WO2026028155A1 - Méthodes de traitement de la fasciite plantaire - Google Patents

Méthodes de traitement de la fasciite plantaire

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Publication number
WO2026028155A1
WO2026028155A1 PCT/IB2025/057817 IB2025057817W WO2026028155A1 WO 2026028155 A1 WO2026028155 A1 WO 2026028155A1 IB 2025057817 W IB2025057817 W IB 2025057817W WO 2026028155 A1 WO2026028155 A1 WO 2026028155A1
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WIPO (PCT)
Prior art keywords
collagenase
units
treatment
day
gpa
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Pending
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PCT/IB2025/057817
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English (en)
Inventor
Saji VIJAYAN
James P. TURSI
Luis A. ORTEGA
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Endo Biologics Ltd
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Endo Biologics Ltd
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Publication of WO2026028155A1 publication Critical patent/WO2026028155A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24003—Microbial collagenase (3.4.24.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24007—Interstitial collagenase (3.4.24.7), i.e. matrix metalloprotease 1 or MMP1

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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 plantar fascia. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • (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.
  • the total dose injected comprises between about 0.2 mg to about 1.0 mg.
  • FIG. 1 is a schema depicting the clinical study flow of Study EN3835-108 PFA.
  • FIG. 2 is the clinical study schematic diagram of Study EN3835-108 PFA.
  • FIG. 3 illustrates where the three injection sites are marked in a triangular shape.
  • 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.
  • NRS Observed Mean Pain Intensity Numerical Ranking Scale
  • 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 a line 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • biosimilar 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 that the 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.
  • 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.
  • Day 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.
  • 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.
  • plantar fasciitis 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.
  • ‘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.
  • treatment session is synonymous with “treatment visit” and includes a single visit to a doctor’s office in which the pharmaceutical formulation is administered.
  • Pain Intensity Numeric Rating Scale 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:
  • 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:
  • 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:
  • 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:
  • CGIC Scale Clinician Global Impression of Change Scale
  • FFI Froot Function Index
  • 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.
  • PGIC Patient Global Impression of Change
  • 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;
  • Kcat molecules of substrate cleaved per second
  • 1/ Kcat The microseconds required to cleave a molecule of substrate
  • Collagenase I activity 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 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.
  • “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.
  • 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.
  • the collagenase I comprises the amino acid sequence of SEQ ID NO: 1.
  • 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.
  • the collagenase II comprises the amino acid sequence of SEQ ID NO: 2. Table 1. Sequences
  • the collagenase may comprise ColQl having a sequence comprising the below: YSMADLNKMNDQELVETLGCIKWHQITDLFQFNEDAKAFYKDKGKMQVI IDELAHRGSTFTRDDSKGIQTFTEVLRS AFYLAFYNNELSELNERSFQDKCLPALKAIAKNPNFKLGTAEQDTWSAYGKLI SNASSDVETVQYASNILKQYNDN FNTYVNDRMKGQAIYDIMQGIDYDIQSYLIEARKEANETMWYGKVDGFINEINRIALLNEVTPENKWLVNNGIYFAS RLGKFHSNPNKGLEWTQAMHMYPRLSEPYFVAVEQITTNYNGKDYSGNTVDLEKIRKEGKEQYLPKTYTFDDGSIV FKTGDKVSEEKIKRLYWAAKEVKAQYHRVIGNDKALEPGNADDILTIVIYNSPEEYQLNRQLYGYETN
  • the collagenase can comprise a mixture of collagenase
  • 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 collagenase
  • 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.
  • the collagenase can comprise collagenase Clostridium histolyticum (CCH).
  • CCH 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' 1 o KM: About 4.1 to 410 nanoMolar o Kcat: About 1.1 to 107 sec' 1 o 1/Kcat: About 376 to 37,222 microseconds o Kcat/Kju: About 5,140 to 508,814 mM ⁇ sec' 1 and 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' 1 o KM: About 0.03 to 3.1 mM o Kcat: About 93 to 9,179 sec' 1 o 1/Kcat: About 4 to 428 microseconds o Kcat/KM: About 60 to 5,934 mM ⁇ sec' 1
  • 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 3.8 min' 1 o KM: About 2.07 x 10' 4 mM o Kcat: About 53 sec' 1 o 1/Kcat: About 18,799 microseconds o WKM: About 256,977 mM ⁇ sec' 1 and 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' 1 o KM: About 1.6 mM o Kcat: About 4,636 sec' 1 o 1/Kcat: About 216 microseconds o kcat/KM: About 2,997 mM ⁇ sec' 1
  • the collagenase I and collagenase II can have the following characteristics:
  • Collagenase I SRC microplate assay
  • Vmax About 0.08 to 7.70 min' 1
  • o KM About 4.1 to 410 nanoMolar
  • Kcat About 1.1 to 107 sec' 1
  • 1/Kcat About 376 to 37,222 microseconds
  • o Kcat/K M About 5,140 to 508,814 mM ⁇ sec' 1
  • Collagenase II (GPA microplate assay) o Vmax: About 0.3 to 30.5 min' 1 o KM: About 0.03 to 3.1 mM o Kcat: About 93 to 9,179 sec' 1 o 1/Kcat: About 4 to 428 microseconds o Kcat/KM: About 60 to 5,934 mM ⁇ sec' 1
  • the collagenase I and collagenase II can have the following characteristics:
  • Collagenase I (SRC assay): o Vmax: About 3.8 min' 1 o KM: About 2.07 x 10' 4 mM o Kcat: About 53 sec' 1 o 1/Kcat: About 18,799 microseconds o WKM: About 256,977 mM ⁇ sec' 1
  • Collagenase II (GPA assay): o Vmax: About 15.4 min' 1 o KM: About 1.6 mM o Kcat: About 4,636 sec' 1 o 1/Kcat: About 216 microseconds o kcat/KM: About 2,997 mM ⁇ sec' 1
  • 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.
  • the disclosed methods can comprise administering the pharmaceutical composition during a single treatment session or during multiple treatment sessions.
  • the number 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.
  • 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.
  • 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.
  • 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.
  • 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 is administered 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.
  • 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.
  • the collagenase I may have a potency of about 500 SRC units/mg to about 30,000 SRC units/mg.
  • 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 collagen
  • 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
  • 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 to about 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).
  • 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
  • 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).
  • 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 GPA units, 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 1,000 G
  • 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).
  • 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-
  • 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).
  • 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.
  • 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).
  • 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.
  • about 600 ABC units to about 33,750 ABC units are administered in a single treatment session.
  • the amount of collagenase administered in a subsequent treatment session can be greater than, less than, or the same as the amount of 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.
  • 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.
  • 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.
  • 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.
  • the collagenase may be in the form of a pharmaceutical formulation comprising collagenase and pharmaceutically acceptable excipients.
  • excipients may include sterile water or sodium chloride/calcium chloride for injection, pH adjusting agents and stabilizers.
  • 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.
  • 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 0.9 mg/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
  • 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.
  • 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.
  • 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.
  • 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.
  • a total volume of about 0.1 ml to about 1.0 ml can be administered to each affected area.
  • 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.
  • the pharmaceutical formulation can comprise the collagenase and a pharmaceutically acceptable carrier.
  • 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 standard pharmaceutical 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).
  • the pharmaceutical formulation is XIAFLEX®.
  • the pharmaceutical formulation comprises 0.9% sodium chloride and 0.03% calcium chloride dihydrate in water.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, 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.
  • the subject 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.
  • an antiseptic sterile solution e.g. 70% isopropyl alcohol
  • anesthetics as needed, before injection of collagenase.
  • the clinician 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.
  • 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.
  • the needle tip passes through the fascia, the needle should be partially withdrawn until the tip is within the fascia.
  • the clinician 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.
  • 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.
  • the clinician 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.
  • 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.
  • 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.
  • the collagenase comprises:
  • (b) means for breaking down collagen Type I and/or, collagen Type III.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RPF PRO Plantar Fasciitis Patient Reported Outcome
  • 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.
  • 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.
  • 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.
  • 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.
  • 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”). Overall mean 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.
  • 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.
  • 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).
  • FFI Foot Function Index
  • 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:
  • 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:
  • 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:
  • a negative change from baseline indicates pain improvement, i.e., lower scores are better.
  • the subjects’ baseline values are obtained before treatment with collagenase.
  • 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.
  • PGIC Patient Global Impression of Change
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SRC soluble rat-tail tendon collagen
  • 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.
  • 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:
  • Mi.eucine Slope of the leucine standard curve
  • 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.
  • F-Enzyme Buffer Dilute F-TC Assay Buffer by combining 4 mL with 16 mL water. Store at 2-8 °C.
  • 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)
  • 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.
  • 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.
  • 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.
  • the leucine standard curve is prepared according to the following table.
  • the sample is diluted to 0.01 mg/mL with F-Enzyme Buffer in two stages vortexed gently to mix.
  • 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,
  • 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.
  • the luminescence spectrometer is set up with the following instrument parameters. The fluorescence of each preparation is read with 1 hour of derivatization.
  • F(net) Mean Collagenase Sample (EM480) - Blank (EM480)
  • 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 (R 2 ) are determined. Do not force through zero.
  • the above SRC assay may be employed to analyze the specific activity of any collagenase.
  • 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”).
  • substrate soluble rat-tail collagen
  • Tripeptide GPA (Bachem H3615 or equivalent)
  • 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.
  • 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) .
  • assay buffer for example, 20 pL 4 mg/mL GPA in 980 pL assay buffer
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 10 6
  • 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.
  • 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.
  • 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 the enzymatic 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:
  • 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.
  • V2 volume of water needed to produce a 10 mM stock solution (mL)
  • 1 mM Leucine Working Stock Solution Thaw a vial of 10 mM Leucine Stock Solution and dilute to 1 mM by combining 150 pL with 1350 pL water. Mix well prior to use.
  • 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.
  • 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.
  • 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).
  • the leucine standard curve is prepared according to the following table.
  • the collagenase sample is diluted to 0.01 mg/mL with f-Appel’s Buffer in two stages and vortexed gently to mix.
  • 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.
  • a set of potency curves are prepared for each collagenase sample as follows:
  • 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.
  • 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 pL of 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.
  • the fluorometer is set up with the following instrument parameters and the fluorescence of each preparation is read with 1 hour of derivatization.
  • each leucine standard (X-axis) is plotted against the fluorescence response at 480 nm (Y -axis).
  • the slope (m), coefficient of determination (R 2 ), 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 (R 2 ) for the resulting linear curves are determined.
  • This method is similar to the GPA assay above, except it is performed in a microplate.
  • 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”).
  • zGPGGPA carbenzoxy-glycyl-L-prolyl- glycyl-glycyl-L-propyl-L-alanine
  • substrate carbenzoxy-glycyl-L-prolyl- glycyl-glycyl-L-propyl-L-alanine
  • Peptide substrate (zGPGGPA) (Bachem M1260 or equivalent)
  • Tripeptide GPA (Bachem H3615 or equivalent)
  • 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).
  • assay buffer for example, 20 pL 4 mg/mL tripeptide GPA in 980 pL assay buffer.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • reaction 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.
  • Anorogenic reagent fluorescamine.
  • 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.
  • 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.
  • the collagenase sample specific activity can be calculated as follows:
  • GPA Microplate Assay Units ((Slope of Collagenase Sample) / (Slope of Tripeptide GPA x incubation time)) x 10 6 .
  • 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.
  • 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 activity of 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).
  • 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.
  • 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.
  • 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.
  • reaction tubes are capped and the contents are mixed gently but thoroughly.
  • reaction tubes are placed in a 37°C water bath and incubated for 22 ⁇ 0.5 hours.
  • 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 .
  • the reaction tubes are removed from the water bath. The actual time the incubation ends is recorded.
  • 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.
  • 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.
  • 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.
  • 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 the time that the last sample is read, in hours, are recorded. All readings are to be done within a 1-hour interval.
  • 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.
  • 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.
  • Step A 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.
  • 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.
  • BTC units activity in nmol leu eq/min x collagen correction factor.
  • BTC unit/mL (Activity in BTC units)/( Sample volume used in m )
  • BTC unit/mg (Activity in BTC units/mL)/(Protein Concentration in mg/mL) Conversions
  • ABC units BTC units x 1.09.
  • the study 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.
  • Age and Sex Be an ambulatory male or female 18 to 65 years of age.
  • 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).
  • 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.
  • opioids e.g., codeine, heroin, hydrocodone, hydromorphone, morphine, or oxycodone
  • 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.
  • IRT Interactive Response Technology
  • 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).
  • 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.
  • an antiseptic sterile solution e.g. 70% isopropyl alcohol
  • 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.
  • 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.”
  • NCI National Cancer Institute
  • CCAE Common Terminology Criteria for Adverse Events
  • ADL activities of daily living. 7. Method for Effectiveness Analysis:
  • Effectiveness parameters in the Full Analysis Set 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Exploratory Endpoint 3 Mean Change from Baseline to Day 84/EOS/ET in the Pain Intensity NRS Scores Between Treatment Groups.
  • 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.
  • Primary Endpoint To Assess the Incidence, Severity, and Duration of TEAEs.
  • the primary estimand is defined by the following:
  • Treatment condition was either EN3835 or placebo, in the doses described for the following groups:
  • 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.
  • 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.
  • SOC System Organ Class
  • PT preferred term
  • duration of TEAE was summarized by treatment group as frequency and percentages of participants.
  • 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 Analyzed
  • 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%).
  • 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.
  • RPF PRO Pain Intensity NRS, Foot Pain Severity Interference with PFA Scale, and the Foot Pain Frequency Impact in PFA Scale
  • RPF PRO Recalcitrant Plantar Fasciitis Patient Reported Outcome
  • NRS Observed Mean Pain Intensity Numerical Ranking Scale
  • 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 a line 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.
  • Pain Intensity NRS Score ranges from 0 to 10, with 0 signifying “No Pain” to 10 signifying “Pain as Bad as You Can Imagine.”
  • 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.
  • 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.
  • 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).
  • a responder was defined as a participant with a response of “Very Satisfied” or “Quite Satisfied” at the specified study visit.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • Age and Sex Be an ambulatory male or female 18 to 75 years of age (inclusive).
  • Conservative therapies may include rest, physical therapy, splinting/bracing, orthotics, icing, physiotherapy, acetaminophen, corticosteroids, or nonsteroidal anti-inflammatory drugs (NSAIDs).
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • 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.
  • EOS End of study
  • 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.
  • 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.
  • Table 24 Study Intervention Administration Method a Each injection delivers 1 aliquot.
  • 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.
  • TEAEs should be managed per the investigator’s standard of care, with concomitant medications and/or therapies.
  • 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.
  • IRT interactive response technology
  • 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.
  • 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.
  • Pain Intensity Numeric Rating Scale 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”).
  • FFI Foot Function Index
  • 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:
  • 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:
  • 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:
  • 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), 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:
  • 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 Pain Intensity NRS scores range from 0 (“No Pain”) to 10 (“Worst Pain Imaginable”).
  • 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”).
  • 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”).
  • 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”).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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”).
  • ADL activities of daily living
  • CAM controlled ankle movement
  • Testing 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 and associated 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.
  • Analysis Sets For the purposes of analysis, the following analysis sets are defined:
  • 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.
  • 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 observation carried 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.
  • LSM Least square mean
  • Tipping Point Analysis A sensitivity analysis utilizing a tipping -point approach will be implemented to assess the robustness of the treatment effect.
  • MMRM mixed effect model for repeated measures
  • This MMRM model will include treatment group, study visit, 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.
  • 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.
  • 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:
  • Target Population Adult participants with plantar fasciitis who are included in the FAS.
  • 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.
  • 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.
  • 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:
  • Target Population Adult participants with plantar fasciitis who are included in the FAS.
  • 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 MI of 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ADP average daily (24-hour) pain
  • 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.
  • 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 “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).
  • Foot Function Index 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.
  • 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.
  • PGIC Patient Global Impression of Change
  • 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 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.
  • 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.

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Abstract

L'invention divulgue des méthodes de traitement de la fasciite plantaire (PFA) chez un sujet, qui consiste à injecter une formulation pharmaceutique contenant de la collagénase dans une ou plusieurs zones affectées par la fasciite plantaire dans le pied du sujet pour ainsi traiter la fasciite plantaire.
PCT/IB2025/057817 2024-08-01 2025-07-31 Méthodes de traitement de la fasciite plantaire Pending WO2026028155A1 (fr)

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