Methods of treating plantar fasciitis

Abstract

Disclosed herein are methods of treating plantar fasciitis (PFA) in a subject comprising injecting a pharmaceutical formulation comprising collagenase into one or more plantar fasciitis affected areas in the subject's foot to thereby treat the plantar fasciitis.

Description

METHODS OF TREATING PLANTAR FASCIITISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 678,192 filed on August 1, 2024, which is incorporated herein by reference in its entirety to the full extent permitted by law.FIELD

[0002] Disclosed herein are methods of treating plantar fasciitis and the use of collagenase in the treatment of plantar fasciitis.BACKGROUND

[0003] Plantar fasciitis (“PF A”) is the most common cause of plantar heel pain, accounting for 11% to 15% of all foot complaints. Cheney N, Sharpe BD, Lauf J, Long J. Controversies in surgical treatment of recalcitrant plantar fasciitis. Tech Foot Ankle Surg. 2021;20(2):86-102 (“Cheney et al, 2021”). It is characterized by inferior heel pain, often with the first step out of bed in the morning that tends to decrease with continued activity. Pain recurs when activity resumes after a period of rest. Motley, Plantar fasciitis / fasciosis. Clin Podiatr Med Surg. 2021;38(2): 193-200. Generally, the duration of symptoms helps to define the 3 phases of plantar fasciitis: acute, subacute, and chronic. Acute plantar fasciitis refers to the initial 4 to 6 weeks after onset. It can be either traumatic in etiology or due to mechanical overload. Subacute plantar fasciitis is usually present for approximately 6 to 12 weeks, and chronic plantar fasciitis is present for more than 3 months. A further characterization of chronic plantar fasciitis is refractory / recalcitrant. Recalcitrant plantar fasciitis is best defined as chronic plantar fasciitis that has not improved with appropriate intervention for more than 6 months and is much more difficult to successfully treat. Schneider HP, Baca JM, Carpenter BB, Dayton PD, Fleischer AE, Sachs BD. American College of Foot and Ankle Surgeons Clinical Consensus Statement: Diagnosis and Treatment of Adult Acquired Infracalcaneal Heel Pain. J Foot Ankle Surg. 20I8;57(2):370-38I.

[0004] The etiology of plantar fasciitis is not fully understood. The plantar fascia (or aponeurosis) is a dense connective tissue, comprised primarily of fibrocytes that serve as a link between the origin on the plantar medial tubercle of the calcaneus and the forefoot. The proximal attachment to the calcaneus is fibrocartilaginous. Currently, the treatment for acute and chronic plantar fasciitis is similar with varying degrees of success and includes conservative treatments including physical therapy (strapping, stretching, rolling, massage), non-steroid anti-inflammatory drugs, corticosteroid injections, platelet-rich plasma injections, autologous whole blood injections, botulinum toxin injection, orthotics, heel cups, night splints, electrotherapy, needling, proliferation therapy, and extracorporeal shock wave therapy. Latt LD, Jaffe DE, Tang Y, Talj anovic MS. Evaluation and treatment of chronic plantar fasciitis. Foot Ankle Orthop. 2020;5(l):2473011419896763; Motley, 2021.

[0005] While non-operative treatments (stretching, physical therapy, orthotics) have a lower risk of complications than invasive options, other treatments, including corticosteroids, can cause adverse events (AEs). These include a 2.4% rate of plantar fascial rupture and soft tissue related AEs, including postinjection flare, skin atrophy, and fat pad atrophy. Cheney et al., 2021; Cole BJ, Schumacher HR Jr. Injectable corticosteroids in modem practice. J Am Acad Orthop Surg. 2005; 13(1): 37-46. Plantar fasciitis that has not improved with appropriate intervention for greater than 6 months is considered chronic and is much more difficult to treat successfully. Schneider et al, 2018. Surgical interventions for plantar fasciitis include plantar fasciotomy (open and endoscopic and / or complete or partial) during which the plantar fascia is released. Yanbin X, Haikun C, Xiaofeng J, Wanshan Y, Shuangping L. Treatment of chronic plantar fasciitis with percutaneous latticed plantar fasciotomy. J Foot Ankle Surg. 2015;54(5): 856-859; Schneider et al, 2018; Latt et al, 2020. While surgical techniques may vary between surgeons, plantar fascial release / fasciotomy generally includes a medial longitudinal incision centered on the anterior inferior aspect of the calcaneus approximately 3 to 4 cm below the medial malleolus. The origin of the plantar fascia is identified and isolated by blunt dissection from the superficial fatty layer and the deep first muscular layer. The entire plantar fascia is then divided from medial to lateral sides using either scissors or a scalpel to disrupt the fascia. Postoperatively, the foot is bandaged; patients are provided crutches and are instructed to be partial weightbearing for 2 weeks at which point a wound check is performed. Patients gradually increase weightbearing as tolerated over a further 2- to 4-week period. Benton-Weil W, Borrelli AH, Weil LS Jr, Weil LS Sr. Percutaneous plantar fasciotomy: a minimally invasive procedure for recalcitrant plantar fasciitis. J Foot Ankle Surg. 1998;37(4):269-272; Wheeler P, Boyd K, Shipton M. Surgery for patients with recalcitrant plantar fasciitis: good results At short-, medium-, and long-term follow-up. Orthop J Sports Med. 2014;2(3):2325967114527901.

[0006] The potential risks of surgery include biomechanical changes including flattening of the longitudinal arch and heel hypoesthesia, worsening or persistent heel pain from reflex sympathetic dystrophy, and potential complications associated with rupture of the plantarfascia. These biomechanical changes may not present acutely but may cause long-term sequelae and progression of deformity. Relapses after surgery are common due to pain-induced by postoperative scarring. Sammarco GJ, Helfrey RB. Surgical treatment of recalcitrant plantar fasciitis. Foot Ankle Int. 1996;17(9):520-526; Yanbin et al, 2015. Other risks of surgery include injury to the lateral plantar nerve, lateral column pain, and wound complications. Cheney et al, 2021. There is a need for a non-surgical treatment option in plantar fasciitis to avoid the above disadvantages of current treatments.SUMMARY

[0007] Disclosed herein are methods of treating plantar fasciitis in a subject, the methods comprising injecting a pharmaceutical formulation comprising collagenase into one or more plantar fasciitis affected areas in the subject’s foot to thereby treat the plantar fasciitis. Also disclosed herein is the use of collagenase in the preparation of a medicament for treating plantar fasciitis in a subject, wherein the medicament is prepared for injection into one or more plantar fasciitis affected areas in the subject’s foot.

[0008] In certain embodiments, the present disclosure is to a method of treating plantar fasciitis in a subject, the method comprising: injecting a pharmaceutical formulation comprising collagenase into a plantar fasciitis affected area to treat the plantar fasciitis in an amount sufficient to treat the plantar fasciitis. In one embodiment, the total dose administered is an amount sufficient to alleviate the pain associated with plantar fasciitis. Such dose may comprise between about 0.2 mg to about 2.0 mg collagenase. The collagenase may comprise, in some embodiments, collagenase I activity, collagenase II activity, or a combination thereof. In certain embodiments, the collagenase has a potency of about 5 SRC units / mg to about 180,000 SRC units / mg, or a potency of about 500 SRC units / mg to about 30,000 SRC units / mg, or a potency of about 10,000 GPA units / mg to about 400,000 GPA units / mg, or a potency of about 10,000 GPA units / mg to about 200,000 GPA units / mg.

[0009] In other embodiments, the collagenase comprises a mixture of collagenase I and collagenase II activity, wherein the collagenase I potency is about 5 SRC units / mg to about 180,000 SRC units / mg, and the collagenase II potency is about 10,000 GPA units / mg to about 400,000 GPA units / mg. Further, the collagenase may comprise collagenase I and collagenase II from Clostridium histolyticum. The collagenase I may comprise the amino acid sequence of SEQ ID NO: 1. Alternatively, the collagenase may comprise a collagenase II where, in some embodiments, the collagenase II has a potency of about 10,000 GPA units / mg to about 400,000 GPA units / mg, or about 10,000 GPA units / mg to about 300,000 GPA units / mg. Thecollagenase II may comprise the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the collagenase may comprise a mixture of collagenase I and collagenase II where, in some embodiments, the collagenase I has a potency of about 5 SRC units / mg to about SRC 180,000 units / mg, and the collagenase II has a potency of about 10,000 GPA units / mg to about 400,000 GPA units / mg.

[0010] In another embodiment, about 0.2 mg to about 0.6 mg of collagenase is injected to the affected area, or about 0.2 mg of collagenase is injected to the affected area, or about 0.3 mg of collagenase is injected to the affected area, or about 0.6 mg of collagenase is injected to the affected area. In certain embodiments, the method comprises the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase, which results in an improvement in Pain Intensity NRS Score. In the Pain Intensity NRS Score, the injection results in a negative change from baseline of about 0.0 to -2.0 at Day 7 after treatment, or about -0.5 to about -4 at Day 14 after treatment, or about -1 to about -5 at Day 28 after treatment, or about -1.5 to about -6 at one or more of Days 42, 56, or 84 after treatment.

[0011] In another embodiment, the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Foot Pain Severity Interference in PFA Scale. In the Foot Pain Severity Interference in PFA Scale, the injection results in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

[0012] In certain embodiments, the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Foot Pain Frequency Impact in PFA Scale. In the Foot Pain Frequency Impact in PFA Scale, the injection results in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

[0013] In another embodiment, the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Subject Satisfaction with Treatment Scale. In the Subject Satisfaction with Treatment Scale, the injection injected to a plurality of subjects results in at least 25%, or at least 35%, or at least 45%, or at least 55% of the subjects reporting they are “Quite Satisfied” or “Very Satisfied” at one or more of Days 7, 14, 28, 42, 56, or 84 following collagenase treatment.

[0014] In further embodiments, the present disclosure is directed to a method of treating plantar fasciitis in a subject, comprising injecting a collagenase enzyme to a plantar fasciitis affected area, the collagenase enzyme comprising:(a) means for binding or recruiting collagen Type I and Type III; and(b) means for breaking down collagen Type I and / or, collagen Type III, wherein a dose of collagenase injected comprises an amount sufficient to treat the plantar fasciitis.In certain embodiments, the total dose injected comprises between about 0.2 mg to about 1.0 mg.

[0015] Additional embodiments of the present processes, compositions, methods of treatment and the like will be apparent from the following description, drawings, examples, and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment or aspect. Additional 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

[0016] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, there are shown in the drawings exemplary embodiments of the methods; however, the methods are not limited to the specific embodiments disclosed. Pursuant to 37 C.F.R. § 1.84, the patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. In the drawings:

[0017] FIG. 1 is a schema depicting the clinical study flow of Study EN3835-108 PFA.

[0018] FIG. 2 is the clinical study schematic diagram of Study EN3835-108 PFA.

[0019] FIG. 3 illustrates where the three injection sites are marked in a triangular shape.

[0020] FIG. 4 is a line graph showing the Observed Mean Pain Intensity Numerical Ranking Scale (NRS) Score by treatment group (Day 1 injection of placebo or about 0.2 mg,0.34 mg, or 0.6 mg collagenase) wherein the Pain Intensity NRS Score is measured at Days 1, 7, 14, 28, 42, 56, and 84.

[0021] FIG. 5 is a line graph showing the Mean Change from Baseline in Pain Intensity NRS Score by treatment group (Day 1 injection of placebo or about 0.2 mg, 0.34 mg, or 0.6 mg collagenase) wherein the NRS Score is measured at Days 1, 7, 14, 28, 42, 56, and 84.

[0022] FIG. 6 is a line graph of the data plotted in FIG. 4 with the vertical axis adjusted for clarity.

[0023] FIG. 7 is a line graph of the data plotted in FIG. 5 with the vertical axis adjusted for clarity.

[0024] FIG. 8 is a line graph showing Mean Subject Satisfaction by treatment group (Day 1 injection of placebo or about 0.2 mg, 0.34 mg, or 0.6 mg collagenase) wherein the Mean Subject Satisfaction is measured at Days 1, 7, 14, 28, 42, 56, and 84.

[0025] FIG. 9 is a line graph of the data plotted in FIG. 8 with the vertical axis adjusted for clarity.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0026] The disclosed methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods.

[0027] Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

[0028] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where a range ofnumerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. All ranges are inclusive and combinable. The disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values.

[0029] When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0030] It is to be appreciated that certain features of the disclosed methods which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.A. DEFINITIONS

[0031] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0032] As used herein, the singular forms “a,” “an,” and “the” include the plural.

[0033] The term “about” when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. Thus, the term “about” is used to encompass variations of ± 10% or less, variations of ± 5% or less, variations of ± 1% or less, variations of ± 0.5% or less, or variations of ± 0. 1% or less from the specified value.

[0034] The term “administering” and similar terms indicate a procedure by which the pharmaceutical formulation is injected into a subject such that the area(s) of the foot affected by plantar fasciitis is contacted with the pharmaceutical formulation.

[0035] The term “biosimilar” (of an approved reference product / biological drug, i.e., reference listed drug) refers to a biological product that is highly similar to the reference product notwithstanding minor differences in clinically inactive components with no clinically meaningful differences between the biosimilar and the reference product in terms of safety, purity and potency, based upon data derived from (a) analytical studies that demonstrate thatthe biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components; (b) animal studies (including the assessment of toxicity); and / or (c) a clinical study or studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity, and potency in one or more appropriate conditions of use for which the reference product is licensed and intended to be used and for which licensure is sought for the biosimilar. The biosimilar may be an interchangeable product that may be substituted for the reference product at the pharmacy without the intervention of the prescribing healthcare professional. To meet the additional standard of “interchangeability,” the biosimilar is to be expected to produce the same clinical result as the reference product in any given patient and, if the biosimilar is administered more than once to an individual, the risk in terms of safety or diminished efficacy of alternating or switching between the use of the biosimilar and the reference product is not greater than the risk of using the reference product without such alternation or switch. The biosimilar utilizes the same mechanisms of action for the proposed conditions of use to the extent the mechanisms are known for the reference product. The condition or conditions of use prescribed, recommended, or suggested in the labeling proposed for the biosimilar have been previously approved for the reference product. The route of administration, the dosage form, and / or the strength of the biosimilar are the same as those of the reference product and the biosimilar is manufactured, processed, packed or held in a facility that meets standards designed to assure that the biosimilar continues to be safe, pure and potent. The biosimilar may include minor modifications in the amino acid sequence when compared to the reference product, such as N- or C-terminal truncations that are not expected to change the biosimilar performance.

[0036] The term “comprising” is intended to include examples encompassed by the terms “consisting essentially of’ and “consisting of’; similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0037] The term “Day” as used herein refers to the timing of when a subject receives a collagenase dose and dates of follow-up visits, i.e., Day 1 is when a subject receives a dose of collagenase, Day 7 is 6 days after Day 1, Day 14 is 13 days after Day 1, etc.

[0038] The term “subject” as used herein is intended to mean any animal, in particular, mammals. The methods are applicable to human and nonhuman animals, although most preferably with humans. “Subject” and “patient” can be used interchangeably herein.

[0039] The term “plantar fasciitis” or “PFA” refers to inflammation of a thick band of tissue that connects the heel bone to the toes. It is one of the most common causes of heel pain.The most common symptom is a stabbing pain in the bottom of the foot near the heel. Pain is usually the worst in the morning. Generally, the duration of symptoms helps to define the 3 phases of plantar fasciitis: acute, subacute, and chronic. Acute plantar fasciitis refers to the initial 4 to 6 weeks after onset. It can be either traumatic in etiology or due to mechanical overload. Subacute plantar fasciitis is usually present for approximately 6 to 12 weeks, and chronic plantar fasciitis is present for more than 3 months. Refractory or recalcitrant plantar fasciitis refers to chronic plantar fasciitis that has not improved with appropriate intervention for more than 6 months and is more difficult to successfully treat.

[0040] ‘ ‘Treat,” “treatment,” and like terms include one or more of the following: reducing and / or eliminating the pain or discomfort associated with plantar fasciitis, reducing and / or eliminating the underlying cause of plantar fasciitis, reducing and / or eliminating the likelihood of the occurrence of plantar fasciitis, improving the subject’s ability to stand, walk, and / or place weight on the foot, and inducing and / or improving the subject’s overall comfort.

[0041] The term “treatment session” is synonymous with “treatment visit” and includes a single visit to a doctor’s office in which the pharmaceutical formulation is administered.

[0042] The term “Pain Intensity Numeric Rating Scale (NRS)” or “Pain Intensity NRS” refers to a patient-reported outcome assessment using a single-item NRS ranging from 0 (“No Pain”) to 10 (“Worst Pain Imaginable”) to capture the intensity for foot pain related to plantar fasciitis, as follows:Pain Intensity Numeric Rating Scale (NRS)Please choose the response below that best describes your foot pain in the past 24 hours.□ □ □ □ □ □ □ □ □ □ a0 1 2 3 4 5 6 7 8 9 10Wherein 0 is “no pain” and 10 is “worst pain imaginable.”

[0043] The term “Foot Pain Severity Interference in PFA Scale” refers to a patient- reported outcome assessment where each study participant is asked to characterize the severity of foot pain when trying to perform activities of daily living. The severity of foot pain is characterized on a 5-point ordinal scale ranging from 0 indicating "None" to 4 indicating "Very Severe." Activities of daily living include:1. Taking the first steps in the morning2. After standing for a long time3. Walking after sitting for a long time4. Walking after standing for a long time5. Walking a short distance (for example, in and / or around the home)6. Walking longer distances (outside the home, more than a block)7. At the end of the day

[0044] The term “Foot Pain Frequency Impact in PFA Scale” refers to a patient- reported outcome assessment where each study participant is asked to characterize the frequency of foot pain on 5 -point ordinal scale during 3 activities of daily living. The scale ranges from 0 indicating "Never" to 4 indicating "Always." The activities of daily living include:1. Sleep (e.g., falling asleep and / or staying asleep)2. Ability to complete daily routine activities (e.g., housework, yardwork, running errands)3. Ability to participate in recreational activities (e.g., exercise, running, traveling)

[0045] The term “Subject Satisfaction with Treatment” refers to a patient-reported outcome assessment using a multipoint scale to assess his / her satisfaction with treatment of their plantar fasciitis on a 5-point scale ranging from -2 (“Very Dissatisfied”) to +2 (“Very Satisfied”) as follows:-2 Very Dissatisfied-1 Quite Dissatisfied0 Neither Satisfied nor Dissatisfied+1 Quite Satisfied+2 Very Satisfied

[0046] The term “Clinician Global Impression of Change Scale,” or “CGIC Scale” refers to when an investigator determines the degree of improvement with treatment per the affected foot on a 7-point scale ranging from -3 (“Very Much Worse”) to +3 (“Very Much Improvement”) as follows:-3 Very Much Worse-2 Much Worse-1 Minimally Worse0 No Change+1 Minimal Improvement+2 Much Improvement+3 Very Much Improvement

[0047] The term “Foot Function Index (FFI)” or “FFI” refers generally to a patient- reported outcome assessment using a multipoint scale to assess foot pain, disability, and difficulty in participants with ankle and foot disorders originally created and validated for rheumatoid arthritis of the foot and ankle. FFI can be adapted (as described in the Examples herein) to assess pain, activity difficulty, and activity limitation. The measure is relevant and comprehensive for patients with plantar fasciitis and is easy to understand and complete.

[0048] The term “Patient Global Impression of Change (PGIC) — Foot Pain” or “PGIC” refers to a patient-reported outcome assessment using a multipoint scale to assess the change in the overall severity of their foot pain in the past week on a 7-point scale, ranging from to +3 (“Very Much Improvement”) to -3 (“Very Much Worse”) as follows:+3 Very Much Improvement+2 Much Improvement+1 Minimal Improvement0 No Change-1 Minimally Worse-2 Much Worse-3 Very Much Worse

[0049] As used herein, “collagenase” refers to any of the following: (a) collagenase (including mutants) having activity as defined by EC 3.4.24.3 (www.brenda- enzymes.org / enzyme.php?ecno=3.4.24.3 (accessed August 24, 2020); (b) collagenase produced by fermentation of Clostridium histolyticum (also known as Hathewayci histolytica),' (c) CCH (as described herein); (d) collagenase having at least 50% sequence alignment with collagenase I (also referred as class I collagenase) as determined by BLAST; (e) collagenase having at least 50% sequence alignment with collagenase II (also referred as class II collagenase) as determined by BLAST; (f) collagenase produced by fermentation of other source organisms (z.e., wm-Clostridium histolyticum), e.g., mammalian, crustacean, fungal, bacterial, or microbial collagenase; (g) collagenase obtained by recombinant techniques; (h) collagenase with a molecular mass from about 65 kDa to about 130 kDa; (i) collagenase designated as collagenase I (col I) or collagenase II (col II); (j) mixtures of collagenase I and II; (k) collagenase from strain JCM 1403 (ATCC 19401) or derivatives thereof; (1) collagenase from strain ATCC 21000 or derivatives thereof; (m) collagenase from ATCC 69334 or derivatives thereof; (n) collagenase from C. perfringens,' (o) collagenase from Vibrio alginolyticus,' (p) collagenase from Streptomyces,' (q) collagenase from Pseudomonas,' (r) collagenase fromAchromobacter iophagus (s) collagenase described by Worthington Biochemical Corp. (www.Worthington-biochem.com; “Product Highlights”); (t) collagenase described by Sigma- Aldrich (www.sigma-aldrich.com); (u) a collagenase having one or more of the following characteristics:• Vmax (min-1) of about 0.08 to 7.70 (SRC assay; as described in Int’l Pub. No. W02020 / 058755), or about 0.3 to 30.5 (GPA assay; as described in Int’l Pub. No. W02020 / 058755);• KM of about 4.1 to 410 nM (SRC assay), or about 0.03 to 3.1 mM (GPA assay);• Kcat (sec-1) of about 1. 1 to 107 (SRC assay), or about 93 to 9, 179 (GPA assay);• 1 / Kcat (microseconds) of about 376 to 37,222 (SRC assay), or about 4 to 428 (GPA assay);• Kcat / KM (mM^sec-1) of about 5, 140 to 508,814 (SRC assay), or about 60 to 5,934 (GPA assay);• A molecular mass from about 60 kDa to about 130 kDa, or about 70 kDa to about 130 kDa, or about 80 kDa to about 120 kDa, or about 90 kDa to about 120 kDa, or about 100 kDa to about 110 kDa;• A purity by area of at least 80% as measured by reverse phase HPLC (high pressure liquid chromatography);• Potency (i.e., specific activity) of about 500 to about 30,000 SRC units / mg;• Potency of about 5,000 to about 30,000 f-SRC units / mg;• Potency of about 100,000 to about 400,000 GPA units / mg;• Potency of about 175,000 to about 500,000 f-GPA units / mg;• Potency of about 5,000 to about 25,000 ABC units / mg;• Less than or equal to 1% by area of an impurity selected from the group consisting of clostripain, gelatinase, and leupeptin; or• Less than or equal to 1 cfu / mL bioburden, wherein:Vmax = maximal rateKM = [Substrate] at 50% of VmaxKcat = molecules of substrate cleaved per second1 / Kcat = The microseconds required to cleave a molecule of substrate;(v) collagenase described by Nordmark Arzneimittel GmbH & Co. KG; (w) collagenase from strain 004; (x) equivalents, biosimilars, or mixtures of any of the collagenases recited herein;(y) biosimilars of the collagenase component of XIAFLEX® or biosimilars of XIAFLEX®; (z) ColQl (see, e.g., Hoppe, Brandstetter, Schonauer, Biochemical characterization of a collagenase from Bacillus cereus strain QI, Sci. Rep. 11:4187 (2021); (aa) collagenase (s) in Family M9, such as M09.001 -collagenase V (Vibrio spp.) (Vibrio alginolyticus), MEROPS Accession MER001172 (peptidase unit: 76-531), or in Subfamily M9A or M9B, or mixtures of one or more of a collagenase in Family M9; or (bb) collagenase derived from Clostridium histolyticum either by fermentation or through recombinant expression in a suitable host cell.

[0050] “Collagenase I activity,” as used herein, refers to the activity of Class I Collagenases, which exhibit high collagenolytic activity and low peptidase activity, making them particularly effective at breaking down collagen and gelatin substrates. They show a preference for collagen and gelatin as a substrate. “Collagenase II activity,” as used herein, refers to the activity of Class II Collagenases, which exhibit low collagenolytic activity and higher peptidase activity, meaning they are more efficient at cleaving small peptides. Class I and II collagenases work synergistically to degrade native collagen, having complementary activity that enhances overall collagen breakdown.

[0051] “Potency,” as used herein, refers to the level of collagenase activity per mg of material as determined by one or more enzyme assays, including those described in the Examples section herein.

[0052] Non-limiting examples of collagenases that may be used in the disclosure herein are described in U.S. Patent No. 7,811,560, U.S. Patent No. 9,757,435, U.S. Patent No. 9,744,138, and Int’l Pub. No. WO2012 / 125948. In some embodiments, the collagenase can comprise a collagenase I. A suitable collagenase I includes, for example, a collagenase I comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some aspects, the collagenase I comprises the amino acid sequence of SEQ ID NO: 1.

[0053] In some embodiments, the collagenase can comprise a collagenase II. A suitable collagenase II includes, for example, a collagenase II comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some aspects, the collagenase II comprises the amino acid sequence of SEQ ID NO: 2.Table 1. Sequences

[0054] In certain embodiments, the collagenase may comprise ColQl having a sequence comprising the below:YSMADLNKMNDQELVETLGCIKWHQITDLFQFNEDAKAFYKDKGKMQVI IDELAHRGSTFTRDDSKGIQTFTEVLRS AFYLAFYNNELSELNERSFQDKCLPALKAIAKNPNFKLGTAEQDTWSAYGKLI SNASSDVETVQYASNILKQYNDN FNTYVNDRMKGQAIYDIMQGIDYDIQSYLIEARKEANETMWYGKVDGFINEINRIALLNEVTPENKWLVNNGIYFAS RLGKFHSNPNKGLEWTQAMHMYPRLSEPYFVAVEQITTNYNGKDYSGNTVDLEKIRKEGKEQYLPKTYTFDDGSIV FKTGDKVSEEKIKRLYWAAKEVKAQYHRVIGNDKALEPGNADDILTIVIYNSPEEYQLNRQLYGYETNNGGIYIEET GTFFTYERTPEQSIYSLEELFRHEFTHYLQGRYEVPGLFGRGDMYQNERLTWFQEGNAEFFAGSTRTNNWPRKSI I SGLSSDPASRYTAERTLFAKYGSWDFYNYSFALQSYLYTHQFETFDKIQDLIRANDVKNYDAYRENLSKDPKLNKEY QEYMQQLIDNQDKYNVPAVADDYLAEHAPKSLTAVEKEMTETLPMKDAKMTKHSSQFFNTFTLEGTYTGSVTKGESE DWNAMSKKVNEVLEQLAQKEWSGYKTVTAYFVNYRVNSSNEFEYDWFHGIAKDDGENKAPTVNINGPYNGLVKEGI QFKSDGSKDEDGKIVSYLWDFGDGRTSTEVNPVHVYEREGSYKVALIVKDDKGKESKSETTVTVKDGSLTESEPNNR PEEANRIGLNTTIKGSLIGGDHTDVYTFNVASAKDIDI SVLNEYGIGMTWVLHHESDMQNYAAYGQANGNHIEANFN AKPGEYYLYVYKYDNGDGTYKLSVK(SEQ ID NO: 3. Source: htps: / / www.uniprot.Org / uniprotkb / B9J3S4 / entry#sequences accessed July 17, 2024)

[0055] In some embodiments, the collagenase can comprise a mixture of collagenaseI and collagenase II. The collagenase can comprise, for example, a mixture of a collagenase I comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a collagenaseII comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some aspects, the collagenase comprises a mixture of the collagenase I comprising the amino acid sequence of SEQ ID NO: 1 and the collagenase II comprising the amino acid sequence of SEQ ID NO: 2. Suitable mixtures of the collagenase I and collagenase II include, for example, a collagenase I: collagenase II mass ratio of 0.1: 1, 0.25: 1, 0.5: 1, 0.75: 1, 1: 1, 1.1: 1, 1.25: 1, 1.5: 1, 1.75: 1, 2: 1, 1:0.1, 1:0.25, 1:0.5; 1:0.75, 1: 1.1, 1: 1.25, 1: 1.5, 1: 1.75, or 1:2. Each of the collagenase I and collagenase II may have a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as measured by, for example, reverse phase HPLC.

[0056] In some embodiments, the collagenase can comprise collagenase Clostridium histolyticum (CCH). “CCH,” as used herein, refers to collagenase Clostridium histolyticum containing a mixture of collagenase I (SEQ ID NO: 1) and collagenase II (SEQ ID NO: 2) in an approximate 1 : 1 mass ratio. CCH is obtained by the fermentation of Clostridium histolyticum (also known as Hathewayci histolytica).The collagenase can have type I collagenase activity and type II collagenase activity, wherein: the type I collagenase activity has one or more of the following characteristics based on the SRC microplate assay o Vmax: About 0.08 to 7.70 min'1o KM: About 4.1 to 410 nanoMolar o Kcat: About 1.1 to 107 sec'1o 1 / Kcat: About 376 to 37,222 microseconds o Kcat / Kju: About 5,140 to 508,814 mM^sec'1and the type II collagenase activity has one or more of the following characteristics based on the GPA microplate assay o Vmax: About 0.3 to 30.5 min'1o KM: About 0.03 to 3.1 mM o Kcat: About 93 to 9,179 sec'1o 1 / Kcat: About 4 to 428 microseconds o Kcat / KM: About 60 to 5,934 mM^sec'1The collagenase can have type I collagenase activity and type II collagenase activity, wherein: the type I collagenase activity has one or more of the following characteristics based on the SRC microplate assay o Vmax: About 3.8 min'1o KM: About 2.07 x 10'4mM o Kcat: About 53 sec'1o 1 / Kcat: About 18,799 microseconds o WKM: About 256,977 mM^sec'1and the type II collagenase activity has one or more of the following characteristics based on the GPA microplate assay o Vmax: About 15.4 min'1o KM: About 1.6 mM o Kcat: About 4,636 sec'1o 1 / Kcat: About 216 microseconds o kcat / KM: About 2,997 mM^sec'1

[0057] In some embodiments, the collagenase I and collagenase II can have the following characteristics:Collagenase I (SRC microplate assay) o Vmax: About 0.08 to 7.70 min'1o KM: About 4.1 to 410 nanoMolar o Kcat: About 1.1 to 107 sec'1o 1 / Kcat: About 376 to 37,222 microsecondso Kcat / KM: About 5,140 to 508,814 mM^sec'1Collagenase II (GPA microplate assay) o Vmax: About 0.3 to 30.5 min'1o KM: About 0.03 to 3.1 mM o Kcat: About 93 to 9,179 sec'1o 1 / Kcat: About 4 to 428 microseconds o Kcat / KM: About 60 to 5,934 mM^sec'1

[0058] In some embodiments, the collagenase I and collagenase II can have the following characteristics:Collagenase I (SRC assay): o Vmax: About 3.8 min'1o KM: About 2.07 x 10'4mM o Kcat: About 53 sec'1o 1 / Kcat: About 18,799 microseconds o WKM: About 256,977 mM^sec'1Collagenase II (GPA assay): o Vmax: About 15.4 min'1o KM: About 1.6 mM o Kcat: About 4,636 sec'1o 1 / Kcat: About 216 microseconds o kcat / KM: About 2,997 mM^sec'1B. INTRODUCTION

[0059] Disclosed herein are methods of treating plantar fasciitis in a subject, the methods comprising injecting a pharmaceutical formulation comprising collagenase into an area of the subject’s foot affected by plantar fasciitis to thereby treat the plantar fasciitis. The disclosed methods can be used to treat plantar fasciitis in one or both of the subject’s feet. Suitable methods of administering the pharmaceutical formulation comprising collagenase include, for example, one or more injections of the pharmaceutical formulation into each of the affected areas. The methods can comprise one, two, three, four, five, six, seven, eight, nine, ten, or more than ten injections of the pharmaceutical formulation into each of the affected areas of the foot or feet.

[0060] The disclosed methods can comprise administering the pharmaceutical composition during a single treatment session or during multiple treatment sessions. Thenumber of treatment sessions will depend, in part, on the size of the affected area and severity of the plantar fasciitis as well as the response to the first or subsequent treatment sessions. The methods can comprise administering the pharmaceutical composition during one, two, three, four, five, six, seven, eight, nine, ten, or more than ten treatment sessions. The injections of the pharmaceutical formulation into each of the one or more affected areas can be administered in a single treatment session, over multiple treatment sessions, or in each treatment session. For example, two injections can be given in a single treatment session, one injection can be given in a first treatment session and one injection can be given in a second treatment session, or two injections can be given in a first treatment session and two injections can be given in a second treatment session.C. DOSES OF COLLAGENASE

[0061] The dose of collagenase administered in a single treatment session depends, in part, on the size of the affected area, the severity of the plantar fasciitis, the volume administered per injection, and the anticipated number of treatment sessions. Suitable doses of collagenase administered in a single treatment session include, for example, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.10 mg, about 0.12 mg, about 0.20 mg, about 0.24 mg, about 0.30 mg, about 0.35 mg, about 0.40 mg, about 0.45 mg, about 0.50 mg, about 0.60 mg, about 0.70 mg, about 0.80 mg, about 0.90 mg, about 1.0 mg, about 1.35 mg, about 1.8 mg, about 2.0 mg, about 2.7 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 6.0 mg, or more than about 6.0 mg. In some embodiments, a dose of about 0.12 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 0.2 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 0.3 mg collagenase is administered per treatment session. In some embodiments, a dose of about 0.6 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 1.0 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 1.25 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 1.35 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 1.5 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 1.8 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 2.0 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 2.5 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 2.7 mg of collagenase isadministered per treatment session. In some embodiments, a dose of about 3.0 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 4.0 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 5.0 mg of collagenase is administered per treatment session. In some embodiments, a dose of about 6.0 mg of collagenase is administered per treatment session.

[0062] In some embodiments, about 0.2 mg to about 0.6 mg of collagenase is administered to each of the one or more affected areas in a first treatment session. In some embodiments, about 0.3 mg to about 0.6 mg of collagenase is administered to each of the one or more affected areas in a first treatment session.

[0063] The collagenase I may have a potency of about 500 SRC units / mg to about 30,000 SRC units / mg. In some embodiments, the potency is about 500 SRC units / mg to about 25,000 SRC units / mg, or about 500 SRC units / mg to about 20,000 SRC units / mg, or about 500 SRC units / mg to about 15,000 SRC units / mg, or about 500 SRC units / mg to about 12,500 SRC units / mg, or about 500 SRC units / mg to about 10,000 SRC units / mg, or about 500 SRC units / mg to about 7,500 SRC units / mg, or about 500 SRC units / mg to about 5,000 SRC units / mg, or about 500 SRC units / mg to about 2,500 SRC units / mg, or about 500 SRC units / mg to about 2,000 SRC units / mg, or about 500 SRC units / mg to about 1,000 SRC units / mg, wherein “mg” refers to the amount of collagenase I present in a composition (as distinct from excipients and other constituents).

[0064] Suitable amounts of collagenase I administered in a single treatment session include about 5 SRC units to about 180,000 SRC units. In some embodiments, about 5 SRC units, about 25 SRC units, about 35 SRC units, about 50 SRC units, about 60 SRC units, about 75 SRC units, about 100 SRC units, about 200 SRC units, about 225 SRC units, about 250 SRC units, about 450 SRC units, about 500 SRC units, about 675 SRC units, about 750 SRC units, about 1,000 SRC units, about 2,100 SRC units, about 2,500 SRC units, about 3,600 SRC units, about 5,000 SRC units, about 10,000 SRC units, about 13,500 SRC units, about 15,000 SRC units, about 25,000 SRC units, about 27,000 SRC units, about 40,500 SRC units, about 50,000 SRC units, about 75,000 SRC units, about 100,000 SRC units, about 150,000 SRC units, about 175,000 SRC units, about 180,000 SRC units are administered in a single treatment session. In some embodiments, about 60 SRC units to about 40,500 SRC units are administered in a single treatment session.

[0065] The collagenase I may have a potency of about 5,000 f-SRC units / mg to about 30,000 f-SRC units / mg. In some embodiments, the potency is about 5,000 f-SRC units / mg toabout 25,000 f-SRC units / mg, or about 5,000 f-SRC units / mg to about 20,000 f-SRC units / mg, or about 5,000 f-SRC units / mg to about 15,000 f-SRC units / mg, or about 5,000 f-SRC units / mg to about 12,500 f-SRC units / mg, or about 5,000 f-SRC units / mg to about 10,000 f-SRC units / mg, or about 5,000 f-SRC units / mg to about 7,500 f-SRC units / mg, wherein “mg” refers to the amount of collagenase I present in a composition (as distinct from excipients and other constituents).

[0066] Suitable amounts of collagenase I administered in a single treatment session include about 50 f-SRC units to about 180,000 f-SRC units. In some embodiments, about 50 f- SRC units, about 75 f-SRC units, about 100 f-SRC units, about 150 f-SRC units, about 200 f- SRC units, about 250 f-SRC units, about 350 f-SRC units, about 500 f-SRC units, about 600 f- SRC units, about 750 f-SRC units, about 1,000 f-SRC units, about 1,500 f-SRC units, about 2,000 f-SRC units, about 2,100 f-SRC units, about 2,250 f-SRC units, about 2,500 f-SRC units, about 3,600 f-SRC units, about 4,500 f-SRC units, about 5,000 f-SRC units, about 6,750 f-SRC units, about 7,500 f-SRC units, about 10,000 f-SRC units, about 13,500 f-SRC units, about 15,000 f-SRC units, about 20,000 f-SRC units, about 25,000 f-SRC units, about 27,000 f-SRC units, about 40,500 f-SRC units, about 50,000 f-SRC units, about 100,000 f-SRC units, about 150,000 f-SRC units, about 180,000 f-SRC units are administered in a single treatment session. In some embodiments, about 600 f-SRC units to about 40,500 f-SRC units are administered in a single treatment session.

[0067] The collagenase II may have a potency of about 10,000 GPA units / mg to about 400,000 GPA units / mg, or about 10,000 GPA units / mg to about 350,000 GPA units / mg, or about 10,000 GPA units / mg to about 300,000 GPA units / mg, or about 10,000 GPA units / mg to about 250,000 GPA units / mg, or about 10,000 GPA units / mg to about 200,000 GPA units / mg, or about 10,000 GPA units / mg to about 150,000 GPA units / mg, wherein “mg” refers to the amount of collagenase II present in a composition (as distinct from excipients and other constituents).

[0068] Suitable amounts of collagenase II administered in a single treatment session include about 1,000 GPA units to about 2,400,000 GPA units. In some embodiments, about 1,000 GPA units, about 2,500 GPA units, about 5,000 GPA units, about 7,000 GPA units, about 10,000 GPA units, about 12,000 GPA units, about 15,000 GPA units, about 25,000 GPA units, about 28,000 GPA units, about 45,000 GPA units, about 48,000 GPA units, about 50,000 GPA units, about 75,000 GPA units, about 90,000 GPA units, about 100,000 GPA units, about 135,000 GPA units, about 150,000 GPA units, about 180,000 GPA units, about 200,000 GPAunits, about 250,000 GPA units, about 300,000 GPA units, about 360,000 GPA units, about 400,000 GPA units, about 500,000 GPA units, about 540,000 GPA units, about 600,000 GPA units, about 1,000,000 GPA units, about 2,000,000 GPA units, about 2,400,000 GPA units are administered in a single treatment session. In some embodiments, about 12,000 GPA units to about 540,000 GPA units are administered in a single treatment session.

[0069] The collagenase II may have a potency of about 175,000 f-GPA units / mg to about 500,000 f-GPA units / mg, or about 175,000 f-GPA units / mg to about 450,000 f-GPA units / mg, or about 175,000 f-GPA units / mg to about 400,000 f-GPA units / mg, or about 175,000 f-GPA units / mg to about 350,000 f-GPA units / mg, or about 175,000 f-GPA units / mg to about 300,000 f-GPA units / mg, or about 175,000 f-GPA units / mg to about 250,000 f-GPA units / mg, or about 175,000 f-GPA units / mg to about 200,000 f-GPA units / mg, wherein “mg” refers to the amount of collagenase II present in a composition (as distinct from excipients and other constituents).

[0070] Suitable amounts of collagenase II administered in a single treatment session include about 1,750 f-GPA units to about 3,000,000 f-GPA units. In some embodiments, about 1,750 f-GPA units, about 2,500 f-GPA units, about 5,000 f-GPA units, about 7,000 f-GPA units, about 10,000 f-GPA units, about 12,250 f-GPA units, about 15,000 f-GPA units, about 21,000 f-GPA units, about 25,000 f-GPA units, about 28,000 f-GPA units, about 35,000 f-GPA units, about 50,000 f-GPA units, about 60,000 f-GPA units, about 75,000 f-GPA units, about 78,750 f-GPA units, about 100,000 f-GPA units, about 150,000 f-GPA units, about 157,500 f-GPA units, about 200,000 f-GPA units, about 225,000 f-GPA units, about 236,250 f-GPA units, about 250,000 f-GPA units, about 300,000 f-GPA units, about 400,000 f-GPA units, about 450,000 f- GPA units, about 500,000 f-GPA units, about 600,000 f-GPA units, about 675,000 f-GPA units, about 1,000,000 f-GPA units, about 2,000,000 f-GPA units, about 3,000,000 f-GPA units are administered in a single treatment session. In some embodiments, about 21,000 f-GPA units to about 675,000 f-GPA units are administered in a single treatment session.

[0071] The collagenase I and / or collagenase II can have a specific activity of about 5,000 BTC units / mg to about 25,000 BTC units / mg, or about 5,000 BTC units / mg to about 20,000 BTC units / mg, or about 5,000 BTC units / mg to about 17,500 BTC units / mg, or about 5,000 BTC units / mg to about 15,000 BTC units / mg, or about 5,000 BTC units / mg to about 10,000 BTC units / mg, or about 5,000 BTC units / mg to about 7,500 BTC units / mg, wherein “mg” refers to the amount of collagenase(s) present in a composition (as distinct from excipients and other constituents).

[0072] Suitable amounts of collagenase I and / or collagenase II administered in a single treatment session include about 50 BTC units to about 150,000 BTC units. In some embodiments, about 50 BTC units, about 100 BTC units, about 150 BTC units, about 200 BTC units, about 250 BTC units, about 350 BTC units, about 500 BTC units, about 600 BTC units, about 750 BTC units, about 1,000 BTC units, about 1,250 BTC units, about 1,500 BTC units, about 1,750 BTC units, about 2,000 BTC units, about 2,250 BTC units, about 2,500 BTC units, about 3,000 BTC units, about 4,500 BTC units, about 5,000 BTC units, about 6,750 BTC units, about 10,000 BTC units, about 11,250 BTC units, about 15,000 BTC units, about 22,500 BTC units, about 25,000 BTC units, about 33,750 BTC units, about 50,000 BTC units, about 100,000 BTC units, about 150,000 BTC units are administered in a single treatment session. In some embodiments, about 600 BTC units to about 33,750 BTC units are administered in a single treatment session.

[0073] The collagenase I and / or collagenase II can have a specific activity of about 5,000 ABC units / mg to about 25,000 ABC units / mg, or about 5,000 ABC units / mg to about 20,000 ABC units / mg, or about 5,000 ABC units / mg to about 17,500 ABC units / mg, or about 5,000 ABC units / mg to about 15,000 ABC units / mg, or about 5,000 ABC units / mg to about 12,500 ABC units / mg, or about 5,000 ABC units / mg to about 10,000 ABC units / mg, or about 5,000 ABC units / mg to about 7,500 ABC units / mg, wherein “mg” refers to the amount of collagenase(s) present in a composition (as distinct from excipients and other constituents).

[0074] Suitable amounts of collagenase I and / or collagenase II administered in a single treatment session include about 50 ABC units to about 150,000 ABC units. In some embodiments, about 50 ABC units, about 100 ABC units, about 150 ABC units, about 200 ABC units, about 250 ABC units, about 350 ABC units, about 500 ABC units, about 600 ABC units, about 750 ABC units, about 1,000 ABC units, about 1,250 ABC units, about 1,500 ABC units, about 1,750 ABC units, about 2,000 ABC units, about 2,250 ABC units, about 2,500 ABC units, about 3,000 ABC units, about 4,500 ABC units, about 5,000 ABC units, about 6,750 ABC units, about 10,000 ABC units, about 11,250 ABC units, about 15,000 ABC units, about 22,500 ABC units, about 25,000 ABC units, about 33,750 ABC units, about 50,000 ABC units, about 100,000 ABC units, about 150,000 ABC units are administered in a single treatment session. In some embodiments, about 600 ABC units to about 33,750 ABC units are administered in a single treatment session.

[0075] The amount of collagenase administered in a subsequent treatment session (or multiple subsequent treatment sessions) can be greater than, less than, or the same as the amountof collagenase administered in the first treatment session. In some embodiments, about 0. 1 mg to about 1.0 mg of collagenase is administered to each of the one or more affected areas in a subsequent treatment session. In some embodiments, about 0.2 mg to about 0.6 mg of collagenase is administered to each of the one or more affected areas in a subsequent treatment session. In some embodiments, about 0.3 mg of collagenase is administered to each of the one or more affected areas in a subsequent treatment session.

[0076] In some embodiments, about 0.1 mg to about 1.0 mg of collagenase is administered to each of the one or more affected areas in a first treatment session and about 0.1 mg to about 1.0 mg of collagenase is administered to each of the one or more affected areas in a subsequent treatment session.

[0077] In some embodiments, about 0.2 mg to about 0.6 mg of collagenase is administered to each of the one or more affected areas in a first treatment session and about 0.2 mg to about 0.6 mg of collagenase is administered to each of the one or more affected areas in a subsequent treatment session.

[0078] The number of injections administered to each affected area in a single treatment session is based, in part, on the size and severity of the affected area of plantar fasciitis. One, two, three, four, five, six, seven, eight, nine, ten, or more than ten injections can be administered to each affected area in a single treatment session.D. FORMULATIONS

[0079] The collagenase may be in the form of a pharmaceutical formulation comprising collagenase and pharmaceutically acceptable excipients. Such excipients may include sterile water or sodium chloride / calcium chloride for injection, pH adjusting agents and stabilizers.

[0080] One non-limiting example is XIAFLEX®, supplied commercially by Applicant as single-use glass vials containing 0.9 mg of collagenase as a sterile, lyophilized powder for reconstitution. Sterile diluent for reconstitution is also provided in a single-use glass vial. Inactive ingredients include hydrochloric acid, sucrose, and tromethamine. The diluent contains calcium chloride dihydrate in 0.9% sodium chloride. XIAFLEX® Prescribing Information (2018).

[0081] Suitable concentrations of the collagenase in the pharmaceutical formulation include about 0.01 mg / ml, about 0.025 mg / ml, about 0.050 mg / ml, about 0.075 mg / ml, about 0.1 mg / ml, about 0.125 mg / ml, about 0.150 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9mg / ml, about 1.0 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, about 2.0 mg / ml, about 2.1 mg / ml, about 2.25 mg / ml, about 2.3 mg / ml, about 2.4 mg / ml, about 2.5 mg / ml, about 2.6 mg / ml, about 2.7 mg / ml, about 2.8 mg / ml, about 2.9 mg / ml, about 3.0 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 10.0 mg / ml, or greater than about 10.0 mg / ml. The concentration of collagenase in the pharmaceutical formulation can be from about 0.01 mg / ml to about 10.0 mg / ml, from about 0.01 mg / ml to about 5.0 mg / ml, from about 0.01 mg / ml to about 2.5 mg / ml, from about 0.01 mg / ml to about 1.0 mg / ml, from about 0.01 mg / ml to about 0.5 mg / ml, from about 0.01 mg / ml to about 0. 1 mg / ml, from about 0.05 mg / ml to about 10.0 mg / ml, from about 0.1 mg / ml to about 10 mg / ml, from about 0.5 mg / ml to about 10 mg / ml, from about 1.0 mg / ml to about 10 mg / ml, from about 2.5 mg / ml to about 10 mg / ml, or from about 5.0 mg / ml to about 10 mg / ml. The collagenase can have a concentration of about 0.6 mg / ml to about 2.25 mg / ml. In some embodiments, the collagenase can have a concentration of about 0.6 mg / ml. In some embodiments, the collagenase can have a concentration of about 1.2 mg / ml. In some embodiments, the collagenase can have a concentration of about 2.25 mg / ml.

[0082] Suitable volumes of each injection include, for example, about 0.1 ml, about 0.2 ml, about 0.3 ml, about 0.4 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1.0 ml, or greater than about 1.0 ml. In some embodiments, each injection has a volume of about 0. 1 ml to 0.3 ml. In some embodiments, each injection has a volume of about 0.2 ml.

[0083] A total volume of about 0.1 ml to about 1.0 ml can be administered to each affected area. In some embodiments, a total volume of about 0.2 ml to about 0.6 ml can be administered to each affected area. A total volume of about 0.2 ml can be administered to each affected area. A total volume of about 0.3 ml can be administered to each affected area. A total volume of about 0.4 ml can be administered to each affected area. A total volume of about 0.5 ml can be administered to each affected area. A total volume of about 0.6 ml can be administered to each affected area.

[0084] The pharmaceutical formulation can comprise the collagenase and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with the collagenase, allows the collagenase to retain its biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. Preferred diluents for parenteral administration are phosphate buffered saline or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000). In some embodiments, the pharmaceutical formulation is XIAFLEX®. In some embodiments, the pharmaceutical formulation comprises 0.9% sodium chloride and 0.03% calcium chloride dihydrate in water.E. METHODS OF TREATMENT— INJECTION TECHNIQUES AND DOSING

[0085] The methods of treating plantar fasciitis in a subject can comprise injecting a pharmaceutical formulation comprising collagenase into one or more plantar fasciitis affected areas in one or both of the subject’s feet to treat the plantar fasciitis, wherein a total dose of collagenase administered per treatment session comprises between about 0.1 mg to about 2.0 mg. The plantar fasciitis may be acute, subacute, and chronic, including recalcitrant plantar fasciitis.

[0086] In some embodiments, the subject has bilateral plantar fasciitis and the methods comprise injecting a maximum dose of about 0.1 mg to about 2.0 mg of collagenase to both feet in a first treatment session. In such embodiments, the methods can comprise injecting a maximum dose of about 0.1 mg to about 1.0 mg of collagenase per foot in a first treatment session. In some aspects, a maximum dose of about 0.2 mg to about 0.9 mg of collagenase is injected per foot in a first treatment session. About 0.2 mg to about 0.9 mg of collagenase can be administered to each foot in a subsequent treatment session.

[0087] In some embodiments, the subject has unilateral plantar fasciitis and the methods comprises injecting a maximum dose of about 0.2 mg to about 1.25 mg of collagenase to the foot in a first treatment session. About 0.2 mg to about 1.25 mg of collagenase can be administered to the foot in a subsequent treatment session.

[0088] In one, non-limiting, exemplary injection technique, the following steps are provided:1. Site marking utilizing the palpation guided technique: The clinician locates the subject’s medial calcaneal tuberosity via palpation, and using a surgical marker, places a reference mark on the palpated site. Approximately 2.5 centimeters (cm) distal (toward the toes) to the medial calcaneal tuberosity, the clinician marks the first injection site, centeredbetween the midline of the foot and the medial border of the plantar fascia medial band. The remaining 2 injection sites should be marked in the triangular shape depicted in Figure 3 approximately 0.5 cm spacing between sites.2. The subject then receives 3 aliquots as 3 injections of collagenase formulation administered during the first treatment session as follows: a. The injection site area of the foot should be disinfected with an antiseptic sterile solution (e.g., 70% isopropyl alcohol) and allowed to dry, to prevent potential infection. If desired, apply anesthetics as needed, before injection of collagenase. b. While the participant is sitting in a podiatry chair or on the examination table with their leg extended in front of them, the clinician secures the affected foot with her non-dominant hand, and grasps the toes close to the metatarsal heads and dorsiflex to palpate the plantar fascia. c. Using a surgical marker, the clinician places a reference mark on the palpated site at the calcaneal tuberosity. She marks the first injection site approximately 2.5 cm distal (toward the toes) to the calcaneal tuberosity reference mark, centered between the midline of the foot and the medial border of the plantar fascia band. The remaining two (2) injection sites should be marked in a triangular shape, maintaining approximately 0.5 cm spacing between the sites (as illustrated in Figure 3). d. With the clinician’s dominant hand, she performs the first of the three (3) 0.1 mb injections using the administration syringe previously prepared with the collagenase formulation. The direction of the injection should be perpendicular to the skin and medial band of the fascia. The clinician ensures the needle is placed intrafascially by resistance upon insertion, taking care to avoid hitting the calcaneus. In the event the needle tip passes through the fascia, the needle should be partially withdrawn until the tip is within the fascia. e. Prior to injection, the clinician should be sure to withdraw the plunger of the syringe to ensure that it is not injected intravascularly. If there are signs of blood coming inside the syringe, the steps are restarted making sure to select a new injection site. If there was negative aspiration, continue with the next step. f. The clinician slowly depresses the plunger to the 0.2 m mark to administer the first 0.1 mb injection of study intervention within the plantar fascia. After each injection, the clinician holds the syringe stable with the plunger depressed for approximately 10 seconds, as tolerated by the subject. This will allow the drug to penetrate and absorb into the injected fascial area. The needle is then removed slowly while keeping the plunger depressed.g. For the remaining two (2) injections, the clinician repeats the above steps with the same needle and syringe (depressing plunger to 0.1 mL for the second injection and to 0.0 mL for the third injection). h. The clinician then places a soft gauze bandage held in place by dressing tape, over the injected area of the foot.

[0089] Other injection volumes may be employed to deliver the desired dose of collagenase. It will also be understood that the subject may receive about 0.2 mg to about 0.6 mg collagenase administered as 3 injections, or about 0.2 mg collagenase administered as 3 injections, or about 0.3 mg collagenase administered as 3 injections, or about 0.6 mg administered as 3 injections.

[0090] In further embodiments, the present disclosure is directed to a method of treating plantar fasciitis in a subject, comprising injecting a collagenase enzyme to a plantar fasciitis affected area in an amount sufficient to treat the plantar fasciitis, the collagenase enzyme comprises means for degrading collagen. In another embodiment, the collagenase comprises:(a) means for binding or recruiting collagen Type I and Type III; and / or(b) means for breaking down collagen Type I and / or, collagen Type III.

[0091] In other embodiments, the collagenase comprises means for digesting native collagen fibrils under physiological conditions of pH, temperature and ionic strength, and which act by cleaving the helical part of the collagen molecule. In certain aspects, the collagenase has means for cleaving polypeptide chains that make up the collagen triple helix structure at various loci thereby leading to solubilization from the collagen fibril. The collagenases described herein may have means to cleave Type I collagen and / or Type III collagen by binding the Type I and / or Type III collagen, unwinding the local triple helix, and sequential cutting of individual chains inside the catalytic cleft.

[0092] In certain embodiments, the total dose comprises between about 0.2 mg to about 2.0 mg. The collagenase may comprise a collagenase I where, in some embodiments, the collagenase I has a potency of about 5 SRC units / mg to about 180,000 SRC units / mg, or about 500 SRC units / mg to about 30,000 units / mg. The collagenase I may comprise the amino acid sequence of SEQ ID NO: 1. Alternatively, the collagenase may comprise a collagenase II where, in some embodiments, the collagenase II has a potency of about 10,000 GPA units / mg to about 400,000 GPA units / mg, or about 10,000 GPA units / mg to about 300,000 GPA units / mg. The collagenase II may comprise the amino acid sequence of SEQ ID NO: 2. In yet anotherembodiment, the collagenase may comprise a mixture of collagenase I and collagenase II where, in some embodiments, the collagenase I has a potency of about 5 SRC units / mg to about SRC 180,000 units / mg, and the collagenase II has a potency of about 10,000 GPA units / mg to about 400,000 GPA units / mg.F. THERAPEUTIC ENDPOINTS AND MEASUREMENTS OF EFFICACY

[0093] The collagenase treatment as disclosed herein is effective in decreasing the pain and discomfort caused by plantar fasciitis. Efficacy can be assessed by any suitable patient-reported or physician-reported outcome tool. In certain embodiments, a statistically significant number of subjects experience an improvement in decreasing the pain and discomfort caused by plantar fasciitis. In other examples, efficacy can be assessed by the Recalcitrant Plantar Fasciitis Patient Reported Outcome (RPF PRO) Instrument, which comprises the Pain Intensity NRS, Foot Pain Severity Interference in PFA Scale, Foot Pain Frequency Impact in PFA Scale and Subject Satisfaction Treatment Scale.

[0094] Pain Intensity NRS: As described above, the range for the Pain Intensity NRS Score is 10, where 0 is no pain and 10 is worst pain imaginable. A negative change from baseline indicates pain improvement, i.e., lower scores are better. The subjects’ baseline values are obtained before treatment with collagenase. In some embodiments, the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA results in a negative change from baseline of about 0.0 to -2.0 at Day 7 after treatment, or about -0.5 to about -4 at Day 14 after treatment, or about -1 to about -5 at Day 28 after treatment, or about -1.5 to about -6 at one or more of Days 42, 56, or 84 after treatment.

[0095] Foot Pain Severity Interference in PFA Scale: As described above, the severity of foot pain is characterized on a 5-point scale ranging from 0 (“None”) to 4 (“Very Severe”). Overall mean scores are derived as the average of responses to all seven (7) foot pain severity interference questions. A negative change from baseline indicates pain improvement, i.e., lower scores are better. The subjects’ baseline values are obtained before treatment with collagenase. In some embodiments, the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA results in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

[0096] Foot Pain Frequency Impact in PFA Scale: As described above, frequency of foot pain is characterized on a 5-point scale ranging from 0 (“Never”) to 4 (“Always”). Overallmean scores are derived as the average of responses to all three (3) foot pain frequency questions. A negative change from baseline indicates pain improvement, i.e., lower scores are better. The subjects’ baseline values are obtained before treatment with collagenase. In some embodiments, the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA results in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

[0097] Subject Satisfaction Treatment Scale: In some embodiments, at least 25%, or at least 35%, or at least 45%, or at least 55% of subjects in a patient population report they are “Quite Satisfied” or “Very Satisfied” at one or more of Days 7, 14, 28, 42, 56, or 84 following a collagenase treatment of about 0.2 mg to about 1.0 mg (total dose).

[0098] Foot Function Index (FFI): As described above, each subject is asked to complete the FFI, which assesses the impact of foot pathology on 3 domains of pain, difficulty, and activity limitation. The FFI is completed at screening (Day -28 to Day -1), Day 1, Day 15 (±3 days), Day 29 (±3 days), Day 43 (±3 days), Day 57 (±3 days) and Day 85 (EOS) / ET) (±3 days). The subject completes the scale prior to evaluation by the investigator. The FFI Activity Limitation Subscale consists of 3 items and measures limitations in activities in the past week because of their feet, such as staying off one foot or both feet. It is scored on a 5-point verbal rating scale as follows:0 - Never1 - Rarely2 - Sometimes3 - Often4 - Always

[0099] The FFI Difficulty Subscale consists of 9 items and measures difficulty performing various functional activities in the past week, because of their feet, such as difficulty climbing stairs. It is scored on a 5-point verbal rating scale as follows:0 - No difficulty1 - A little difficulty2 - Some difficulty3 - Much difficulty4 - A lot of difficulty

[0100] The FFI Pain Subscale consists of 9 items and measures the severity of foot pain during the past week in different situations, such as walking barefoot versus walking with shoes. For the pain subscale, if the participant marks “does not wear orthotics” items pertaining to orthotics are not scored and are not included in the total score. It is scored on a 5 -point verbal rating scale as follows:0 - None1 - Mild2 - Moderate3 - Severe4 - Extreme

[0101] A negative change from baseline indicates pain improvement, i.e., lower scores are better. The subjects’ baseline values are obtained before treatment with collagenase. In some embodiments, the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA results in a negative change from baseline of about 0.0 to - 1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

[0102] Patient Global Impression of Change (PGIC) — Foot Pain: As described above, PGIC refers to a patient-reported outcome assessment using a multipoint scale to assess the change in the overall severity of their foot pain in the past week on a 7-point scale, ranging from to +3 (“Very Much Improvement”) to -3 (“Very Much Worse”). A positive change from baseline indicates pain improvement, i.e., higher scores are better. The subjects’ baseline values are obtained before treatment with collagenase. In some embodiments, the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA results in a positive change from baseline of about 0.0 to +1.0 at Day 7 after treatment, or about +0.2 to about +2.5 at Day 14 after treatment, or about +0.5 to about +3 at Day 28 after treatment, or about +0.75 to about +3.5 at Day 42 after treatment, or about +1.0 to +4 at one or more of Days 56 or 84 after treatment.

[0103] Clinician Global Impression of Change Scale, or CGIC Scale: As described above, CGIC Scale refers to when an investigator determines the degree of improvement with treatment per the affected foot on a 7-point scale ranging from -3 (“Very Much Worse”) to +3 (“Very Much Improvement”). A positive change from baseline indicates pain improvement, i.e., higher scores are better. The subjects’ baseline values are obtained before treatment with collagenase. In some embodiments, the injection of a total dose of about 0.2 mg to about 1.0 mgto treat PFA results in a positive change from baseline of about 0.0 to +1.0 at Day 7 after treatment, or about +0.2 to about +2.5 at Day 14 after treatment, or about +0.5 to about +3 at Day 28 after treatment, or about +0.75 to about +3.5 at Day 42 after treatment, or about +1.0 to +4 at one or more of Days 56 or 84 after treatment.

[0104] Durability: In another aspect, the treatment method evaluates the durability of the above effects. The treatments described herein result in subjects demonstrating durability of effect at 6 months and 12 months. In other embodiments, there is a statistically significant number of subjects demonstrating durability of effect at 6 months and 12 months. In other embodiments, at least about 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 100% of patients demonstrate such durability.EXAMPLES

[0105] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.A. ENZYME ASSAYS1. Collagenase I Potency as Measured by SRC Assay (Cuvette)

[0106] The SRC assay is primarily used to measure the potency of collagenase I. The general methodology is as follows. Leucine standards and collagenase sample solutions are prepared. The first step of the assay involves an enzymatic reaction in which soluble rat-tail tendon collagen (SRC) is digested by the collagenase. The second step involves the subsequent measurement of liberated peptide fragments / amino acids with the Anorogenic derivative fluorescamine. The assay follows the methodology below, but a person of ordinary skill in the art will appreciate that certain modifications (e.g., dilution concentrations and times) may be made.

[0107] Collagenase and leucine standard samples are treated with reagents in order to tag the generated GPA with fluorescamine. The leucine standards and collagenase samples are allowed to incubate at room temperature for 10 minutes prior to determining the fluorescence of each solution at 392 and 480 nm excitation and emission wavelengths, respectively. The resulting slopes of the leucine and collagenase sample curves are then used to calculate potency units as follows:Potency (f-SRC units / mg) = (Msampie / Mi.eucme) x (DF / T) x CF where:Msampie = Slope of the collagenase sample potency curveMi.eucine = Slope of the leucine standard curveDF = Dilution Factor (1500 pL / 100 mL = 15)T = Reaction time (2.5 hr x 60 min / 1 hr = 150 min)CF = Conversion factor (1000 j g / 1 mg = 1000)

[0108] An exemplary SRC assay is set forth below: Buffers and Reagents

[0109] The following buffers / reagents are used:1. F-TC Assay Buffer, pH 7.2 (22g HEPES [4-(2 -Hydroxy ethyl)- 1- piperazineethane sulfonic acid], 4.4 g calcium acetate)2. F-Enzyme Buffer, pH 7.23. 200 mM Borate, pH 9.04. 10 mM Leucine Stock Solution5. 1 mM Leucine Working Stock Solution6. 1 mM Fluorescamine Solution in Acetone7. 2 mg / mL Rat Tail Collagen in 0.02N acetic acid Preparation of Solutions

[0110] Solutions are prepared as follows:

[0111] F-TC Assay Buffer: Dissolve 22 g HEPES and 4.4 g calcium acetate in approximately 900 mL of water. Adjust pH to 7.2 with sodium hydroxide and QS to IL with water. Store at 2-8 °C.

[0112] F-Enzyme Buffer: Dilute F-TC Assay Buffer by combining 4 mL with 16 mL water. Store at 2-8 °C.

[0113] lOmM Leucine Stock Solution: Dissolve 65.5 mg of leucine in 50 mL of water. Leucine must be weighed directly into a 100 mL (or equivalent) glass beaker on the scale. Weigh out approximately 65 mg (target weight) of leucine into the beaker. Based on the weight of leucine weighed, calculate the amount of water to add to the beaker using the equation below. Add the calculated volume of water to the beaker and mix thoroughly to ensure the leucine is fully dissolved. Dispense in to 1 mL aliquots. Store at less than or equal to - 20 °C.V2(mL) = C2(mg) x VI (50 mL)Cl (65.5mg)Where:C2 = mass of leucine weighed (mg)VI = 50 (mL of water)Cl = 65.5 (mg of leucine)V2 = volume of water needed to produce a 10 mM stock solution (mL)

[0114] 1 mM Leucine Working Stock Solution: Thaw a vial of 10 mM Leucine Stock Solution and dilute to 1 mM by combining 150 pL with 1350 pL water. Mix well prior to use.

[0115] 0.5 N HC1: Dilute HC1 to 0.5 N with water and mix well. Store at room temperature. Alternatively, commercially available 0.5 N HC1 may be used.

[0116] 0.02 N Acetic Acid: Combine 1 mL of 1 N Acetic Acid with 49 mL of water and mix well. Store at room temperature.

[0117] 200 mM Borate, pH 9.0: Dissolve 2.4 g boric acid in approximately 150 mL water. Adjust the pH to 9.0 using sodium hydroxide. QS to 200 mL with water and mix well. Store at 2-8 °C.

[0118] 1 mM Fluorescamine Solution: Dissolve 15 mg of fluorescamine with 50 mL acetone and swirl to dissolve. Store at 2-8 °C protected from light.

[0119] Substrate Solution (2 mg / mL Rat Tail Collagen): Dilute sock rat tail collagen to 2 mg / mL with 0.02 N acetic acid. Store at 2-8 °C.

[0120] The leucine standard curve is prepared according to the following table.Table 2: Preparation of the leucine standard curve

[0121] 100 pL of each Leucine Standard is then transferred into separate centrifuge tubes for detection of fluorescamine.Collagenase Sample and Blanks Preparation

[0122] The sample is diluted to 0.01 mg / mL with F-Enzyme Buffer in two stages vortexed gently to mix. The following is an example dilution scheme:1. 100 pL x 1.0 mg / mL -> 1000 pL = 0.1 mg / mL2. 100 pL x 0.1 mg / mL -> 1000 pL = 0.01 mg / mL

[0123] The diluted samples are maintained at room temperature until use.

[0124] Blanks are prepared according to the following table by first combining the sample and 0.5 N hydrochloric acid to inactivate the enzyme prior to addition of buffers and substrate.

[0125] Collagenase samples in labeled tubes according to the following table. Tubes 1,2, 4 and 6 are prepared from one preparation and tubes 3, 5 and 7 from the duplicate preparation.Table 3 : Blank and Collagenase Sample Preparations

[0126] The tubes are capped and vortexed gently to mix. The potency curve preparations are incubated in a 25 °C ± 3 °C water bath for 2.5 hours. At the end of incubation, the potency curve tubes are removed from the water bath. 750pL of 0.5 N HC1 is added to each preparation and vortexed thoroughly to mix. The preparations may be stored at 2-8 °C for up to 22 hours prior to detection.Detection / Fluorometer Setup

[0127] The leucine standards are prepared as described above.

[0128] The luminescence spectrometer is set up with the following instrument parameters. The fluorescence of each preparation is read with 1 hour of derivatization.Table 4: Parameters and SettingsCalculations

[0129] The concentration of each leucine standard (X-axis) is plotted against the fluorescence response at 480 nm (Y -axis). The slope (m), coefficient of determination (R2), andthe mean fluorescence of each duplicate preparation are determined. Do not force through zero. The net fluorescence of each collagenase sample preparation is calculated as follows:F(net) = Mean Collagenase Sample (EM480) - Blank (EM480)

[0130] The amount of the collagenase sample in each preparation is plotted (X-axis) against the net fluorescence (Y -axis). The slopes (m) and coefficient of determination (R2) are determined. Do not force through zero.

[0131] The potency of the collagenase is determined as follows:Potency (f-SRC units / mg) = (Msampie / M eucine) x (DF / T) x CFWhere:Msampie = Slope of the collagenase sample potency curveMi.eucine = Slope of the leucine standard curveDF = Dilution Factor (1500 pL / 100 mL = 15)T = Reaction time (2.5 hr x 60 min / 1 hr = 150 min)CF = Conversion factor (1000 pg / 1 mg = 1000)

[0132] The above SRC assay may be employed to analyze the specific activity of any collagenase.2. SRC Microplate Assay for the Determination of Collagenase I Activity in a Collagenase Sample

[0133] This method is similar to the SRC assay above, except it is performed in a microplate. Like the SRC assay above, the microplate assay measures the collagenase activity towards soluble rat-tail collagen (SRC) substrate (hereafter, “substrate”). The assay follows the methodology below, but a person of ordinary skill in the art will appreciate that certain modifications may be made.Buffers and Reagents

[0134] The following buffers and reagents are used:1. Soluble Rat Collagen Substrate (BD Biosciences 354236)2. Tripeptide GPA (Bachem H3615 or equivalent)3. Fluorescamine (Acros 191675000 or equivalent)4. Purified Water (Millipore, Milli-Q-Plus 18.2 MQ system or equivalent)5. 1 M HEPES buffer (Gibco 15630-080 or equivalent)6. 1 M Calcium Acetate (Ca^FECh ) (Emerald Biosciences EBS-100-CAAC or equivalent)7. Surfact-Amps 20™ (10% Tween solution) (Pierce Cat.#28320 or equivalent)8. 1.0 N Acetic acid (Sigma 318590 or equivalent)9. 0.5 N Hydrochloric Acid (VWR 101223-134 or equivalent)10. Boric acid (Sigma B7660 or equivalent)11. 2.5 N Sodium hydroxide (J.T Baker 5666-02 or equivalent)12. Acetone (Sigma 270725 or equivalent)Preparation of Solutions

[0135] Preparation of assay buffer (50 mM HEPES pH 7.1 / 0.05% Tween 20 / 5 mM (Ca(C2H3C>2)2): An amount of 50 mL 1 M HEPES is pipetted into 800 mL DI water. 5 mL 1 M (Ca(C2H3C>2)2) and 5 mL Surfact-Amps (10% Tween 20) are added. The pH is checked and adjusted to 7.1 ± 0.1 if necessary. A sufficient quantity of water is added to adjust the volume to 1 L and the solution is filtered through a 0.22 micron filter. This assay buffer can be stored at room temperature for up to 3 months.

[0136] Preparation of 0. 1 N NaOH: An amount of 2 mL of 2.5 N NaOH is added into 48 mL DI water. This solution can be stored at room temperature for up to 3 months.

[0137] Preparation of 4 mg / mL tripeptide GPA stock: An amount of 400 mg (± 1 mg) of GPA tripeptide is dissolved into 10 mL 0.1 N NaOH and vortexed until totally dissolved. A sufficient quantity of assay buffer is added to make the volume 100 mL and the solution is dispensed into 0.5 mL aliquots and stored at -70°C. The 4 mg / mL tripeptide GPA stock can be stored at -70°C for up to one year.

[0138] Preparation of 0.02 N acetic acid: An amount of 1 mL of 1.0 N acetic acid is added to 40 mL of purified water. A sufficient amount of purified water is added to adjust the volume to 50 mL. This solution can be stored at room temperature for up to 1 year.

[0139] Preparation of 2 mg / mL SRC substrate stock solution: An amount of 23.3 mL 0.02 N acetic acid is added directly to the vial in which substrate is supplied (supplied in one nonlimiting example as 100 mg SRC at 3.75 mg / mL). Other concentrations of SRC substrate may be used. The calculation is below:100 mg 3.75 mg / mL = 26.7 mL;Total vol (mL) = (3.75 mg / mL x 26.7 mL) / 2 mg / mL;Total vol (50.0 mL) - 26.7 mL = 23.3mL

[0140] The solutions are mixed thoroughly by inversion and can be stored at 2-8°C for up to 3 months.

[0141] Preparation of 0.6 mg / mL SRC substrate working solution: An amount of 4.2 mL of assay buffer is added to a 15 mL conical tube. Then, 1.8 mL of 2 mg / mL SRC substratestock solution is added and the solution is mixed by inversion. This solution should be prepared immediately before addition to plate.

[0142] Preparation of 120 mM Boric Acid pH 9.0: An amount of 7.4 g (± 0.5 g) of the boric acid is dissolved in 800 mb DI water. The solution is titrated with NaOH to pH 9.0 and sufficient DI water is added to adjust the volume to IL. This solution can be stored at room temperature for up to 3 months.

[0143] Preparation of 1 mM Fluorescamine in Acetone: An amount of 28 ± 2 mg Fluorescamine is dissolved in 100 mL acetone. This solution needs to be freshly prepared and protected from light and moisture.Preparation of Tripeptide GPA Standard and Serial Dilution

[0144] A 0.08 mg / mL (329 pM) tripeptide GPA standard is prepared by making a 50- fold dilution of the 4 mg / mL tripeptide GPA stock in assay buffer (for example, 20 pL 4 mg / mL GPA in 980 pL assay buffer) . In the assay plate, row A, 200 pL of 329 pM tripeptide GPA standard is pipetted into Al and A7. An amount of 100 pL assay buffer is pipetted into A2-A6 and A8- A12.

[0145] For the tripeptide GPA standard serial dilution, an amount of 100 pL is transferred from Al into A2, mixed, an amount of 100 pL is transferred from A2 into A3, and repeated until A5. An amount of 100 pL is taken out from A5 well so that its final volume is 100 pL. The A6 well contains buffer only.

[0146] For the second tripeptide GPA standard serial dilution, an amount of 100 pL is transferred from A7 into A8, mixed, an amount of 100 pL is transferred from A8 into A9, and repeated until Al l. An amount of 100 pL is taken out from Al l well so that its final volume is 100 pL. The A12 well contains buffer only.Preparation of Collagenase Test Samples

[0147] For collagenase samples (e.g., a lyophilized collagenase drug product), the sample is allowed to come to room temperature for at least 10 minutes and reconstituted to form a 3.0 pg / mL stock solution. Different concentrations may be used. A test collagenase sample (T1 A) is prepared from the stock solution by diluting with assay buffer. The procedure is repeated to prepare triplicate test samples (T1A, TIB, TIC).Discussion

[0148] In this method, 50 pL of increasing concentrations of the test collagenase samples are mixed with 50 pL of excess substrate (0.2 mg / mL final concentration) in a 96-well plate. An amount of 50 pL of assay buffer is added to rows C-G in a U-bottomed, 96 well polypropylenereaction plate. 150 pL of collagenase samples are pipetted into row B. Then, a 1 / 1.5 serial dilution is performed using a multi-channel pipette, by transferring 100 pL of collagenase sample from row B into row C, mixing and repeating the process until row G is reached. An amount of 100 pL is removed and discarded from row G. The Blank is prepared in row H by pipetting 50 pL assay buffer to row H. This row contains no enzyme. The following table contains the final collagenase concentrations after adding 50 pL substrate to row B through row H.Table 5 : Assay Target Concentrations After Substrate AdditionCollagenase Reaction

[0149] The incubator and temperature probe are turned on to a temperature 22 ± 1°C prior to the addition of substrate to the plates. An amount of 50 pL 0.6 mg / mL SRC substrate is added to each well from row B to row H, added column by column then mixed. The reaction start time begins after the substrate is added to the first column. The plate is covered and placed in the 22 ± 1°C incubator for a total reaction time of 45 ± 5 minutes. To quench the reaction, 100 pL of 0.5 N HC1 is added into each well of the dilution plate, column by column, and mixed. Reaction time ends after the HC1 is added to the first column.Detection

[0150] An amount of 195 pL of 120 mM Borate pH 9.0 is added to each well of a Microplate Greiner polypropylene black reading plate. 30 pL of the quenched reaction mixture is transferred from the reaction plate into the corresponding wells of the reading plate and mixed well. Then, 75 pL of 1 mM Fluorescamine is added to each well of the reading plate (using a polypropylene tray to dispense Fluorescamine / acetone) and mixed immediately after every addition. The plate is read within 15 minutes after Fluorescamine addition with a Molecular Devices M2 fluorescence plate reader using the following settings: Excitation 380 nm, Emission 473 nm, cutoff 455 nm, 6 reads / well, PMT medium.

[0151] The concentration of GPA (pM) versus the emission at 473 nm and the concentration of collagenase (ng / mL) versus the emission at 473 nm are plotted. For each plot, a linear regression is fitted with no fixed parameters. For collagenase samples, the zero point data are excluded from the linear fit and the entire triplicate data set for each sample is used to generate the plot. The slopes for the tripeptide GPA standard and collagenase samples are determined. Specific Activity and Relative Potency Determination

[0152] The collagenase sample specific activity can by calculated as follows:SRC Microplate Assay Units = ((Slope of Collagenase Sample) / (Slope of Tripeptide GPA x incubation time)) x 106

[0153] The specific activity of the collagenase test sample is determined from the slope of the tripeptide GPA standard and calculated by the curve-fitting program. Using the microplate method, different concentrations of substrate and different times may be used to calculate enzyme kinetics according to Michaelis-Menten.3. Collagenase II Potency as Measured by GPA Assay (Cuvette)

[0154] The GPA assay is primarily used to measure the potency of collagenase II (a class II collagenase). The first step of the assay involves an enzymatic reaction involving the digestion of the substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-prolyl-L-alanine (zGPGGPA) by a collagenase sample into two peptides: carbobenzoxy-gly cyl -L-prolyl-gly cine (zGPG) and glycyl- prolyl-L-alanine (GPA). The second step involves the subsequent measurement of liberated GPA with the Anorogenic derivative fluorescamine. The assay follows the methodology below, but a person of ordinary skill in the art will appreciate that certain modifications (e.g., dilution concentrations and times) may be made.

[0155] The general methodology is as follows. Leucine standards are prepared. A collagenase sample is obtained and solutions are prepared to be used in the first step for theenzymatic cleavage of zGPGGPA (hereafter “substrate”) by collagenase. Following this step, the collagenase-treated samples (containing the liberated GPA) and leucine standards are treated at room temperature for a period of time with fluorescamine in order to fluorescently tag the free amino groups of the generated GPA and leucine molecules, respectively. The fluorescence emission of each solution at 480 nm is measured following excitation at 392 nm. The resulting slopes of the leucine and collagenase sample curves are then used to calculate potency units as follows:Potency (f-GPA units / mg) = (Msampie / Mi.eucme) x (DF / T) where:Msampie = Slope of the collagenase sample potency curve Mi.eucine = Slope of the leucine standard curve DF = Dilution FactorT = Reaction time

[0156] Additional, non-limiting details regarding the GPA assay methodology are set forth below.Buffers and Reagents

[0157] The following buffers and reagents are used:1. f-Appel’s Buffer, pH 7.2 (55mM HEPES,100mM calcium acetate)2. 1 mM Leucine Working Stock Solution3. 200 mM Borate, pH 9.04. 0.5 mM Fluorescamine Solution in Acetone5. 2 mg / mL zGPGGPA Substrate in f-Appel’s Buffer Solution Preparation

[0158] Solutions are prepared as follows:

[0159] f-Appel’s Buffer: Dissolve 13.0 g HEPES and 17.6 g calcium acetate in approximately 800 mL of water. Adjust pH to 7.2 with sodium hydroxide and QS to IL with water. Store at 2-8 degrees C.

[0160] lOmM Leucine Stock Solution: Dissolve 65.5 mg of leucine in 50 mL of water. Leucine must be weighed directly into a 100 mL (or equivalent) glass beaker on the scale. Weigh out approximately 65 mg (target weight) of leucine into the beaker. Based on the weight of leucine, calculate the amount of water to add to the beaker using the equation below. Add the calculated volume of water to the beaker and mix thoroughly to ensure the leucine is fully dissolved. Dispense into 1 mL aliquots. Store at less than or equal to 20 degrees C.V2(mL) = C2(mg) x VI (50 mL)Cl (65.5mg) where:C2 = mass of leucine weighed (mg)VI = 50 (mL of water)Cl = 65.5 (mg of leucine)V2 = volume of water needed to produce a 10 mM stock solution (mL)

[0161] 1 mM Leucine Working Stock Solution: Thaw a vial of 10 mM Leucine Stock Solution and dilute to 1 mM by combining 150 pL with 1350 pL water. Mix well prior to use.

[0162] 0.5 N HC1: Dilute HC1 to 0.5 N with water and mix well. Store at room temperature. Alternatively, commercially available 0.5 N HC1 may be used.

[0163] 200 nM Borate, pH 9.0: Dissolve 2.4 g boric acid in approximately 150 mL water. Adjust the pH to 9.0 using sodium hydroxide. QS to 200 mL with water and mix well. Store at 2-8 degrees C.

[0164] 0.5 mM Fluorescamine Solution: Mix 15 mg of fluorescamine with 100 mL acetone and swirl to dissolve. Store at 2-8 degrees C protected from light.

[0165] Substrate Solution (2 mg / mL zGPGGPA): Prepare substrate at 2 mg / mL with f- Appel’s buffer. Dissolve on a mechanical shaker / rotator, allowing sufficient time for complete dissolution (about 15 minutes).Leucine Standard Curve

[0166] The leucine standard curve is prepared according to the following table.Table 6: Preparation of the leucine standard curve.“LI” means Leucine standard sample 1, “L2” means Leucine standard sample 2, etc.

[0167] 100 pL of each Leucine Standard is then transferred into separate tubes for detection of fluorescamine.Collagenase Sample Preparation

[0168] The collagenase sample is diluted to 0.01 mg / mL with f-Appel’s Buffer in two stages and vortexed gently to mix. The following is an example dilution scheme:1. 100 pL x 1.01000 pL = 0.1 mg / mL2. 100 pL x 0.11000 pL = 0.01 mg / mLBlank Preparations

[0169] Blanks are prepared by combining 45 pL of the diluted preparation with 500 pL of 0.5 N hydrochloric acid to inactivate the enzyme. 455 pL of zGPGGPA substrate solution is added and vortexed to mix thoroughly. 100 pL of each blank is transferred into separate tubes for detection of impurities that may react with fluorescamine.Potency Curves

[0170] A set of potency curves are prepared for each collagenase sample as follows:Table 7

[0171] The tubes containing substrate and buffer are warmed in a water bath at 25 °C for a minimum of 15 minutes. A second set of tubes is labeled and 50 pL of 0.5 N hydrochloric acid is added to each. The diluted collagenase sample preparations (0.01 mg / mL) is added to the tubes according to the below table for a 10-minute incubation, and the tubes are mixed and returned to the water bath. The incubation period is started upon addition of the first preparation to the prewarmed substrate.Table 8: Sample Preparation

[0172] The preparations are removed from the water bath with 1-2 minutes remaining on the 10-minute incubation and vortexed gently to mix. Ten minutes after addition of the first preparation to the substrate, 50 pL is transferred from each tube into the tubes containing 50 pLof 0.5 N HC1. The preparations should be added directly to the acid to quench the digestion. Each tube is vortexed to mix well after quenching all preparations.Detection

[0173] 400 pL of 200 mM Borate Buffer and 500 pL of 0.5 mM Fluorescamine Solution is added to all detection tubes containing 100 pL of each preparation (blanks, collagenase sample potency curves, and leucine standards). The tubes are vortexed thoroughly to mix and are incubated at room temperature for a minimum of 10 minutes.Fluorometer Setup

[0174] The fluorometer is set up with the following instrument parameters and the fluorescence of each preparation is read with 1 hour of derivatization.Table 9Calculations

[0175] The concentration of each leucine standard (X-axis) is plotted against the fluorescence response at 480 nm (Y -axis). The slope (m), coefficient of determination (R2), and mean fluorescence of each potency curve preparation is determined. Collagenase sample and leucine potency curves are calculated by plotting the concentration of each preparation (X-axis) against the mean fluorescent response at 480 nm (Y-axis). The slope (m) and coefficient of determination (R2) for the resulting linear curves are determined.Determine Potency of Collagenase Sample

[0176] Potency is determined using the following equation:Potency (f-GPA units / mg) = (Msampie / Mi.eucme) x (DF / T)Where:Msampie = Slope of the collagenase sample potency curveMi.eucine = Slope of the leucine standard curve DF = Dilution Factor (1100 pL / 50 pL = 22) T = Reaction time (10 minutes)

[0177] This method is similar to the GPA assay above, except it is performed in a microplate. Like the assay above, the microplate assay measures the proteolytic activity of collagenase samples in the enzymatic cleavage of the substrate carbenzoxy-glycyl-L-prolyl- glycyl-glycyl-L-propyl-L-alanine (zGPGGPA) (hereafter, “substrate”). The assay follows the methodology below, but a person of ordinary skill in the art will appreciate that certain modifications (e.g., dilution concentrations and times) may be made.Buffers and Reagents

[0178] The following buffers and reagents are used:1. Peptide substrate (zGPGGPA) (Bachem M1260 or equivalent)2. Tripeptide GPA (Bachem H3615 or equivalent)3. Fluorescamine (Acros 191675000 or equivalent)4. Purified Water (Milli-Q-Plus 18.2 MQ system or equivalent)5. I M HEPES buffer (Gibco 15630-080 or equivalent)6. Surfact-Amps 20™ (10% Tween solution) (Pierce Cat.#28320 or equivalent)7. I M Calcium Acetate (Ca^FECh ) (Emerald Biosciences Cat.#EBS-100-CAAC or equivalent)8. Boric acid (Sigma B7660 or equivalent)9. 2.5 N NaOH (J.T Baker 5666-02 or equivalent)10. 0.5 N Hydrochloric Acid (VWR 101223-134 or equivalent)11. Acetone (Sigma 270725 or equivalent)Preparation of Solutions

[0179] Preparation of assay buffer (50 mM HEPES pH 7.1 / 0.05% Tween 20 / 5 mM (Ca(C2H3C>2)2): An amount of 50 mL 1 M HEPES is pipetted into 800 mL DI water. 5 mL 1 M (Ca(C2H3C>2)2) and 5 mL Surfact-Amps (10% Tween 20) are added. The pH is checked and adjusted to 7. 1 ± 0.05 if necessary. A sufficient quantity of water is added to adjust the volume to 1 L and the solution is filtered through a 0.22 micron filter. This assay buffer can be stored at room temperature for up to 3 months.

[0180] Preparation of 0. 1 N NaOH: An amount of 2 mL of 2.5 N NaOH is added into 48 mL DI water. This solution can be stored at room temperature for up to 3 months.

[0181] Preparation of 4 mg / mL tripeptide GPA stock solution: An amount of 400 mg (± 1 mg) of tripeptide GPA is dissolved into 10 mL 0.1 N NaOH and vortexed until totallydissolved. A sufficient quantity of assay buffer is added to make the volume 100 mL and the solution is dispensed into 0.5 mL aliquots and stored at -70°C. The 4 mg / mL tripeptide GPA stock can be stored at -70°C for up to one year.

[0182] Preparation of 4 mg / mL (6.8 mM) peptide substrate zGPGGPA: An amount of 400 mg (+ 1 mg) of the peptide substrate zGPGGPA is dissolved into 10 mL 0.1 N NaOH and vortexed until totally dissolved. A sufficient quantity of assay buffer is added to make the volume 100 mL. This solution can be stored at 4°C for up to 3 months.

[0183] Preparation of 120 mM Boric Acid pH 9.0: An amount of 7.4 g (± 0.5 g) of the boric acid is dissolved into 800 mL DI water. The solution is titrated with NaOH to pH 9.0. A sufficient quantity of DI water is added to adjust the volume to 1 liter. This solution can be stored at room temperature for up to 3 months.

[0184] Preparation of 1 mM Fluorescamine in Acetone: An amount of 28 ± 2 mg Fluorescamine is dissolved in 100 mL acetone. This solution needs to be freshly prepared and protected from light and moisture.Preparation of Tripeptide GPA Standard and Serial Dilution

[0185] A 0.08 mg / mL (329 pM) tripeptide GPA standard is prepared by making a 50- fold dilution of the 4 mg / mL tripeptide GPA stock in assay buffer (for example, 20 pL 4 mg / mL tripeptide GPA in 980 pL assay buffer). In the assay plate, row A, 200 pL of 329 pM tripeptide GPA standard is pipetted into Al and A7. An amount of 100 pL assay buffer is pipetted into A2- A6 and A8-A12.

[0186] For the tripeptide GPA standard serial dilution, an amount of 100 pL is transferred from Al into A2, mixed, an amount of 100 pL is transferred from A2 into A3, and repeated until A5. An amount of 100 pL is taken out from A5 so that its final volume is 100 pL. The A6 well contains buffer only.

[0187] For the second tripeptide GPA standard serial dilution, an amount of 100 pL is transferred from A7 into A8, mixed, an amount of 100 pL is transferred from A8 into A9, and repeated until well Al l. An amount of 100 pL is taken out from Al 1 so that its final volume is 100 pL. The A12 well contains buffer only.Preparation of Collagenase Samples

[0188] For collagenase samples (e.g., a lyophilized collagenase drug product), the sample is allowed to come to room temperature for at least 10 minutes and is reconstituted to form a 500 ng / mL stock solution. Different concentrations may be used. A test collagenase sample(T1A) is prepared from the stock solution by diluting with assay buffer. The procedure is repeated to prepare triplicate test samples (T1A, TIB, TIC).Discussion

[0189] In this method, 50 pL of increasing concentrations of the collagenase test samples are mixed with 50 pL of excess substrate (2.0 mg / mL final concentration) in a 96-well plate. An amount of 50 pL of assay buffer is added to rows C-G in a U-bottomed, 96 well polypropylene reaction plate. 150 pL of collagenase samples are pipetted into row B. Then, a 1 / 1.5 serial dilution is performed using a multi-channel pipette, by transferring 100 pL of collagenase sample from row B into row C, mixing and repeating the process until row G is reached. An amount of 100 pL is removed and discarded from row G. The below table contains the final collagenase concentrations after adding 50 pL substrate to row B through row H.

[0190] The Blank is prepared in row H by pipetting 50 pL assay buffer to row H. This row contains no enzyme. Exemplary concentrations are shown in the table below.Assay Target Concentrations After Substrate AdditionTable 10Collagenase Reaction

[0191] The zGPGGPA substrate is cleaved by class II collagenases into zGPG and GPA during a 15-minute incubation time at room temperature. The incubator and temperature probe are turned on to a temperature 22 ± 1°C prior to the addition of substrate to the plates. To column 1- 12 in row B-H, 50 pL 4 mg / mL (6.8 mM) zGPGGPA substrate is added column by column, then mixed. The reaction start time begins after the substrate is added to the first column. The plate is covered and placed in the 22 ± 1°C incubator for a total reaction time of 15 ± 1 minutes.

[0192] After incubation, the reaction is quenched by the addition of hydrochloric acid, and the amount of released GPA peptide is quantitated after reacting the free amino terminus of the peptide with the Anorogenic reagent, fluorescamine. To quench the reaction, 100 pL of 0.5 N HC1 is added into each well from row A to row H, added column by column, and then mixed. Reaction time ends after the HC1 is added to the first column.Detection

[0193] An amount of 195 pL of 120 mM Borate pH 9.0 is added to each well of a Microplate Greiner polypropylene black reading plate. 30 pL of the quenched reaction mixture is transferred from the reaction plate into the corresponding wells of the reading plate and mixed well. Then, 75 pL of 1 mM Fluorescamine is added to each well of the reading plate (using a polypropylene tray to dispense Fluorescamine / acetone) and mixed immediately after every addition. The plate is read within 15 minutes after Fluorescamine addition with a Molecular Devices M2 fluorescence plate reader using the following settings: Excitation 380 nm, Emission 473 nm, cutoff 455 nm, 6 reads / well, PMT medium.

[0194] The concentration of GPA (pM) versus the emission at 473 nm and the concentration of collagenase (ng / mL) versus the emission at 473 nm are plotted. For each plot, a linear regression is fitted with no fixed parameters. For collagenase test samples, the zero point data are excluded from the linear fit and the entire triplicate data set for each sample is used to generate the plot. The slopes for the tripeptide GPA standard and collagenase samples are determined.Potency Determination

[0195] The collagenase sample specific activity can be calculated as follows:

[0196] GPA Microplate Assay Units = ((Slope of Collagenase Sample) / (Slope of Tripeptide GPA x incubation time)) x 106.

[0197] The specific activity of the collagenase test sample is determined from the slope of the tripeptide GPA standard and calculated by the curve-fitting program. Using the microplate method, different concentrations of substrate and different times may be used to calculate enzyme kinetics according to Michaelis-Menten.5. Collagenase Potency In BTC Unit Assay

[0198] The bovine tendon collagen (BTC) assay is based on the procedures of Mandi et al., Arch. Biochem. Biophys. 74: 465-475 (1958), as modified by Keller and Mandi, 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 the activityof both collagenase I and II. The BTC assay is colorimetric and utilizes ninhydrin to detect the peptides produced by collagenase I and II degradation of BTC. This reaction is also run at pH 7.2, but for 22 h at 37° C in tris (hydroxymethyl) aminomethane (TRIS) buffer containing 10 mM divalent calcium ion. Since bovine tendon collagen is an insoluble substrate, it is important that it be finely divided. Trypsin is run as a control in order to account for the presence of denatured collagen or other protein impurities. The assay is run in the presence of calcium ions, which are required for collagenase activity. The number of peptides solubilized is determined by reacting the N-terminal amino group of the peptides with ninhydrin and measuring colorimetrically the amount of adjunct formed (Rosen 1957).Reagents and Solutions

[0199] The following reagents and buffers are used:1. Collagen Substrate (collagen)2. Deionized (DI) Water (water)3. Tris Assay Buffer4. Trypsin Stock Solution5. 0.5 M HC16. Leucine Standard Assay Solution (1 mM leucine)7. Rosen Buffer8. 3% Ninhydrin9. 50% IsopropanolIncubation

[0200] The reaction tubes are set up and labeled as follows: three tubes for the trypsin controls, six tubes for the Reference Solution and six tubes for each sample under test. 10 ± 1 mg collagen is weighed out in the order indicated in the below table and the weighed collagen is placed into each reaction tube.Table 11

[0201] For samples under test, the amount of enzyme should contain an activity between 1.6 to 5.7 nmol leucine equivalent (leu eq) / min per reaction tube (ACT). Undissolved samples should first be dissolved in Tris assay buffer before they are used in the assay. The concentration (before adding to the reaction tubes) should be no less than 0.0065 mg / mL.

[0202] The reaction tubes are set up to have a matrix pattern as shown in the below table. The following table assumes 2 under test samples. If more or less samples are run, the number of reaction tubes are adjusted, but the pattern is retained. Constant volumes are listed in the table.Table 12: The Matrix Pattern* Suggested maximum number of samples is 3.

[0203] The reaction tubes are capped and the contents are mixed gently but thoroughly.The reaction tubes are placed in a 37°C water bath and incubated for 22 ± 0.5 hours.Quenching And Filtration

[0204] A filtrate tube is labeled to correspond to each reaction tube incubated. A funnel containing a folded filter paper is placed onto each labeled filtrate tube . At the end of the incubationperiod, the reaction tubes are removed from the water bath. The actual time the incubation ends is recorded.

[0205] The reaction tubes are uncapped and the reaction is quenched by dispensing 2 mL 0.5 M HC1 into each reaction tube. The contents of the tubes is mixed thoroughly and fdtered into the appropriate fdtrate tube.

[0206] The previous two steps need to be finished as quickly as possible because undigested collagen could be dissolved by HC1 in a short time. The filtrate may be stored refrigerated in covered filtrate tubes for up to 95.5 hours before color development.Color Development

[0207] Boiling tubes are set up and labeled as follows: six tubes for the water and the leucine controls (Step 1) and two tubes for each filtrate tub (Step 1). The following amounts of water and leucine standard assay solution are placed into the six leucine control tubes.Table 13

[0208] 0.8 mL of water is pipetted into each boiling tube (Step 2). 0.2 mL of filtrate is pipetted from each sample into the appropriately labeled boiling tubes. 0.5 mL of Rosen buffer is dispensed into each boiling tube. Under a containment hood, 0.5 mL of 3% ninhydrin is dispensed into each boiling tube. The contents of each tube is mixed thoroughly on a vortex mixer. The boiling tubes are placed in a boiling water bath in a fume hood and boiled for 15 ± 1 minutes. At the end of the boiling period, the boiling tubes are removed from the water bath. Under a containment hood, 5.0 mL of 50% isopropanol is dispensed into each boiling tube and the contents are mixed thoroughly. The boiling tubes are allowed to reach ambient temperature (at least 10 minutes) before reading the absorbances.Reading of Absorbances

[0209] The absorbances of the tubes are read while working under a containment hood. The spectrophotometer is turned on and allowed to warm up. The wavelength of the spectrophotometer is set to 570 nm and the spectrophotometer is zeroed against 50% isopropanol. The absorbances (A570) of the water, leucine, trypsin controls, and the samples under test are read. The time that the first sample is read, in hours, is recorded. The readings as 1000 X A570 and thetime that the last sample is read, in hours, are recorded. All readings are to be done within a 1-hour interval.Calculations Principles

[0210] The total reading time, in minutes, and the total time of incubation are calculated. The total reading time should be less than 60 minutes and the total time of incubation should be between 1290 - 1350 minutes. Using the linear least square method, the slope "b" and correlation coefficient "r" for leucine standards are calculated (x = nmol leucine vs y = A570 reading). The unit for "b" value is As7o / nmol leucine. The "b" value is recorded to two decimal places, b value for leucine should be between 2.88 - 3.33. The average reading for the trypsin controls (T) is calculated. The average reading for the trypsin controls (T) should be 221 - 338. This average to the nearest whole number (Step A) is recorded. The average duplicated sample A570 reading for each reaction tube is calculated. This number is recorded to the nearest whole number. The average trypsin (Step A) is subtracted from the average sample A570 reading to get the net sample reading.

[0211] The activity (ACT) per tube, in nmol leu eq / min, is calculated as follows:ACT (nmol leu eq / min) = ((Net sample reading)(20)) / ((b)(Time in minutes)) where 20 is the dilution factor for the amount of reaction mixture developed and "b" is the slope of the leucine standard curve. This number is recorded to one decimal point. The activity per tube of the samples under test should be 1.6 - 5.7 nmol leu eq / min.

[0212] The activity in BTC units is calculated as follows:BTC units = activity in nmol leu eq / min x collagen correction factor.

[0213] The activity in BTC units / mL of the sample is calculated as follows:BTC unit / mL = (Activity in BTC units) / ( Sample volume used in m )

[0214] The specific activity of the sample in BTC units / mm is calculated as follows: BTC unit / mg = (Activity in BTC units / mL) / (Protein Concentration in mg / mL) Conversions

[0215] The conversion of BTC units to ABC units is:ABC units = BTC units x 1.09.

[0216] The conversion of ABC units to SRC units is:SRC units = ABC units x 6.3

[0217] The conversion of ABC units to mg is:1 ABC unit = 0.000058 mg of collagenase6. Other Assays

[0218] Assay methods utilizing labelled collagen have been reported by Gisslow et al., Anal. Biochem., 68: 70-78 (1975); Robertson et al., Clinica Chimica Acta, 42: 43-45 (1972); Sakamoto et al., A New Method for the Assay of Tissue Collagenase (36297) (1972); and the Worthington Biochemical Corp. Assay (www_worthington-biochem_com / CLS / assay) (accessed July 3, 2019).B. STUDY EN3835-108 PFA1. Overview

[0219] This was a multi-center, proof-of-concept study that was conducted to evaluate the safety, tolerability, immunogenicity, and effectiveness of escalating single doses of EN3835 in the treatment of adult participants with recalcitrant plantar fasciitis. Participants were screened to assign approximately 60 participants into 4 treatment groups (3 treatment groups receiving EN3835 and 1 treatment group receiving placebo). EN3835 and placebo were administered via intrafascial injection in different doses and concentrations.

[0220] The study was conducted in 3 sequential parts, Parts A, Al, and B, with delayed initiation of Part B, pending a safety assessment after all participants in Part Al had completed the Day 7 Visit. After providing informed consent, and completing screening assessments, participants received a study intervention on Day 1, with follow-up visits on Days 7, 14, 28, 42, 56, and 84. At each follow-up visit, safety was assessed by reporting of treatment-emergent adverse events (TEAEs), vital signs, and concomitant medications / procedures. Effectiveness was assessed by participant completion of a Pain Intensity NRS, Foot Pain Severity Interference in PFA Scale, Foot Pain Frequency Impact in PFA Scale, and Subject Satisfaction with Treatment Scale. Immunogenicity was assessed at Baseline and at Day 84.

[0221] During Part A, participants were randomized 1: 1 to receive either EN3835 as 3 aliquots of 0.1 mb to provide a total dose of 0.34 mg or matching placebo. Participants were blinded to study intervention assignment. During Part Al, participants received the same dose of EN3835 administered in Part A in an open-label manner. Safety data were continuously reviewed by the medical monitor. Once the last participant enrolled in Part Al completed their Day 7 Visit, an Internal Safety Review Committee (ISRC) review of the data occurred. The frequency of treatment-related serious adverse events (SAEs) and treatment-related severe adverse events (AEs) were used to determine if enrollment could commence into Part B of the study. Until the ISRC completed its assessment, enrollment into Part B was held, but continued screening waspermissible during this time. During Part B of the study, participants received EN3835 as 3 aliquots of 0.1 mL to provide a total dose of 0.60 mg. EN3835 was administered in an open-label manner.

[0222] For participants enrolled in Part A of the study, the study comprised a Screening Period of up to 28 days, a Treatment Period of 1 day, and a Follow-up Period of approximately 83 days. The total duration that participants were expected to enroll in the study was approximately 112 days. For participants enrolled in Parts Al and B of the study, the study comprised a Screening Period of up to 45 days. Total duration of study participation for Parts Al and B participants was approximately 129 days. All participants attended follow-up visits and completed final study assessments on Day 84 of the study. A schema depicting study flow is presented in Figure 1 and a study schematic diagram is presented in Figure 2.2. Selection of Study Population — Inclusion Criteria

[0223] For inclusion into the study, participants were required to fulfill all the following criteria at the Screening Visit and on Day 1 :• Age and Sex: Be an ambulatory male or female 18 to 65 years of age.• Disease Characteristic: Had no significant medical history or examination findings related to the participant's heel or plantar area to be treated, which in the investigator's opinion, would make the participant unsuitable for study intervention administration.• Type of Participant: Had recalcitrant plantar fasciitis in only one foot. o Had a diagnosis of plantar fasciitis with at least 6 months of symptoms prior to the Screening Visit. o Symptoms had not responded to at least 6 months of conservative therapies, (conservative therapies may have included rest, physical therapy, splinting / bracing, orthotics, icing, physiotherapy, acetaminophen, corticosteroids, orNSAIDs).• Had recalcitrant plantar fasciitis with medial heel pain (must score minimum of 5 on the Pain Intensity NRS) in the foot to be treated.• Agreed to not initiate or change use of orthotics or inserts during the study period.• Had been willing and able to comply with all protocol required visits and assessments.• If female, been of nonchildbearing potential (history of hysterectomy, bilateral oophorectomy, bilateral tubal ligation, or postmenopausal with no history of menstrual flow in the 12 months prior to the Screening Visit); or, if of childbearing potential, be nonpregnant, nonlactating and agree to use effective contraception when with a male partner for the duration of the study and for 28 days after treatment. Acceptable forms of contraception include hormonal measures (oral contraceptive pills, contraceptive patch,contraceptive ring, or injections), intrauterine devices, double barrier method (condom plus diaphragm, condom or diaphragm plus spermicidal gel or foam, surgical sterilization of the male partner), and abstinence.• Agreed not to use opioids (e.g., codeine, heroin, hydrocodone, hydromorphone, morphine, or oxycodone) during the study period and has not used opioids 2 weeks before the Screening Visit.3. Study Intervention / Selection of Injection Site

[0224] For each group in the study, 3 injections sites were identified on the heel of the foot. The injection sites were spaced 0.5 cm from each other and marked. Complete instructions were provided in the Injection Guide in the Study Operations Manual (summarized below). Topical anesthetics (e.g., cream, ointment, gel, spray) could have been applied before injection of study intervention at the point of maximum tenderness provided it had not interfered with proper marking of injection sites or with administration of the study intervention. At the investigator’s discretion, a local nerve block could have been administered before injection of the study intervention, according to local institutional practice. The anesthetic used was to be captured as a concomitant medication.

[0225] a. Study Intervention Administered: Details of the study intervention and administration are presented in Table 14. Study intervention was administered via intrafascial injections using a palpation guided technique.Table 14: Study Intervention Administration*Approx. 0.34 mg administered equates to approx. 0.2 mg in the results below because there was dead space in the syringes used to inject the collagenase.

[0226] Study intervention was administered after reconstitution with diluent. Intrafascial injections of study intervention into the plantar fascia was administered, as detailed in Table 14, using a palpation guided technique in one treatment in three (3) aliquots following participant entry into the Interactive Response Technology (IRT) system, further details provided in Table 15.

[0227] b. Identity of Investigational Products: EN3835 (XIAFLEX®) and its placebo are sterile lyophilized powders that are reconstituted with a sterile diluent made of 0.9% sodium chloride and 0.03% calcium chloride dihydrate in water. EN3835 / placebo was supplied in glass vials. All participants administered EN3835 received EN3835 lot 54853 (Par Sterile Products LLC for Auxilium Pharmaceuticals LLC) and sterile diluent lot 50790 (Par Sterile Products LLC for Auxilium Pharmaceuticals LLC). All participants who were administered placebo received lot 347477 (manufactured by Par Sterile Products LLC for Auxilium Pharmaceuticals LLC) and sterile diluent lot 50790 (manufactured by Par Sterile Products LLC for Auxilium Pharmaceuticals LLC).

[0228] c. Instructions for Injection Site Marking Utilizing the Palpation Guided Technique: Locate the medial calcaneal tuberosity via palpation, and using a surgical marker, place a reference mark on the palpated site. Approximately 2.5 centimeters (cm) distal (toward the toes) to the medial calcaneal tuberosity mark the first injection site, centered between the midline of the foot and the medial border of the plantar fascia medial band. The remaining 2 injection sites should be marked in the triangular shape depicted in Figure 3 approximately 0.5 cm spacing between sites.

[0229] During Part A, participants were randomized 1: 1 to receive either EN3835 as 3 aliquots of 0. 1 mL to provide a total dose of 0.34 mg or matching placebo. Participants received 3 aliquots as 3 injections administered on Day 1. During Part Al, participants received the same dose in an open-label manner. In Part B, participants received 0.60 mg of EN3835 administered as 3 aliquots in an open-label manner.Table 15: Dose of Study Intervention■ Each aliquot was 1 injection: participants received 3 aliquots as 3 injections. In Part A, a 1-mL syringe with a 25- gauge x 1.5-inch needle was used for each injection. In Parts Al and B, a 1-mL syringe with a 25-gauge x 1-inch needle was used for all 3 injections.NA = Not applicable d. Instructions for Three (3) Aliquot Intrafascial Injection:

[0230] 1. Prepare one single administration syringe as instructed.

[0231] 2. The injection site area of the foot should be disinfected with an antiseptic sterile solution (e.g., 70% isopropyl alcohol) and allowed to dry, to prevent potential infection. If desired, apply anesthetics as needed, before injection of study intervention. While the participant is sitting in a podiatry chair or on the examination table with their leg extended in front of them, secure the affected foot with your non-dominant hand, and grasp the toes close to the metatarsal heads and dorsiflex to palpate the plantar fascia.

[0232] 3. Using a surgical marker, place a reference mark on the palpated site at the calcaneal tuberosity. Mark the first injection site approximately 2.5cm distal (toward the toes) to the calcaneal tuberosity reference mark, centered between the midline of the foot and the medial border of the plantar fascia band. The remaining two (2) injection sites should be marked in a triangular shape, maintaining approximately 0.5cm spacing between the sites (as illustrated in Figure 3).

[0233] 4. With your dominant hand, perform the first of the three (3) 0.1 m injections using the administration syringe previously prepared (step 1 above). The direction of the injection should be perpendicular to the skin and medial band of the fascia. Ensure the needle is placed intrafascially by resistance upon insertion, taking care to avoid hitting the calcaneus. In the event the needle tip passes through the fascia, partially withdraw the needle until the tip is within the fascia.

[0234] 5. Prior to injection, be sure to withdraw the plunger of the syringe to ensure that you do not inject intravascularly. If there are signs of blood coming inside the syringe, restart making sure to select a new injection site. If there was negative aspiration, continue with step 5.

[0235] 6. Slowly depress the plunger to the 0.2 m mark to administer the first 0. 1 m injection of study intervention within the plantar fascia. After each injection, hold the syringestable with plunger depressed for approximately 10 seconds, as tolerated by the participant. This will allow the study intervention to penetrate and absorb into the injected fascial area. Remove the needle slowly while keeping the plunger depressed.

[0236] 7. For the remaining two (2) injections, repeat steps 4, 5 with the same needle and syringe (depressing plunger to 0.1 mb for the second injection and to 0.0 mb for the third injection).

[0237] 8. Place a soft gauze bandage held in place by dressing tape, over the injected area of the foot.

[0238] 9. After the study intervention administration is complete, the participant should lay supine for posttreatment vital signs assessments, and then remain in either the podiatry chair or examination table with leg extended in front of them. The participant should not move the injected foot for 30 minutes.

[0239] e. Instructions for Care Procedures After Injection: To evaluate the participant for possible immediate immunological adverse events (AEs), the participant will remain in direct observation of medical personnel who are skilled in the management of acute allergic reactions for the first 30 minutes after receiving the injection(s) of study intervention. The participant may be discharged from the study unit after a 30-minute observation period provided:• The participant exhibits no sign of an immunological or other clinically significant systemic or local AE.• The participant's vital signs have remained stable throughout the 30-minute observation period.• Apply a soft gauze bandage held in place by a self-adherent elastic wrap to the treated area.• Instruct the participant when to remove the dressing and to inspect the treated foot for edema, bruising, etc. as per standard of care.• After the observation period has concluded, participants should be encouraged to resume normal daily activities including walking and bearing weight on the treated foot as tolerated.

[0240] 4. Method of Assigning Participants to Treatment Groups: In Part A, participants were randomized according to an interactive response technology (IRT) system to receive the study interventions in a 1: 1 ratio and to ensure balance across treatment groups. Once all participants in Part Al completed the Day 7 Visit, an ISRC reviewed and provided a recommendation that the study may continue . Enrollment then commenced into Part B . Safety data was continuously reviewed by the medical monitor. A participant randomized (Part A) or assignedto study intervention in the IRT system (Parts Al and B) was considered enrolled in the study. Part A was conducted in a single-blind manner. Parts Al and B were conducted in an open-label manner.

[0241] a. Doses Used in the Study: The range of EN3835 doses administered in a single treatment session was 0.34 mg to 0.60 mg. Study intervention (EN3835) was injected at a pre specified location in the medial band of the plantar fascia.

[0242] The study was conducted in 3 sequential parts (A, Al, and B) where Parts A and Al investigated the lowest dose (0.34 mg), and Part B investigated the highest dose (0.60 mg). The ISRC reviewed safety data through Day 7 after dosing between Parts Al and B. Part B enrolled after the ISRC delivered an able-to-proceed decision. This dose range provided information on the safety, tolerability, and effectiveness of EN3835 versus placebo and informed the doses selected for use in the subsequent clinical studies.

[0243] b. Selection and Timing of Dose for Each Participant: Participants were assigned to receive a single dose of either EN3835 or placebo during this study. All participants were expected to attend all follow-up visits (Days 7, 14, 28, 42, 56) and remain in the study until the Day 84 (EOS) Visit.

[0244] c. Blinding: During Part A of this study, participants (but not the investigators or the sponsor) were blinded to treatment assignment. Parts Al and B were unblinded.

[0245] 5. Effectiveness and Safety Variables: The following measures were used to assess effectiveness during this study: the Pain Intensity NRS, Foot Pain Severity Interference in PFA Scale, the Foot Pain Frequency Impact in PFA Scale, and the Subject Satisfaction with Treatment Scale.

[0246] a. Recalcitrant Plantar Fasciitis Patient Reported Outcome Instrument: At Screening (Day -45 to Day -1), Days 1, 7, 14, 28, 42, 56, and 84, each participant was asked to complete questionnaires addressing symptom experience related to pain using the Recalcitrant Plantar Fasciitis Patient Reported Outcome (RPF PRO) Instrument. The questionnaires were completed in the following order:

[0247] i. Pain Intensity NRS: At Screening (Day -45 to Day -1), Days 1, 7, 14, 28, 42, 56, and 84, each participant was asked to describe the worst severity of foot pain in the past week, on a Pain Intensity NRS of 0 to 10, with 0 signifying “No Pain” to 10 “Pain as Bad as You Can Imagine.”

[0248] ii. Foot Pain Severity Interference in PFA Scale: At Screening (Day -45 to Day - 1), Days 1, 7, 14, 28, 42, 56, and 84, each participant was asked to characterize the severity of footpain when trying to perform activities of daily living. The severity of foot pain is characterized on a 5-point ordinal scale ranging from 0 indicating "None" to 4 indicating "Very Severe." Activities of daily living include:1. Taking the first steps in the morning2. After standing for a long time3. Walking after sitting for a long time4. Walking after standing for a long time5. Walking a short distance (for example, in and / or around the home)6. Walking longer distances (outside the home, more than a block)7. At the end of the day

[0249] iii. Foot Pain Frequency Impact in PFA Scale: At Screening (Day -45 to Day - 1), Days 1, 7, 14, 28, 42, 56, and 84, each participant was asked to characterize the frequency of foot pain on 5-point ordinal scale during 3 activities of daily living. The scale ranges from 0 indicating "Never" to 4 indicating "Always." The activities of daily living include:1. Sleep (e.g., falling asleep and / or staying asleep)2. Ability to complete daily routine activities (e.g., housework, yardwork, running errands)3. Ability to participate in recreational activities (e.g., exercise, running, traveling)

[0250] iv. Subject Satisfaction with Treatment Scale: At At Screening (Day -45 to Day -1), Days 1, 7, 14, 28, 42, 56, and 84, each participant was asked to rate his / her satisfaction with treatment of their plantar fasciitis on a 5-point scale ranging from -2 ("Very Dissatisfied") to +2 ("Very Satisfied") as follows:-2 Very Dissatisfied- 1 Quite Dissatisfied0 Neither Satisfied nor Dissatisfied+1 Quite Satisfied+2 Very Satisfied

[0251] 6. Safety Assessments: All safety assessments were performed throughout the study. Additional (unscheduled) safety assessments were performed as needed. To ensure participant safety and protect data integrity, Applicant, in accordance with the FDA Guidance on Conduct of Clinical Trials of Medical Products during COVID- 19 Public Health Emergency (March 2020, updated 30 August 2021), allowed remote visits for certain safety assessments. Additionally, participants impacted by the health emergency were allowed to continue in the study and complete remaining assessments when the investigational sites were open.

[0252] Assessment of the Severity of Local Injection Site Reactions: Based on clinical assessments, investigators classified and assessed the severity of local injection site reactions (ISRs) during the study according to Table 16.Table 16: Assessment and Grading of the Severity of Injection Site ReactionsSource: Adapted from the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) Version 5 and FDA Guidance for Industry Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials, September 1997.ADL = activities of daily living.7. Method for Effectiveness Analysis:

[0253] Effectiveness parameters in the Full Analysis Set (FAS) were analyzed, summarized, and listed by treatment group. The FAS included all participants who received at least one injection of EN3835 or placebo, and had at least one Pain Intensity NRS measure. This analysis set will be used for all efficacy analyses. Effectiveness parameters in this study included assessments of the Pain Intensity NRS, Foot Pain Severity Interference in PFA Scale, Foot Pain Frequency Impact in PFA Scale, and Subject Satisfaction with Treatment Scale. Since the primary objective of this study was to assess safety and tolerability, assessment of effectiveness was considered secondary or exploratory. The estimands for the secondary and exploratory endpoints are included in Table 17.Table 17: Estimands for the Secondary and Exploratory EndpointsTable 17: Estimands for the Secondary and Exploratory Endpoints (Continued)8, Method for Secondary Analysis:

[0254] a. Secondary Endpoint 1 : Mean Change from Baseline to Days 7, 14, 28, 42, and 56 in the Pain Intensity NRS Scores between Treatment Groups. There were no sensitivity or supportive analyses for this endpoint. As shown in FIG. 4, observed mean Pain Intensity NRS scores in the FAS are presented by study visit and treatment group using a line graph with Pain Intensity NRS scores (y-axis), and study visit along the x-axis. As shown in FIG. 5, the mean changes from Baseline in the Pain Intensity NRS scores in the FAS are presented by study visit and treatment group using a line graph with the mean change from Baseline score (y-axis) and visit along the x-axis.

[0255] b. Secondary Endpoint 2: The Proportion of Participants Reporting “Quite Satisfied” or “Very Satisfied” by Treatment Group on Days 7, 14, 28, 42, and 56 on the Subject Satisfaction with Treatment Scale.9, Exploratory Endpoints

[0256] Exploratory Endpoint 1 : Mean Change from Baseline to Each Study Visit in the Foot Pain Severity Interference PFA Scale Overall Score and for Each Individual Question on the Scale for Participants Treated with EN3835 or Placebo.

[0257] Exploratory Endpoint 2: Mean Change from Baseline to Each Study Visit on the Foot Pain Frequency Impact in PFA Scale Overall Score and for Each Individual Question for Participants Treated with EN3835 or Placebo.

[0258] Exploratory Endpoint 3 : Mean Change from Baseline to Day 84 / EOS / ET in the Pain Intensity NRS Scores Between Treatment Groups.

[0259] Exploratory Endpoint 4: The Proportion of Participants Reporting "Quite Satisfied" or "Very Satisfied" on the Subject Satisfaction with Treatment Scale.by Treatment Group on Day 84.10, Primary Safety Analysis

[0260] Primary Endpoint: To Assess the Incidence, Severity, and Duration of TEAEs. The primary estimand is defined by the following:• The primary clinical question of interest was: In participants with recalcitrant plantar fasciitis, what is the rate of incidence, severity, and duration of TEAEs in each treatment group throughout the study?• Treatment condition: Study intervention was either EN3835 or placebo, in the doses described for the following groups:• Part A: o Three 0.1-mL aliquots (0.34 mg dose) of EN3835o Three 0.1-mL aliquots of placebo• Part Al: o Three 0.1-mL aliquots (0.34 mg dose) of EN3835• Part B: o Three 0.1-mL aliquots (0.60 mg dose) of EN3835• Target Population: Adult participants (male or female 18 to 65 years of age) with recalcitrant plantar fasciitis in only 1 foot with medial heel pain (must score a minimum of 5 on the Pain Intensity NRS) that are included in the Safety Population.• Variable (Endpoint) of interest: Incidence, severity, and duration of treatment-emergent adverse events.• Treatments being compared: Rate of incidence, severity, and duration of TEAEs were compared for all treatment groups, but specific comparisons were conducted for the following: o Three 0.1-mL aliquots (0.34 mg dose) ofEN3835 (Part A) vs 3 aliquots of matching placebo (Part A)• The rate of incidence, severity, and duration of TEAEs were assessed in the following groups: o Three 0.1-mL aliquots (0.34 mg dose) of EN3835 (Part Al) o Three 0.1-mL aliquots (0.60 mg dose) of EN3835 (Part B)• Intercurrent Event: The treatment policy strategy was adopted for the analysis.• Use of rescue medication was the potential intercurrent event that could have occurred. All observed data was used for the analysis irrespective of the use of prohibited medications or concomitant medications.• Population Level-Summary measure: AEs were summarized by System Organ Class (SOC), preferred term (PT), severity, and duration of TEAE. The analysis was summarized by treatment group as frequency and percentages of participants.11. Effectiveness EvaluationData Sets Analyzed

[0261] The Safety Population comprised all 62 participants enrolled in the study. The FAS comprised 61 participants enrolled in the study. All but one participant was included in the FAS; this participant was excluded from the FAS because of a missing post injection Pain Intensity NRS score.Table 18: Data Sets AnalyzedDemographic and Other Baseline Characteristics

[0262] The mean (SD) age of participants was 49.8 (8.3) years old. More than three- quarters of participants enrolled were between 40 to 60 years old (47 participants; 77%) and were female (47 participants; 77%). Most participants were white (56 participants; 91.8%), not Hispanic or Latino (53 participants; 86.9%), and overweight (mean [SD] BMI 29.63 [3.93] kg / m2). Recalcitrant plantar fasciitis was reported more frequently in left foot (36 participants; 59%) than the right foot (25 participants; 41%).

[0263] Effectiveness Results and Tabulations of Individual Participant Data- Analysis of Effectiveness — The analysis of effectiveness in this study included the changes from Baseline and the differences between treatment groups in the scales comprising the RPF PRO (i.e., Pain Intensity NRS, Foot Pain Severity Interference with PFA Scale, and the Foot Pain Frequency Impact in PFA Scale), and the Subject Satisfaction with Treatment Scale.

[0264] Recalcitrant Plantar Fasciitis Patient Reported Outcome (RPF PRO) Instrument: Change from Baseline in the Pain Intensity NRS. Per the inclusion criteria, enrolled participants were required to have recalcitrant plantar fasciitis with a Pain Intensity NRS score > 5. At Baseline, the mean (SD) Pain Intensity NRS scores were similar between treatment groups. All treatment groups had mean pain scores > 7 at Screening and Baseline, indicating severe pain (See FIG. 4, line graph showing the Observed Mean Pain Intensity Numerical Ranking Scale (NRS) Score by treatment group (Day 1 injection of placebo or about 0.2 mg, 0.34 mg, or 0.6 mg collagenase) wherein the NRS Score is measured at Days 1, 7, 14, 28, 42, 56, and 84; Table 19, below).

[0265] FIG. 4 is a line graph showing the Observed Mean Pain Intensity Numerical Ranking Scale (NRS) Score by treatment group (Day 1 injection of placebo or about 0.2 mg, 0.34 mg, or 0.6 mg collagenase) wherein the Pain Intensity NRS Score is measured at Days 1, 7, 14, 28, 42, 56, and 84. FIG. 5 is a line graph showing the Mean Change from Baseline in Pain Intensity NRS Score by treatment group (Day 1 injection of placebo or about 0.2 mg, 0.34 mg, or 0.6 mg collagenase) wherein the NRS Score is measured at Days 1, 7, 14, 28, 42, 56, and 84. FIG. 6 is aline graph of the data plotted in FIG. 4 with the vertical axis adjusted for clarity. FIG. 7 is a line graph of the data plotted in FIG. 5 with the vertical axis adjusted for clarity.

[0266] After receiving injections of study intervention on Day 1, by Day 7, there was an initial increase (worsening) in the Pain Intensity NRS scores in all treatment groups, apart from the placebo treatment group. After this initial peak on Day 7, Pain Intensity NRS scores decreased from Baseline over time in all treatment groups (Table 12) with the reduction in pain most pronounced in the EN3835 treatment groups. On Day 84 / EOS / ET, the mean (SD) observed scores were 5.0 (3.37) in the Part A placebo treatment group, 3.6 (2.59) in the Part A EN3835 0.34-mg treatment group, 2.9 (2.50) in the Part Al EN3835 0.34-mg treatment group, and 3.8 (3.27) in the Part B EN3835 0.60-mg treatment group.

[0267] The magnitude of change in the mean (SD) Pain Intensity NRS scores from Baseline to Day 84 / EOS / ET was similar between the 3 EN3835 treatment groups, which was higher than in the placebo treatment group:• Part A Placebo: -2.8 (3.14)• Part A EN3835 0.34 mg: -4.1 (2.98)• Part Al EN3835 0.34 mg: -4.3 (2.43)• Part B EN3835 0.60 mg: -4.0 (3.29)Table 19: Observed and Mean Change from Baseline in Pain Intensity NRS Score byStudy Visit and Treatment Group (FAS)Table 19: Observed and Mean Change from Baseline in Pain Intensity NRS Score by StudyVisit and Treatment Group (FAS) (Continued)■ Baseline was defined as the last non-missing measurement / assessment prior to the first dose of study intervention. SD = Standard Deviation, EOS = End of Study, ET = Early Termination.Note: Pain Intensity NRS Score ranges from 0 to 10, with 0 signifying “No Pain” to 10 signifying “Pain as Bad as You Can Imagine.”

[0268] Change from Baseline in the Foot Pain Severity Interference in PFA Scale: At Baseline, the mean (SD) Foot Pain Severity Interference score in all treatment groups, indicated that “Moderate” to “Severe” foot pain due to plantar fasciitis that interferes with their activities. After receiving treatment with study intervention on Day 1, on Day 7, there was an initial increase (worsening) in the overall mean Foot Pain Severity Interference in PFA Scale scores in all treatment groups, apart from the Part A placebo and EN3835 0.34 mg treatment group, where the pain scores decreased (improved) slightly. After Day 7, Foot Pain Severity Interference in PFA Scale scores decreased over time in all treatment groups with the reduction in pain most pronounced in the EN3835 treatment groups (Table 20, below).

[0269] On Day 84 / EOS / ET, the observed overall mean (SD) Foot Pain Severity Interference in PFA Scale scores remained stable at 1.580 (0.9724) in the Part A placebo treatmentgroup, 1.325 (0.7586) in the Part A EN3835 0.34 mg treatment group, 1.047 (1.0144) in the Part Al EN3835 0.3 -mg treatment group, and 1.331 (0.9567) in the Part B EN3835 0.60 mg treatment group. The magnitude of change in the overall mean (SD) Foot Pain Severity Interference in PFA Scale scores from Baseline to Day 84 / EOS / ET was similar between the 3 EN3835 treatment groups (Table 13):• Part A Placebo: -1.022 (1.1391)• Part A EN3835 0.34 mg: -1.369 (0.7624)• Part Al EN3835 0.34 mg: -1.439 (0.8687)• Part B EN3835 0.60 mg: -1.716 (1.1566)Table 20: Observed and Mean Change from Baseline on Foot Pain SeverityInterference in PFA Scale (Overall Mean) by Study Visit and Treatment Group (FAS)Table 20: Observed and Mean Change from Baseline on Foot Pain SeverityInterference in PFA Scale (Overall Mean) by Study Visit and Treatment Group (FAS) (Continued)■ Baseline was defined as the last non-missing measurement / assessment prior to the first dose of study intervention. SD = Standard Deviation, EOS = End of Study, ET = Early Termination.Note: The severity of foot pain was characterized on a 5-point ordinal scale ranging from 0 indicating “None” to 4 indicating “Very Severe.” The overall mean score was derived as the average of responses to all 7 foot pain severity interference questions. If a response to any individual question was missing, then overall mean score was considered missing for the respective study visit.

[0270] Change from Baseline on Foot Pain Severity Interference in PFA Scale by Question: The observed and mean change from Baseline for each question on Foot Pain Severity Interference in PFA Scale by study visit and treatment group are presented in Table 21 below. There was a noteworthy improvement at Day 84 / EOS / ET in the response to level of pain participants experienced after taking the first steps in the morning. At Baseline, more than half of all participants in all treatment groups, reported “Severe” or “Very Severe” Pain after taking the first steps in the morning. At Day 84 / EOS / ET, fewer than 25% of participants in all treatment groups reported “Severe” or “Very Severe” pain, with the greatest mean [SD] improvement fromBaseline on this question, observed in the Part A 0.34 mg treatment group (-1.7 [0.91]) and in the Part B EN3835 0.60 mg treatment group (-1.9 [1.18]).

[0271] There was also a noteworthy improvement at Day 84 / EOS / ET in the response to level of pain participants experienced at the end of the day. At Baseline, more than half of all participants in all treatment groups, reported “Severe” or “Very Severe” Pain after taking the first steps in the morning. At Day 84 / EOS / ET, < 20% of participants in all treatment groups reported “Severe” or “Very Severe” pain, with the greatest mean [SD] improvement from Baseline on this question, observed in the Part A 0.34 mg treatment group (-1.5 [0.94]) and in the Part B EN3835 0.60 mg treatment group (-1.8 [1.24]).

[0272] Change from Baseline on Foot Pain Frequency Impact in PFA Scale: At Baseline, mean (SD) scores on the Foot Pain Frequency Impact Scale indicated that participants in all treatment groups indicated that the frequency of their foot pain “Sometimes” interferes with sleep, daily routine activities, and recreational activities. After receiving study intervention on Day 1 there was an initial decrease (improvement) in the overall Foot Pain Frequency Impact in PFA Scale scores on Day 7 in all treatment groups except for the Part B EN3835 0.60 mg treatment group. After Day 7, the overall mean Foot Pain Frequency Impact in PFA Scale scores decreased over time in all treatment groups with the reduction in pain most pronounced in the EN3835 treatment groups (Table 21 below).

[0273] On Day 84 / EOS / ET, the observed mean (SD) Foot Pain Frequency Impact in PFA Scale scores were 1.286 (1.0112) in the Part A placebo treatment group, 1.072 (0.9717) in the Part A EN3835 0.34 mg treatment group, 0.913 (1.1028) in the Part Al EN3835 0.34 mg treatment group, and 0.896 (1.0380) in the Part B EN3835 0.60 mg treatment group (Table 21 below).

[0274] The magnitude of change in the overall mean (SD) Foot Pain Frequency Impact in PFA Scale scores from Baseline to Day 84 / EOS / ET was similar between the placebo, Part A and Al EN3835 treatment groups and highest in the Part B EN3835 treatment group:• Part A Placebo: -1.284 (1.0283)• Part A EN3835 0.34 mg: -1.260 (0.8994)• Part Al EN3835 0.34 mg: -1.376 (0.9747)• Part B EN3835 0.60 mg: -1.916 (1.1579)Table 21 : Observed and Mean Change from Baseline on Foot Pain Frequency Impact inPFA Scale (Overall Mean) by Study Visit and Treatment Group (FAS)Table 21 : Observed and Mean Change from Baseline on Foot Pain Frequency Impact in PFA Scale (Overall Mean) by Study Visit and Treatment Group (FAS) (Continued)■ Baseline was defined as the last non-missing measurement / assessment prior to the first dose of study intervention. Note: The frequency of foot pain was characterized on 5-point ordinal scale. The scale ranges from 0 indicating “Never” to 4 indicating “Always.” The overall mean score was derived as the average of responses to all 3, foot pain frequency impact questions. If a response to any individual question was missing, then overall mean score was considered as missing for the respective study visit.

[0275] Change from Baseline on Foot Pain Frequency Impact in PFA Scale by Question: There was a noteworthy change from Baseline to Day 84 / EOS / ET in the response to change in frequency on foot pain when falling asleep or staying asleep. At Baseline, approximately one quarter of participants indicated that their frequency of pain in plantar fasciitis, was “Often” or “Always” a factor in falling asleep and / or staying asleep. At Day 84 / EOS / ET, fewer than 10% of participants in the EN3835 treatment groups reported that their frequency of pain was “Often” or “Always” a factor in falling / and or staying asleep, with the greatest magnitude of improvement noted in the EN3835 0.60 mg treatment group (Table 21). The observed and mean change fromBaseline for all questions on the Foot Pain Frequency Impact in PFA Scale is presented by study group and treatment group in Table 21.

[0276] Subject Satisfaction with Treatment Scale: By Day 84 / EOS / ET, more than half of all participants receiving EN3835 were considered responders on the Subject Satisfaction with Treatment Scale (Part A EN3835 0.34 mg treatment group: 8 participants; 57. 1%, Part Al EN3835 0.34 mg treatment group: 10 participants; 66.7%, Part B EN3835 0.60 mg treatment group: 9 participants; 56.3%). This was notably higher than the responder rate in the Part A placebo treatment group (5 participants; 35.7%) (Table 22 below).Table 22: Summary of Subject Satisfaction with Treatment Scores by Treatment Group and by Study Visit (FAS)Table 22: Summary of Subject Satisfaction with Treatment Scores by Treatment Group and by Study Visit (FAS) (Continued)■ A responder was defined as a participant with a response of “Very Satisfied” or “Quite Satisfied” at the specified study visit.Note: Subject Satisfaction with Treatment Scale is ranging from -2 (“Very Dissatisfied”) to +2 (“Very Satisfied”). Percentages are based on the number of participants (‘n’) at each study visit.

[0277] FIG. 8 is a line graph showing Mean Subject Satisfaction by treatment group (Day 1 injection of placebo or about 0.2 mg, 0.34 mg, or 0.6 mg collagenase) wherein the Mean Subject Satisfaction is measured at Days 1, 7, 14, 28, 42, 56, and 84. FIG. 9 is a line graph of the data plotted in FIG. 8 with the vertical axis adjusted for clarity.12. Effectiveness Conclusions

[0278] EN3835 resulted in an improvement in the change from Baseline throughout the study in all 3 scales comprising the RPF PRO (the Pain Intensity NRS, the Foot Pain Severity Interference in PFA Scale, the Foot Pain Frequency Impact), and in the Subject Satisfaction with Treatment Scale. Changes from Baseline were consistent amongst the Part A single-blind, placebocontrol treatment group and the Parts Al and B EN3835 open-label treatment groups.

[0279] Improvement was reported in the Pain Intensity NRS scores. The magnitude of change from Baseline to Day 84 / EOS / ET in the mean (SD) Pain Intensity NRS scores was similar between the 3 EN3835 treatment groups; this was higher than in the placebo treatment group:• Part A Placebo: -2.8 (3.14)• Part A EN3835 0.34 mg: -4.1 (2.98)• Part Al EN3835 0.34 mg: -4.3 (2.43)• Part B EN3835 0.60 mg: -4.0 (3.29)

[0280] After the Day 7 Visit, there was an improvement at all timepoints during the study, in all treatment groups with the reduction in pain most pronounced in the EN3835 treatment groups. Improvement was reported in the Foot Pain Severity Interference in PFA Scale scores. The magnitude of change from Baseline to Day 84 / EOS / ET in the overall mean (SD) Foot Pain Severity Interference in PFA Scale scores was similar between the 3 EN3835 treatment groups:• Part A Placebo: -1.022 (1.1391)• Part A EN3835 0.34 mg: -1.369 (0.7624)• Part Al EN3835 0.34 mg: -1.439 (0.8687)• Part B EN3835 0.60 mg: -1.716 (1.1566)

[0281] After the Day 7 Visit, Foot Pain Severity Interference in PFA Scale scores decreased over time all treatment groups with the reduction in foot pain severity interference most pronounced in the EN3835 treatment groups. Improvement was reported in the overall Foot Pain Frequency Impact in PFA Scale scores. The magnitude of change from Baseline to Day 84 / EOS / ET in the mean (SD) Foot Pain Frequency Impact in PFA Scale scores was similar between the placebo, Part A and Al EN3835 treatment groups and highest in the Part B EN3835 treatment group:• Part A Placebo: -1.284 (1.0283)• Part A EN3835 0.34 mg: -1.260 (0.8994)• Part Al EN3835 0.34 mg: -1.376 (0.9747)• Part B EN3835 0.60 mg: -1.916 (1.1579)

[0282] After the Day 7 Visit, the overall mean Foot Pain Frequency Impact in PFA Scale scores decreased overtime in all treatment groups with the reduction in foot pain frequency impact most pronounced in the EN3835 treatment groups. On the Subject Satisfaction with Treatment Scale, by Day 84 / EOS / ET, more than half of all participants receiving EN3835 were considered responders, reporting "Very Satisfied" or "Quite Satisfied" (Part A EN3835 0.34 mg treatment group: 8 participants; 57.1%, Part Al EN3835 0.34 mg treatment group: 10 participants; 66.7%, Part B EN3835 0.60-mg treatment group: 9 participants; 56.3%). This was notably higher than the responder rate in the Part A placebo treatment group (5 participants; 35.7%).13. Discussion and Overall Conclusions

[0283] This study was a Phase 1 proof-of-concept study of EN3835 and placebo to assess the safety, effectiveness, and tolerability of single doses of study intervention in the treatment of plantar fasciitis nonresponsive to conservative measures. The safety profde of EN3835 was consistent with the known AE profde for other indications for which XIAFLEX is approved or being investigated (XIAFLEX® Prescribing Information; EN3835 Investigators Brochure). Most TEAEs were ISRs that were mild to moderate in severity, assessed as treatment-related, and resolved within 21 days. Almost all participants enrolled completed the study, with no SAEs, AESIs, or participants withdrawing from the study due to TEAEs. The frequency, severity, and duration of TEAEs was related to the dose, with a higher frequency, severity, and duration of AEs observed in the highest dose group (EN3835 0.60 mg treatment group). There were no clinically meaningful changes in hematology and biochemistry parameter values. In the Part B EN3835 0.60 mg treatment group, the proportion of participants that tested positive for anti-AUX-I and anti- AUX-II antibodies and neutralizing antibodies was lower than what was observed after a single cycle of EN3835 0.58 mg in the treatment of Dupuytren’s Contracture (DC) (XIAFLEX® Prescribing Information).

[0284] Treatment with EN3835 in participants with plantar fasciitis resulted in an improvement in the change from Baseline throughout the study in all 3 scales comprising the RPF PRO (the Pain Intensity NRS, the Foot Pain Severity Interference in PFA Scale, the Foot Pain Frequency Impact), and in the Subject Satisfaction with Treatment Scale. Changes from Baseline in effectiveness measures were consistent amongst the Part A single-blind, placebo-controlled treatment group and the Parts Al and B EN3835 open-label treatment groups. Overall, improvements were more pronounced in the EN3835 treatment groups than in the placebo treatment group. Participants that received EN3835 reported a change in pain intensity on the Pain Intensity NRS by Day 84 / EOS / ET. The magnitude of change observed is considered clinically meaningful to an individual participant (See, e.g., Dworkin RH, Turk DC, Wyrwich KW, et al. Interpreting the clinical importance of treatment outcomes in chronic pain clinical trials: IMMPACT recommendations. J Pain. 2008;9(2): 105-121). There was also improvement in the Foot Pain Severity Interference Scale scores and Foot Pain Frequency Impact Scale scores at Day 84 / EOS / ET. Finally, more than half of all EN3835 -treated participants were considered responders on the Subject Satisfaction with Treatment Scale. Overall, the safety and effectiveness profile of EN3835 in the treatment of recalcitrant plantar fasciitis was favorable and warrants further evaluation.C. STUDY EN3835-227 PFA1. Overview

[0285] This is an ongoing Phase 2, multicenter, double-blind, placebo-controlled, randomized, dose-ranging study to assess the efficacy, safety and tolerability of EN3835 (XIAFLEX® (collagenase Clostridium histolyticum)) in the treatment of plantar fasciitis (PF A). As detailed below, approximately 225 subjects will be randomized into 3 treatment groups (EN3835 0.3 mg, EN3835 0.6 mg, and placebo [75:75:75]) in a 1: 1: 1 ratio. EN3835 and placebo will be administered via intrafascial injection. After receiving study intervention on Day 1, participants will be encouraged to resume normal daily activities, including walking, and bearing weight on the treated foot. Participants will receive a study intervention on Day 1, with follow-up visits on Days 15, 29, 43, 57, and 85. Rescue analgesic medication will be provided only for the treatment of breakthrough pain due to plantar fasciitis. Participants will be counseled to take rescue medications to treat breakthrough pain due to plantar fasciitis, only. Should rescue analgesic medication be required, the Pain Intensity NRS score should be documented in an eDiary before each dose of rescue medication. Additionally, at approximately the same time every day, between 6:00 PM and 10:00 PM local time, participants will record the ADP score on the Pain Intensity NRS regardless of the daily rescue analgesic medication intake. The dose, frequency, amount, and timing of the rescue analgesic medication taken will be documented in the eDiary every time it is taken.

[0286] Inclusion Criteria: To be eligible to participate in the study, at the screening visit, the participant must:

[0287] Age and Sex: Be an ambulatory male or female 18 to 75 years of age (inclusive).

[0288] Disease Characteristics:• Have no significant medical history or examination findings related to the foot to be treated, which in the investigator's opinion, would make the participant unsuitable for study intervention administration.• Have unilateral plantar fasciitis.• Have a diagnosis of plantar fasciitis for > 6 months, which has not responded to conservative therapies. Conservative therapies may include rest, physical therapy, splinting / bracing, orthotics, icing, physiotherapy, acetaminophen, corticosteroids, or nonsteroidal anti-inflammatory drugs (NSAIDs).• Have current foot pain due to plantar fasciitis.

[0289] Type of Participant: Agree to not use prohibited medication, and not use any medication to treat plantar fasciitis pain throughout the study, except as permitted per the protocol. Further, participants must be willing and able to comply with all protocol requirements, required visits, procedures, and assessments.

[0290] For all participants, at the time points described herein, the efficacy of the study intervention will be assessed on the Pain Intensity NRS, the FFI Scale, PGIC Foot Pain Scale, the CGIC Scale, the Subject Satisfaction with Treatment Scale, and rescue medication use. Safety will be assessed by evaluating the incidence, severity, and duration of AEs, TEAEs, AESIs, SAEs, changes in vital signs and clinical laboratory values.

[0291] All participants will complete the study at the end of study (EOS) (Day 85) Visit, during which efficacy and safety will be assessed. The study will comprise a Screening Period of up to 28 days, a Treatment Period of 1 day, and a Follow-up Period of approximately 84 days. The total duration that participants are expected to enroll in the study is approximately 113 days. The EOS is defined as the completion of the final assessment for the last participant. All participants are expected to remain in the study until Day 85, regardless of whether the participant received a complete treatment (3 injections) of study intervention.

[0292] Based on the largest diameter of width or length of the affected area collected on ultrasound, subjects will receive 1 or 2 intralesional injections of EN3835 per affected area (Table 14). During the Initial Treatment Period subjects with affected areas of < 1.5 cm will receive 1 injection of EN3835 per affected area. Subjects with affected areas of > 1.5 cm will receive 2 injections of EN3835 per affected area. After discharge from the inpatient unit, subjects will be encouraged to resume normal daily activities including weight bearing and walking on the treated foot / feet.2. Identity of Investigational Products

[0293] CCH, or EN3835 (previously known as AA4500), comprises a mixture of 2 collagenases, AUX-I and AUX-II in an approximate 1: 1 mass ratio. These collagenases are isolated and purified from the fermentation of the bacterium Clostridium histolyticum. Collagenase AUX-I is a single polypeptide chain consisting of approximately 1000 amino acids. It has an observed molecular weight of 114 kDa. It belongs to the Class I Clostridium histolyticum collagenases. Collagenase AUX-II is a single polypeptide chain comprising approximately 1000 amino acids. It has an observed molecular weight of 113 kDa. It belongs to the Class II Clostridium histolyticum collagenases. These 2 collagenases are not immunologically cross-reactive and have different specificities, such that together they become synergistic, providing a very broad hydrolyzing reactivity toward collagen.3. Study Interventions AdministeredTable 23

[0294] Participants will be randomized in a 1: 1: 1 ratio to 1 of 3 treatment groups to receive either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. These will be administered in 0. 1-mL aliquots as 3 separate injections (Table 23).Table 24: Study Intervention Administration MethodaEach injection delivers 1 aliquot.4. Injection of Study Intervention

[0295] EN3835 or placebo will be administered at a volume of approximately 0.1 mb per injection for a total volume of 0.3 mb per treatment. Study intervention will be administered as an intrafascial injection into the plantar proximal portion of the medial band of the plantar fascia using a palpation guided technique. A 1-mL syringe with a 25-gauge x 1-inch needle will be used for each injection.

[0296] Approximately 30 minutes following injection, the foot will be bandaged, and participants are encouraged to resume normal daily activities including walking and bearingweight as tolerable. TEAEs should be managed per the investigator’s standard of care, with concomitant medications and / or therapies.5. Rescue Analgesic Medication

[0297] On Day 1, eligible participants will be provided with acetaminophen as rescue analgesic medication for breakthrough pain due to plantar fasciitis. Details of the rescue analgesic medication provided is detailed in Table 24.Table 25: Study Intervention - Rescue Analgesic Medication Administration■ Participants will record the Pain Intensity NRS score prior taking rescue analgesic medication.

[0298] Rescue analgesic medication will be dispensed via the interactive response technology (IRT) at Day 1, resupplied, and collected at subsequent visits until the Day 85 Visit. At each visit, participants will be instructed to bring the remaining rescue analgesic medication for drug accountability to be performed by the site.

[0299] Acetaminophen caplets / tablets / capsules will be issued by the sponsor in its approved marketed product (carton, bottle, documents) labeled for clinical use. Participants will be instructed to take up to 2 x 500 mg caplets / tablets / capsules of acetaminophen every 6 hours as needed for breakthrough plantar fasciitis pain. Participants will be counseled not to exceed 3000 mg (6 caplets / tablets / capsules) in a 24-hour period unless directed by the investigator.

[0300] Further, participants will be instructed to take rescue medications only to treat breakthrough pain due to plantar fasciitis. Should rescue analgesic medication be required, the Pain Intensity NRS score should be documented in an eDiary every time a dose of rescue medication is taken. Additionally, at approximately the same time every day, between 6:00 PM and 10:00 PM local time, participants will record the ADP score on the Pain Intensity NRS. The dose, frequency, amount, and timing of the rescue analgesic medication taken will be documented in the eDiary every time it is taken. Participants will be counseled that many over-the-counter (OTC) medications contain acetaminophen. Use of acetaminophen or other pain medications forother types of pain medications for other types of pain or illness should be recorded as a concomitant medication on the appropriate electronic case report (eCRF) page.6. Efficacy Assessments

[0301] Efficacy will be assessed by the participant through the following PRO assessments, including Pain Intensity NRS, FFI, PGIC Foot Pain, and the Subject Satisfaction with Treatment Scales. Efficacy will also be assessed by the investigator via the CGIC Scale. Rescue analgesic medication use will also be assessed.

[0302] Pain Intensity Numeric Rating Scale (NRS): Completed by the subjects daily. Each participant will be asked to describe their foot pain in the past 24 hours, on a Pain Intensity NRS ranging from 0 (“No Pain”) to 10 (“Worst Pain Imaginable”). Participants will also be asked to describe their foot pain, immediately before taking rescue medication, on a Pain Intensity NRS ranging from 0 (“No Pain”) to 10 (“Worst Pain Imaginable”).

[0303] Foot Function Index (FFI): Each participant will be asked to complete the FFI, which assesses the impact of foot pathology on 3 domains of pain, difficulty, and activity limitation. The FFI will be completed at screening (Day -28 to Day -1), Day 1, Day 15 (±3 days), Day 29 (±3 days), Day 43 (±3 days), Day 57 (±3 days) and Day 85 (EOS) / ET) (±3 days). The participant will complete the scale prior to evaluation by the investigator. The FFI Activity Limitation Subscale consists of 3 items and measures limitations in activities in the past week because of their feet, such as staying off one foot or both feet. It is scored on a 5 -point verbal rating scale as follows:0 - Never1 - Rarely2 - Sometimes3 - Often4 - Always

[0304] The FFI Difficulty Subscale consists of 9 items and measures difficulty performing various functional activities in the past week, because of their feet, such as difficulty climbing stairs. It is scored on a 5-point verbal rating scale as follows:0 - No difficulty1 - A little difficulty2 - Some difficulty3 - Much difficulty4 - A lot of difficulty

[0305] The FFI Pain Subscale consists of 9 items and measures the severity of foot pain during the past week in different situations, such as walking barefoot versus walking with shoes. For the pain subscale, if the participant marks “does not wear orthotics” items pertaining to orthotics are not scored and are not included in the total score. It is scored on a 5 -point verbal rating scale as follows:0 - None1 - Mild2 - Moderate3 - Severe4 - Extreme

[0306] Patient Global Impression of Change (PGIC) — Foot Pain: At Day 15 (±3 days), Day 29 (±3 days), Day 43 (±3 days), Day 57 (±3 days) and Day 85 (EOS) / ET) (±3 days), prior to evaluation by the investigator, each participant will be asked to describe the change in the overall severity of their foot pain in the past week on a 7-point scale, ranging from to +3 (“Very Much Improvement”) to -3 (“Very Much Worse”) as follows:+3 Very Much Improvement+2 Much Improvement+ 1 Minimal Improvement0 No Change-1 Minimally Worse-2 Much Worse-3 Very Much Worse

[0307] Subject Satisfaction with Treatment Scale: At Day 15 (±3 days), Day 29 (±3 days), Day 43 (±3 days), Day 57 (±3 days) and Day 85 (EOS) / ET) (±3 days),, prior to evaluation by the investigator), each participant will be asked to rate his / her satisfaction with treatment of their plantar fasciitis on a 5-point scale ranging from -2 (“Very Dissatisfied”) to +2 (“Very Satisfied”) as follows:-2 Very Dissatisfied-1 Quite Dissatisfied0 Neither Satisfied nor Dissatisfied+1 Quite Satisfied+2 Very Satisfied

[0308] Clinician Global Impression of Change (CGIC) Scale: At Day 15 (±3 days), Day 29 (±3 days), Day 43 (±3 days), Day 57 (±3 days) and Day 85 (end of study (EOS) / end of treatment (ET) (±3 days), the investigator will determine the degree of improvement in the participant's plantar fasciitis, compared with baseline (prior to initiation of treatment), on a 7-point scale ranging from -3 (“Very Much Worse”) to +3 (“Very Much Improvement”) as follows:-3 Very Much Worse-2 Much Worse-1 Minimally Worse0 No Change+ 1 Minimal Improvement+2 Much Improvement+3 Very Much Improvement7. Objectives, Endpoints and Estimands

[0309] Primary Endpoint: To assess the overall improvement in the intensity of foot pain due to plantar fasciitis receiving different doses of EN3835 or placebo on the Pain Intensity Numeric Rating Scale (NRS) at Week 12. Such improvement is measured by mean change from Baseline to Week 12 in the weekly mean of the average daily (24-hour) pain (ADP) score as measured on the Pain Intensity NRS in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The Pain Intensity NRS score ranges from 0 (“No Pain”) to 10 (“Worst Pain Imaginable”).

[0310] Key Secondary Endpoint #1: To assess the overall improvement on the Foot Function Index a (FFI) Difficulty Subscale in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. Such improvement is measured by mean change from Baseline to Day 85 in the FFI Difficulty Subscale score in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The FFI Difficulty Subscale score ranges from 0 to 36, with the individual score on each of the 9 items ranging from 0 (“No Difficulty”) to 4 (“A Lot of Difficulty”).

[0311] Key Secondary Endpoint #2: To assess the overall improvement on the FFI Activity Limitation Subscale in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. Such improvement is measured by mean change from Baseline to Day 85 in the FFI Activity Limitation Subscale score in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The FFI Activity Limitation Subscale scoreranges from 0 to 12, with the individual score on each of the 3 items ranging from 0 (“Never”) to 4 (“Always”).

[0312] Secondary Endpoint #1: To assess the overall improvement in foot pain on the Pain Intensity NRS in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. Such improvement is measured by mean change from Baseline to Weeks 2, 4, 6, 8 and 12, in the weekly mean of the ADP score measured on the Pain Intensity NRS in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The proportion of participants with > 30% decrease from Baseline to Week 12 in the weekly mean of the ADP score as measured on the Pain Intensity NRS in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The proportion of participants with > 50% decrease from Baseline to Week 12 in the weekly mean of the ADP score as measured on the Pain Intensity NRS in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The Pain Intensity NRS scores range from 0 (“No Pain”) to 10 (“Worst Pain Imaginable”).

[0313] Secondary Endpoint #2: To assess the overall improvement on the FFI Difficulty Subscale in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. Such improvement is measured by mean change from Baseline to Days 15, 29, 43, 57, and 85 in the FFI Difficulty Subscale score in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The FFI Difficulty Subscale score ranges from 0 to 36, with the individual score on each of the 9 items ranging from 0 (“No Difficulty”) to 4 (“A Lot of Difficulty”).

[0314] Secondary Endpoint #3: To assess the overall improvement on the FFI Activity Limitation Subscale in participants with plantar fasciitis receiving different doses of EN3835 or placebo overtime. Such improvement is measured by mean change from Baseline to Days 15, 29, 43, 57, and 85 in the FFI Activity Limitation Subscale score in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The FFI Activity Limitation Subscale score ranges from 0 to 12, with the individual score on each of the 3 items ranging from 0 (“Never”) to 4 (“Always”).

[0315] Secondary Endpoint #4: To assess the overall improvement on the FFI Pain Subscale in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. Such improvement is measured by mean change from Baseline to Days 15, 29, 43, 57, and 85 in the FFI Pain Subscale score in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The FFI Pain Subscale score ranges from 0 to 36, with the individual score on each of the 9 items ranging from 0 (“None”) to 4 (“Extreme”).

[0316] Secondary Endpoint #5: To assess the overall improvement on the FFI a Scale in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. Such improvement is measured by mean change from Baseline to Days 15, 29, 43, 57, and 85 in the FFI Total score in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The FFI Total score ranges from 0 to 84, with a higher score indicating worse pain, difficulty, and activity limitation.

[0317] Secondary Endpoint #6: To assess rescue analgesic medication use in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. An evaluation will be made of the proportion of participants with plantar fasciitis that used rescue analgesic medication during the study receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The total amount (mg) of rescue analgesic medication used during the study in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo will be assessed.

[0318] Secondary Endpoint #7 : To assess the patient global impression of change on the PGIC Foot Pain Scale with treatment in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. An evaluation will be made of the proportion of participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo reporting “Minimal Improvement” (+1), “Much Improvement” (+2), and “Very Much Improvement” (+3) on the PGIC Foot Pain Scale. The PGIC Foot Pain Scale is a 7-point scale ranging from -3 (“Very Much Worse”) to +3 (“Very Much Improvement”) on Days 15, 29, 43, 57, and 85.

[0319] Secondary Endpoint #8: To assess investigator assessment of improvement on the CGIC Scale in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. An evaluation will be made of the proportion of participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo for whom the investigator reported “Minimal Improvement” (+1), “Much Improvement” (+2), or “Very Much Improvement” (+3) on the CGIC Scale. The CGIC Scale is a 7-point scale ranging from -3 (“Very Much Worse”) to +3 (“Very Much Improvement”) on Days 15, 29, 43, 57, and 85.

[0320] Secondary Endpoint #9: To assess participant satisfaction with treatment in participants with plantar fasciitis receiving different doses of EN3835 or placebo over time. An evaluation will be made of the proportion of participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo reporting “Quite Satisfied” or “Very Satisfied” on the Subject Satisfaction with Treatment Scale on Days 15, 29, 43, 57, and 85. The Subject Satisfaction with Treatment Scale is a 5-point scale ranging from -2 (“Very Dissatisfied”) to +2 (“Very Satisfied”).8. Safety Assessment and Endpoint

[0321] To assess the safety and tolerability of different doses of EN3835 and placebo when administered to participants with plantar fasciitis throughout the study. An evaluation will be made of the incidence, severity, and duration of treatment-emergent adverse events (TEAEs), adverse events of special interest (AESIs), and serious adverse events (SAEs) throughout the study in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The change from Baseline in vital signs will be assessed at Days 15, 29, 43, 57, and 85 in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. The change from Baseline in clinical laboratory values will be assessed at Day 85 in participants with plantar fasciitis receiving either EN3835 0.3 mg, 0.6 mg, or placebo.

[0322] Based on clinical assessments, investigators should classify and assess the severity of local injection site reactions during the study according to Table 25 below. The table does not comprise a comprehensive list of injection site reactions. Local injection site reactions will be reported as TEAEs in the eCRF.Table 26: Assessment and Grading of the Severity of Injection Site ReactionsSource: Adapted from the National Cancer Institute Common Terminology Criteria for Adverse Events Version 5 and FDA Guidance for Industry Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials, September 1997.ADL = activities of daily living; CAM = controlled ankle movement9. Statistical Considerations

[0323] This section is a summary of the planned statistical analyses of the most important endpoints including the primary endpoint.

[0324] Statistical Hypotheses: For the primary estimand, the change from Baseline toWeek 12 in the weekly mean of the ADP score on the Pain Intensity NRS, the aim is to obtain the estimates for each treatment group at Week 12.• Multiplicity Adjustments: o For the primary estimand, the primary comparisons will be tested sequentially based on “gatekeeping” procedures to control the type I error. The order of the testing will be: o EN3835 0.6 mg vs placebo o EN3835 0.3 mg vs placeboThe significance tests for key secondary endpoints will also be conducted in sequential order to preserve the overall type-I error rate < 0.05 in the following order, including the comparison order for each:1. Key secondary endpoint # 1 o EN3835 0.6 mg vs placebo o EN3835 0.3 mg vs placebo2. Key secondary endpoint #2 o EN3835 0.6 mg vs placebo o EN3835 0.3 mg vs placeboTesting of the first key secondary endpoint will be conducted if the results of both primary comparisons are statistically significant. Testing of these key secondary endpoints andassociated pairwise comparisons will be conducted in a hierarchical manner defined above until the p value > 0.05. For the other secondary endpoints, no multiplicity adjustment(s) will be made. The nominal p values will be reported for these endpoints.

[0325] Analysis Sets: For the purposes of analysis, the following analysis sets are defined:Table 27Statistical Analyses

[0326] Primary Analysis — Definition of Endpoint: The primary endpoint is defined as the mean change from Baseline to Week 12 in the weekly mean of the ADP score as measured on the Pain Intensity NRS in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo.• Primary Endpoints / Estimand Analysis: The primary estimand includes the following components: o Treatment of Interest: The randomized study intervention (0.3-mg dose of EN3835, 0.6-mg dose of EN3835, or placebo) o Target Population: Adult participants with plantar fasciitis who are included in the FAS. o Variable (Endpoint) of Interest: Change from Baseline to Week 12 in the weekly mean of the ADP score as measured on the Pain Intensity NRS. o Intercurrent Events: The intercurrent events during this study include discontinuation of study intervention or withdrawal from the study, rescue medication use, prohibited medication use, and surgical procedures for plantar fasciitis. A treatment policy and composite strategy will be used for handling the intercurrent events as specified below:■ Intercurrent events due to discontinuation of study intervention or withdrawal from the study due to an AE or lack of efficacy (LOE) will be handled using a composite strategy. The multiple imputation (MI) of the baseline observationcarried forward (BOCF) of the weekly mean of ADP scores on the Pain Intensity NRS will be used to impute the weekly mean of Week 12.■ Intercurrent events due to discontinuation of study intervention or withdrawal from the study due to reasons other than an AE or LOE (e.g., withdrew consent or loss to follow-up) will be handled using a treatment policy strategy. If applicable, the weekly mean of Week 12 will be imputed using an MI method.■ Intercurrent events due to any prohibited medication used or surgical procedure for plantar fasciitis will be handled using a composite strategy. The MI of the BOCF of the weekly mean of ADP scores on the Pain Intensity NRS will be used to impute the weekly mean of Week 12.■ Intercurrent events due to any excessive rescue medication use will be handled using a composite strategy. The MI of the BOCF of the weekly mean of ADP scores on the Pain Intensity NRS will be used to impute the weekly mean of Week 12. Excessive rescue medication use is defined as a participant that used > 2000 mg acetaminophen / average daily dose over a 12-week period and > 5 out of 7 days rescue medication use at Week 12. If a participant used rescue medication and did not meet the excessive use criteria, it will be considered as limited rescue medication use.■ Intercurrent events due to any limited rescue medication use will be handled using a treatment policy strategy. If applicable, the weekly mean of Week 12 will be imputed using an MI method. o Population Level-Summary Measure: Least square mean (LSM) estimates for treatment difference between each EN3835 treatment group (EN3835 0.3 mg and placebo and EN3835 0.6 mg and placebo) will be reported.

[0327] After imputation, the changes from Baseline will be analyzed using an analysis of covariance (ANCOVA) model with treatment group as fixed effect, baseline score as the covariate. The results from the imputed datasets will be combined using Rubin's method. The summary measure, LSM estimate for treatment difference between EN3835 treatment groups and placebo and their corresponding 95% confidence intervals (Cis), will be reported.

[0328] Tipping Point Analysis: A sensitivity analysis utilizing a tipping -point approach will be implemented to assess the robustness of the treatment effect.

[0329] Another sensitivity analysis will be performed based on all observed data (no imputation) up to Week 12. The changes from Baseline will be analyzed using a mixed effect model for repeated measures (MMRM). This MMRM model will include treatment group, studyvisit, treatment by visit interaction as fixed effects, and baseline as a covariate. The repeated measure is the participant visits. The dependent variable will be the change from Baseline at each visit. The summary measure, LSM estimate for the treatment difference at Week 12 between each EN3835 treatment group and placebo and the corresponding 95% Cis, will be reported. An unstructured covariance structure will be used in the model. If the model does not converge using the unstructured covariance structure, an autoregressive covariance structure will be used instead.

[0330] ANCOVA with a Different Variance Estimate: The third sensitivity analysis will be performed using the same imputed datasets from the primary analysis, but deviations from the ANCOVA assumptions will be explored, including regression with Huber-White sandwich errors, and accounting for heteroskedasticity across treatment arms. The summary measure, the LSM estimate forthe treatment difference at Week 12 between each EN3835 group and placebo and the corresponding 95% Cis will be reported.

[0331] Key Secondary Endpoint #1 Analysis: Key secondary endpoint #1 is defined as the mean change from Baseline to Day 85 in the FFI Difficulty Subscale score in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. Key secondary estimand #1 includes the following components:• Treatment of Interest: The randomized study intervention (0.3 mg dose of EN3835, 0.6 mg dose of EN3835, or placebo)• Target Population: Adult participants with plantar fasciitis who are included in the FAS.• Variable (Endpoint) of Interest: Change from Baseline to Day 85 in the FFI Difficulty Subscale score.• Intercurrent Events: The intercurrent events during this study include discontinuation of study intervention or withdrawal from the study, rescue medication use, prohibited medication use, and surgical procedures for plantar fasciitis. A treatment policy and composite strategy will be used for handling the intercurrent events as specified below: o Intercurrent events due to discontinuation of study intervention or withdrawal from the study due to an AE or LOE will be handled using a composite strategy. The MI of the BOCF on the FFI Difficulty Subscale will be used to impute the score of Day 85. o Intercurrent events due to discontinuation of study intervention or withdrawal from the study due to reasons other than an AE or LOE (e.g., withdrew consent or loss to followup) will be handled using a treatment policy strategy. If applicable, the FFI Difficulty Subscale score of Day 85 will be imputed using an MI method.o Intercurrent events due to any prohibited medication used or surgical procedure for plantar fasciitis will be handled using a composite strategy. The MI of the BOCF on the FFI Difficulty Subscale will be used to impute the score of Day 85. o Intercurrent events due to any excessive rescue medication use will be handled using a composite strategy. The MI of the BOCF on the FFI Difficulty Subscale will be used to impute the score of Day 85. Excessive rescue medication use is defined as a participant that used > 2000 mg acetaminophen / average daily dose over a 12-week period and > 5 out of 7 days rescue medication use at Week 12. If a participant used rescue medication and did not meet the excessive use criteria, it will be considered as limited rescue medication use. o Intercurrent events due to any limited rescue medication use will be handled using a treatment policy strategy. If applicable, the FFI Difficulty Subscale score of Day 85 will be imputed using an MI method.• Population Level-Summary Measure: LSM estimates for treatment difference between each EN3835 treatment group (EN3835 0.3 mg and placebo and EN3835 0.6 mg and placebo) will be reported.

[0332] The method of analysis (including sensitivity analysis and handling of missing data) for key secondary endpoint # 1 will follow the same method of analysis used for the primary estimand.

[0333] Key Secondary Endpoint #2 Analysis: Key secondary endpoint #2 is defined as the mean change from Baseline to Day 85 in the FFI Activity Limitation Subscale score in participants with plantar fasciitis receiving either EN3835 0.3 mg, EN3835 0.6 mg, or placebo. Key secondary estimand #2 includes the following components:• Treatment of Interest: The randomized study intervention (0.3 mg dose of EN3835, 0.6 mg dose of EN3835, or placebo)• Target Population: Adult participants with plantar fasciitis who are included in the FAS.• Variable (Endpoint) of Interest: Change from Baseline to Day 85 in the FFI Activity Limitation Subscale score.• Intercurrent Events: The intercurrent events during study include discontinuation of study intervention or withdrawal from the study, rescue medication use, prohibited medication use, and surgical procedures for plantar fasciitis. A treatment policy and composite strategy will be used for handling the intercurrent events as specified below: o Intercurrent events due to discontinuation of study intervention or withdrawal from the study due to an AE or LOE will be handled using a composite strategy. The MIof the BOCF on the FFI Activity Limitation Subscale will be used to impute the Day 85 score. o Intercurrent events due to discontinuation of study intervention or withdrawal from the study due to reasons other than an AE or LOE (e.g., withdrew consent or loss to follow-up) will be handled using a treatment policy strategy. If applicable, the FFI Activity Limitation Subscale score of Day 85 will be imputed using an MI method. o Intercurrent events due to any prohibited medication used or surgical procedure for plantar fasciitis will be handled using a composite strategy. The MI of the BOCF on the FFI Activity Limitation Subscale will be used to impute the score of Day 85. o Intercurrent events due to any excessive rescue medication use will be handled using a composite strategy. The MI of the BOCF on the FFI Activity Limitation Subscale will be used to impute the score of Day 85. Excessive rescue medication use is defined as a participant that used 2000 mg acetaminophen / average daily dose over a 12-week period and 5 out of 7 days rescue medication use at Week 12. If a participant used rescue medication and did not meet the excessive use criteria, it will be considered as limited rescue medication use. o Intercurrent events due to any limited rescue medication use will be handled using a treatment policy strategy. If applicable, the FFI Activity Limitation Subscale score of Day 85 will be imputed using an MI method.• Population Level-Summary Measure: LSM estimates for treatment difference between each EN3835 treatment group (EN3835 0.3 mg and placebo and EN3835 0.6 mg and placebo) will be reported.

[0334] The method of analysis (including sensitivity analysis and handling of missing data) for key secondary endpoint #2 will follow the same method of analysis used for the primary estimand.

[0335] Secondary Endpoints Analyses: The other secondary endpoints include the mean changes from Baseline to study visits (Days 15, 29, 43, 57, and 85) for FFI Subscale scores, FFI Total score, and the mean changes from Baseline in the weekly average in the ADP score on the Pain Intensity NRS at Weeks 2, 4, 6, 8 and 12. Summary statistics will be provided for each parameter by treatment group. These endpoints will be analyzed using the MMRM method based on observed data. The LSM estimates for each EN3835 treatment group and placebo difference at each visit and their corresponding 95% Cis will be reported.

[0336] In addition, the proportion of participants with a > 30% or a > 50% decrease from Baseline at Week 12 in the weekly mean ADP score as measured on the Pain Intensity NRS will be compared between EN3835 treatment groups to placebo using the Cochran-Mantel-Haenszel (CMH) method. The proportion of participants that used rescue analgesic medication overall will be compared between EN3835 treatment groups to placebo using the CMH method. In addition, the amount (mg) of rescue analgesic medication used overall will be analyzed using an ANOVA to compare treatment groups. Response rates for the PGIC Foot Pain, CGIC, and participant satisfaction with treatment will be compared between each EN3835 treatment group and placebo using a CMH test at each scheduled study visit. A participant will be defined as a PGIC Foot Pain Scale responder or CGIC Scale responder if their response is “Minimal Improvement,” “Much Improvement,” or “Very Much Improvement;” otherwise, the participant will be defined as a nonresponder. A participant will be defined as a participant satisfaction with treatment responder if their response is “Quite Satisfied” or “Very Satisfied;” otherwise, the participant will be defined as a non-responder. All secondary efficacy endpoints will be summarized by treatment group and study visit (if applicable) using appropriate descriptive statistics.10. Results

[0337] Pain Intensity NRS: As described above, the range for the Pain Intensity NRS Score is 10, where 0 is no pain and 10 is worst pain imaginable. A negative change from baseline indicates pain improvement, i.e., lower scores are better. The subjects’ baseline values are obtained before treatment with collagenase. Applicant expects that there will be a reduction in the weekly mean of the average daily (24-hour) pain (ADP) score as measured on the Pain Intensity NRS at week 12 versus baseline. For example, the reduction may comprise: (a) at least about a 0.5, 1.0, 1.5 or 2.0 point reduction in the weekly mean of the ADP score at week 12 versus baseline; (b) a reduction in the weekly mean of the ADP score at Weeks 2, 4, 6, 8 and 12 versus baseline; or (c) at least about a 0.5, 1.0, 1.5 or 2.0 point reduction in the weekly mean of the ADP score at Weeks 2, 4, 6, 8 and 12 versus baseline. Applicant further expects that the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA will result in a negative change from baseline of about 0.0 to -2.0 at Day 7 after treatment, or about -0.5 to about -4 at Day 14 after treatment, or about - 1 to about -5 at Day 28 after treatment, or about - 1.5 to about -6 at one or more of Days 42, 56, or 84 after treatment.

[0338] Subject Satisfaction Treatment Scale: Applicant expects that a treated patient reports “Quite Satisfied” or “Very Satisfied” on the Subject Satisfaction with Treatment Scale on Days 15, 29, 43, 57, and 85. For example, that at least 25%, or at least 35%, or at least 45%, or at least 55% of subjects in a patient population will report they are “Quite Satisfied” or “VerySatisfied” at one or more of Days 7, 14, 28, 42, 56, or 84 following a collagenase treatment of about 0.2 mg to about 1.0 mg (total dose).

[0339] Foot Function Index (FFI): As described above, each subject is asked to complete the FFI, which assesses the impact of foot pathology on 3 domains of pain, difficulty, and activity limitation. A negative change from baseline indicates pain improvement, i.e., lower scores are better. The subjects’ baseline values are obtained before treatment with collagenase. Applicant expects improvement in one or more of the 3 domains. For example: (a) a reduction in one or more of the FFI Difficulty Subscale, FFI Activity Limitation Subscale or FFI Pain Subscale scores at Day 85 versus baseline; (b) at least about a 1.0, 2.0 or 3.0 point reduction in the FFI Difficulty Subscale score at Day 85 versus baseline; (c) at least about a 1.0, 2.0 or 3.0 point reduction in the FFI Activity Limitation Subscale score at Day 85 versus baseline; (d) a reduction in the FFI Difficulty Subscale score at Days 15, 29, 43, 57, and 85 versus baseline; (e) at least about a 1.0, 2.0 or 3.0 point reduction in the FFI Difficulty Subscale score at Days 15, 29, 43, 57, and 85 versus baseline; (f) a reduction in the FFI Activity Limitation Subscale score at Days 15, 29, 43, 57, and 85 versus baseline; (g) at least about a 1.0, 2.0 or 3.0 point reduction in the FFI Activity Limitation Subscale score at Days 15, 29, 43, 57, and 85 versus baseline; (h) a reduction in the FFI Pain Subscale score at Days 15, 29, 43, 57, and 85 versus baseline; (i) at least about a 1.0, 2.0 or 3.0 point reduction in the FFI Pain Subscale score at Days 15, 29, 43, 57, and 85 versus baseline; (j) a reduction in the FFI Total score at Days 15, 29, 43, 57, and 85 versus baseline; or (k) at least about a 1.0, 2.0 or 3.0 point reduction in the FFI Total score at Days 15, 29, 43, 57, and 85 versus baseline. Applicant further expects that the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA will result in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

[0340] Patient Global Impression of Change (PGIC) — Foot Pain: As described above, PGIC refers to a patient-reported outcome assessment using a multipoint scale to assess the change in the overall severity of their foot pain in the past week on a 7-point scale, ranging from to +3 (“Very Much Improvement”) to -3 (“Very Much Worse”). A positive change from baseline indicates pain improvement, i.e., higher scores are better. The subjects’ baseline values are obtained before treatment with collagenase. Applicant expects treated patients will exhibit an at least one point positive change on the PGIC Foot Pain Scale. Applicant further expects that the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA will result in a positive change from baseline of about 0.0 to +1.0 at Day 7 after treatment, or about +0.2 to about +2.5 atDay 14 after treatment, or about +0.5 to about +3 at Day 28 after treatment, or about +0.75 to about +3.5 at Day 42 after treatment, or about +1.0 to +4 at one or more of Days 56 or 84 after treatment.

[0341] Clinician Global Impression of Change Scale, or CGIC Scale: As described above, CGIC Scale refers to when an investigator determines the degree of improvement with treatment per the affected foot on a 7-point scale ranging from -3 (“Very Much Worse”) to +3 (“Very Much Improvement”). A positive change from baseline indicates pain improvement, i.e., lower scores are better. The subjects’ baseline values are obtained before treatment with collagenase. Applicant expects treated patients will exhibit an at least one point positive change on the CGIC Scale. Applicant further expects that the injection of a total dose of about 0.2 mg to about 1.0 mg to treat PFA will result in a positive change from baseline of about 0.0 to +1.0 at Day 7 after treatment, or about +0.2 to about +2.5 at Day 14 after treatment, or about +0.5 to about +3 at Day 28 after treatment, or about +0.75 to about +3.5 at Day 42 after treatment, or about +1.0 to +4 at one or more of Days 56 or 84 after treatment.

[0342] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

[0343] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.

Claims

CLAIMSWhat is claimed:

1. A method of treating plantar fasciitis in a subj ect, the method comprising : inj ecting a pharmaceutical formulation comprising collagenase into a plantar fasciitis affected area in a total dose sufficient to treat the plantar fasciitis.

2. The method of claim 1, wherein the total dose of collagenase administered comprises between about 0.2 mg to about 2.0 mg.

3. The method of claim 1, wherein the collagenase comprises collagenase I activity, collagenase II activity, or a combination thereof.

4. The method of claim 3, wherein the collagenase has a potency of about 5 SRC units / mg to about 180,000 SRC units / mg.

5. The method of claim 4, wherein the collagenase has a potency of about 500 SRC units / mg to about 30,000 SRC units / mg.

6. The method of claim 3, wherein the collagenase has a potency of about 10,000 GPA units / mg to about 400,000 GPA units / mg.

7. The method of claim 6, wherein the collagenase has a potency of about 10,000 GPA units / mg to about 200,000 GPA units / mg.

8. The method of claim 1 , wherein the collagenase comprises a mixture of collagenase I and collagenase II activity, wherein the collagenase I potency is about 5 SRC units / mg to about 180,000 SRC units / mg, and the collagenase II potency is about 10,000 GPA units / mg to about 400,000 GPA units / mg.

9. The method of claim 1, wherein the collagenase comprises collagenase I and collagenase II from Clostridium histolyticum.

10. The method of claim 9, wherein the collagenase I comprises an amino acid sequence of SEQ ID NO: 1 and the collagenase II comprises an amino acid sequence of SEQ ID NO: 2.

11. The method of claim 1 , wherein the collagenase comprises CoIQ 1.

12. The method of claim 11, wherein the ColQl comprises an amino acid sequence of SEQ ID NO: 3.

13. The method of claim 1, wherein about 0.2 mg to about 0.6 mg of collagenase is injected to the affected area.

14. The method of claim 13, wherein about 0.2 mg of collagenase is injected to the affected area.

15. The method of claim 13, wherein about 0.3 mg of collagenase is injected to the affected area.

16. The method of claim 13, wherein about 0.6 mg of collagenase is injected to the affected area.

17. The method of claim 1, wherein the collagenase has a concentration of about 0.6 mg / ml to about 2.25 mg / ml.

18. The method of claim 17, wherein the collagenase has a concentration of about 0.6 mg / ml, about 1.2 mg / ml, or about 2.25 mg / ml.

19. The method of claim 1, wherein the collagenase is injected in one or more injections.

20. The method of claim 19, wherein each injection has a volume of about 0.1 ml to 0.3 ml.

21. The method of claim 19, wherein each injection has a volume of about 0.2 ml.

22. The method of claim 3, wherein about 5 SRC units to about 180,000 SRC units of collagenase are administered in one or more injections.

23. The method of claim 3, wherein the collagenase has a potency of about 5,000 f- SRC units / mg to about 30,000 f-SRC units / mg.

24. The method of claim 23, wherein about 50 f-SRC units to about 180,000 f-SRC units of collagenase are administered in one or more injections.

25. The method of claim 3, wherein about 1,000 GPA units to about 2,400,000 GPA units of collagenase are administered in one or more injections.

26. The method of claim 1, wherein the collagenase has a potency of about 175,000 f- GPA units / mg to about 500,000 f-GPA units / mg.

27. The method of claim 26, wherein about 1,750 f-GPA units to about 3,000,000 f- GPA units of collagenase are administered in one or more injections.

28. The method of claim 1, wherein the collagenase comprises collagenase I and / or collagenase II having a specific activity of about 5,000 BTC units / mg to about 25,000 BTC units / mg.

29. The method of claim 28, wherein about 50 BTC units to about 150,000 BTC units of collagenase I and / or collagenase II are administered in one or more injections.

30. The method of claim 1, wherein the collagenase comprises collagenase I and / or collagenase II having a specific activity of about 5,000 ABC units / mg to about 25,000 ABC units / mg.

31. The method of claim 30, wherein about 50 ABC units to about 150,000 ABC units of collagenase I and / or collagenase II are administered in one or more injections.

32. The method of claim 1, wherein treating the plantar fasciitis results in an improvement in Pain Intensity NRS Score.

33. The method of claim 1, wherein the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Pain Intensity NRS Score.

34. The method of claim 33, wherein the injection results in a negative change from baseline of about 0.0 to -2.0 at Day 7 after treatment, or about -0.5 to about -4 at Day 14 after treatment, or about - 1 to about -5 at Day 28 after treatment, or about - 1.5 to about -6 at one or more of Days 42, 56, or 84 after treatment.

35. The method of claim 1, wherein treating the plantar fasciitis results in an improvement in Foot Pain Severity Interference in PFA Scale.

36. The method of claim 1, wherein the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Foot Pain Severity Interference in PFA Scale.

37. The method of claim 36, wherein the injection results in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

38. The method of claim 1, wherein treating the plantar fasciitis results in an improvement in Foot Pain Frequency Impact in PFA Scale.

39. The method of claim 1, wherein the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Foot Pain Frequency Impact in PFA Scale.

40. The method of claim 39, wherein the injection results in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

41. The method of claim 1, wherein treating the plantar fasciitis results in an improvement in Subject Satisfaction with Treatment Scale.

42. The method of claim 1, wherein the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Subject Satisfaction with Treatment scale.

43. The method of claim 42, wherein the injection injected to a plurality of subjects results in at least 25%, or at least 35%, or at least 45%, or at least 55% of the subjects reporting they are “Quite Satisfied” or “Very Satisfied” at one or more of Days 7, 14, 28, 42, 56, or 84 following collagenase treatment.

44. A method of treating plantar fasciitis in a subject, comprising injecting a collagenase enzyme to a plantar fasciitis affected area at a total dose sufficient to treat the plantar fasciitis, the collagenase enzyme comprising:(a) means for binding or recruiting collagen Type I and Type III; and(b) means for breaking down collagen Type I and / or, collagen Type III.

45. The method of claim 44, wherein a total dose of collagenase injected comprises between about 0.2 mg to about 2.0 mg.

46. The method of claim 44, wherein the collagenase comprises collagenase I activity, collagenase II activity, or a combination thereof.

47. The method of claim 46, wherein the collagenase has a potency of about 5 SRC units / mg to about 180,000 SRC units / mg.

48. The method of claim 47, wherein the collagenase has a potency of about 500 SRC units / mg to about 30,000 SRC units / mg.

49. The method of claim 46, wherein the collagenase has a potency of about 10,000 GPA units / mg to about 400,000 GPA units / mg.

50. The method of claim 49, wherein the collagenase has a potency of about 10,000 GPA units / mg to about 200,000 GPA units / mg.

51. The method of claim 44, wherein the collagenase comprises a mixture of collagenase I and collagenase II activity, wherein the collagenase I potency is about 5 SRC units / mg to about 180,000 SRC units / mg, and the collagenase II potency is about 10,000 GPA units / mg to about 400,000 GPA units / mg.

52. The method of claim 44, wherein the collagenase comprises collagenase I and collagenase II from Clostridium histolyticum.

53. The method of claim 52, wherein the collagenase I comprises an amino acid sequence of SEQ ID NO: 1 and the collagenase II comprises an amino acid sequence of SEQ ID NO: 2.

54. The method of claim 44, wherein the collagenase comprises CoIQ 1.

55. The method of claim 54, wherein the ColQl comprises an amino acid sequence of SEQ ID NO: 3.

56. The method of claim 44, wherein about 0.2 mg to about 0.6 mg of collagenase is injected to the affected area.

57. The method of claim 56, wherein about 0.2 mg of collagenase is injected to the affected area.

58. The method of claim 56, wherein about 0.3 mg of collagenase is injected to the affected area.

59. The method of claim 56, wherein about 0.6 mg of collagenase is injected to the affected area.

60. The method of claim 44, wherein the collagenase has a concentration of about 0.6 mg / ml to about 2.25 mg / ml.

61. The method of claim 60, wherein the collagenase has a concentration of about 0.6 mg / ml, about 1.2 mg / ml, or about 2.25 mg / ml.

62. The method of claim 44, wherein the collagenase is injected in one or more injections.

63. The method of claim 62, wherein each injection has a volume of about 0.1 ml to 0.3 ml.

64. The method of claim 62, wherein each injection has a volume of about 0.2 ml.

65. The method of claim 46, wherein about 5 SRC units to about 180,000 SRC units of collagenase are administered in one or more injections.

66. The method of claim 46, wherein the collagenase has a potency of about 5,000 f- SRC units / mg to about 30,000 f-SRC units / mg.

67. The method of claim 66, wherein about 50 f-SRC units to about 180,000 f-SRC units of collagenase are administered in one or more injections.

68. The method of claim 46, wherein about 1,000 GPA units to about 2,400,000 GPA units of collagenase are administered in one or more injections.

69. The method of claim 44, wherein the collagenase has a potency of about 175,000 f-GPA units / mg to about 500,000 f-GPA units / mg.

70. The method of claim 69, wherein about 1,750 f-GPA units to about 3,000,000 f- GPA units of collagenase are administered in one or more injections.

71. The method of claim 44, wherein the collagenase comprises collagenase I and / or collagenase II having a specific activity of about 5,000 BTC units / mg to about 25,000 BTC units / mg.

72. The method of claim 71, wherein about 50 BTC units to about 150,000 BTC units of collagenase I and / or collagenase II are administered in one or more injections.

73. The method of claim 44, wherein the collagenase comprises collagenase I and / or collagenase II having a specific activity of about 5,000 ABC units / mg to about 25,000 ABC units / mg.

74. The method of claim 73, wherein about 50 ABC units to about 150,000 ABC units of collagenase I and / or collagenase II are administered in one or more injections.

75. The method of claim 44, wherein treating the plantar fasciitis results in an improvement in Pain Intensity NRS Score.

76. The method of claim 44, wherein the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Pain Intensity NRS Score.

77. The method of claim 76, wherein the injection results in a negative change from baseline of about 0.0 to -2.0 at Day 7 after treatment, or about -0.5 to about -4 at Day 14 after treatment, or about - 1 to about -5 at Day 28 after treatment, or about - 1.5 to about -6 at one or more of Days 42, 56, or 84 after treatment.

78. The method of claim 44, wherein treating the plantar fasciitis results in an improvement in Foot Pain Severity Interference in PFA Scale.

79. The method of claim 44, wherein the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Foot Pain Severity Interference in PFA Scale.

80. The method of claim 79, wherein the injection results in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

81. The method of claim 44, wherein treating the plantar fasciitis results in an improvement in Foot Pain Frequency Impact in PFA Scale.

82. The method of claim 44, wherein the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Foot Pain Frequency Impact in PFA Scale.

83. The method of claim 82, wherein the injection results in a negative change from baseline of about 0.0 to -1.0 at Day 7 after treatment, or about -0.2 to about -2.5 at Day 14 after treatment, or about -0.5 to about -3 at Day 28 after treatment, or about -0.75 to about -3.5 at Day 42 after treatment, or about -1.0 to -4 at one or more of Days 56 or 84 after treatment.

84. The method of claim 44, wherein treating the plantar fasciitis results in an improvement in Subject Satisfaction with Treatment Scale.

85. The method of claim 44, wherein the injection of a total dose of about 0.2 mg to about 0.6 mg of collagenase results in an improvement in Subject Satisfaction with Treatment scale.

86. The method of claim 85, wherein the injection injected to a plurality of subjects results in at least 25%, or at least 35%, or at least 45%, or at least 55% of the subjects reporting they are “Quite Satisfied” or “Very Satisfied” at one or more of Days 7, 14, 28, 42, 56, or 84 following collagenase treatment.

87. A method of injecting collagenase into a plantar fasciitis affected area in a foot of a human subject affected thereby, comprising: a. marking a first injection site approximately centered between the midline of the foot and the medial border of the plantar fascia medial band; b. marking two other injection sites in an approximately triangular shape; and c. injecting in each of the three sites an aliquot of a collagenase formulation.

88. The method of claim 87, wherein a clinician places a reference mark on a palpated site at the calcaneal tuberosity.

89. The method of claim 88, wherein the clinician marks the first injection site approximately 2.5 cm distal toward the toes to the calcaneal tuberosity reference mark, approximately centered between the midline of the foot and the medial border of the plantar fascia band.

90. The method of claim 87, wherein there is approximately 0.25 cm to approximately 1 cm spacing between each of the three sites.

91. The method of claim 89, wherein the remaining two injection sites are marked in a triangular shape, maintaining approximately 0.5 cm spacing between the sites.

92. The method of claim 87, wherein the direction of the injection is perpendicular to the skin and medial band of the fascia.

93. The method of claim 91, wherein the clinician slowly depresses the plunger to administer each of the injections of collagenase formulation within the plantar fascia.

94. The method of claim 93 , wherein after each inj ection, the clinician holds the syringe stable with the plunger depressed for approximately 10 seconds to allow the collagenase formulation to penetrate and absorb into the injected fascial area after which the needle is then removed slowly while keeping the plunger depressed.

95. The method of claim 91, wherein the injection volume of each of the three injections is approximately 0.1 mb.

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