Thermosensitive Hydrogel Composition

Abstract

An embodiment of the present invention provides a biocompatible, thermosensitive hydrogel composition for subcutaneous or intramuscular injection, the hydrogel comprising a poloxamer, such as poloxamer 338, poloxamer 188, or poloxamer 407, or a mixture thereof, and the composition includes a therapeutic protein or antigen embedded in the composition for sustained release.

Description

[Technical Field]

[0001] The present disclosure is in the field of thermosensitive hydrogels. More specifically, the present disclosure relates to the localized delivery of therapeutic proteins or antigens via thermosensitive hydrogels, their composition, formulations, application methods, and uses. [Background technology]

[0002] Allergic diseases, such as asthma, rhinitis, eczema, and food allergies, have reached epidemic proportions worldwide. Hypersensitivity reactions in these diseases are based on the formation of immunoglobulin E (IgE) antibodies against allergens, which are essentially harmless protein antigens. Treatment of allergic diseases by administering to patients hypoallergenic versions of the desired allergen has been proposed, for example, in WO 2009 / 153414.

[0003] A recent trend in the treatment of allergic symptoms is to induce tolerance using allergen specific desensitization instead of allergen avoidance, which is often not possible or only treats the symptoms. Current desensitization therapies are based on allergens purified from natural sources, and batch-to-batch variations in both the amount of allergen components and the presence of other non-allergenic protein substances can lead to problems related to finding and maintaining the appropriate dosage and efficacy of treatment. These problems result in the potential risk of anaphylactic side effects and sensitization to new allergens (neosensitization). The use of recombinant hypoallergenic variants for desensitization eliminates the drawbacks associated with batch-to-batch variations and minimizes the amount of other protein components that may cause side effects or undesired activation of the immune system against these impurity proteins.

[0004] WO 2012 / 143374 discloses mutant polypeptides useful as hypoallergens. Recombinant birch pollen Bet v 1 polypeptides were produced containing mutations at selected amino acid positions that reduced or completely reduced their ability to trigger sensitized mast cells or basophils, resulting in a variety of allergic responses, while retaining the ability to induce the production of protective IgG antibodies.

[0005] WO 2019 / 135027 discloses modified Equ c 1 polypeptides and the use of such polypeptides as hypoallergenic variants for desensitization to equine allergies. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2009 / 153414 [Patent Document 2] International Publication No. 2012 / 143374 [Patent Document 3] International Publication No. 2019 / 135027

[0007] [Patent Document 1] Zhang K., Shi X. et al. 2015. Poloxamer-based in situ hydrogels for controlled delivery of hydrophilic macromolecules after intramuscular injection in rats. Drug Delivery; 22(3): 375-382. [Patent Document 2] Scholl I., Weissenbock A., Forster-Waldl ​​E., Untersmayr E., Walter F., Willheim M., Boltz-Nitulescu G., Scheiner O., Gabor F. and Jensen-Jarolim E. 2004. Allergen-loaded biodegradable poly(D,L-lactic-co-glycolic) acid nanoparticles down-regulate an ongoing Th2 response in the BALB / c mouse model. Clin Exp Allergy 2004; 34:315-321. [Patent Document 3] Storni et al. Vaccine against peanut allergy based on engineered virus-like particles displaying single major peanut allergens. (2020) J Allergy Clin Immunol; 145:1240-53. Summary of the Invention [Problem to be solved by the invention]

[0008] For the treatment of allergies, allergen-specific immunotherapy using hypoallergenic variants has the potential to restore lasting immune tolerance, but improvements in tolerance-promoting dosage formulations are needed to increase therapeutic efficacy and reduce the side effects of this approach. [Means for solving the problem]

[0009] According to a first aspect of the present invention, there is provided a biocompatible thermosensitive hydrogel composition for subcutaneous or intramuscular injection. The composition comprises a 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) poloxamer, such that the composition contains 15-20% (w / w) 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.

[0010] According to a second aspect of the present invention, there is provided a method for treating an allergic or autoimmune disease, comprising administering a biocompatible thermosensitive hydrogel composition to a patient in need thereof, the administration being by subcutaneous or intramuscular injection.

[0011] According to a third aspect of the present invention there is provided the use of a biocompatible thermosensitive hydrogel composition for the manufacture of a medicament for the treatment of an allergy or an autoimmune disease.

[0012] The invention is defined by the features of the independent claims. Some particular embodiments are defined in the dependent claims. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1. (A) Dissolution of a hydrogel composition containing hypoallergenic DM-101, and (B) DM-101 release from a hydrogel composition containing 5% P188 and 18% P407. A hydrogel composition with 10% P188 and 20% P407 was used as a control. Released protein was measured by ELISA. Hypoallergenic DM-101 contains two modifications (N28K and E101K) in its amino acid sequence compared to the wild-type birch allergen Bet v 1 (Bet v 1.0101) (see WO 2012 / 143374). [Figure 2]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. [Figure 3] Figure 3. Stability of correctly folded hypoallergenic hippocampal allergen DM101 in different mixtures of simple buffer and poloxamer. The amount of DM101 was measured from solutions after storage at +4 and room temperature for the indicated times (0-6 months) using an immunoassay that is highly folding-sensitive, i.e., measures only correctly folded DM101. (AE) Gel dissolution time and (FJ) DM-101 release. [Figure 4] Figure 4. Characterization of P188 / P338 hydrogel compositions. (A) Dissolution time and (B) DM-101 release of 5% P188 / 18% P407, 2% P188 / 18% P388, and 0% P188 / 16% P338 from the hydrogels. [Figure 5] Figure 5. Viscosity of P188 / P338 hydrogel compositions. The viscosity of different hydrogel compositions was measured. The values ​​on the x-axis relate to the percentage (w / w) of poloxamer named below the value. [Figure 6]Figure 6. Mouse skin prick test. Darker color (i.e., Evans blue dye) visualizes epidermal inflammation. To determine the effect of different poloxamer formulations on the allergenicity of DM-101 mice, they were sensitized to Birch allergen. Sets of mice were sensitized intraperitoneally with wild-type Bet v 1 and alum adjuvant. After successful sensitization, i.e., demonstrating a positive IgE response, mice were challenged with different formulations of DM-101, using wild-type Bet v 1 as a positive control or plain buffer as a negative control. 30 μl of a 20 mg DM-101 / ml solution was administered. Results shown are 30 minutes after intradermal injection. Formulations: F1: DM-101 in diluent buffer (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, pH 7.4); F2: DM-101 in 0.05% P188 in diluent buffer; F3: DM-101 in 5% P188 / 18% P407 in diluent buffer; F4: DM-101 (1 mg / ml) with aluminum adjuvant (5 mg / ml) in diluent buffer; PBS: negative control; Bet v 1: positive control containing wild-type birch allergen. C48 / 80: mast cell degranulating compound 48 / 80 (20 mg / ml; Sigma) as a positive control. Results: F3 induced the mildest reaction in all mice. Positive skin reactions were observed in F1 and F2. Positive skin reactions were also observed in F4, but were less severe than in F1 and F2, but more pronounced than in F3. [Figure 7] Figure 7. Body temperature after subcutaneous injection. The effect of poloxamer formulation on the anaphylactic potency of DM-101 is clearly demonstrated. Both poloxamer formulation candidates reduce the anaphylactic potency of DM-101 by approximately four-fold. The difference between the 5% P188 / 18% P407 formulation and the 16% P338 formulation is minimal. Body temperature after subcutaneous injection. (A) Temperature curves of control and allergic animals challenged with formulation 1. (B) Temperature curves of allergic animals challenged with formulations 3 and 5. [Figure 8]Figure 8. Release of the allergen 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 an o-phthalaldehyde (OPA) assay according to the manufacturer's instructions (Thermo Scientific). BLG behaves almost identically to DM-101, although the total amount of released allergen measured with Ara h 2 remains slightly lower. DETAILED DESCRIPTION OF THE INVENTION

[0014] (Embodiment) The present invention discloses a thermosensitive hydrogel composition for subcutaneous or intramuscular injection, which contains a therapeutic protein or antigen, the release of which is controlled by the hydrogel composition.

[0015] (Thermosensitive biocompatible hydrogel) The term "biocompatible hydrogel" refers to a hydrogel that does not produce toxic (or cytotoxic) or harmful effects or products and is not itself immunogenic, which is essential so that the hydrogel itself does not induce an adverse reaction during processing.

[0016] The composition comprises a poloxamer, a nonionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Because the length of the polymer blocks can be customized, many different poloxamers exist. Poloxamers are sold under the trade names Pluronic, Synperonic, and Kolliphor. In some embodiments, the composition comprises poloxamer 338 (P338, Pluronic F108). In other embodiments, the composition comprises a mixture of poloxamer 388 and poloxamer 188 (P188, Pluronic F68). In yet other embodiments, the poloxamer is a mixture of poloxamer 407 (P407, Pluronic F127) and poloxamer 188. The compositions contain 15%, 18%, 20%, 22%, up to 25% (w / w) poloxamer, such as 15%, 16%, 17%, 18%, 19%, 20% (w / w) poloxamer 338 or poloxamer 407.

[0017] 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 yet other embodiments, 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.

[0018] The term "thermosensitive" refers to a composition whose physical state is temperature-dependent. In some embodiments of the present disclosure, the poloxamer hydrogel is in a liquid state at temperatures ranging from 4°C to 25°C and forms a gel at temperatures ranging from 30°C to 37°C. The gelation time is less than 150 seconds at 37°C. Preferably, the gel forms in 60 seconds, more preferably 30 seconds, at 37°C. In some preferred embodiments, the hydrogel is in a liquid state at storage temperatures ranging from 4°C to 8°C. In other preferred embodiments, the hydrogel is in a liquid state at room temperature, 20°C to 25°C.

[0019] The thermosensitive behavior of hydrogels allows for easy injection of the composition at room temperature and the formation of in situ hydrogel implants at 37°C. In situ gel-forming drug delivery systems provide a means by which controlled-release depots can be physically inserted into target sites without the use of surgery. These systems avoid the use of large needles or microsurgery; they are injected as low-viscosity solutions and convert to gel or solid depots in the body.

[0020] (Therapeutic proteins and antigens) The biocompatible hydrogel compositions of the present disclosure contain at least one therapeutic protein or antigen embedded within the composition. As used herein, the term "antigen" includes a moiety or molecule containing an epitope to which a binding agent (e.g., an antibody) can bind. The term "epitope" is a term well known in the art and refers to a specific region of an antigen to which an agent (e.g., an antibody) can bind. An antigenic determinant can be a linear, conformational, nonlinear, or discontinuous antigenic determinant. Those skilled in the art will understand that, in the case of polypeptide antigens, the presence of an antigenic determinant 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 specific amino acid sequence regardless of the folding of said sequence. In other embodiments, an agent binds to an epitope only if it has a specific three-dimensional structure.

[0021] In some embodiments, up to 1%, 2%, 5%, 8%, 10%, 12%, 15%, 16%, 17%, 18%, 19%, or 20% of the therapeutic protein or antigen is released from the composition in 30 minutes at 37° C. Release is determined as the percentage of protein in the sample relative to the starting composition at a particular time point. This can be determined, for example, by 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 least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the therapeutic protein or antigen is released from the composition in 300 minutes, preferably in 240 minutes, and more preferably in 180 minutes at 37° C.

[0022] The advantage of such a timed release is that proteins or antigens that potentially provoke a harmful 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 for immune system activation in a time frame that the patient can be monitored by a medical professional, and on the other hand, the protein is not released from the composition so quickly that it could lead to a harmful immune response.

[0023] 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.

[0024] In some embodiments, the allergen is plant pollen or a genetically engineered hypoallergenic form thereof. Such plant pollen includes, but is not limited to, pollen from trees such as birch, alder, ash, aspen, cedar, juniper, maple, olive, or oak; pollen from grasses such as rye or timothy; or pollen from weeds such as mugwort or ragweed. In a preferred embodiment, the allergen is birch or timothy pollen or a genetically engineered hypoallergenic form thereof. In other embodiments, the allergen is an animal protein or a genetically engineered hypoallergenic form 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 derived from horses or a genetically engineered hypoallergenic form thereof.

[0025] In a preferred embodiment, the allergen is the birch allergen Bet v 1, or a genetically engineered hypoallergen thereof. In other preferred embodiments, the allergen is the horse allergen Equ cc 1, the peanut allergen Ara h 2, or a genetically engineered hypoallergen thereof.

[0026] In other embodiments, the therapeutic protein or antigen is an autoantigen. Autoantigens include one or more purified native autoantigens, one or more recombinant autoantigens or derivatives thereof, and one or more fragments of native or recombinant autoantigens. As used herein, the term "autoantigen" (also known as self-antigen) refers to an antigen that is a normal tissue component but is nevertheless the target of a humoral or cell-mediated immune response, as in autoimmune diseases. Autoantigens can be, for example, proteins, complexes of proteins, DNA or RNA, either single- or double-stranded, or glycoproteins. 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).

[0027] As used herein, the term "fragment" includes naturally occurring polypeptides (either degradation products, synthetically synthesized peptides, or recombinant peptides) and modified peptides, which may have modifications that, for example, make 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. Fragments may also include further extensions, deletions, substitutions, or insertions. The term "peptide" as used herein refers to any chain of amino acid residues, regardless of its length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0028] (Use of biocompatible hydrogel compositions) The biocompatible hydrogel compositions of the present disclosure enable sustained local delivery of therapeutic proteins or antigens. Sustained delivery results in prolonged and delayed presentation of the protein or antigen. The hydrogel can serve as a depot for sufficient amounts of an allergen or autoantigen, or fragment thereof, tolerance-promoting adjuvants, and, optionally, a tolerance-promoting active immune modulator. The goal is to present the protein or antigen to the immune system in a controlled manner to avoid a harmful immune response. To further avoid a harmful immune response, the hydrogel composition can also contain additional compounds or agents that reduce the sensitivity of allergy-related immune cells to stimulation by allergens. Examples of such compounds or agents are antihistamines and anti-IgE antibodies, such as omalizumab.

[0029] Additionally, the hydrogel composition can protect therapeutic proteins or antigens from enzymatic degradation in the body, thus enabling the administration of sensitive molecules such as RNA.

[0030] In some embodiments, the biocompatible hydrogel compositions are for use in allergen tolerance-promoting immunotherapy or in the treatment of autoimmune diseases, including, but not limited to, type 1 diabetes, rheumatoid arthritis, autoimmune uveitis, and multiple sclerosis. In other embodiments, the biocompatible hydrogel compositions are for use in the treatment of allergic diseases, such as allergic conjunctivitis, allergic rhinitis, and allergic asthma.

[0031] In some embodiments, the antigen comprises a live virus, a live bacterium, a killed virus, a killed bacterium, a nucleic acid, a protein subunit of an infectious agent, or a mixture thereof. In a preferred embodiment, the hydrogel composition of the present disclosure is a vaccine.

[0032] Biocompatible hydrogel compositions, in some embodiments, include a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, 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, and a buffer such as acetate, citrate, or phosphate.

[0033] The present disclosure further provides a method for preventing or treating an allergic or autoimmune disease, comprising administering the biocompatible thermosensitive hydrogel composition to a patient in need thereof. Administration is preferably by subcutaneous or intramuscular injection. Subcutaneous injections are preferably administered at weekly or biweekly intervals as needed.

[0034] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to an animal treated with one or more exemplary compounds taught herein, including, but not limited to, monkeys, humans, birds, cats, dogs, horses, rodents, cattle, pigs, sheep, goats, livestock mammals, sport mammals, and pet mammals. A suitable subject for various embodiments can be any animal, including humans, suspected of having, diagnosed with, or at risk of developing a disease that can be ameliorated, treated, or prevented by administration of one or more exemplary compounds described herein.

[0035] The present disclosure further provides the use of the biocompatible thermosensitive hydrogel composition for the manufacture of a medicament for the prevention or treatment of an allergic or autoimmune disease.

[0036] In one embodiment, the hydrogel comprises 5% P188 and 18% P407 in a diluent comprising 0.2 mg / mL DM-101 in 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4.

[0037] In another embodiment, the hydrogel comprises 16% P338 in a diluent comprising 0.2 mg / mL DM-101 in 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4.

[0038] Unless otherwise specified, experimentally measured or determined properties herein are measured or determined at room temperature, which is 25° C. unless otherwise specified.

[0039] It is to be understood that the disclosed embodiments of the invention are not limited to the particular structures, processing steps, or materials disclosed herein, but extend to equivalents thereof as would be recognized by one of ordinary skill in the art. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0040] Throughout this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment.

[0041] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list were individually identified as a separate and unique member. Accordingly, individual members of such lists should not be construed as de facto equivalents of other members of the same list, but should be construed solely based on their presentation in a common grouping, without any indication to the contrary. Additionally, various embodiments and examples of the present invention may be referred to herein, along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives should not be construed as de facto equivalents of each other, but should be considered as separate and distinct representations of the present invention.

[0042] Furthermore, the described functions, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as example lengths, widths, shapes, etc., to provide a thorough understanding of embodiments of the present invention. However, one skilled in the art will recognize that the present invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other examples, well-known structures, materials, or operations will not be shown or described in detail to avoid obscuring aspects of the present invention.

[0043] While the foregoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those skilled in the art that numerous modifications in embodiment, use, and detail can be made without the exercise of inventive faculty and without departing from the principles and concepts of the present invention. Accordingly, the present invention is not intended to be limited except as by the claims set forth below.

[0044] The verbs "to comprise" and "to include" are used in this specification as open limitations which neither exclude nor require the presence of unrecited features. Features recited in the dependent claims may be freely combined with each other unless expressly stated otherwise. Furthermore, it is to be understood that throughout this specification the use of "a" or "an", i.e., the singular, does not exclude the plural. [Example]

[0045] (Experimental Department) DM-101 Formulations 1. DM-101 at 125, 250 or 500 μg / ml in dilution buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0046] DM-101 Formulation 3. 250, 500, 1000, 2000, or 4000 μg / ml DM-101 in 5% P188 / 18% P407 in dilution buffer (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0047] DM-101 Formulation 5. 250, 500, 1000, 2000 or 4000 μg / ml DM-101 in 16% P338 in dilution buffer (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0048] 5% P188 / 18% P407, peanut allergen rAra h2 purified as described by Storni et al. (2020) in a total volume of 3 ml. 0.3 ml of β-lactoglobulin, BLG, Sigma, L3908 (2 mg / ml) or Storni et al. (2020) (2 mg / ml), 0.5 ml of 30% P188, and 1.8 ml of 30% P407 in 0.4 ml of diluent buffer (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0049] rAra h2 / BLG in 16% P338, total volume 3 ml. 0.3 ml β-lactoglobulin, BLG, Sigma, L3908 (2 mg / ml) or rAra h2 purified as described in Storni et al. 2020 (2 mg / ml), and 1.6 ml 30% P338 in 1.1 ml diluent buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).

[0050] Example 1. A 30% (w / w) P407 stock solution was prepared in diluent (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, pH 7.4) in a total volume of 200 mL as follows: 140 g of diluent was weighed into a sterile Schott bottle, 60 g of P407 was weighed and added to the bottle containing the diluent in small batches with constant stirring, and the solution was mixed at 4°C until the poloxamer was completely dissolved.

[0051] A 30% (w / w) P188 stock solution was prepared in diluent (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, pH 7.4) in a total volume of 100 mL. 70 g of diluent was weighed into a sterile Schott bottle. 30 g of P188 was weighed into the bottle. The solution was mixed at room temperature until the poloxamer was completely dissolved.

[0052] A hydrogel containing 5% (w / w) P188, 18% (w / w) P407, and 0.2 mg / mL DM-101 was prepared in a total volume of 100 mL by adding 60 mL of cold 30% (w / w) P407 stock to a sterile Schott bottle. 16.7 mL of cold 30% (w / w) P188 stock solution was then added to the bottle. 3.34 mL of cold diluent was then added. Finally, 20 mL of 1 mg / mL DM-101 solution was added. The solution was mixed by inverting the bottle or gently vortexing.

[0053] Example 2 A 30% (w / w) P338 stock solution was prepared in diluent (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, pH 7.4) in a total volume of 200 mL as follows: 140 g of diluent was weighed into a sterile Schott bottle, 60 g of P407 was weighed and added in small batches to the bottle containing the diluent under constant agitation, and the solution was mixed at 4° C. until the poloxamer was completely dissolved.

[0054] A hydrogel containing 16% (w / w) P338 and 0.2 mg / mL DM-101 was prepared in a total volume of 93.75 mL. 50 mL of cold 30% (w / w) P338 stock was added to a sterile Schott bottle. 25 mL of cold diluent was added to the bottle. Finally, 18.75 mL of 1 mg / mL DM-101 solution was added. The solution was mixed by inverting the bottle or gently vortexing.

[0055] Gelation time. The sample was incubated at room temperature for 30 minutes. 0.5 mL of the sample was taken into a 1 mL syringe using a 25 G needle and injected into a glass vial. The vial was placed in a water bath at 33°C or 37°C, and gelation was monitored at 10-second intervals. The gelation time was the time when no solution moved when the vial was inverted.

[0056] Viscosity. The viscosity was measured using a rotational rheometer (AR-G2, TA instruments). The viscosity varied depending on the shear rate over the temperature range of 0.5-500°C at 22°C. -1, measured at 5 points per decade.

[0057] Release from DM-101 hydrogels. Samples were placed in a 37°C water bath until a clear gel was formed. 150 μl of diluent was carefully added onto the surface of the gel. At 30-minute intervals, the diluent was removed, and the amount of release medium and vial weight were measured. At each time point, fresh diluent (150 μl) was added to the surface of the gel. Sampling continued until the gel was completely dissolved. Dissolution was estimated by visual inspection. DM-101 was determined from the samples using a sandwich-type Bet v 1 ELISA according to the manufacturer's protocol (Bet v 1 ELISA 2.0EP, Indoor Biotechnologies). Samples were diluted to a standard range of 0.19–100 ng / ml.

[0058] Reversibility of the hydrogel. A 0.5 mL sample was incubated at 37°C for 15 minutes to form a gel, and then transferred to 4°C for 15 minutes to return the sample to the solution phase. This cycle was repeated three times, after which the release of DM-101 was analyzed as described above.

[0059] Skin prick testing. Skin prick testing was performed as described by Scholl et al. (2004).

[0060] Viscosity of hydrogel-formulated DM-101. The goal was to obtain an appropriate gelation temperature (T sol-gel To reduce the viscosity of the solvent while maintaining the viscosity of the solution, various combinations of P188 and P407 were tested to form hydrogels. The results are shown in Table 1. [Table 1]

[0061] Protein release from 5% P188 / 18% P407 hydrogels. The results are shown in Figure 1B and Table 2. [Table 2]

[0062] Hydrogel Viscosity and Gelation. Gel-forming ability is tested in a 37°C water bath. See Table 3. [Table 3]

[0063] Reversibility of the hydrogels. The reversibility of the hydrogels was tested. Samples underwent three gel-sol cycles before analyzing the release of DM-101 by ELISA. See Figure 2 for the results.

[0064] Gel-forming ability of P188 and P338 hydrogels. The gelation times at 37°C and 33°C are shown in Table 4. [Table 4]

[0065] The release from DM-101 P188 / P407, P188 / P338 and P338 hydrogels is shown in Figure 4 and Table 5. [Table 5]

[0066] The viscosities of P188 / P407, P188 / P338 and P338 hydrogels are shown in Figure 5 .

[0067] Allergenicity of formulations in mice. Figure 6: Skin prick test. Figure 7: Body temperature measurement after subcutaneous administration. The results are excellent, showing that both poloxamer formulations 3 and 5 allow at least a four-fold higher DM-101 dose rate (i.e., at least 200-400 µg) compared to buffer formulation 1 (25-100 µg).

[0068] (Conclusion) The objective of this disclosure was to develop a novel formulation for use in allergen tolerance-promoting immunotherapy. Specific objectives were: 1) to protect the allergen from adsorption to surfaces (vials, syringes, etc.) when delivered at low concentrations; 2) to protect the allergen's native fold from partial or total denaturation; 3) to achieve a depot effect over several hours upon allergen exposure to the immune system; and 5) to dissolve the depot-forming agent within several hours. This was achieved using poloxamer as a stabilizing compound and its ability to form a thermosensitive hydrogel as a depot agent.

[0069] In the experiments described herein, various poloxamer compositions were tested for gelation temperature and gelation time to determine the optimal concentration for the desired use. The results are shown in Table 1 and Figure 5. Viscosity measurements are important because highly viscous solutions are difficult to deliver with a hypodermic needle at room temperature. Viscosities below 150 can be easily handled with a 25 G needle, which was also verified in experiments using mice. The gelation and viscosity of the present compositions were not affected by successive cycles of gelation and dissolution (Figure 2).

[0070] The release time of allergens from the gels was measured in vitro as described in Figures 1B and 8B. Optimal release times (>60%–80% release in 4 h) were obtained using selected poloxamer mixtures. Release times were determined using the model hypoallergen DM-101 as well as the milk allergen BLG and peanut allergen Ara h2, demonstrating generality (Figure 8B).

[0071] The dissolution time of the poloxamer composition was shown to be several hours in vitro (Figures 1A and 8A).

[0072] Figure 3 shows the storage stability measurements of correctly folded DM-101 in different mixtures of poloxamers. The amount of DM-101 was measured from the solutions after storage at +4 for the indicated times (0 to 6 months), using an immunoassay that is folding-sensitive, i.e., measures only correctly folded DM-101. The dissolution times of the stored gels (3A-3E) and the release of correctly folded DM-101 (3F-3J) were shown to be highly stable.

[0073] The controlled release of allergens from the poloxamer compositions of the present invention reduces adverse effects in sensitized mice, thus allowing for lower or higher concentrations of allergens in immunotherapy. This is illustrated by the skin prick test results in Figure 6. The results show that DM-101 embedded in the poloxamer formulation of the present invention (sample F3) exhibits fewer signs of inflammation compared to DM-101 in simple buffer (sample F1).

[0074] In another study (see Figure 7), mice sensitized to an adverse allergic reaction were injected with DM-101 in a plain buffer (Formulation 1) and a poloxamer formulation (Formulations 3 and 5). The anaphylactic response was measured as a decrease in the body temperature of the mice. Injection of 50 μg of DM-101 (Formulation 1) in plain buffer resulted in a significant decrease in body temperature, whereas 200 μg or more was required to produce a similar response as the poloxamer formulations (Formulations 3 and 5).

[0075] Toxicity and Safety. The biocompatible thermosensitive hydrogel compositions described herein are intended for subcutaneous or intramuscular injection of active pharmaceutical ingredients (e.g., proteins for allergen immunotherapy). Therefore, the formulation must be safe and nontoxic for clinical use. Safety and toxicity were evaluated in a 12-week repeated-dose toxicity study in New Zealand White rabbits in accordance with applicable international and European guidelines. This nonclinical toxicity study was conducted in a GLP-compliant laboratory for a 16.5% poloxamer 338 formulation containing DM-101 as the active ingredient. In conclusion, once-weekly subcutaneous administration of the poloxamer 338 formulation with DM-101 for 12 weeks was well tolerated in New Zealand White rabbits at the tested DM-101 dose level of 0.1 mg / week. The dose volume administered to rabbits (0.5 mL) was the same as that intended for use in clinical trials. Therefore, this toxicity study also demonstrated the safety of subcutaneous administration of the excipient poloxamer 338 at clinical dose levels in rabbits.

[0076] Thus, this set of examples demonstrates that independent criteria for formulation purposes are met.

Claims

1. A biocompatible thermosensitive hydrogel composition for subcutaneous or intramuscular injection comprising a 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, and the composition contains up to 25% (w / w) of the poloxamer, thereby comprising 15-20% (w / w) of poloxamer 338 or poloxamer 407; and the composition comprises a therapeutic protein or antigen embedded in the composition.

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

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

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

5. 5. The biocompatible hydrogel composition of claim 4, wherein the therapeutic protein or antigen is released from the composition in 240 minutes, preferably 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. 7. The biocompatible hydrogel composition of claim 6, wherein the allergen is plant pollen such as birch or timothy grass pollen, or a genetically modified hypoallergenic form thereof, or the allergen is animal protein or a genetically modified hypoallergenic form thereof.

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

9. 8. The biocompatible hydrogel composition of claim 7, wherein the allergen is horse allergen Equ c 1, peanut allergen Ara h 2, or a genetically engineered hypoallergen thereof.

10. 6. The biocompatible hydrogel composition of claim 1, wherein the antigen comprises a live virus, a live bacterium, a killed virus, a killed bacterium, a nucleic acid, a protein subunit of an infectious agent, or a mixture thereof.

11. The biocompatible hydrogel composition of claim 10 , wherein the composition is a vaccine.

12. 12. The biocompatible hydrogel composition according to any 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. 12. The biocompatible hydrogel composition according to any of claims 1 to 11, wherein the composition comprises 15-19% (w / w) of poloxamer 338, preferably 15-17% (w / w) of poloxamer 338, more preferably 16% of poloxamer 338.

14. 12. 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. 15. The biocompatible hydrogel composition of any one of claims 1 to 14, wherein the composition comprises a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, 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, and a buffer such as acetate, citrate or phosphate.

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. The biocompatible hydrogel composition of any one of claims 1 to 16 for use in allergen tolerance promoting immunotherapy or in the treatment of autoimmune diseases.

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

19. Use of the biocompatible thermosensitive hydrogel composition according to any one of claims 1 to 16 for the manufacture of a medicament for treating an allergic or autoimmune disease.

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