a well-defined small molecular weight hexapeptide as a substrate and produces two well-defined tripeptides. The GPA assay produces a fluorescent signal and is very sensitive. Finally, the GPA assay is suitable for Michaelis-Menten kinetic analysis because it uses a single substrate and reaction conditions (10-minute incubation), which are close to the initial enzyme velocity. The SRC assay is very suitable for collagen-degrading enzymes with a collagen-binding domain, while the GPA assay is very suitable for collagen-degrading enzymes without a collagen-binding domain, commonly known as gelatinases.

[0269] a. GPA unit assay method and specific activity unit

[0270] i. Collagenase titer determined by the GPA assay (in cuvette)

[0271] The GPA assay is mainly used to measure the potency of class II collagenase. The first step of the assay involves an enzymatic reaction that includes digestion of the substrate carboxyl-glycyl-L-prolyl-glycyl-prolyl-L-alanine (ZGPGPA) into two peptides: carboxyl-glycyl-L-prolyl-glycine (zGPG) and glycyl-prolyl-L-alanine (GPA) by a collagenase sample. The second step involves subsequent measurement of the released GPA with the fluorescent derivative fluorescamine. The assay follows the method below, but those of ordinary skill in the art will understand that certain modifications (such as dilution concentration and time) can be made on the premise of achieving the experimental purpose.

[0272] The general method is as follows. Prepare a leucine standard. Obtain a collagenase sample and prepare a solution for the first step of the enzymatic cleavage of zGPGGPA (hereinafter referred to as "substrate") by the collagenase. After this step, treat the collagenase-treated sample (containing the released GPA) and the leucine standard with fluorescamine at room temperature for a period of time to fluorescently label the free amino groups in the resulting GPA and leucine molecules, respectively. After excitation at 392 nm, measure the fluorescence emission of each solution at 480 nm. The slopes of the leucine and collagenase sample curves are used to calculate the titer units as follows:

[0273] Titer (f-GPA units / mg) = (M 样品 / M 亮氨酸 ) x (DF / T)

[0274] Where:

[0275] M 样品 = slope of the collagenase titer curve

[0276] M 亮氨酸 = slope of the leucine standard curve

[0277] DF = Dilution Factor

[0278] T = Reaction Time

[0279] In addition, non - limiting details of the GPA assay method are presented below:

[0280] Buffers and Reagents

[0281] 1. f - Appel buffer, pH 7.2 (55 mM HEPES, 100 mM calcium acetate)

[0282] 2. 1 mM leucine working stock solution

[0283] 3. 200 mM borate, pH 9.0

[0284] 4. Fluorescamine solution in 0.5 mM acetone

[0285] 5. 2 mg / mL zGPGGPA substrate in f - Appel's buffer

[0286] Solution Preparation

[0287] Solutions are prepared as follows:

[0288] f - Appel 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 degrees Celsius.

[0289] 10 mM leucine stock solution: Dissolve 65.5 mg of leucine in 50 mL of water. Leucine must be weighed directly onto a scale in a 100 - mL (or equivalent) glass beaker. Weigh approximately 65 mg (target weight) of leucine into the beaker. Calculate the volume of water to add to the beaker using the following formula. Add the calculated volume of water to the beaker and mix well to ensure complete dissolution of leucine. Aliquot into 1 - mL portions. Store at or below 20 degrees Celsius.

[0290] V2 (mL)= C2 (mg) x V1(50mL)

[0291] C1 (65.5 mg)

[0292] Where:

[0293] C2 = Weight of leucine weighed (mg)

[0294] V1 = 50 (mL of water)

[0295] C1 = 65.5 (mg of leucine)

[0296] V2 = Volume of water required to produce 10 mM stock solution (m1)

[0297] 1 mM Leucine working stock solution: Thaw a vial of 10 mM leucine stock solution and dilute to 1 mM by combining 150 μL and 1350 μL of water. Stir well before use.

[0298] 0.5 N HCl: Dilute HCl to 0.5 N with water and mix well. Store at room temperature. Alternatively, commercially available 0.5 N HCl can be used.

[0299] 200 nM Borate, pH 9.0: Dissolve 2.4 grams of boric acid in approximately 150 mL of water. Adjust the pH to 9 using sodium hydroxide. QS to 200 mL with water and mix well. Store at 2 - 8 °C.

[0300] 0.5 mM Fluorescamine solution: Mix 15 mg of fluorescamine with 100 mL of acetone and stir to dissolve. Store protected from light at 2 - 8 °C.

[0301] Substrate solution (2 mg / mL zGPGGPA): Prepare 2 mg / mL of the substrate with f - Appel buffer. Dissolve in the presence of a mechanical shaker / rotator, allowing sufficient time for complete dissolution (about 15 minutes).

[0302] Leucine standard curve

[0303] Prepare the leucine standard curve according to Table 7

[0304] Table 7. Preparation of leucine standard curve

[0305]

[0306] “L1” refers to leucine standard sample 1

[0307] Then, 100 μL of each leucine standard was transferred separately into tubes and the fluorescamine in them was detected.

[0308] Collagenase sample preparation

[0309] Dilute the collagenase sample to 0.01 mg / mL with f - Appel buffer in two steps and then gently stir to mix. The following is an exemplary dilution protocol:

[0310] 1. 100 μL x 1.0 mg / mL → 1000 μL = 0.1 mg / mL

[0311] 2. 100 μL x 0.1 mg / mL → 1000 μL = 0.01 mg / mL

[0312] Blank Preparation

[0313] The blank was prepared by mixing 45 μL of the dilution preparation with 500 μL of 0.5 N hydrochloric acid to inactivate the enzyme. 455 μL of the zGPGGPA substrate solution was added and stirred well. 100 μL of the blank sample was transferred to a separate test tube to detect impurities that might react with fluorescamine.

[0314] Potency Curve

[0315] A set of potency curves was prepared for each collagenase sample as follows:

[0316] Test tube 2mg / mL substrate solution f-Appel buffer (μL) 1-2 1 45 3-4 1 30 5-6 1 15

[0317] The test tubes containing the substrate and buffer were heated in a water bath at 25 °C for at least 15 minutes. Label the second set of test tubes and add 50 μL of 0.5 N hydrochloric acid to each test tube. Add the diluted collagenase sample preparation (0.01 mg / mL) to the test tubes according to Table 8 and incubate for 10 minutes. After mixing, return to the 25 °C water bath. Start the incubation phase after adding the first preparation to the preheated substrate.

[0318] Table 8. Sample Preparation

[0319] Preparation Test tube Sample (μL) Titer curve 1-2 55 Titer curve 3-4 70 Titer curve 5-6 85

[0320] When there was 1 - 2 minutes remaining in the 10 - minute incubation, remove the preparation from the water bath and gently vortex - mix. 10 minutes after adding the first preparation to the substrate, transfer 50 μL from each test tube to a test tube containing 50 μL of 0.5 N HCl. These preparations should be added directly to the acid to inhibit digestion. After quenching all the preparations, vortex each test tube to mix well.

[0321] Detection

[0322] Add 400 μL of 200 mM borate buffer and 500 μL of 0.5 mM fluorescamine solution to all the detection test tubes containing 100 μL of each preparation (blank, collagenase sample potency curve, and leucine standard). Stir well. Let the test tubes incubate at room temperature for at least 10 minutes.

[0323] Fluorometer Settings

[0324] Set the fluorometer with the following instrument parameters and read the fluorescence of each preparation 1 hour after derivatization.

[0325] Parameter Setting Excitation wavelength 392nm Excitation wavelength 480nm Integration 5.0sec. Slit (Ex&Em) 5.0nm (bandpass filter) Path length 3mm

[0326] Calculation

[0327] Plot the concentration of each leucine standard (X-axis) against the fluorescence response at 480 nm (Y-axis). Determine the slope (m) and the coefficient of determination (R2). Measure the average fluorescence of each titer curve preparation. Prepare the collagenase sample and leucine titer curves by plotting the concentration of each preparation (X-axis) against the average fluorescence response at 480 nm (Y-axis). Determine the slope (m) and the coefficient of determination (R2) of the resulting linear curve.

[0328] Determine the titer of the collagenase sample

[0329] Titer (f-GPA units / mg) = (M 样品 / M 亮氨酸 ) x (DF / T)

[0330] Where:

[0331] M 样品 = slope of the collagenase sample titer curve

[0332] M 亮氨酸 = slope of the leucine standard curve

[0333] DF = dilution factor (1100 μL / 50 μL = 22)

[0334] T = reaction time (10 minutes)

[0335] ii. GPA microplate assay for determining type II collagenase activity in a collagenase sample

[0336] This method is similar to the above GPA, except that it is carried out in a microplate. Like the above determination, the microplate assay measures the proteolytic activity of the collagenase sample in the enzymatic cleavage of the substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-L-alanyl-L-alanine (zGPGGPA) (hereinafter referred to as "substrate"). This experiment follows the following method, but those of ordinary skill in the art will understand that certain modifications (such as dilution concentration and time) can be made within the scope of achieving the purpose of this experiment.

[0337] Reagents

[0338] 1. Peptide substrate (zGPGGPA) (Bachem M1260 or equivalent)

[0339] 2. Tripeptide GPA (Bachem H3615 or equivalent)

[0340] 3. Fluorescamine (Acros 191675000 or equivalent)

[0341] 4. Purified water (Milli-Q-Plus 18.2 MΩ system or equivalent)

[0342] 5. 1M HEPES buffer (Gibco 15630-080 or equivalent)

[0343] 6. Surfact-Amps 20 TM (10% Tween solution) (Pierce Cat.#28320 or equivalent)

[0344] 7. 1M calcium acetate (Ca(C2H3O2)2) (Emerald Biosciences Cat.#EBS-100-CAAC or equivalent)

[0345] 8. Boric acid (Sigma B7660 or equivalent)

[0346] 9. 2.5N NaOH (J.T Baker 5666-02 or equivalent)

[0347] 10. 0.5N hydrochloric acid (VWR 101223-134 or equivalent)

[0348] 11. Acetone (Sigma 270725 or equivalent)

[0349] Preparation of solutions

[0350] (i) Preparation of experimental buffer (50 mM HEPES pH 7.1 / 0.05% Tween 20 / 5 mM (Ca(C2H3O2)2): Pipette 50 mL of 1M HEPES into 800 mL of deionized water. Add 5 mL of 1M (Ca(C2H3O2)2) and 5 mL of Surfact-Amps (10% Tween 20). Check the pH and adjust to 7.1 ± 0.05 if necessary. Add sufficient water to adjust the volume to 1 L and filter the solution through a 0.22 micron filter. This experimental buffer can be stored at room temperature for 3 months.

[0351] (ii) Preparation of 0.1N NaOH: Add 2 mL of 2.5N NaOH to 48 mL of deionized water. This solution can be stored at room temperature for 3 months.

[0352] (iii) Preparation of 4 mg / mL tripeptide GPA stock solution: Dissolve 400 mg (±1 mg) of tripeptide GPA in 10 mL of 0.1N NaOH and rotate until completely dissolved. Add sufficient experimental buffer to make the volume 100 mL and divide the solution into 0.5 mL aliquots and store at -70 °C. The 4 mg / mL tripeptide GPA stock can be stored at -70 °C for one year.

[0353] (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 sodium hydroxide and vortex until completely dissolved. Add sufficient assay buffer to bring the volume to 100 mL. This solution can be stored at 4 °C for 3 months.

[0354] (v) Preparation of 120 mM boric acid at pH 9.0: Dissolve 7.4 g (±0.5 g) of boric acid in 800 mL of deionized water. Titrate the solution to pH 9.0 with NaOH. Add sufficient deionized water to adjust the volume to 1 liter. This solution can be stored at room temperature for 3 months.

[0355] (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.

[0356] Preparation and serial dilution of tripeptide GPA standard

[0357] Prepare a 0.08 mg / mL (329 μM) tripeptide GPA standard by diluting the 4 mg / mL tripeptide GPA stock solution 50-fold in assay buffer (e.g., dilute 20 μL of 4 mg / mL tripeptide GPA in 980 μL of assay buffer). Using a pipette, transfer 200 μL of the 329 μM tripeptide GPA standard to wells A1 and A7 in row A of the assay plate. Using a pipette, transfer 100 μL of assay buffer to wells A2 - A6 and A8 - A12.

[0358] For the tripeptide GPA standard serial dilution, transfer 100 μL from well A1 to well A2, mix, transfer 100 μL from well A2 to well A3, and repeat until well A5. Remove 100 μL from well A5 to give a final volume of 100 μL. Well A6 contains only buffer.

[0359] For the second tripeptide GPA standard serial dilution, transfer 100 μL from well A7 to well A8, mix, transfer 100 μL from well A8 to well A9, and repeat until well A11. Remove 100 μL from well A11 to give a final volume of 100 μL. Well A12 contains only buffer.

[0360] Preparation of collagenase samples

[0361] For collagenase samples (e.g., lyophilized collagenase drug product), cool the sample to room temperature over at least ten minutes and reconstitute to a 500 ng / mL stock solution. Different concentrations can be used. Dilute the stock solution with assay buffer to prepare the collagenase samples to be tested (T1A). Repeat this procedure to prepare three test samples (T1A, T1B, T1C).

[0362] Discussion

[0363] In this method, 50 μL of the collagenase sample with increasing concentrations is mixed with 50 μL of an excess substrate (final concentration 2.0 mg / mL) in a 96-well plate. 50 μL of the assay buffer is added to rows C-G of a U-bottom 96-well polypropylene reaction plate. 150 μL of the collagenase sample is pipetted into row B. Then, serial dilutions of 1 / 1.5 are made using a multi-channel pipette, transferring 100 μL of the collagenase sample from row B to row C, mixing and repeating the process until row G is reached. 100 μL is removed and discarded from row G. Table 9 contains the final collagenase concentrations after adding 50 μL of substrate to rows B-H.

[0364] The blank for row H is prepared by pipetting 50 μL of the assay buffer into row H. This row contains no enzyme. Exemplary concentrations are shown in Table 9.

[0365] Table 9. Experimental target concentrations after adding substrate

[0366]

[0367] Collagenase reaction

[0368] The zGPGGPA substrate is cleaved by class II collagenase into zGPG and GPA during a 15-minute incubation at room temperature. The incubator and temperature probe are turned on (temperature was 22 ± 1 °C before adding the substrate to the culture plate). 50 μL of 4 mg / mL (6.8 mM) zGPGGPA substrate is added column-wise to columns 1-12 of rows B-H and then mixed. The start time of the reaction is counted after the substrate is added to the first column. The culture plate is covered and placed in a 22 ± 1 °C incubator for a total reaction time of 15 ± 1 minutes.

[0369] After incubation, the reaction is quenched by adding hydrochloric acid, and the amount of released GPA peptide is quantified after reacting the free amino terminus of the peptide with the fluorogenic reagent fluorescamine. To stop the reaction, 100 μL of 0.5 N HCl is added from row A to each well of rows H, column-wise, and then mixed. The reaction time ends after the HCl is added to the first column.

[0370] Detection

[0371] Add 195 μL of 120 mM borate (pH 9.0) to each well of a Greiner polypropylene black microplate reader plate. Transfer 30 μL of the quenched reaction mixture from the reaction plate to the corresponding well of the reader plate and mix well. Then, add 75 μL of 1 mM fluorescamine to each well of the reader plate (dispense fluorescamine / acetone using a polypropylene tray) and mix immediately after each addition. Read the plate within 15 minutes after fluorescamine addition using a Molecular Devices M2 fluorescence plate reader, using the following settings: excitation 380 nm, emission 473 nm, cut-off 455 nm, 6 reads / well, PMT medium.

[0372] Curves were plotted for GPA concentration (μM) vs. emission at 473 nm and collagenase concentration (ng / mL) vs. emission at 473 nm. For each plot, the linear regression fit had no fixed parameters. For the collagenase test samples, zero point data was excluded from the linear fit and plots were generated using the entire triplicate data set for each sample. The slopes of the tripeptide GPA standards and the collagenase samples were determined.

[0373] Potency determination

[0374] Calculate the specific activity of the collagenase sample as follows:

[0375] GPA microplate assay unit = ((collagenase sample slope) / (tripeptide GPA slope x incubation time)) x 10 6 .

[0376] The specific activity of the collagenase sample to be tested is determined by the slope of the tripeptide GPA standard and calculated by a curve fitting program. According to the Michaelis-Menton equation, using the microplate method, enzyme kinetics can be calculated with different substrate concentrations and different times.

[0377] iii. Collagenase potency determined by the GPA experiment

[0378] The collagenase effective in the disclosure herein may have a potency of about 100,000 to about 300,000 GPA units / mg or about 175,000 to about 300,000 f-GPA units / mg. In other embodiments, the potency may be about 70,000 to about 400,000 GPA units / mg, or about 100,000 to about 375,000 GPA units / mg, or about 125,000 to about 350,000 GPA units / mg, or about 150,000 to about 325,000 GPA units / mg, or about 175,000 to about 300,000 GPA units / mg, or about 200,000 to about 275,000 GPA units / mg. Alternatively, the potency may be about 70,000 to about 400,000 f-GPA units / mg, or about 100,000 to about 375,000 f-GPA units / mg, or about 125,000 to about 350,000 f-GPA units / mg, or about 150,000 to about 325,000 f-GPA units / mg, or about 175,000 to about 300,000 f-GPA units / mg, or about 230,000 to about 430,000 f-GPA units / mg, or about 200,000 to about 275,000 f-GPA units / mg. The collagenase may also have an activity of about 30,100 to 87100 or about 43,000 to 67,000 GPA microplate assay units. The above GPA can be used to analyze the specific activity of any collagenase.

[0379] b. SRC unit analysis method and specific activity unit

[0380] i. Collagenase potency determined by SRC assay (cuvette)

[0381] The SRC experiment is mainly used to measure the potency of class I collagenase. The general method is as follows. Prepare leucine standard and collagenase sample solutions. The first step of the experiment involves the enzymatic reaction of collagenase digesting soluble rat tail tendon collagen (SRC). The second step involves subsequently measuring the released peptide fragments / amino acids with the fluorescent derivative fluorescamine. The experiment follows the following method, but those of ordinary skill in the art will understand that certain modifications (such as dilution concentration and time) can be made in order to achieve the experimental purpose.

[0382] Treat this collagenase and leucine standard sample with reagents so as to label the generated GPA with fluorescein. The leucine standard and collagenase samples are incubated at room temperature for 10 minutes, and then the fluorescence of each solution is measured at excitation and emission wavelengths of 392 and 480 nm, respectively. The slopes of the leucine and collagenase sample curves are used to calculate the potency units as follows:

[0383] Potency (f-GPA units / mg) = (M 样品 / M 亮氨酸 )x(DF / T)x CF

[0384] Wherein:

[0385] M 样品 = Slope of the collagenase titer curve

[0386] M 亮氨酸 = Slope of the leucine standard curve

[0387] DF = Dilution factor (1500 μL / 100 mL = 15)

[0388] T = Reaction time (2.5 hr x 60 min / 1 hr = 150 min)

[0389] CF = Conversion factor (1000 μg / 1 mg = 1000)

[0390] In addition, non-limiting details of the SRC experimental method are described below.

[0391] Buffers and Reagents

[0392] 1. F-TC experimental buffer, pH 7.2 (22 g HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 4.4 g, calcium acetate)

[0393] 2. F-enzyme buffer, pH 7.2

[0394] 3. 200 mM borate, pH 9.0

[0395] 4. 10 mM leucine stock solution

[0396] 5. 1 mM leucine working stock solution

[0397] 6. Fluorescamine solution in 1 mM acetone

[0398] 7. Rat tail collagen at a concentration of 2 mg / mL in 0.02 N acetic acid

[0399] Solution Preparation

[0400] Prepare the solutions according to the following steps

[0401] F-TC experimental 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 adjust the QS to 1 L with water. Store at 2 - 8 °C.

[0402] F-enzyme-buffer: Dilute the F-TC experimental buffer by combining 4 ml of the F-TC experimental buffer with 16 mL of water. Store at 2 - 8 °C.

[0403] 10 mM Leucine Stock Solution: Dissolve 65.5 mg of leucine in 50 mL of water. Leucine must be weighed directly onto the scale of a 100 mL (or equivalent size) glass beaker. Weigh approximately 65 mg (target weight) of leucine into the beaker. Calculate the volume of water to add to the beaker using the following formula based on the weight of leucine weighed. Add the calculated volume of water to the beaker and mix well to ensure complete dissolution of leucine. Aliquot into 1 mL portions. Store at a temperature of -20 °C or lower.

[0404]

[0405] where

[0406] C2 = mass of leucine weighed (mg)

[0407] V1 = 50 (mL of water)

[0408] C1 = 65.5 (mg of leucine)

[0409] V2 = volume of water required to produce 10 mM stock solution (mL)

[0410] 1 mM Leucine Working Stock Solution: Thaw a vial of 10 mM leucine stock solution and mix 150 μL with 1350 μL of water to dilute to 1 mM. Stir well before use.

[0411] 0.5 N HCl: Dilute HCl with water to 0.5 N and mix well. Store at room temperature. Alternatively, commercially available 0.5 N HCl can be used.

[0412] 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.

[0413] 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. Add water to QS to 200 mL and mix well. Store at 2 - 8 °C.

[0414] 1 mM Fluorescamine Solution: Dissolve 15 mg of fluorescamine in 50 μL of acetone and stir to dissolve. Store protected from light at 2 - 8 °C.

[0415] Substrate Solution (2 mg / mL Rat Tail Collagen): Dilute rat tail collagen to 2 mg / mL with 0.02 N acetic acid. Store at 2 - 8 °C.

[0416] Leucine Standard Curve

[0417] Prepare a leucine standard curve according to Table 10.

[0418] Table 10. Preparation of Leucine Standard Curve

[0419]

[0420] Then transfer 100 μL of each leucine standard to separate centrifuge tubes for fluorescein detection.

[0421] Collagenase Sample and Blank Preparation

[0422] Dilute the collagenase sample to 0.01 mg / mL with F-enzyme buffer in two steps and then gently vortex to mix. The following is an exemplary dilution protocol:

[0423] 1. 100 μL x 1.0 mg / mL → 1000 μL = 0.1 mg / mL

[0424] 2. 100 μL x 0.1 mg / mL → 1000 μL = 0.01 mg / mL

[0425] Maintain the diluted samples at room temperature until use.

[0426] Prepare the blank by first combining the sample with 0.5 N hydrochloric acid to inactivate the enzyme, and then adding buffer and substrate according to Table 11.

[0427] Label each collagenase sample tube according to Table 11. Tubes 1, 2, 4, and 6 are prepared from one preparation and tubes 3, 5, and 7 are prepared from a duplicate preparation.

[0428] Table 11. Blank and Collagenase Sample Preparation

[0429]

[0430] Cover the tubes and gently rotate to mix. Incubate the potency curve preparations in a 25 °C ± 3 °C water bath for 2.5 hours. After incubation, remove the potency curve tubes from the water bath. Add 750 μL of 0.5 N HCl to each preparation and rotate thoroughly to mix. The preparations can be stored at 2 - 8 °C for 22 hours before detection.

[0431] Detection / Fluorometer Settings

[0432] Prepare the leucine standard as described above.

[0433] Set the fluorometer with the following instrument parameters and read the fluorescence of each preparation 1 hour after derivatization.

[0434] Parameter Setting Excitation wavelength 392nm Excitation wavelength 480nm Integration 5.0sec. Slit (Ex&Em) 5.0nm (bandpass filter) Path length 3mm

[0435] Calculation

[0436] Plot the concentration of each leucine standard (X-axis) against the fluorescence response at 480 nm (Y-axis). Determine the slope (m) and the coefficient of determination (R2). Measure the average fluorescence of each potency curve preparation. Do not force zero. Determine the average fluorescence of each replicate preparation. Calculate the net fluorescence of each collagenase sample preparation

[0437] F(net) = average collagenase sample (EM 480 ) - blank (EM 480 )

[0438] Plot the concentration (X-axis) of the collagenase sample in each preparation against the net fluorescence (Y-axis). Determine the slope (m) and the determination factor (R 2 ). Do not force zero.

[0439] Determine the potency of the collagenase sample

[0440] Potency (f-SRC units / mg) = (M 样品 / M 亮氨酸 ) x (DF / T) x CF

[0441] Where:

[0442] M 样品 = slope of the collagenase sample potency curve

[0443] M 亮氨酸 = slope of the leucine standard curve

[0444] DF = dilution factor (1500 μL / 100 mL = 15)

[0445] T = reaction time (2.5 hr x 60 minutes / 1 hr = 150 min)

[0446] CF = conversion factor (1000 μg / 1 mg = 1000)

[0447] The above SRC experiment can be used to analyze the specific activity of any collagenase as an alternative.

[0448] ii. SRC microplate assay for determining type I collagenase activity in a collagenase sample

[0449] This method is similar to the above SRC, except that it is performed in a microplate. Like the above SRC experiment, the microplate assay measures collagenase activity in the cleavage of a soluble rat-tail collagen (SRC) substrate (hereinafter referred to as "substrate"). This experiment follows the following method, but those of ordinary skill in the art will understand that certain modifications (such as dilution concentration and time) can be made within the scope of achieving the purpose of this experiment.

[0450] Reagents

[0451] 1. Soluble rat-tail collagen substrate (BD Biosciences 354236)

[0452] 2. Tripeptide GPA (Bachem H3615 or equivalent)

[0453] 3. Fluorescamine (Acros 191675000 or equivalent)

[0454] 4. Purified water (Milli-Q-Plus 18.2 MΩ system or equivalent)

[0455] 5. 1M HEPES buffer (Gibco 15630-080 or equivalent)

[0456] 6. 1M Calcium acetate (Ca(C2H3O2)2) (Emerald Biosciences Cat.# EBS-100-CAAC or equivalent)

[0457] 7. Surfact-Amps 20 TM (10% Tween solution) (Pierce Cat.# 28320 or equivalent)

[0458] 8. 1.0N Acetic acid (Sigma 318590 or equivalent)

[0459] 9. 0.5N Hydrochloric acid (VWR 101223-134 or equivalent)

[0460] 10. Boric acid (Sigma B7660 or equivalent)

[0461] 11. 2.5N Sodium hydroxide (J.T Baker 5666-02 or equivalent)

[0462] 12. Acetone (Sigma 270725 or equivalent)

[0463] Preparation of solutions

[0464] (i) Preparation of experimental buffer (50 mM HEPES pH 7.1 / 0.05% Tween 20 / 5 mM (Ca(C2H3O2)2): Pipette 50 mL of 1M HEPES into 800 mL of deionized water. Add 5 mL of 1M (Ca(C2H3O2)2) and 5 mL of Surfact-Amps (10% Tween 20). Check the pH and adjust to 7.1 ± 0.1 if necessary. Add sufficient water to adjust the volume to 1 L and filter the solution through a 0.22 micron filter. This experimental buffer can be stored at room temperature for 3 months.

[0465] (ii) Preparation of 0.1N NaOH: Add 2 mL of 2.5N NaOH to 48 mL of deionized water. This solution can be stored at room temperature for 3 months.

[0466] (iii) Preparation of 4mg / mL tripeptide GPA stock solution: Dissolve 400 mg (±1 mg) of tripeptide GPA in 10 mL of 0.1N NaOH and rotate until completely dissolved. Add sufficient experimental buffer to make the volume 100 mL, divide the solution into 0.5 mL aliquots, and store at -70 °C. The 4mg / mL tripeptide GPA stock can be stored at -70 °C for one year.

[0467] (iv) Preparation of 0.02N acetic acid: Add 1 mL of 1.0N acetic acid to 40 mL of pure water. Add sufficient pure water to adjust the volume to 50 mL. This solution can be stored at room temperature for one year.

[0468] (v) Preparation of 2mg / mL SRC substrate stock solution: Add 23.3 mL of 0.02N acetic acid directly to the tube supplying the substrate (100 mg of SRC supplied at a concentration of 3.75 mg / mL in a non-limiting example). Other concentrations of SRC substrate can be used. The calculation is as follows:

[0469] 100 mg ÷ 3.75 mg / mL = 26.7 mL;

[0470] Total vol (mL) = (3.75 mg / mL x 26.7 mL) / 2 mg / mL;

[0471] Total vol (50.0 mL) - 26.7 mL = 23.3 mL

[0472] Mix the solution well by inverting and can be stored at 2 - 8 °C for 3 months.

[0473] (vi) Preparation of 0.6mg / mL SRC substrate working solution: Add 4.2 mL of experimental buffer to a 15 mL conical tube. Then, add 1.8 mL of 2mg / mL SRC substrate stock solution and mix the solution by inverting. This solution should be prepared immediately before adding to the plate.

[0474] (vii) Preparation of 120mM boric acid with a pH of 9.0: Dissolve 7.4 g (±0.5 g) of boric acid in 800 mL of deionized water. Titrate the solution with NaOH to pH 9.0 and add sufficient deionized water to adjust the volume to 1 L. This solution can be stored at room temperature for 3 months.

[0475] (viii) Preparation of Fluorescamine at a concentration of 1 mM 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.

[0476] Preparation and serial dilution of the tripeptide GPA standard

[0477] Prepare a 0.08 mg / mL (329 μM) tripeptide GPA standard by diluting a 4 mg / mL tripeptide GPA stock solution 50-fold in the experimental buffer (e.g., dilute 20 μL of 4 mg / mL tripeptide GPA in 980 μL of experimental buffer). In row A of the experimental plate, pipette 200 μL of the 329 μM tripeptide GPA standard into A1 and A7. Pipette 100 μL of the experimental buffer into A2 - A6 and A8 - A12.

[0478] For the tripeptide GPA standard serial dilutions, transfer 100 μL from A1 to A2, mix, transfer 100 μL from A2 to A3, and repeat until A5. Remove 100 μL from the A5 well to give a final volume of 100 μL. Well A6 contains only buffer.

[0479] For the second tripeptide GPA standard serial dilution, transfer 100 μL from A7 to A8, mix, transfer 100 μL from A8 to A9, and repeat until well A11. Remove 100 μL from the A11 well to give a final volume of 100 μL. Well A12 contains only buffer.

[0480] Preparation of the collagenase samples to be tested

[0481] For the collagenase samples (e.g., lyophilized collagenase drug product), cool the sample to room temperature over at least ten minutes and reconstitute it into a 3.0 μg / mL stock solution. Different concentrations can be used. Dilute the stock solution with the experimental buffer to prepare the collagenase samples to be tested (T1A). Repeat this procedure to prepare three test samples (T1A, T1B, T1C).

[0482] Discussion

[0483] In this method, 50 μL of the collagenase sample with increasing concentrations is mixed with 50 μL of an excess substrate (final concentration of 2.0 mg / mL) in a 96-well plate. 50 μL of the experimental buffer is added to rows C-G of a U-bottom 96-well polypropylene reaction plate. 150 μL of the collagenase sample is pipetted into row B. Then, serial dilutions of 1 / 1.5 are performed using a multi-channel pipette, transferring 100 μL of the collagenase sample from row B to row C, mixing, and repeating the process until row G is reached. 100 μL is removed and discarded from row G. The H row blank is prepared by pipetting 50 μL of the experimental buffer into row H. This row does not contain enzyme. Table 12 contains the final collagenase concentrations after adding 50 μL of substrate to rows B-H.

[0484] Table 12. Experimental target concentrations after adding substrate

[0485]

[0486] Collagenase reaction

[0487] Open the incubator and the temperature probe (temperature is 22 ± 1 °C before adding the substrate to the culture plate). Add 50 μL of 0.6 mg / mL SRC substrate to each well of rows B-H, adding column by column and then mixing. The reaction start time begins after the substrate is added to the first column. Cover the culture plate and place it in an incubator at 22 ± 1 °C for a total reaction time of 45 ± 5 minutes. After incubation, quench the reaction by adding 100 μL of 0.5 N hydrochloric acid to each well of the dilution plate, adding column by column and then mixing. The reaction time ends after the HCl is added to the first column.

[0488] Detection

[0489] Add 195 μL of 120 mM borate (pH 9.0) to each well of a microplate Greiner polypropylene black reading plate. Transfer 30 μL of the quenched reaction mixture from the reaction plate to the corresponding well of the reading plate and mix well. Transfer 30 μL of the annealed reaction mixture from the reaction plate to the corresponding well of the reading plate and mix well. Then, add 75 μL of 1 mM fluorescamine to each well of the reading plate (using a polypropylene tray to dispense fluorescamine / acetone) and mix immediately after each addition. Read the plate within 15 minutes after adding fluorescamine using a Molecular Devices M2 fluorescence plate reader with the following settings: excitation 380 nm, emission 473 nm, cutoff 455 nm, 6 reads / well, PMT medium.

[0490] Plotted the relationship curves of GPA concentration (μM) vs. emission at 473 nm and collagenase concentration (ng / mL) vs. emission at 473 nm. For each graph, the linear regression fit had no fixed parameters. For the collagenase test samples, zero-point data was excluded from the linear fit, and the plot was generated using the entire three-dataset for each sample. Determined the slopes of the tripeptide GPA standard and the collagenase samples.

[0491] Determination of specific activity and related potency

[0492] The specific activity of the collagenase samples was calculated as follows:

[0493] SRC microplate assay unit = ((collagenase sample slope) / (tripeptide GPA slope x incubation time)) x 10 6

[0494] The specific activity of the collagenase samples to be tested was determined by the slope of the tripeptide GPA standard and calculated by a curve fitting program. According to the Michaelis-Menton equation, using the microplate method, enzyme kinetics can be calculated with different substrate concentrations and different times.

[0495] iii. Potency of collagenase determined by SRC assay

[0496] The potency of the collagenase effective in the content disclosed herein is about 500 to 15,000 SRC units / mg. In certain embodiments, the potency is about 500 to about 12,500 SRC units / mg, or about 700 to about 10,000 SRC units / mg, or about 1,000 to about 7,500 SRC units / mg, or 1,500 to about 6,000 SRC units / mg, or about 2,500 to about 5,000 SRC units / mg. Alternatively, the potency can be about 5,000 to about 35,000 f-SRC units / mg, or about 10,000 to about 30,000 f-SRC units / mg, or about 13,000 to about 23,000 f-SRC units / mg, or about 15,000 to about 25,000 f-SRC units / mg. The collagenase can also have a potency of about 980 to 3,510 or about 1,400 to 2,700 SRC microplate assay units.

[0497] c. Potency of collagenase in BTC unit assay

[0498] The bovine tendon collagenase assay is based on the method described by Mandl et al. (1958) and modified by Keller and Mandl (1963). Since bovine tendon collagen is an insoluble substrate, it is important to isolate it finely. Trypsin is run as a control to account for the presence of denatured collagen or other protein impurities. The experiment is carried out in the presence of calcium ions required for collagenase activity. The number of soluble peptides is determined by reacting the N-terminal amino groups of the peptides with ninhydrin and measuring the amount of the auxochrome formed colorimetrically (Rosen 1957).

[0499] The aim of this method is to detect the specific activity of collagenase using a collagen substrate.

[0500] Reagents and Solutions

[0501] 1. Collagen Substrate (Collagen)

[0502] 2. Deionized Water (Water)

[0503] 3. Tris Assay Buffer

[0504] 4. Trypsin Stock Solution

[0505] 5. 0.5 M HCl

[0506] 6. Leucine Standard Assay Solution (1 mM Leucine)

[0507] 7. Rosen Buffer

[0508] 8. 3% Ninhydrin

[0509] 9. 50% Isopropanol

[0510] Assay

[0511] Set up and label reaction tubes as follows: Three tubes for trypsin control, six tubes for reference solutions, and six tubes for each sample to be tested. Label each tube and open the lids. Weigh 10 ± 1 mg of collagen in the order shown in Table 13 and place the weighed collagen into each reaction tube.

[0512] Table 13. Weighing Order and Reaction Tube Numbers

[0513] Weighing order Reaction test tube # 1 1 2 4 3 6 4 8 5 10 6 12 7 14 8 16 9 18 10 20 11 2 12 3 13 5 14 7 15 9 16 11 17 13 18 15 19 17 20 19 21 21

[0514] For the test samples, the enzyme activity in each reaction tube (ACT) should be between 1.6 and 5.7 nmol leu eq / min. Undissolved samples should first be dissolved in Tris assay buffer and then used for assay analysis. The concentration (before addition to the reaction tube) should be not less than 0.0065 mg / mL.

[0515] Set up the reaction tubes according to Table 14 so that they have a matrix pattern. The following table assumes 2 samples to be tested. If running more or fewer samples, adjust the number of reaction tubes, but keep the pattern. Table 14 shows the case where the volume remains constant.

[0516] Table 14. Matrix pattern

[0517]

[0518] *The maximum recommended number of samples is 3

[0519] Cover the reaction tubes. Mix the contents gently but thoroughly. Place the reaction tubes in a water bath at 37 °C. Incubate for 22 ± 0.5 hours. Record the actual time a when the culture starts, 37 °C, the number of water baths used, the batch number of the collagen lipid, the collagen correction factor for the batch used, and the batch numbers of all solutions used

[0520] Quench and filter

[0521] Label the filtrate tubes to correspond to each reaction tube that was cultured. Place a funnel with a folded filter paper on each labeled filtrate tube. At the end of the culture phase, remove the reaction tubes from the water bath. Record the actual time when the culture ends.

[0522] Open the reaction tubes and discard the lids. Quench the reaction by dispensing 2 mL of 0.5 M HCl into each reaction tube. Mix the contents of the tubes well. Filter the contents of each reaction tube into the appropriate filtration tube.

[0523] The first two steps need to be completed as soon as possible because undigested collagen can be dissolved by hydrochloric acid in a short time. Before color development, the filtrate can be refrigerated in a covered filtrate tube for up to 95.5 hours. Record the refrigeration and storage time.

[0524] Color development

[0525] Set up and label the boiling tubes as follows: 6 tubes for water and leucine control (step 1), 2 tubes for each filtrate bucket (step 1). Place the following amounts of water and leucine standard analytical solution into the six leucine control tubes.

[0526] Test tube # 1 2 3 4 5 6 Water (μL) 1000 900 850 800 750 700 Leucine (μL) 0 100 150 200 250 300 Leucine (nmol) 0 100 150 200 250 300

[0527] Transfer 0.8 mL of water to each boiled test tube (Step 2) using a pipette. Pipette 0.2 mL of filtrate from each sample into appropriately labeled boiled test tubes. Add 0.5 mL of Rosen buffer to each boiled test tube. Dispense 0.5 mL of 3% ninhydrin into each boiled test tube under a fume hood. Thoroughly mix the contents of each tube on a vortex mixer. Place the boiled test tubes in a boiling water bath in a fume hood. Boil for 15 ± 1 minute. At the end of the boiling period, remove the boiled test tubes from the water bath. Dispense 5.0 mL of 50% isopropanol into each boiled test tube under a fume hood and thoroughly mix the contents. Allow the boiled test tubes to reach ambient temperature (at least 10 minutes) before taking absorbance readings.

[0528] Absorbance reading

[0529] While working under a fume hood, take the absorbance of each test tube. Turn on the spectrophotometer and allow it to warm up. Set the wavelength of the spectrophotometer to 570 nm. Zero the spectrophotometer with 50% isopropanol. Take the absorbance (A 570 ) of water, leucine, trypsin control, and test samples. Record the time in hours when the first sample is read. Record the readings as 1000X A 570 , and record the time (in hours) when the last sample is read. All readings should be completed within a 1-hour interval.

[0530] Calculation principle

[0531] Calculate the total reading time and total incubation time in minutes. The total reading time should be less than 60 minutes, and the total incubation time should be between 1290 - 1350 minutes. Using linear least squares, calculate the slope "b" and correlation coefficient "r" of the leucine standard (x = nmol leucine vs y = A 570 readings). The unit of the "b" value is A 570 / nmol leucine. Record the "b" value to two decimal places. The b value of leucine should be between 2.88 - 3.33. Calculate the average reading (T) of the trypsin control. The average reading of the trypsin control (T) should be 221 - 338. Record this average to the nearest whole number (Step A). The average of the duplicate samples A 570 readings for each reaction tube. Record this number to the nearest whole number. Subtract the average trypsin (Step A) from the average sample A 570 readings to obtain the net sample reading.

[0532] Calculate the activity (ACT) of each test tube in nmol leu eq / min as follows:

[0533]

[0534] In the formula, 20 is the dilution coefficient of the amount of the formed reaction mixture, and "b" is the slope of the leucine standard curve. Record this number to one decimal place. The activity of each test sample tube should be 1.6 - 5.7 nmol leu eq / min.

[0535] Calculate the activity in BTC units as follows:

[0536] BTC unit = Activity in nmol leu eq / min x Collagen correlation coefficient.

[0537] Calculate the activity of the sample in BTC units / mL as follows:

[0538]

[0539] Calculate the specific activity of the sample in BTC units / mm as follows:

[0540]

[0541] Conversion of BTC units to ABC units:

[0542] ABC unit = BTC unit x 1.09

[0543] i. BTC units and ABC units

[0544] Various collagenase compositions can be used, in which the collagenase has a specific activity of about 5,000 BTC units / mg to about 25,000 BTC units / mg, or about 10,000 BTC units / mg to about 25,000 BTC units / mg, or about 15,000 BTC units / mg, or about 17,500 BTC units / mg, or about 20,000 BTC units / mg, or about 22,500 BTC units / mg, or about 9,175 BTC units / 0.58 mg, or 15,817 BTC units / mg, where "mg" refers to the amount of collagenase present in the composition (different from excipients and other components).

[0545] In addition, various collagenase compositions can be used, wherein 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, wherein "mg" refers to the amount of collagenase present in the composition (different from excipients and other components).

[0546] d. Other experiments

[0547] The experimental methods used to label collagen are described in 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). Other methods also include the experimental methods of Worthington Biochemical Corporation (http: / / www.worthington-biochem.com / CLS / assay.html) (accessed on July 3, 2019).

[0548] 4. Collagenase dose

[0549] Regarding the collagenase dosage used herein, the present invention provides a therapeutically effective amount of collagenase, which is sufficient to bind and dissolve the septum upon subcutaneous injection, thereby reducing the appearance of subcutaneous cellulite compared to the pre-treatment baseline.

[0550] In one embodiment, the collagenase can be injected in a single or divided dose in an amount of about 0.01 mg to about 20 mg. In another embodiment, the collagenase can be injected in a single or divided dose in an amount of about 0.05 mg to about 15 mg. In another embodiment, the collagenase can be injected in a single or divided dose in an amount of about 0.10 mg to about 10 mg. In another embodiment, the collagenase can be injected in a single or divided dose in an amount of about 0.15 mg to about 5 mg. In another embodiment, the collagenase can be injected in a single or divided dose in an amount of about 0.20 mg to about 3 mg. In another embodiment, the collagenase can be injected in a single or divided dose in an amount of about 0.25 mg to about 2 mg. In still 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, 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, 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, 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, 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, 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.

[0551] In one embodiment, according to the measurement results of the SRC experiment, the collagenase has a V of about 2.6 min -1 to 5.2 min -1 of max In other embodiments, according to the measurement results of the SRC experiment, the collagenase has a V of about 3.0 min -1 to 5.0 min -1 of max In other embodiments, according to the measurement results of the SRC experiment, the collagenase has a V of about 3.4 min -1 to 4.8 min -1 of max In still another embodiment, according to the measurement results of the SRC experiment, the collagenase has a V of about 3.5 min -1 to 4.5 min -1 of max In still another embodiment, according to the measurement results of the SRC experiment, the collagenase has about 2.0 min -1 about 2.1 min -1 about 2.2 min -1 about 2.3 min -1 about 2.4 min -1 about 2.5 min -1 about 2.6 min -1 about 2.7 min -1 about 2.8 min -1 about 2.9 min -1 about 3.0 min -1 about 3.1 min -1 about 3.2 min -1, approximately 3.3 min -1 , approximately 3.4 min -1 , approximately 3.5 min -1 , approximately 3.6 min -1 , approximately 3.7 min -1 , approximately 3.8 min -1 , approximately 3.9 min -1 , approximately 4.0 min -1 , approximately 4.1 min -1 , approximately 4.2 min -1 , approximately 4.3 min -1 , approximately 4.4 min -1 , approximately 4.5 min -1 , approximately 4.6 min -1 , approximately 4.7 min -1 , approximately 4.8 min -1 , approximately 4.9 min -1 , approximately 5.0 min -1 , approximately 5.1 min -1 , approximately 5.2 min -1 , approximately 5.3 min -1 , approximately 5.4 min -1 , approximately 5.5 min -1 , approximately 5.6 min -1 , approximately 5.7 min -1 , approximately 5.8 min -1 , approximately 5.9 min -1 , or approximately 6.0 min -1 of V max . In other embodiments, according to the measurement results of the SRC experiment, the collagenase has approximately 0.7 min -1 to 7.6 min -1 , or approximately 1 to 6, or approximately 2 to 5, or approximately 3 to 4 min -1 of V max .

[0552] In one embodiment, according to the measurement results of the GPA experiment, the collagenase has approximately 135 min -1 to 268 min -1 of V max . In other embodiments, according to the measurement results of the GPA experiment, the collagenase has approximately 150 min -1 to 250 min -1 of V max . In other embodiments, according to the measurement results of the GPA experiment, the collagenase has approximately 175 min-1 to 225 min -1 of V max 。In still another embodiment, according to the measurement results of the GPA experiment, the collagenase has about 130 min -1 , about 135 min -1 , about 140 min -1 , about 145 min -1 , about 150 min -1 , about 155 min -1 , about 160 min -1 , about 165 min -1 , about 170 min -1 , about 175 min -1 , about 180 min -1 , about 185 min -1 , about 190 min -1 , about 195 min -1 , about 200 min -1 , about 205 min -1 , about 210 min -1 , about 215 min -1 , about 220 min -1 , about 225 min -1 , about 230 min -1 , about 235 min -1 , about 240 min -1 , about 245 min -1 , about 250 min -1 , about 255 min -1 , about 260 min -1 , about 265 min -1 , about 270 min -1 , about 275 min -1 , or about 280 min -1 of V max 。In other embodiments, according to the measurement results of the GPA experiment, the collagenase has about 4 min -1 to 400 min -1 of V max , or according to the measurement results of the GPA experiment, the collagenase has about 0.3 to 30.5, or about 10 to 375, or about 20 to 350, or about 50 to 300, or about 100 to 275 min -1 of V max 。

[0553] In one embodiment, according to the measurement results of the SRC experiment, the K of the collagenase m is about 75 mM to 147 mM. In other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase m is about 80 mM to 140 mM. In other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase m is about 85 mM to 130 mM. In other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase m is about 90 mM to 120 mM. In still other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase m is 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. In other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase m is about 4.4 mM to 437 mM, or according to the measurement results of the SRC experiment, the K of the collagenase m is about 5 to 400, or about 20 to 375 or about 50 to 325, or about 100 to 275, or about 150 to 250 mM, or about 4.1 to 410 sodium moles.

[0554] In one embodiment, according to the measurement results of the GPA experiment, the K of the collagenase m is about 0.03 mM to 3.1 mM. In other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase m is about 1.00 mM to 1.60 mM. In other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase m is about 1.10 mM to 1.50 mM. In other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase mis from about 1.15 mM to 1.40 mM. In still other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase m is 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. In other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase m is from about 0.027 mM to 2.7 mM, or according to the measurement results of the GPA experiment, the K of the collagenase m is from about 0.1 to 2, or from about 0.5 to 1.5 or from about 1 to 1.35 mM.

[0555] In one embodiment, according to the measurement results of the SRC experiment, the K of the collagenase cat is between about 36 sec -1 and 671 sec -1 In other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase cat is between about 50 sec -1 and 600 sec -1 In other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase cat is between about 60 sec -1 and 500 sec -1 In other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase cat is between about 70 sec -1 and 400 sec -1 In still another embodiment, according to the measurement results of the SRC experiment, the K of the collagenase cat is between about 100 sec -1 and 350 sec -1Between. In other embodiments, according to the measurement results of the SRC experiment, the Kcat of the collagenase is about 30 sec -1 , about 40 sec -1 , about 50 sec -1 , about 60 sec -1 , about 70 sec -1 , about 80 sec -1 , about 90 sec -1 , about 100 sec -1 , about 110 sec -1 , about 120 sec -1 , about 130 sec -1 , about 140 sec -1 , about 150 sec -1 , about 160 sec -1 , about 170 sec -1 , about 180 sec -1 , about 190 sec -1 , about 200 sec -1 , about 210 sec -1 , about 220 sec -1 , about 230 sec -1 , about 240 sec -1 , about 250 sec -1 , about 260 sec -1 , about 270 sec -1 , about 280 sec -1 , about 290 sec -1 , about 300 sec -1 , about 310 sec -1 , about 320 sec -1 , about 330 sec -1 , about 340 sec -1 , about 350 sec -1 , about 360 sec -1 , about 370 sec -1 , about 380 sec -1 , about 390 sec -1 , about 400 sec -1 , about 410 sec -1 , about 420 sec -1 , about 430 sec -1 , about 440 sec -1 , about 450 sec -1 , about 460 sec -1, about 470 sec -1 , about 480 sec -1 , about 490 sec -1 , about 500 sec -1 , about 510 sec -1 , about 520 sec -1 , about 530 sec -1 , about 540 sec -1 , about 550 sec -1 , about 560 sec -1 , about 570 sec -1 , about 580 sec -1 , about 590 sec -1 , about 600 sec -1 , about 610 sec -1 , about 620 sec -1 , about 630 sec -1 , about 640 sec -1 , about 650 sec -1 , about 660 sec -1 , about 670 sec -1 , about 680 sec -1 , about 690 sec -1 , about 700 sec -1 , about 710 sec -1 , about 720 sec -1 , about 730 sec -1 , about 740 sec -1 , about 750 sec -1 or about 760 sec -1 。In other embodiments, according to the measurement results of the SRC experiment, the K of the collagenase cat is between about 1 sec -1 and 107 sec -1 , or according to the measurement results of the SRC experiment, between about 10 and 100, or about 20 and 80, or about 30 and 70, or about 40 and 60 sec -1 .

[0556] In one embodiment, according to the measurement results of the GPA experiment, the K of the collagenase cat is between about 90 sec -1 and 10000 sec -1 , or between 41,000 sec -1 and 81,000 sec -1Between. In other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase cat is about 45,000 sec -1 to 75,000 sec -1 Between. In other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase cat is about 50,000 sec -1 to 70,000 sec -1 Between. In other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase cat is about 55,000 sec -1 to 65,000 sec -1 Between. In still another embodiment, according to the measurement results of the GPA experiment, the K of the collagenase cat is about 35,000 sec -1 about 37,500 sec -1 about 40,000 sec -1 about 42,500 sec -1 about 45,000 sec -1 about 47,500 sec -1 about 50,000 sec -1 about 52,500 sec -1 about 55,000 sec -1 about 57,500 sec -1 about 60,000 sec -1 about 62,500 sec -1 about 65,000 sec -1 about 67,500 sec -1 about 70,000 sec -1 about 72,500 sec -1 about 75,000 sec -1 about 77,500 sec -1 about 80,000 sec -1 about 82,500 sec -1 or about 85,000 sec -1 。In other embodiments, according to the measurement results of the GPA experiment, the K of the collagenase cat is about 1215 sec -1 to about 120,000 sec -1between, or based on the measurement results of the GPA experiment, between approximately 2,000 and 100,000, or between approximately 10,000 and 90,000, or between approximately 20,000 and 80,000, or between approximately 30,000 and 70,000, or between approximately 40,000 and 60,000 sec -1 between.

[0557] In one embodiment, based on the measurement results of the SRC experiment, the 1K of the collagenase cat is between approximately 376 and 38,000 μsec, or between approximately 14,000 μsec and approximately 28,000 μsec. In other embodiments, based on the measurement results of the SRC experiment, the 1K of the collagenase cat is between approximately 16,000 μsec and approximately 26,000 μsec. In one embodiment, based on the measurement results of the SRC experiment, the 1 / K of the collagenase cat is between approximately 18,000 μsec and approximately 24,000 μsec. In one embodiment, based on the measurement results of the SRC experiment, the 1 / K of the collagenase cat is between approximately 20,000 μsec and approximately 22,000 μsec. In still another embodiment, based on the measurement results of the SRC experiment, the 1 / K of the collagenase catis 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. In other embodiments, according to the determination result of the SRC experiment, 1 / K of the collagenase cat is between about 370 μsec and about 36,700 μsec, or according to the determination result of the SRC experiment, 1 / K of the collagenase catBetween approximately 750 and 30,000, or between approximately 2,500 and 25,000, or between approximately 5,000 and 20,000, or between approximately 10,000 and 18,000, or approximately 15,000 μsec.

[0558] In one embodiment, according to the measurement results of the GPA experiment, the 1 / K of the collagenase cat Is between approximately 4 and 430 μsec. In other embodiments, according to the measurement results of the GPA experiment, the 1 / K of the collagenase cat Is between approximately 14 μsec and approximately 23 μsec. In another embodiment, according to the measurement results of the GPA experiment, the 1 / K of the collagenase cat Is between approximately 16 μsec and approximately 21 μsec. In still another embodiment, according to the measurement results of the GPA experiment, the 1 / K of the collagenase catis approximately 10.0 μsec, approximately 10.2 μsec, approximately 10.4 μsec, approximately 10.6 μsec, approximately 10.8 μsec, approximately 11.0 μsec, approximately 11.2 μsec, approximately 11.4 μsec, approximately 11.6 μsec, approximately 11.8 μsec, approximately 12.0 μsec, approximately 12.2 μsec, approximately 12.4 μsec, approximately 12.6 μsec, approximately 12.8 μsec, approximately 13.0 μsec, approximately 13.2 μsec, approximately 13.4 μsec, approximately 13.6 μsec, approximately 13.8 μsec, approximately 14.0 μsec, approximately 14.2 μsec, approximately 14.4 μsec, approximately 14.6 μsec, approximately 14.8 μsec, approximately 15.0 μsec, approximately 15.2 μsec, approximately 15.4 μsec, approximately 15.6 μsec, approximately 15.8 μsec, approximately 16.0 μsec, approximately 16.2 μsec, approximately 16.4 μsec, approximately 16.6 μsec, approximately 16.8 μsec, approximately 17.0 μsec, approximately 17.2 μsec, approximately 17.4 μsec, approximately 17.6 μsec, approximately 17.8 μsec, approximately 18.0 μsec, approximately 18.2 μsec, approximately 18.4 μsec, approximately 18.6 μsec, approximately 18.8 μsec, approximately 19.0 μsec, approximately 19.2 μsec, approximately 19.4 μsec, approximately 19.6 μsec, approximately 19.8 μsec, approximately 20.0 μsec, approximately 20.2 μsec, approximately 20.4 μsec, approximately 20.6 μsec, approximately 20.8 μsec, approximately 21.0 μsec, approximately 21.2 μsec, approximately 21.4 μsec, approximately 21.6 μsec, approximately 21.8 μsec, approximately 22.0 μsec, approximately 22.2 μsec, approximately 22.4 μsec, approximately 22.6 μsec, approximately 22.8 μsec, approximately 23.0 μsec, approximately 23.2 μsec, approximately 23.4 μsec, approximately 23.6 μsec, approximately 23.8 μsec, approximately 24.0 μsec, approximately 24.2 μsec, approximately 24.4 μsec, approximately 24.6 μsec, approximately 24.8 μsec, approximately 25.0 μsec, approximately 25.2 μsec, approximately 25.4 μsec, approximately 25.6 μsec, approximately 25.8 μsec, approximately 26.0 μsec, approximately 26.2 μsec, approximately 26.4 μsec, approximately 26.8 μsec, approximately 27.0 μsec, approximately 27.2 μsec or approximately 27.4 μsec. In other embodiments, according to the measurement results of the GPA experiment, the 1 / K of the collagenase catBetween about 0.3 μsec and about 32 μsec, or according to the measurement results of the GPA experiment, the 1 / K of the collagenase cat is between about 1 and 30, or about 5 and 25, or about 10 and 20, or about 15 μsec.

[0559] In one embodiment, according to the measurement results of the SRC experiment, the K of the collagenase cat / K m is between about 5,140 mM -1 sec -1 and about 508,814 mM -1 sec -1 In another embodiment, according to the measurement results of the SRC experiment, the K of the collagenase cat / K m is between about 0.50 mM -1 sec -1 and about 7.75 mM -1 sec -1 In another embodiment, according to the measurement results of the SRC experiment, the K of the collagenase cat / K m is between about 0.75 mM -1 sec -1 and about 7.00 mM -1 sec -1 In yet another embodiment, according to the measurement results of the SRC experiment, the K of the collagenase cat / K m is between about 1.00 mM -1 sec -1 and about 6.00 mM -1 sec -1 In yet another embodiment, according to the measurement results of the SRC experiment, the K of the collagenase cat / K m is about 0.10 mM -1 sec -1 about 0.20 mM -1 sec -1 about 0.30 mM -1 sec -1 about 0.40 mM -1 sec -1 about 0.50 mM -1 sec -1 about 0.60 mM -1 sec -1 about 0.70 mM -1 sec -1, approximately 0.80 mM - 1 sec -1 , approximately 0.90 mM -1 sec -1 , approximately 1.00 mM -1 sec -1 , approximately 1.10 mM -1 sec -1 , approximately 1.20 mM -1 sec -1 , approximately 1.30 mM -1 sec -1 , approximately 1.40 mM -1 sec -1 , approximately 1.50 mM -1 sec -1 , approximately 1.60 mM -1 sec -1 , approximately 1.70 mM - 1 sec -1 , approximately 1.80 mM -1 sec -1 , approximately 1.90 mM -1 sec -1 , approximately 2.00 mM -1 sec -1 , approximately 2.10 mM -1 sec -1 , approximately 2.20 mM -1 sec -1 , approximately 2.30 mM -1 sec -1 , approximately 2.40 mM -1 sec -1 , approximately 2.50 mM -1 sec -1 , approximately 2.60 mM - 1 sec -1 , approximately 2.70 mM -1 sec -1 , approximately 2.80 mM -1 sec -1 , approximately 2.90 mM -1 sec -1 , approximately 3.00 mM -1 sec -1 , approximately 3.10 mM -1 sec -1 , approximately 3.20 mM -1 sec -1 , approximately 3.30 mM-1 sec -1 , approximately 3.40 mM -1 sec -1 , approximately 3.50 mM - 1 sec -1 , approximately 3.60 mM -1 sec -1 , approximately 3.70 mM -1 sec -1 , approximately 3.80 mM -1 sec -1 , approximately 3.90 mM -1 sec -1 , approximately 4.00 mM -1 sec -1 , approximately 4.10 mM -1 sec -1 , approximately 4.20 mM -1 sec -1 , approximately 4.30 mM -1 sec -1 , approximately 4.40 mM - 1 sec -1 , approximately 4.50 mM -1 sec -1 , approximately 4.60 mM -1 sec -1 , approximately 4.70 mM -1 sec -1 , approximately 4.80 mM -1 sec -1 , approximately 4.90 mM -1 sec -1 , approximately 5.00 mM -1 sec -1 , approximately 5.10 mM -1 sec -1 , approximately 5.20 mM -1 sec -1 , approximately 5.30 mM - 1 sec -1 , approximately 5.40 mM -1 sec -1 , approximately 5.50 mM -1 sec -1 , approximately 5.60 mM -1 sec -1 , approximately 5.70 mM -1 sec -1 , approximately 5.80 mM -1sec -1 , approximately 5.90 mM -1 sec -1 , approximately 6.00 mM -1 sec -1 , approximately 6.10 mM -1 sec -1 , approximately 6.20 mM - 1 sec -1 , approximately 6.30 mM -1 sec -1 , approximately 6.40 mM -1 sec -1 , approximately 6.50 mM -1 sec -1 , approximately 6.60 mM -1 sec -1 , approximately 6.70 mM -1 sec -1 , approximately 6.80 mM -1 sec -1 , approximately 6.90 mM -1 sec -1 , approximately 7.00 mM -1 sec -1 , approximately 7.10 mM - 1 sec -1 , approximately 7.20 mM -1 sec -1 , approximately 7.30 mM -1 sec -1 , or approximately 7.40 mM -1 sec -1 . In other embodiments, according to the measurement results of the SRC experiment, the K cat / K m is between approximately 0.0048 mM -1 sec -1 and approximately 0.47 mM -1 sec -1 , or according to the measurement results of the SRC experiment, the K cat / K m is between approximately 0.009 and approximately 0.3, or approximately 0.01 and approximately 0.25, or approximately 0.1 and approximately 0.25 mM -1 sec -1 .

[0560] In one embodiment, according to the measurement results of the GPA experiment, the K cat / K mBetween approximately 60 mM - 1 sec -1 and approximately 6000 mM -1 sec -1 In another embodiment, according to the measurement results of the GPA experiment, the K cat / K m is between approximately 30,000 mM -1 sec -1 and approximately 85,000 mM -1 sec -1 In another embodiment, according to the measurement results of the GPA experiment, the K cat / K m is between approximately 36,000 mM -1 sec -1 and approximately 77,000 mM -1 sec -1 In yet another embodiment, according to the measurement results of the GPA experiment, the K cat / K m is between approximately 40,000 mM -1 sec -1 and approximately 70,000 mM -1 sec -1 In yet another embodiment, according to the measurement results of the GPA experiment, the K cat / K m is approximately 40,000 mM -1 sec -1 , approximately 42,000 mM -1 sec -1 , approximately 44,000 mM -1 sec -1 , approximately 46,000 mM -1 sec -1 , approximately 48,000 mM -1 sec -1 , approximately 50,000 mM -1 sec -1 , approximately 52,000 mM -1 sec -1 , approximately 54,000 mM -1 sec -1 , approximately 56,000 mM -1 sec -1 , approximately 58,000 mM -1 sec -1 , approximately 60,000 mM -1sec -1 , about 62,000 mM -1 sec -1 , about 64,000 mM -1 sec -1 , about 66,000 mM -1 sec -1 , about 68,000 mM -1 sec -1 , about 70,000 mM -1 sec -1 , about 72,000 mM -1 sec -1 , about 74,000 mM -1 sec -1 , about 76,000 mM -1 sec -1 , about 78,000 mM -1 sec -1 , about 80,000 mM -1 sec -1 , about 82,000 mM -1 sec -1 , about 84,000 mM -1 sec -1 , about 86,000 mM -1 sec -1 , about 88,000 mM -1 sec -1 , about 90,000 mM -1 sec -1 , about 92,000 mM -1 sec -1 , about 94,000 mM -1 sec -1 , or about 96,000 mM -1 sec -1 . In other embodiments, according to the measurement results of the GPA experiment, the K cat / K m is between about 900 mM -1 sec -1 and about 90,000 mM -1 sec -1 . Or according to the measurement results of the SRC experiment, the K cat / K m is between about 2,000 and about 80,000, or about 10,000 and about 70,000, or about 20,000 and about 60,000 mM -1 sec-1 , or about 30,000 to 50,000, or about 40,000 to 45,000 mM -1 sec -1 between.

[0561] In one embodiment, the molecular weight of the collagenase is between about 60 kDa and about 130 kDa. In other embodiments, the molecular weight of the collagenase is between about 70 kDa and about 130 kDa. In other embodiments, the molecular weight of the collagenase is between about 80 kDa and about 120 kDa. In still another embodiment, the molecular weight of the collagenase is between about 90 kDa and about 120 kDa. In other embodiments, the molecular weight of the collagenase is between about 100 kDa and about 110 kDa. In still another embodiment, the molecular weight of the collagenase is about 55 kDa, about 57 kDa, about 60 kDa, about 62 kDa, about 65 kDa, about 67 kDa, about 70 kDa, about 72 kDa, about 75 kDa, about 77 kDa, about 80 kDa, about 82 kDa, about 85 kDa, about 87 kDa, about 90 kDa, about 92 kDa, about 95 kDa, about 97 kDa, 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.

[0562] In one embodiment, the purity of the collagenase determined by reverse HPLC is at least 80%. In other embodiments, the purity of the collagenase determined by reverse HPLC is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In still another embodiment, the collagenase may comprise less than or equal to 1% clostripain by area. In other embodiments, the collagenase further comprises less than or equal to 1% gelatinase by area. In other embodiments, the collagenase may further comprise less than or equal to 1% leupeptin by area. In still another embodiment, the collagenase comprises less than or equal to 1 cfu / mL bioburden.

[0563] In one embodiment, the titer (i.e., specific activity) of the collagenase is between about 500 and about 30,000 SRC units / mg. In other embodiments, the titer of the collagenase is between about 2,500 and about 25,000 SRC units / mg. In other embodiments, the titer of the collagenase is between about 5,000 and about 20,000 SRC units / mg. In yet another embodiment, the titer of the collagenase is about 500, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, about 25,000, about 25,500, about 26,000, about 26,500, about 27,000, about 27,500, about 28,000, about 28,500, about 29,000, about 29,500 or about 30,000 SRC units / mg.

[0564] In one embodiment, the titer (i.e., specific activity) of the collagenase is between about 5,000 and about 30,000 f-SRC units / mg. In other embodiments, the titer of the collagenase is between about 7,500 and about 25,000 f-SRC units / mg. In other embodiments, the titer of the collagenase is between about 10,000 and about 20,000 f-SRC units / mg. In yet another embodiment, the titer of the collagenase is about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, 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.

[0565] In one embodiment, the titer (i.e., specific activity) of the collagenase is between about 100,000 and about 400,000 GPA units / mg. In other embodiments, the titer of the collagenase is between about 150,000 and about 350,000 GPA units / mg. In other embodiments, the titer of the collagenase is between about 200,000 and about 300,000 GPA units / mg. In yet another embodiment, the titer of the collagenase is about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about 300,000, about 310,000, about 320,000, about 330,000, about 340,000, about 350,000, about 360,000, about 370,000, about 380,000, about 390,000 or about 400,000 GPA units / mg.

[0566] In one embodiment, the potency of the collagenase is between about 175,000 and about 500,000 f-GPA units / mg. In other embodiments, the potency of the collagenase is between about 250,000 and about 450,000 f-GPA units / mg. In other embodiments, the potency of the collagenase is between about 300,000 and about 400,000 GPA units / mg. In still another embodiment, the potency of the collagenase is about 175,000, about 185,000, about 195,000, about 205,000, about 215,000, about 225,000, about 235,000, about 245,000, about 255,000, about 265,000, about 275,000, about 285,000, about 295,000, about 305,000, about 315,000, about 325,000, about 335,000, about 345,000, about 355,000, about 365,000, about 375,000, about 385,000, about 395,000, about 405,000, about 415,000, about 425,000, about 435,000, about 445,000, about 455,000, about 465,000, about 475,000, about 485,000, or about 495,000 f-GPA units / mg.

[0567] In one embodiment, the potency of the collagenase is between about 5,000 and about 25,000 ABC units / mg. In one embodiment, the potency of the collagenase is between about 7,500 and about 20,000 ABC units / mg. In one embodiment, the potency of the collagenase is between about 10,000 and about 17,500 ABC units / mg. In other embodiments, the potency of the collagenase is about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, or about 25,000 ABC units / mg.

[0568] In some embodiments, the collagenase present in the composition includes collagenase I and collagenase II, and the ratio of collagenase I to collagenase II is about 1:1. Other ratios of collagenase I to collagenase II can also be used, for example, 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1. The purity of each of collagenase I and collagenase II is at least 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% by area, as measured by reverse HPLC.

[0569] In other embodiments, the collagenase composition comprises CCH, and the CCH comprises AUX I and AUX II in a ratio of approximately 1:1. Other ratios of AUX I and AUX II may also be used, such as 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1. The purity of each of AUX I and AUX II is at least 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% by area, as measured using reverse HPLC.

[0570] In other embodiments, the collagenase composition may be liquid or may be reconstituted from a lyophilized form with a diluent. The dose of the mixture is determined only by the amount of collagenase present, not the diluent, and may include from about 0.1 mg to about 20 mg in one or more injections. In other embodiments, the dose administered is about 0.06 mg, 0.48 mg, 0.84 mg, 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg, 5.04 mg, 5.88 mg, 6.72 mg, 7.56 mg, or 8.4 mg in a single injection or multiple injections.

[0571] For example, about 0.06 mg, 0.48 mg, 0.84 mg, or 1.68 mg is administered in about 12 injections. The volume of the collagenase composition injected can range from 0.01 mL to 3 mL per injection, or about 0.2 mL to 150 mL for the total treatment visit. In a specific embodiment, the above dose is the dose of the collagenase composition comprising CCH. In other embodiments, the above dose is the dose of the collagenase composition having one or more of the following characteristics:

[0572] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[0573] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[0574] ·K cat (sec -1) is from about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[0575] · 1 / K cat , the microseconds are from about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[0576] · K cat / K M , mM -1 sec -1 is 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[0577] · The molecular weight is from 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.

[0578] · Measured by reverse HPLC (high performance liquid chromatography), the purity is at least 80% by area

[0579] · The potency (i.e., specific activity) is from about 500 to about 30,000 SRC units / mg

[0580] · The potency is from about 5,000 to about 30,000 f-SRC units / mg

[0581] · The potency is from about 100,000 to about 400,000 GPA units / mg

[0582] · The potency is from about 175,000 to about 500,00 f-GPA units / mg

[0583] · The potency is from about 5,000 to about 25,000 ABC units / mg

[0584] · By area, the content of impurities is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[0585] · A bioburden of less than or equal to 1 cfu / mL.

[0586] In other embodiments, in each treatment area, approximately 0.84 mg of CCH is divided among approximately 12 equal aliquots of injections (approximately 0.07 mg x 12 injections = approximately 0.84 mg). In some cases, the above treatment of 0.84 mg is performed every 10 - 40 days and can be divided into 2, 3, 4, or 5 clinic visits. In other cases, 0.84 mg is injected into more than one treatment area during one clinic visit or every 10 - 40 days during 2, 3, 4, or 5 clinic visits. In other embodiments, there are more than five treatment visits (clinic visits).

[0587] In another aspect, in one or more injections, the amount of collagenase that can be injected into the treatment area at each treatment visit is about 0.001 mg to 20 mg, which can be in one injection or multiple injections. For example, these doses are evenly divided into 3 to about 100 injections. These collagenases are in liquid form or reconstituted from lyophilized solids with a diluent. The dose of collagenase is measured by the amount of collagenase, regardless of the diluent, and can include about 0.1 mg to 1 mg, or 0.25 mg to 0.75 mg, or 0.1 mg to 2 mg, or 0.25 mg to 1.75 mg, or 0.5 mg to 1 mg, 0.1 mg to 3 mg, or 0.25 mg to 2.75 mg, or 0.5 mg to 2.5 mg, or 0.75 mg to 2.25 mg, or 1 mg to 2 mg, or 0.1 mg to 4 mg, or 0.25 mg to 3.75 mg, or 0.5 mg to 3.5 mg, or 0.75 mg to 3 mg, or 1 mg to 3 mg. In other embodiments, the dose is about 0.001 mg, 0.01 mg, 0.04 mg, 0.05 mg, 0.07 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 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 per injection or multiple injections.

[0588] In other embodiments, the doses administered in one or more injections are about 0.06 mg, 0.48 mg, 0.84 mg, 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg or 5.04 mg. In another embodiment, 0.06 mg, 0.48 mg, 0.84 mg, 1.68 mg, 2.52 mg, 3.36 mg, 4.2 mg or 5.04 mg are administered in 12 injections to the treatment area. In other embodiments, the dose of collagenase is divided into 3 or more injections. The volume of the collagenase composition for each injection can be between 0.01 mL and 3 mL, or the total volume for each treatment visit is about 1 mL to 150 mL.

[0589] In one aspect, the above AUX I and II mixtures (“CCH”) can be injected in one or more injections in an amount of about 0.01 mg to 10 mg of collagenase per treatment visit. For example, the dose is evenly distributed in about 3 to about 50 injections. The collagenase can be in liquid form or can be reconstituted from a lyophilized form with a diluent. The dose of the mixture is measured by the amount of collagenase, regardless of the diluent, and can include about 0.1 mg to 1 mg, or 0.25 mg to 0.75 mg, or 0.1 mg to 2 mg, or 0.25 mg to 1.75 mg, or 0.5 mg to 1 mg, 0.1 mg to 3 mg, or 0.25 mg to 2.75 mg, or 0.5 mg to 2.5 mg, or 0.75 mg to 2.25 mg, or 1 mg to 2 mg, or 0.1 mg to 4 mg, or 0.25 mg to 3.75 mg, or 0.5 mg to 3.5 mg, or 0.75 mg to 3 mg, or 1 mg to 3 mg, or about 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 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 in one or more injections. In other embodiments, the dose of CCH administered contains 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 per injection or multiple 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 distributed into 12 injections for administration. The volume of the injected collagenase composition can range from 0.01 ml to 3 ml per injection, or a total of about 1 ml to 80 ml is administered per medical visit.

[0590] The dose of collagenase can also be expressed in mg per injection (again, not considering the diluent), for example, from about 0.001 mg to 0.5 mg per injection, from about 0.01 mg to about 5 mg per injection, or from about 0.005 mg to about 0.1 mg per injection, or about 0.005 mg, 0.04 mg or 0.07 mg per injection.

[0591] In some aspects, the present invention contemplates injecting from about 500 ABC units to about 50,000 ABC units per treatment visit, or from about 10,000 ABC units to about 25,000 ABC units per treatment visit. In other embodiments, the dose of collagenase per injection is about 50 ABC units to about 2,500 ABC units, or about 85 ABC units to about 2,000 ABC units, or about 1,500 ABC units to about 1,750 ABC units, or about 200 ABC units to about 1,500 ABC units, or about 300 ABC units to 1,500 ABC units, or about 300 ABC units to about 1,250 ABC units, or about 500 ABC units to about 1,000 ABC units.

[0592] In certain embodiments, the dose is based on the following specific activities:

[0593]

[0594] * Calculate the milligrams by multiplying the specific activity in SRC units and SRC units / mg by the reciprocal of the specific activity in SRC units and SRC units / mg. For example, when the dose is 500 SRC units and the specific activity is 500 SRC units / mg, the milligram amount equivalent to a 500 SRC unit dose is (500 SRC units) * (1 / (500 SRC units / mg)) = 1.00 mg

[0595] The milligrams of SRC units and ABC units / mg specific activity are calculated as follows: Multiply the SRC units by 6.3 ABC units / SRC unit, and then multiply by the reciprocal of the ABC units / mg specific activity. For example, when the dose is 500 SRC units and the specific activity is 10,000 ABC units / 0.58 mg, the milligram amount equivalent to 500 SRC units is (500 SRC units) * (6.3 ABC units / SRC unit) * (1 / (10,000 ABC units / 0.58 mg)) = 0.18 mg.

[0596] In some aspects, the present invention contemplates an amount of collagenase to be injected of from about 5,000 BTC units to about 25,000 BTC units, or from about 10,000 BTC units to about 25,000 BTC units, or about 15,000 BTC units, or about 17,500 BTC units, or about 20,000 BTC units, or about 22,500 BTC units, or about 9,175 BTC units, about 15,817 BTC units.

[0597] 5. Preparation

[0598] The CCH or other collagenase may be present in the form of a pharmaceutical composition, which includes CCH or collagenase and a pharmaceutically acceptable excipient. These excipients may include sterile water for injection or sodium chloride or calcium chloride, a pH adjuster, and a stabilizer.

[0599] A non-limiting example is commercially available from the applicant in the form of a disposable glass vial containing 0.9 mg CCH as a sterile lyophilized powder for reconstitution. A sterile diluent for reconstitution is also provided in the disposable glass vial. The inactive ingredients include hydrochloric acid, sucrose, and tromethamine. The diluent contains calcium chloride dihydrate in 0.9% sodium chloride. Prescribing Information (2018).

[0600] In other embodiments, the CCH is present in the form of a sterile lyophilized powder, including 0.92 mg CCH, sucrose, Tris, mannitol, and hydrochloric acid, in a 5 ml vial. The sterile diluent for reconstitution may include water for injection, normal saline, or 0.6% sodium chloride and calcium chloride dihydrate at a concentration of 0.03% in water for injection, filled into a separate 5 ml vial.

[0601] The collagenase or CCH may also be filled into vials of other sizes, for example, 10 ml, 15 ml, 20 ml, or 30 ml. Other pH adjusters, sugars, polyols, and stabilizers may be found in Rowe et a1., Handbook of Pharmaceutical Excipients (5 th Ed.).

[0602] 6. Treatment methods: injection techniques and dosage regimens

[0603] The above-mentioned collagenase composition can be effectively used in a method for treating or reducing the severity of cellulite in the human body. The present invention relates to a method for reducing the severity of cellulite in a human patient, the method comprising: providing a composition comprising at least one collagenase; and injecting a therapeutically effective amount of the composition into one or more dimples, wherein the patient exhibits a reduction in the severity of cellulite compared to the baseline severity before treatment. As further detailed below, the composition can be administered by various injection techniques and the efficacy can be measured by several scales and other measurement tools.

[0604] The applicant has previously described related treatment methods and hereby incorporates by reference in their entirety their patent applications herein, such as PCT patent application PCT / US2018 / 020551 (published on September 7, 2018, with publication number WO2018 / 160905); US Provisional Application No. 62 / 697376, filed on July 12, 2018, titled "Injection Techniques for Treating Edematous Fibrosclerotic Polyneuropathy"; US Provisional Application No. 62 / 733046, filed on September 18, 2018, titled "Composition and Method for Quadrant Single-Dose Collagenase Treatment of Edematous Fibrosclerotic Polyneuropathy"; and US Patent Application Publication No. US20180327731, filed on March 28, 2018, titled "Method for Producing Collagenase".

[0605] The administration of the collagenase composition described herein can be bilateral (two thighs or two buttocks) or to all 4 quadrants (two buttocks and two thighs) of a single subject during a treatment visit. Such treatment visits may be conducted every 10 - 40 days, for 2, 3, 4, or 5 treatment visits over the course of a year.

[0606] Table 15 outlines various injection parameters of collagenase and related techniques for treating patients.

[0607] Table 15. Various injection parameters of collagenase and related techniques for treating patients

[0608]

[0609]

[0610]

[0611] Table 16 outlines five non-limiting examples of injection techniques and treatments contemplated by the present invention, which correspond to Figures 7-11 the injection techniques shown.

[0612]

[0613] The following provides a detailed introduction to Treatments I to V.

[0614] Treatment I: Superficial injection of collagenase, in three equal parts

[0615] As Figure 7 shown, in this embodiment ("Treatment I"), when the subject is in the prone position, collagenase is subcutaneously injected perpendicular to the long axis of the pit. Each injection consists of a single skin injection of collagenase, divided into three 0.1 mL equal parts (total injection volume of 0.3 mL) for injection. During each treatment visit, 8 syringes (4 syringes for each treatment area) are prepared for administration. Each syringe contains 0.9 mL of collagenase composition (injected 3 times per syringe). The dose for each subject may vary, and the total dose for each treatment area is approximately 0.5 mg to about 5 mg of collagenase.

[0616] More specifically, the following steps are carried out:

[0617] · If the pit is an elongated trough-shaped pit (position a), the needle is perpendicular to the skin surface and perpendicular to the long axis of the pit, then the needle is pushed all the way in (1 / 2 inch), and 0.1 mL of collagenase composition is injected by gently pushing the syringe plunger. In most cases, the plane containing injection points A, B, and C will be parallel to the long axis of the subject's body.

[0618] · Gently withdraw the needle and reposition it so that it forms an angle of approximately 45° with the skin surface, towards the subject's head (position B), push the needle all the way in, and inject 0.1 mL of collagenase composition by gently pushing the syringe plunger. Position B is preferably towards the subject's head.

[0619] · Gently withdraw the needle and reposition it so that it forms an angle of approximately 45° with the skin surface, towards the subject's feet (position C), push the needle all the way in, and inject 0.1 mL of collagenase composition by gently pushing the syringe plunger. Position B is preferably towards the subject's feet.

[0620] · Withdraw the needle completely from the injection site and move to the next injection site.

[0621] · A syringe containing 0.9 mL of collagenase composition is sufficient for three injection site injections.

[0622] · Four (4) 0.9 mL syringes are used at each treatment site (each buttock or each thigh), and a total of 12 injections of 0.3 mL (3 equal parts, each 0.1 mL) are made at 12 injection sites.

[0623] · During each treatment visit, a total of 24 injections are made in two treatment areas (2 buttocks or 2 thighs).

[0624] Treatment II: superficial injection of collagenase, 1 portion

[0625] As Figure 8 shown, in this example (“Treatment II”), when the subject is in the prone position, subcutaneous injection with collagenase is performed. Each injection consists of a single skin injection of collagenase, i.e., a single superficial injection of 0.3 mL aliquots. During each treatment visit, 8 syringes (4 syringes for each treatment area) are prepared for administration. Each syringe contains 0.9 mL of collagenase (injected 3 times per syringe). The dose for each subject is approximately 0.5 mg to 5 mg of total collagenase dose in each treatment area.

[0626] During each treatment visit, 8 syringes (4 syringes for each treatment area) are prepared for administration. Each syringe contains 0.9 mL of collagenase composition (injected 3 times per syringe). More specifically, the following procedure is followed:

[0627] · Position the needle at an angle of approximately 30° to the skin surface at the injection site, towards the subject's head, push the needle all the way in (1 / 2 inch), and inject 0.3 mL of collagenase composition by gently pushing the syringe plunger.

[0628] · Withdraw the needle completely from the injection site and move to the next injection site.

[0629] · A syringe containing 0.9 mL of collagenase composition is sufficient for three injection site injections.

[0630] · Four (4) 0.9 mL syringes are used at each treatment site (each buttock or each thigh), and a total of 12 injections of 0.3 mL (3 aliquots, 0.1 mL each) are made at 12 injection sites.

[0631] · At each treatment visit, a total of 24 injections are made in two treatment areas (2 buttocks or 2 thighs).

[0632] Treatment III: deep injection of collagenase, 1 portion

[0633] As Figure 9 shown, in this example (“Treatment III”), when the subject is in the prone position, subcutaneous injection with collagenase is performed. Each injection consists of a single skin injection of collagenase, i.e., a single deep injection of 0.3 mL aliquots. During each treatment visit, 8 syringes (4 syringes for each treatment area) are prepared for administration. Each syringe contains 0.9 mL of collagenase (injected 3 times per syringe). The dose for each subject is approximately 0.5 mg to 5 mg of total collagenase dose in each treatment area.

[0634] More specifically, the following procedure is followed:

[0635] ·Position the needle at an angle of approximately 30° to the skin surface at the injection site, towards the subject's head, push the needle all the way in (1 inch), and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0636] ·Position the needle at an angle of approximately 30° to the skin surface at the injection site, towards the subject's head, push the needle all the way in (1 inch), and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0637] ·A syringe containing 0.9 ml of the collagenase composition is sufficient for three injection site injections.

[0638] ·Use four (4) 0.9 ml syringes at each treatment site (each buttock or each thigh) and inject a total of 12 times 0.3 ml (3 equal portions, 0.1 ml each) at 12 injection sites.

[0639] ·Discard the used needles and syringes.

[0640] ·At each treatment visit, a total of 24 injections are performed in two treatment areas (2 buttocks or 2 thighs).

[0641] Treatment IV: Deep and shallow injections of collagenase, 5 equal portions

[0642] As Figure 10 shown, in Treatment IV, when the subject is in the prone position, subcutaneous injections are performed using collagenase. Each injection consists of a single skin injection of collagenase, with five 0.3 ml equal portions injected each time (total injection volume is 1.5 mL). During each treatment visit, 24 syringes are prepared (12 syringes for each treatment area) for administration. Each syringe contains 1.5 ml of collagenase (0.3 mL is injected each time per syringe, for 5 injections). The dose for each subject in each treatment area is in the range of approximately 0.5 mg to 5 mg of total collagenase dose.

[0643] During each treatment visit, 24 syringes are prepared (12 syringes for each treatment area) for administration. Each syringe contains 1.5 ml of collagenase (0.3 mL is injected each time per syringe, for 5 injections). More specifically, follow the following procedure:

[0644] ·Position the needle at an angle of approximately 30° to the skin surface at the injection site, towards the subject's head, push the needle all the way in (1 inch), and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0645] · Gently withdraw the needle (1 / 2 inch) and maintain an angle of approximately 30° with the skin surface. Reposition it towards the subject's head (Position B). Then, inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0646] · Maintain the needle at an angle of approximately 30° with the skin surface. Reposition the needle at the midpoint between Position A and Position B (Position C, towards the subject's shoulder) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0647] · Maintain the needle at an angle of approximately 30° with the skin surface. Reposition the needle precisely opposite Position B (Position D) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0648] · Maintain the needle at an angle of approximately 30° with the skin surface. Reposition the needle at the midpoint between Position A and Position D (Position E, towards the subject's shoulder) and inject 0.3 mL of the collagenase composition by gently pushing the syringe plunger.

[0649] · A 3 - ml syringe containing 1.5 mL of the collagenase composition is sufficient for injection at one injection site.

[0650] · At each treatment site (each buttock or each thigh), use twelve (12) 3 - ml syringes and inject a total of 12 times with 1.5 ml (5 equal portions,

[0651] each portion being 0.3 ml, for a total dose of 18 mL).

[0652] · At each treatment visit, in two treatment areas (2 buttocks or 2 thighs)

[0653] a total of 24 injections are made.

[0654] Treatment V: Superficial injection of collagenase, 4 equal portions

[0655] As Figure 11 shown, in this example (“Treatment V”), when the subject is in the prone position, subcutaneous injection with collagenase is performed. Each injection consists of a single skin injection of collagenase, i.e., four deep injections of 0.3 - ml equal portions (total injection volume of 1.2 mL). During each treatment visit, 24 syringes are prepared (12 syringes for each treatment area) for administration. Each syringe contains 1.2 mL of collagenase (4 equal portions of 0.3 mL each). The dose for each subject is approximately 0.5 mg to approximately 5 mg of the total collagenase dose in each treatment area.

[0656] During each treatment visit, prepare 24 syringes (12 syringes for each treatment area) for administration. Each syringe contains 1.2 mL of collagenase composition (0.3 mL per of 4 equal parts). More specifically, follow the following procedure:

[0657] · Position the needle at an angle of approximately 30° to the skin surface at the injection site, towards the subject's side, push the needle all the way in (1 / 2 inch), and inject 0.3 mL of collagenase composition by gently pushing the syringe plunger.

[0658] · Maintain the needle at an angle of approximately 30° to the skin surface, reposition the needle at position A to an angle of approximately 60° with respect to position A and towards the patient's shoulder (position B), and inject 0.3 mL of collagenase composition by gently pushing the syringe plunger.

[0659] · Maintain the needle at an angle of approximately 30° to the skin surface, reposition the needle at an angle of approximately 60° with respect to position B and towards the patient's other shoulder (position C), and inject 0.3 mL of collagenase composition by gently pushing the syringe plunger.

[0660] · Maintain the needle at an angle of approximately 30° to the skin surface, reposition the needle at position A and towards the other side of the subject (position D), and inject 0.3 mL of collagenase composition by gently pushing the syringe plunger.

[0661] · A 3 mL syringe containing 1.2 mL of collagenase composition is sufficient for injection at one injection site.

[0662] · Use twelve (12) 3 mL syringes (each containing 1.2 mL of collagenase composition) at each treatment site (each buttock or each thigh), and inject a total of 12 times 1.2 mL (4 equal parts, 0.3 mL each, total dose 14.4 mL) at 12 injection sites.

[0663] · A total of 24 injections are performed in two treatment areas (2 buttocks or 2 thighs).

[0664] In a further embodiment, the collagenase is injected into the affected area as Figure 12 described. The injection interval varies within a range of approximately 0.1 cm to approximately 15 cm, or approximately 1 cm to approximately 10 cm, or approximately 0.5 cm to approximately 2 cm. As Figure 12 shown, each injection is divided into three 0.1 mL equal parts (0.3 mL per injection) and used. The first equal part is administered with the needle perpendicular to the skin surface. For the second and third equal parts, the needle is slightly withdrawn and positioned at an angle of approximately 45° to the left and approximately 45° to the right of the vertical axis.

[0665] AsFigures 7-11 The treatments I to V shown and Figure 12 the injection techniques shown can use different doses, injection angles, volumes, number of syringes, injection depths, and other parameters, as detailed in Tables 15 and 16. All such variations are included within the scope of the present invention.

[0666] In another embodiment, approximately 0.84 mg of CCH is divided into 12 equal - dose injections and injected into the affected area, such as a quadrant (i.e., right buttock or left buttock or right thigh or left thigh) (approximately 0.07 mg x 12 injections = approximately 0.84 mg CCH). In some cases, a 0.84 - milligram treatment is administered every 10 - 40 days, for 2, 3, 4, or 5 treatments. In other cases, 0.84 mg is injected into more than one affected area or quadrant every 10 - 40 days, for 2, 3, 4, or 5 treatments.

[0667] In certain embodiments, as shown in Table 17, the patient is administered collagenase.

[0668] Table 17. Collagenase dose and volume

[0669]

[0670] a 0.3 mL of collagenase is injected each time, divided into three equal parts and 0.1 mL of each part is administered.

[0671] Further, in certain embodiments, the parameters for treating the subject are provided in Tables 18 and 19.

[0672] Table 18. CCH lyophilized preparation parameters

[0673]

[0674] Table 19. CCH dilution parameters

[0675]

[0676] In one embodiment, the osmotic pressure of the recombinant product is about 50 to about 1,000, about 100 to about 900, about 200 to about 800, about 300 to about 700, about 400 to about 600, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1,000 mOsm / kg. In yet another embodiment, the osmotic pressure of the recombinant product is about 512 mOsm / kg, about 275 mOsm / kg, about 281 mOsm / kg, or about 227 mOsm / kg.

[0677] In addition to the above methods, the present invention also provides a method for treating or reducing EFP in a subject in need, wherein the method has at least one of the following advantages compared to the conventional procedures and treatments for EFP:

[0678] a. Easy to administer by a doctor;

[0679] b. Shorter treatment time;

[0680] c. Unexpected efficacy, considering that doctors generally believe that improvement of aesthetic conditions is difficult to achieve;

[0681] d. Does not require administration of hyaluronidase;

[0682] e. Does not require heating;

[0683] f. Does not require administration of laser;

[0684] g. No dissection;

[0685] h. Does not require anesthesia (although there may be bruising);

[0686] i. Does not require wearing a compression garment; and

[0687] j. Does not use vacuum.

[0688] In other embodiments, the method for treating or reducing subcutaneous cellulite does not require the severity of the subcutaneous cellulite to be treated, i.e., for example, collagenase treatment is safe and effective regardless of the prevalence or severity of cellulite.

[0689] F. Phase 4 - End of treatment and determination of efficacy

[0690] The treatment methods described herein effectively treat subcutaneous cellulite through various measures described below. As used herein, "day" refers to the study day; thus, for example, day 22 is 21 days after the first injection, and so on. Additionally, in one embodiment, "day" is + / - 7 days.

[0691] 1. Efficacy of CR-PCSS and PR-PCSS assays

[0692] Improvement in each patient at any visit refers to an increase of at least 1 level or grade compared to baseline or any previous score or rating. The average score or rating of a group of patients at any one visit is increased by approximately 0.1 compared to the baseline examination or any previous average score or rating. A responder is a patient who has an increase of at least 25% in the maximum total score or rating from the baseline examination results. In certain embodiments, as determined by measurement using CR-PCSS and / or PR-PCSS, the treatment methods described in detail above produce one or more of the following efficacy endpoints:

[0693] 1. The score in the CR-PCSS and / or PR-PCSS rating is increased by at least 0.1 compared to baseline.

[0694] 2. As clinically evaluated by a clinician of the target thigh, there is at least a 2-grade improvement in the severity of CR-PCSS at day 22, 43, 71, 90, 180, 365, or 730 days starting from baseline (the "first day" before treatment).

[0695] 3. As evaluated by the subject observing digital images of the target thigh, there is at least a 2-grade improvement in the severity of PR-PCSS at day 22, 43, 71, 90, 180, 365, or 730 days starting from baseline (the first day).

[0696] 4. Improvement demonstrated by a 2-grade composite response at day 22, 43, 71, 90, 180, 365, or 730 days is defined as the subject having at least a 2-grade improvement in the severity of CR-PCSS compared to baseline and at least a 2-grade improvement in the severity of PR-PCSS compared to baseline.

[0697] 5. As clinically evaluated by a clinician of the target thigh, there is at least a 1-grade improvement in the severity of subcutaneous cellulite in CR-PCSS compared to baseline at day 22, 43, 71, 90, 180, 365, or 730 days.

[0698] 6. As evaluated by the subject observing digital images of the target thigh, there is at least a 1-grade improvement in the severity of subcutaneous cellulite in PR-PCSS compared to baseline at day 22, 43, 71, 90, 180, 365, or 730 days.

[0699] 7. At day 22, day 43, day 71, day 90, day 180, day 365, or day 730, improvement demonstrated by a Grade 1 composite response is defined as at least a 1-grade improvement in severity in the CR-PCSS compared to baseline and at least a 1-grade improvement in severity in the PR-PCSS compared to baseline for the subject.

[0700] 8. In the patient population where all CR-PCSS scores are moderate or severe, improvement in at least one treatment area is statistically significant compared to placebo, where the improvement is one or more of those in 1 to 7 above.

[0701] 9. The treatment maintained improvement levels compared to pre-treatment baseline in at least 5% of patients within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the initial dose. In other cases, the treatment showed improvement in at least 5% of patients compared to pre-treatment baseline and showed more improvement over time. Some treatments led to improvement in at least 10%, or 20%, or 30%, or 40% or 50% of patients at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with more improvement.

[0702] 10. The improvement relative to baseline observed in the CR-PCSS at day 180 was consistent in the left and right thighs.

[0703] 11. In the patient population where all CR-PCSS scores are moderate or severe, the median time to the earliest Grade 2 CR-PCSS and / or PR-PCSS improvement in at least one treatment area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0704] 12. In the patient population where all CR-PCSS scores are moderate or severe, the median time to the earliest Grade 1 CR-PCSS and / or PR-PCSS improvement in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0705] 13. In the patient population where all CR-PCSS scores are moderate or severe, the mean scores of the subjects' CR-PCSS and / or PR-PCSS separated from placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[0706] 14. Among the patient population with all CR-PCSS scores being moderate or severe, the percentage of subjects having a grade 2 composite response measured by CR-PCSS and / or PR-PCSS in at least one treatment area on day 71 is about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0707] 15. Among the patient population with all CR-PCSS scores being moderate or severe, the percentage of subjects achieving a grade 1 composite response measured by CR-PCSS and / or PR-PCSS in at least one treatment area on day 71 is about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0708] 16. Among the patient population with all CR-PCSS scores being moderate or severe, more than one-third, one-half, two-thirds, or three-fourths of the patients have a CR-PCSS and / or PR-PCSS response of at least grade 1 in at least one treatment area on day 71 after treatment, where the CR-PCSS results are independent of age, BMI, or skin color.

[0709] 17. The severity of subcutaneous cellulite rapidly decreases within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment.

[0710] In other embodiments, about 1 mg to about 20 mg of collagenase is injected into at least one treatment area during at least one treatment visit to produce one or more of the results in items 1 to 17 above, where the collagenase has one or more of the following characteristics:

[0711] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[0712] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[0713] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[0714] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[0715] ·K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[0716] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[0717] · The purity determined by reverse HPLC (high pressure liquid chromatography) is at least 80% by area

[0718] · The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[0719] · The potency is about 5,000 to about 30,000 f-SRC units / mg

[0720] · The potency is about 100,000 to about 400,000 GPA units / mg

[0721] · The potency is about 175,000 to about 500,00 f-GPA units / mg

[0722] · The potency is about 5,000 to about 25,000 ABC units / mg

[0723] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[0724] · The bioburden is less than or equal to 1 cfu / mL

[0725] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17

[0726] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17, wherein the collagenase has one or more of the following characteristics

[0727] · V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[0728] ·K M is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[0729] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0730] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment)

[0731] ·K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or about 60 to 5,934 (GPA experiment)

[0732] ·The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[0733] ·The purity determined by reverse HPLC (high pressure liquid chromatography) is at least 80% by area

[0734] ·The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[0735] ·The potency is about 5,000 to about 30,000 f-SRC units / mg

[0736] ·The potency is about 100,000 to about 400,000 GPA units / mg

[0737] ·The potency is about 175,000 to about 500,00 f-GPA units / mg

[0738] ·The potency is about 5,000 to about 25,000 ABC units / mg

[0739] ·The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[0740] ·A bioburden of less than or equal to 1 cfu / mL

[0741] In another embodiment, injecting from about 1 mg to about 20 mg of collagenase, according to any one of Treatments I through V, results in one or more of the above Results 1 through 17.

[0742] In certain embodiments, at least one treatment visit, from about 1 mg to about 20 mg of a collagenase I and collagenase II in a ratio of about 1:1 is injected into at least one treatment area and results in one or more of the above Results 1 through 17, wherein the collagenase I and collagenase II have the following characteristics:

[0743] · Type I

[0744] ο Experiment: SRC microplate

[0745] ο Vmax, min -1 : from about 0.08 to 7.70

[0746] ο K M : from about 4.1 to 410 nanomoles

[0747] ο K cat , sec -1 : from about 1.1 to 107

[0748] ο 1 / K cat , microseconds: from about 376 to 37,222

[0749] ο K cat / K M , mM -1 sec -1 : from about 5,140 to 508,814

[0750] · Type II

[0751] ο Experiment: GPA microplate

[0752] ο V max , min -1 : from about 0.3 to 30.5

[0753] ο K M , mM: from about 0.03 to 3.1

[0754] ο K cat , sec -1 : from about 93 to 9,179

[0755] ο 1 / K cat , microseconds: from about 4 to 428

[0756] ο K cat / K M , mM -1 sec-1 : From about 60 to 5,934

[0757] Other ratios may also be used (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be designated as AUX - I and AUX - II, respectively.

[0758] In certain embodiments, at least once during a treatment visit, from about 1 mg to about 20 mg of collagenase I and collagenase II in a ratio of about 1:1 are injected into at least one treatment area and one or more of the above results 1 to 17 are obtained, wherein the collagenase I and collagenase II have the following characteristics:

[0759] · Type I

[0760] ο Experiment: SRC microplate

[0761] ο Vmax, min -1 : From about 0.08 to 7.70

[0762] ο K M : From about 4.1 to 410 nanomoles

[0763] ο K cat , sec -1 : From about 1.1 to 107

[0764] ο 1 / K cat , microseconds: From about 376 to 37,222

[0765] ο K cat / K M , mM -1 sec -1 : From about 5,140 to 508,814

[0766] · Type II

[0767] ο Experiment: GPA microplate

[0768] ο V max , min -1 : From about 0.3 to 30.5

[0769] ο K M , mM: From about 0.03 to 3.1

[0770] ο K cat , sec -1 : From about 93 to 9,179

[0771] ο1 / K cat , microseconds: approximately 4 to 428

[0772] οK cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[0773] Other ratios may also be used (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be designated as AUX - I and AUX - II, respectively.

[0774] In other embodiments, in response to the above treatment, a subject is a grade 2 CR - PCSS responder, which means that at the evaluation time point, the subject shows a CR - PCSS rating that is at least 2 grades higher (change of - 2, - 3, or - 4) than the baseline. A grade 1 CR - PCSS responder means that at the evaluation time point, the subject's CR - PCSS score is at least 1 grade higher (change of - 1, - 2, - 3, or - 4) than the baseline. A subject is a grade 2 PR - PCSS responder, which means that at the evaluation time point, the subject's PR - PCSS score is at least 2 grades higher (change of - 2, - 3, or - 4) than the baseline. A grade 1 PR - PCSS responder means that at the evaluation time point, the subject's PR - PCSS score is at least 1 grade higher (change of - 1, - 2, - 3, or - 4) than the baseline. In other aspects, a grade 2 composite responder means that the subject is both a grade 2 PR - PCSS responder and a grade 2 CR - PCSS responder at the evaluation time point. A grade 1 composite responder means that at the evaluation time point, the subject is both a grade 1 PR - PCSS responder and a grade 1 CR - PCSS responder.

[0775] 2. Efficacy measured by the Hexsel Cellulite Severity Scale (Hexsel CSS)

[0776] In Hexsel CSS, improvement in an individual patient at any visit means an increase of at least 1 level or 1 grade relative to the baseline or any previous score. Improvement in a group of patients at any visit is an improvement of approximately 0.1 from the baseline or any previous Hexsel CSS mean score or rating. A responder is a patient whose maximum total score or rating is at least 25% higher than the baseline findings. In certain embodiments, the treatment methods detailed above produce one or more of the following efficacy endpoints measured by Hexsel CSS:

[0777] 1. In all patient groups with a baseline Day 1 Hexsel CSS score, injection of collagenase into at least one treatment area during at least one treatment visit resulted in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[0778] · From severe to moderate (score changing from 11 - 15 to 6 - 10)

[0779] · From severe to mild (score changing from 11 - 15 to 1 - 5)

[0780] · From severe to zero (score changing from 11 - 15 to 0)

[0781] · From moderate to mild (score changing from 6 - 10 to 1 - 5)

[0782] · From moderate to zero (score changing from 6 - 10 to 0)

[0783] · From mild to zero (score changing from 1 - 5 to 0)

[0784] 2. Clinician on - site assessment of the target thigh showed that, starting from baseline (pre - treatment "Day 1"), at Day 22, 43, 71, 90, 180, 365, or 730 days, there was at least a 2 - grade improvement in the severity of Hexsel CSS.

[0785] 3. Subject - observed digital images of the target thigh showed that, starting from baseline (Day 1), at Day 22, 43, 71, 90, 180, 365, or 730 days, there was at least a 2 - grade improvement in the severity of Hexsel CSS.

[0786] 4. Improvement demonstrated by a 2 - grade composite response at Day 22, 43, 71, 90, 180, 365, or 730 days was defined as a clinician - evaluated improvement in the subject's Hexsel CSS severity by at least 2 grades compared to baseline, and a subject - evaluated improvement in severity by at least 2 grades compared to baseline.

[0787] 5. Clinician on - site assessment of the target thigh showed that, starting from baseline (Day 1), at Day 22, 43, 71, 90, 180, 365, or 730 days, there was at least a 1 - grade improvement in the severity of cellulite in Hexsel CSS compared to baseline.

[0788] 6. Subject - observed digital images of the target thigh showed that, starting from baseline (Day 1), at Day 22, 43, 71, 90, 180, 365, or 730 days, there was at least a 1 - grade improvement in the severity of cellulite in Hexsel CSS compared to baseline.

[0789] 7. Improvement demonstrated by a Level 1 composite response at Day 22, Day 43, Day 71, Day 90, Day 180, Day 365, or Day 730 is defined as at least a 1-level improvement in the severity of the subject in the Hexsel CSS compared to baseline, as evaluated by the clinician, and at least a 1-level improvement in the severity compared to baseline, as evaluated by the subject.

[0790] 8. In the patient population where all Hexsel CSS scores are moderate or severe, the improvement in at least one treatment area is statistically significant compared to placebo, where the improvement is one or more of those described in 2 to 7 above.

[0791] 9. The treatment maintained an improved level compared to the pre-treatment baseline in at least 5% of patients within 71 days of the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the initial dose. In other cases, the treatment resulted in at least 5% of patients showing improvement compared to the pre-treatment baseline and showing more improvement over time. Some treatments led to at least 10%, or 20%, or 30%, or 40% or 50% of patients showing improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with more improvement.

[0792] 10. The improvement relative to baseline observed in the Hexsel CSS at Day 180 was consistent across the left and right thighs.

[0793] 11. In the patient population where all Hexsel CSS scores are moderate or severe, the median time to the earliest occurrence of a 2-level Hexsel CSS improvement in at least one treatment area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0794] 12. In the patient population where all Hexsel CSS scores are moderate or severe, the median time to the earliest occurrence of a 1-level Hexsel CSS improvement in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0795] 13. In the patient population where all Hexsel CSS scores are moderate or severe, the mean score of the subject's Hexsel CSS separated from placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[0796] 14. Among patient populations with all Hexsel CSS scores being moderate or severe, the percentage of subjects having a grade 2 composite response measured by Hexsel CSS in at least one treatment area at day 71 is about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0797] 15. Among patient populations with all Hexsel CSS scores being moderate or severe, the percentage of subjects achieving a grade 1 composite response measured by Hexsel CSS in at least one treatment area at day 71 is about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0798] 16. Among patient populations with all Hexsel CSS scores being moderate or severe, more than one - third, one - half, two - thirds, or three - fourths of the patients have a Hexsel CSS response of at least grade 1 in at least one treatment area at day 71 after treatment, where the Hexsel CSS results are independent of age, BMI, or skin color.

[0799] 17. The severity of subcutaneous cellulite rapidly decreases within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment.

[0800] In other embodiments, about 1 mg to about 20 mg of collagenase is injected into at least one treatment area during at least one treatment visit to produce one or more of the results in items 1 to 17 above, where the collagenase has one or more of the following characteristics:

[0801] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[0802] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[0803] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[0804] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[0805] ·Kcat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[0806] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[0807] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[0808] · The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[0809] · The potency is about 5,000 to about 30,000 f-SRC units / mg

[0810] · The potency is about 100,000 to about 400,000 GPA units / mg

[0811] · The potency is about 175,000 to about 500,000 f-GPA units / mg

[0812] · The potency is about 5,000 to about 25,000 ABC units / mg

[0813] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[0814] · The bioburden is less than or equal to 1 cfu / mL

[0815] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17

[0816] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17, wherein the collagenase has one or more of the following characteristics

[0817] · V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[0818] · KM , is from about 4.1 to 410 nanomoles (SRC experiment), or, from about 0.03 to 3.1 mM (GPA experiment)

[0819] ·K cat (sec -1 ) is from about 1.1 to about 107 (SRC experiment), or, from about 93 to 9,179 (GPA experiment)

[0820] ·1 / K cat , microseconds is from about 376 to 37,222 (SRC experiment), or, from about 4 to 428 (GPA experiment)

[0821] ·K cat / K M , mM -1 sec -1 is from about 5,140 to 508,814 (SRC experiment), or, from about 60 to 5,934 (GPA experiment)

[0822] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[0823] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[0824] · The potency (i.e., specific activity) is from about 500 to about 30,000 SRC units / mg

[0825] · The potency is from about 5,000 to about 30,000 f-SRC units / mg

[0826] · The potency is from about 100,000 to about 400,000 GPA units / mg

[0827] · The potency is from about 175,000 to about 500,00 f-GPA units / mg

[0828] · The potency is from about 5,000 to about 25,000 ABC units / mg

[0829] · The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[0830] · A bioburden of less than or equal to 1 cfu / mL

[0831] In another embodiment, injecting from about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V produces one or more of the above Results 1 to 17.

[0832] In certain embodiments, in at least one treatment visit, from about 1 mg to about 20 mg of a mixture of type I collagenase and type II collagenase in a ratio of about 1:1 is injected into at least one treatment area and produces one or more of the above Results 1 to 17, wherein the type I collagenase and type II collagenase have the following characteristics:

[0833] · Type I

[0834] ο Experiment: SRC microplate

[0835] ο Vmax, min -1 : about 0.08 to 7.70

[0836] ο K M : about 4.1 to 410 nanomoles

[0837] ο K cat , sec -1 : about 1.1 to 107

[0838] ο 1 / K cat , microseconds: about 376 to 37,222

[0839] ο K cat / K M , mM -1 sec -1 : about 5,140 to 508,814

[0840] · Type II

[0841] ο Experiment: GPA microplate

[0842] ο V max , min -1 : about 0.3 to 30.5

[0843] ο K M , mM: about 0.03 to 3.1

[0844] ο K cat , sec -1 : about 93 to 9,179

[0845] ο 1 / K cat , microseconds: about 4 to 428

[0846] ο K cat / K M , mM -1 sec-1 : From about 60 to 5,934

[0847] Other ratios can also be used, (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, type I and type II collagenases can be represented as AUX - I and AUX - II, respectively.

[0848] In certain embodiments, at least once during a treatment visit, a ratio of about 1 mg to about 20 mg of type I and type II collagenases at a ratio of about 1∶1 is injected into at least one treatment area and one or more of the above results 1 to 17 are obtained, wherein the type I and type II collagenases have the following characteristics:

[0849] ·Type I

[0850] ο Experiment: SRC microplate

[0851] ο Vmax, min -1 : From about 0.08 to 7.70

[0852] ο K M : From about 4.1 to 410 nanomoles

[0853] ο K cat , sec -1 : From about 1.1 to 107

[0854] ο 1 / K cat , microseconds: From about 376 to 37,222

[0855] ο K cat / K M , mM -1 sec -1 : From about 5,140 to 508,814

[0856] ·Type II

[0857] ο Experiment: GPA microplate

[0858] ο V max , min -1 : From about 0.3 to 30.5

[0859] ο K M , mM: From about 0.03 to 3.1

[0860] ο K cat , sec -1 : From about 93 to 9,179

[0861] ο1 / K cat , microseconds: approximately 4 to 428

[0862] οK cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[0863] Other ratios can also be used, (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenases can be represented as AUX-I and AUX-II, respectively.

[0864] 3. Efficacy determined by the Hexsel Depression Depth Score

[0865] In the Hexsel depression depth score, improvement in an individual patient at any visit is at least a 1-level or 1-grade increase relative to baseline or any previous score. Improvement in a group of patients at any one visit is that the mean Hexsel depression depth score or rating is increased by approximately 0.1 compared to baseline or any previous mean Hexsel depression depth score or rating. Responders are patients who have at least a 25% increase in the maximum total score or rating from baseline findings. In certain embodiments, the treatment methods detailed above produce one or more of the following efficacy endpoints measured by the Hexsel depression depth score:

[0866] 1. In all patient populations with a Hexsel depression depth score on day 1 of baseline examination, injection of collagenase into at least one treatment area during at least one treatment visit results in a statistically significant number of patients meeting one or more of the following efficacy endpoints:

[0867] · Changing from deep depression (3) to moderate depression (2)

[0868] · Changing from deep depression (3) to shallow depression (1)

[0869] · Changing from deep depression (3) to no depression (0)

[0870] · Changing from moderate depression (2) to shallow depression (1)

[0871] · Changing from moderate depression (2) to no depression (0)

[0872] · Changing from shallow depression (1) to no depression (0)

[0873] 2. Clinically evaluated by the clinician of the target thigh, at least a 2-grade improvement in the severity of the Hexsel depression depth score at day 22, day 43, day 71, day 90, day 180, day 365, or day 730 starting from baseline (the "first day" before treatment).

[0874] 3. Evaluated by the subject observing digital images of the target thigh, at least a 2-grade improvement in the severity of the Hexsel depression depth score at day 22, day 43, day 71, day 90, day 180, day 365, or day 730 starting from baseline (the first day).

[0875] 4. The improvement demonstrated by a 2-grade composite response at day 22, day 43, day 71, day 90, day 180, day 365, or day 730 is defined as at least a 2-grade improvement in the severity of the Hexsel depression depth score compared to baseline as evaluated by the clinician for the subject, and at least a 2-grade improvement in the severity of the Hexsel depression depth score compared to baseline as evaluated by the subject.

[0876] 5. Evaluated by the subject observing digital images of the target thigh, at least a 1-grade improvement in the severity of subcutaneous cellulite in the Hexsel depression depth score compared to baseline at day 22, day 43, day 71, day 90, day 180, day 365, or day 730 starting from baseline (the first day).

[0877] 6. Evaluated by the subject observing digital images of the target thigh, at least a 1-grade improvement in the severity of subcutaneous cellulite in the Hexsel depression depth score compared to baseline at day 22, day 43, day 71, day 90, day 180, day 365, or day 730 starting from baseline (the first day).

[0878] 7. The improvement demonstrated by a 1-grade composite response at day 22, day 43, day 71, day 90, day 180, day 365, or day 730 is defined as at least a 1-grade improvement in the severity of the Hexsel depression depth score compared to baseline as evaluated by the clinician for the subject, and at least a 1-grade improvement in the severity of the Hexsel depression depth score compared to baseline as evaluated by the subject.

[0879] 8. In the patient population where all Hexsel CSS scores are moderate or severe, compared to the placebo, the improvement in at least one treatment area is statistically significant, where the improvement is one or more of those in items 2 to 7 above.

[0880] 9. The treatment maintained an improved level compared to the pre-treatment baseline in at least 5% of patients within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained this level 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, the treatment showed an improvement in at least 5% of patients compared to the pre-treatment baseline and showed more improvement over time. Some treatments led to improvement in at least 10%, or 20%, or 30%, or 40% or 50% of patients at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with more improvement.

[0881] 10. At day 180, the improvement relative to baseline observed in the Hexsel depression depth score was consistent on the left and right thighs.

[0882] 11. In the patient population where all Hexsel depression depth scores were moderate or severe, the median time to the earliest improvement in the Hexsel depression depth score of grade 2 in at least one treatment area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0883] 12. In the patient population where all Hexsel depression depth scores were moderate or severe, the median time to the earliest improvement in the Hexsel depression depth score of grade 1 in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0884] 13. In the patient population where all Hexsel depression depth scores were moderate or severe, the mean score of the Hexsel depression depth score of the subjects separated from the placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[0885] 14. In the patient population where all Hexsel depression depth scores were moderate or severe, the percentage of subjects with a composite response of grade 2 measured by the Hexsel depression depth score in at least one treatment area at day 71 was approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or greater than 50%.

[0886] 15. Among patient populations in which all Hexsel depression depth scores are moderate or severe, the percentage of subjects having a Grade 1 composite response in at least one treatment area as measured by the Hexsel depression depth score at Day 71 is about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0887] 16. Among patient populations in which all Hexsel depression depth scores are moderate or severe, more than one-third, one-half, two-thirds, or three-fourths of the patients have a Hexsel depression depth score response of at least Grade 1 in at least one treatment area at Day 71 after treatment, where the Hexsel depression depth score results are independent of age, BMI, or skin color.

[0888] 17. The severity of cellulite rapidly decreases within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment.

[0889] 18. In one or more treatment areas, the change in score from baseline to Day 71 ranges from approximately -0.1 to approximately -2.0.

[0890] 19. In a patient population with statistical significance, the least squares (LS) mean of one or more treatment areas is about -0.1 to about -1.5 (95% confidence interval (CI)).

[0891] In other embodiments, about 1 mg to about 20 mg of collagenase is injected into at least one treatment area during at least one treatment visit to produce one or more of the results in Items 1 to 19 above, where the collagenase has one or more of the following characteristics:

[0892] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[0893] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[0894] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[0895] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[0896] ·K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment),

[0897] or, about 60 to 5,934 (GPA experiment)

[0898] · Molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[0899] · Purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[0900] · Potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[0901] · Potency is about 5,000 to about 30,000 f-SRC units / mg

[0902] · Potency is about 100,000 to about 400,000 GPA units / mg

[0903] · Potency is about 175,000 to about 500,00 f-GPA units / mg

[0904] · Potency is about 5,000 to about 25,000 ABC units / mg

[0905] · Purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[0906] · Bioburden of less than or equal to 1 cfu / mL

[0907] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 19.

[0908] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 19, wherein the collagenase has one or more of the following characteristics:

[0909] · V max (min -1) is from approximately 0.08 to 7.70 (SRC experiment), or from approximately 0.3 to 30.5 (GPA experiment)

[0910] ·K M , is from about 4.1 to 410 nanomoles (SRC experiment), or from about 0.03 to 3.1 mM (GPA experiment)

[0911] ·K cat (sec -1 ) is from approximately 1.1 to approximately 107 (SRC experiment), or from about 93 to 9,179 (GPA experiment)

[0912] ·1 / K cat , microseconds is from approximately 376 to 37,222 (SRC experiment), or from approximately 4 to 428 (GPA experiment)

[0913] ·K cat / K M , mM -1 sec -1 is from approximately 5,140 to 508,814 (SRC experiment), or from approximately 60 to 5,934 (GPA experiment)

[0914] · The molecular weight is in the range 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

[0915] · The purity determined by reverse HPLC (high pressure liquid chromatography) is at least 80% by area

[0916] · The titer (i.e., specific activity) is from approximately 500 to approximately 30,000 SRC units / mg · The titer is from approximately 5,000 to approximately 30,000 f-SRC units / mg

[0917] · The titer is from approximately 100,000 to approximately 400,000 GPA units / mg

[0918] · The titer is from approximately 175,000 to approximately 500,00 f-GPA units / mg

[0919] · The titer is from approximately 5,000 to approximately 25,000 ABC units / mg

[0920] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[0921] · A bioburden of less than or equal to 1 cfu / mL

[0922] In another embodiment, injecting from about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V results in one or more of Results 1 to 19 above.

[0923] In certain embodiments, from about 1 mg to about 20 mg of a mixture of type I collagenase and type II collagenase in a ratio of about 1:1 is injected into at least one treatment area using any one of Treatments I to V and results in one or more of Results 1 to 19 above, wherein the type I collagenase and type II collagenase have the following characteristics:

[0924] · Type I

[0925] ο Experiment: SRC microplate

[0926] ο Vmax, min -1 : about 0.08 to 7.70

[0927] ο K M : about 4.1 to 410 nanomoles

[0928] ο K cat , see -1 : about 1.1 to 107

[0929] ο 1 / K cat , microseconds: about 376 to 37,222

[0930] ο K cat / K M , mM -1 sec -1 : about 5,140 to 508,814

[0931] · Type II

[0932] ο Experiment: GPA microplate

[0933] ο V max , min -1 : about 0.3 to 30.5

[0934] ο K M , mM: about 0.03 to 3.1

[0935] ο K cat , sec -1 : about 93 to 9,179

[0936] ο 1 / K cat , microseconds: about 4 to 428

[0937] ο K cat / K M , mM-1 sec -1 : about 60 to 5,934

[0938] Other ratios can also be used (e.g., 0.1-2:1, or 0.25-2:1, or 0.5-2:1, or 0.75-2:1, or 1:0.1-2, or 1:0.25-2, or 1:0.5-2, or 1:0.75-2, or 1:0, or 0:1). Further, type I and type II collagenases can be represented as AUX-I and AUX-II, respectively.

[0939] In certain embodiments, a ratio of about 1 mg to about 20 mg of type I and type II collagenases at a ratio of about 1:1 is injected into at least one treatment area using any one of treatments I to V and one or more of the above results 1 to 19 are obtained, wherein the type I and type II collagenases have the following characteristics:

[0940] · Type I

[0941] ο Experiment: SRC microplate

[0942] ο VmaX, min -1 : about 0.08 to 7.70

[0943] ο K M : about 4.1 to 410 nanomoles

[0944] ο K cat , sec -1 : about 1.1 to 107

[0945] ο 1 / K cat , microseconds: about 376 to 37,222

[0946] ο K cat / K M , mM -1 sec -1 : about 5,140 to 508,814

[0947] · Type II

[0948] ο Experiment: GPA microplate

[0949] ο V max , min -1 : about 0.3 to 30.5

[0950] ο K M , mM: about 0.03 to 3.1

[0951] ο K cat , sec-1 : Approximately 93 to 9,179

[0952] ο1 / K cat , microseconds: approximately 4 to 428

[0953] οK cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[0954] Other ratios may also be used (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be denoted as AUX-I and AUX-II, respectively.

[0955] 4. Efficacy measured using the Likert Aesthetic Scale

[0956] In the Richter scale, the improvement of each patient at any visit refers to an increase of at least 1 level or 1 grade compared to the baseline or any previous score or rating. The average score or rating of a group of patients at any one visit is increased by approximately 0.1 compared to the baseline examination or any previous average score or rating. In certain embodiments, as determined by Richter scale scoring measurement, the treatment methods described in detail above produce one or more of the following efficacy endpoints:

[0957] 1. In a population of patients with cellulite, injection of collagenase into at least one treatment area during at least one treatment visit results in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[0958] · Richter scale score of "Improved" (1)

[0959] · Richter scale score of "Significantly improved" (2)

[0960] · Richter scale score of "Very significantly improved" (3)

[0961] 2. Clinician on-site assessment of the target thigh shows that, starting from the baseline ("Day 1" before treatment), on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 365th day, or 730th day, the appearance of the treatment area shown by the Richter scale score has at least a 2 - level improvement compared to before treatment.

[0962] 3. As evaluated by the subject's observation of digital images of the target thigh, on days 22, 43, 71, 90, 180, 365, or 730, the appearance of the treatment area shown by the Likert scale score had at least a 2-level improvement compared to before treatment.

[0963] 4. As evaluated by on-site assessment by a clinician of the target thigh, on days 22, 43, 71, 90, 180, 365, or 730, the appearance of the treatment area shown by the Likert scale score had at least a 1-level improvement compared to before treatment.

[0964] 5. As evaluated by the subject's observation of digital images of the target thigh, on days 22, 43, 71, 90, 180, 365, or 730, the appearance of the treatment area shown by the Likert scale score had at least a 1-level improvement compared to before treatment.

[0965] 6. In patients with subcutaneous cellulite, the improvement in the Likert scale score in at least one treatment area was statistically significant, where the improvement was one or more of those in items 2 to 7 above.

[0966] 7. The treatment enabled at least 5% of the patients to maintain the improvement level compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the first administration. In other cases, at least 5% of the patients treated showed improvement compared to the pre-treatment baseline and showed more improvement over time. Some treatments led to at least 10%, or 20%, or 30%, or 40% or 50% of the patients showing improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with more improvement.

[0967] 8. The improvement relative to the baseline observed in the Likert scale score on day 180 was consistent between the left and right thighs.

[0968] 9. In all patient groups with subcutaneous cellulite, the median time for the earliest appearance of a 2-level improvement in the Likert scale score in at least one treatment area was about 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[0969] 10. In all patient groups with subcutaneous cellulite, the median time for the earliest appearance of a 2-level improvement in the Likert scale score 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.

[0970] 11. In all patient populations with cellulite, the mean score on the subject Likert scale separated from placebo 21 days after the first treatment and showed continued and significant improvement after subsequent treatments.

[0971] 12. In all patient populations with cellulite, the percentage of subjects with a grade 2 response in at least one treatment area measured by the Likert scale at day 71 was approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0972] 13. In all patient populations with cellulite, the percentage of subjects with a grade 1 response in at least one treatment area measured by the Likert scale at day 71 was approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[0973] 14. In all patient populations with cellulite, more than one - third, one - half, two - thirds, or three - fourths of the patients had a Likert scale score response of at least grade 1 in at least one treatment area at day 71 after treatment, where the Likert scale score results were independent of age, BMI, or skin color.

[0974] 15. The severity of cellulite decreased rapidly within 7, 14, 21, 30, 35, 40, 45, or 50 days after the first treatment.

[0975] In other embodiments, in other embodiments, injecting from about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produced one or more of the results in items 1 to 15 above, where the collagenase had one or more of the following characteristics:

[0976] ·V max (min -1 ) was from about 0.08 to 7.70 (SRC experiment), or, from about 0.3 to 30.5 (GPA experiment)

[0977] ·K M , was from about 4.1 to 410 nanomoles (SRC experiment), or, from about 0.03 to 3.1 mM (GPA experiment)

[0978] ·K cat (sec -1 ) was from about 1.1 to about 107 (SRC experiment), or, from about 93 to 9,179 (GPA experiment)

[0979] ·1 / K cat, in microseconds, it is about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[0980] ·K cat / K M , mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[0981] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[0982] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[0983] · The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[0984] · The potency is about 5,000 to about 30,000 f-SRC units / mg

[0985] · The potency is about 100,000 to about 400,000 GPA units / mg

[0986] · The potency is about 175,000 to about 500,00 f-GPA units / mg

[0987] · The potency is about 5,000 to about 25,000 ABC units / mg

[0988] · The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[0989] · A bioburden of less than or equal to 1 cfu / mL

[0990] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 15

[0991] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 15, wherein the collagenase has one or more of the following characteristics

[0992] ·V max (min -1) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[0993] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[0994] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[0995] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment)

[0996] ·K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or about 60 to 5,934 (GPA experiment)

[0997] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[0998] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[0999] · The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[1000] · The potency is about 5,000 to about 30,000 f-SRC units / mg

[1001] · The potency is about 100,000 to about 400,000 GPA units / mg

[1002] · The potency is about 175,000 to about 500,00 f-GPA units / mg

[1003] · The potency is about 5,000 to about 25,000 ABC units / mg

[1004] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1005] Bioburden less than or equal to 1 cfu / mL

[1006] In another embodiment, injecting about 1 mg to about 20 mg of collagenase, according to any one of Treatments I to V, results in one or more of Results 1 to 15 above.

[1007] In certain embodiments, at least once during a treatment visit, about 1 mg to about 20 mg of a mixture of Type I and Type II collagenase in a ratio of about 1:1 is injected into at least one treatment area and results in one or more of Results 1 to 15 above, wherein the Type I and Type II collagenase have the following characteristics:

[1008] · Type I

[1009] ο Experiment: SRC microplate

[1010] ο Vmax, min -1 : about 0.08 to 7.70

[1011] ο K M : about 4.1 to 410 nanomoles

[1012] ο K cat , sec -1 : about 1.1 to 107

[1013] ο 1 / K cat , microseconds: about 376 to 37,222

[1014] ο K cat / K M , mM -1 sec -1 : about 5,140 to 508,814

[1015] · Type II

[1016] ο Experiment: GPA microplate

[1017] ο V max , min -1 : about 0.3 to 30.5

[1018] ο K M , mM: about 0.03 to 3.1

[1019] ο K cat , sec -1 : about 93 to 9,179

[1020] ο 1 / K cat , microseconds: about 4 to 428

[1021] ο Kcat / K M ,mM -1 sec -1 :About 60 to 5,934

[1022] Other ratios may also be used, (e.g., 0.1-2∶1, or 0.25-2∶1, or 0.5-2∶1, or 0.75-2∶1, or 1∶0.1-2, or 1∶0.25-2, or 1∶0.5-2, or 1∶0.75-2, or 1∶0, or 0∶1). Further, type I and type II collagenases may be represented as AUX-I and AUX-II, respectively.

[1023] 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 into at least one treatment area using any one of Treatments I to V and one or more of the above results 1 to 17 are obtained, wherein the type I and type II collagenases have the following characteristics:

[1024] ·Type I

[1025] ο Experiment: SRC microplate

[1026] ο Vmax, min -1 :About 0.08 to 7.70

[1027] ο K M :About 4.1 to 410 nanomoles

[1028] ο K cat ,sec -1 :About 1.1 to 107

[1029] ο 1 / K cat ,microsecond: About 376 to 37,222

[1030] ο K cat / K M ,mM -1 sec -1 :About 5,140 to 508,814

[1031] ·Type II

[1032] ο Experiment: GPA microplate

[1033] ο V max ,min -1 :About 0.3 to 30.5

[1034] ο K M ,mM: About 0.03 to 3.1

[1035] oK cat , sec -1 : Approximately 93 to 9,179

[1036] o1 / K cat , microseconds: Approximately 4 to 428

[1037] oK cat / K M , mM -1 sec -1 : Approximately 60 to 5,934

[1038] Other ratios can also be used, (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases can be represented as AUX - I and AUX - II, respectively.

[1039] 5. Pit analysis

[1040] In certain embodiments, treating subcutaneous cellulite with collagenase can reduce the dimple size parameters as described below:

[1041] · Depth : Reduction by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%,

[1042] or 2.5%, or 2%, or 1%

[1043] · Width : Reduction by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%,

[1044] or 2.5%, or 2%, or 1%

[1045] · Length : Reduction by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%, or 2.5%, or 2%, or 1%

[1046] · Total volume: Reduced by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%, or 2.5%, or 2%, or 1%

[1047] · Surface area : Reduced by approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20%, or 15%, or 10%, or 5%, or 2.5%, or 2%, or 1%

[1048] In certain embodiments, the treatment maintained an improved level in at least 5% of patients compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the initial administration. In other cases, the treatment showed improvement in at least 5% of the patients compared to the pre-treatment baseline and showed more improvement over time. Some treatments resulted in at least 10%, or 20%, or 30%, or 40% or 50% of the patients showing improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with more improvement.

[1049] In other embodiments, injecting about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit can reduce the size parameter of at least one pit by at least 5%, or at least 10% or at least 20%, wherein the collagenase has one or more of the following characteristics:

[1050] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1051] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[1052] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1053] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment)

[1054] ·K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment),

[1055] or, about 60 to 5,934 (GPA experiment)

[1056] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1057] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[1058] · The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[1059] · The potency is about 5,000 to about 30,000 f-SRC units / mg

[1060] · The potency is about 100,000 to about 400,000 GPA units / mg

[1061] · The potency is about 175,000 to about 500,00 f-GPA units / mg

[1062] · The potency is about 5,000 to about 25,000 ABC units / mg

[1063] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1064] · A bioburden of less than or equal to 1 cfu / mL

[1065] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V can reduce the size parameter of at least one pit by at least 5%, or at least 10%, or at least 20%.

[1066] In other embodiments, injecting about 1 mg to about 20 mg of collagenase into at least one treatment area according to Treatments I to V can reduce the size parameter of at least one pit by at least 5%, or at least 10%, or at least 20%, wherein the collagenase has one or more of the following characteristics:

[1067] · V max(min -1 ) is from about 0.08 to 7.70 (SRC experiment), or from about 0.3 to 30.5 (GPA experiment)

[1068] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[1069] ·K cat (sec -1 ) of about 1.1 to 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1070] ·1 / K cat , microseconds are about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment)

[1071] ·K cat / K M , mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or about 60 to 5,934 (GPA experiment)

[1072] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1073] · The purity determined by reverse HPLC (high - pressure liquid chromatography) is at least 80% by area

[1074] · The potency (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[1075] · The potency is about 5,000 to about 30,000 f - SRC units / mg

[1076] · The potency is about 100,000 to about 400,000 GPA units / mg

[1077] · The potency is about 175,000 to about 500,00 f - GPA units / mg

[1078] · The potency is about 5,000 to about 25,000 ABC units / mg

[1079] · The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1080] ·Bioburden less than or equal to 1 cfu / mL

[1081] In another embodiment, injecting about 1 mg to about 20 mg of CCH according to any one of Treatments I to V can reduce the size parameter of at least one pit by at least 5%, or at least 10%, or at least 20%.

[1082] In certain embodiments, at least one treatment area is injected with about 1 mg to about 20 mg of type I collagenase and type II collagenase in a ratio of about 1:1 during at least one treatment visit, which can reduce the size parameter of at least one pit by at least 5%, or at least 10%, or at least 20%, wherein the collagenase has one or more of the following characteristics:

[1083] ·Type I

[1084] ο Experiment: SRC microplate

[1085] ο Vmax, min -1 : About 0.08 to 7.70

[1086] ο K M : About 4.1 to 410 nanomoles

[1087] ο K cat ,sec -1 : About 1.1 to 107

[1088] ο 1 / K cat ,microsecond: About 376 to 37,222

[1089] ο K cat / K M ,mM -1 sec -1 : About 5,140 to 508,814

[1090] ·Type II

[1091] ο Experiment: GPA microplate

[1092] ο V max ,min -1 : About 0.3 to 30.5

[1093] ο K M ,mM: About 0.03 to 3.1

[1094] ο K cat ,sec -1 : About 93 to 9,179

[1095] ο 1 / Kcat , microseconds: approximately 4 to 428

[1096] °K cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[1097] Other ratios can also be used (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases can be represented as AUX - I and AUX - II, respectively.

[1098] In certain embodiments, injecting at least one of Treatments I to V a ratio of approximately 1 mg to approximately 20 mg of type I and type II collagenases at a ratio of approximately 1:1 into at least one treatment area can reduce the size parameter of at least one pit by at least 5%, or at least 10%, or at least 20%, wherein the type I and type II collagenases have the following characteristics:

[1099] · Type I

[1100] ° Experiment: SRC microplate

[1101] ° Vmax, min -1 : approximately 0.08 to 7.70

[1102] ° K M : approximately 4.1 to 410 nanomoles

[1103] ° K cat , see -1 : approximately 1.1 to 107

[1104] ° 1 / K cat , microseconds: approximately 376 to 37,222

[1105] ° K cat / K M , mM -1 sec -1 : approximately 5,140 to 508,814

[1106] · Type II

[1107] ° Experiment: GPA microplate

[1108] ° V max , min -1 : approximately 0.3 to 30.5

[1109] οK M ,mM: approximately 0.03 to 3.1

[1110] οK cat ,see -1 : approximately 93 to 9,179

[1111] ο1 / K cat ,microseconds: approximately 4 to 428

[1112] οK cat / K M ,mM -1 sec -1 : approximately 60 to 5,934

[1113] Other ratios can also be used, (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases can be represented as AUX - I and AUX - II, respectively.

[1114] 6. Efficacy measured by the Subject Global Aesthetic Improvement Scale (S-GAIS) and the Investigator Global Aesthetic Improvement Scale (I-GAIS) of efficacy

[1115] According to the measurements of S - GAIS and I - GAIS, the above - described treatment methods can achieve improved treatment effects. A grade 2 S - GAIS responder is a subject with an S - GAIS score of at least 2 (+2 or +3) at the evaluation time point. A grade 1 S - GAIS responder is a subject with an S - GAIS score of at least 1 (+1, +2 or +3) at the evaluation time point. A grade 2 I - GAIS responder is a subject with an I - GAIS score of at least 2 (+2 or +3) at the evaluation time point. A grade 1 I - GAIS responder is a subject with an I - GAIS score of at least 1 (+1, +2 or +3) at the evaluation time point. Improvement in an individual patient at any visit means at least a 1 - level or 1 - step improvement compared to the baseline or any previous score. The average score or rating of a group of patients at any visit is about 0.1 higher than the baseline examination or any previous average score or rating.

[1116] In certain embodiments, by S - GAIS and / or I - GAIS measurements, the treatment methods described in detail above result in one or more of the following efficacy endpoints:

[1117] 1. In a patient population with subcutaneous cellulite, injecting collagenase into at least one treatment area during at least one treatment visit results in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[1118] · The S-GAIS and / or I-GAIS score is "Improved" (+1)

[1119] · The S-GAIS and / or I-GAIS score is "Markedly Improved" (+2)

[1120] · The S-GAIS and / or I-GAIS score is "Very Markedly Improved" (+3)

[1121] 2. Clinician on-site assessment of the target thigh shows that at least a grade 2 improvement can be observed in the I-GAIS on day 22, day 43, day 71, day 90, day 180, day 365, or day 730.

[1122] 3. Subject assessment of digital images of the target thigh shows that at least a grade 2 improvement can be observed in the S-GAIS on day 22, day 43, day 71, day 90, day 180, day 365, or day 730.

[1123] 4. Improvement demonstrated by a grade 2 composite response on day 22, day 43, day 71, day 90, day 180, day 365, or day 730 is defined as the clinician assessing that the severity of the subject in the I-GAIS has improved by at least 2 grades compared to baseline, and the subject assessing that the severity of the subject in the S-GAIS has improved by at least 2 grades compared to baseline.

[1124] 5. Clinician on-site assessment of the target thigh shows that at least a grade 1 improvement can be observed in the I-GAIS on day 22, day 43, day 71, day 90, day 180, day 365, or day 730.

[1125] 6. Subject assessment of digital images of the target thigh shows that at least a grade 1 improvement can be observed in the S-GAIS on day 22, day 43, day 71, day 90, day 180, day 365, or day 730.

[1126] 7. Improvement demonstrated by a grade 1 composite response on day 22, day 43, day 71, day 90, day 180, day 365, or day 730 is defined as the clinician assessing that the severity of the subject in the I-GAIS has improved by at least 1 grade compared to baseline, and the subject assessing that the severity of the subject in the S-GAIS has improved by at least 1 grade compared to baseline.

[1127] 8. Among the patient population with subcutaneous cellulite, in the patient population with all scores being moderate or severe, the improvement in I-GAIS and / or S-GAIS in at least one treatment area is statistically significant compared to the placebo, where the improvement is one or more of those in items 2 to 7 above.

[1128] 9. The treatment enabled at least 5% of the patients to maintain the improvement level compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the initial administration. In other cases, the treatment enabled at least 5% of the patients to show improvement compared to the pre-treatment baseline and showed more improvement over time. Some treatments led to at least 10%, or 20%, or 30%, or 40% or 50% of the patients showing improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first administration, with more improvement.

[1129] 10. The improvement relative to the baseline observed in I-GAIS and / or S-GAIS on the 180th day was consistent on the left and right thighs.

[1130] 11. In all patient populations with subcutaneous cellulite, the median time for the earliest improvement in the 2-level I-GAIS and / or S-GAIS scores in at least one treatment area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1131] 12. In all patient populations with subcutaneous cellulite, the median time for the earliest improvement in the 1-level I-GAIS and / or S-GAIS scores in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1132] 13. In all patient populations with subcutaneous cellulite, the average scores 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 after subsequent treatments.

[1133] 14. In all patient populations with subcutaneous cellulite, the percentage of subjects with a 2-level response measured by I-GAIS and / or S-GAIS in at least one treatment area on the 71st day was approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or greater than 50%.

[1134] 15. Among all patient populations with subcutaneous cellulite, the percentage of subjects with a grade 1 response in at least one treatment area as measured by I-GAIS and / or S-GAIS at day 71 is approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1135] 16. Among all patient populations with subcutaneous cellulite, among all patient populations with a moderate or severe Hexsel CSS score, more than one-third, one-half, two-thirds, or three-fourths of the patients have a measurement score response of at least grade 1 in at least one treatment area at day 71 after treatment, where the measurement score results of I-GAIS and / or S-GAIS are independent of age, BMI, or skin color.

[1136] 17. The severity of subcutaneous cellulite rapidly decreases within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment.

[1137] In other embodiments, injecting from about 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produces one or more of the results in items 1 to 17 above, where the collagenase has one or more of the following characteristics:

[1138] ·V max (min -1 ) is from about 0.08 to 7.70 (SRC experiment), or, from about 0.3 to 30.5 (GPA experiment)

[1139] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[1140] ·K cat (sec -1 ) is from about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[1141] ·1 / K cat , microseconds is from about 376 to 37,222 (SRC experiment), or, from about 4 to 428 (GPA experiment)

[1142] ·K cat / K M ,mM -1 sec -1 is from about 5,140 to 508,814 (SRC experiment), or, from about 60 to 5,934 (GPA experiment)

[1143] · having a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1144] · having a purity of at least 80% by area as determined by reverse HPLC (high performance liquid chromatography)

[1145] · having a potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1146] · having a potency of about 5,000 to about 30,000 f-SRC units / mg

[1147] · having a potency of about 100,000 to about 400,000 GPA units / mg

[1148] · having a potency of about 175,000 to about 500,000 f-GPA units / mg

[1149] · having a potency of about 5,000 to about 25,000 ABC units / mg

[1150] · having a purity of impurities of less than or equal to 1% by area, said impurities selected from the group consisting of clostripain, gelatinase, and leupeptin

[1151] · having a bioburden of less than or equal to 1 cfu / mL

[1152] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17.

[1153] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17, wherein the collagenase has one or more of the following characteristics:

[1154] · V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[1155] · K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[1156] · K cat (sec -1) is from about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[1157] ·1 / K cat , in microseconds is from about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[1158] ·K cat / K M , mM -1 sec -1 is from about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[1159] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[1160] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[1161] · The potency (i.e., specific activity) is from about 500 to about 30,000 SRC units / mg

[1162] · The potency is from about 5,000 to about 30,000 f-SRC units / mg

[1163] · The potency is from about 100,000 to about 400,000 GPA units / mg

[1164] · The potency is from about 175,000 to about 500,00 f-GPA units / mg

[1165] · The potency is from about 5,000 to about 25,000 ABC units / mg

[1166] · The purity of the impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1167] · A bioburden of less than or equal to 1 cfu / mL

[1168] In another embodiment, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17

[1169] In certain embodiments, in at least one treatment visit, a ratio of approximately 1 mg to approximately 20 mg of type I collagenase and type II collagenase that is approximately 1:1 is injected into at least one treatment area and one or more of the above results 1 to 17 are obtained, wherein the type I collagenase and type II collagenase have the following characteristics:

[1170] ·Type I

[1171] ο Experiment: SRC microplate

[1172] ο Vmax, min -1 : Approximately 0.08 to 7.70

[1173] ο K M : Approximately 4.1 to 410 nanomoles

[1174] ο K cat ,sec -1 : Approximately 1.1 to 107

[1175] ο 1 / K cat ,microsecond: Approximately 376 to 37,222

[1176] ο K cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1177] ·Type II

[1178] ο Experiment: GPA microplate

[1179] ο V max ,min -1 : Approximately 0.3 to 30.5

[1180] ο K M ,mM: Approximately 0.03 to 3.1

[1181] ο K cat ,sec -1 : Approximately 93 to 9,179

[1182] ο 1 / K cat ,microsecond: Approximately 4 to 428

[1183] ο K cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1184] Other ratios can also be used (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, type I and type II collagenases can be represented as AUX-I and AUX-II, respectively.

[1185] In certain embodiments, any one of Treatments I to V is used to inject a ratio of approximately 1 mg to approximately 20 mg of type I collagenase and type II collagenase at a ratio of approximately 1∶1 into at least one treatment area and obtain one or more of the above results 1 to 17, wherein the type I collagenase and type II collagenase have the following characteristics:

[1186] ·Type I

[1187] ο Experiment: SRC microplate

[1188] ο Vmax, min -1 : Approximately 0.08 to 7.70

[1189] ο K M : Approximately 4.1 to 410 nanomoles

[1190] ο K cat ,sec -1 : Approximately 1.1 to 107

[1191] ο 1 / K cat ,microsecond: Approximately 376 to 37,222

[1192] ο K cat / K M ,mM -1 Sec -1 : Approximately 5,140 to 508,814

[1193] ·Type II

[1194] ο Experiment: GPA microplate

[1195] ο V max ,min -1 : Approximately 0.3 to 30.5

[1196] ο K M ,mM: Approximately 0.03 to 3.1

[1197] ο K cat ,sec -1 : Approximately 93 to 9,179

[1198] ο 1 / K cat, microseconds: approximately 4 to 428

[1199] οK cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[1200] Other ratios may also be used (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, type I and type II collagenases may be designated as AUX-I and AUX-II, respectively.

[1201] 7. Efficacy of PR-CIS assay

[1202] As determined by PR-CIS, the treatment methods described in detail above can achieve improved treatment effects. The total PR-CIS score is the sum of six items on the scale. Item 1 in PR-CIS asks the subject about their satisfaction with the appearance of cellulite, which is reversed by subtracting the evaluated value reported by the subject from 10. The total PR-CIS score can range from 0 to 60, and the higher the value, the greater the negative impact of cellulite. For the total PR-CIS score, a responder is a subject whose total PR-CIS score is at least 12 points lower than the baseline at the evaluation time point. For the individual PR-CIS impact score, a response is an improvement of at least 2 score intervals from the baseline at each time point. Additionally, a responder is any patient who shows at least a 20% increase in the maximum total score compared to the baseline examination. Improvement for an individual patient at any visit means an increase of at least 1 level or grade compared to the baseline or any previous score. The average score or rating for a group of patients at any visit is about 0.1 higher than the baseline examination or any previous average score or rating. Additionally, improvement means a change of at least 1 level out of 60 relative to the baseline.

[1203] In certain embodiments, based on the results of the PR-CIS determination, the treatment methods described in detail above can achieve one or more of the following efficacy endpoints:

[1204] 1. In a population of patients with subcutaneous cellulite, injecting collagenase into at least one treatment area during at least one treatment visit will result in a statistically significant number of patients meeting one or more of the following efficacy endpoints:

[1205] · The PR-CIS shows improvement in at least one area selected from the group of happiness, annoyance, self-awareness, embarrassment, looking old, looking overweight / unhealthy.· At one or more assessment time points, the total PR-CIS score is at least 12 points lower than the baseline.

[1206] · The PR-CIS impact score shows improvement of at least 2 score intervals from the baseline at one or more assessment time points.

[1207] · Improvement means a change of at least 1 level out of 60 relative to the baseline.

[1208] 2. For the target thigh, starting from the baseline (the "first day" before treatment), on the 22nd, 43rd, 71st, 90th, 180th, 365th or 730th day, the severity measured by the PR-CIS is improved by at least 12 points.

[1209] 3. In the patient population with subcutaneous cellulite, starting from the baseline (the "first day" before treatment), on the 22nd, 43rd, 71st, 90th, 180th, 365th or 730th day, the severity measured by the PR-CIS shows a statistically significant improvement compared to the placebo, which means an improvement of at least 12 points.

[1210] 4. The treatment enables at least 5% of the patients to maintain the improvement level compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintain this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the initial administration. In other cases, the treatment enables at least 5% of the patients to show improvement compared to the pre-treatment baseline and show more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40% or 50% of the patients showing improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with more improvement.

[1211] 5. The improvement observed in the PR-CIS score relative to the baseline on the 180th day is consistent on the left and right thighs.

[1212] 6. In all patient populations with subcutaneous cellulite, in at least one treatment area, the median time for the total PR-CIS score measured at one or more assessment time points to be at least 12 points lower than the baseline 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.

[1213] 7. In all patient groups with cellulite, the mean score of the subjects on the PR-CIS separated from that of the placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[1214] 8. In all patient groups with cellulite, more than one-third, one-half, two-thirds, or three-fourths of the patients had a reduction of at least 12 in the total PR-CIS score compared to the baseline at one or more assessment time points on day 71 after treatment, where the scoring results of the PR-CIS were independent of age, BMI, or skin color.

[1215] 9. The severity of cellulite decreased rapidly within 7, 14, 21, 30, 35, 40, 45, or 50 days after the first treatment visit.

[1216] In other embodiments, about 1 mg to about 20 mg of collagenase is injected into at least one treatment area during at least one treatment visit to produce one or more of the results in Items 1 to 9 above, where the collagenase has one or more of the following characteristics:

[1217] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1218] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[1219] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1220] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment)

[1221] ·K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or about 60 to 5,934 (GPA experiment)

[1222] · The molecular weight is in the range 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,

[1223] · The purity determined by reverse HPLC (high pressure liquid chromatography) is at least 80% by area

[1224] · The potency (i.e., specific activity) is approximately 500 to approximately 30,000 SRC units / mg

[1225] · The potency is approximately 5,000 to approximately 30,000 f-SRC units / mg

[1226] · The potency is approximately 100,000 to approximately 400,000 GPA units / mg

[1227] · The potency is approximately 175,000 to approximately 500,00 f-GPA units / mg

[1228] · The potency is approximately 5,000 to approximately 25,000 ABC units / mg

[1229] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1230] · A bioburden of less than or equal to 1 cfu / mL

[1231] In other cases, injecting approximately 1 mg to approximately 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 9.

[1232] In other cases, injecting approximately 1 mg to approximately 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 9, wherein the collagenase has one or more of the following characteristics:

[1233] · V max (min -1 ) is approximately 0.08 to 7.70 (SRC experiment), or, approximately 0.3 to 30.5 (GPA experiment)

[1234] · K M , is approximately 4.1 to 410 nanomoles (SRC experiment), or, approximately 0.03 to 3.1 mM (GPA experiment)

[1235] · K cat (sec -1) is from about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[1236] ·1 / K cat , in microseconds is from about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[1237] ·K cat / K M , mM -1 sec -1 is from about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[1238] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1239] · The purity determined by reverse HPLC (high pressure liquid chromatography) is at least 80% by area

[1240] · The titer (i.e., specific activity) is from about 500 to about 30,000 SRC units / mg

[1241] · The titer is from about 5,000 to about 30,000 f-SRC units / mg

[1242] · The titer is from about 100,000 to about 400,000 GPA units / mg

[1243] · The titer is from about 175,000 to about 500,00 f-GPA units / mg

[1244] · The titer is from about 5,000 to about 25,000 ABC units / mg

[1245] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1246] · The bioburden is less than or equal to 1 cfu / mL

[1247] In another embodiment, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 9.

[1248] In certain embodiments, in at least one treatment visit, a ratio of approximately 1 mg to approximately 20 mg of type I collagenase and type II collagenase that is approximately 1:1 is injected into at least one treatment area and one or more of the above results 1 to 9 are obtained, wherein the type I collagenase and type II collagenase have the following characteristics:

[1249] ·Type I

[1250] ο Experiment: SRC microplate

[1251] ο Vmax, min -1 : Approximately 0.08 to 7.70

[1252] ο K M : Approximately 4.1 to 410 nanomoles

[1253] ο K cat , sec -1 : Approximately 1.1 to 107

[1254] ο 1 / K cat , microseconds: Approximately 376 to 37,222

[1255] ο K cat / K M , mM -1 sec -1 : Approximately 5,140 to 508,814

[1256] ·Type II

[1257] ο Experiment: GPA microplate

[1258] ο V max , min -1 : Approximately 0.3 to 30.5

[1259] ο K M , mM: Approximately 0.03 to 3.1

[1260] ο K cat , sec -1 : Approximately 93 to 9,179

[1261] ο 1 / K cat , microseconds: Approximately 4 to 428

[1262] ο K cat / K M , mM -1 sec -1 : Approximately 60 to 5,934

[1263] Other ratios can also be used (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, type I and type II collagenases can be represented as AUX-I and AUX-II, respectively.

[1264] In certain embodiments, from about 1 mg to about 20 mg of a mixture of type I and type II collagenases in a ratio of about 1∶1 is injected into at least one treatment area using any one of Treatments I to V and one or more of the above results 1 to 9 are obtained, wherein the type I and type II collagenases have the following characteristics:

[1265] ·Type I

[1266] ο Experiment: SRC microplate

[1267] ο Vmax, min -1 : about 0.08 to 7.70

[1268] ο K M : about 4.1 to 410 nanomoles

[1269] ο K cat ,sec -1 : about 1.1 to 107

[1270] ο 1 / K cat ,microsecond: about 376 to 37,222

[1271] ο K cat / K M ,mM -1 sec -1 : about 5,140 to 508,814

[1272] ·Type II

[1273] ο Experiment: GPA microplate

[1274] ο V max ,min -1 : about 0.3 to 30.5

[1275] ο K M ,mM: about 0.03 to 3.1

[1276] ο K cat ,sec -1 : about 93 to 9,179

[1277] ο 1 / K cat, microseconds: approximately 4 to 428

[1278] οK cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[1279] Other ratios can also be used (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, type I and type II collagenases can be represented as AUX-I and AUX-II, respectively.

[1280] 8. Efficacy of the PR-CIS short form assay

[1281] Measurement by the PR-CIS short form demonstrated that the treatment methods described in detail above can achieve improved treatment effects. The total PR-CIS short form score is the sum of five items on the scale. Item 1 in the PR-CIS asks the subject about their satisfaction with the appearance of cellulite and will be reversed by subtracting the assessment reported by the subject from 10. The total score of the PR-CIS short form can range from 0 to 50, with higher numbers indicating a greater negative impact of cellulite. For the total score of the PR-CIS short form, a responder is a subject whose total PR-CIS score is at least 10 points lower than the baseline at the assessment time point. For the individual PR-CIS short form impact score, a response is an improvement of at least 2 score intervals from the baseline at each time point. In addition, a responder is any patient who shows at least a 20% increase in the maximum total score compared to the baseline examination. Improvement for an individual patient at any visit means at least a 1-level or 1-grade increase compared to the baseline or any previous score. The average score or rating for a group of patients at any visit is approximately 0.1 higher than the baseline examination or any previous average score or rating. In addition, improvement is a level change of at least 1 point from the baseline of 50 points.

[1282] In certain embodiments, as determined by the PR-CIS short form, the treatment methods described above are capable of achieving one or more of the following effects:

[1283] 1. In a patient population with subcutaneous cellulite, injecting collagenase into at least one treatment area during at least one treatment visit can result in a statistically significant number of patients meeting one or more of the following efficacy endpoints:

[1284] · The PR-CIS short form shows improvement in at least one area selected from the group consisting of happiness, annoyance, self-consciousness, embarrassment, looking old, looking overweight / unhealthy

[1285] · At one or more assessment time points, the total score of the PR-CIS short form is at least 10 points lower than the baseline.

[1286] · The impact score of the PR-CIS short form shows an improvement of at least 2 score intervals from the baseline at one or more assessment time points.

[1287] · Further, improvement means a change of at least 1 level out of 50 relative to the baseline.

[1288] 2. For the target thigh, at 22 days, 43 days, 71 days, 90 days, 180 days, 365 days or 730 days starting from the baseline (the "first day" before treatment), the severity measured by the PR-CIS short form is improved by at least 12 points.

[1289] 3. In the patient population with cellulite, at 22 days, 43 days, 71 days, 90 days, 180 days, 365 days or 730 days starting from the baseline (the "first day" before treatment), a statistically significant improvement in severity measured by the PR-CIS short form compared to the placebo means an improvement of at least 10 points.

[1290] 4. The treatment enabled at least 5% of the patients to maintain the improvement level compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the initial administration. In other cases, the treatment enabled at least 5% of the patients to show improvement compared to the pre-treatment baseline and showed more improvement over time. Some treatments led to at least 10%, or 20%, or 30%, or 40% or 50% of the patients to show improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first administration, with more improvement.

[1291] 5. The improvement relative to the baseline observed in the PR-CIS short form score on the 180th day was consistent on the left and right thighs.

[1292] 6. In all patient populations with cellulite, in at least one treatment area, the median time for the total score measured by the PR-CIS short form to be reduced by at least 10 compared to the baseline at one or more assessment time points 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.

[1293] 7. In all patient groups with subcutaneous cellulite, the mean score of the PR-CIS short form in the subjects separated from the placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[1294] 8. In all patient groups with subcutaneous cellulite, more than one-third, one-half, two-thirds, or three-fourths of the patients had a reduction of at least 10 in the total score of the PR-CIS short form compared to the baseline at one or more assessment time points on day 71 after treatment, where the scoring results of the PR-CIS were independent of age, BMI, or skin color.

[1295] 9. The severity of subcutaneous cellulite decreased rapidly within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment visit.

[1296] In other embodiments, from about 1 mg to about 20 mg of collagenase is injected into at least one treatment area during at least one treatment visit to produce one or more of the results of items 1 to 9 above, where the collagenase has one or more of the following characteristics

[1297] ·V max (min -1 ) is from about 0.08 to 7.70 (SRC experiment), or from about 0.3 to 30.5 (GPA experiment)

[1298] ·K M , is from about 4.1 to 410 nanomoles (SRC experiment), or from about 0.03 to 3.1 mM (GPA experiment)

[1299] ·K cat (sec -1 ) is from about 1.1 to about 107 (SRC experiment), or from about 93 to 9,179 (GPA experiment)

[1300] ·1 / K cat , microseconds is from about 376 to 37,222 (SRC experiment), or from about 4 to 428 (GPA experiment)

[1301] ·K cat / K M ,mM -1 sec -1 is from about 5,140 to 508,814 (SRC experiment), or from about 60 to 5,934 (GPA experiment)

[1302] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1303] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[1304] · The titer (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[1305] · The titer is about 5,000 to about 30,000 f-SRC units / mg

[1306] · The titer is about 100,000 to about 400,000 GPA units / mg

[1307] · The titer is about 175,000 to about 500,00 f-GPA units / mg

[1308] · The titer is about 5,000 to about 25,000 ABC units / mg

[1309] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1310] · The bioburden is less than or equal to 1 cfu / mL

[1311] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 9.

[1312] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 9, wherein the collagenase has one or more of the following characteristics:

[1313] · V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1314] · K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[1315] · K cat (sec -1) is from about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[1316] ·1 / K cat , in microseconds is from about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[1317] ·K cat / K M , mM -1 sec -1 is from about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[1318] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[1319] · The purity determined by reverse HPLC (high pressure liquid chromatography) is at least 80% by area

[1320] · The potency (i.e., specific activity) is from about 500 to about 30,000 SRC units / mg

[1321] · The potency is from about 5,000 to about 30,000 f-SRC units / mg

[1322] · The potency is from about 100,000 to about 400,000 GPA units / mg

[1323] · The potency is from about 175,000 to about 500,00 f-GPA units / mg

[1324] · The potency is from about 5,000 to about 25,000 ABC units / mg

[1325] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1326] · The bioburden is less than or equal to 1 cfu / mL

[1327] In another embodiment, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 9

[1328] In certain embodiments, in at least one treatment visit, a ratio of about 1 mg to about 20 mg of type I collagenase and type II collagenase that is about 1:1 is injected into at least one treatment area and one or more of the above results 1 to 9 are obtained, wherein the type I collagenase and type II collagenase have the following characteristics:

[1329] ·Type I

[1330] ο Experiment: SRC microplate

[1331] ο Vmax, min -1 : about 0.08 to 7.70

[1332] ο K M : about 4.1 to 410 nanomoles

[1333] ο K cat ,sec -1 : about 1.1 to 107

[1334] ο 1 / K cat ,microsecond: about 376 to 37,222

[1335] ο K cat / K M ,mM -1 sec -1 : about 5,140 to 508,814

[1336] ·Type II

[1337] ο Experiment: GPA microplate

[1338] ο V max ,min -1 : about 0.3 to 30.5

[1339] ο K M ,mM: about 0.03 to 3.1

[1340] ο K cat ,sec -1 : about 93 to 9,179

[1341] ο 1 / K cat ,microsecond: about 4 to 428

[1342] ο K cat / K M ,mM -1 sec -1 : about 60 to 5,934

[1343] Other ratios can also be used (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, type I and type II collagenases can be represented as AUX-I and AUX-II, respectively.

[1344] In certain embodiments, any one of Treatments I to V is used to inject about 1 mg to about 20 mg of type I and type II collagenases in a ratio of about 1∶1 into at least one treatment area and obtain one or more of the above results 1 to 9, wherein the type I and type II collagenases have the following characteristics:

[1345] ·Type I

[1346] ο Experiment: SRC microplate

[1347] ο Vmax, min -1 : about 0.08 to 7.70

[1348] ο K M : about 4.1 to 410 nanomoles

[1349] ο K cat ,sec -1 : about 1.1 to 107

[1350] ο 1 / K cat ,microsecond: about 376 to 37,222

[1351] ο K cat / K M ,mM -1 sec -1 : about 5,140 to 508,814

[1352] ·Type II

[1353] ο Experiment: GPA microplate

[1354] ο V max ,min -1 : about 0.3 to 30.5

[1355] ο K M ,mM: about 0.03 to 3.1

[1356] ο K cat ,sec -1 : about 93 to 9,179

[1357] ο 1 / K cat, microseconds: approximately 4 to 428

[1358] °K cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[1359] Other ratios may also be used (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be designated as AUX - I and AUX - II, respectively.

[1360] 9. Efficacy determined by the Subject Self-Rating Scale (SSRS)

[1361] Based on measurements by the SSRS, the treatment methods described in detail above produce improved treatment effects. An SSRS responder is a subject who is at least slightly satisfied (slightly satisfied [4], very satisfied [5], or extremely satisfied [6]) with the appearance of cellulite on the thigh on day 71. In addition, a responder is any patient who has at least a 17% improvement in the maximum total score compared to baseline. Improvement for an individual patient at any visit refers to an increase of at least 1 level or grade compared to baseline or any previous score. The average score or rating for a group of patients at any visit is approximately 0.1 higher than the baseline examination or any previous average score or rating. In certain embodiments, the following one or more efficacy endpoints are produced by the treatment method as measured by the SSRS rating:

[1362] 1. In a population of patients with subcutaneous cellulite, injection of collagenase into at least one treatment area during at least one treatment visit results in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[1363] · SSRS score of "fairly satisfied"

[1364] · SSRS score of "satisfied"

[1365] · SSRS score of "very satisfied"

[1366] 2. For the target thigh, on days 22, 43, 71, 90, 180, 365, or 730 starting from baseline (day 1 before treatment), the severity as measured by the SSRS score is improved by at least 2 grades.

[1367] 3. For the target thigh, on the 22nd, 43rd, 71st, 90th, 180th, 365th or 730th day starting from the baseline (the "first day" before treatment), the severity measured by the SSRS score improved by at least 1 level.

[1368] 4. In the patient population with subcutaneous cellulite, the improvement measured by the SSRS score in at least one treatment area was statistically significant compared to the placebo, where the improvement was one or more of those in 2 to 3 above.

[1369] 5. The treatment enabled at least 5% of the patients to maintain the improvement level compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintained this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the first administration. In other cases, the treatment enabled at least 5% of the patients to show improvement compared to the pre-treatment baseline and showed more improvement over time. Some treatments led to at least 10%, or 20%, or 30%, or 40% or 50% of the patients to show improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first administration, with more improvement.

[1370] 6. The improvement relative to the baseline observed in the SSRS score on the 180th day was consistent on the left and right thighs.

[1371] 7. In all patient populations with subcutaneous cellulite, the median time for the earliest occurrence of a 2-level SSRS score improvement in at least one treatment area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1372] 8. In all patient populations with subcutaneous cellulite, the median time for the earliest occurrence of a 1-level SSRS score improvement in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1373] 9. In all patient populations with subcutaneous cellulite, the average SSRS score of the subjects separated from the placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[1374] 10. In all patient populations with subcutaneous cellulite, the percentage of subjects with a 2-level response measured by the SSRS score in at least one treatment area on the 71st day was approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or greater than 50%.

[1375] 11. Among all patient populations with subcutaneous cellulite, the percentage of subjects with a Grade 1 response as measured by the SSRS score in at least one treatment area at Day 71 is about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1376] 12. Among all patient populations with subcutaneous cellulite, more than one-third, one-half, two-thirds, or three-fourths of the patients have a SSRS measurement score response of at least Grade 1 in at least one treatment area at Day 71 after treatment, where the measurement score results of the SSRS are independent of age, BMI, or skin color.

[1377] 13. The severity of subcutaneous cellulite rapidly decreases within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment visit.

[1378] In other embodiments, about 1 mg to about 20 mg of collagenase is injected into at least one treatment area during at least one treatment visit to produce one or more of the results in Items 1 to 13 above, where the collagenase has one or more of the following characteristics:

[1379] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[1380] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[1381] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[1382] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[1383] ·K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[1384] · having a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1385] · having a purity of at least 80% by area as determined by reverse HPLC (high performance liquid chromatography)

[1386] · having a potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1387] · having a potency of about 5,000 to about 30,000 f-SRC units / mg

[1388] · having a potency of about 100,000 to about 400,000 GPA units / mg

[1389] · having a potency of about 175,000 to about 500,00 f-GPA units / mg

[1390] · having a potency of about 5,000 to about 25,000 ABC units / mg

[1391] · having a purity of impurities of less than or equal to 1% by area, said impurities selected from the group consisting of clostripain, gelatinase, and leupeptin

[1392] · having a bioburden of less than or equal to 1 cfu / mL

[1393] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 13.

[1394] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 13, wherein the collagenase has one or more of the following characteristics.

[1395] · V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[1396] · K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[1397] · K cat (sec -1) is from about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[1398] · 1 / K cat , microseconds is from about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[1399] · K cat / K M , mM -1 sec -1 is from about 5,140 to 508,814 (SRC experiment),

[1400] or, about 60 to 5,934 (GPA experiment)

[1401] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1402] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[1403] · The potency (i.e., specific activity) is from about 500 to about 30,000 SRC units / mg

[1404] · The potency is from about 5,000 to about 30,000 f-SRC units / mg

[1405] · The potency is from about 100,000 to about 400,000 GPA units / mg

[1406] · The potency is from about 175,000 to about 500,00 f-GPA units / mg

[1407] · The potency is from about 5,000 to about 25,000 ABC units / mg

[1408] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1409] · A biological burden of less than or equal to 1 cfu / mL

[1410] In another embodiment, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 13.

[1411] In certain embodiments, during at least one treatment visit, a ratio of approximately 1 mg to approximately 20 mg of type I collagenase and type II collagenase that is approximately 1:1 is injected into at least one treatment area and one or more of the above results 1 to 13 are obtained, wherein the type I collagenase and type II collagenase have the following characteristics:

[1412] · Type I

[1413] ο Experiment: SRC microplate

[1414] ο Vmax, min -1 : Approximately 0.08 to 7.70

[1415] ο K M : Approximately 4.1 to 410 nanomoles

[1416] ο K cat ,sec -1 : Approximately 1.1 to 107

[1417] ο 1 / K cat ,microsecond: Approximately 376 to 37,222

[1418] ο K cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1419] · Type II

[1420] ο Experiment: GPA microplate

[1421] ο V max ,min -1 : Approximately 0.3 to 30.5

[1422] ο K M ,mM: Approximately 0.03 to 3.1

[1423] ο K cat ,sec -1 : Approximately 93 to 9,179

[1424] ο 1 / K cat ,microsecond: Approximately 4 to 428

[1425] ο K cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934

[1426] Other ratios may also be used (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, type I and type II collagenases may be denoted as AUX-I and AUX-II, respectively.

[1427] In certain embodiments, any one of Treatments I to V is used to inject a ratio of approximately 1 mg to approximately 20 mg of type I and type II collagenases at approximately 1∶1 into at least one treatment area and obtain one or more of the above results 1 to 13, wherein the type I and type II collagenases have the following characteristics:

[1428] ·Type I

[1429] ο Experiment: SRC microplate

[1430] ο Vmax, min -1 : Approximately 0.08 to 7.70

[1431] ο K M : Approximately 4.1 to 410 nanomoles

[1432] ο K cat ,sec -1 : Approximately 1.1 to 107

[1433] ο 1 / K cat ,microsecond: Approximately 376 to 37,222

[1434] ο K cat / K M ,mM -1 sec -1 : Approximately 5,140 to 508,814

[1435] ·Type II

[1436] ο Experiment: GPA microplate

[1437] ο V max ,min -1 : Approximately 0.3 to 30.5

[1438] ο K M ,mM: Approximately 0.03 to 3.1

[1439] ο K cat ,sec -1 : Approximately 93 to 9,179

[1440] ο 1 / K cat, microseconds: approximately 4 to 428

[1441] °K cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[1442] Other ratios may also be used (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases may be designated as AUX - I and AUX - II, respectively.

[1443] 10. Efficacy measured based on the subject's satisfaction with cellulite treatment (SSCT)

[1444] Based on measurements by SSCT, the treatment methods described in detail above produce improved treatment effects. In the SSCT assessment, subjects with a response of "satisfactory" or "very satisfactory" to cellulite on the thigh on day 71 were considered responders showing treatment efficacy. Improvement in an individual patient at any visit refers to an increase of at least 1 level or grade compared to the baseline or any previous score. The average score or rating of a group of patients at any visit is increased by approximately 0.1 compared to the baseline examination or any previous average score or rating. Further, for SSCT, improvement is at least 0.1 compared to the placebo. In certain embodiments, the following one or more efficacy endpoints are produced by the treatment methods as measured by SSCT rating:

[1445] 1. In a patient population with subcutaneous cellulite, injection of collagenase into at least one treatment area during at least one treatment visit results in a statistically significant number of such patients meeting the following one or more efficacy endpoints:

[1446] · SSCT score of "satisfactory"

[1447] · SSCT score of "very satisfactory"

[1448] 2. For the target thigh, on days 22, 43, 71, 90, 180, 365, or 730, the SSCT score increases (e.g., 1 to 2, etc.).

[1449] 3. In a patient population with subcutaneous cellulite, on days 22, 43, 71, 90, 180, 365, or 730, the increase in the SSCT score in at least one treatment area is statistically significant compared to the placebo.

[1450] 4. The treatment maintained improved outcomes in at least 5% of patients compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained this level 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, the treatment showed improvement in at least 5% of patients compared to the pre-treatment baseline and showed further improvement over time. Some treatments resulted in at least 10%, or 20%, or 30%, or 40% or 50% of patients showing improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first administration, with further improvement.

[1451] 5. The improvement relative to baseline observed in the SSCT score on day 180 was consistent in the left and right thighs.

[1452] 6. In all patient groups with subcutaneous cellulite, the median time for the earliest improvement in the SSCT score in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1453] 7. In all patient groups with subcutaneous cellulite, the mean SSRS score of the subjects separated from the placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[1454] 8. In all patient groups with subcutaneous cellulite, the percentage of subjects with an improved SSCT score in at least one treatment area on day 71 was approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or greater than 50%.

[1455] 9. In all patient groups with subcutaneous cellulite, more than one-third, one-half, two-thirds or three-fourths of the patients had an improved SSCT score in at least one treatment area on day 71 after treatment, and the measured SSRS score results were independent of age, BMI or skin color.

[1456] 10. The severity of subcutaneous cellulite rapidly decreased within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days or 50 days after the first treatment visit.

[1457] In other embodiments, injecting approximately 1 mg to about 20 mg of collagenase into at least one treatment area during at least one treatment visit produced one or more of the results in items 1 to 10 above, where the collagenase had one or more of the following characteristics:

[1458] ·V max (min -1 ) is approximately 0.08 to 7.70 (SRC experiment), or approximately 0.3 to 30.5 (GPA experiment)

[1459] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[1460] ·K cat (sec -1 ) is approximately 1.1 to approximately 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1461] ·1 / K cat , microseconds is approximately 376 to 37,222 (SRC experiment), or approximately 4 to 428 (GPA experiment)

[1462] ·K cat / K M ,mM -1 sec -1 is approximately 5,140 to 508,814 (SRC experiment),

[1463] or approximately 60 to 5,934 (GPA experiment)

[1464] · The molecular weight is in the range 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,

[1465] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[1466] · The titer (i.e., specific activity) is approximately 500 to approximately 30,000 SRC units / mg

[1467] · The titer is approximately 5,000 to approximately 30,000 f-SRC units / mg

[1468] · The titer is approximately 100,000 to approximately 400,000 GPA units / mg

[1469] · The titer is approximately 175,000 to approximately 500,00 f-GPA units / mg

[1470] · The titer is approximately 5,000 to approximately 25,000 ABC units / mg

[1471] · The purity of the impurity by area is less than or equal to 1%, and the impurity is selected from the group consisting of clostripain, gelatinase, and leupeptin

[1472] · A biological burden of less than or equal to 1 cfu / mL

[1473] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 10.

[1474] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 10, wherein the collagenase has one or more of the following characteristics:

[1475] · V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1476] · K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[1477] · K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1478] · 1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment)

[1479] · K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment),

[1480] or about 60 to 5,934 (GPA experiment)

[1481] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1482] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[1483] ·The titer (i.e., specific activity) is about 500 to about 30,000 SRC units / mg

[1484] ·The titer is about 5,000 to about 30,000 f-SRC units / mg

[1485] ·The titer is about 100,000 to about 400,000 GPA units / mg

[1486] ·The titer is about 175,000 to about 500,00 f-GPA units / mg

[1487] ·The titer is about 5,000 to about 25,000 ABC units / mg

[1488] ·The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1489] ·A bioburden of less than or equal to 1 cfu / mL

[1490] In another embodiment, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 10

[1491] In certain embodiments, at least once during a treatment visit, about 1 mg to about 20 mg of a 1:1 ratio of type I collagenase and type II collagenase is injected into at least one treatment area and produces one or more of the above results 1 to 10, wherein the type I collagenase and type II collagenase have the following characteristics

[1492] ·Type I

[1493] ο Experiment: SRC microplate

[1494] ο Vmax, min -1 : about 0.08 to 7.70

[1495] ο K M : about 4.1 to 410 nanomoles

[1496] ο K cat ,sec -1 : about 1.1 to 107

[1497] ο 1 / K cat ,microsecond: about 376 to 37,222

[1498] ο K cat / K M ,mM -1 sec-1 : Approximately 5,140 to 508,814

[1499] ·Type II

[1500] ο Experiment: GPA microplate

[1501] ο V max , min -1 : Approximately 0.3 to 30.5

[1502] ο K M , mM: Approximately 0.03 to 3.1

[1503] ο K cat , see -1 : Approximately 93 to 9,179

[1504] ο 1 / K cat , microseconds: Approximately 4 to 428

[1505] ο K cat / K M , mM -1 sec -1 : Approximately 60 to 5,934

[1506] Other ratios can also be used, (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, Type I and Type II collagenases can be represented as AUX-I and AUX-II, respectively.

[1507] In certain embodiments, any one of Treatments I to V is used to inject approximately 1 mg to approximately 20 mg of a mixture of Type I and Type II collagenases in a ratio of approximately 1:1 into at least one treatment area and obtain one or more of the above results 1 to 10, wherein the Type I and Type II collagenases have the following characteristics:

[1508] ·Type I

[1509] ο Experiment: SRC microplate

[1510] ο Vmax, min -1 : Approximately 0.08 to 7.70

[1511] ο K M : Approximately 4.1 to 410 nanomoles

[1512] ο K cat , sec -1 : Approximately 1.1 to 107

[1513] ο1 / K cat ,microsecond: approximately 376 to 37,222

[1514] οK cat / K M ,mM -1 sec -1 :approximately 5,140 to 508,814

[1515] ·Type II

[1516] οExperiment: GPA microplate

[1517] οV max ,min -1 :approximately 0.3 to 30.5

[1518] οK M ,mM: approximately 0.03 to 3.1

[1519] οK cat ,see -1 :approximately 93 to 9,179

[1520] ο1 / K cat ,microsecond: approximately 4 to 428

[1521] οK cat / K M ,mM -1 sec -1 :approximately 60 to 5,934

[1522] Other ratios can also be used, (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, Type I and Type II collagenases can be represented as AUX - I and AUX - II, respectively.

[1523] 11. Efficacy of the Thigh Cellulite Efficacy Scale (CR-TCES; PR-TCES)

[1524] Improvement in an individual patient at any visit refers to an increase of at least 1 level or 1 grade relative to the baseline or any previous score. Improvement in a group of patients at any visit is an improvement of approximately 0.1 compared to the baseline or any previous mean score. Responders are patients who show an increase of at least 20% in the maximum total score or rating from the baseline findings. In certain embodiments, the treatment methods detailed above produce one or more of the following efficacy endpoints measured by CR - TCES and / or PR - TCES:

[1525] 1. The CR-TCES and / or PR-TCES score improves by at least 0.1 compared to the baseline.

[1526] 2. For the target thigh, the evaluation is conducted on-site by a clinician at day 22, 43, 71, 90, 180, 365, or 730 days starting from the baseline (the "first day" before treatment), and the severity measured by the CR-TCES score improves by at least 2 grades.

[1527] 3. For the target thigh, the evaluation is conducted on-site by a clinician at day 22, 43, 71, 90, 180, 365, or 730 days starting from the baseline (the "first day" before treatment), and the severity measured by the PR-TCES score improves by at least 2 grades.

[1528] 4. The improvement demonstrated by a grade 2 composite response at day 22, 43, 71, 90, 180, 365, or 730 days is defined as the severity in CR-TCES improving by at least 2 grades compared to the baseline, and the severity in PR-TCES improving by at least 2 grades compared to the baseline.

[1529] 5. Starting from the baseline (the first day), at day 22, 43, 71, 90, 180, 365, or 730 days, the severity of cellulite in CR-TCES evaluated on-site by a clinician of the target thigh improves by at least 1 grade compared to the baseline.

[1530] 6. Starting from the baseline (the first day), at day 22, 43, 71, 90, 180, 365, or 730 days, the severity of cellulite in PR-TCES evaluated on-site by a clinician of the target thigh improves by at least 1 grade compared to the baseline.

[1531] 7. The improvement demonstrated by a grade 1 composite response at day 22, 43, 71, 90, 180, 365, or 730 days is defined as the severity in CR-TCES improving by at least 1 grade compared to the baseline, and the severity in PR-TCES improving by at least 1 grade compared to the baseline.

[1532] 8. In the patient population where all CR-TCES scores are moderate or severe, compared to the placebo, the improvement in at least one treatment area is statistically significant, where the improvement is one or more of the above 1 to 7.

[1533] 9. The treatment maintained an improved level in at least 5% of patients compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of patients maintained this level 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, the treatment showed improvement in at least 5% of patients compared to the pre-treatment baseline and showed more improvement over time. Some treatments led to improvement in at least 10%, or 20%, or 30%, or 40% or 50% of patients at 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first dose, with additional improvement.

[1534] 10. The improvement relative to baseline observed in the CR-TCES and / or PR-TCES scores on day 180 was consistent across the left and right thighs.

[1535] 11. In the patient population where all CR-TCES scores were moderate or severe, the median time to the earliest improvement in 2-level CR-TCES and / or PR-TCES scores in at least one treatment area was approximately 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1536] 12. In the patient population where all CR-TCES scores were moderate or severe, the median time to the earliest improvement in 1-level CR-TCES and / or PR-TCES scores in at least one treatment area was approximately 15 days, or 20 days, or 30 days, or 40 days, or 50 days, or 60 days, or 70 days, or 80 days, or 90 days.

[1537] 13. In the patient population where all CR-TCES scores were moderate or severe, the mean scores of the subjects' CR-TCES and / or PR-TCES separated from the placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[1538] 14. In the patient population where all CR-TCES scores were moderate or severe, the percentage of subjects achieving a 2-level composite response measured by CR-TCES and / or PR-TCES in at least one treatment area at day 71 was approximately 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or greater than 50%.

[1539] 15. Among the patient population with all CR-TCES scores being moderate or severe, the percentage of subjects achieving a grade 1 composite response measured by CR-TCES and / or PR-TCES in at least one treatment area at day 71 is about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or greater than 50%.

[1540] 16. Among the patient population with all CR-TCES scores being moderate or severe, more than one-third, one-half, two-thirds, or three-fourths of the patients have at least a grade 1 CR-TCES and / or at least a grade 1 PR-TCES response in at least one treatment area at day 71 after treatment, where the measured scoring results of CR-TCES are independent of age, BMI, or skin color.

[1541] 17. The severity of subcutaneous cellulite rapidly decreases within 7 days, 14 days, 21 days, 30 days, 35 days, 40 days, 45 days, or 50 days after the first treatment visit.

[1542] In other embodiments, about 1 mg to about 20 mg of collagenase is injected into at least one treatment area during at least one treatment visit to produce one or more of the results in items 1 to 17 above, where the collagenase has one or more of the following characteristics:

[1543] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment)

[1544] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or, about 0.03 to 3.1 mM (GPA experiment)

[1545] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or,

[1546] about 93 to 9,179 (GPA experiment)

[1547] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[1548] ·K cat / K M , mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment),

[1549] Alternatively, about 60 to 5,934 (GPA assay)

[1550] · having a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa

[1551] · having a purity of at least 80% by area as determined by reverse HPLC (high pressure liquid chromatography)

[1552] · having a potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg

[1553] · having a potency of about 5,000 to about 30,000 f-SRC units / mg

[1554] · having a potency of about 100,000 to about 400,000 GPA units / mg

[1555] · having a potency of about 175,000 to about 500,000 f-GPA units / mg

[1556] · having a potency of about 5,000 to about 25,000 ABC units / mg

[1557] · having a purity of impurities of less than or equal to 1% by area, said impurities being selected from the group consisting of clostripain, gelatinase, and leupeptin

[1558] · having a bioburden of less than or equal to 1 cfu / mL

[1559] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17.

[1560] In other cases, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17, wherein the collagenase has one or more of the following characteristics:

[1561] · V max (min -1 ) is about 0.08 to 7.70 (SRC assay), or, about 0.3 to 30.5 (GPA assay)

[1562] · K M , is about 4.1 to 410 nanomoles (SRC assay), or, about 0.03 to 3.1 mM (GPA assay)

[1563] ·K cat (sec -1 ) is from about 1.1 to about 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment)

[1564] ·1 / K cat , in microseconds is from about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment)

[1565] ·K cat / K M , mM -1 sec -1 is from about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment)

[1566] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1567] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[1568] · The potency (i.e., specific activity) is from about 500 to about 30,000 SRC units / mg · The potency is from about 5,000 to about 30,000 f-SRC units / mg

[1569] · The potency is from about 100,000 to about 400,000 GPA units / mg

[1570] · The potency is from about 175,000 to about 500,00 f-GPA units / mg

[1571] · The potency is from about 5,000 to about 25,000 ABC units / mg

[1572] · The purity of impurities is less than or equal to 1% by area, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin

[1573] · A bioburden of less than or equal to 1 cfu / mL

[1574] In another embodiment, injecting about 1 mg to about 20 mg of collagenase according to any one of Treatments I to V will produce one or more of the above results 1 to 17.

[1575] In certain embodiments, in at least one treatment visit, a ratio of approximately 1 mg to approximately 20 mg of type I collagenase and type II collagenase that is approximately 1:1 is injected into at least one treatment area and one or more of the above results 1 to 10 are obtained, wherein the type I collagenase and type II collagenase have the following characteristics:

[1576] · Type I

[1577] ο Experiment: SRC microplate

[1578] ο Vmax, min -1 : Approximately 0.08 to 7.70

[1579] ο K M : Approximately 4.1 to 410 nanomoles

[1580] ο K cat , see -1 : Approximately 1.1 to 107

[1581] ο 1 / K cat , microseconds: Approximately 376 to 37,222

[1582] ο K cat / K M , mM -1 sec -1 : Approximately 5,140 to 508,814

[1583] · Type II

[1584] ο Experiment: GPA microplate

[1585] ο V max , min -1 : Approximately 0.3 to 30.5

[1586] ο K M , mM: Approximately 0.03 to 3.1

[1587] ο K cat , sec -1 : Approximately 93 to 9,179

[1588] ο 1 / K cat , microseconds: Approximately 4 to 428

[1589] ο K cat / K M , mM -1 sec -1 : Approximately 60 to 5,934

[1590] Other ratios can also be used, (e.g., 0.1 - 2∶1, or 0.25 - 2∶1, or 0.5 - 2∶1, or 0.75 - 2∶1, or 1∶0.1 - 2, or 1∶0.25 - 2, or 1∶0.5 - 2, or 1∶0.75 - 2, or 1∶0, or 0∶1). Further, type I and type II collagenases can be represented as AUX-I and AUX-II, respectively.

[1591] 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 into at least one treatment area using any one of Treatments I to V and one or more of the above results 1 to 17 are obtained, wherein the type I and type II collagenases have the following characteristics:

[1592] ·Type I

[1593] ο Experiment: SRC microplate

[1594] ο Vmax, min -1 : about 0.08 to 7.70

[1595] ο K M : about 4.1 to 410 nanomoles

[1596] ο K cat ,sec -1 : about 1.1 to 107

[1597] ο 1 / K cat ,microsecond: about 376 to 37,222

[1598] ο K cat / K M ,mM -1 sec -1 : about 5,140 to 508,814

[1599] ·Type II

[1600] ο Experiment: GPA microplate

[1601] ο V max ,min -1 : about 0.3 to 30.5

[1602] ο K M ,mM: about 0.03 to 3.1

[1603] ο K cat ,sec -1 : about 93 to 9,179

[1604] ο 1 / K cat, microseconds: approximately 4 to 428

[1605] °K cat / K M , mM -1 sec -1 : approximately 60 to 5,934

[1606] Other ratios can also be used, (e.g., 0.1 - 2:1, or 0.25 - 2:1, or 0.5 - 2:1, or 0.75 - 2:1, or 1:0.1 - 2, or 1:0.25 - 2, or 1:0.5 - 2, or 1:0.75 - 2, or 1:0, or 0:1). Further, type I and type II collagenases can be represented as AUX - I and AUX - II, respectively.

[1607] 12. Efficacy of Body-Q

[1608] According to the Body - Q measurements, the treatment methods described in detail above produce improved treatment effects. For cellulite, a total of 16 scale items measure the patient response over the past week time period, with options ranging from "not at all" to "very bothersome", and are combined into a Flesch - Kincaid grade reading level. The scores vary in the range from 16 (very bothersome) to 64 (not at all). For the total Body - Q score, a responder is a patient whose total Body - Q score increases by at least 16 points compared to the baseline at the assessment time point. For each of the Body - Q impact scores, a response is an improvement of at least one score interval compared to the baseline at each time point. In an alternative embodiment, the number of items scored can be greater than or less than 16, and the responder is a patient with an improvement in the maximum total score of at least 25% compared to the baseline.

[1609] In certain embodiments, the treatment methods detailed above produce one or more of the following efficacy endpoints as measured by Body - Q:

[1610] 1. In a patient population with cellulite, injecting collagenase into at least one treatment area during at least one treatment visit results in a statistically significant number of such patients meeting one or more of the following efficacy endpoints:

[1611] · Body - Q shows improvement in at least one aspect selected from the group consisting of:

[1612] ° at one or more assessment time points, the total score measured by Body - Q is at least 16 points higher than the baseline;

[1613] ° for each of the Body - Q impact scores, there is at least a 1 - point improvement compared to the baseline at each time point; and

[1614] οImprove by at least 25% of the maximum total score starting from the baseline.

[1615] · At one or more evaluation time points, the total Body-Q score increases by at least 16 compared to the baseline.

[1616] · The Body-Q impact score improves by at least 1 point at each time point compared to the baseline.

[1617] · The average change in identifying cellulite by Body-Q compared to the baseline occurs on the 90th day and / or the 180th day.

[1618] 2. For the target thigh, the improvement in severity starting from the baseline (the "first day" before treatment) is that the Body-Q score increases by at least 16 points on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 365th day or 730th day.

[1619] 3. In the patient population with cellulite, for the target thigh, the Body-Q measurement results starting from the baseline (the first day before treatment) on the 22nd day, 43rd day, 71st day, 90th day, 180th day, 365th day or 730th day show that the improvement in severity is statistically significant compared to the placebo, that is, at least 16 points increase.

[1620] 4. The treatment enables at least 5% of the patients to maintain the improvement level compared to the pre-treatment baseline within 71 days after the initial dose. In some cases, at least 10%, or 20%, or 30%, or 40%, or 50% of the patients maintain this level for at least 6 months, or 9 months, or 12 months, or 18 months, or 2 years or 3 years, or 4 years, or 5 years after the first administration. In other cases, the treatment enables at least 5% of the patients to show improvement compared to the pre-treatment baseline and show more improvement over time. Some treatments result in at least 10%, or 20%, or 30%, or 40% or 50% of the patients showing improvement 1 month, or 3 months, or 6 months, or 9 months, or 12 months, or 18 months, or 24 months after the first administration, with more improvement.

[1621] 5. On the 180th day after the first injection, the improvement observed in the Body-Q score is consistent on the left and right thighs compared to the baseline.

[1622] 6. In all patient populations with cellulite, the median time for the total Body-Q score to increase by at least 16 compared to the baseline 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.

[1623] 7. Among all patient groups with subcutaneous cellulite, the mean Body-Q score of the subjects separated from the placebo 21 days after the first treatment and showed continuous and significant improvement after subsequent treatments.

[1624] 8. Among all patient groups with subcutaneous cellulite, more than one-third, one-half, two-thirds, or three-fourths of the patients had a total Body-Q score increase of at least 16 compared to baseline at at least one evaluation time point in at least one treatment area on day 71 after treatment, where the measured Body-Q score results were independent of age, BMI, or skin color.

[1625] 9. The severity of subcutaneous cellulite decreased rapidly within 7, 14, 21, 30, 35, 40, 45, or 50 days after the first treatment visit.

[1626] In other embodiments, about 1 mg to about 20 mg of collagenase is injected into at least one treatment area during at least one treatment visit to produce one or more of the results in items 1 to 9 above, where the collagenase has one or more of the following characteristics:

[1627] ·V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or about 0.3 to 30.5 (GPA experiment)

[1628] ·K M , is about 4.1 to 410 nanomoles (SRC experiment), or about 0.03 to 3.1 mM (GPA experiment)

[1629] ·K cat (sec -1 ) is about 1.1 to about 107 (SRC experiment), or about 93 to 9,179 (GPA experiment)

[1630] ·1 / K cat , microseconds is about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment)

[1631] ·K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or about 60 to 5,934 (GPA experiment)

[1632] · The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa,

[1633] · The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area

[1634] · The potency (i.e...

Claims

1. A method for reducing the severity of cellulite in the subcutaneous fat of the thighs of human patients, comprising the following steps: a. Provide a collagenase composition having at least two of the following characteristics: i.V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, Approximately 0.3 to 30.5 (GPA experiment); ii.K M , from about 4.1 to 410 nanomoles (SRC assay), or, from about 0.03 to 3.1 mM (GPA assay); iii.K cat (sec -1 ) is from about 1.1 to about 107 (SRC experiment), or, from about 93 to 9,179 (GPA experiment); iv. 1 / K cat , in microseconds, is approximately 376 to 37,222 (SRC experiment), or, approximately 4 to 428 (GPA experiment); v.K cat / K M ,mM -1 sec -1 was approximately 5,140 to 508,814 (SRC experiment), or, approximately 60 to 5,934 (GPA experiment); vi. Molecular weight is 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; vii. Purity determined by reverse-phase HPLC (high-pressure liquid chromatography) is at least 80% by area; viii. Potency is approximately 5,000 to approximately 30,000 f-SRC units / mg; ix. Potency is approximately 175,000 to approximately 500,000 f-GPA units / mg; x. Potency is approximately 5,000 to approximately 25,000 ABC units / mg; xi. Impurities less than or equal to 1% by area, said impurities selected from the group consisting of clostripain, gelatinase, and leupeptin; xii. Bioburden less than or equal to 1 cfu / mL; And b. Inject a therapeutically effective amount of the collagenase composition according to a treatment regimen selected from the group consisting of Treatments I to V, wherein the improvement in the appearance of cellulite is established by a scale or other measurement tool selected from the following group: Hexsel Cellulite Severity Scale (Hexsel CSS), Hexsel Depression Depth Scale, Likert Scale, Pit Analysis, Clinician-Reported Photographic Digital Cellulite Severity Scale (CR-PCSS), Patient-Reported Photographic Digital Cellulite Severity Scale (PR-PCSS), Investigator Global Aesthetic Improvement Scale (I-GAIS), Subject Global Aesthetic Improvement Scale (S-GAIS), Patient-Reported Cellulite Impact Scale (PR-CIS), Simplified Version of PR-CIS, Patient Self-Assessment Form (SSRS), Subject Satisfaction Questionnaire for Cellulite Treatment (SSCT), Clinician's Assessment of Cellulite Severity (photographic or other images), Body-Q, Thigh Cellulite Efficacy Scale (PR-TCES; CR-TCES), and any valid image digital or other clinical use and / or patient scale for evaluating cellulite severity, improvement, and / or patient satisfaction.

2. The method according to claim 1, wherein The collagenase is injected according to Treatment IV.

3. The method according to claim 1, wherein, The collagenase composition includes AUX-I and AUX-II and has the following characteristics: a. AUX-I (SRC experiment): i.V max ,min -1 : about 0.08 to 7.70 ii.K M : about 4.1 to 410 nanomoles iii.K cat , sec -1 : from about 1.1 to about 107 iv. 1 / K cat , microseconds: approximately 376 to 37,222 v.K cat / K M ,mM -1 sec -1 : approximately 5,140 to 508,814 b. AUX-II (GPA experiment) i.V max , min -1 : about 0.3 to 30.5 ii.K M , mM: about 0.03 to 3.1 iii.K cat , sec -1 : approximately 93 to 9,179 iv. 1 / K cat , microseconds: approximately 4 to 428 v.K cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934.

4. The method according to claim 1, wherein the collagenase composition includes AUX-I and AUX-II and has the following characteristics: a. AUX-I (SRC experiment): i.Vmax, min -1 : Approximately 3.8 ii.K M , mM: approximately 2.07x10 -4 iii.K cat ,sec -1 : about 53 iv. 1 / K cat , microseconds: approximately 18,799 v.k cat / K M ,mM -1 sec -1 : Approximately 256,977 b. AUX II (GPA experiment): i.Vmax, min -1 : Approximately 15.4 ii.K M , mM: approximately 1.6 iii.K cat , sec -1 : approximately 4,636 iv. 1 / K cat , microseconds: approximately 216 v.k cat / K M ,mM -1 sec -1 : Approximately 2,997.

5. The method according to claim 1, wherein the composition includes at least 3 characteristics.

6. The method according to claim 1, wherein the composition comprises at least 4 features.

7. The method according to claim 1, wherein the composition comprises at least 6 features.

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

9. The method according to claim 1, wherein the composition comprises CCH.

10. The method according to claim 1, wherein the composition has an efficacy of about 10,000 ABC units / 0.58 mg and the therapeutically effective amount is from about 1 mg to 20 mg.

11. The method according to claim 1, wherein the composition has an efficacy of from about 15,000 ABC units / mg to 20,000 ABC units / mg and the therapeutically effective amount is from about 1 mg to 20 mg.

12. The method according to claim 1, wherein the therapeutically effective amount is from about 1 mg to 10 mg and the composition has an efficacy of from about 20,000 to about 30,000 f-SRC units / mg or from about 175,000 to about 300,000 f-GPA units / mg.

13. The method according to claim 1, wherein the treatment is administration to a patient population, and the treatment enables at least 5% of the patients to maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

14. The method according to claim 13, wherein at least 10% of the patients maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

15. The method according to claim 13, wherein at least 20% of the patients maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

16. The method according to claim 1, wherein when administered to a patient population, the treatment enables at least 5% of the patients to have an improved effect compared to the pre-treatment baseline, and the improvement becomes more significant over time.

17. The method according to claim 1, wherein determined by CR-PCSS and / or PR-PCSS measurement, the treatment produces one or more of the following efficacy endpoints: a. Clinician on-site assessment of the target thigh, at day 22, 43, 71, 90, 180 or 365 starting from the baseline (pre-treatment "day one"), with at least a 2-grade improvement in the severity of CR-PCSS; b. Subject's observation and evaluation of digital images of the target thigh, at day 22, 43, 71, 90, 180 or 365 starting from the baseline (day one), with at least a 2-grade improvement in the severity of PR-PCSS; c. Improvement demonstrated by a 2-grade composite response at day 22, 43, 71, 90, 180 or 365, defined as the subject having at least a 2-grade improvement in severity in CR-PCSS compared to the baseline and at least a 2-grade improvement in severity in PR-PCSS compared to the baseline; d. On the 22nd day, 43rd day, 71st day, 90th day, 180th day, or 365th day, the severity of cellulite in the CR-PCSS is improved by at least 1 grade compared to the baseline as evaluated on-site by a clinician of the target thigh; e. On the 22nd day, 43rd day, 71st day, 90th day, 180th day, 365th day, the severity of cellulite in the PR-PCSS is improved by at least 1 grade compared to the baseline as evaluated by the subject observing digital images of the target thigh; f. On the 22nd day, 43rd day, 71st day, 90th day, 180th day, or 365th day, the improvement demonstrated by a Grade 1 composite response is defined as the severity of the subject in the CR-PCSS being improved by at least 1 grade compared to the baseline, the severity in the PR-PCSS being improved by at least 1 grade compared to the baseline; and g. In the patient population with all CR-PCSS scores being moderate or severe, the improvement in at least one treatment area was statistically significant compared with placebo, where the improvement is one or more of the above a to f.

18. The method according to claim 1, wherein it is determined by measurement using the Hexsel dimple depth scale, and the treatment produces one or more of the following efficacy endpoints: a. As evaluated on-site by a clinician of the target thigh, starting from the baseline (the "first day" before treatment), on the 22nd day, 43rd day, 71st day, 90th day, 180th day, or 365th day, the severity of the Hexsel dimple depth score is improved by at least 2 grades; b. As evaluated by the subject observing digital images of the target thigh, starting from the baseline (the first day), on the 22nd day, 43rd day, 71st day, 90th day, 180th day, or 365th day, the severity of the Hexsel dimple depth score is improved by at least 2 grades; c. The improvement demonstrated by a Grade 2 composite response on the 22nd day, 43rd day, 71st day, 90th day, 180th day, or 365th day is defined as the severity of the subject in the Hexsel dimple depth score being improved by at least 2 grades compared to the baseline as evaluated by the clinician, and the severity being improved by at least 2 grades compared to the baseline as evaluated by the subject; d. Starting from the baseline (the first day), on the 22nd day, 43rd day, 71st day, 90th day, 180th day, or 365th day, as evaluated on-site by a clinician, the severity of cellulite in the Hexsel dimple depth score of the target thigh is improved by at least 1 grade compared to the baseline; e. Starting from the baseline (the first day), on the 22nd day, 43rd day, 71st day, 90th day, 180th day, or 365th day, as evaluated by the subject observing digital images of the target thigh, the severity of cellulite in the Hexsel dimple depth score is improved by at least 1 grade compared to the baseline; f. Starting from the baseline (the first day), on the 22nd day, 43rd day, 71st day, 90th day, 180th day, or 365th day, the improvement demonstrated by a Grade 1 composite response is defined as the severity of the subject in the Hexsel dimple depth score being improved by at least 1 grade compared to the baseline as evaluated by the clinician, and the severity being improved by at least 1 grade compared to the baseline as evaluated by the subject; and g. In the patient population where all Hexsel CSS scores are moderate or severe, The improvement in at least one treatment area was statistically significant compared to placebo, where the improvement is one or more of a to f above.

19. The method according to claim 1, wherein the treatment determined by pit analysis produces one or more of the following efficacy endpoints: a. The depth is reduced by at least 5%; b. The width is reduced by at least 5%; c. The length is reduced by at least 5%; d. The total volume is reduced by at least 5%; and e. The surface area is reduced by at least 5%.

20. A method for reducing the severity of cellulite in the thighs of a human patient, comprising the steps of: a. Providing a collagenase composition having at least two of the following characteristics: i.V max (min -1 ) is about 0.08 to 7.70 (SRC experiment), or, about 0.3 to 30.5 (GPA experiment); ii.K M , from about 4.1 to 410 nanomoles (SRC assay), or, from about 0.03 to 3.1 mM (GPA assay); iii.K cat (sec -1 ) is about 1.1 to 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment); iv. 1 / K cat , in microseconds is about 376 to 37,222 (SRC experiment), or, about 4 to 428 (GPA experiment); v.K cat / K M ,mM -1 sec -1 is about 5,140 to 508,814 (SRC experiment), or, about 60 to 5,934 (GPA experiment); vi. The molecular weight is in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa; vii. The purity determined by reverse HPLC (high performance liquid chromatography) is at least 80% by area; viii. The potency (i.e., specific activity) is about 5000 to about 30,000 SRC units / mg; ix. The potency is about 175,000 to about 500,00 f-GPA units / mg; x. The potency is about 5,000 to about 25,000 ABC units / mg; xi. The purity of impurities by area is less than or equal to 1%, and the impurities are selected from the group consisting of clostripain, gelatinase, and leupeptin; xii. The bioburden is less than or equal to 1 cfu / mL; and b. Injecting a therapeutically effective amount of the collagenase composition according to a treatment regimen selected from the group consisting of Treatments I to V, wherein the color intensity of the bruise is significantly reduced or alleviated between about 3 days and 20 days of the treatment visit.

21. The method according to claim 20, wherein, Inject collagenase according to Treatment IV.

22. The method according to claim 20, wherein, The collagenase composition includes AUX-I and AUX-II and has the following characteristics: a. AUX-I (SRC experiment): i.V max (min -1 ):Approximately 0.08 to 7.70 ii.K M : about 4.1 to 410 nanomoles iii.K cat , sec -1 : from about 1.1 to about 107 iv. 1 / K cat , microseconds: approximately 376 to 37,222 v.K cat / K M ,mM -1 sec -1 : approximately 5,140 to 508,814 b. AUX-II (GPA experiment) i.V max , min -1 : approximately 0.3 to 30.5 ii.K M , mM: about 0.03 to 3.1 iii.K cat , sec -1 : approximately 93 to 9,179 iv. 1 / K cat , microseconds: approximately 4 to 428 v.K cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934.

23. The method according to claim 20, wherein The collagenase composition includes AUX-I and AUX-II and has the following characteristics: a. AUX-I (SRC experiment): i.V max , min -1 : Approximately 3.8 ii.K M , mM: approximately 2.07x10 -4 iii.K cat , sec -1 : approximately 53 iv. 1 / K cat , microseconds: approximately 18,799 v.k cat / K M ,mM -1 sec -1 : Approximately 256,977 b. AUX II (GPA experiment): i.V max ,min -1 : Approximately 15.4 ii.K M , mM: approximately 1.6 iii.K cat , sec -1 : approximately 4,636 iv. 1 / K cat , microsecond: approximately 216 v.k cat / K M ,mM -1 sec -1 : Approximately 2,997.

24. The method according to claim 20, wherein The composition includes at least 3 characteristics.

25. The method according to claim 20, wherein The composition includes at least 4 characteristics.

26. The method according to claim 20, wherein, The composition includes at least 5 characteristics.

27. The method according to claim 20, wherein The composition includes one or more collagenases in an amount of about 1 mg to 20 mg.

28. The method according to claim 20, wherein The composition includes CCH.

29. The method according to claim 20, wherein, The composition has an efficacy of about 10,000 ABC units / 0.58 mg and the therapeutically effective amount is about 1 mg to 20 mg.

30. The method according to claim 20, wherein The composition has an efficacy 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.

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

32. The method according to claim 20, wherein The treatment is administration to a patient population, and the treatment enables at least 5% of the patients to maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

33. The method according to claim 32, wherein at least 10% of the patients maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

34. The method according to claim 32, wherein at least 20% of the patients maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

35. The method according to claim 20, wherein When administered to a patient population, the treatment enables at least 5% of the patients to have an improved effect compared to the pre-treatment baseline, and the improvement becomes more significant over time.

36. A method for reducing the severity of cellulite in the thighs of a human patient, comprising the steps of: a. providing a collagenase composition having at least two of the following characteristics: i.V max (min -1 ) is from about 0.08 to 7.70 (SRC experiment), or, from about 0.3 to 30.5 (GPA experiment); ii.K M , from about 4.1 to 410 nanomoles (SRC experiment), or, from about 0.03 to 3.1 mM (GPA experiment); iii.K cat (sec -1 ) of about 1.1 to 107 (SRC experiment), or, about 93 to 9,179 (GPA experiment); iv. 1 / K cat , in microseconds is about 376 to 37,222 (SRC experiment), or about 4 to 428 (GPA experiment); v.K cat / K M ,mM -1 sec -1 are approximately 5,140 to 508,814 (SRC experiment), or, approximately 60 to 5,934 (GPA experiment); vi. a molecular weight in the range of about 60 kDa to about 130 kDa, or about 70 to about 130 kDa, or about 80 to about 120 kDa, or about 90 to about 120 kDa, or about 100 to about 110 kDa: vii. a purity determined by reverse HPLC (high performance liquid chromatography) of at least 80% by area; viii. a potency (i.e., specific activity) of about 5000 to about 30,000 SRC units / mg; ix. a potency 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. a purity of impurities by area of less than or equal to 1%, the impurities selected from the group consisting of clostripain, gelatinase, and leupeptin; xii. a bioburden of less than or equal to 1 cfu / mL; and b. injecting a therapeutically effective amount of the collagenase composition according to a treatment regimen selected from the group consisting of Treatments I to V, wherein the appearance of cellulite is improved as determined by the Thigh Subcutaneous Cellulite Efficacy Scale (PR-TCES and / or CR-TCES).

37. The method according to claim 36, wherein, Inject collagenase according to Treatment IV.

38. The method according to claim 36, wherein, The collagenase composition comprises AUX-I and AUX-II and has the following characteristics: a. AUX-I (SRC experiment): i.V max (min -1 ):About 0.08 to 7.70 ii.K M : about 4.1 to 410 nanomoles iii.K cat , sec -1 : from about 1.1 to about 107 iv. 1 / K cat , microseconds: approximately 376 to 37,222 v.K cat / K M ,mM -1 sec -1 : approximately 5,140 to 508,814 b. AUX-II (GPA experiment) i.V max , min -1 : about 0.3 to 30.5 ii.K M , mM: from about 0.03 to 3.1 iii.K cat , sec -1 : approximately 93 to 9,179 iv. 1 / K cat , microseconds: approximately 4 to 428 v.K cat / K M ,mM -1 sec -1 : Approximately 60 to 5,934.

39. The method according to claim 36, wherein, The collagenase composition comprises AUX-I and AUX-II and has the following characteristics: a. AUX-I (SRC experiment): i.V max , min -1 : Approximately 3.8 ii.K M , mM: approximately 2.07 x 10 -4 iii.K cat ,sec -1 :about 53 iv. 1 / K cat , microseconds: approximately 18,799 v.k cat / K M ,mM -1 sec -1 : Approximately 256,977 b. AUX II (GPA experiment): i.V max , min -1 : Approximately 15.4 ii.K M , mM: approximately 1.6 iii.K cat , sec -1 : approximately 4,636 iv. 1 / K cat , microseconds: approximately 216 v.k cat / K M ,mM -1 sec -1 : Approximately 2,997.

40. The method according to claim 36, wherein The composition comprises at least 3 characteristics.

41. The method according to claim 36, wherein The composition comprises at least 4 characteristics.

42. The method according to claim 36, wherein, The composition comprises at least 5 characteristics.

43. The method according to claim 36, wherein, The composition comprises one or more collagenases in an amount of about 1 mg to 20 mg.

44. The method according to claim 36, wherein, The composition comprises CCH.

45. The method according to claim 36, wherein The efficacy of the composition is approximately 10,000 ABC units / 0.58 mg and the therapeutically effective amount is approximately 1 mg to 20 mg.

46. The method according to claim 36, wherein The efficacy of the composition is approximately 15,000 ABC units / mg to 20,000 ABC units / mg and the therapeutically effective amount is approximately 1 mg to 20 mg.

47. The method according to claim 36, wherein The therapeutically effective amount is approximately 1 mg to 10 mg and the efficacy of the composition is approximately 20,000 to approximately 30,000 f-SRC units / mg or approximately 175,000 to approximately 300,000 f-GPA units / mg.

48. The method according to claim 36, wherein The treatment is administered to a patient population, and the treatment enables at least 5% of the patients to maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

49. The method according to claim 48, wherein at least 10% of the patients maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

50. The method according to claim 48, wherein at least 20% of the patients maintain an improved level compared to the pre-treatment baseline at least 71 days after administration of the initial dose.

51. The method according to claim 48, wherein, When administered to a patient population, the treatment enables at least 5% of the patients to have an improved effect compared to the pre-treatment baseline, and the improvement becomes more significant over time.

52. The method according to claim 36, wherein, As determined by CR-TCES and / or PR-TCES, the treatment produces one or more of the following efficacy endpoints: a. Clinician on-site assessment of the target thigh shows that the severity of the CR-TCES score improves by at least 2 levels at day 22, day 43, day 71, day 90, day 180, or day 365 starting from the baseline (pre-treatment "day 1"). b. Subject observation of digital images of the target thigh shows that the severity of the PR-TCES score improves by at least 2 levels at day 22, day 43, day 71, day 90, day 180, or day 365 starting from the baseline (day 1). c. Improvement demonstrated by a level 2 composite response at day 22, day 43, day 71, day 90, day 180, or day 365, which is defined as the severity improving by at least 2 levels compared to the baseline in CR-TCES measurement and the severity improving by at least 2 levels compared to the baseline in PR-TCES measurement; d. Clinician on-site assessment of the target thigh shows that the severity of the CR-TCES score improves by at least 1 level at day 22, day 43, day 71, day 90, day 180, or day 365 starting from the baseline (day 1). e. Subject observation of digital images of the target thigh shows that the severity of the PR-TCES score improves by at least 1 level at day 22, day 43, day 71, day 90, day 180, or day 365 starting from the baseline (day 1). f. Improvement demonstrated by a Grade 1 composite response on Day 22, Day 43, Day 71, Day 90, Day 180, or Day 365 is defined as at least a 1-grade improvement in severity compared to baseline in CR-TCES measurements and at least a 1-grade improvement in severity compared to baseline in PR-TCES measurements; g. In the patient population where all CR-TCES scores are moderate or severe, improvement in at least one treatment area is statistically significant compared to placebo, where the improvement is one or more of a to f above.

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