Compositions and formulation methods for sustained local release of antifibrotic agents

Abstract

The present disclosure describes methods for making injectable, sustained-release nanocrystals for the localized delivery of anti-fibrotic drugs in tissues in a sustained manner.

Description

[Background technology]

[0001] (Federally Sponsored Research and Development) This invention was made with government support under grants CA190040, DK107806, and EB017742 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0002] (Background technology) Abnormal tissue healing processes can cause tissue fibrosis, which is characterized by excessive extracellular deposition of collagen and other extracellular matrix components. In the gastrointestinal (GI) tract, fibrosis usually begins with epithelial damage, such as digestive injury to the distal esophagus or inflammatory bowel disease (IBD). Fibrosis in the GI tract can narrow the lumen and form strictures, which can lead to intestinal obstruction, intestinal surgery, and bowel loss. There are no FDA-approved drugs for the treatment of GI tract fibrosis, and therefore, the only current treatments are surgical resection or endoscopic dilation. However, many strictures recur after treatment, leading to further morbidity and mortality. Summary of the Invention

[0003] In some aspects, the presently disclosed subject matter provides a formulation comprising a plurality of sulconazole nanocrystals and one or more stabilizers.

[0004] In some embodiments, the one or more stabilizers are selected from polyvinyl alcohol (PVA), hyaluronic acid (HA), carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), sodium cholate (CHA), cellulose derivatives, polysaccharides, polyethylene glycol, poloxamers, and combinations thereof. In certain embodiments, the poloxamer comprises poloxamer 407.

[0005] In some embodiments, the concentration of sulconazole is about 10 to about 500 mg / mL. In certain embodiments, the formulation comprises: (a) about 1.5% to about 5% PVA, (b) about 0.5% to 1% HA, (c) about 1% to about 2% CMC, (d) about 1% to about 5% HPMC, and (e) about 2% to about 6% poloxamer 407.

[0006] In some embodiments, the formulation is lyophilized.

[0007] In some embodiments, the presently disclosed subject matter provides a precursor formulation comprising a formulation and a plurality of grinding beads. In particular embodiments, the plurality of grinding beads comprises zirconium oxide beads. In more particular embodiments, the formulation comprises about 500 mg of sulconazole, about 2.0 g of 0.5 mm zirconium oxide beads, and about 1 mL of 2% (w / v) poloxamer 407.

[0008] In another aspect, the presently disclosed subject matter provides a method for treating or preventing fibrosis or intestinal restenosis in the gastrointestinal (GI) tract of a subject in need thereof, the method comprising administering a formulation of the present disclosure to the subject. In some aspects, the formulation is administered by injection. In certain aspects, the injection comprises intraperitoneal (IP) injection or subcutaneous (SC) injection. In more specific aspects, the formulation is injected near the site of stricture. In even more specific aspects, the formulation is injected near the site of stricture after a surgical or endoscopic procedure.

[0009] In certain embodiments, the fibrosis is associated with inflammatory bowel disease (IBD). In particular embodiments, the inflammatory bowel disease is selected from Crohn's disease (CD), ulcerative colitis (UC), and combinations thereof.

[0010] In some embodiments, administration of the formulation modulates acute healing responses and / or interrupts one or more pathological fibrotic tissue remodeling processes. In some embodiments, the method comprises reducing the thickness of the collagen layer in the small intestine of a subject. In some embodiments, administration of the formulation results in a folded, flexible epithelial structure that is more similar to healthy tissue.

[0011] In certain embodiments, the administration of the formulation is sustained release administration.

[0012] In some embodiments, the concentration of sulconazole in the formulation ranges from about 100 mg / mL to about 500 mg / mL. In some embodiments, the volume of the formulation injected ranges from about 10 μL to about 100 μL. In some embodiments, the formulation is injected at a dose of about 100 mg / kg to about 1875 mg / kg of sulconazole.

[0013] Certain aspects of the subject matter of the present disclosure have been described above; other aspects that are covered in whole or in part by the subject matter of the present disclosure will become apparent as the specification proceeds, as fully described herein below and in connection with the accompanying examples and figures.

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application will be provided by the Office upon request and payment of the necessary fee.

[0015] Thus, to describe the subject matter of the present disclosure in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale. [Brief explanation of the drawings]

[0016] [Figure 1]Figure 1A shows that sulconazole inhibits the expression of key fibrotic genes in CCD-18Co and LX2 cell lines activated by TGFβ1. TGF-β-stimulated CCD-18Co colonic fibroblasts were left untreated (untreated) or treated with pirfenidone (4 mM) or sulconazole (10 µM) and then stained for alpha-smooth muscle actin (α-SMA, red), cytoplasmic type 1 collagen (COL1A1, green), and cell nuclei (blue). Figure 1B shows that sulconazole inhibits the expression of key fibrotic genes in CCD-18Co and LX2 cell lines activated by TGFβ1. Western blot analysis showed that 10 µM sulconazole-treated stimulated CCD-18Co and LX2 cells had decreased expression of α-SMA and COL1A1 compared to stimulated untreated cells (untreated). Figure 1C shows that sulconazole inhibits the expression of key fibrotic genes in CCD-18Co and LX2 cell lines activated by TGFβ1. RT-PCR demonstrated that sulconazole reduced the expression of α-SMA (ACTA2) at much lower drug concentrations (1-10 μM) in CCD-18Co (n=3-5) and LX2 (n=3) cells compared with pirfenidone (4 mM). The dotted line represents normalization to stimulated, untreated control cells. Data are expressed as mean ± SEM, and *p<0.05 compared to untreated control cells. [Figure 2]Figure 2A shows the characterization of sulconazole nanocrystals (sul-NC). This is a transmission electron microscope (TEM) image of 500 mg / mL Sul-NC. The scale bar represents 500 nm. Figure 2B shows the characterization of sulconazole nanocrystals (sul-NC). This is a bar graph showing the size of 500 mg / mL Sul-NC across nine batches. The average across batches (Ave.) is displayed as mean ± SD. Figure 2C shows the characterization of sulconazole nanocrystals (sul-NC). Sulconazole release from Sul-NC was compared to free sulconazole under accelerated in vitro conditions in a rapid equilibrium dialysis device. Data are shown as mean ± SD. Figure 2D shows the characterization of sulconazole nanocrystals (sul-NC). The stability of 500 mg / mL Sul-NC was evaluated by particle size after storage at room temperature or 4°C for 112 days. Data are shown as mean ± SD. [Figure 3] Schematic diagram of the bleomycin-induced mouse skin fibrosis model. A small area on the back of the mouse was shaved, and five injection sites were marked. Bleomycin was injected into different sectors, from 1 to 5, every other day. Sul-NC (50 mg / kg or 150 mg / kg) was injected into sector 5 once a week. [Figure 4]Figure 4A shows that Sul-NC reduced fibrillar collagen deposition and skin thickening in a bleomycin-induced mouse fibrosis model. Representative tissue sections are shown from mice (n = 5) with bleomycin-induced skin fibrosis after weekly injections of Sul-NC (50 mg / kg or 150 mg / kg), IP injections of sulconazole (free Sul, 10 mg / kg) every other day (IP every 2 days), or daily oral administration of pirfenidone (100 mg / kg). Mice not receiving bleomycin are shown as "sham" mice, and mice injected with bleomycin vehicle are shown as "vehicle." H&E staining of skin tissue sections shows the dermal thickness of each treatment group (representative dermal areas are indicated by double-sided arrows). Figure 4B shows that Sul-NC reduced fibrillar collagen deposition and skin thickening in a bleomycin-induced mouse fibrosis model. Mason's trichrome staining of tissue sections shows the thickness and collagen layer of samples corresponding to each treatment group. Scale bar = 100 μm. [Figure 5] Weekly injection of Sul-NC reduces dermal thickening in a bleomycin-induced mouse fibrosis model. Quantification of dermal thickness in a bleomycin-induced dermal fibrosis model (n=5) following weekly injection of Sul-NC (50 mg / kg or 150 mg / kg), IP injection of sulconazole (free Sul, 10 mg / kg) every other day (IP every 2 days), or daily oral administration of pirfenidone (100 mg / kg). Mice not treated with bleomycin are designated as sham, and mice treated with bleomycin vehicle are designated as vehicle. Data are presented as mean ± SD, with *p<0.01 compared to vehicle. [Figure 6] The experimental procedure for creating a mouse model of small intestinal fibrosis to test the efficacy of Sul-NC in preventing fibrosis is described below. A 6 mm section of intestine was excised from one C57BL / 6 mouse and cut into equal 1 mm-long fragments. Each fragment was then implanted subcutaneously into the neck of another mouse. Seven days after implantation, the grafts were excised to assess fibrosis. [Figure 7]Figure 7A shows that Sul-NC reduces collagen deposition in a mouse model of intestinal fibrosis. Representative Masson's trichrome-stained intestinal tissue sections from PBS (vehicle) treatment (yellow arrows indicate collagen capsules). Figure 7B shows that Sul-NC reduces collagen deposition in a mouse model of intestinal fibrosis. Representative Masson's trichrome-stained intestinal tissue sections from a single injection of 50 mg / kg of Sul-NC (n = 3-5) (yellow arrows indicate collagen capsules). Figure 7C shows that Sul-NC reduces collagen deposition in a mouse model of intestinal fibrosis. Representative Masson's trichrome-stained intestinal tissue sections from a single injection of 150 mg / kg of Sul-NC (n = 3-5) (yellow arrows indicate collagen capsules). Figure 7D shows that Sul-NC reduces collagen deposition in a mouse model of intestinal fibrosis. Representative Masson's trichrome-stained intestinal tissue sections (yellow arrows indicate collagen capsules) from mice (n = 3–5) orally administered pirfenidone at 100 mg / kg three times daily for 7 days. Figure 7E shows that Sul-NC reduces collagen deposition in a mouse model of intestinal tissue fibrosis. The thickness of the collagen layer in small intestinal grafts was significantly reduced by a single injection of Sul-NC and daily oral pirfenidone treatment. Data are shown as mean ± SEM, and *p < 0.01 compared to untreated controls. [Figure 8] Figure 8A shows that Sul-NC is effective in preventing fibrosis in a porcine esophageal stricture model. Representative endoscopic images of the esophageal lumen at the stricture site (30, 40, and 50 cm from the incisor) from pigs treated with vehicle (2% F127) or Sul-NC are shown. Figure 8B shows that Sul-NC is effective in preventing fibrosis in a porcine esophageal stricture model. X-ray images of strictures (yellow arrows point to the esophageal lumen) after treatment with vehicle or Sul-NC are shown. Figure 8C shows that Sul-NC is effective in preventing fibrosis in a porcine esophageal stricture model. Luminal diameters were measured from tissue sections from pigs treated with vehicle (n = 12 strictures from 5 pigs) or Sul-NC (n = 9 strictures from 5 pigs). Data are shown as mean ± SEM. *p < 0.05 compared to vehicle. [Figure 9] Figure 1 shows that Sul-NC is effective in preventing fibrosis in a porcine esophageal stricture model. Representative images show the esophageal lumen of pigs treated with vehicle or Sul-NC at the stricture site compared with healthy tissue sections from unaffected areas of the esophagus. Scale bar represents 5 mm. [Figure 10] Figures 10A, 10B, and 10C show experiments in which Sul-NC was formulated at 500 mg / mL in 2% F127 and stored at room temperature (RT) or 4°C, or lyophilized without cryoprotectant and stored at RT. The particle size of Sul-NC was measured over 168 days. Figure 10B shows experiments in which Sul-NC was formulated at 500 mg / mL in 2% F127 and stored at room temperature (RT) or 4°C, or lyophilized without cryoprotectant and stored at RT. The PDI of Sul-NC was measured over 168 days. Figure 10C shows the particle size (Figure 10A), PDI (Figure 10B), and zeta potential (Figure 10C) of Sul-NC were measured over 168 days. The native formulation showed good stability under storage at room temperature and 4°C. The zeta potential of Sul-NC was measured over 168 days. Data are shown as mean ± SD. [Figure 11]Figures 11A, 11B, and 11C show experiments in which sul-nc was formulated at 500 mg / ml in 2% w / w F127 and then either left undiluted or diluted 1:10 in either 2% F127 or water before lyophilization. The lyophilized powder was then reconstituted with water and characterized. Particle size measurements were performed after reconstitution with water. Dilution before lyophilization did not improve particle characteristics after reconstitution. Data are shown as mean ± SD. 1: undiluted, lyophilized; 2: diluted 1:10 in 2% F127, lyophilized; 3: diluted 1:10 in water, lyophilized. Figure 11B shows experiments in which sul-nc was formulated at 500 mg / ml in 2% w / w F127 and then either left undiluted or diluted 1:10 in either 2% F127 or water before lyophilization. PDI measurements were performed after reconstitution with water. Dilution before lyophilization did not improve particle properties after reconstitution. Data are presented as mean ± SD. 1: No dilution, lyophilized; 2: Dilution 1:10 in 2% F127, lyophilized; 3: Dilution 1:10 in water, lyophilized. Figure 11C shows an experiment in which sul-nc was formulated at 500 mg / ml in 2% w / w F127 and then either left undiluted or diluted 1:10 in either 2% F127 or water before lyophilization. Zeta potential measurements were performed after reconstitution with water. Dilution before lyophilization did not improve particle properties after reconstitution. Data are presented as mean ± SD. 1: No dilution, lyophilized; 2: Dilution 1:10 in 2% F127, lyophilized; 3: Dilution 1:10 in water, lyophilized. [Figure 12]Figures 12A and 12B show experiments in which Sul-NC was formulated at 25 mg / mL in 0.25%, 0.5%, or 1% (w / v) hyaluronic acid (HA) and stored at 4°C. Sul-NC (Figure 12A) particle size was measured over 8 days. Sul-NC formulated with HA was larger than that formulated with 2% F127, likely due to the higher molecular weight and increased viscosity of the hyaluronic acid. Sul-NC formulated with 0.5% hyaluronic acid showed stable size over time. Figure 12B shows experiments in which Sul-NC was formulated at 25 mg / mL in 0.25%, 0.5%, or 1% (w / v) hyaluronic acid (HA) and stored at 4°C. Zeta potential was measured over 8 days. The particle zeta potential trended negative as the hyaluronic acid concentration increased, due to the polyanionic nature of the polymer. Data are presented as mean ± SD. [Figure 13] Figure 13A shows an experiment in which Sul-NC was formulated in 5% (w / v) hydroxypropyl methylcellulose (HPMC) at sulconazole concentrations of 50 mg / mL, 100 mg / mL, or 200 mg / mL and stored at 4°C. The particle size of Sul-NC was measured over a 21-day period. Figure 13B shows an experiment in which Sul-NC was formulated in 5% (w / v) hydroxypropyl methylcellulose (HPMC) at sulconazole concentrations of 50 mg / mL, 100 mg / mL, or 200 mg / mL and stored at 4°C. The zeta potential of Sul-NC was measured over a 21-day period. Lower sulconazole concentrations in the HPMC coating were shown to produce stable particles with near-neutral zeta potentials. Data are shown as mean ± SD. [Figure 14]Figure 14A shows an experiment in which Sul-NC was formulated in 5% (w / v) polyvinyl alcohol (PVA) at 50 mg / mL, 100 mg / mL, or 200 mg / mL and stored at 4 °C. The particle size of Sul-NC was measured over 21 days. Similar to HPMC, the PVA-coated Sul-NC at 50 mg / mL and 100 mg / mL was stable with a near-neutral zeta potential. Overall, the particle size was larger than that of the 2% F127 formulation. Figure 14B shows an experiment in which Sul-NC was formulated in 5% (w / v) polyvinyl alcohol (PVA) at 50 mg / mL, 100 mg / mL, or 200 mg / mL and stored at 4 °C. The zeta potential of Sul-NC was measured over 21 days. Similar to HPMC, the PVA-coated Sul-NC at 50 mg / mL and 100 mg / mL was stable with a near-neutral zeta potential. Overall, particle size was larger than with 2% F127. Data are presented as mean ± SD. DETAILED DESCRIPTION OF THE INVENTION

[0017] The presently disclosed subject matter is described more fully below with reference to the accompanying figures, which illustrate some, but not all, embodiments of the invention. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated figures. Therefore, it should be understood that the presently disclosed subject matter is not to be limited to the particular embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0018] In some embodiments, the present disclosure provides a formulation comprising a plurality of sulconazole nanocrystals and one or more stabilizers.

[0019] In certain embodiments, the particle size of the sulconazole nanocrystals ranges from about 100 to about 600 nm, including about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 nm, and any integer or sub-integer therebetween. In certain embodiments, the particle size of the sulconazole nanocrystals is about 200 nm.

[0020] In some embodiments, the one or more stabilizers are selected from polyvinyl alcohol (PVA), hyaluronic acid (HA), carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), sodium cholate (CHA), poloxamer, and combinations thereof. As used herein, the term "poloxamer" refers to a nonionic triblock copolymer comprising a central hydrophobic chain of polyoxypropylene (i.e., poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (i.e., poly(ethylene oxide)), i.e., poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO).

[0021] Poloxamers have the following general structure:

[0022] [ka]

[0023] Each a is an integer from about 2 to about 130, and each b is an integer from about 15 to about 67. Poloxamers are also known under the trade names Pluronic®, Synperonic™, Kolliphor®, and Lutrol® F. Due to the variability of each "a" and "b" in the chemical structure of the poloxamers described above, many different poloxamers exist, with molecular weights and weight ratios of ethylene oxide to propylene oxide varying from 1100 to 14,000 and 1:9 to 8:2, respectively, each with different properties.

[0024] Poloxamers typically begin with the letter "P" (for poloxamer) followed by three digits. Multiplying the first two digits by 100 gives the approximate molecular weight of the polyoxypropylene core, and multiplying the last digit by 10 gives the percentage of polyoxyethylene content. For example, a poloxamer designated "P407" has a polyoxypropylene molecular weight of 4000 g / mol and a polyoxyethylene content of 70%.

[0025] Furthermore, the naming convention for commercial embodiments, such as the Pluronic® brand name, begins with a letter defining its physical form at room temperature (L = liquid, P = paste, F = flake (solid)), followed by two or three digits. The first digit (or two digits for three-digit numbers) of the numerical designation is multiplied by 300 to indicate the approximate molecular weight of the polyoxypropylene core, and the last digit is multiplied by 10 to indicate the percentage of polyoxyethylene content. For example, Pluronic® F127 is a flake (or solid) poloxamer having an approximate polyoxypropylene core weight of about 3600 and a polyoxyethylene content of 70%. Pluronic® F127 is also known as Synperonic™ PE / F-127, Kolliphor® P407, and Poloxamer 407. In certain embodiments, the poloxamer is Pluronic® F127 (Poloxamer 407).

[0026] Other suitable poloxamers include, but are not limited to, the Pluronic® family of poloxamers (Pluronic® P84, P85, F88, F98, F108, P102, P103, P104, P105, P123, F108, etc.).

[0027] In one embodiment, the concentration of sulconazole is about 10 to about 500 mg / mL. In a specific embodiment, the formulation comprises: (a) about 1.5% to about 5% PVA, (b) about 0.5% to 1% HA, (c) about 1% to about 2% CMC, (d) about 1% to about 5% HPMC, and (e) about 2% to about 6% poloxamer 407.

[0028] In certain embodiments, the formulation is lyophilized. In certain embodiments, the lyophilized formulation further comprises a cryoprotectant. In other embodiments, the lyophilized formulation does not comprise a cryoprotectant.

[0029] In other embodiments, the presently disclosed subject matter provides a precursor formulation comprising the above-described formulation and a plurality of grinding beads. In certain embodiments, the plurality of grinding beads comprises zirconium oxide beads. The zirconium oxide beads may be used to homogenize the formulation during mixing and then removed from the formulation by filtration prior to injection. In more specific embodiments, the formulation comprises about 500 mg of sulconazole, about 2.0 g of 0.5 mm zirconium oxide beads, and about 1 mL of 2% (w / v) poloxamer 407.

[0030] The present invention provides a method for treating or preventing fibrosis or intestinal restenosis in a subject, the method comprising administering the above-described formulation to the subject. As used herein, the term "stricture" refers to an abnormal narrowing of a bodily passageway, such as due to inflammation, cancer, or scar tissue formation. In some embodiments, the stricture is in the intestine of the subject. In other embodiments, the stricture is in the large intestine of the subject. In other embodiments, the stricture is in the esophagus of the subject.

[0031] In certain embodiments, administration of the formulation is by injection. In certain embodiments, injection comprises intraperitoneal (IP) injection or subcutaneous (SC) injection.

[0032] In some embodiments, the formulation is injected near the stenosis site. In certain embodiments, the formulation is injected near the stenosis site after surgery or endoscopic surgery. As used herein, the term "proximal" refers to a position about 2 cm from the stenosis site, including about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 cm.

[0033] In certain embodiments, the fibrosis is associated with inflammatory bowel disease (IBD). In certain embodiments, the inflammatory bowel disease is selected from Crohn's disease (CD), ulcerative colitis (UC), and combinations thereof.

[0034] In some embodiments, the formulation modulates acute healing responses and / or interrupts one or more pathological fibrotic tissue remodeling processes. In some embodiments, the method comprises reducing the thickness of the collagen layer in the small intestine of a subject. In some embodiments, administration of the formulation results in a folded, flexible epithelial structure that is more similar to healthy tissue.

[0035] In certain embodiments, administration of the formulation is sustained release administration such that the drug is released in vivo for more than 3 days, including more than 3 days, more than 7 days, more than 30 days, more than 60 days, and more than 90 days.

[0036] In some embodiments, the concentration of sulconazole in the formulation ranges from about 100 mg / mL to about 500 mg / mL. In some embodiments, the volume of the formulation injected ranges from about 10 μL to about 100 μL. In some embodiments, the formulation is injected at a dose of about 100 mg / kg to about 1875 mg / kg of sulconazole.

[0037] As used herein, the term "treatment" can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of a disease, disorder, or condition to which the term applies, or one or more symptoms or signs of the disease, disorder, or condition. Prevention refers to not causing a disease, disorder, condition, or its symptoms or signs, or worsening of its severity. Thus, the compounds of the present disclosure can be administered prophylactically to prevent or reduce the occurrence or recurrence of a disease, disorder, or condition.

[0038] While the "subject" treated by many embodiments of the disclosed methods is desirably a human subject, it should be understood that the methods described herein are effective on all vertebrate species intended to be encompassed by the term "subject." Accordingly, a "subject" can include a human subject for medical purposes, such as treatment of an existing condition or disease or prophylactic treatment to prevent the onset of a condition or disease, or an animal subject for medical, veterinary, or developmental purposes. Suitable animal subjects include mammals, including, but not limited to, primates such as humans, monkeys, and apes; bovines, such as cows and oxen; ovines, such as goats; caprines, such as pigs and domestic pigs; equines, such as horses, donkeys, and zebras; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; rodents, including mice, rats, and the like. The animal may be a transgenic animal. In some embodiments, the subject is a human, including, but not limited to, a fetal, neonatal, infant, juvenile, or adult subject. Additionally, a "subject" can also include a patient suffering from or suspected of suffering from a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein. The term "subject" also refers to an organism, tissue, cell, or collection of cells from a subject.

[0039] Generally, an "effective amount" of an active agent refers to the amount necessary to elicit a desired biological response. As will be understood by those skilled in the art, the effective amount of an agent can vary depending on factors such as the desired biological endpoint, the agent being delivered, the configuration of the pharmaceutical composition, and the drug target.

[0040] The term "combination" is used in the broadest sense to mean administering to a subject at least two agents, more specifically, the formulations of the present disclosure, in combination with a second agent or therapy. More specifically, the term "in combination" refers to the simultaneous administration of two (or more) active agents or therapies to treat a single disease. As used herein, active agents or therapies can be administered in combination in a single dosage form, simultaneously in separate dosage forms, or in separate dosage forms that are administered alternately or sequentially on the same or different days. In one embodiment of the presently disclosed subject matter, active agents or therapies are combined and administered in a single dosage form. In another embodiment, active agents or therapies are administered in separate dosage forms (e.g., when it is desired to change the amount of one while maintaining the amount of the other). A single dosage form can contain additional active agent therapies for the treatment of a disease condition.

[0041] Additionally, formulations of the present disclosure combined with additional therapeutic agents or therapies may further be administered with adjuvants, alone or in combination with one or more therapeutic agents, that enhance the stability of the agent, in certain embodiments facilitate administration of pharmaceutical compositions containing them, enhance dissolution or dispersion, enhance inhibitory activity, include other active ingredients, provide adjunctive therapy, etc. Advantageously, such combination therapies allow for the administration of lower amounts of conventional therapeutic agents, thereby avoiding potential toxicity and side effects that may occur when those agents are used as monotherapies.

[0042] The timing of administering the formulation of the present disclosure in combination with additional therapeutic agents or therapies can be changed, as long as the beneficial effect of the combination of these agents is achieved.Therefore, the term "in combination" refers to administering the formulation of the present disclosure and the additional therapeutic agents or therapies described herein simultaneously, sequentially, or in combination.Therefore, the subject who is administered the combination of the formulation of the present disclosure and the additional therapeutic agents or therapies can receive the formulation of the present disclosure and the additional therapeutic agents or therapies simultaneously (i.e., simultaneously) or at different times (i.e., consecutively, in any order, on the same day or different days), as long as the combined effect of both drugs is achieved in the subject.

[0043] When administered sequentially, the agents may be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes, or more of each other. In other embodiments, sequentially administered agents may be administered within 1, 5, 10, 15, 20 days, or more of each other. When a formulation of the present disclosure and an additional agent or therapy are administered simultaneously, they may be administered to a subject as separate pharmaceutical compositions, each comprising either a formulation of the present disclosure or at least one additional therapeutic agent, or they may be administered to a subject as a single pharmaceutical composition comprising both agents.

[0044] When administered in combination, the effective concentration of each agent to induce a particular biological response may be lower than the effective concentration of each agent when administered alone, thereby allowing for a reduction in the dose of one or more agents compared to the dose required when the agent is administered as a single agent. The effects of multiple agents are not necessarily additive or synergistic, but may be. Agents may be administered multiple times.

[0045] In some embodiments, administering two or more agents in combination can result in a synergistic effect. As used herein, the terms "synergy," "synergistic," "synergistically," and derivatives thereof, such as "synergistic effect" or "synergistic combination" or "synergistic composition," refer to a situation in which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of each agent when administered individually.

[0046] Synergy can be expressed in terms of the "synergy index (SI)," which can generally be determined from the ratio determined by the following formula, as described in F.C. Kull et al., Applied Microbiology 9, 538 (1961): Q a / Q A +B / Q B = Synergy Index (SI) wherein: Q A is the concentration of component A acting alone to produce the endpoint relative to component A, Q a is the concentration of component A in the mixture that produced the endpoint, Q B is the concentration of component B acting alone to produce the endpoint relative to component B, Q b is the concentration of component B in the mixture that produced the endpoint.

[0047] Generally, Q a / Q A and Q b / Q BA sum greater than 1 indicates antagonism, a sum equal to 1 indicates additive action, and a sum less than 1 demonstrates synergy. The lower the SI, the greater the synergistic effect exhibited by that particular mixture. Thus, a "synergistic combination" exhibits greater activity than would be expected based on the observed activity of the individual components when used alone. Furthermore, a "synergistically effective amount" of a component refers to the amount of that component needed to elicit a synergistic effect, for example, with another therapeutic agent present in the composition.

[0048] Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, a reference to "a subject" includes a plurality of subjects unless the context clearly dictates otherwise (e.g., a plurality of subjects).

[0049] Throughout this specification and claims, the terms "comprises," "comprises," and "comprising" are used in a non-exclusive sense unless the context otherwise requires. Similarly, the term "comprises" and its grammatical variations are intended to be non-limiting, and the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0050] For purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, properties, and other numerical values used in the specification and claims are to be understood as being modified in all instances by the term "about," even if the value, amount, or range does not explicitly state otherwise. Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are not, and need not be, exact, but may be approximate and / or may be made larger or smaller as necessary to reflect tolerances, conversion factors, rounding, measurement error, and the like, as well as other factors known to those of ordinary skill in the art, depending upon the desired properties sought to be obtained by the subject matter of the present disclosure. For example, the term "about" when referring to a value can be meant to encompass variations in some embodiments of ±100%, in some embodiments of ±50%, in some embodiments of ±20%, in some embodiments of ±10%, in some embodiments of ±5%, in some embodiments of ±1%, in some embodiments of ±0.5%, and in some embodiments of ±0.1% from the specified amount, as appropriate for practicing the disclosed methods or using the disclosed compositions.

[0051] Furthermore, the term "about," when used in connection with one or more numerical values or numerical ranges, is understood to refer to all such numerical values, including all numerical values within the range, and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers contained within that range, including, for example, integers and fractions thereof (e.g., recitation of 1 to 5 includes 1, 2, 3, 4, 5, as well as fractions thereof such as 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range. [Example]

[0052] The following examples are included to provide guidance to those skilled in the art for practicing representative embodiments of the presently disclosed subject matter. Given this disclosure and the general level of skill in the art, those skilled in the art will appreciate that the following examples are intended to be merely illustrative and that numerous changes, modifications, and variations can be adopted without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples below are for illustrative purposes only and should not be construed in any way as limiting the ability to prepare the disclosed compounds by other methods. [Example]

[0053] Injectable sustained-release antifibrotic nanoformulations for preventing fibrosis and intestinal restenosis

[0054] (1.1 Overview) The subject matter of the present disclosure includes an injectable formulation for sustained delivery of an antifibrotic drug to the stricture site to prevent recurrence after surgical or endoscopic treatment. Screening of a small molecule drug library revealed the potent antifibrotic properties of sulconazole, an FDA-approved drug for the treatment of fungal infections. Because free sulconazole has a short lifespan that is insufficient for treating fibrosis during wound healing, sulconazole nanocrystals (Sul-NC) were formulated for sustained release. Importantly, Sul-NC was found to dramatically increase the maximum tolerated dose in mice and provide superior or equivalent fibrosis prevention efficacy with fewer administrations in mouse models of skin and intestinal fibrosis. In a porcine model of esophageal stricture, a single injection of Sul-NC after balloon dilation prevented restenosis, as measured by a significant increase in esophageal diameter compared to placebo.

[0055] (1.2 Background technology) Normal tissue repair involves immune and mesenchymal cell activation, extracellular matrix (ECM) deposition, and tissue remodeling (Rieder et al., 2007). However, severe or repeated injury, such as in chronic inflammation, can lead to excessive ECM accumulation and fibrotic tissue formation (Rieder et al., 2007). Fibrosis can affect any organ and can lead to the destruction of normal tissue architecture or organ dysfunction or failure (Henderson et al., 2020). Myofibroblasts, which express high levels of α-smooth muscle actin (α-SMA), are contractile, and play a central role in fibrosis and scar formation (Rieder et al., 2007). Chronic inflammation leads to impaired mucosal tissue repair and myofibroblast proliferation (Rieder, 2013; Rieder and Fiocchi, 2009). Therefore, targeting myofibroblast proliferation pathways has been proposed as a strategy for developing effective antifibrotic drugs. Henderson et al., 2020; Bollong et al., 2017; Rosenbloom et al., 2013. To date, only two antifibrotic medications are generally approved by the FDA: pirfenidone and nintedanib for the treatment of idiopathic pulmonary fibrosis (Raghu and Selman, 2015). However, to date, there are no antifibrotic medications for other indications, including intestinal fibrosis. Therefore, effective antifibrotic therapies represent a critical unmet need.

[0056] In the gastrointestinal (GI) tract, inflammatory bowel disease (IBD) is a major cause of intestinal fibrosis and exemplifies significant morbidity due to intestinal fibrosis (Latella and Rieder, 2017). IBD is divided into two major subtypes, Crohn's disease (CD) and ulcerative colitis (UC), and affects more than 3 million people in the United States (Dahlhamer et al., 2015). Fibrosis is the underlying mechanism for the development of intestinal strictures, which narrow the intestine (Rieder et al., 2007; Schmoyer et al., 2021), occurring in 27%–54% of patients with Crohn's disease and 1.5%–11.2% of patients with UC (Le Berre et al., 2020). Such strictures often require surgical or endoscopic treatment and almost always recur due to ongoing fibrosis (Rieder et al., 2007). Recurrent fibrostenosis typically leads to multiple surgeries, bowel shortening, stomas, reduced quality of life, and high healthcare costs (Lan et al., 2018). Because the pro-fibrosis mechanism in IBD is driven by inflammation, there was great hope that potent anti-inflammatory therapies developed over the past 20 years would reduce the incidence of stricture / fibrosis complications. However, this hope has not come to fruition (Cosnes et al., 2005; D'Amico et al., 2020; Li et al., 2019; Moschen et al., 2019). There appears to be an urgent need for dedicated anti-fibrotic treatments developed and formulated to address the demands of GI tract fibrostenosis.

[0057] Intestinal strictures offer an opportunity for localized targeted therapy to reduce the potential for systemic complications. While not wishing to be bound by any particular theory, it is believed that local injection of antifibrotic drugs during endoscopic dilation may modulate wound healing and fibrosis processes while reducing the risk of systemic side effects. Notably, previous efforts using single injections of drugs such as corticosteroids and mitomycin C have been largely ineffective (Madadi-Sanjani et al., 2018; East et al., 2007). This may be due in part to their short duration of action before elimination from the body. While not wishing to be bound by any particular theory, sustained-release formulations may be optimal to positively impact tissue remodeling processes, such as fibrosis, which occur over days to weeks.

[0058] The presently disclosed subject matter demonstrates that the topical antifungal agent sulconazole has potent antifibrotic effects. Again, without wishing to be bound by any particular theory, it is believed that providing sustained and effective concentrations of sulconazole locally at the tissue site may modulate the acute healing response and interrupt the process of remodeling pathological fibrotic tissue. The presently disclosed subject matter demonstrates that a sulconazole nanocrystal (Sul-NC) formulation was highly effective in preventing fibrosis in rodent models of skin and intestinal fibrosis and a novel porcine model of esophageal stricture. (Li et al., 2021). Finally, the presently disclosed subject matter demonstrates that Sul-NC is well tolerated and safe. The data of the presently disclosed subject matter establish a foundation to support further preclinical studies leading to the development of Sul-NC as an antifibrotic agent.

[0059] (1.3 Results) 1.3.1 Identification of Sulconazole as an Antifibrotic Agent by High-Throughput Microscopy-Based Screening To identify potential antifibrotic agents, we used a high-throughput microscopy-based drug screening approach using transforming growth factor β (TGF-β)-activated human primary colon fibroblasts (CCD-18Co). Next, a panel of 1,586 FDA-approved small molecule drugs was screened for their ability to reduce α-SMA and type 1 collagen (COL1A1) expression by fluorescent immunocytochemistry. Pirfenidone was included in this screen based on its known antifibrotic activity and was found to be effective at a dose of 4 mM (Figure 1A). Notably, sulconazole similarly reduced α-SMA and COL1A1 staining at a much lower dose of 10 μM (Figure 1A). Next, we used both activated CCD-18Co cells and human hepatic stellate cells (LX2) in confirmatory Western blot analysis, which showed that sulconazole significantly reduced α-SMA and COL1A1 protein production (Figure 1B). Next, we analyzed α-SMA expression by RT-PCR in both activated CCD-18Co cells and LX-2 cells. Sulconazole significantly reduced ACTA2 expression in activated CCD-18Co cells and LX-2 cells, even at concentrations as low as 1 μM and 5 μM, respectively, whereas pirfenidone showed no effect until concentrations reached 4 mM (Figure 1C). These in vitro results suggest that sulconazole is a potent antifibrotic candidate, acting at much lower concentrations than pirfenidone.

[0060] 1.3.2 Sulconazole can be formulated as a sustained-release preparation A series of generally regarded as safe (GRAS) stabilizers were tested at various concentrations for use in nanomilling sulconazole. The resulting nanocrystals varied in size and polydispersity, with larger molecular weight polymeric stabilizers, such as hyaluronic acid (HA), generally resulting in larger particle sizes and higher polydispersities (Table 1).

[0061] Table 1. Particle size, polydispersity index (PDI), and zeta potential of sulconazole when formulated with a range of drug concentrations (10-200 mg / mL) and 0.5-5% polyvinyl alcohol (PVA), hyaluronic acid (HA), carboxymethylcellulose (CMC), and hydroxypropylmethylcellulose (HPMC). Data are expressed as mean ± SD. NR = not recorded.

[0062] [Table 1]

[0063] Incorporation of Pluronic F127 generally resulted in smaller and more uniform particle sizes, even as the concentration of sulconazole increased from 10 mg / mL to 500 mg / mL (see Table 2).

[0064] Table 2. Sul-NC particle size, polydispersity index (PDI), and ζ-potential when formulated at a range of drug concentrations (10–500 mg / mL) and 2–6% F127. Data are expressed as mean ± SD.

[0065] [Table 2]

[0066] Similar particle sizes were obtained with 2–6% Pluronic F127, but 2% was selected for the final formulation due to its low viscosity, favorable for injection through an endoscopic needle. Electron microscopy of 500 mg / mL Sul-NC showed nonspherical particles with cubic edges, reflecting the drug's crystallinity (Figure 2A). Particle sizes were consistent across nine batches manufactured on different days, ranging from 196 to 261 nm (average across batches: 232 ± 19 nm) (Figure 2B). Using a rapid equilibrium dialysis system for accelerated in vitro drug release, we confirmed that free sulconazole was not impeded by the dialysis membrane and that Sul-NC dissolved within 7–8 days (Figure 2C). Furthermore, 500 mg / mL Sul-NC was stable for 112 days of storage at room temperature or 4°C, as assessed by particle size measurement (Figure 2D).

[0067] 1.3.3 Sul-NC increased the maximum tolerated dose (MTD) of sulconazole in vivo To evaluate the safety of Sul-NC and determine the dose for efficacy experiments, we determined the maximum tolerated dose (MTD) of free sulconazole and Sul-NC. First, three groups of five mice were intraperitoneally (IP) injected with a single dose of 15 mg / kg, 30 mg / kg, or 40 mg / kg of free sulconazole. All mice survived (Table 3). However, when the IP dose was increased to 50 mg / kg, only three of nine mice (33%) survived (Table 3). Similarly, when sulconazole was administered subcutaneously (SC) at 50 mg / kg, only two of four mice (50%) survived (Table 3). All non-surviving mice died within 1 to 4 days of injection (not shown).

[0068] Table 3. Determination of the maximum tolerated dose of free sulconazole injected intraperitoneally (IP) or subcutaneously (SC) at different doses. Mice that did not survive died within 1–4 days after injection.

[0069] [Table 3]

[0070] Next, we investigated the effect of the nanoformulation on the tolerability of subcutaneously injected sulconazole. Previously, we observed that injection volume can affect the rate and extent of drug absorption from crystalline formulations. Therefore, we also tested different injection volumes (10–100 μL) and Sul-NC concentrations (100–500 mg / mL) (Hsueh et al., 2021). We found that the combination of higher injection volumes (50–100 μL) and lower sulconazole concentrations (100–200 mg / mL) resulted in higher mortality (Table 4). For example, after subcutaneous injection of 100 μL of the 100 mg / mL formulation (500 mg / kg), only two out of three mice (66%) survived, whereas injection of 50 μL of the 500 mg / mL formulation (1250 mg / kg) resulted in 100% survival (Table 4). This observation suggests that increasing the formulation concentration and decreasing the injection volume slows the rate of systemic drug absorption. However, when the Sul-NC dose was increased to 1,875 mg / kg, only 2 of 4 mice (50%) survived, and at 2,500 mg / kg, 0 of 4 mice (0%) survived. Smaller injection volumes (<75 μL) improved tolerability, but concentration may be a limiting factor at such high doses. Overall, a high dose of 1,250 mg / kg Sul-NC was well tolerated, but survival was reduced when free sulconazole was administered at only 50 mg / kg.

[0071] Table 4. Determination of the maximum tolerated dose of Sul-NC injected subcutaneously (SC) at various concentrations and injection volumes. Mice that did not survive died within 1–4 days after injection.

[0072] [Table 4]

[0073] To further investigate systemic drug toxicity, free sulconazole or Sul-NC was injected before blood samples were collected and markers of liver and kidney function were assessed. Mice received a single subcutaneous dose of 312.5 mg / kg, 625 mg / kg, or 1,250 mg / kg of Sul-NC at 500 mg / mL, respectively. Plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatine remained within normal ranges after 6 hours in all mice injected with Sul-NC (Table 5). However, a single subcutaneous injection of the free drug at 50 mg / kg elevated blood levels of ALT and BUN, suggesting liver and kidney damage (Table 5). This difference in tolerance reflects the sustained-release properties of the nanocrystals and suggests that the Sul-NC formulation is safe even when used at high concentrations.

[0074] 1.3.4 Sul-NC is an effective antifibrotic agent in rodent skin and intestinal fibrosis models Animal models of GI tract fibrosis / stenosis are lacking (Li et al., 2021). To obtain preliminary evidence of the efficacy of Sul-NC as an antifibrotic agent in vivo, a well-described and reproducible bleomycin-induced rodent skin fibrosis model was used (Yamamoto et al., 1999). To induce fibrosis, bleomycin was injected into five sites within a 1 x 1 cm area on the back of mouse skin every other day for 4 weeks, as previously described (Figure 3). (Yamamoto et al., 1999). Five groups of mice (n = 5 each) were administered free sulconazole (Free Sul) at 10 mg / kg IP every other day, Sul-NC at 50 mg / kg or 150 mg / kg SC once weekly, PBS (vehicle) SC once weekly, or pirfenidone at 100 mg / kg orally daily. Histological sections of skin samples excised from the bleomycin-injected sites were examined by H&E or Masson's trichrome staining (Figure 4), revealing a significant decrease in dermal thickness in all treatments compared to vehicle (Figure 5). Furthermore, Masson's trichrome staining confirmed that the dermal thickness was due to the deposition of collagen and other ECM proteins (blue staining in the figure). Of note, there were notable differences in dose and frequency of administration when comparing pirfenidone (daily administration, 2,800 mg / kg total dose) with Sul-NC (once weekly, 200 mg / kg total dose).

[0075] We employed an intestinal transplant model (Figure 6) previously used to verify the antifibrotic effects of oral pirfenidone (Meier et al., 2016). A single injection of Sul-NC at a dose of 50 mg / kg or 150 mg / kg was performed at the transplant site and compared with oral pirfenidone at 100 mg / kg three times daily, a dose previously described as effective in this model. Both 150 mg / kg and 150 mg / kg significantly reduced collagen deposition (thickness of the collagen layer) compared with vehicle-injected animals (13.2 ± 1.1 μm) and were comparable to pirfenidone administered orally three times daily (9.1 ± 0.3 μm) (Figure 7). Again, there were notable differences in dose and frequency of administration when comparing pirfenidone (two doses per day, 2,100 mg / kg total) with Sul-NC (single dose, 50 mg / kg).

[0076] 1.3.5 Sul-NC is effective in a porcine GI tract stenosis model A clinically relevant porcine esophageal stricture model involving endoscopic balloon dilation followed by stricture reformation was recently established (Li et al., 2021). Two weeks after induction, strictures exceeding 6 mm in diameter were first endoscopically balloon dilated to 10 mm and then injected with either vehicle or Sul-NC. Two weeks after treatment, the stricture site was examined using endoscopy (Figure 8A) and contrast-enhanced radiography (Figure 8B). Significantly larger luminal openings were observed in Sul-NC-treated pigs (6.3 ± 0.5 mm) compared with vehicle (1.2 ± 0.5 mm) (Figure 8C). Histological sections stained with Masson's trichrome and Sirius red demonstrated significantly reduced collagen deposition in Sul-NC-injected sites compared with vehicle controls (Figure 9). Furthermore, Sul-NC-treated tissue exhibited a folded, flexible epithelial structure more similar to unaffected areas or healthy esophageal tissue (Figure 9). These results confirmed the antifibrotic effect of Sul-NC formulation in pigs.

[0077] (1.4 Discussion) One of the major unmet clinical needs in the management of inflammatory bowel disease (IBD) is the management of patients with Crohn's disease with anastomotic strictures. Within 20 years of diagnosis, up to 54% of patients with Crohn's disease develop fibrotic strictures (Le Berre et al., 2020). Endoscopic dilation is possible only in select cases; the remaining cases require surgery, leading to bowel loss, morbidity, and mortality. However, strictures almost always recur, resulting in further surgery, abdominal adhesions, bowel loss, bowel shortening, and / or other complications (Latella and Rieder, 2017). The seemingly constant clinical course of these patients is, at least in part, due to the lack of effective antifibrotic medications.

[0078] Recently, high-throughput screening of hundreds to thousands of compounds has identified many potential antifibrotic drugs. These drugs include itraconazole (chemically modified to CBR-096-4) (Bollong et al., 2017), haloperidol (Rehman et al., 2019), and several other azole antifungal agents (oxiconazole, clotrimazole, and butoconazole) (Braun et al., 2020). However, to our knowledge, the efficacy of these antifibrotic drugs in the GI tract has not been verified, nor has their formulation for topical treatment been focused on. The mechanism by which sulconazole exerts its antifibrotic effects is currently unknown, but its known bactericidal activity appears to be mediated by the inhibition of fungal cytochrome P450 enzymes (Fromtling, 1988; Georgopapadakou and Walsh, 1996; Monk et al., 2020; Vanden Bossche et al., 1988).

[0079] In human lymphoblastoid and hepatocyte cells, sulconazole and other antifungal imidazole derivatives demonstrated nonspecific inhibition of P450 enzymes (Zhang et al., 2002). Evidence suggests that P450 inhibition or downregulation may have a protective effect against tissue damage and inflammation caused by toxic chemicals such as ethanol and carbon tetrachloride (Nagappan et al., 2019; Song et al., 2021). However, because P450 enzymes play a critical role in drug metabolism and detoxification, global inhibition is undesirable and may be associated with systemic side effects, further promoting formulations for localized treatment.

[0080] Early efforts toward localized treatments to prevent fibrosis in the GI tract involved the direct injection of anti-inflammatory drugs such as triamcinolone (East et al., 2007) or antifibrotic nucleic acids (Suzuki and Yoneyama, 2017) into inflamed tissues. However, these attempts did not lead to clinically meaningful results. This is likely because a single injection of a drug or nucleic acid in solution has a very short duration of action before it is eliminated from the body. In contrast, tissue remodeling processes such as fibrosis occur over days to weeks, necessitating the development of sustained-release formulations.

[0081] However, due to sulconazole's low water solubility, various approaches for sustained drug release, such as encapsulation in a polymer matrix, have been employed. Considering the limited volume that can be directly injected into intestinal tissue and the need to load the maximum possible amount of drug for the longest-lasting therapeutic effect, formulation as particles with a pure drug core can achieve higher drug loadings than encapsulation (Hsueh et al., 2021; Farah et al., 2019). For example, the 500 mg / mL Sul-NC formulation described here contains approximately 96% drug by weight. Reducing the amount of excipients is also advantageous for minimizing the potential for accumulation of material that could cause injection-site reactions or fibrotic responses. Furthermore, increasing the drug concentration and reducing the injection volume have been observed to lead to an increased maximum tolerated dose of Sul-NC via subcutaneous injection in mice (approximately 1,250 mg / kg compared to 50 mg / kg of free sulconazole), likely due to a reduced absorption surface area and slower dissolution rate (Hsueh et al., 2021).

[0082] Although more detailed pharmacokinetic and safety studies are needed, there is a rationale for repurposing a drug typically used for topical administration as an infusion into the GI tract. Previous pharmacokinetic studies have shown that systemic absorption of topically applied sulconazole is much higher than that of other azoles, ranging from 8.7 to 11.3% for a 9-g dose (two 4.5-g doses administered 12 hours apart) (Franz and Lehman, 1988). Furthermore, fecal excretion is one of the major clearance routes, suggesting that topical sulconazole administration is associated with relatively high levels of drug exposure already in the intestine (Franz and Lehman, 1988). Furthermore, potential toxicity of sulconazole is likely associated with accumulation in the liver, which is even higher with systemic administration. Liver and kidney function was evaluated during Sul-NC administration. Doses up to 1,250 mg / kg showed no detectable changes in blood levels of ALT, AST, BUN, or creatinine 7 days after infusion.

[0083] The subject matter of this disclosure is the first to demonstrate the successful development of a nanocrystalline formulation of sulconazole with localized, sustained-release, antifibrotic effects in the GI tract. One potential limitation of this study is the paucity of animal models of GI tract fibrosis.

[0084] Furthermore, GI tract fibrosis can be triggered by a variety of stimuli. However, to alleviate some of these concerns, we tested the efficacy of Sul-NC in three animal models in which fibrosis is induced by different stimuli: chemical (cutaneous bleomycin model), ischemia (intestinal transplant model), and thermal (porcine GI stenosis model). While none of these models fully reproduces GI tract stenosis in patients, the fact that Sul-NC was effective across these models, involving various organ systems and inducers, is reassuring and provides a foundation for further research targeting the specific etiology of GI tract fibrosis. Furthermore, although maximum tolerated dose experiments were performed in rodents, further research should focus on similar experiments after infusion into the GI tract, as systemic absorption may differ from subcutaneous injection. Therefore, scaled-up, systemic preclinical studies regarding pharmacokinetics, toxicity, and maximum tolerated dose are warranted.

[0085] (1.5 demand) There is a lack of available drugs for the prevention of pathological fibrosis. Several indications, such as intestinal strictures in inflammatory bowel disease (IBD), can be treated by localized injection into tissues with reduced risk of systemic side effects. However, sustained-release approaches are necessary to combat disease processes that progress over weeks to months. The subject matter of the present disclosure demonstrates that the antifungal drug sulconazole exhibits potent antifibrotic properties. A sulconazole nanocrystalline formulation (Sul-NC) was developed and demonstrated dramatic increases in the maximum tolerated dose and fibrosis prevention in mouse models of skin and intestinal fibrosis and in a patient-like porcine esophageal stricture model. A thorough understanding of the pharmacokinetics, toxicity, MTD, and mechanism of action of the Sul-NC formulation may allow its repurposing as an antifibrotic drug for clinical trials.

[0086] 1.6 Materials and Methods (1.6.1 Materials) Human primary colon fibroblasts (CCD-18Co, CRL-1459) were purchased from ATCC (Manassas, VA). Human hepatic stellate cells (LX2) were a gift from Dr. S.L. Friedman (Xu et al., 2005). TGF-β, rapid equilibrium dialysis (RED) device inserts (8K MWCO), PBS, TBS, ultrapure water, ReverAid First Strand cDNA Synthesis Kit (1622), Pierce BCA Protein Assay Kit (23227), DAPI (D1306), DMEM, alpha-MEM, FBS, 100 μm sterile cell strainer (22363549), Tris base powder (BP152-500), penicillin-streptomycin, and SYBR Green PCR Master Mix Kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). α-SMA antibody (C-6198), polyethylene glycol 300 (PEG300) (81160), trifluoroacetic acid, and Tween 80 were purchased from Sigma Aldrich (St. Louis, MO, USA). Type 1 collagen antibody (ab138492) and anti-α-SMA antibody (ab5694) were obtained from Abcam (Waltham, MA, USA). Tylose MH 300 (93800) was procured from Millipore Sigma (St. Louis, MO, USA). High-performance liquid chromatography (HPLC)-grade acetonitrile and water were purchased from Fisher Scientific (Hampton, NH, USA). TRIzol reagent (15596026) and normal goat serum (31873) were obtained from Invitrogen (Waltham, MA, USA). RIPA buffer (9806) was obtained from Cell Signaling Technology (Danvers, MA, USA). Complete protease inhibitor cocktail (1183617001) was obtained from Roche.Mini-Protean TGX gels (4–15%), 1X Tris / glycine / SDS buffer (161-0772), nitrocellulose membranes, and 1X Tris / glycine with methanol (161-0771) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Intercept (TBS) blocking buffer (927-60001), IRDye® 800CW goat anti-rabbit IgG secondary antibody (RRID AB_2651127), and IRDye® 680RD goat anti-mouse IgG secondary antibody were purchased from Li-Cor (Lincoln, NE, USA). Sulconazole nitrate (K466) was purchased from AK Scientific (Union City, CA, USA). Pluronic F127 (Kolliphor P407) was purchased from BASF (Geismar, LA, USA). Polyvinyl alcohol (PVA, 78 kDa, 88 mol% hydrolyzed) and uranyl acetate (98%, ACS reagent) were purchased from Polysciences Inc. (Warrington, PA). Hydroxypropylmethylcellulose (HPMC, 3,550 mPa·s, USP grade HY124) and carboxymethylcellulose sodium salt (CMC, 173 kDa) were purchased from Spectrum (Gardena, CA). Hyaluronic acid sodium salt (HA, 1-2 MDa) was purchased from Carbosynth (San Diego, CA). Ultrathin (UL) carbon-coated 400-mesh copper grids (EMS CF400-Cu-UL) were purchased from Electron Microscopy Sciences (Hatfield, PA). 0.5 mm zirconium oxide beads were purchased from Next Advance (Troy, NY). 2.0 mL Eppendorf tubes (T20-100) were obtained from Stellar Scientific. PCR primers were purchased from IDT (Coralville, Iowa, USA). Insulin syringes were purchased from BD (Franklin Lakes, New Jersey).Bleomycin (B3972) and pirfenidone (TCP1871) were purchased from TCI (Portland, OR, USA), and the drug library was obtained from Johns Hopkins University.

[0087] (1.6.2 Cell culture) CCD-18Co cells were cultured using alpha-MEM supplemented with 10% FBS and 1% penicillin-streptomycin according to the manufacturer's protocol. The LX2 cell line was maintained in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were tested for mycoplasma (Young et al., 2010) before each experiment. Cell lines were maintained in a humidified incubator at 37°C with 5% CO2.

[0088] 1.6.3 High-Throughput Microscopy-Based Drug Screening Human primary colon fibroblasts (CCD-18Co) were seeded in 96-well flat-bottom plates. After 24 hours, cells were washed and starved for 24 hours in alpha-MEM medium without FBS. The medium was then replaced with alpha-MEM supplemented with 10% FBS containing 5 ng / ml TGF-β and activated for 48 hours. The cells were then washed again, and fresh medium containing 5 ng / ml TGF-β and 10 μM of a drug from a library of 1,586 FDA-approved small molecules was applied to the cells. After 4 days, cells were fixed with 10% neutral formalin for 15 minutes and permeabilized with 0.5% Triton X-100 in PBS. Then, cells were blocked with 10% goat serum for 1 hour, and α-SMA and cell nuclei were stained. A Keyence BZ-X700 high-throughput microscope was used for high-throughput immunofluorescence scanning. All drugs in the library were first screened at 10 μM in CCO-18Co cells to assess their effect on α-SMA production. Drugs that caused a visible decrease in fluorescent signal were then screened again at 5 μM and 10 μM to assess their effect on α-SMA and type 1 collagen production. Software (Keyence Bz-X700 Analyzer software) was programmed to capture three sets of images from each well, including the red channel for Alexa Fluor 594 (α-SMA), the green channel for Alexa Fluor 488 (type 1 collagen), and the blue channel for DAPI (nuclei), at a fixed exposure and exposure time. Images were analyzed using Image J2 Fiji (NIH, Bethesda, MD) to determine the efficacy of drug treatment, as assessed by the reduction in the intensity of α-SMA and collagen staining per cell. Three images were averaged for each drug condition.

[0089] (1.6.4 Western Blot) LX2 and CCD-18Co cells were seeded in 6-well plates. After 24 hours, cells were washed and starved for 24 hours in alpha-MEM medium without FBS. The medium was then replaced with alpha-MEM supplemented with 10% FBS containing 5 ng / ml TGF-β and activated for 48 hours. Cells were then washed again and incubated for 96 hours in fresh medium containing 5 ng / ml TGF-β and 10 μM sulconazole (dissolved in DMSO at 100 mM and diluted to 10 μM in cell culture medium). Cells were then washed three times with cold PBS and treated with protein lysis RIPA buffer with a complete protease inhibitor cocktail. Protein concentration was measured using the Pierce BCA Protein Assay Kit. 20 μg of total protein lysate from each treatment group was loaded onto each well of a Mini-Protean TGX gel (4–15%) and electrophoresed using 1X Tris / Glycine / SDS buffer. Proteins were then transferred to a nitrocellulose membrane using 1X Tris / Glycine with methanol. The membrane was blocked with Intercept (TBS) blocking buffer for 1 hour at room temperature and then incubated overnight at 4°C with anti-α-SMA antibody (1:1000) and anti-COL1A1 antibody (1:1000). The membrane was washed three times for 10 minutes with TBS-0.1% Tween and incubated with IRDye-secondary antibody (1:10,000) for 1 hour at room temperature. The membrane was then washed three times for 10 minutes with TBS-0.1% Tween and rinsed with 1X TBS to remove residual Tween 20. The membrane was then scanned using an Odyssey Image System (Li-Cor, Lincoln, NE, USA).

[0090] 1.6.5 Development and Characterization of Sulconazole Nanocrystals (Sul-NC) Sul-NC was formulated using a wet bead nanomilling method as previously described (Hoang et al., 2019; Date et al., 2018; Date et al., 2021). Wet bead milling was performed using a laboratory-scale tissue homogenizer (TissueLyser LT, Qiagen Inc., Germantown, MD). To determine the optimal sulconazole formulation—one that is relatively uniform in particle size, stable at room temperature, and injectable through a small-gauge endoscopic needle—various stabilizers were used. These stabilizers included polyvinyl alcohol (PVA), hyaluronic acid (HA), carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), and Pluronic® F127 (F127) (Table 1). The final formulation approach for animal administration consisted of 500 mg of sulconazole, 2.0 g of 0.5 mm zirconium oxide beads, and 1 mL of 2% (w / v) Pluronic F127 solution in a 2 mL Eppendorf tube. The contents were milled for 10 hours at 3,000 vibrations per minute in a refrigerated room at 4 °C. The mixture was then passed through a 100 μm cell strainer to separate the milling beads. The milling time was optimized to obtain particles of approximately 200 nm, for a final formulation of 5 hours. For porcine studies, 30 tubes were prepared and combined prior to characterization. The particle size, polydispersity index (PDI), and surface charge (ζ potential) of Sul-NC were measured using a Malvern Zetasizer Nano ZS (scattering angle 173°) (Malvern, Westborough, MA). For particle size and polydispersity index measurements, Sul-NC was diluted 1:100 with ultrapure water. For ζ-potential measurements, Sul-NC was diluted 1:40 with 10 mM NaCl (pH 7). The morphology of Sul-NC was measured by transmission electron microscopy (TEM). Sul-NC (500 mg / mL, 8 μL) was adsorbed onto glow-discharged (EMS GloQube, Hatfield, PA) ultrathin (UL) carbon-coated 400-mesh copper grids (EMS CF400-Cu-UL) by levitation for 2 min.The grids were rinsed with three drops of Tris-buffered saline (TBS) (approximately 40 μL per drop, 1 min each), negatively stained with two successive drops of 1% uranyl acetate (UAT) with tylose, and then quickly aspirated. The grids were imaged on a Hitachi 7600 TEM (Tokyo, Japan) operating at 80 kV with an AMT XR80 CCD detector (8 megapixels) (Woburn, MA) (or a Philips CM120 (Cambridge, MA)). For stability testing, 500 mg / mL sulconazole samples were left on the benchtop (room temperature) or in a refrigerator (4 °C) for up to 112 days. To assess particle stability, particle size was measured periodically as described above.

[0091] 1.6.5 In vitro drug release Sul-NC (5 mg / mL, 50 μL) was placed in a rapid equilibrium dialysis (RED) device with an 8 kDa molecular weight cutoff (n = 3). The outer reservoir was filled with 1 mL of 0.5% Tween 80 in phosphate-buffered saline (PBS-T) solution. Samples were incubated on an orbital shaker with temperature control at 37 °C and 300 rpm. Every 24 h, 1 mL of the solution was withdrawn and replenished with 1 mL of fresh PBS-T solution. For free sulconazole, 50 μL of a solution containing sulconazole at the measured solubility limit (240 μg / mL) was added to the dialysis tubing and equilibrated with 1 mL of PBS-T solution in the outer reservoir (n = 3). To quantify the released sulconazole, the solution was transferred to an autosampler vial and analyzed by high-performance liquid chromatography (Prominence LC2030, Shimadzu, Columbia, MD). Separation was performed at room temperature using an isocratic flow on a Luna™ 5 μm C18(2) 100Å LC column, 250 × 4.6 mm (Phenomenex, Torrance, CA). Mobile phase A was water containing 0.1% trifluoroacetic acid (TFA), and mobile phase B was acetonitrile containing 0.1% TFA. The isocratic flow consisted of 70% mobile phase A and 30% mobile phase B at a flow rate of 1 mL / min for 10 min. The retention time of sulconazole was 1.8 min, with a λmax of 210 nm. A calibration curve for sulconazole was calculated using the area under the curve at RT = 1.8 min over the range of 0.5–50 μg / mL. The amount of drug was quantified and used to calculate the percentage accumulation. Release curves were plotted using GraphPad Prism 9 (San Diego, CA).

[0092] (1.6.6 Animal Welfare Statement) All animal experiments were approved and conducted in accordance with the guidelines of the Johns Hopkins University Animal Care and Use Committee. All procedures were performed in an AAALAC-accredited facility in compliance with the Guidelines for the Care and Use of Laboratory Animals and the Animal Welfare Act. C57BL / 6J male and female mice (5–8 weeks old) were obtained from Jackson Laboratory (Bar Harbor, ME). Yorkshire pigs (Sus scrofa domestica, female, 35–50 kg) were purchased from Archer Farms (Darlington, MD).

[0093] (1.6.7 Maximum Tolerated Dose (MTD) Study) Five- to eight-week-old male and female C57BL / 6J mice were subcutaneously injected with increasing doses of free drug or Sul-NC until visible toxicity and / or death were observed. Each treatment group contained 3 to 9 mice. Free sulconazole was dissolved in DMSO at 100 mg / mL and then diluted to 10 mg / mL with polyethylene glycol 300 (PEG300). Free sulconazole was injected by IP or SC injection at doses of 15 mg / kg, 30 mg / kg, 40 mg, and 50 mg / kg (Table 3). Sul-NC was administered subcutaneously (SC) at various concentrations and injection volumes at doses ranging from 250 to 2,500 mg / kg (Table 4). Mice were observed daily for up to 7 days after injection and were euthanized if they became obviously ill due to drug toxicity. The MTD was injected, and blood was collected for liver and kidney function tests (Table 5).

[0094] 1.6.8 Skin Fibrosis Model Male C57BL / 6J mice aged 5–8 weeks were used for the bleomycin-induced skin fibrosis model. The upper back was shaved and a specific 1 cm 2One hundred microliters of bleomycin (0.5 mg / ml in PBS) was injected subcutaneously every other day into one of five sectors within the area (see Figure 3). Bleomycin injections were administered periodically from positions 1 to 5 over a period of four weeks (Chakraborty et al., 2020). Mice receiving intestinal transplants were randomly divided into four groups for treatment, with each group containing three to five mice. Treatments were performed as follows: IP injection of 10 mg / kg free sulconazole in 50 μL of PEG300 every 2 days (14 total IP injections); SC injection of 50 μL (50 mg / kg) sulconazole every 7 days (4 total SC injections); SC injection of 50 μL (150 mg / kg) sulconazole every 7 days (4 total SC injections); or oral administration of pirfenidone (100 mg / kg in 100 μL of PEG300) once daily. The dose of free sulconazole was selected to minimize potential systemic toxicity while simultaneously providing repeated administration to offset rapid drug clearance. Sul-NC was injected only into sector 5. After 28 days, dermis was harvested, sectioned, and stained at the Johns Hopkins Reference Histology Laboratory. Five representative images were taken for each mouse to quantify dermal thickness. For each image, five different regions were measured by a blinded observer using Image J Fiji (NIH, Bethesda, MD), and data were analyzed using GraphPad Prism 9 (San Diego, CA, USA).

[0095] (1.6.9 Mouse intestinal transplant model) Male C57BL / 6J mice aged 5–8 weeks were used for the intestinal transplantation model with minor modifications (Figure 6). This model follows the same procedure as Meier et al., 2016. Briefly, a 6 cm segment of small intestine proximal to the ileocecal valve was excised from a donor mouse in a sterile biosafety cabinet. The tissue was washed three times with cold saline, carefully cut into 1 cm fragments, and placed in a Petri dish containing cold saline. The back of the neck of the recipient mouse was shaved. A small incision was made in the skin above the neck, and the donor intestinal fragment was placed in a subcutaneous pocket. The skin was then sutured with 3-0 nylon sutures. For infection prevention, the mice were administered cefazolin (300 mg / kg) intraperitoneally. The mice receiving the intestinal transplant were randomly divided into four groups for treatment, with each group containing 3–5 mice. Sul-NC was injected subcutaneously at 50 or 150 mg / kg (50 μL volume) near the transplanted tissue using an insulin injection needle. As a positive control, we used pirfenidone (100 mg / kg in 100 μL of PEG300, orally administered three times daily) at a dose previously shown to significantly reduce collagen deposition around donor tissue (Meier et al., 2016). Seven days later, the transplanted tissues were harvested for sectioning and Masson's Trichrome staining at the Johns Hopkins Reference Histology Laboratory. To quantify collagen thickness, four representative images were taken for each tissue. For each image, a masked observer measured 10 different regions using Image J Fiji (NIH, Bethesda, MD). Data were analyzed using GraphPad Prism 9 (San Diego, CA, USA).

[0096] (1.6.10 Quantitative RT-PCR (qRT-PCR)) RNA was extracted using TRIzol reagent as previously described (Li et al., 2017). RNA was reverse transcribed into cDNA using the ReverAid First Strand cDNA Synthesis Kit. The SYBR Green PCR Master Mix kit was used for real-time PCR using a QuantStudio 3 PCR machine (Applied Biosystems, Waltham, MA). Details of the primers used are listed in Table 6. Each reaction was run in triplicate for each primer set using a no-template negative control. QuantStudio Design and Analysis Software 1.5.2 was used to analyze the data using the ΔΔCT method.

[0097] [Table 5]

[0098] (1.6.11 Porcine esophageal stricture model) Argon plasma coagulation (APC) was performed as previously described (Li et al., 2021). Pigs were housed in the large animal facility at the Johns Hopkins University School of Medicine and fed a standard commercial pig diet with water available ad libitum. Pigs were acclimated for at least 1 week before treatment.

[0099] Esophagogastroduodenoscopy (EGD) was performed using a forward-looking single-channel upper gastrointestinal endoscope equipped with a videoscope system (EG-27i10 Video Gastroscope with 2.8 working channels, Standard HD+, PENTAX Medical, USA). APC was performed using an APC electrosurgical device equipped with an APC generator (VIO 300D) and an APC2 unit (ERBE, Tübingen, Germany). Briefly, a 2-cm section of the marginal mucosa was excised to create strictures at 30 cm, 40 cm, and 50 cm from the incisors (Li et al., 2021). Strictures were induced in a total of 11 pigs, but one pig died on day 12 due to esophageal stricture perforation at the 50 cm position. Veterinary staff monitored the pigs daily, and their weights were measured weekly. When the pigs could no longer tolerate solid food, they were given liquid feed (Ensure®). Buprenorphine and carprofen were administered to control pain and fever as directed by veterinary staff.

[0100] (1.6.12 Treatment of Esophageal Strictures with Local Sul-NC Injection via EGD) Fourteen days after APC treatment, pigs were sedated and underwent fluoroscopic EGD to assess stricture formation and measure luminal diameter. Of the 30 ablation areas in 10 pigs, 23 strictures were less than 6 mm and were included in further analysis. Pigs were randomly assigned to either a vehicle control group (n = 5) or a treatment group (n = 5). The 23 strictures included in the study were divided into 12 in the control group and 9 in the Sul-NC group. The strictures were then balloon-dilated to 10 mm and injected with vehicle (2% F127) or Sul-NC, as previously described in Li et al., 2021. In the first two pigs receiving Sul-NC (n = 3 strictures), 5 mL of a 500 mg / mL concentration was injected in four separate 1.25 mL increments around the periphery of the stricture (total dose of 150 mg / kg). This concentration proved difficult to pass through the endoscope needle, and the nanocrystals repeatedly clogged the needle. Furthermore, fluid leaked from the tissue immediately after injection. Therefore, various dilutions were evaluated in vitro to determine the maximum concentration that could be easily administered through an endoscope needle. Adding 2% F127 to the Sul-NC formulation to a dilution of 300 mg / mL eliminated the needle clogging effect. Therefore, the remaining three pigs (n = 6 strictures) received 3 mL of 300 mg / mL Sul-NC in 6 x 0.5 mL injections around each stricture site (total dose 54 mg / kg). The control group received 6 x 0.5 mL injections of 2% F127 around each stricture site. 14 days after treatment, the pigs were sedated, an endoscope was placed in the esophagus immediately adjacent to the stricture, Omnipaque 240 was injected through a catheter advanced through the endoscope, and fluoroscopic images were obtained to assess stricture re-formation. Fluoroscopy-guided EGD was then performed to assess stricture reformation internally and measure luminal diameter. Because some strictures were too narrow to allow EGD passage, the pigs were euthanized, the esophagus removed, and stricture diameters were measured ex vivo. Additionally, healthy tissue specimens were obtained from uninvolved areas of the esophagus. Tissue specimens were placed in 10% neutral-buffered formalin before paraffin embedding, sectioning (4 mm), and staining at the Johns Hopkins Reference Histology Laboratory.Slides were stained with hematoxylin and eosin (H&E), Masson's trichrome, or picrosirius red according to standard protocols.

[0101] (1.6.14 Statistical analysis) Data were presented as mean ± SD or mean ± SEM for each graphical representation. Graphs were generated using GraphPad Prism 9. A two-tailed Student's t-test (for comparisons of two groups) or a one-way analysis of variance (ANOVA) test (for comparisons of three or more groups) was used to determine statistical significance by P value. A P value of <0.05 or 0.01 was considered statistically significant.

[0102] (1.6.14 Abbreviations) gastrointestinal tract - GI, inflammatory bowel disease - IBD, Crohn's disease - CD, ulcerative colitis - UC, extracellular matrix - ECM, transforming growth factor - TGF-β, alpha-smooth muscle actin - α-SMA, sulconazole nanocrystals - sul-NC, EGD - esophagogastroduodenoscopy - EGD, argon plasma coagulation - APC, generally regarded as safe - GRAS, polyvinyl alcohol - PVA, hyaluronic acid - HA, carboxymethylcellulose - CMC, pluronic F127 - F127, transmission electron microscopy - TEM, maximum tolerated dose - MTD, intraperitoneal - IP, alanine aminotransferase - ALT, aspartate aminotransferase - AST, blood urea nitrogen - BUN, polydispersity index - PDI, trifluoroacetic acid - TFA, rapid equilibrium dialysis - RED [Example]

[0103] Formulated with sulconazole nanocrystals

[0104] (2.1 Method) Sul-NC was formulated using a wet bead-based milling method. Nanomilling was performed using a laboratory-scale tissue homogenizer (TissueLyser LT, Qiagen Inc., Germantown, MD). Various stabilizers were used in the formulation, including polyvinyl alcohol (PVA, 78 kDa, 88 mol% hydrolyzed), hyaluronic acid (HA, 1–2 MDa), hydroxypropyl methylcellulose (HPMC, 3,550 mPa·s), and Pluronic F127 (Coliphor P407, F127). The final formulation contained sulconazole (50–500 mg / mL according to the instructions), 2.0 g of 0.5 mm zirconium oxide beads, and 1 mL of stabilizer solution in a 2 mL Eppendorf tube. The contents were milled at 3,000 vibrations per minute for 10 hours in a refrigerated room at 4°C. The mixture was then passed through a 100 μm cell strainer to separate the milling beads. The particle size, polydispersity index (PDI), and surface charge (ζ-potential) of Sul-NC were measured using a Malvern Zetasizer Nano ZS (scattering angle 173°) (Malvern, Westboro, MA). For particle size and polydispersity index measurements, Sul-NC was diluted 1:100 with ultrapure water, and for ζ-potential measurements, Sul-NC was diluted 1:40 with 10 mM NaCl (pH 7). Particles formulated with HA, PVA, and HPMC were stored at 4°C during stability testing. To freeze-dry the particles, samples were processed neat or diluted 1:10 with water or 2% F127 as specified, then flash-frozen in liquid nitrogen and placed in a 750 mL glass freeze-drying flask (Flask No. 7542700, Labconco, Kansas City, MO) connected to a Labconco Freezone 4.5 Plus freeze-drying apparatus (Kansas City, MO). Samples were stored in the freeze-dryer at -74°C and 0.110 mBar for 24 hours. As previously mentioned, freeze-dried samples were reconstituted in water before being characterized for size, PDI, and zeta potential as described above.

[0105] (2.2 Results) Sul-NCs formulated at 500 mg / mL in 2% F127 were stored at room temperature or 4°C and the size, zeta potential, and PDI were measured over a period of 168 days (Figure 10). The particles showed good stability up to day 112, after which the zeta potential and PDI began to fluctuate, suggesting potential metastability (although the overall particle size remained relatively stable).

[0106] Alternatively, Sul-NCs were lyophilized either undiluted or diluted with either water or 2% F127 (w / w), then reconstituted in water. Samples diluted in water had smaller standard deviations in size and PDI than those diluted in F127, suggesting that dilution with water may improve stability. However, samples not diluted before lyophilization had the smallest overall size standard deviation, a PDI of approximately 0.3, and a nearly neutral zeta potential. This suggests that for best post-reconstitution stability, it may be optimal to not dilute the particles before lyophilization (Figure 11). Alternatively, the use of a cryoprotectant may further enhance particle stability throughout the lyophilization process.

[0107] Other excipients were also suitable for formulating sulconazole particles, but generally resulted in larger average particle sizes. Formulation of sulconazole with HA resulted in larger particles with a more negative zeta potential due to the anionic nature of HA (Figure 12). Higher HA concentrations resulted in larger particle size and greater instability, likely due to the use of higher molecular weight HA. Particle size can be reduced by using lower molecular weight HA as a stabilizer. Particles milled with 5% HPMC remained stable for up to 14 days of storage, and after that, the lower concentrations of sulconazole, 50 mg / mL and 100 mg / mL, appeared to be more stable than the 200 mg / mL formulation (Figure 13). Formulation of sulconazole with 5% PVA produced stable particles approximately 400 nm in size with a nearly neutral zeta potential for up to 14 days. Similarly, lower concentrations of sulconazole, 50 mg / mL and 100 mg / mL, showed some improvement in stability compared to 200 mg / mL sulconazole (Figure 14). [Example]

[0108] Preparation and characterization of sulconazole microcrystals (Sul-MC)

[0109] Sulconazole nitrate (25 mg / mL) was weighed and dissolved in acetonitrile. The suspension was heated at 75°C until completely dissolved, then cooled to room temperature and dried and recrystallized under vacuum for 24 hours. Sulconazole crystals were milled by wet bead milling using a TissueLyser (TissueLyser LT, Qiagen Inc., Germantown, MD) in the presence of 1.0 mm Zr beads at 25–40 OS / s for 5–30 minutes. Various stabilizers were screened to evaluate their effect on the size uniformity, ease of redispersion, sedimentation, and injectability through small-gauge needles of sulconazole microcrystals (Sul-MC). The stabilizers screened included carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), polyvinyl alcohol (PVA), hyaluronic acid (HA), and sodium cholate (CHA) as a small molecule surfactant at various concentrations and with various molecular weights (Tables 7 and 8). The mixture was then passed through a 100 μm cell strainer to separate the crushed beads. Particle size was measured using a Multisizer 4e Coulter Counter (Beckman Coulter, Indiana, USA), and particle size and crystalline structure were confirmed using a scanning electron microscope (SEM, MCP Thermo Scientific Helios G4 UC).

[0110] [Table 6]

[0111] Various cellulose derivatives (CMC, HPMC, and HEC) were evaluated as viscosity enhancers to stabilize various concentrations of Sul-MC using wet bead milling (Table 7). The resulting Sul-MC particles varied in size. While not wishing to be bound by any particular theory, it is believed that higher stabilizer concentrations result in smaller particle sizes of Sul-MC. This observation is likely due to the higher frictional forces exerted by beads in a more viscous dispersion medium. Despite being within the desired size range for injection through a small-gauge needle, Sul-MC milled in the presence of cellulose derivatives or PVA was not easily dispersible and formed a precipitate that could not easily pass through a small-gauge needle within 24 hours.

[0112] [Table 7]

[0113] HA was screened at various molecular weights as a stabilizer for Sul-MC during wet bead milling, with or without the low-molecular-weight surfactant CHA (Table 8). While high-molecular-weight HA (e.g., 2.5 mDa) yielded Sul-MC in an acceptable size range, the resulting dispersions were very difficult to pass through small-gauge (26 G) needles for injection. Furthermore, phase separation was observed after 24 hours of storage at 4°C.

[0114] Therefore, lower molecular weight HA (e.g., 100 kDa or 500 kDa) was tested at various concentrations. Increasing the HA concentration resulted in a smaller particle size of Sul-MC, likely due to increased viscosity. Similarly, adding CHA decreased the viscosity of the medium and increased Sul-MC particle size. By increasing the milling time (30 min) and vibration cycle (40 OS), stable Sul-MC formulations in the desired size range were observed. Furthermore, Sul-MC easily passed through a small-gauge needle for administration. Furthermore, the size and crystalline structure of these Sul-MC particles were also confirmed by SEM images. Importantly, the particle size of Sul-MC was reproducible across two replicates (R1 and R2), as shown in Table 8.

[0115] (References) All publications, patent applications, patents, and other references mentioned herein are indicative of the level of ordinary skill in the art to which the subject matter of this disclosure pertains. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference were specifically and individually indicated to be incorporated by reference. Although numerous patent applications, patents, and other references are referenced herein, it should be understood that such reference does not constitute an admission that any of these documents form part of the general knowledge in the art. [Prior art documents] [Non-patent literature]

[0116] [Non-Patent Document 1] Rieder F, Brenmoehl J, Leeb S, et al. Wound healing and fibrosis in intestinal disease. Gut 2007;56:130-9. [Non-patent document 2] Henderson NC, Rieder F, Wynn TA. Fibrosis: from mechanisms to medicines. Nature 2020;587:555–566.

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[0117] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will understand that certain changes and modifications may be practiced within the scope of the appended claims.

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