Temperature-sensitive hydrogel composition

Abstract

In accordance with an aspect of an embodiment of the present invention, there is provided a biocompatible temperature-sensitive hydrogel composition for subcutaneous or intramuscular injection. The hydrogel includes a poloxamer, wherein the poloxamer is a poloxamer 338, a poloxamer 188, or a poloxamer 407, or a mixture thereof. The composition includes a therapeutic protein or antigen embedded in the composition for sustained release.

Description

Technical Field

[0001] The present disclosure belongs to the field of thermosensitive hydrogels. More specifically, the present disclosure relates to local delivery of therapeutic proteins or antigens through thermosensitive hydrogels, constituent materials, preparations, application methods and uses of thermosensitive hydrogels. Background Art

[0002] Allergic diseases, such as asthma, rhinitis, eczema and food allergies, are reaching epidemic proportions in the world. The hypersensitivity reactions of these diseases are based on the formation of immunoglobulin E (IgE) antibodies, which are in principle directed against harmless protein antigens, allergens. For example, in WO2009153414, it has been suggested to treat allergic diseases by administering to patients hypoallergenic variants of the desired allergen.

[0003] Recently, the trend of treating all allergic symptoms has been towards the development of active tolerance induction using allergen-specific desensitization, rather than avoiding allergens, because this is usually impossible, or only treating symptoms. Current desensitization therapy is based on allergens purified from natural sources, wherein batch-to-batch differences in the amount of allergen components and the presence of other non-allergenic protein substances may lead to problems associated with finding and maintaining the correct dosage and effective treatment. These problems can lead to severe anaphylactic side effects and potential risks of sensitization to new allergens (neosensitization). Desensitization using recombinant hypoallergenic variants will remove the shortcomings associated with batch-to-batch differences and minimize the amount of other protein components that may cause side effects or unnecessary activation of the immune system to these impurity proteins.

[0004] WO2012143374 discloses mutant polypeptides for use as hypoallergenic agents. The recombinant birch pollen Bet v 1 polypeptides produced contain mutations at selected amino acid positions to reduce or completely eliminate their ability to trigger sensitized mast cells or basophils leading to various allergic reactions, but retain their ability to induce the production of protective IgG antibodies.

[0005] WO2019135027 discloses a modified Equ c 1 polypeptide and the use of such a polypeptide as a hypoallergenic variant for desensitization to horse allergies.

[0006] For the treatment of allergies, allergen-specific immunotherapy with hypoallergenic variants has the potential to restore durable immune tolerance, however, improved tolerance-promoting dosage formulations are needed to increase the therapeutic efficacy and reduce the side effects of this approach. Summary of the invention

[0007] According to a first aspect of the present invention, a biocompatible thermosensitive hydrogel composition for subcutaneous or intramuscular injection is provided. The composition comprises poloxamer, wherein the poloxamer is poloxamer 338 or a mixture thereof with poloxamer 188, or the poloxamer is a mixture of poloxamer 407 and poloxamer 188. The composition contains up to 25% (w / w) of poloxamer, so that the composition comprises 15-20% (w / w) of poloxamer 338 or poloxamer 407. The composition comprises a therapeutic protein or antigen embedded in the composition. Preferably, the composition comprises two or more therapeutic proteins or antigens.

[0008] According to a second aspect of the present invention, there is provided a method for treating allergy or autoimmune disease, the method comprising administering a biocompatible thermosensitive hydrogel composition to a patient in need thereof, wherein the administration is performed by subcutaneous or intramuscular injection.

[0009] According to a third aspect of the present invention, there is provided use of a biocompatible thermosensitive hydrogel composition in preparing a medicament for treating allergies or autoimmune diseases.

[0010] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1. (A) Dissolution of a hydrogel composition comprising the hypoallergenic DM-101, and (B) release of DM-101 from a hydrogel composition comprising 5% P188 and 18% P407. A hydrogel composition of 10% P188 and 20% P407 was used as a control. Released protein was determined by ELISA. Compared to the wild-type birch allergen Bet v 1 (Bet v 1.0101), the hypoallergenic DM-101 contains two modifications (N28K and E101K) in its amino acid sequence, see WO2012143374.

[0012] Figure 2. Reversibility capabilities of hydrogel compositions. (A) Gel dissolution time and (B) DM-101 release from 5% P188 and 18% P407 hydrogel compositions were measured after three sol-gel cycles.

[0013] Figure 3. Stability of the correctly folded hypoallergenic birch allergen DM 101 in common buffer and different poloxamer mixtures. The amount of DM101 was measured from the solution after storage at +4 and room temperature for the indicated time (0-6 months) and with a folding-sensitive immunoassay (i.e. only correctly folded DM 101 was measured). (AE) Gel dissolution time and (FJ) DM-101 release.

[0014] Figure 4. Properties of P188 / P338 hydrogel compositions. (A) Dissolution time and (B) DM-101 release from hydrogels 5% P188 / 18% P407, 2% P188 / 18% P388, and 0% P188 / 16% P338.

[0015] Figure 5 .Viscosity of P188 / P338 hydrogel compositions. The viscosity of different hydrogel compositions was determined. The values ​​on the x-axis relate to the percentage of poloxamer (w / w) and are named below them.

[0016] Figure 6 . Mouse skin prick test. Darker colors (i.e. Evans blue dye) visualize inflammation of the skin. In order to determine the effect of different poloxamer formulations on the allergenicity of DM-101, the mice were sensitized to birch allergen. One group of mice was sensitized intraperitoneally with wild-type Bet v 1 and alum adjuvant. After successful sensitization, i.e. showing a positive IgE response, the mice were challenged with different formulations of DM 101, using wild-type Bet v 1 as a positive control or normal buffer as a negative control. 30 μl of a solution of 20 mg DM-101 / ml was administered. The results shown are the results 30 minutes after intradermal injection. Formulations: F1: DM-101 in dilution buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4); F2: DM-101 in 0.05% P188 in dilution buffer; F3: DM-101 in 5% P188 / 18% P407 in dilution buffer; F4: DM-101 (1 mg / ml) with aluminum adjuvant (5 mg / ml) in dilution buffer; PBS: negative control, Bet v 1: positive control containing wild-type birch allergen. C48 / 80: Mast cell degranulation compound 48 / 80 (20 mg / mL; Sigma) was used as a positive control. Results: F3 showed the mildest reaction of all mice. Positive skin responses were seen with F1 and F2. Positive skin responses were also seen with F4, but these responses were milder than with F1 and F2, but more pronounced than with F3.

[0017] Figure 7. Body temperature after subcutaneous injection. The effect of poloxamer formulation on the severe allergic potency of DM-101 is clearly demonstrated. The two poloxamer formulation candidates reduced the severe allergic potency of DM-101 by approximately 4-fold. There was little difference between the 5% P188 / 18% P407 and 16% P338 formulations. Body temperature after subcutaneous injection. (A) Temperature profiles of control animals and allergic animals challenged with formulation 1. (B) Temperature profiles of allergic animals challenged with formulations 3 and 5.

[0018] Figure 8. Release of the allergens β-lactoglobulin (BLG) and recombinant Ara h 2 from 5% P188 / 18% P407 and 16% P338 hydrogels. The amount of released protein was determined using the o-phthalaldehyde (OPA) assay according to the manufacturer's instructions (Thermo Scientific). BLG behaved very similarly to DM-101, but the total amount of released allergen was still slightly lower for Ara h 2. DETAILED DESCRIPTION

[0019] The invention discloses a thermosensitive hydrogel composition for subcutaneous or intramuscular injection. The composition comprises a therapeutic protein or antigen. The release of the therapeutic protein or antigen is controlled by the hydrogel composition.

[0020] Thermosensitive biocompatible hydrogel

[0021] The term "biocompatible hydrogel" refers to a hydrogel that does not produce toxic (or cytotoxic) or deleterious effects or products and is not itself immunogenic. This is necessary so that during treatment, the hydrogel itself does not cause a rejection response.

[0022] The composition includes a poloxamer, which is a nonionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) and two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) on either side. Because the length of the polymer block can be customized, there are many different poloxamers. Poloxamers are sold under the trade names Pluronic, Synperonic, and Kolliphor. In some embodiments, the composition includes poloxamer 338 (P338, Pluronic F108). In other embodiments, the composition includes a mixture of poloxamer 388 and poloxamer 188 (P188, Pluronic F68). In another embodiment, the poloxamer is a mixture of poloxamer 407 (P407, Pluronic F127) and poloxamer 188. The composition contains up to 15%, 18%, 20%, 22%, 25% (w / w) of poloxamer such that the composition comprises 15%, 16%, 17%, 18%, 19%, 20% (w / w) of poloxamer 338 or poloxamer 407.

[0023] In some embodiments, the biocompatible hydrogel composition comprises 4-6% (w / w) poloxamer 188 and 17-19% (w / w) poloxamer 407, preferably 5% (w / w) poloxamer 188 and 18% (w / w) poloxamer 407. In other embodiments, the biocompatible composition comprises 15-19% (w / w) poloxamer 338, preferably 15-17% (w / w) poloxamer 338, more preferably 16% (w / w) poloxamer 338. In another embodiment, the biocompatible hydrogel composition comprises 1-3% (w / w) poloxamer 188 and 15-19% (w / w) poloxamer 338, preferably 2% (w / w) poloxamer 188 and 18% (w / w) poloxamer 338.

[0024] The term "thermosensitive" refers to a composition in which the physical state of the composition depends on the temperature. In some embodiments of the present disclosure, the poloxamer hydrogel is in a liquid state in the temperature range of 4°C to 25°C and forms a gel at 30-37°C. At 37°C, the gelation time is less than 150 seconds. Preferably, at 37°C, the gel is formed within 60 seconds, more preferably within 30 seconds. In some preferred embodiments, the hydrogel is in a liquid state in the storage temperature range of 4°C to 8°C. In other preferred embodiments, the hydrogel is in a liquid state at room temperature 20-25°C.

[0025] The thermosensitive behavior of the hydrogel allows the composition to be easily injected at room temperature and to form an in situ hydrogel implant at 37°C. In situ gel-forming drug delivery systems provide a method by which a controlled-release reservoir can be physically inserted into a target site without the use of surgery. These systems avoid the use of large needles or microsurgery, and they are injected as low-viscosity solutions that convert into a gel or solid reservoir in vivo.

[0026] Therapeutic proteins and antigens

[0027] The biocompatible hydrogel composition of the present disclosure includes at least one therapeutic protein or antigen embedded in the composition. As used herein, the term "antigen" includes a portion or molecule containing an epitope to which a binding agent (e.g., an antibody) can bind. The term "epitope" is a well-known term in the art and refers to a specific region of an antigen to which an agent (e.g., an antibody) can bind. An epitope can be a linear, conformational, nonlinear, or discontinuous epitope. In the case of a polypeptide antigen, it will be understood by those skilled in the art that the presence of an epitope may or may not depend on the secondary, tertiary, or quaternary structure of the polypeptide. For example, in some embodiments, an agent can bind to a certain amino acid sequence independently of the folding of the sequence. In other embodiments, an agent binds to an epitope only when it has a certain three-dimensional structure.

[0028] In some embodiments, at 37°C, up to 1%, 2%, 5%, 8%, 10%, 12%, 15%, 16%, 17%, 18%, 19%, 20% of the therapeutic protein or antigen is released from the composition within 30 minutes. The release is measured as the percentage of protein in the sample relative to the starting composition at a specific time point. This can be measured by, for example, enzyme-linked immunosorbent assay, SDS-PAGE, protein concentration measurement or mass spectrometry or any other suitable method for determining protein content in a sample. In other embodiments, at 37°C, within 300 minutes, preferably within 240 minutes, more preferably within 180 minutes, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of the therapeutic protein or antigen is released from the composition.

[0029] The advantage of such timed release is that proteins or antigens that potentially cause an undesirable immune response can be presented to the immune system in a controlled manner. On the one hand, the protein or antigen is released from the composition quickly enough to activate the immune system within a time frame that a healthcare professional can monitor the patient, and on the other hand, the protein is not released from the composition too quickly, otherwise it will cause an undesirable immune response.

[0030] In some embodiments, the embedded therapeutic protein is an allergen, preferably a hypoallergen, more preferably a genetically engineered hypoallergen. The allergen can be a purified protein, a recombinant protein, a pollen extract, or an extract from animal hair, dander, saliva or urine.

[0031] In some embodiments, the allergen is plant pollen or a genetically engineered hypoallergen thereof. Such plant pollen includes, but is not limited to, pollen from trees such as birch, alder, ash, poplar, cedar, juniper, maple, olive, or oak; grasses such as rye or timothy; or weeds such as mugwort or ragweed. In a preferred embodiment, the allergen is pollen from birch or timothy, or a genetically engineered hypoallergen thereof. In other embodiments, the allergen is an animal protein, or a genetically engineered hypoallergen thereof. Such animal proteins include, but are not limited to, proteins from horses, dogs, cats, rodents, and rabbits. In a preferred embodiment, the allergen is from a horse or a genetically engineered hypoallergen thereof.

[0032] In a preferred embodiment, the allergen is birch allergen Bet v 1, or a genetically engineered hypoallergen thereof. In other preferred embodiments, the allergen is horse allergen Equ c 1, peanut allergen Ara h 2, or a genetically engineered hypoallergen thereof.

[0033] In other embodiments, the therapeutic protein or antigen is an autoantigen. Autoantigens include one or more purified natural autoantigens, one or more recombinant autoantigens or derivatives thereof, and one or more fragments of natural or recombinant autoantigens. As used herein, the term "autoantigen" (also referred to as autoantigen) is an antigen that is a target of a humoral or cell-mediated immune response (such as in an autoimmune disease) despite being a normal tissue component. For example, an autoantigen can be a protein, a protein complex, DNA or RNA, a single or double chain or a glycoprotein. Examples of autoantigens include, but are not limited to, insulin, proinsulin, glutamic acid decarboxylase, myelin basic protein (MBP), type II collagen, thyroid peroxidase, or retinol binding protein-3 (RBP-3).

[0034] As used herein, the term "fragment" includes natural polypeptides (degradation products, synthetically synthesized peptides or recombinant peptides) and modified peptides, which may have modifications, such as making the peptide more stable or less immunogenic. Such modifications include, but are not limited to, cyclization, N-terminal modifications, C-terminal modifications, peptide bond modifications, backbone modifications, and residue modifications. The fragment may also include other extensions, deletions, substitutions, or insertions. The term "peptide" refers herein to any chain of amino acid residues, regardless of its length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0035] Uses of biocompatible hydrogel compositions

[0036] The biocompatible hydrogel compositions disclosed herein allow for sustained local delivery of therapeutic proteins or antigens. Sustained delivery results in the extension and slowing of the presentation of the protein or antigen. The hydrogel can serve as a reservoir for sufficient amounts of allergens or autoantigens or fragments thereof, tolerance-promoting adjuvants, and optional tolerance-promoting active immunomodulators. The purpose is to present the protein or antigen to the immune system in a controlled manner to avoid adverse immune responses. In order to further avoid adverse immune responses, the hydrogel compositions may also include other compounds or agents that make allergy-related immune cells less sensitive to allergen stimulation. Examples of such compounds or agents are antihistamines and anti-IgE antibodies, such as Omalizumab.

[0037] In addition, the hydrogel composition can protect therapeutic proteins or antigens from enzymatic degradation in vivo.Thus, the hydrogel composition of the present disclosure can allow for administration of sensitive molecules, such as RNA.

[0038] In some embodiments, the biocompatible hydrogel composition is used for immunotherapy to promote allergen tolerance or treatment of autoimmune diseases. Autoimmune diseases include, but are not limited to, diseases such as type I diabetes, rheumatoid arthritis, autoimmune uveitis, and multiple sclerosis. In other embodiments, the biocompatible hydrogel composition is used to treat allergic diseases such as allergic conjunctivitis, allergic rhinitis, and allergic asthma.

[0039] In some embodiments, the antigen comprises live viruses, live bacteria, inactivated viruses, inactivated bacteria, nucleic acids, protein subunits of infectious agents, or mixtures thereof. In preferred embodiments, the hydrogel composition of the present disclosure is a vaccine.

[0040] In some embodiments, the biocompatible hydrogel composition includes a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, an antioxidant such as ascorbic acid or sodium bisulfite, a chelating agent such as ethylenediaminetetraacetic acid (EDTA), a preservative, a buffer such as acetate, citrate or phosphate.

[0041] The present invention further provides a method for preventing or treating allergies or autoimmune diseases, comprising administering a biocompatible thermosensitive hydrogel composition to a patient in need thereof. Administration is preferably performed by subcutaneous or intramuscular injection. Subcutaneous injection is preferably performed at intervals of one or two weeks as required.

[0042] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to animals treated with one or more exemplary compounds taught herein, including but not limited to simians, humans, birds, felines, canines, equines, rodents, cattle, pigs, sheep, goats, mammalian farm animals, mammalian sports animals, and mammalian pets. Suitable subjects for various embodiments can be any animal, including humans suspected of having, diagnosed with, or at risk of developing a disease that can be improved, treated, or prevented by administering one or more exemplary compounds described herein.

[0043] The present disclosure further provides use of the biocompatible thermosensitive hydrogel composition in preparing a medicament for preventing or treating allergies or autoimmune diseases.

[0044] In one embodiment, the hydrogel comprises 5% P188 and 18% P407 in a diluent of 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4 containing 0.2 mg / mL DM-101.

[0045] In another embodiment, the hydrogel comprises 16% P338 in a diluent of 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4 containing 0.2 mg / mL DM-101.

[0046] Unless otherwise stated, the properties experimentally measured or determined herein are measured or determined at room temperature. Unless otherwise stated, room temperature is 25°C.

[0047] It should be understood that the disclosed embodiments of the present invention are not limited to the specific structures, process steps or materials disclosed herein, but can be extended to equivalents thereof that will be recognized by those of ordinary skill in the relevant art. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limiting.

[0048] References throughout the specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" that appear in various paragraphs throughout the specification do not necessarily all refer to the same embodiment.

[0049] As used herein, for convenience, multiple items, structural elements, constituent elements and / or materials may be presented in a common list. However, these lists should be understood as each member in the list being individually identified as a separate and unique member. Therefore, without an indication to the contrary, any single member in the list should not be interpreted as the de facto equivalent of any other member in the same list based solely on its presentation in a common group. In addition, various embodiments and examples of the present invention, together with alternatives to its various components, may be mentioned herein. It should be understood that such embodiments, examples and alternatives should not be interpreted as de facto equivalents to each other, but should be regarded as separate and autonomous representations of the present invention.

[0050] In addition, the described features, structures or characteristics can be combined in any suitable manner in one or more embodiments. In the following description, many specific details, such as examples of length, width, shape, etc., are provided to provide a thorough understanding of embodiments of the present invention. However, those skilled in the relevant art will recognize that the present invention can be implemented without one or more specific details, or implemented using other methods, components, materials, etc. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid obscuring various aspects of the present invention.

[0051] Although the foregoing examples illustrate the principles of the present invention in one or more specific applications, it is obvious to those skilled in the art that various modifications may be made in the details of form, use and implementation without the exercise of inventive ability and without departing from the principles and concepts of the present invention. Therefore, the present invention is not intended to be limited except as set forth below.

[0052] The verbs "to comprise" and "to include" are used in this document as open limitations, which neither exclude nor require the existence of features that are not recited. Unless explicitly stated otherwise, the features recited in the dependent claims may be freely combined with each other. Furthermore, it should be understood that the use of "a" or "an" throughout this document, i.e. in the singular, does not exclude the plural.

[0053] Examples

[0054] DM-101 Formulations 1. 125, 250 or 500 μg / ml DM-101 in dilution buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0055] DM-101 Formulation 3. 250, 500, 1000, 2000 or 4000 μg / ml DM-101 in 5% P188 / 18% P407 in dilution buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0056] DM-101 Formulation 5. 250, 500, 1000, 2000 or 4000 μg / ml DM-101 in 16% P338 in dilution buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0057] Ara h 2 / BLG in 5% P188 / 18% P407, 3 ml total volume. 0.3 ml β-lactoglobulin, BLG, Sigma, L3908 (2 mg / ml) or peanut allergen rAra h 2 (2 mg / ml) purified as described in Storni et al. 2020, 0.5 ml 30% P188 and 1.8 ml 30% P407 in 0.4 ml of dilution buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0058] Ara h 2 / BLG in 16% P338, 3 ml total volume. 0.3 ml β-lactoglobulin, BLG, Sigma, L3908 (2 mg / ml) or rAra h 2 purified as described in Storni et al. 2020 (2 mg / ml), and 1.6 ml 30% P338 in 1.1 ml of dilution buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0059] Example 1. A 30% (w / w) P407 stock solution was prepared in a total volume of 200 mL in diluent (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) as follows: 140 g of diluent was weighed into a sterile Schott bottle, 60 g of P407 was weighed and added in small portions to the bottle containing the diluent under constant stirring, and the solution was mixed at 4°C until the poloxamer was completely dissolved.

[0060] Prepare a 30% (w / w) P188 stock solution in diluent (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) in a total volume of 100 mL. Weigh 70 g of diluent into a sterile Schott bottle. Weigh 30 g of P188 into the bottle. Mix the solution at RT until the poloxamer is completely dissolved.

[0061] Prepare a hydrogel with 5% (w / w) P188, 18% (w / w) P407, and 0.2 mg / mL DM-101 in a total volume of 100 mL. Add 60 mL of cold 30% (w / w) P407 stock solution to a sterile Schott bottle. Add 16.7 mL of cold 30% (w / w) P188 stock solution to the bottle. Then add 3.34 mL of cold diluent. Finally, add 20 mL of 1 mg / mL DM-101 solution. Mix the solution by inverting the bottle or gently vortexing.

[0062] Example 2. A 30% (w / w) P338 stock solution was prepared in a total volume of 200 mL in diluent (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) as follows: 140 g of the diluent was weighed into a sterile Schott bottle, 60 g of P407 was weighed and added in small portions to the bottle containing the diluent under constant stirring, and the solution was mixed at 4°C until the poloxamer was completely dissolved.

[0063] Prepare a hydrogel with 16% (w / w) P338 and 0.2 mg / mL DM-101 in a total volume of 93.75 mL. Add 50 mL of cold 30% (w / w) P338 stock solution to a sterile Schott bottle. Then add 25 mL of cold diluent to the bottle. Finally, add 18.75 mL of 1 mg / mL DM-101 solution. Mix the solution by inverting the bottle or gently vortexing.

[0064] Gel time. Incubate the sample at room temperature for 30 minutes. Draw 0.5 mL of the sample into a 1 mL syringe using a 25G needle and inject into a glass vial. Place the vial in a 33°C or 37°C water bath and monitor gelation every 10 seconds. The time when the solution does not move even if the vial is turned over is considered the gel time.

[0065] Viscosity. Viscosity was measured using a rotational rheometer (AR-G2, TA Instruments). Viscosity was measured at 22 °C as a function of shear rate in the range of 0.5–500 s -1 , taking 5 points for every ten orders of magnitude.

[0066] DM-101 is released from the hydrogel. Place the sample in a water bath at 37°C until a clear gel is formed. Carefully add 150 μl of diluent to the surface of the gel. Remove the diluent every 30 minutes and measure the amount of release medium and the weight of the vial. At each time point, add fresh diluent (150 μl) to the surface of the gel. Continue sampling until the gel is completely dissolved. Assess dissolution by visual inspection. DM-101 is measured from the sample using a sandwich Bet v 1 ELISA according to the manufacturer's protocol (Bet v 1 ELISA 2.0 EP, Indoor Biotechnologies). Dilute the sample to the standard range of 0.19-100 ng / ml.

[0067] Reversibility of hydrogels. 0.5 mL of sample was incubated at 37°C for 15 min to form a gel and moved to 4°C for 15 min to return the sample to the solution phase. This cycle was repeated three times before analyzing the release of DM-101 as described above.

[0068] Skin prick test. Skin prick testing was performed as described by et al. (2004).

[0069] The viscosity of DM-101 prepared by hydrogel. The purpose is to test different combinations of P188 and P407 to form hydrogels in order to maintain a sufficient gelation temperature (T 溶胶-凝胶 ) while reducing the viscosity of the solution. The results are shown in Table 1.

[0070] Table 1.

[0071]

[0072] *The T sol-gel values ​​shown are determined as in Zhang K., Shi X. et al. 2014. Information is shown in Table 1.

[0073] Protein release from 5% P188 / 18% P407 hydrogel. Figure 1B and are shown in Table 2.

[0074] Table 2. 10 / 20 hydrogel refers to 20% P188 and 20% P407. 5 / 18 hydrogel refers to 5% P188 and 18% P407.

[0075]

[0076] Viscosity and gelation of hydrogels. The gel-forming ability was tested in a 37°C water bath. See Table 3.

[0077] Table 3.

[0078]

[0079]

[0080] Reversibility of the hydrogel. The reversibility of the hydrogel was tested. The samples underwent three gel-sol cycles before analyzing the release of DM-101 by ELISA. The results are shown in Figure 2.

[0081] Gel-forming ability of P188 and P338 hydrogels. The gelation times at 37°C and 33°C are shown in Table 4.

[0082] Table 4.

[0083]

[0084] The release of DM-101 from P188 / P407, P188 / P338, and P338 hydrogels is shown in FIG. 4 and Table 5.

[0085] Table 5.

[0086]

[0087]

[0088] Figure 5 The viscosities of P188 / P407, P188 / P338, and P338 hydrogels are shown in FIG.

[0089] Allergenicity of the preparations in mice. Figure 6 , Skin prick test. Figure 7, sc. Body temperature measurement after challenge. The results are very good, indicating that poloxamer formulations 3 and 5 allow at least four times higher DM-101 dose rates (i.e. at least 200-400 μg) compared to buffered formulation 1 (25-100 μg).

[0090] in conclusion

[0091] The purpose of the present disclosure is to develop a novel formulation for immunotherapy to promote allergen tolerance. Specific objectives are 1) to protect allergens from being adsorbed to surfaces (vials, syringes, etc.) when delivered at low concentrations; 2) to protect the natural folding of allergens from partial or complete denaturation; 3) to obtain a reservoir effect of allergens within hours of exposure to the immune system; and 5) to dissolve the reservoir forming agent within a few hours. This has been achieved by using poloxamers as stabilizing compounds and their ability to form thermosensitive hydrogels as reservoir agents.

[0092] In the experiments of the present disclosure, the gelation temperature and gelation time of various poloxamer compositions were tested to determine the optimal concentration for the desired application. The results are shown in Tables 1 and Figure 5The viscosity measurement is important because solutions that are too viscous at room temperature are difficult to deliver through a hypodermic needle. Viscosities below 150 can be easily handled with a 25G needle, which has also been verified in experiments with mice. The gelation and viscosity of the present composition are not affected by continuous gelation and dissolution cycles (Figure 2).

[0093] like Figure 1B and 8B As shown, the release time of the allergens from the gel was measured in vitro. The best release time was obtained with the selected poloxamer mixture (more than 60%-80% in 4 hours). The release time was determined not only with the model hypoallergen DM-101, but also with the milk allergen BLG and the peanut allergen Ara h 2 to show the universality ( Figure 8B ).

[0094] The time for the poloxamer composition to dissolve in vitro was shown to be several hours (Figures 1A and 8A).

[0095] In Fig. 3, the measurement results of storage stability of correctly folded DM-101 in different poloxamer mixtures are shown. After storing for a specified time (0-6 months) at +4, the amount of DM-101 is measured from the solution and measured with an immunoassay sensitive to folding (i.e., only measuring correctly folded DM-101). The dissolution time (3A-3E) of the stored gel and the release (3F-3J) of correctly folded DM-101 show high stability.

[0096] The controlled release of allergens from the present poloxamer compositions reduces side effects in sensitized mice, thus allowing higher concentrations of hypoallergenic or allergenic agents in immunotherapy. Figure 6 This is demonstrated by the skin prick test results of . The results show that DM-101 embedded in the poloxamer formulation of the present invention (sample F3) exhibits less signs of inflammation than DM-101 in a common buffer (sample F1).

[0097] In another test for adverse allergic responses (see Figure 7), sensitized mice were injected with DM-101 in a plain buffer (Formulation 1) and DM-101 in a poloxamer formulation (Formulations 3 and 5). The severe allergic response was measured as a decrease in the body temperature of the mice. As can be seen, a significant decrease in body temperature was seen when 50 μg of DM-101 (Formulation 1) was injected in a plain buffer, while more than 200 μg of DM-101 (Formulations 3 and 5) were required to produce a similar response with the poloxamer formulation.

[0098] Toxicity and safety. The biocompatible thermosensitive hydrogel compositions described herein are intended for subcutaneous or intramuscular injection of active pharmaceutical ingredients (such as proteins for allergen immunotherapy). Therefore, the formulation must be safe and non-toxic in clinical applications. According to applicable international and European guidelines, a 12-week repeated dose toxicity study was conducted in New Zealand white rabbits to evaluate safety and toxicity. This nonclinical toxicity study was conducted by a GLP-compliant testing agency for a 16.5% poloxamer 338 formulation with DM-101 as the active ingredient. It was concluded that at the DM-101 dose level of 0.1 mg / week studied, the poloxamer 338 formulation with DM-101 administered subcutaneously once a week for 12 weeks was well tolerated in New Zealand white rabbits. The dose volume (0.5 mL) administered to rabbits was the same as the dose volume intended to be used in clinical trials. Therefore, this toxicity study also demonstrated that the subcutaneous administration of excipient poloxamer 338 at clinical dose levels in rabbits is safe.

[0099] Therefore, this set of examples demonstrates that the independent criteria for the formulation objectives are met.

[0100] Citation List

[0101] Patent Literature

[0102] WO 2009153414

[0103] WO 2012143374

[0104] WO 2019135027

[0105] Non-patent literature

[0106] Zhang K., Shi

[0107] I., A., E.,Untersmayr E.,Walter F.,Willheim M.,Boltz-Nitulescu G.,Scheiner O.,Gabor F.and Jensen-JarolimE.2004.Allergen-loaded biodegradable poly(D,L-lactic-co-glycolic)acidnanoparticles down-regulate an ongoing Th2 response in the BALB / c mousemodel.Clin Exp Allergy 2004;34:315–321.

[0108] Storni et al.Vaccine against peanut allergy based on engineeredvirus-like particles displaying single major peanut allergens.(2020)J AllergyClin Immunol;145:1240-53。

Claims

1. A biocompatible thermosensitive hydrogel composition for subcutaneous or intramuscular injection, comprising poloxamer, wherein: The poloxamer is poloxamer 338 or a mixture thereof with poloxamer 188, or the poloxamer is a mixture of poloxamer 407 and poloxamer 188, the composition contains up to 25% (w / w) of the poloxamer, such that the composition comprises 15-20% (w / w) of poloxamer 338 or poloxamer 407; and wherein the composition comprises a therapeutic protein or antigen embedded in the composition.

2. The biocompatible hydrogel composition according to claim 1, wherein: The composition is in an injectable liquid state at 4-25°C and forms a gel within 60 seconds, preferably within 30 seconds at 30-37°C.

3. The biocompatible hydrogel composition according to claim 2, wherein: At 37°C, up to 20% of the therapeutic protein or antigen is released from the composition within 30 minutes.

4. The biocompatible hydrogel composition according to claim 3, wherein: At least 60% of the therapeutic protein or antigen is released from the composition within 180-300 minutes at 37°C.

5. The biocompatible hydrogel composition according to claim 4, wherein: The therapeutic protein or antigen is released from the composition within 240 minutes, preferably within 180 minutes.

6. The biocompatible hydrogel composition according to any one of claims 1 to 5, wherein: The therapeutic protein is an allergen, preferably a hypoallergen, more preferably a genetically engineered hypoallergen.

7. The biocompatible hydrogel composition according to claim 6, wherein: The allergen is a plant pollen, such as pollen from birch or timothy grass, or a genetically engineered hypoallergenic version thereof, or wherein the allergen is an animal protein or a genetically engineered hypoallergenic version thereof.

8. The biocompatible hydrogel composition according to claim 7, wherein: The allergen is birch allergen Bet v 1 or a genetically engineered hypoallergen thereof.

9. The biocompatible hydrogel composition according to claim 7, wherein: The allergen is horse allergen Equc 1, peanut allergen Ara h 2 or a genetically engineered hypoallergen thereof.

10. The biocompatible hydrogel composition according to any one of claims 1 to 5, wherein: The antigens include live viruses, live bacteria, inactivated viruses, inactivated bacteria, nucleic acids, protein subunits of infectious agents or mixtures thereof.

11. The biocompatible hydrogel composition according to claim 10, wherein: The composition is a vaccine.

12. The biocompatible hydrogel composition according to any one of claims 1 to 11, wherein: The composition comprises 4-6% (w / w) of Poloxamer 188 and 17-19% (w / w) of Poloxamer 407, preferably 5% (w / w) of Poloxamer 188 and 18% (w / w) of Poloxamer 407.

13. The biocompatible hydrogel composition according to any one of claims 1 to 11, wherein: The composition comprises 15-19% (w / w) of Poloxamer 338, preferably 15-17% (w / w) of Poloxamer 338, and more preferably 16% (w / w) of Poloxamer 338.

14. The biocompatible hydrogel composition according to any one of claims 1 to 11, wherein: The composition comprises 1-3% (w / w) of Poloxamer 188 and 15-19% (w / w) of Poloxamer 338, preferably 2% (w / w) of Poloxamer 188 and 18% (w / w) of Poloxamer 338.

15. The biocompatible hydrogel composition according to any one of claims 1 to 14, wherein: The composition includes a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), preservatives, buffers such as acetates, citrates or phosphates.

16. The biocompatible hydrogel composition according to any one of claims 1 to 15, further comprising an anti-IgE antibody and / or an antihistamine.

17. Use of the biocompatible hydrogel composition according to any one of claims 1 to 16 for immunotherapy to promote allergen tolerance or for treating autoimmune diseases.

18. A method for treating allergies or autoimmune diseases, comprising administering an effective amount of the biocompatible thermosensitive hydrogel composition according to any one of claims 1 to 16 to a patient in need thereof, wherein the administration is performed by subcutaneous or intramuscular injection.

19. Use of the biocompatible thermosensitive hydrogel composition according to any one of claims 1 to 16 in preparing a medicament for treating allergies or autoimmune diseases.

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