Uses of jellyfish collagen

Abstract

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

Description

Technology Field

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

[0002] The vocal folds (VF) play a crucial role in voice communication and glottal function, and unilateral true VF paralysis (UVFP) disrupts these functions. UVFP is known to be three times more widespread than bilateral vocal fold paralysis. UVFP exhibits a multifactorial etiology, with approximately 80% of cases involving idiopathic, secondary heart and lung diseases, or non-thyroid malignancies. Thyroidectomy is a major risk factor for UVFP, accounting for the remaining approximately 20% of cases (Reference [Spataro et al, 2014. Otolaryngol. Head Neck Surg., 151:286-293]). Indeed, due to the increased incidence and early diagnosis of thyroid disease, thyroidectomy rates nearly doubled from 2005 to 2020, and more than 10,000 patients annually in the United States require treatment for UVFP. Because the recurrent laryngeal nerve (RLN) is closely connected to the thyroid gland, accidental injury or transection may occur during thyroidectomy.

[0003] Injection medialization laryngoplasty (IL) is the primary treatment for UVFP and aims to restore glottal closure and phonation. Generally, a volume-enhancing substance is injected into the thyroarytenoid space on the lateral side of the paralyzed vulva and brought closer to the contralateral vulva. This process is referred to as mediation. The results of mediation are transient and depend on the substance used and the type of immune response. This procedure has a history of over 100 years and was pioneered by Bruening in 1911 using paraffin. Because synergistic RLN resternation can occur within 6 to 8 months after VFP, IL was initially performed as a temporary measure. More recent outcome data suggest that resternation may not warrant additional problems associated with microsurgery when compared to IL (Reference [Siu et al, 2016. Laryngoscope, 126:1616-1624]).

[0004] Over the years, the field of biomaterials has progressively shifted toward compounds that can be injected relatively easily with smaller internal diameter needles under local anesthesia, are tolerable, do not migrate, and require fewer clinic visits (Reference [Eppley and Dadvand, 2006. Plast. Reconstr. Surg., 118:98e-106e]). Additionally, various variations of the Bruening protocol and various biomaterials have been described (Reference [Li et al, 2016. Biomaterials, 108:91-110; Mallur and Rosen, 2010. Clin. Exp. Otorhinolaryngol., 3:177-182]). Each biomaterial aims to strike an optimal balance between ease and convenience of injection, duration of laryngeal closure, and the degree of recovered phonation. In some cases, rapid reabsorption of the injectable agent may be desirable when there is a possibility of re-nerve distribution, while in others, a longer-acting material may be preferred. Three widely used compounds in ILs are calcium hydroxyapatite and its carboxymethylcellulose carriers, cross-linked hyaluronic acid (e.g., Restylane® brands), and undifferentiated human cadaveric acellular dermis (MACD) (e.g., Cymetra® brands). In addition to Cymetra® and Restylane®, various materials have been introduced to the market that attempt to balance the restoration of glottal function with vocalization. Examples include bovine gelatin (Gelfoam®), carboxymethylcellulose (Radiesse®), bovine collagen (Zyplast® and Zyderm®), calcium-hydroxyapatite, fat, and fascia. While Restylane® and Cymetra® are the most commonly used IL products, their relatively short shelf life necessitates multiple visits to the laryngology clinic. Furthermore, since Cymetra® is manufactured from human cadaver skin, it is prone to batch-to-batch variability, and bovine materials pose a risk of prion contamination.Silk (Gulka et al, 2019. Laryngoscope, 129:1856-1862) and tissue-mimicking nanofiber hybrids (Latifi. et al , 2018. Sci Rep. Other materials such as , 8:1047]) were also considered for use in IL.

[0005] As mentioned above, the glottal closure benefits of currently used IL biomaterials are generally short-lived, and because the quality of phonation also changes, multiple visits to the clinic are required. Thus, there is a need for an IL product that can be safely administered in a doctor's office, yields consistent results, provides long-term glottal closure benefits (thus reducing the need for repeated medical visits), and restores proper phonation. Therefore, a biomaterial suitable for the treatment of IL-induced vocal cord paralysis without exhibiting the aforementioned disadvantages would be particularly advantageous.

[0006] The present 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 discovered that a composition containing jellyfish collagen is useful for the treatment of IL-induced vocal cord paralysis as an alternative to currently used biomaterials (e.g., cross-linked hyaluronic acid and human cadaver undifferentiated acellular dermis) and exhibits not only excellent results in terms of vocal cord mediation, but also other excellent features including minimal in vivo material migration, increased glottal benefits with minimal tissue pathology, low immunogenicity, tissue regenerative benefits (e.g., angiogenesis), and reduced risk of virus delivery and / or disease / prion transmission. This is a completely unexpected finding considering the other physicochemical properties of jellyfish collagen compared to conventional biomaterials used in the treatment of IL-induced vocal cord paralysis.

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

[0008] A second aspect of the present invention relates to a method for treating vocal cord paralysis, wherein the method comprises the step of administering a composition containing jellyfish collagen to a subject requiring treatment.

[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. Brief explanation of the drawing

[0010] Embodiments of the present invention will now be described merely as examples with reference to the accompanying drawings: Fig. 1 ... shows a table summarizing the rabbit study and key results. The "general keys" of the table are as follows: + = High reliability; + / - = Medium confidence level; - = Low reliability; ADSC = Adipose mesenchymal stem cells; IL = Injection-mediated laryngoplasty; VFP = Vocal cord paralysis. The "Histological Key" in the table is as follows: A = Adipocyte infiltration; HN = Histiocytic nodule (granuloma); LN = Lymphocytic nodules; MA = Muscle atrophy; MD / F = Myoclonus death / fibrosis. Fig. 2 It shows the rheological characteristics of the injected material. (A) Rheological characteristics of MX-JC solution exhibiting non-Newtonian, liquid dilution behavior. (B) Comparison of rheological properties of Cymetra® (275 mg / mL), MX-JC (225 mg / mL) and Restylane® (20 mg / mL). Fig. 3 This shows MRI images and volume calculations 4 weeks after injection-mediated laryngoplasty (IL). upper panel: MRI images corresponding to the largest parameterized ellipsoid for all 11 animals tested. The rabbit numbers listed in the table of Fig. 1 are indicated on each panel. lower panel : The counted "slice" volumes for all animals are displayed in order from highest to lowest. The X-axis corresponds to the "slice number" and the y-axis represents the volume in microliters (μL). (A) . Undifferentiated cross-linked jellyfish collagen (MX-JC) + adipose-derived mesenchymal stem cells (ADSC). (B) . MX-JC Standalone. (C) . Cross-linked hyaluronic acid (X-HA) (Restylane® brand). (D) . Undifferentiated acellular dermis (MACD) (Cymetra® brand). Fig. 4 This shows MRI images and volume calculations 12 weeks after injection-mediated laryngoplasty (IL). upper panel : MRI images corresponding to the largest parameterized ellipsoid for all 11 animals tested. The rabbit numbers listed in the table of Figure 1 are indicated on each panel. lower panel : The counted "slice" volumes for all animals are displayed in order from highest to lowest. The X-axis corresponds to the "slice number" and the y-axis represents the volume in microliters (μL). (A) . Undifferentiated cross-linked jellyfish collagen (MX-JC) + adipose-derived mesenchymal stem cells (ADSC). (B) . MX-JC Standalone. (C). Cross-linked hyaluronic acid (X-HA) (Restylane® brand). (D) . Undifferentiated acellular dermis (MACD) (Cymetra® brand). Fig. 5 Is (A) IL after 4 weeks and (B)This shows the percentile grade analysis of oval volume after 12 weeks of IL. Oval volumes were ordered from highest to lowest and graded on a scale of 100 to 1. Grade 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. Fig. 6 This shows the histological analysis of tissue sections. Representative slides of each group at 4 weeks and 12 weeks after IL are displayed using 10X and 100X magnification (R# = rabbit number). Specific details for implementing the invention

[0011] In the following detailed description, a number of specific details are provided to provide a complete understanding of the invention. Those skilled in the art will understand that embodiments of the invention may be practiced without these specific details while remaining within the scope of the claims.

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

[0013] In a second aspect, the present invention provides a method for treating vocal cord paralysis, said method comprising the step of administering a composition containing jellyfish collagen to a subject requiring treatment.

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

[0015] In some embodiments, the 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 of the recurrent laryngeal nerves (RLN) that causes a disruption of nerve impulses to the laryngeal muscles. Paralysis of one RLN is referred to as unilateral true vocal cord paralysis (UVFP), and paralysis of both RLNs is referred to as bilateral vocal cord paralysis (BVFP). Possible causes of vocal cord paralysis include, but are not limited to, general trauma to the head and neck region, nerve damage during surgery, congenital conditions, infectious causes (e.g., viral or bacterial infections), endocrine diseases (e.g., thyroid disease), systemic nervous system diseases, and certain cancers.

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

[0018] "Injection-mediated laryngoplasty (IL)" involves injecting filler material into the thyroid anterior space at a lateral position of the paralyzed vocal cord to bring it closer to the opposite vocal cord. This procedure aims to improve the patient's voice and glottal closure.

[0019] In some embodiments, jellyfish collagen is its ateloform. The term "ateloform" includes the meaning of a low-immunogenic derivative of collagen obtained by removing N- 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, jellyfish collagen is its teloform. "Teloform" includes the meaning of collagen extracted under acidic conditions that produce soluble collagen containing telopeptides.

[0021] In some embodiments, jellyfish collagen is thiolized. The term 'thiolized' refers to jellyfish collagen into which -SH groups or 'thiol' groups are introduced by reacting with thiols.

[0022] In some embodiments, jellyfish collagen is methacrylated. "Methacrylated" includes collagen to which methacrylate groups have been added to produce collagen methacrylamide.

[0023] In a preferred embodiment, jellyfish collagen is cross-linked. In the context of the invention, the term 'cross-linking' refers to the connection of two independent collagen molecules through a covalent bond.

[0024] Any cross-linking agent known to cross-link under conditions in which collagen microfibrils are formed is considered to be a suitable cross-linking agent for use in the present invention. For example, the jellyfish collagen of the present invention may be cross-linked using cross-linking agents such as EDC, Genipin, 1,4-BDDGE, polyethylene glycol (PEG), and mucochloric acid. Preferably, the cross-linking agent is EDC. The EDC may be present at a concentration of 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 source of jellyfish collagen may be derived from the subphylum Scyphozoa. In some embodiments, the source of jellyfish collagen is Rhizostoma pulmo (but is not limited to the following): Rhizostomas pulmo ), Lofilema Esculentum( Rhopilema esculentum ), Lofilema Nomadica( Rhopilema nomadica ), Stomolopus meleagris( Stomolophus meleagrisThe order Rhizostomeae, including ), and Cassiopeia andromeda ( Cassiopea andromeda Cassiopeia species (upside-down jellyfish), including ) and Aurelia species ( Aurelia sp. ), and other species, such as Nemophilema nomurai ( Nemopilema nomurai ), Lofilema Esculentum( Rhopilema esculentum ), Lofilema Nomadica( Rhopilema nomadica ), Stomolopus meleagris( Stomolophus meleagris It may be selected from the group consisting of the order Semaeostomeases, including ), or any combination thereof. Preferably, the source of jellyfish collagen is Rhizostomas pulmo. Accordingly, the collagen may comprise 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%, and at least 99% Rhizostomas pulmo collagen.

[0026] Jellyfish can be formulated in powder or freeze-dried forms such as micronized powder, hydrogel, paste, membrane, scaffold, solution, sponge matrix, and nanofiber electrospun matrix. A 'hydrogel' is a highly absorbent material consisting of a network of hydrophilic polymer chains. The term 'paste' refers to a semi-solid formulation. In a preferred embodiment, jellyfish collagen is in the form of micronized powder.

[0027] In some embodiments, the jellyfish collagen has a particle size of 1 μm to 1000 μm, preferably 100 μm to 500 μm, and more preferably 200 μm to 400 μm. Jellyfish collagen may 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, much more preferably 200 to 300 mg / mL, and most preferably 200 mg / mL to 250 mg / mL. Jellyfish collagen may be 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 composition containing jellyfish collagen according to the present invention may further include adipose-derived mesenchymal stem cells (ADSCs) in some embodiments. Protocols for the preparation of ADSCs are well known in the art and can be performed by those skilled in the art as a matter of course. For example, a method for preparing ADSCs is described in the literature [Oldenburg et al, 2018. Laryngoscope, 128(1): 160-167].

[0030] A composition comprising jellyfish collagen according to the present invention may further comprise pharmaceutically acceptable excipients and / or carriers, and / or pharmaceutically active ingredients. The excipients and carriers may improve the stability of the pharmaceutically active ingredient, which may or may not have a conjugated active substance, or the jellyfish collagen, and / or improve its biopharmaceutical properties. Examples of suitable pharmaceutically acceptable excipients and carriers may include monosaccharides such as sterile water, lidocaine, olive oil, ethyl oleate, glycol, fructose, glucose, and galactose; non-reducing disaccharides such as sucrose, lactose, and trehalose; non-reducing oligosaccharides such as raffinose and melesitose; 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 the above excipients or carriers (or any other suitable equivalents) are also considered. Any other material having a similar effect is also understood to be suitable.

[0031] There are various methods for 'isolating' or 'purifying' jellyfish collagen from its anatomical environment. Many of these will be well known and common to those skilled in the art. For example, collagen can be purified from jellyfish by acid extraction, in which various anatomical parts of the jellyfish are immersed in an acidic solution. 'Immersion' or 'immersed' refers to the process of incubating the jellyfish in an acidic solution for a sufficient amount of time to release collagen molecules. Another 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 for the degradation of the anatomical environment to release collagen molecules. The exact temperature, pH, and incubation time of the enzymatic extraction method depend on the proteolytic enzyme used. The most suitable conditions will be well known to those skilled in the art. As a non-limiting example, the enzyme pepsin may be incubated with the jellyfish under acidic conditions to release collagen molecules. It is considered that any enzyme may be used in the enzymatic extraction method, and the above examples are not limited in any way.

[0032] Subsequently, the collagen may be further isolated or purified from impure contaminants of the acid or enzymatic extraction method by a number of different means. For example, insoluble contaminants may be removed by centrifugation. If a purer collagen source is required, the isolated collagen may be gel-filtered, or alternative chromatographic methods may be used to purify the collagen molecules from other soluble contaminants of the extraction process. The exact method of further purification is not particularly limited. Any method that is well known and commonly used by protein biochemists may be adopted to obtain purified or isolated jellyfish collagen. This step may also enable the transfer of jellyfish collagen into a suitable storage buffer to obtain a suitable solution of purified jellyfish collagen. This can be achieved by first equilibrating the chromatographic apparatus with a suitable storage buffer prior to purification. There are many well-known alternative methods that can be used for this purpose. Preferably, the collagen used in the present invention has a purity of 70% to 99%, where purity refers to the weight percentage of the solution attributable to collagen molecules. More preferably, the collagen solution has a purity of at least 95%, 96%, 97%, 98%, or 99%.

[0033] The present invention is now further described with reference to the following examples and studies.

[0034] Examples

[0035] Example 1: Study Design and Animals

[0036] Materials and Methods

[0037] 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 (average weight upon arrival 3.12 ± 0.18 kg) were divided into four groups of N = 6 each based on power calculations (1-β = 80%, p<0.05) using the results of a previous study (Oldenburg et al, 2018. Laryngoscope, 128(1): 160-167).

[0038] The four test groups are as follows:

[0039] Group 1: Rabbits provided with undifferentiated cross-linked jellyfish collagen (MX-JC) and adipose-derived mesenchymal stem cells (ADSC).

[0040] Group 2: A rabbit provided with MX-JC.

[0041] Group 3: Rabbits provided with cross-linked hyaluronic acid (X-HA) (Restylane®).

[0042] Group 4: Rabbits provided with undifferentiated acellular dermis (MACD) (Cymetra®).

[0043] The protocol is described in detail elsewhere (References [Oldenburg et al, 2018. Laryngoscope, 128(1): 160-167; Oldenburg et al, 2017. Laryngoscope, 127(5): E166-E169]). Briefly, rabbits were acclimatized for 2 weeks prior to surgery for sequential RLN section and fat harvesting. Anesthesia was induced by intramuscular injection of ketamine 42 mg / kg, xylazine 6 mg / kg, and acepromazine 1.2 mg / kg, and maintained with 1 to 2% isoflurane. Buprenorphine 0.18 mg / kg and carprofen 1.5 mg / kg were used for postoperative pain control. The left RLN was blunt-handled along the inferior thyroid artery to confirm the location, and a 1 cm section was removed. A fat fragment approximately 1.5 cm in diameter was collected from the annular trachea region and placed in sterile saline. Only the fat from Group 1 was further processed for ADSC expansion and injection. Two weeks later, the animals were re-anesthetized for IL, the larynx was exposed, and the needle position and injection delivery were visualized using an image-guided endoscope. IL was administered using a 23-gauge needle with the goal of delivering the substance laterally to the vocalization process of the arytenoid cartilage. 100 μL was injected into each animal using a 1 mL syringe. MX-JC (225 mg / mL) was prepared from dry powder using pH-buffered saline at the time of injection and preheated to 37°C to facilitate mixing. For Group 1, 1 x 10⁶ of the prepared MX-JC was added prior to injection. 6Canine ADSCs were mixed with 5 ng / mL of TGF-β2 and delivered 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 note-taking and video recording were performed for both surgery and IL to obtain details such as the degree of RLN paralysis, needle placement, net volume of injected material, and complications occurring during surgery or postoperative recovery. Overall health status was monitored by weighing the animals upon arrival, after surgery, and once a week thereafter.

[0044] The preparation of ADSCs was performed as previously described (Reference [Oldenburg et al, 2018. Laryngoscope, 128(1): 160-167]). Briefly, adipose tissue was sliced ​​with a scalpel and digested with 3 to 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 incubated at 37°C for 1.5 hours with occasional shaking, and then separated from the adipose layer by centrifugation at 500 g for 5 minutes. The pellet was washed with phosphate-buffered saline, filtered through a 70 μm sieve to remove large particulate matter, centrifuged at 500 g for 5 minutes to prepare, and finally resuspended in 5 to 10 mL of A-MEM medium for overnight incubation at 37°C, 5% CO2, and 95% humidity. The next day, unattached material was removed and fresh medium was added. The medium was replaced every 2 to 3 days. Cells were subcultured at a 1:2 ratio at 60 to 80% confluence. Subculture 3 cells were cryopreserved for 24 to 48 hours prior to IL and newly expanded. A total of 1 x 10⁶ were used only in Group 1 rabbits. 6The ADSCs were co-injected with MX-JC.

[0045] result

[0046] Animal data are summarized in the table in Figure 1. One animal in Group 4 dies from anesthesia and was not replaced. Other animals did not die due to complications of the surgical procedure, postoperative care, or protocol. Experimental success rates were determined by video and simultaneous recording analysis. For RLN surgery, 21 / 23 animals (approximately 91%) exhibited paralysis of the left VF. One animal exhibited partial VFP, and one did not exhibit VFP (Group 3). Injections delivering 100 μL into the left thyroid anterior space were scored as "high confidence," while all others were scored as "medium-low confidence." Approximately 70% of all injections were "high confidence." Volumes exceeding 100 μL and deviations from material extrusion and / or delivery sites accounted for 22% and 8%, respectively, of the "medium-low confidence" injections. Because co-injecting MX-JC and ADSC is difficult, IL injections were administered to 5 animals (83%) in Group 1, which was of "medium-low confidence." Regardless of treatment, rabbits were fed normally throughout the experiment and body weight increased as expected (data not shown).

[0047] Example 2: Viscosity and Rheology Tests

[0048] Materials and Methods

[0049] To aid in understanding the behavior of the materials during injection, the rheological properties of three compounds were evaluated using a DHR-1 Discovery Hybrid Rheometer (TA Instruments, New Castle, DE, USA) equipped with a 40 mm parallel Peltier plate structure. Dynamic viscoelasticity was measured as a function of frequency in the linear viscoelastic region using 1.0 mL of sample. Test temperature = 39°C; absorption time = 0 sec; shear rate = 0.1 to 500 s -1 ; Max equilibrium time = 60 seconds; Sampling period = 30 seconds.

[0050] result

[0051] 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 appropriate viscosity, concentrations ranging from 60 to 300 mg / ml were tested for tactile consistency and rheological properties. Overall, viscosity tended to decrease asymptotically at higher shear rates as concentration increased (Fig. 2a). MX-JC, Restylane®, and Cymetra® exhibited non-Newtonian liquid dilution behavior. The strain curves of Cymetra® (275 mg / mL) and MX-JC (225 mg / mL) were nearly parallel, while the curve of Restylane® (20 mg / mL) showed the highest resistance to dilution (Fig. 2b). This rheological analysis confirmed that MX-JC and Cymetra® possessed similar liquid dilution characteristics.

[0052] Example 3: MRI Analysis

[0053] Materials and Methods

[0054] MRI analysis was performed on the material obtained from the animal described in Example 1. NMR experiments were performed 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. The specimen removed from the fixative was gently dried on tissue paper, transferred to a 20 mm tube, and securely fixed to the center of the tube with a custom Teflon holder. The tube was filled with Fluorinert FC-770 (3M, St. Paul, MN), a perfluorinated aproton solvent that helps improve the homogeneity of the area around the specimen but does not contribute to the background signal. Images were acquired at the core temperature of the gradient coil between 21°C and 25°C. To visualize the specimen, a RARE (Rapid Acquisition with Refocused Echoes) sequence was used with the following parameters:

[0055] a) Repetition time: 4000 ms;

[0056] b) Echo time: 10.37 ms;

[0057] c) RARE argument: 12;

[0058] d) FOV: 16 cm X 16 cm;

[0059] e) Matrix: 160 X 160;

[0060] f) Slice thickness: 0.5 mm;

[0061] g) Planar resolution: 100 um / pxl;

[0062] h) Segment thickness: 0.5 mm; and

[0063] i) Acquisition time: 10 minutes 24 seconds.

[0064] The section and 3D protocols were compared for maximum feature resolution. The section protocol consisted of 26 0.5 mm digital "sections" of the larynx starting from the cricothyroid cartilage and moving upward. After performing an initial short scan (3 to 5 sections) to confirm the start and end positions of the parameterized material, a "digital box" containing the large-scale material was constructed and sectioned as described above. Using the 3D protocol, 400 images were acquired overnight at 0.005 mm intervals.

[0065] Statistical analysis

[0066] The group size for 80% statistical power and p≤0.05 was determined as follows:

[0067]

[0068] In the above formula, "s" is the standard deviation and "d" is the difference between population means (Lehr, 1992. Stat. Med., 11(8): 1099-1102). For volumetric analysis, each of the 26 MRI digital "sections" from all 23 animals was scored separately by three independent observers to help limit observational bias. MRI images were converted to DICOM format, and volumetric analysis was performed using Analyze software (Mayo Clinic) or ImageJ. If necessary, ellipsoids were verified by histology. To determine statistical differences between groups, ellipsoids were sorted by size and graded. Kruskal-Wallis ANOVA of group grades was performed with a statistical significance threshold of p ≤ 0.05. Individual group differences were compared with the Mann-Whitney U test, and statistical significance was set at p ≤ 0.05.

[0069] result

[0070] Twenty-six MRI images of the larynx were recorded for each animal, spaced 0.5 mm apart by moving upward from the annular point. The images depict the parametric ellipsoids generated by the injected material, as well as the uninjected tissues located above and below the injection site. Overall, the injected material was present in 46% of all MRI images at 4 weeks and 29% at 12 weeks, which may decrease by approximately 37% (p=0.002) over time due to reabsorption. The reabsorption rate varied for each material (see the table in Fig. 1 and volume data calculations below). The highest reabsorption rate was in Group 3 (58%), followed by Group 4 (47%), Group 1 (31%), and finally Group 2 (29%). Volumes in μL were calculated by multiplying the ellipsoid area calculated using Analyze or ImageJ by the digital "slice" thickness (0.5 mm) (Figs. 3 and 4). MRI images show the size and location of the largest ellipsoid for each animal sacrificed at 4 weeks (Figs. 3a, 3b, 3c, and 3d, upper panel) and 12 weeks (Figs. 4a, 4b, 4c, and 4d, upper panel). Volume data are displayed for each animal in order from highest to lowest. The X-axis represents the image number, and the y-axis represents the volume in μL (Figs. 3 and 4, lower panel).

[0071] Group 1 data consisted of 31 ellipsoids at week 4 and 23 at week 12 (N=54); Group 2, 41 and 18 (N=59); Group 3, 33 and 24 (N=57); and Group 4, 27 and 26 (N=53). When the week 4 and week 12 data were reduced, the volume of material remaining after IL (mean ± standard deviation) 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 1, 3, and 4 (p<0.0009). To further compare differences in ellipsoid size across all groups, and because they are related to other biomaterial characteristics and laryngeal tissue responses, the inventors graded all ellipsoid volumes in decreasing order from largest to smallest and expressed them on a scale of 100 (highest) to 1 (lowest). The data were analyzed for individual group differences compared using Kruskal-Wallis ANOVA and Mann-Whitney U tests of the grades (p=0.0004). Grade data at 4 and 12 weeks after IL are shown in Figure 5. Group 2 ellipsoid volumes were consistently larger than the other three groups.

[0072] In summary, MX-JC was shown to have a longer residence time in the thyropydilate space than Restylane®, Cymetra®, or MX-JC injected with ADSCs. Specifically, MX-JC was found to last longer than Cymetra® and Restylane® at both 4 and 12 weeks after IL (i.e., Cymetra® lasted more than 40% longer, and Restylane® lasted more than 100% longer).

[0073] Example 4: Histological Analysis

[0074] Materials and Methods

[0075] Histological analysis was performed on the material obtained from the animal described in Example 1. After fixation in 4% paraformaldehyde for at least 72 hours, the laryngeal blocks were embedded in paraffin for sectioning. Three blocks of the same size were initially cut. Up to 72 5 μm tissue sections from each block were placed on Corning glass slides, ensuring that all three blocks were represented on each section. Hematoxylin and eosin staining were performed as previously described (References [Oldenburg et al, 2017. Laryngoscope, 127(5): E166-E169; Voss et al, 2018. Laryngoscope, 128(12): E402-E408]). Briefly, the slides were deparaffinized, stained in Harris hematoxylin solution for 10 minutes, and then washed under running tap water for 5 minutes. After 1 to 5 seconds, the specimen was separated in 1% acid alcohol, washed with tap water for 1 minute, and blued in 0.2% ammonia water or saturated lithium carbonate solution for 3 minutes. The specimen was washed on a slide in tap water for 5 minutes, immersed 10 times in 95% alcohol, counterstained in eosin solution for 1 minute, and then dehydrated once with 95% ethanol, twice with 100% alcohol, and for 5 minutes. Finally, the specimen was mounted in a xylene medium.

[0076] result

[0077] Representative histological data are shown in Fig. 6 and summarized in the table presented in Fig. 1.

[0078] MX-JC was stained with H&E as a clear, net-like substance surrounded by a layer of inflammatory cells. This was most distinct in Group 2 after 4 weeks of IL, while Group 1 showed relatively little inflammatory infiltration. By 12 weeks, the inflammatory response in Group 2 had significantly subsided. 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 distinguishable at 4 weeks but less distinct at 12 weeks due to reabsorption. This substance had relatively few surrounding inflammatory cells. Cymetra® was similar in appearance to MX-JC, appearing as a very clear nodular shape that was easily visible at both 4 and 12 weeks.

[0079] To confirm the characteristics of local tissue changes and inflammatory responses to the injected substances, slides were examined by a veterinary pathologist, and the pathologist's observations are summarized in the table in Figure 1. First, a certain 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 it was not possible to distinguish between the injected ADSCs and proliferating local tissue adipocytes. Third, in the case of Group 3, there was extensive myoclonus and fibrosis associated with Restylane® injection. Fourth, two types of inflammatory nodules appeared in MX-JC and Cymetra®. In the former case, the substance was surrounded by histiocytes and exhibited a T-cell inflammatory response, while in the latter case, the nodules were composed mainly of plasma cells exhibiting a B-cell immune response.

[0080] In previous studies (Oldenburg et al, 2017. Laryngoscope, 127(5): E166-E169), the administration of ADSCs with Cymetra® primarily induced a lymphocytic inflammatory response. These results were confirmed by the results of this study (see table in Figure 1). It should be noted that the previously observed immunological responses were of relatively similar intensity in both the Cymetra® alone group and the Cymetra® + ADSC group (Oldenburg et al, 2017. Laryngoscope, 127(5): E166-E169), indicating that the lymphocytic inflammatory response was not suppressed by growth factors and cytokines secreted by ADSCs, which are typically associated with immunosuppressive effects. In contrast, in this study, MX-JC triggered a T-cell mediated immune response characterized by dendritic and macrophage infiltration that was significantly downregulated by the co-injection of ADSCs (comparison of histological data between Group 1 and Group 2 in Figure 6). Unlike that observed in Group 3, the T cell-mediated response triggered in Group 2 decreased significantly by 12 weeks without obvious features of tissue destruction (excluding deneurogenic effects) at 4 and 12 weeks.

Claims

Claim 1 A pharmaceutical composition for use in the treatment of vocal fold paralysis, wherein the pharmaceutical composition comprises jellyfish collagen. Claim 2 A pharmaceutical composition according to claim 1, wherein the vocal fold paralysis is unilateral true vocal fold paralysis (UVFP) or bilateral vocal fold paralysis (BVFP). Claim 3 A pharmaceutical composition according to claim 1 or 2, wherein the treatment of vocal cord paralysis is performed by injection medialisation laryngoplasty (IL). Claim 4 A pharmaceutical composition according to claim 1 or 2, wherein the jellyfish collagen is in its atelo form. Claim 5 A pharmaceutical composition according to claim 1 or 2, wherein the jellyfish collagen is in its telo form. Claim 6 A pharmaceutical composition according to claim 1 or 2, wherein the jellyfish collagen is cross-linked. Claim 7 A pharmaceutical composition according to claim 1 or 2, wherein the jellyfish collagen is thiolized. Claim 8 A pharmaceutical composition according to claim 1 or 2, wherein the jellyfish collagen is methacrylated. Claim 9 A pharmaceutical composition according to claim 1 or 2, wherein the source of jellyfish collagen is derived from the subphylum Scyphozoa. Claim 10 In paragraph 1 or 2, the source of jellyfish collagen is Rhizostoma pulmo ( Rhizostomas pulmo ), Lofilema Esculentum( Rhopilema esculentum ), Lofilema Nomadica( Rhopilema nomadica ), Stomolopus meleagris( Stomolophus meleagris ), Aurelia species ( Aurelia sp. ), Cassiopeia Andromeda( Cassiopea andromeda ), Nemophilema Nomurai( Nemopilema nomurai A pharmaceutical composition selected from the group consisting of ), or any combination thereof. Claim 11 A pharmaceutical composition according to claim 1 or 2, wherein the jellyfish collagen is in the form of a micronized powder. Claim 12 A pharmaceutical composition according to claim 11, wherein the finely powder has a particle size of 1 μm to 1000 μm. Claim 13 A pharmaceutical composition according to claim 1 or 2, wherein the jellyfish collagen is at a concentration of 1 to 500 mg / mL. Claim 14 A pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition further comprises adipose-derived mesenchymal stem cells (ADSC). Claim 15 A pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition further comprises a pharmaceutical active ingredient. Claim 16 A pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier. Claim 17 delete

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