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 used for the treatment of vocal cord paralysis.

Background Art

[0002] The vocal folds (VF) play an important role in voice communication and glottal function, and unilateral vocal fold paralysis (UVFP) inhibits these functions. UVFP is said to occur three times more frequently than bilateral vocal fold paralysis. UVFP has a multifactorial etiology, and about 80% of cases are either idiopathic, secondary to cardiac and pulmonary diseases, or non-thyroid malignancies. Thyroidectomy is a major risk factor for UVFP and accounts for the remaining approximately 20% of cases (Spataro et al., 2014 Otolaryngol. Head Neck Surg., 151:286-293). In fact, thyroidectomy has almost doubled between 2005 and 2020 due to an increase in incidence and the implementation of early diagnosis of thyroid diseases, and in the United States, more than 10,000 patients require treatment for UVFP every year. Since the recurrent laryngeal nerve (RLN) runs closely related to the thyroid gland, it can be accidentally damaged or transected during thyroidectomy.

[0003] Injective medialization laryngoplasty (IL) is the first-choice treatment for UVFP and aims to restore glottal closure and phonation. Generally, a volume adjuster is injected into the thyroid cavity outside the VF for the purpose of bringing the paralyzed VF closer to the contralateral VF. This process is called "medial movement". The effect of medial movement is temporary and varies depending on the material used and the type of immune reaction. This technique has a history of over 100 years and was developed by Brunning in 1911 using paraffin. Since RLN reinnervation may occur 6 to 8 months after VFP, IL was initially performed as a temporary measure. Recent outcome data suggest that reinnervation may not be able to address 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 towards compounds that can be relatively easily injected during local anesthesia using small-bore needles, are well-tolerated, do not migrate, and require fewer follow-up visits (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 voice recovery. In some cases, it is desirable for the injectable agent to be absorbed quickly when there is a possibility of renervation, but in other cases, a longer-lasting material is preferable. Three compounds widely used in laryngeal ligaments (ILs) are calcium hydroxyapatite and its carboxymethylcellulose carrier, cross-linked hyaluronic acid (e.g., Restylane® brand), and micronized human cadaver acetolary dermis (MACD) (e.g., Cymetra® brand). In addition to Cymetra® and Restylane®, various other materials have been introduced to the market to restore vocal function and articulation in a balanced way. Examples include bovine gelatin (Gelfoam®), carboxymethylcellulose (Radiesse®), bovine collagen (Zyplast®, Zyderm®), calcium hydroxyapatite, fat, and fascia. Restylane® and Cymetra® are the most commonly used IL products, but their effects are relatively short-lived, requiring frequent visits to a laryngologist. Furthermore, because Cymetra® is prepared from human cadaver skin, it is prone to batch-to-batch variability, while bovine materials pose a risk of prion contamination.Furthermore, materials such as silk (Gulka et al, 2019 Laryngoscope, 129:1856-1862) and tissue-mimicking nanofibril hybrids (Latifi et al, 2018 Sci Rep., 8:1047) are also being considered for use in IL.

[0005] As described above, the glottal closure effect of currently used IL biomaterials is generally short-lived, requires frequent clinic visits, and results in inconsistent voice quality. Therefore, there is a need for an IL product that can be safely administered in the examination room, provides stable effects, offers long-term glottal closure (resulting in reduced need for follow-up visits), and restores proper vocalization. Accordingly, a biomaterial suitable for treating vocal cord paralysis caused by IL without exhibiting the aforementioned drawbacks would be particularly advantageous. [Overview of the project]

[0006] This invention relates to jellyfish collagen for use in the treatment of vocal cord paralysis. As is evident from the data presented below, the inventors have surprisingly found that compositions containing jellyfish collagen are useful in treating IL-induced vocal cord paralysis as an alternative to currently used biomaterials (e.g., cross-linked hyaluronic acid and micronized cell-free dermis from human cadavers), demonstrating superior results in internalization of vocal cord folds, as well as other superior properties (including minimal mass transfer in vivo, increased glottal benefits with minimal histopathology, 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, given the different physicochemical properties of jellyfish collagen compared to the biomaterials commonly used in the treatment of IL-induced vocal cord paralysis.

[0007] Therefore, 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 for treating vocal cord paralysis, the method comprising administering a composition containing jellyfish collagen to a person in need thereof.

[0009] A third aspect of the present invention relates to the use of jellyfish collagen for the manufacture of a pharmaceutical for the treatment of vocal cord paralysis. Hereinafter, embodiments of the present invention will be described illustratively with reference to the attached figures. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows a table summarizing the rabbit studies and main findings. In the table, the symbols generally mean the following: +=high confidence; + / -=medium confidence; -=low confidence; ADSC=adipocyte mesenchymal stem cells; IL=injection-intermediate laryngoplasty; VFP=vocal cord paralysis. The symbols in the "Histological Structure" column of the table have the following meanings: A=adipocyte infiltration, HN=histiocytic nodule (granuloma), LN=lymphocytic nodule, MA=muscle atrophy, MD / F=myocyte death / fibrosis. [Figure 2] Figure 2 shows the rheological characteristics of the injected materials. (A) The rheological characteristic of the MX-JC solution is that it exhibits non-Newtonian liquefaction behavior. (B) A 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 injected medial laryngoplasty (IL). Upper panel: MRI images correspond to the largest internalized ellipsoid for all 11 animals tested. The rabbit numbers listed in the table in Figure 1 are shown in each panel. Lower panel: Digital “slice” volumes for all animals, arranged from highest to lowest. The X-axis corresponds to the “number of slices,” and the Y-axis represents volume in microliters (μL). (A). Miniaturized 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). Miniaturized cell-free dermis (MACD) (Cymetra® brand). [Figure 4] Figure 4 shows MRI images and volume calculations 12 weeks after injection internal laryngoplasty (IL). Upper panel: MRI images correspond to the largest internalized ellipsoid for all 11 animals tested. The rabbit numbers listed in the table in Figure 1 are shown in each panel. Lower panel: Digital "slice" volumes for all animals are ordered from highest to lowest. The X-axis corresponds to the "number of slices," and the Y-axis represents volume in microliters (μL). (A). Miniaturized 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). Miniaturized cell-free dermis (MACD) (Cymetra® brand). [Figure 5] Figure 5 shows the percentile rank analysis results for ellipsoid volumes at (A) 4 weeks after IL and (B) 12 weeks after IL. The ellipsoid volumes were ranked from highest to lowest and assigned a rank on a scale from 100 to 1. Rank data was 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 the histological analysis of tissue sections. Representative slides from each group at 4 weeks and 12 weeks post-IL are shown at 10x and 100x magnification (R# = rabbit number). [Modes for carrying out the invention]

[0011] In the following detailed description, numerous specific examples are described in detail to provide a complete understanding of the present invention. Those skilled in the art will understand that embodiments of the present invention can still be carried out without the specific detailed description, while remaining within the scope of the claims.

[0012] In a first embodiment, the present invention provides a composition for use in the treatment of vocal cord paralysis, the composition containing jellyfish collagen.

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

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

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

[0016] "Vocal cord paralysis" refers to damage to one or both of the recurrent laryngeal nerves (RLNs), resulting in the disruption of 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 (viral and bacterial infections, etc.), endocrine disorders (thyroid disorders, etc.), systemic neurological disorders, and certain cancers.

[0017] In some embodiments, the treatment of vocal cord paralysis is performed by injection medialization laryngoplasty (IL).

[0018] "Injection medialization laryngoplasty (IL)" means injecting a filler into the paraglottic space located outside the paralyzed vocal cord for the purpose of bringing it closer to 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 atelocollagen form. "Ateloform" means a low-immunogenic derivative of collagen obtained by removing the N-terminal and C-terminal telopeptide components known to induce antigenicity in humans. Telopeptides are generally removed by treating collagen with type I pepsin.

[0020] In some embodiments, the jellyfish collagen is in a telogenized form. "Telogenized form" means collagen that is extracted under acidic conditions and produces soluble collagen containing telopeptides.

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

[0022] In some embodiments, the jellyfish collagen is methacrylated. "Methacrylate" means collagen to which a methacrylic acid group has 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 bonded via a covalent bond.

[0024] Any crosslinking agent known to crosslink under conditions in which collagen fibrils are formed may be a suitable crosslinking agent for use in the present invention. For example, the jellyfish collagen of the present invention can be crosslinked using a crosslinking agent such as EDC, genipin, 1,4-BDDGE, polyethylene glycol (PEG), or mucochloroic 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 can be from the subphylum Chordata. In some examples, the raw materials for jellyfish collagen can be selected from a group consisting of the order Rhopilema, including the jellyfish Rhopilema esculentum, the genus Rhopilema nomadica, Stomolophus meleagris, Cassiopeia (upside-down jellyfish), including Cassiopeia andromeda, the order Semaostomase, including the genus Aurelia, Nemopilema nomurai, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, or other species such as combinations thereof. Preferably, the raw material for jellyfish collagen is the jellyfish *Crocidolomia japonica*. Therefore, the collagen may consist 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%, and at least 92%. Or, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% *Crocidolomia japonica* collagen.

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

[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 particle sizes 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, and 375 μm to 400 μm.

[0028] In some embodiments, the jellyfish 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 mg / mL 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] The jellyfish collagen-containing composition according to the present invention may further contain adipose-derived mesenchymal stem cells (ADSCs) in some embodiments. Protocols for the preparation of ADSCs are well known in the art and can be followed as a routine matter 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] The jellyfish collagen composition according to the present invention may further contain pharmaceutically acceptable excipients and / or carriers, as well as a pharmaceutically active ingredient. The excipients and carriers may or may not contain the active substance and can enhance the stability of the pharmaceutically active ingredient or jellyfish collagen and / or improve the biopharmaceutical profile. Examples of suitable pharmaceutically acceptable excipients and carriers include sterile water, lidocaine, olive oil, monosaccharides such as ethyl oleate, glycol, fructose, glucose, and galactose; non-reducing disaccharides such as sucrose, lactose, and trehalose; non-reducing oligosaccharides such as raffinose and melegitose; non-reducing starch-derived polysaccharide products such as maltodextrin, dextran, and cyclodextrin; and non-reducing alditols such as mannitol and xylitol. Further suitable excipients include cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, and / or polyvinylpyrrolidone. A mixture of two or more of the above excipients or carriers (or other suitable equivalents) is also conceivable. Other substances with similar effects are understood to be suitable as well.

[0031] There are several methods for "isolating" or "purifying" jellyfish collagen from its anatomical environment. Many of these are well-known and routine to those skilled in the art. For example, one method for purifying collagen from jellyfish is acid extraction, which involves immersing different anatomical parts of the jellyfish in an acidic solution. "Bathing" or "bathed" refers to the process of incubating the jellyfish in an acidic solution for a sufficient amount of time to release collagen molecules. An alternative method of collagen purification is enzymatic extraction, in which the jellyfish is incubated with at least one proteolytic enzyme for a sufficient amount of time under conditions favorable to the degradation of the anatomical environment in order to release collagen molecules. The precise temperature, pH, and incubation time for enzymatic extraction vary depending on the proteolytic enzyme used. The most suitable conditions should be well-known to those skilled in the art. As a non-limiting example, collagen molecules can be released by incubating the enzyme pepsin with jellyfish under acidic conditions. It is assumed that any enzyme can be used in enzymatic extraction, and the examples above are not intended to be limiting.

[0032] Subsequently, collagen can be further separated or purified from undesirable contaminants of the acid or enzymatic extraction method by 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 the purification of collagen molecules from other soluble contaminants of the extraction process. The exact method of further purification is not particularly limited. Any method well known and routinely used by protein biochemists can be adapted for the purpose of obtaining purified or isolated jellyfish collagen. This step also makes it possible to transfer the jellyfish collagen to a desired storage buffer to obtain a desired solution of purified jellyfish collagen. This can be achieved by first equilibrating the chromatographic apparatus with the desired storage buffer before purification. There are many alternative well-known methods that can be used for this purpose. Preferably, the collagen used in this invention has a purity of 70% to 99%, where purity means weight % representing the proportion of collagen molecules in the solution. More preferably, the collagen solution has a purity of at least 95%, 96%, 97%, 98%, or 99%. [Examples]

[0033] Next, the present invention will be further described with reference to the following examples and discussions. Example 1: Study design and animals material and method Following protocol approval by the Institutional Animal Care and Use Committee (IACUC A4201), a total of 24 three-week-old female New Zealand white rabbits (average weight upon arrival: 3.12 ± 0.18 kg) were divided into four groups of N=6 each based on a power calculation using previous findings (1-β=80%, p<0.05) (Oldenburg et al, 2018. Laryngoscope, 128(1):160-167).

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

[0035] The protocol is 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 acclimatized two weeks prior to the RLN resection and fat harvesting surgeries, which were performed sequentially. Anesthesia was administered intramuscularly with ketamine 42 mg / kg, xylazine 6 mg / kg, and acepromazine 1.2 mg / kg, maintained with 1-2% isoflurane. Buprenorphine 0.18 mg / kg and carprofen 1.5 mg / kg were used to control postoperative pain. The left RLN was located by blunt dissection along the inferior thyroid artery, and a 1 cm section was excised. A fat fragment approximately 1.5 cm in diameter was harvested from the annular thyroid gland and placed in sterile saline. Only the fat from group 1 was further processed for ADSC inflation and injection. Two weeks later, the animals were anesthetized again for IL, the larynx was exposed, and image-guided endoscopy was used to visualize needle position and injection delivery. IL was performed with a 23-gauge needle, ensuring the material reached the lateral aspect of the vocal process of the thyroid cartilage. 100 μL was injected into each animal using a 1 mL syringe. MX-JC (225 mg / mL) was reconstituted with pH-buffered saline as a dry powder at the time of injection, and then warmed to 37°C to facilitate mixing. In Group 1, the reconstituted MX-JC was 1X10 before injection. 6ADSC and 5 ng / mL of TGF-β2 were mixed and administered to the same animals from which the samples were collected. Cymetra® (275 mg / mL) and Restylane® (20 mg / mL) were injected according to the manufacturer's specifications. Note-taking and video recording were performed simultaneously for both the surgery and IL to permanently preserve details such as the degree of RLN paralysis, needle position, net amount of injected substance, and complications that occurred during surgery or postoperative recovery. The animals' weight was measured upon arrival, postoperatively, and once a week thereafter, and their overall health was observed.

[0036] ADSCs were prepared as previously described (Oldenburg et al, 2018 Laryngoscope, 128(1):160-167). Briefly, adipose tissue was finely dissected 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 isolated from the adipose layer by incubation at 37°C for 1.5 hours with occasional shaking, followed by centrifugation at 500 g for 5 minutes. The pellets were washed with phosphate-buffered saline, sieved 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. The pellets were incubated overnight at 37°C, 5% CO2, and 95% humidity. The following day, non-adherent material was removed and fresh medium was added. Medium changes were performed every 2-3 days. Cells were subculturified at a 1:2 ratio with 60-80% confluence. Three subculturinated cells were cryopreserved and freshly grown for 24-48 hours before intracellular feeding. Only rabbits in Group 1 received a total of 1 x 10⁴ cells. 6 These ADSCs were injected together with MX-JC.

[0037] result Animal data are summarized in the table in Figure 1. One animal in Group 4 died under anesthesia and was not replaced. There were no deaths from surgical procedures, postoperative care, or protocol complications in the other animals. The success rate of the experiment was determined by analyzing video and time-lapse notes. In RLN surgery, paralysis of the left VF was observed in 21 out of 23 animals (~91%). One animal showed partial VFP, and one did not show VFP (Group 3). Injections of 100 μL into the left thymic cavity were classified as "high confidence," and others as "medium to low confidence." Approximately 70% of all injections were "high confidence." Injections exceeding 100 μL, material spillage, and deviation from the injection site accounted for 22% and 8%, respectively ("medium to low confidence"). Due to the difficulty of simultaneous injection of ADSC and MX-JC, 5 animals (83%) in Group 1 received IL injections with "medium to low confidence." Regardless of the treatment, the rabbits ate normally during the experiment and gained weight as expected (data not shown).

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

[0039] result MX-JC with an average particle size of 300 μm was homogenized in phosphate-buffered saline using two interconnected syringe assemblies. To produce an injectable with the desired viscosity, concentrations ranging from 60 to 300 mg / ml were tested for tactile consistency and rheological properties. Overall, with increasing concentration, viscosity tended to decrease asymptotically as the shear rate increased (Figure 2A). MX-JC, Restylane®, and Cymetra® exhibited non-Newtonian liquid thinning behavior. The deformation curves for Cymetra® (275 mg / mL) and MX-JC (225 mg / mL) were nearly parallel, while the curve for Restylane® (20 mg / mL) showed the least viscosity reduction (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 materials obtained from the animals described in Example 1. NMR experiments were conducted using an Avance III 300 MHz (7T) wide-bore NMR spectrometer equipped with micro-imaging accessories (Bruker, BioSpin, Billerica, MA) and a 20 mm diameter volume coil. After removing the specimen from the fixative, it was lightly dried with tissue paper, transferred to a 20 mm tube, and securely fixed in the center of the tube with a custom-made Teflon holder. The tube was filled with Fluorinert FC-770 (3M, St. Paul, Minnesota), a perfluorinated, proton-free solvent that helps improve the uniformity of the magnetic field around the sample but does not contribute to background signaling. Images were acquired when the core temperature of the gradient coil was between 21°C and 25°C. A RARE (Rapid Acquisition with Refocused Echoes) sequence was used to visualize the specimens, with the following parameters set. a) Repetition time: 4,000 ms. b) Echo duration: 10.37 ms. c) Rare factor: 12; d) FOV: 16cm x 16cm; 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] The slice protocol and the 3D protocol were compared to determine the maximum feature resolution. The slice protocol involved creating 26 0.5 mm digital sections of the larynx, starting from the cricothyroid cartilage and moving upwards. An initial short scan (3-5 slices) was performed to identify the start and end positions of the intermediate material, after which a "digital box" containing the bulking material was constructed and sliced ​​as described above. In the 3D protocol, 400 images were acquired overnight at 0.005 mm intervals.

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

number

[0043] result For each animal, 26 larynx MRI images were recorded at 0.5 mm intervals and moved upward starting from the cricoid cartilage landmark. The images show uninjected tissue located above and below the injection site, as well as the inwardly 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, indicating a decrease of approximately 37% (p=0.002), which is likely due to absorption over time. The absorption rate differed depending on the material (see the table in Figure 1 and the volume data calculations described below). The highest absorption rate was in group 3 (58%), followed by group 4 (47%), group 1 (31%), and finally group 2 (29%). The volume (μL) was calculated by multiplying the area of ​​the ellipsoid calculated in Analyze or ImageJ by the thickness of the digital "slice" (0.5 mm) (Figures 3 and 4). The MRI images show the size and location of the largest ellipsoid for each animal sacrificed at week 4 (Figures 3A, 3B, 3C, 3D, upper panel) and week 12 (Figures 4A, 4B, 4C, and 4D, upper panel). The volume data for each animal is displayed in descending order. The X-axis represents the number of images, and the Y-axis represents the volume (μL) (Figures 3 and 4, lower panel).

[0044] Regarding the ellipsoids, Group 1 had 31 at week 4 and 23 at week 12 (N=54), Group 2 had 41 at week 4 and 18 at week 12 (N=59), Group 3 had 33 at week 4 and 24 at week 12 (N=57), and Group 4 had 27 at week 4 and 26 at week 12 (N=52). When the data from 4 weeks and 12 weeks were folded, the amount of material remaining after IL (mean ± SD) 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 of Group 2 was statistically significantly higher than that of Groups 13 and 4 (p<0.0009). To further compare the differences in ellipsoid size across all groups, in relation to the characteristics of the biomaterial and the response of the laryngeal tissue, all ellipsoids were ranked in ascending order of volume and shown on a scale from 100 (highest) to 1 (lowest). The data were analyzed using a one-way ANOVA based on the Kruskal-Wallis rank (p=0.0004) and the Mann-Whitney U test for comparison of group differences. Rank data at 4 and 12 weeks post-IL are shown in Figure 5. The ellipsoid volume in Group 2 was consistently larger than that of the other three groups.

[0045] The above results demonstrate that MX-JC has a longer residence time in the thyroid cavity than MX-JC co-injected with Restylane®, Cymetra®, or ADSCs. Specifically, MX-JC was found to last longer than Cymetra® and Restylane® at both 4 weeks and 12 weeks after IL administration (Cymetra® was more than 40% superior, and Restylane® was more than 100% superior).

[0046] Example 4: Histological analysis material and method Histological analysis was performed on materials obtained from the animals described in Example 1. After fixing with 4% paraformaldehyde for a minimum of 72 hours, laryngeal blocks were embedded in paraffin to prepare sections. Initially, three blocks of the same size were cut. Up to 72 5 μm tissue sections were placed on Corning microscope slides from each block, 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, the slides were deparaffinized, stained with Harris hematoxylin solution for 10 minutes, and then washed with tap water for 5 minutes. The samples were differentiated with 1% acidic alcohol for 1-5 seconds, rinsed with tap water for 1 minute, and then blued with 0.2% ammonia water or saturated lithium carbonate aqueous solution for 3 minutes. After rinsing with tap water slides for 5 minutes and immersing in 95% alcohol 10 times, the samples were counterstained with eosin solution for 1 minute, and dehydrated by alternating between 95% ethanol once and 100% alcohol twice for 5 minutes. Finally, the samples were mounted on xylene medium.

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

[0048] MX-JC was stained with H&E and appeared as a distinct reticular substance surrounded by a layer of inflammatory cells. This was most pronounced in Group 2 at 4 weeks post-IL, while relatively little inflammatory infiltration was observed in Group 1. By 12 weeks, the inflammatory response in Group 2 had subsided considerably. Restylane® appeared as a bluish crystalline substance, as previously reported (Zeitels et al, 2019 Ann. Otol. Rhinol. Laryngol., 128(3_suppl):71S-81S), and was easily identifiable at 4 weeks, but appeared to become difficult to identify at 12 weeks due to absorption. This material contained relatively few surrounding inflammatory cells. Cymetra® was similar in appearance to MX-JC and presented as a distinct nodular shape that was easily identifiable at both 4 and 12 weeks.

[0049] To identify the nature of local tissue changes and the inflammatory response to the injected material, veterinary pathologists examined the slides, and their observations were summarized in the table in Figure 1. Firstly, some degree of muscle atrophy secondary to VF denervation was observed in all groups. Secondly, adipocyte infiltration was observed in all groups, not just group 1, but it was not possible to distinguish between the injected ADSCs and proliferating local tissue adipocytes. Thirdly, in group 3, widespread myocyte death and fibrosis associated with Restylane® injection were observed. And fourthly, two different types of inflammatory nodules were observed with MX-JC and Cymetra. In the former case, the material was surrounded by histiocytes and showed a T-cell inflammatory response, while in the latter case, the nodules were mainly composed of plasma cells showing a B-cell immune response.

[0050] Previous studies (Oldenburg et al, 2017 Laryngoscope, 127(5):E166-E169) showed that administration of ADSCs with Cymetra® resulted in a lymphocyte-dominant inflammatory response. These findings were confirmed in the results of this study (see table in Figure 1). Furthermore, the immunological responses observed previously were of relatively similar intensity in both the Cymetra®-only group and the Cymetra® + ADSC group (Oldenburg et al, 2017 Laryngoscope, 127(5):E166-E169), indicating that the lymphocyte inflammatory response is not weakened by growth factors or cytokines that have traditionally been associated with the immunosuppressive effects of ADSCs. In contrast, in this study, MX-JC induced a T-cell-mediated immune response characterized by dendritic cell and macrophage infiltration that was significantly downregulated by co-injection with ADSCs (compare the histological data of Group 1 and Group 2 in Figure 6). The T-cell-mediated response induced in Group 2 also differed from that observed in Group 3, lacking the clear features of tissue destruction (excluding the effects of denervation) at weeks 4 and 12, and significantly decreased 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. The pharmaceutical composition described in 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 subphylum Chordata.

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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