Sustained release of purified collagenase clostridium histolyticum

Abstract

Formulations, their fabrication methods, and methods of treating fibrosis include tunable hydrogels loaded with collagenase for breaking down collagen and thereby reducing stiffness in fibrotic tissue. The formulations may be tuned for topical application or injection, and provide localized and sustained treatment of fibrotic conditions.

Description

Atty. Dkt. No. 125141.04914.MGH2025-111-02Sustained Release of Purified Collagenase Clostridium HistolyticumCross Reference to Related Applications

[0001] The present application is based on, claims priority to, and incorporates herein by reference in its entirety for all purposes, US Provisional Application Serial No. 63 / 726,451, filed November 29, 2024.Statement of Government Support

[0002] Not Applicable.Background

[0003] Fibrosis is a condition that develops slowly but eventually leads to tissue degeneration, which has devastating consequences for heart, lung, liver, kidney, and skin disorders. It occurs when there is an excessive buildup of fibrous connective tissue in the extracellular matrix (ECM) area of tissues subject to injury or inflammation, leading to increased tissue stiffness. This increased stiffness can cause organ dysfunction and in the case of skin and musculoskeletal disorders severely restrict mobility. The basic components of fibrotic scar tissue are a mixture of fibrotic cells and collagens, especially types I and III. Because the mechanical stiffness of tissue is largely attributable to its collagen network, and the disordered collagen architecture is a defining hallmark of scar tissue, collagenase to partially degrade the collagen network provides a promising approach to soften fibrotic liver, fibrosis in other organs, healing wounds or mature scars. However a previous clinical study (Kang, Norbert et al. “Intra-lesional injections of collagenase are ineffective in the treatment of keloid and hypertrophic scars.” Journal of plastic, reconstructive & aesthetic surgery : JPRAS vol. 59,7 (2006): 693-9) found that intralesional injection of collagenase had no effect on skin scar volume, instead causing side effects including pain, swelling, blistering, ulceration and ecchymosis. Another study (Jin, Bo et al. “Reversibility of experimental rabbit liver cirrhosis by portal collagenase administration.” Laboratory investigation; a journal of technical methods and pathology vol. 85,8 (2005): 992-1002) tested portal perfusion with crude collagenase Clostridium histolyticum (CCH), a mixture of matrix degrading enzymes including both highly specific collagenases as well as proteases with broad target specificity, in a rabbit model of liver cirrhosis and reported mortality greater than 50%.Atty. Dkt. No. 125141.04914.MGH2025-111-02

[0004] Accordingly, there is a need to effectively treat and reverse the buildup of collagen in fibrotic tissue.Summary

[0005] The present disclosure provides systems and methods that overcome the aforementioned drawbacks by providing localized and extended release of collagenase to break down collagen in fibrotic tissues and collagen-mediated disorders.

[0006] In one aspect of the present disclosure, a formulation for treating fibrotic tissue is described. The formulation comprises a tunable hydrogel and a therapeutically effective amount of collagenase associated with the hydrogel.

[0007] In one aspect of the present disclosure, a method of making a formulation for treating fibrotic tissues is described. The method comprises forming a polymer network, loading the polymer network with a therapeutically effective amount of collagenase, and crosslinking the polymer network with collagenase to form a hydrogel.

[0008] In one aspect of the present disclosure, a method of treating fibrotic skin is described.The method comprises forming the formulation as described above, and applying the formulation to an outer surface of the fibrotic skin.

[0009] In one aspect of the present disclosure, a method of treating fibrotic tissue is described. The method comprises forming the formulation as described above, and injecting the formulation into the fibrotic tissue.

[0010] In one aspect of the present disclosure, a formulation for treating fibrotic tissues is described. The formulation comprises a delivery system, and a therapeutically effective amount of collagenase associated with the delivery system.

[0011] These aspects are nonlimiting. Other aspects and features of the systems and methods described herein will be provided below.Brief Description of the Drawings

[0012] The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

[0013] FIG. 1 A shows plots of serum injury markers at one day and 5 days after interstitial infusion of the mouse cirrhotic liver with saline or purified CCH. Purified CCH consists of theAtty. Dkt. No. 125141.04914.MGH2025-111-02 highly specific collagenases from the crude CCH mixture. Liver injury markers ALT and AST are elevated at Id based on a reference range of 20-82 U / L ALT, 31-98 U / L AST established in Otto et al. (“Clinical Chemistry Reference Intervals for C57BL / 6J, C57BL / 6N, and C3HeB / FeJ Mice (Mus musculus).” Journal of the American Association for Laboratory Animal Science : JAALAS vol. 55,4 (2016): 375-86). ALT is normal by day 5, while AST remains slightly above the reference range. There is no significant difference between infusion with saline and purified CCH, suggesting these markers are not increased due to enzyme toxicity. Kidney injury marker creatinine is not elevated at any timepoint. Samples below the 0.2 mg / dL level of detection for creatinine are not represented in that graph.

[0014] FIG. IB shows picrosirius red images of the in vivo efficacy 5 days after interstitial infusion of saline (top) or purified CCH (bottom) in mouse cirrhotic liver. Fibrous bands in the samples that received CCH are thinner and more disconnected. Scale bar = 1 mm.

[0015] FIG. 1C is a plot of the quantification of collagen in saline and CCH infused samples from FIG. IB. Purified CCH caused a 38% decrease in collaged proportionate area.

[0016] FIG. ID shows picrosirius red images of the in vivo efficacy 5 days after interstitial infusion of saline (top) or purified CCH (bottom) in rat cirrhotic liver. Infusion of purified CCH in solution did not improve scarring in this model, which better represents the thicker scar bands found in human cirrhosis. Scale bar = 2.5 mm

[0017] FIG. 2 is a flowchart of making a formulation for treating fibrotic tissue, according to aspects of the present disclosure.

[0018] FIG. 3 is a plot of the enzymatic activity of purified CCH in solution after 7 days and 14 days at 37°C. There is no loss of activity through 14 days. Clostripain and neutral protease found in crude CCH would cause rapid loss of activity as the mixture degrades itself.

[0019] FIG. 4A is a plot of the in vitro release kinetics of purified CCH from an alginate hydrogel, according to aspects of the present disclosure. The data is shown as a percentage of total collagenase loaded in the gel. The majority is released over 8 hours, followed by gradual release of the remaining enzyme load.

[0020] FIG. 4B is a plot of the in vitro release kinetics of purified CCH from an alginate hydrogel with various concentrations of Laponite®, according to aspects of the present disclosure. The data is shown as a percentage of total collagenase loaded in the gel.Atty. Dkt. No. 125141.04914.MGH2025-111-02

[0021] FIG. 5 is a schematic of the different formulation delivery routes tested, according to aspects of the present disclosure. Injection - material was injected intradermally; topical gel - a hydrogel disc was cast and then placed on top of the tissue; topical gel + epidermal channels - channels were created in the epidermis with fractional laser or microneedles to enhance delivery.

[0022] FIG. 6 is a plot of the mechanical properties of ex vivo Yucatan pig skin 5 days after topical application of CCH hydrogel applied to intact epidermis, CCH hydrogel applied to the skin fenestrated with a fractional laser, and blank hydrogel applied to the skin fenestrated with a fractional laser. Curve fit modulus (also referred to as fitted modulus in subsequent figures) was obtained from curve-fitting the stress- stretch curve with the equation— 1), where q is the modulus (Wang, Yuxiang et al. “Hyperelastic Material Properties of Mouse Skin under Compression.” PloS one vol. 8,6 e67439. 18 Jun. 2013). Lower modulus indicates a less stiff material. * p < 0.05.

[0023] FIG. 7 shows plots of the fitted modulus (left) and force to compress to 80% initial height (right) of ex vivo Yucatan pig skin five days after topical application of CCH or control hydrogels with various techniques to permeate the epidermal barrier, according to aspects of the present disclosure.

[0024] FIG. 8 shows plots of the fitted modulus (left) and force to compress to 80% initial height (right) of ex vivo Yucatan pig skin permeated using fractional laser treatment two and four days after topical application of CCH hydrogel with various concentrations of Laponite®.

[0025] FIG. 9 shows plots of mass and the mechanical properties of ex vivo Yucatan pig skin 5 days after topical application of CCH hydrogel with either CO2 or Er:YAG fractional laser treatment at high (350 J / cm2) and low (130 J / cm2) laser fluences. * p < 0.05; ** p < 0.01.

[0026] FIG. 10 is a plot of the rate of change in mass in Yucatan pig skin treated with varying fraction laser types at high (350 J / cm2) and low (130 J / cm2) laser fluences and application of topical CCH hydrogel.

[0027] FIG. 11 show plots of the effects of fractional laser density on Yucatan pig skin treated with topical CCH alginate hydrogel for 2 days after various density of fractional laser treatments.

[0028] FIG. 12 shows picrosirius red and hematoxylin & eosin (H&E) staining progression of Yorkshire pig skin treated with fractional laser and CCH hydrogel, according to aspects of the present disclosure. Top row is 1 day timepoint, bottom row is 3 day timepoint. Scale bar = 1 mm.Atty. Dkt. No. 125141.04914.MGH2025-111-02

[0029] FIG. 13A shows images of various Yorkshire pig skin samples 3 days after injection with CCH hydrogel or blank hydrogel (control), according to aspects of the present disclosure.

[0030] FIG. 13B is a plot of the mass of Yorkshire pig skin samples 3 days after injection with CCH hydrogel or blank hydrogel (control), according to aspects of the present disclosure.

[0031] FIG. 13C shows picrosirius red staining images of samples from FIG. 13A. Scale bar = 500 pm.

[0032] FIG. 14A shows images of various human hypertrophic skin samples one day after injection with CCH hydrogel or blank hydrogel (control), according to aspects of the present disclosure. Scale bars = 250pm.

[0033] FIG. 14B is a plot of the mass of human hypertrophic skin samples one day after injection with CCH hydrogel or blank hydrogel (control), according to aspects of the present disclosure.

[0034] FIG. 15 is a plot of the mechanical properties of ex vivo Yorkshire pig skin 5 days after injection of saline, blank hydrogel, or CCH hydrogel. * p < 0.05.

[0035] FIG. 16 shows plots of the fitted modulus (left) and force to compress to 80% initial height (right) of ex vivo Yorkshire pig skin 5 days after injection of saline, CCH solution, blank hydrogel, or CCH hydrogel. Injecting CCH in solution had no significant effect, while the CCH hydrogel significantly softened the tissue. * p < 0.05.

[0036] FIG. 17 shows plots of the fitted modulus (left) and force to compress to 80% initial height (right) of ex vivo Yucatan pig skin 4 days after injection with CCH in saline (Inject Soln) or injection of CCH alginate hydrogel (Inject Gel).

[0037] FIG. 18 shows plots of the mass (left) and hardness (right) of ex vivo Yucatan pig skin four days after injection of CCH solution, (CCH Soln), CCH hydrogel (CCH Alginate), CCH hydrogel with 0.1% Laponite® (CCH Alginate + 0.1% lap), and CCH hydrogel with 0.25% Laponite® (CCH Alginate + 0.25% lap).

[0038] FIG. 19 is a set of plots comparing mass (left), fitted modulus (center), and force to compress to 80% initial height (right) of ex vivo Yucatan pig skin 7 days after treatment with CCH hydrogel. The hydrogel was either directly injected into the skin (Inject gel), applied topically (Topical gel), or applied topically after microneedling (Topical gel + microneedle). Control groups used a gel that did not contain any collagenase. *p<0.05; **p<0.01.Atty. Dkt. No. 125141.04914.MGH2025-111-02

[0039] FIG. 20 shows Picrosirius red staining images of Yorkshire pig skin after treatment with (A) saline injection control, (B) CCH in solution injection, (C) CCH hydrogel injection, and (D) topical application of CCH hydrogel after fractional laser treatment to disrupt the epidermal barrier. Scale bar = 500 pm

[0040] FIG. 21 is an image of cirrhotic rat liver. The circled portion illustrates an area of the liver injected with CCH alginate hydrogel in vivo, according to aspects of the present disclosure.

[0041] FIG. 22 shows examples of picrosirius red staining images of untreated (top) and CCH alginate hydrogel treated (bottom) cirrhotic rat liver tissue. Scale bar = 1 mm.

[0042] FIG. 23A shows an example of gross damage in cirrhotic rat liver after CCH alginate hydrogel injection in vivo.

[0043] FIG. 23B shows examples of histological damage in cirrhotic rat liver after CCH alginate hydrogel injection in vivo. Scale bar = 1 mm.

[0044] FIG. 24 shows gross cirrhotic rat liver images after controlled injection of alginate hydrogel, according to aspects of the present disclosure.Detailed Description

[0045] Collagen is the major structural constituent of mammalian organisms and makes up a large portion of the total protein content of skin and other parts of the body. Various skin traumas such as burns, surgery, and infection are often characterized by the accumulation of fibrous tissue rich in collagen and having increased proteoglycan content. In addition to the replacement of the normal tissue which has been damaged or destroyed, excessive and disfiguring deposits of new tissue may form during the healing process. Some diseases and conditions are associated with excess collagen deposition and the accumulation of collagen-rich fibrous tissue. Such diseases and conditions may collectively be described herein as “collagen-mediated” or “fibrotic.”

[0046] Collagenases have emerged as a promising treatment option of fibrotic disease. Collagenase Clostridium histolyticum (CCH) is a mixture of enzymes produced by the Clostridium histolyticum bacteria. The crude mixture contains collagenases, clostripain, and neutral protease. The collagenases from this mixture (hereafter referred to as purified CCH) offers a therapeutic with strong ability to lyse type 1 and type 3 collagen which are the most abundant collagen types in fibrotic diseases. This enzyme shows high specificity for collagenAtty. Dkt. No. 125141.04914.MGH2025-111-02 fibrils whereas it does not produce any remarkable alteration in elastic fibers, vascular smooth muscle and axonal myelin sheaths. Purified CCH is a mixture of two synergistic microbial collagenases, class I and class II, which act in combination and cleave the peptide bond of the collagen fibrils.

[0047] Due to its proven effectiveness in some of these fibrotic conditions, purified CCH has been approved as a commercially available enzymatic treatment for at least the treatment of Dupuytren’s contracture and Peyronie’s disease. However, direct injection of purified CCH into the relevant tissue requires several repeat injections. Purified CCH is rapidly inactivated after contact with blood primarily due to the abundant serum protein alpha-2-macroglobulin. Crude CCH also rapidly loses enzyme activity under physiological conditions due to the presence of clostripain and neutral protease which cause the mixture to degrade itself.For example, in skin scarring, injection of purified CCH into hypertrophic scars failed to reduce scar volume. Likewise, the efficacy of purified CCH injection into scar tissue in cirrhotic liver, which shares similar features to Dupuytren’s Contracture - such as densely packed collagen, high cross-linking, and resistance to enzymatic degradation - is limited. Experiments performing one hour of direct infusion of mouse liver with purified CCH is well tolerated, with only mild elevation in hepatocellular enzymes AST and ALT to a similar level as infusion with saline, and caused a reduction in collagen proportionate area of 38% by day 5 (FIGS. 1 A-1C). In contrast, the same experiment in a rat model with more severe scarring, which more closely resembles human cirrhosis, shows that direct infusion of CCH has limited efficacy (FIG. ID), even at increased concentrations and extended infusion times up to 6 hours. This finding may be attributed to interspecies distinctions, where mouse models of cirrhosis develop much thinner and more fragile septa than rats and humans that develop denser and more cross-linked septa.

[0048] To address the limitations of current collagenase treatment approaches, the present disclosure describes formulations and methods for fibrotic tissue remodeling and reducing the stiffness and mechanical stress of fibrotic conditions by local and extended treatment of enzymes for degrading collagen (i.e., collagenases). The examples herein illustrate specific applications of the formulations and methods in fibrotic skin and liver cirrhosis and are intended to be illustrative and non-limiting. The methods and formulations described herein may be applicable in other fibrotic tissues and collagen-mediated disorders, such as, but not limited to pulmonary fibrosis, plantar / palmar fasciitis, tracheal fibrosis, fibroids, gastrointestinal strictures, fibrousAtty. Dkt. No. 125141.04914.MGH2025-111-02 bands, hypertrophic burn scars, keloids, vocal cord fibrosis, morphea, systemic sclerosis, scleromyxedema, mixed connective tissue disease, scarring alopecias, adhesions, restrictive cardiomyopathy, post-radiation sclerosis, eosinophilic fasciitis, cardiac fibrosis (including post- myocardial infarction), peritoneal fibrosis, adhesive capsulitis, muscle fibrosis, foreign body response fibrosis, and others.

[0049] In a non-limiting example, a formulation for extended release for locally treating fibrotic tissue is described comprising a delivery system and fibrosis targeting therapeutic associated with the delivery system. Specifically, the delivery system may include a hydrogel with tunable physical and chemical properties depending on the tissue being treated and the method of administration. For example, the hydrogel may be a solid hydrogel for topical application on the surface of the skin, with or without enhancing skin penetration. Alternatively, the hydrogel may be injectable, such as through a syringe for local delivery to the dermis or internal tissues. The hydrogel comprises a polymer network. The polymer density, single- or multi-polymer composition, degree of crosslinking, polymer molecular weight, polymer linearity, and the like may be modified to produce a hydrogel for its intended treatment tissue.

[0050] The polymer network may comprise natural polymers, synthetic polymers, or combinations thereof. For example, a non-exhaustive list of natural polymers may include alginate, cellulose, chitosan, collagen, hyaluronic acid, gelatin, agarose, starch, dextran, and their derivatives. A non-exhaustive list of synthetic polymers may include polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), poly-2-hydroxyethyl methacrylate (PHEMA), poly-N-isopropyl acrylamide (PNIPAM), polyacrylic acid (PAA), and polyacrylamide (PAAM), poly-lactic-co-glycolic acid (PLGA), poly-urethane, and their derivates.

[0051] In the non-limiting examples shown herein, the polymer network comprises one or more alginate polymers having different molecular weights (MW). For example, the polymer network may be a co-polymer of high MW (HMW) polymers having MW greater than 200 kDa and low MW (LMW) polymer having MW less than 75 kDa. Moreover, the polymer network may be an alginate co-polymer network may comprising an equal amount of LMW polymers and HMW polymers. Alternatively, the polymer network may comprise a 2: 1 - 5: 1 ratio of LMW polymers to HMW polymers. Alternatively, the polymer network may comprise a 2: 1 - 5: 1 ratio of HMW polymers to LMW polymers 1. In the following examples, the polymer network is an alginateAtty. Dkt. No. 125141.04914.MGH2025-111-02 co-polymer network comprising a 3: 1 ratio of LMW polymers to HMW polymers. Specifically, alginate polymers possess high biocompatibility and chelating ability, are biodegradable and non-antigenic, and display long term stability. Moreover, alginate can absorb large quantities of biological liquids and can be purified to prevent immunogenicity, rendering alginate an ideal polymer for hydrogel preparation under mild conditions. Further, hydrogels based on alginate polymer networks may possess structural similarities to ECM in tissues.

[0052] FIG. 2 presents a method 200 of making the tunable hydrogel formulation. The polymer network is formed at step 202 and loaded with a therapeutically effective amount of collagenase at step 204 as described above. At step 206, the hydrogel may be produced by crosslinking the collagenase-loaded polymer network. In the following examples, steps 204 and 206 are combined, such that the therapeutically effective amount of collagenase is pre-mixed with the solution containing the crosslinking agent and subsequently added to polymer network. In the following non-limiting examples, the alginate hydrogel is formed by mixing dissolved alginate polymers at a ratio of 3: 1 of LMW polymers to HMW polymers and subsequently crosslinking the polymer network. Crosslinking the polymer network may include chemical or physical crosslinking approaches. For example, crosslinking the alginate polymer network may include ionic cross-linking with divalent cations (i.e., Ba2+, Fe3+, Ca2+), covalent cross-linking with multifunctional molecules (i.e., poly(acrylamide-co-hydrazide, poly(ethylene glycol)-diamines)), and thermal gelation and in situ copolymerization of thermosensitive polymers (i.e., N- isopropyl acrylamide (NIPAAm)) by UV irradiation. Amphiphilic alginate derivatives obtained by coupling hydrophobic moieties, such as alkyl chains and hydrophobic polymers, to an alginate skeleton may also self-assemble to form hydrogels in aqueous solution. In the following examples, the alginate hydrogel is produced by adding calcium sulfate dihydrate to the polymer network, whereby Ca2+ions are released from bonds between the polymer chains.

[0053] In a non-limiting example, the resulting hydrogel may comprise mechanical properties indicative of a solid gel, a fluid-like injectable gel, or properties therebetween. The alginate hydrogels crosslinked with Ca2+ions may exhibit an elastic modulus in the range of 1-100 kPa. As mentioned briefly above, these mechanical properties of the resulting hydrogel can be finetuned in part by control of the concentration the crosslinking cation.

[0054] The formulation further comprises a therapeutic configured to treat the fibrotic tissue associated with the hydrogel delivery system. In a non-limiting example, the therapeutic may beAtty. Dkt. No. 125141.04914.MGH2025-111-02 an enzyme configured to degrade collagen (i.e., collagenase). Specifically, the collagenase may include at least one of class I or class II collagenase from CCH. In another example, the collagenase purified CCH filtered out of crude CCH. Alternatively, the collagenase may be a recombinant protein. The formulation includes the therapeutic, enzyme, or collagenase in a therapeutically effective amount. As used herein, “therapeutically effective amount” is understood by one of ordinary skill in the art as a quantity sufficient to produce a desired physiological outcome. In a non-limiting example, a therapeutical effective amount may be between 0.5-30 Wiinsch Units / mL CCH (1 Wiinsch Unit is defined as the amount of enzyme that hydrolizes Ipmol of the synthetic Wiinsch substrate per minute at 25°C, pH 7.1).

[0055] In a non-limiting example, the collagenase is associated with the hydrogel by being physically interspersed within the polymer network, chemically linked or functionalized to one or more moieties on the polymers of the polymer network, or a combination thereof. In the following examples, purified CCH may be mixed with calcium sulfate solution and subsequently added to the alginate polymer network to form a CCH-loaded hydrogel formulation. As shown in FIG. 3, the enzymatic activity of purified CCH remains stable at 37°C (whereas crude CCH rapidly loses activity).

[0056] FIG. 4A further illustrates the release profiles of purified CCH loaded into the alginate hydrogel. These results indicate that incorporating purified CCH into a sustained-release hydrogel format could both improve efficacy and mitigate side effects by localizing the enzyme at the desired treatment site, which both ensures a higher concentration of the enzyme at the desired site of action and limits off target exposure, and enabling longer exposure to the treatment enzyme.

[0057] In a non-limiting example, a release extending agent may be added to the formulation to alter the release kinetics of therapeutic, enzyme, or collagenase. In the following examples, the formulation may further comprise synthetic smectite clay such as Laponite® to slow the release of CCH at the target tissue. Specifically, this release extending agent may be mixed with CCH for approximately an hour before mixing with the crosslinking agent and the polymer network. FIG. 4B illustrates the extended-release profiles of CCH loaded into alginate hydrogel with varying concentrations of Laponite®.

[0058] Skin ExampleAtty. Dkt. No. 125141.04914.MGH2025-111-02

[0059] In one aspect of the present disclosure, the formulations described herein may be used to treat scarring in the skin. FIG. 5 illustrates three non-limiting administration approaches for treating fibrotic skin tissue, including injection of the formulation via a syringe, placement of the formulation topically on the surface of the intact skin, or placement of the formulation topically on the surface of fenestrated skin. The resulting skin tissues are evaluated for their changes in mechanical properties after purified CCH treatment in the following results.

[0060] For the topical applications of the formulation, the formulation may include a step of casting the formulation between parallel surfaces to achieve uniform thickness. In a non-limiting example, 60pL the hydrogel formulation for topical application may be cast between sterile glass slides to a thickness of approximately 1mm. After approximately 20 minutes, the cast hydrogel may be placed on top of the tissue to release the therapeutic through the surface.

[0061] In the fenestration example, the skin may be treated to improve penetration of the released purified CCH through the epidermis. In a non-limiting example, fenestration may include, but is not limited to, the application of a fractional laser, puncturing with one or more microneedles, or puncturing with one or more coring needles. Use of a fractional laser may include, but is not limited to, a carbon dioxide (CO2) laser or an Erbium-doped Yttrium Aluminum Garnet (Er:YAG) laser. The fractional laser may be applied at treatment density of 1- 20%. The laser may be applied at sufficient fluence to vaporize channels at least into the epidermis, the depth of such channels being approximately proportional to laser fluence, as described in publications (Wenande, E., Anderson, R. R., & Flaedersdal, M. (2020).Fundamentals of fractional laser-assisted drug delivery: an in-depth guide to experimental methodology and data interpretation. A dvance d drug delivery reviews, 153, 169-184). For example, the laser fluence may be applied at sufficient fluence to vaporize channels in a depth range of 0.01 to 10 mm. For a example a sufficient fluence to vaporize channels in a depth of 0.01-3 mm may be suitable for skin, and up to 10 mm for hypertrophic scars, keloids, and thick fibrotic lesions. In the non-limiting examples provided herein, the fluence may range between 130-350 J / cm2. When using one or more microneedles or coring needles, the needle penetration depth within skin may be up to about 6 mm in length.; for hypertrophic scars, keloids or thick fibrotic lesions the needle penetration depth may be as deep as the scar, keloid, or lesion.Atty. Dkt. No. 125141.04914.MGH2025-111-02

[0062] Alternatively, the other approaches for bypassing the outermost layer of the epidermis (stratum comeum) may include the use of ionic liquids, sonophoresis, or complete removal of the epidermis.

[0063] FIG. 6 shows that Yucatan pig skin was significantly softened after pre-treatment with fractional laser to disrupt the epidermal barrier followed by topical application of CCH hydrogel. Applying the CCH hydrogel over intact epidermis, or pre-treating with fractional laser followed by blank hydrogel (without purified CCH), had no effect.

[0064] FIG. 7 compares the fitted modulus and force to compress to 80% of the original height of Yucatan pig skin of the various fenestration approaches (intact epidermis, microneedle, fractional laser). Intact epidermis prevents delivery of the purified CCH, while both CO2 and Er:YAG fractional laser treatments enable delivery into the tissue. The modulus numbers provided are dimensionless quantities obtained from curve fitting the compression stress-stretch curves.

[0065] FIG. 8 illustrates the effects on the mechanical properties of Yucatan pig skin treated with fractional CO2 laser two days and four days after topical application of CCH hydrogel containing 0%, 0.03% and 0.1% Laponite®. Higher concentrations of Laponite® slow the release of the enzyme, leading to a more gradual change in the mechanical properties.

[0066] FIGS. 9-10 illustrates the effects of varying fractional laser parameters on ex vivo Yucatan pig skin 5 days after topical application of CCH hydrogel or blank hydrogel (control), showing no significant differences in the mechanical properties between types of laser and laser fluences tested.

[0067] FIG. 11 illustrates Yucatan pig skin treated with topical CCH alginate hydrogel or blank hydrogel for 2 days after various densities (5%, 10%, and 20%) of CO2 and Er:YAG fractional laser treatments.

[0068] FIG. 12 shows the topical progression histology of fractional laser treated Yorkshire pig skin at day 1 (top row) and day 3 (bottom row) after topical CCH hydrogel application. Both the picrosirius red (left column) and H&E (right column) stains illustrate a progressive reduction in collagen density spreading deeper into the dermis with time.

[0069] Alternatively, the formulation may be injected into the skin via a syringe below the epidermis. In the following non-limiting examples, the hydrogel may be formed and allowed to set within the syringe for approximately 20 minutes. The formulation may then be administeredAtty. Dkt. No. 125141.04914.MGH2025-111-02 by slowly withdrawing the needle while injecting the hydrogel in order to fdl the space left by the needle.

[0070] FIGS. 13A-13B show ex vivo Yorkshire pig skin 3 days after CCH hydrogel or blank hydrogel (control) injection. The increased compressibility of the CCH hydrogel indicates that purified CCH treated samples were softer than the control sample. FIG. 13 C shows corresponding picrosirius red staining of the pig skin samples treated with either CCH hydrogel or blank hydrogel. The control samples have very dense collagen, while the CCH treated samples show significantly less dense collagen staining.

[0071] FIGS. 14A-14B show human hypertrophic scar samples one day after injection with CCH hydrogel (panels A-C) or blank hydrogel (panels D-F). Human hypertrophic scar tissue decreased in volume (panel A) and stiffness (panel B, side view of tissue being compressed with forceps) after intradermal injection of CCH hydrogel, compared to controls injected with blank gels (panels D, E). Picrosirius red staining confirms reduced collagen in the treated samples (panel C), compared to controls (panel F).

[0072] FIGS. 15 - 16 demonstrate the efficacy of CCH hydrogel compared to CCH in solution. FIG. 15 shows that Yorkshire pig skin was significantly softened 5d after intradermal injection of CCH hydrogel (“Inject CCH Gel”), and this was significantly more effective than the same dose of purified CCH in solution (“Inject CCH soln”). FIG. 16 compares the fitted modulus and force to compress to 80% of the original height of Yorkshire pig skin 5 days after intradermal injection with saline (Control Inject Soln), CCH in solution (CCH Inject Soln), blank hydrogel (Control Inject Gel), and CCH hydrogel (CCH Inject Gel). The modulus numbers provided are dimensionless quantities obtained from curve fitting the compression stress- stretch curves. CCH in solution showed no difference from saline injection, while the CCH hydrogel significantly softened the tissue.

[0073] FIG. 17 compares the fitted modulus and force to compress to 80% of the original height of Yucatan pig skin 4 days after intradermal injections with saline, (Control Inject Soln), CCH in solution (CCH Inject Soln), blank hydrogel (Control Inject Gel), and CCH hydrogel (CCH Inject Gel). The modulus numbers provided are dimensionless quantities obtained from curve fitting the compression stress-stretch curves.

[0074] FIG. 18 shows the mass (left) and hardness (right) in ex vivo Yucatan pig skin 4 days after injection of CCH hydrogel with various concentrations of Laponite®. HigherAtty. Dkt. No. 125141.04914.MGH2025-111-02 concentrations of Laponite® slowed the release of the enzyme, leading to a more gradual change effect. The hardness was measured using a Shore 00 durometer.

[0075] FIG. 19 illustrates the mechanical properties of Yucatan pig skin 7 days after treatment with CCH hydrogel via injections, topical application, and topical application after skin fenestration via microneedling.

[0076] FIG. 20 shows the differences in collagen density in Yorkshire pig skin treated with a saline injection control (panel A), an injection of CCH in solution (panel B), a CCH hydrogel injection (panel C), and a topical application of CCH hydrogel after fractional laser treatment (panel D). Injection of the CCH hydrogel showed reduced collagen density relative to the control and CCH in saline injection. Topical application of CCH hydrogel after fractional laser treatment displays a gradient of collagen density.

[0077] Liver Example

[0078] In one aspect of the present disclosure, the injectable formulations described herein may be used to treat liver cirrhosis. FIG. 21 shows cirrhotic liver with a characteristic nodular appearance due to scar formation. The circled portion of the liver represents the injected area with CCH alginate hydrogel, showing visual improvement on the surface compared to the untreated areas. FIG. 22 shows picrosirius red staining images comparing untreated (top) and CCH alginate hydrogel treated (bottom) liver sections. Injection of CCH alginate hydrogel caused a 31% reduction in the collagen content in the liver (comparing treated and untreated regions of the same liver). CCH hydrogel treated areas contained 14.2% ± 4.6% collagen proportionate area, while untreated areas contained 20.6% ± 4.0% collagen proportionate area. The septa (scar) thickness showed a 20 % reduction, with the treated area resulting a thickness of 33.1 pm ± 12.1pm and the untreated area resulting in a thickness of 41.6 pm ± 6.5pm. Since cirrhotic liver function, and access of therapeutic drugs to the cirrhotic liver, are limited by pressure within the liver as a consequence of the constriction by fibrotic septae, reducing the mechanical stiffness of the cirrhotic liver may improve hepatocellular function while also potentially increasing the efficacy of subsequent liver treatments.

[0079] Despite the reduction in collagen density in treated portions, injection of the alginate hydrogel caused necrosis around some of the injection sites in the cirrhotic liver (FIGS. 23 A- 23B). The discolored white region of the tissue indicates necrosis (FIG. 23A left). The right panel of FIG. 23 A shows a bolus of gel in the corner of the region, which created a vascularAtty. Dkt. No. 125141.04914.MGH2025-111-02 occlusion leading to necrosis of downstream tissue. Picrosirius red staining around a large gel bolus showed extensive disruption of the tissue (FIG. 23B, right panel). This damage is observed even when injecting alginate gels without CCH, indicating the damage is primarily due to a bolus of gel disrupting the tissue, rather than toxicity from the enzyme. This toxicity is due to the stiffness imposed by the high collagen content in cirrhotic liver and the increased pressure on the tissue from the hydrogel injection.

[0080] To address the pressure caused by the injection, the hydrogel may be injected in a controlled manner. For example, the injection volume and speed may be controlled. The injection may be controlled automatically via a motorized injection. The motorized injector may further comprise a controller and feedback potentiometer to make speed and / or volume adjustments. FIG. 24 shows a reduction of the large scale damage by controller-mediated injection, with minor necrosis occurring around the injection sites (arrows).

[0081] Alternatively, as described previously, the hydrogel rheological properties may be tuned to reduce injection pressure while optimizing loading collagenase loading and release kinetics.

[0082] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.

[0083] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0084] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.Atty. Dkt. No. 125141.04914.MGH2025-111-02

[0085] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0086] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0087] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0088] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use an aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

Claims

Atty. Dkt. No. 125141.04914.MGH2025-111-02Claims1. A formulation for treating fibrotic tissue, comprising: a tunable hydrogel; and a therapeutically effective amount of collagenase associated with the hydrogel.

2. The formulation of claim 1, wherein the hydrogel comprises a polymer network.

3. The formulation of claim 2, wherein the polymer network comprises natural polymers, synthetic polymers, or a combination thereof.4 The formulation of claim 2, wherein the polymer network comprises one or more alginate polymers.

5. The formulation of claim 4, wherein the one or more alginate polymers include a mixture of high molecular weight (HMW) polymers greater than 200 kDa and low molecular weight (LMW) polymer less than 75 kDa.

6. The formulation of claim 5, wherein the polymer network comprises a 3 to 1 ratio of LMW polymers to HMW polymers.

7. The formulation of claim 5, wherein the collagenase is interspersed within the polymer network, functionalized to the polymers, or a combination thereof.

8. The formulation of claim 1, wherein the collagenase includes at least one of class I or class II collagenase from Collagenase Clostridium Histolyticum (CCH).

9. The formulation of claim 8, wherein the collagenase is filtered out of crude CCH or produced as recombinant protein.

10. A method of making a formulation for treating fibrotic tissue, comprising: forming a polymer network; loading the polymer network with a therapeutically effective amount of collagenase; andAtty. Dkt. No. 125141.04914.MGH2025-111-02 crosslinking the polymer network loaded with collagenase to form a hydrogel.

11. The method of claim 10, wherein the polymer network comprises natural polymers, synthetic polymers, or a combination thereof.

12. The method of claim 11, wherein the polymer network comprises one or more alginate polymers.

13. The method of claim 12, wherein the one or more alginate polymers include a mixture of high molecular weight (HMW) polymers greater than 200 kDa and low molecular weight (LMW) polymer less than 75 kDa.

14. The method of claim 13, wherein the polymer network comprises a 3 to 1 ratio of LMW polymers to HMW polymers.

15. The method of claim 13, wherein crosslinking the mixture of HMW polymers and LMW polymers includes adding a crosslinking agent to the mixture.

16. The method of claim 15, wherein the crosslinking agent includes divalent cations.

17. The method of claim 16, wherein the crosslinking agent includes a calcium sulfate solution.

18. The method of claim 10, wherein the collagenase includes Collagenase Clostridium Histolyticum (CCH).

19. The method of claim 10, further comprising mixing a release extending agent with the therapeutically effective amount of collagenase prior to loading.

20. A method of treating fibrotic skin, comprising: forming the formulation of claim 1; and applying the formulation to an outer surface of the fibrotic skin.

21. The method of claim 20, wherein the formulation is cast between parallel surfaces for uniform thickness.

22. The method of claim 20, further comprising fenestrating the outer surface of the fibrotic skin before applying the formulation to the outer surface of the fibrotic skin.Atty. Dkt. No. 125141.04914.MGH2025-111-0223. The method of claim 22, wherein fenestrating the outer surface include application of a fractional laser, puncturing with one or more microneedles, or puncturing with one or more coring needles.

24. The method of claim 23, wherein the application of a fractional laser includes a CO2 laser or Er:YAG laser.

25. The method of claim 24, wherein the fractional laser is applied at a treatment density of 5-20%.

26. The method of claim 25, wherein the fractional laser is applied at a fluence sufficient to vaporize channels a depth between 0.01 and 10 mm.

27. The method of claim 23, wherein the one or more microneedles or one or more coring needles are up to 6 mm in length.

28. A method of treating fibrotic tissue, comprising: forming the formulation of claim 1; and injecting the formulation into the fibrotic tissue.

29. The method of claim 28, wherein injecting includes controlling an injection rate or an injection volume of the formulation into the tissue.

30. A formulation for treating fibrotic tissue, comprising: a delivery system; and a therapeutically effective amount of collagenase associated with the delivery system.

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