Uses of jellyfish collagen

Abstract

The present invention relates to jellyfish collagen for use in the treatment of vocal cord paralysis.

Description

[Technical Field]

[0001] The present invention relates to jellyfish collagen for use in the treatment of vocal cord paralysis. [Background technology]

[0002] The vocal cords (VF) play a vital role in speech communication and glottal function, and unilateral true vocal cord paralysis (UVFP) disrupts these functions. UVFP is reportedly three times more common than bilateral vocal cord paralysis. UVFP has a multifactorial etiology, with approximately 80% of cases being either idiopathic, secondary to cardiac or pulmonary disease, or nonthyroid malignancy. Thyroidectomy is the major risk factor for UVFP, accounting for the remaining approximately 20% of cases (Spataro et al., 2014 Otolaryngol. Head Neck Surg., 151:286-293). In fact, thyroidectomies nearly doubled between 2005 and 2020 due to increasing incidence and earlier diagnosis of thyroid disease, with more than 10,000 patients in the United States requiring treatment for UVFP each year. The recurrent laryngeal nerve (RLN) is closely associated with the thyroid gland and can be accidentally injured or severed during thyroidectomy.

[0003] Injection medial laryngoplasty (IL) is the first-line treatment for UVFP, with the goal of restoring glottic closure and speech. Typically, a volume-adjusting material is injected into the thyroid cavity lateral to the VF to bring the paralyzed VF closer to the contralateral VF. This process is called "medialization." The effect of medialization is temporary and varies depending on the material used and the type of immune response. This technique has a history of over 100 years and was developed by Bruning in 1911 using paraffin. Because reinnervation of the RLN can occur 6–8 months after VFP, IL was initially performed as a temporary measure. Recent outcome data suggest that reinnervation may not be able to address the new challenges associated with microneurosurgery when compared with IL (Siu et al., 2016 Laryngoscope, 126:1616–1624).

[0004] Over the years, the field of biomaterials has gradually shifted toward compounds that are relatively easy to inject during local anesthesia using small-bore needles, are well tolerated, do not migrate, and require fewer consultations (Eppley and Dadvand, 2006 Plast. Reconstr. Surg., 118:98e-106e). Furthermore, many variations of the Bruening protocol and numerous biomaterials have been reported (Li et al., 2016 Biomaterials, 108:91-110; Mallur and Rosen, 2010 Clin. Exp. Otorhinolaryngol., 3:177-182). Each biomaterial aims to achieve an optimal balance between ease and convenience of injection, duration of laryngeal closure, and degree of speech recovery. In some cases, rapid absorption of the injectable agent is desirable for potential reinnervation, while in other cases, a more durable material is desirable. Three commonly used compounds for IL are calcium hydroxylapatite and its carboxymethylcellulose carrier, cross-linked hyaluronic acid (e.g., Restylane® brand), and human cadaveric micronized acetal dermis (MACD) (e.g., Cymetra® brand). In addition to Cymetra® and Restylane®, various materials have been introduced to the market to restore balanced glottal capacity and speech production. These include bovine gelatin (Gelfoam®), carboxymethylcellulose (Radiesse®), bovine collagen (Zyplast®, Zyderm®), calcium hydroxylapatite, fat, and fascia. Restylane® and Cymetra® are the most commonly used IL products, but their effectiveness is relatively short-lived, requiring frequent visits to the laryngology office. Additionally, Cymetra® is prepared from human cadaver skin, making it prone to problems of batch-to-batch variability, while bovine material poses the risk of prion contamination.Materials such as silk (Gulka et al, 2019 Laryngoscope, 129:1856-1862) and tissue-mimetic nanofibril hybrids (Latifi et al, 2018 Sci Rep., 8:1047) are also being explored for use in ILs.

[0005] As noted above, the glottal closure effects of currently available IL biomaterials are generally short-lived, require multiple clinic visits, and result in variable voice quality. Therefore, there is a need for IL products that can be safely administered in the office, provide consistent efficacy, provide long-term glottal closure (thus reducing the need for follow-up visits), and restore proper speech. Therefore, biomaterials suitable for use in the treatment of vocal cord paralysis with IL, without exhibiting the drawbacks described above, would be particularly advantageous. Summary of the Invention

[0006] The present invention relates to jellyfish collagen for use in the treatment of vocal cord paralysis. As evidenced by the data presented below, the inventors have surprisingly discovered that compositions containing jellyfish collagen are useful for the treatment of vocal cord paralysis with ILs as an alternative to currently used biomaterials (e.g., cross-linked hyaluronic acid and micronized acellular dermis from human cadavers), while demonstrating superior results in terms of vocal fold medialization, as well as other superior properties, including minimal in vivo mass transfer, increased glottal benefit with minimal tissue pathology, low immunogenicity, tissue regeneration benefits (e.g., angiogenesis), and a lower risk of viral transfer and / or disease / prion transmission. This was a completely unexpected discovery due to the different physicochemical properties of jellyfish collagen compared to common biomaterials used in the treatment of vocal cord paralysis with ILs.

[0007] Thus, a first aspect of the present invention relates to a composition for use in the treatment of vocal cord paralysis, the composition comprising jellyfish collagen.

[0008] A second aspect of the present invention relates to a method of treating vocal cord paralysis, the method comprising administering to a subject in need thereof a composition comprising jellyfish collagen.

[0009] A third aspect of the present invention relates to the use of jellyfish collagen for the manufacture of a medicament for the treatment of vocal cord paralysis. Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: FIG. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows a table summarizing the rabbit studies and key findings. In the table, the symbols generally mean the following: + = high confidence; + / - = medium confidence; - = low confidence; ADSC = adipose mesenchymal stem cells; IL = injection medialization laryngoplasty; VFP = vocal fold paralysis. The symbols for "tissue structure" in the table mean the following: A = adipose infiltration, HN = histiocytic nodule (granuloma), LN = lymphocytic nodule, MA = muscle atrophy, MD / F = muscle cell death / fibrosis. [Figure 2] Figure 2 shows the rheological characteristics of the injected materials. (A) The rheological characteristics of the MX-JC solution show non-Newtonian liquefaction behavior. (B) Comparison of the rheological characteristics of Cymetra® (275 mg / mL), MX-JC (225 mg / mL), and Restylane® (20 mg / mL). [Figure 3]Figure 3 shows MRI images and volume calculations 4 weeks after injection medial laryngoplasty (IL). Upper panel: MRI images correspond to the largest medialized ellipsoid for all 11 animals tested. The rabbit numbers listed in the table in Figure 1 are indicated in each panel. Lower panel: Digital "slice" volumes for all animals, sorted from highest to lowest. The X-axis corresponds to "slice number," and the Y-axis represents volume in microliters (μL). (A) Micronized cross-linked jellyfish collagen (MX-JC) + adipose-derived mesenchymal stem cells (ADSCs). (B) MX-JC only. (C) Cross-linked hyaluronic acid (X-HA) (Restylane® brand). (D) Micronized acellular dermis (MACD) (Cymetra® brand). [Figure 4] Figure 4 shows MRI images and volume calculations 12 weeks after injection medial laryngoplasty (IL). Upper panel: MRI images correspond to the largest medialized ellipsoid for all 11 animals tested. The rabbit numbers listed in the table in Figure 1 are indicated in each panel. Lower panel: Digital "slice" volumes for all animals are ordered from highest to lowest. The X-axis corresponds to "slice number," and the Y-axis represents volume in microliters (μL). (A) Micronized cross-linked jellyfish collagen (MX-JC) + adipose-derived mesenchymal stem cells (ADSCs). (B) MX-JC only. (C) Cross-linked hyaluronic acid (X-HA) (Restylane® brand). (D) Micronized acellular dermis (MACD) (Cymetra® brand). [Figure 5] Figure 5 shows the percentile rank analysis of ellipsoid volume at (A) 4 weeks and (B) 12 weeks after IL. Ellipsoid volumes were ranked from highest to lowest on a scale of 100 to 1. Rank data were extracted for each group, and statistical significance was determined using the Kruskal-Wallis H-test (p<0.001). Individual group differences were compared using the Mann-Whitney U-test. [Figure 6]Figure 6 shows histological analysis of tissue sections. Representative slides from each group at 4 weeks post-IL and 12 weeks post-IL are shown at 10x and 100x magnification (R# = rabbit number). DETAILED DESCRIPTION OF THE INVENTION

[0011] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. Those skilled in the art will understand that embodiments of the present invention may be practiced without the specific details and still remain within the scope of the claims.

[0012] In a first aspect, the present invention provides a composition for use in treating vocal cord paralysis, the composition comprising jellyfish collagen.

[0013] In a second aspect, the present invention provides a method of treating vocal cord paralysis, the method comprising administering to a subject in need thereof a composition comprising jellyfish collagen.

[0014] In a third aspect, the present invention provides the use of jellyfish collagen for the manufacture of a medicament for the treatment of vocal cord paralysis.

[0015] In some cases, vocal cord paralysis is unilateral true vocal cord paralysis (UVFP) or bilateral vocal cord paralysis (BVFP). In a preferred embodiment, the vocal cord paralysis is unilateral true vocal cord paralysis (UVFP).

[0016] "Vocal cord paralysis" refers to damage to one or both recurrent laryngeal nerves (RLNs), disrupting nerve impulses to the laryngeal muscles. When one RLN is paralyzed, it is called unilateral true vocal cord paralysis (UVFP), and when both RLNs are paralyzed, it is called bilateral vocal cord paralysis (BVFP). Causes of vocal cord paralysis include, but are not limited to, head and neck trauma, nerve damage during surgery, congenital disorders, infections (e.g., viral and bacterial infections), endocrine disorders (e.g., thyroid disease), systemic neurological disorders, and certain cancers.

[0017] In some embodiments, vocal cord paralysis is treated by injection medialization laryngoplasty (IL).

[0018] "Injection medialization laryngoplasty (IL)" refers to the injection of a filler material into the thyroarytenoid space at a location lateral to the paralyzed vocal cord with the aim of approximating the contralateral vocal cord. Such a procedure aims to improve the patient's voice and glottal closure.

[0019] In some embodiments, the jellyfish collagen is in an ateloform. "Ateloform" refers to a less immunogenic derivative of collagen obtained by removing the N- and C-terminal telopeptide components known to induce antigenicity in humans. The telopeptides are typically removed by treating the collagen with type I pepsin.

[0020] In some embodiments, the jellyfish collagen is in a telopeptide form, which includes collagen extracted under acidic conditions to produce soluble collagen containing telopeptides.

[0021] In some embodiments, the jellyfish collagen is thiolated. The term "thiolated" is intended to refer to jellyfish collagen that has been reacted with a thiol to introduce an -SH group, i.e., a "thiol" group.

[0022] In some embodiments, the jellyfish collagen is methacrylated. By "methacrylate" we include the meaning of collagen to which methacrylic acid groups have been added to produce collagen methacrylamide.

[0023] In a preferred embodiment, the jellyfish collagen is cross-linked. In the context of the present invention, the term "cross-linked" means that two independent collagen molecules are joined together via a covalent bond.

[0024] Any crosslinking agent known to crosslink under conditions that result in collagen fibril formation is considered suitable for use in the present invention. For example, the jellyfish collagen of the present invention can be crosslinked using crosslinkers such as EDC, genipin, 1,4-BDDGE, polyethylene glycol (PEG), or mucochloric acid. Preferably, the crosslinking agent is EDC. The concentration of EDC may be 0.01% to 5%, 0.05% to 5%, 0.1% to 5%, 0.2% to 5%, 0.3% to 5%, 0.4% to 5%, 0.5% to 5%, 0.6% to 5%, 0.7% to 5%, 0.8% to 5%, 0.9% to 5%, 1% to 5%, 1.5% to 5%, 2% to 5%, 3% to 5%, 3.5% to 5%, 4% to 5%, or 4.5% to 5%. Preferably, the concentration of EDC is 0.5% to 1%.

[0025] The raw materials for jellyfish collagen are: ScyphozoaIn some embodiments, the source of jellyfish collagen can be selected from the group consisting of the order Rhopilema, including Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, and the order Cassiopeia (upside-down jellyfish), including Cassiopeia andromeda, the order Semaostomoides, including Aurelia, and other species such as Nemopilema nomurai, Rhopilema esculentum, Rhopilema nomadica, and Stomolophus meleagris, or combinations thereof. Preferably, the source of jellyfish collagen is Rhopilema esculentum. Thus, the collagen may be composed of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% multicellular jellyfish collagen.

[0026] Jellyfish can be formulated as a powder, such as a micronized powder, a hydrogel, a paste, a membrane, a scaffold, a solution, a sponge matrix, a nanofiber electrospun matrix, or in lyophilized form. A "hydrogel" is a network of hydrophilic polymer chains that results in a highly absorbent material. The term "paste" is intended to refer to a semi-solid formulation. In a preferred embodiment, the jellyfish collagen is in the form of a fine powder.

[0027] In some embodiments, the jellyfish collagen has a particle size of 1 μm to 1,000 μm, preferably 100 μm to 500 μm, and more preferably 200 μm to 400 μm. The jellyfish collagen can have a particle size of 50 μm to 950 μm, 75 μm to 900 μm, 100 μm to 850 μm, 125 μm to 800 μm, 150 μm to 750 μm, 175 μm to 700 μm, 200 μm to 650 μm, 225 μm to 600 μm, 25 μm to 550 μm, 275 μm to 500 μm, 300 μm to 475 μm, 325 μm to 450 μm, 350 μm to 425 μm, or 375 μm to 400 μm.

[0028] In some embodiments, the jellyfish collagen is at a concentration of 1 to 500 mg / mL, preferably 50 to 400 mg / mL, more preferably 100 to 300 mg / mL, even more preferably 200 to 300 mg / mL, and most preferably 200 to 250 mg / mL. The jellyfish collagen is at a concentration of 25 mg / mL to 475 mg / mL, 50 mg / mL to 450 mg / mL, 75 mg / mL to 425 mg / mL, 100 mg / mL to 400 mg / mL, 125 mg / mL to 375 mg / mL, 150 mg / mL to 350 mg / mL, 175 mg / mL to 325 mg / mL, 200 mg / mL to 300 mg / mL, 225 mg / mL to 275 mg / mL, or 200 mg / mL to 250 mg / mL.

[0029] In some embodiments, compositions containing jellyfish collagen according to the present invention can further contain adipose-derived mesenchymal stem cells (ADSCs). Protocols for the preparation of ADSCs are well known in the art and can be routinely followed by those skilled in the art. For example, a method for preparing ADSCs is described in Oldenburg et al., 2018, Laryngoscope, 128(1):160-167.

[0030] Compositions containing jellyfish collagen according to the present invention can further comprise pharmaceutically acceptable excipients and / or carriers, as well as pharmaceutically active ingredients. The excipients and carriers, with or without the active ingredient bound thereto, can enhance the stability and / or biopharmaceutical profile of the pharmaceutically active ingredient or jellyfish collagen. Examples of suitable pharmaceutically acceptable excipients and carriers include sterile water, lidocaine, olive oil, ethyl oleate, glycol, monosaccharides such as fructose, glucose, and galactose; non-reducing disaccharides such as sucrose, lactose, and trehalose; non-reducing oligosaccharides such as raffinose and melezitose; non-reducing starch-derived polysaccharide products such as maltodextrin, dextran, and cyclodextrin; and non-reducing alditols such as mannitol and xylitol. Additional suitable excipients include cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, and / or polyvinylpyrrolidone. Mixtures of two or more of any of the above excipients or carriers (or other suitable equivalents) are also contemplated. It is understood that other substances with similar effects may also be suitable.

[0031] There are multiple methods for "isolating" or "purifying" jellyfish collagen from its anatomical environment. Many of these will be well known and routine to those skilled in the art. For example, to purify collagen from jellyfish, there is the acid extraction method, in which different anatomical regions of the jellyfish are immersed in an acidic solution. "Bathing" or "bathed" refers to the process of incubating the jellyfish in an acid solution for a sufficient time to liberate the collagen molecules. An alternative method of collagen purification is enzymatic extraction, whereby the jellyfish is incubated with at least one proteolytic enzyme under conditions favoring the degradation of the anatomical environment for a sufficient time to liberate the collagen molecules. The exact temperature, pH, and incubation time for the enzymatic extraction method will vary depending on the proteolytic enzyme used. The most suitable conditions will be familiar to those skilled in the art. As a non-limiting example, the enzyme pepsin can be incubated with jellyfish under acidic conditions to liberate the collagen molecules. It is contemplated that any enzyme can be used in the enzymatic extraction method, and the above examples are not intended to be limiting in any way.

[0032] The collagen can then be further separated or purified from undesirable contaminants of the acid or enzyme extraction process using several different methods. For example, insoluble contaminants can be removed by centrifugation. If a purer collagen source is required, the isolated collagen can be subjected to gel filtration or alternative chromatographic techniques that allow for purification of the collagen molecules relative to other soluble contaminants of the extraction process. The exact method of further purification is not particularly limited. Methods familiar and routinely used by protein biochemists can be adapted for the purpose of obtaining purified or isolated jellyfish collagen. This step can also transfer the jellyfish collagen into a desired storage buffer to obtain the desired solution of purified jellyfish collagen. This can be achieved by first equilibrating the chromatography equipment with the desired storage buffer prior to purification. There are many alternative, well-known methods that can be used for this purpose. Preferably, the collagen used in the present invention is 70% to 99% pure, where purity refers to the percentage by weight of collagen molecules in solution. More preferably, the collagen solution has a purity of at least 95%, 96%, 97%, 98%, or 99%. [Example]

[0033] The invention will now be further described with reference to the following examples and discussion. Example 1: Study Design and Animals material and method After protocol approval by the Institutional Animal Care and Use Committee (IACUC A4201), a total of 24 3-week-old female New Zealand White rabbits (mean weight at arrival: 3.12 ± 0.18 kg) were divided into four groups of 6 rabbits each based on a power calculation (1-β = 80%, p < 0.05) using previous findings (Oldenburg et al, 2018. Laryngoscope, 128(1):160-167).

[0034] The four test groups are: Group 1: Rabbits administered finely powdered cross-linked jellyfish collagen (MX-JC) and adipose-derived mesenchymal stem cells (ADSCs). Group 2: A rabbit administered MX-JC. Group 3: Rabbits administered cross-linked hyaluronic acid (X-HA) (Restylane®). Group 4: Rabbits receiving micronized dermis (MACD) (Cymetra®).

[0035] The protocol has been described in detail elsewhere (Oldenburg et al., 2018 Laryngoscope, 128(1):160-167; Oldenburg et al., 2017 Laryngoscope, 127(5):E166-E169). Briefly, rabbits were acclimated for 2 weeks prior to surgery for RLN resection and fat harvest, which were performed sequentially. Anesthesia was achieved with intramuscular administration of ketamine 42 mg / kg, xylazine 6 mg / kg, and acepromazine 1.2 mg / kg, maintained with 1-2% isoflurane. Postoperative pain was controlled with buprenorphine 0.18 mg / kg and carprofen 1.5 mg / kg. The left RLN was located by blunt dissection along the inferior thyroid artery, and a 1 cm section was resected. A fat piece approximately 1.5 cm in diameter was harvested from the cricothyroid region and placed in sterile saline. Only the fat in Group 1 was further processed for ADSC expansion and injection. After two weeks, the animals were anesthetized again for intravenous injection, the larynx was exposed, and image-guided endoscopy was used to visualize needle placement and injection delivery. Intravenous injection was performed with a 23-gauge needle, ensuring that the material reached the lateral side of the vocal cord process of the thyroid cartilage. For each animal, 100 μL was injected using a 1 mL syringe. MX-JC (225 mg / mL) was reconstituted as a dry powder in pH-buffered saline and warmed to 37°C for easy mixing. In Group 1, reconstituted MX-JC was administered at a concentration of 1x10 mL before injection. 6The ADSCs were mixed with 5 ng / mL of TGF-β2 and administered to the same animals from which they were harvested. Cymetra® (275 mg / mL) and Restylane® (20 mg / mL) were injected according to the manufacturer's specifications. Simultaneous notebook and video recordings of both the surgery and IL were performed to permanently record details such as the degree of RLN paralysis, needle position, net volume of injected material, and any complications that occurred during surgery or postoperative recovery. Animals were weighed upon arrival, postoperatively, and weekly thereafter, and their overall health was monitored.

[0036] ADSCs were prepared as previously described (Oldenburg et al., 2018 Laryngoscope, 128(1):160-167). Briefly, adipose tissue was minced with a scalpel and digested with 3–5 mL of 0.15% collagenase type 1 solution (C0130-1G; Sigma, St. Louis, MO) in Advanced MEM® medium (A-MEM, Life Sciences) containing 10% fetal bovine serum (FBS), 1% GlutaMAX, and 1 mg / mL penicillin / streptomycin solution. Cells were separated from the adipose layer by incubation at 37°C for 1.5 h with occasional shaking, followed by centrifugation at 500 g for 5 min. The pellet was washed with phosphate-buffered saline, passed through a 70 μm sieve to remove large particles, reconstituted by centrifugation at 500 g for 5 minutes, and finally resuspended in 5-10 mL of A-MEM medium and incubated overnight at 37°C, 5% CO2, and 95% humidity. The next day, non-adherent material was removed and fresh medium was added. Medium changes were performed every 2-3 days. Cells were passaged 1:2 at 60-80% confluence. Passage 3 cells were cryopreserved and newly expanded for 24-48 hours before IL-1. A total of 1x10 6 ADSCs were injected together with MX-JC.

[0037] result The animal data are summarized in the table in Figure 1. One animal in Group 4 died from anesthesia and was not replaced. No other animals died due to surgical procedures, postoperative care, or protocol complications. Experimental success was determined by analysis of the video and contemporaneous notes. During RLN surgery, left VF paralysis was observed in 21 of 23 animals (~91%). One animal showed partial VFP, and one animal showed no VFP (Group 3). Injections of 100 μL into the left thymic cavity were classified as "high confidence," while others were classified as "medium / low confidence." Approximately 70% of all injections were classified as "high confidence." Injections exceeding 100 μL, overflow of material, and deviation from the injection site accounted for 22% and 8%, respectively ("medium / low confidence"). Due to difficulties in simultaneous injection of ADSCs and MX-JC, five animals (83%) in Group 1 underwent IL injections with "medium / low confidence." Regardless of treatment, rabbits ate normally throughout the experiment and gained weight as expected (data not shown).

[0038] Example 2: Viscosity and Rheology Testing material and method To understand the behavior of the materials during injection, the rheological properties of the three compounds were evaluated using a viscoelasticity measuring device (DHR-1 Discovery Hybrid Rheometer (TA Instruments, New Castle, DE, USA)) equipped with a 40 mm parallel Peltier plate geometry. Dynamic viscoelasticity was measured as a function of frequency in the linear viscoelastic region using 1.0 mL samples. Test temperature = 39°C, immersion time = 0 s, shear rate = 0.1–500 / s. -1 , Maximum equilibration time = 60 s, Sampling time = 30 s.

[0039] result MX-JC with an average particle size of 300 μm was homogenized in phosphate-buffered saline using two interconnected syringe assemblies. To generate injectables of the desired viscosity, concentrations ranging from 60 to 300 mg / ml were tested for tactile consistency and rheological properties. Overall, there was a trend for viscosity to asymptotically decrease at higher shear rates with increasing concentration (Figure 2A). )。 MX-JC, Restylane®, and Cymetra® exhibited non-Newtonian liquid thinning behavior. The deformation curves of Cymetra® (275 mg / mL) and MX-JC (225 mg / mL) were nearly parallel, while the curve of Restylane® (20 mg / mL) was the least resistant to thinning (Figure 2B). This rheological analysis confirmed that MX-JC and Cymetra® have similar liquid thickening properties.

[0040] Example 3: MRI analysis material and method MRI analysis was performed on material obtained from the animals described in Example 1. NMR experiments were performed using an Avance III 300 MHz (7T) wide-bore NMR spectrometer equipped with a microimaging accessory (Bruker, BioSpin, Billerica, MA) and a 20 mm diameter volume coil. After removal from fixative, specimens were gently dried with tissue paper, transferred to 20 mm tubes, and firmly secured to the center of the tube with a custom-made Teflon holder. The tubes were filled with Fluorinert FC-770 (3M, St. Paul, MN), a perfluorinated, proton-free solvent that helps improve magnetic field homogeneity around the sample but does not contribute to background signal. Images were acquired when the gradient coil core temperature was between 21°C and 25°C. To visualize the specimens, a Rapid Acquisition with Refocused Echoes (RARE) sequence was used, with the following parameters: a) Repetition time: 4,000 ms. b) Echo time: 10.37 ms. c) Rare factors: 12; d)FOV:16cmX16cm; e) Matrix: 160X160; f) slice thickness: 0.5 mm; g) In-plane resolution: 100um / pxl; h) slice thickness: 0.5 mm; and i) Acquisition time: 10 minutes 24 seconds.

[0041] A slicing protocol was compared with a 3D protocol to determine maximum feature resolution. The slicing protocol involved creating 26 0.5 mm digital sections of the larynx from the cricothyroid cartilage upward. An initial short scan (3–5 slices) was performed to identify the beginning and end locations of the medialized material, after which a "digital box" containing the medialized material was constructed and sliced ​​as described above. With the 3D protocol, 400 images were acquired overnight at 0.005 mm intervals.

[0042] statistical analysis The group size for 80% statistical power and p ≤ 0.05 was determined as follows:

number

[0043] result For each animal, 26 laryngeal MRI images were recorded at 0.5 mm intervals, moving upward from the cricoid cartilage landmark. The images show uninjected tissue above and below the injection site, as well as the medialized ellipsoid caused by the injected material. Overall, 46% of the MRI images at 4 weeks and 29% of the MRI images at 12 weeks showed the injected material, a decrease of approximately 37% (p=0.002), likely due to absorption over time. Absorption rates varied by material (see the table in Figure 1 and the volumetric data calculations below). Group 3 had the highest absorption rate (58%), followed by Group 4 (47%), Group 1 (31%), and finally Group 2 (29%). The area of ​​the ellipsoid calculated using Analyze or ImageJ was multiplied by the thickness of the digital "slice" (0.5 mm) to calculate the volume (μL) (Figures 3 and 4). MRI images show the size and location of the largest ellipsoid for each animal sacrificed at 4 weeks (Figures 3A, 3B, 3C, and 3D, upper rows) and 12 weeks (Figures 4A, 4B, 4C, and 4D, upper panels). Volume data for each animal are displayed in descending order. The X-axis represents the image number, and the Y-axis represents the volume (µL) (Figures 3 and 4, lower panels).

[0044] For ellipsoids, Group 1 had 31 ellipsoids at week 4 and 23 at week 12 (N = 54), Group 2 had 41 ellipsoids at week 4 and 18 at week 12 (N = 59), Group 3 had 33 ellipsoids at week 4 and 24 at week 12 (N = 57), and Group 4 had 27 ellipsoids at week 4 and 26 at week 12 (N = 52). When collapsing the 4- and 12-week data, the mean (±SD) volume of material remaining after IL was as follows: Group 1, 2.72 ± 1.34 μL; Group 2, 4.06 ± 2.13 μL; Group 3, 1.88 ± 1.25 μL; and Group 4, 2.49 ± 1.44 μL. The mean volume in Group 2 was statistically significantly higher than that in Groups 13 and 4 (p < 0.0009). To further compare the differences in ellipsoid size across all groups, while relating them to biomaterial characteristics and laryngeal tissue response, all ellipsoids were ranked in order of volume, expressed on a scale of 100 (highest) to 1 (lowest). Data were analyzed by Kruskal-Wallis rank-based one-way analysis (p = 0.0004) and Mann-Whitney U test to compare differences between groups. Figure 5 shows the rank data for 4 and 12 weeks after IL. The ellipsoid volume in Group 2 was consistently larger than that of the remaining three groups.

[0045] These results indicate that MX-JC has a longer retention time in the thyroid cavity than Restylane®, Cymetra®, or MX-JC co-injected with ADSCs. Specifically, MX-JC outlasted Cymetra® and Restylane® at both 4 and 12 weeks after IL (Cymetra® was 40% longer, and Restylane® was 100% longer).

[0046] Example 4: Histological analysis material and method Histological analysis was performed on materials obtained from animals described in Example 1. After fixation in 4% paraformaldehyde for a minimum of 72 hours, laryngeal blocks were embedded in paraffin and sectioned. Three equal-sized blocks were initially cut. Up to 72 5-μm tissue sections from each block were placed on Corning slides, ensuring that all three blocks were visible on each slide. Hematoxylin and eosin staining was performed as previously described (Oldenburg et al., 2017 Laryngoscope, 127(5):E166-E169; Voss et al., 2018 Laryngoscope, 128(12):E402-E408). Briefly, slides were deparaffinized and stained with Harris hematoxylin solution for 10 minutes, followed by 5-minute washes in tap water. 1 After differentiation in 1% acidic alcohol for 1-5 seconds and rinsing in tap water for 1 minute, the specimens were blued in 0.2% aqueous ammonia or saturated lithium carbonate solution for 3 minutes. After rinsing in tap water for 5 minutes and dipping in 95% alcohol 10 times, the specimens were counterstained in eosin solution for 1 minute and dehydrated in 95% ethanol once and 100% alcohol twice for 5 minutes. Finally, the specimens were mounted in xylene medium.

[0047] result Representative histological data are shown in Figure 6 and summarized in the table in Figure 1 .

[0048] MX-JC stained with H&E and showed a distinct mesh-like material surrounded by a layer of inflammatory cells. This was most prominent in Group 2 at 4 weeks after IL, while Group 1 showed relatively little inflammatory infiltrate. By 12 weeks, the inflammatory response in Group 2 had subsided considerably. Restylane® appeared as a bluish, crystalline material, as previously reported (Zeitels et al., 2019 Ann. Otol. Rhinol. Laryngol., 128(3_suppl):71S-81S), which was easily identifiable at 4 weeks but appeared less identifiable due to absorption at 12 weeks. This material was relatively free of surrounding inflammatory cells. Cymetra® was similar in appearance to MX-JC, with a clearly defined, easily identifiable nodular shape at both 4 and 12 weeks.

[0049] To identify the nature of local tissue changes and inflammatory responses to the injected material, a veterinary pathologist examined the slides. The pathologist's observations are summarized in the table in Figure 1. First, some degree of muscle atrophy secondary to VF denervation was observed in all groups. Second, adipocyte infiltration was observed in all groups, not just Group 1, but was not distinguishable between injected ADSCs and proliferating local tissue adipocytes. Third, in Group 3, extensive myocyte death and fibrosis were observed following Restylane® injection. Fourth, two distinct types of inflammatory nodules were observed with MX-JC and Cymetra. In the former, the material was surrounded by histiocytes, indicating a T-cell inflammatory response, whereas in the latter, the nodules were primarily composed of plasma cells, indicating a B-cell immune response.

[0050] In a previous study (Oldenburg et al., 2017 Laryngoscope, 127(5):E166-E169), administration of ADSCs with Cymetra® resulted in a lymphocyte-dominated inflammatory response. These findings were confirmed by the results of this study (see table in Figure 1). Furthermore, the immunological responses previously observed were relatively similar in intensity in both the Cymetra® alone and Cymetra® + ADSC groups (Oldenburg et al., 2017 Laryngoscope, 127(5):E166-E169), indicating that the lymphocyte-dominated inflammatory response was not attenuated by growth factors and cytokines that have traditionally been associated with the immunosuppressive effects of ADSCs. In contrast, in the present study, MX-JC induced a T cell-mediated immune response characterized by dendritic cell and macrophage infiltration that was significantly downregulated by co-injection of ADSCs (compare the histological data for Groups 1 and 2 in Figure 6). The T cell-mediated response induced in Group 2 also differed from that observed in Group 3, with no obvious features of tissue destruction (except for the effects of denervation) at weeks 4 and 12, and was significantly reduced by week 12.

Claims

1. 1. A pharmaceutical composition for use in the treatment of vocal cord paralysis, the composition comprising jellyfish collagen.

2. 2. The pharmaceutical composition of claim 1, wherein the vocal cord paralysis is unilateral true vocal cord paralysis (UVFP) or bilateral vocal cord paralysis (BVFP).

3. 2. The pharmaceutical composition of claim 1, wherein the vocal cord paralysis is unilateral true vocal cord paralysis (UVFP).

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the treatment of vocal cord paralysis is by injection medialization laryngoplasty (IL).

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the jellyfish collagen is in its ateloform.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the jellyfish collagen is in its telomorphic form.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the jellyfish collagen is cross-linked.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the jellyfish collagen is thiolated.

9. 9. The pharmaceutical composition of claim 1, wherein the jellyfish collagen is methacrylated.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the source of the jellyfish collagen is from the class Scyphozoa.

11. 11. The pharmaceutical composition according to claim 1, wherein the source of the jellyfish collagen is selected from the group consisting of Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, Aurelia sp., Cassiopea andromeda, Nemopilema nomurai, or any combination thereof.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the jellyfish collagen is in the form of a fine powder.

13. 13. The pharmaceutical composition of claim 12, wherein the fine powder has a particle size of 1 μm to 1,000 μm, or a particle size of 100 μm to 500 μm, or a particle size of 200 μm to 400 μm.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein the jellyfish collagen is at a concentration of 1 to 500 mg / mL, or at a concentration of 50 to 400 mg / mL, or at a concentration of 100 to 300 mg / mL, or at a concentration of 200 to 300 mg / mL, or at a concentration of 200 mg / mL to 250 mg / mL.

15. The pharmaceutical composition according to any one of claims 1 to 14, further comprising adipose-derived mesenchymal stem cells (ADSCs).

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the composition further comprises a pharmaceutically acceptable excipient and / or carrier, and / or a pharmaceutically active ingredient.

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