Protein crosslinks to prevent fibril formation

Abstract

A method is provided for making a protein blocking assembly that is formed by reversibly linking a therapeutic protein to a crosslinking material. The protein blocking assembly contains a chemical structure, which is formed between the multiple bonds between the protein and the crosslinking material, that prevents the interaction of the protein blocking assembly with a second protein due to steric and / or static interference. Furthermore, administration methodologies of administering the protein blocking assembly to a subject are provided, where the protein is cleaved from the protein blocking assembly during or after administration.

Description

PROTEIN CROSSLINKS TO PREVENT FIBRIL FORMATIONRELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 714,411 entitled “PROTEIN CROSSLINKS TO PREVENT FIBRIL FORMATION,” filed October 31, 2024, the entire disclosure of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. 5R01DK123689 awarded by the National Institutes of Health. The government may have certain rights in the invention.BACKGROUND1. Field of the Invention

[0003] Various aspects of the present invention relate generally to therapeutic proteins. More specifically, certain aspects of the invention generally relate to assemblies for preventing protein fibril formation, and methods of making and using the assemblies.2. Description of the Related Art

[0004] Proteins and peptides are classes of pharmaceuticals that are of increasing importance, and that command a growing proportion of newly identified therapeutics. Part of this expansion is due to the diversity of structures that are readily available, as well as the ease of identifying protein-based binding partners for specific therapeutic targets. One of the main problems with proteins is their ability to self-associate. This creates self-associated species with limited efficacy or altered pharmacological properties.

[0005] For example, some therapeutic proteins suffer from the formation of fibrils, regular fiber-like structures that are stabilized by protein-protein interactions and formed when individual proteins (e.g., peptide monomers) repeatedly stick to each other. These fibrils typically have lower solubility than the precursor monomeric proteins, and in addition, lose the biological activity foundin the peptide monomers. Examples of therapeutic proteins that suffer from this phenomenon are insulin and glucagon.

[0006] Insulin is a signaling molecule that also binds to a receptor and induces the uptake of blood glucose. Insulin is soluble at neutral pH, but over time can self-associate to also form fibrils. These fibrils do not retain the ability of monomeric insulin molecules to bind to their target receptor. Thus, insulin bioactivity degrades over time due to fibril formation. In addition to fibril formation, insulin can self-associate to form hexamers. This hexamer formation can be encouraged by including zinc in formulations of insulin. Hexamers are resistant to fibril formation but have their own problems. Specifically, they are large compared to monomeric insulin and so absorb much more slowly than does monomeric insulin, thereby slowing their desired physiological response (blood glucose reduction).

[0007] Previously, others have attempted to deal with protein self-association by altering the primary structure of proteins, such as glucagon and insulin, in order to remove the potential sites of interaction that drive the association. This has worked to a degree but inherently requires the creation of a non-natural protein. Consequently, this introduces a potential source of toxicity, as it introduces a non-native protein into the body. Thus, there are still issues with preventing selfassociation in various therapeutic proteins.SUMMARY

[0008] One or more embodiments of the present disclosure generally concern a method for reversibly preventing a protein from interacting with a second protein. The method generally comprises: (a) providing the protein; (b) providing a crosslinking material; and (c) producing a protein blocking assembly by forming a first bond and a second bond between the crosslinking material and the protein via two different functional groups on the protein. In such embodiments, the protein blocking assembly comprises a chemical structure that prevents interaction of the protein blocking assembly with the second protein due to steric interference or static interference. In addition, the first bond and the second bond are cleavable.

[0009] One or more embodiments of the present disclosure generally concern a method for treating an animal with a protein blocking assembly. Generally, the method comprises: (a) providing a protein blocking assembly comprising a protein bonded to a crosslinking material via a first bond and a second bond, wherein the first bond and the second bond are associated with twodifferent functional groups on the protein, wherein the protein blocking assembly comprises a chemical structure that prevents interaction of the protein blocking assembly with a second protein due to steric interference or static interference; (b) administering the protein blocking assembly to the animal in need of the protein; and (c) cleaving at least the first bond or the second bond within the animal to thereby release the protein from the protein blocking assembly.

[0010] One or more embodiments of the present disclosure generally concern a protein blocking assembly comprising: (a) a protein; and (b) a crosslinking material. In such embodiments, the protein and the crosslinking material are bonded via a first bond and a second bond, wherein the first bond and the second bond are associated with two different functional groups on the protein. Additionally, the protein blocking assembly comprises a chemical structure that prevents interaction of the protein blocking assembly with a second protein due to steric interference or static interference.BRIEF DESCRIPTION OF THE FIGURES

[0011] Embodiments of the present invention are described herein with reference to the following drawing figures, wherein:

[0012] FIG. 1 is an exemplary schematic depicting an interaction between the formed crosslinks of the present disclosure and the mobile segments on certain proteins;

[0013] FIG. 2 is an exemplary schematic depicting an interaction between the formed crosslinks of the present disclosure and certain proteins;

[0014] FIG. 3 depicts isosorbide activation by carbonyldiimidazole (“CDI”);

[0015] FIG. 4 depicts reaction schematics between insulin and activated isosorbide;

[0016] FIG. 5 depicts an analytical HPLC chromatogram for the reaction between activated isosorbide and insulin;

[0017] FIG. 6 is an UHPLC chromatogram of the reaction product between activated isosorbide and insulin;

[0018] FIG. 7 depicts an analytical HPLC chromatogram of the collected isomer fraction from the reaction product between activated isosorbide and insulin;

[0019] FIG. 8 is a mass spectrometer analysis of the collected isomer fraction from the reaction product between activated isosorbide and insulin;

[0020] FIG. 9 depicts the raw mass spectra for isomer 1 from Example 1;

[0021] FIG. 10 depicts the mass spectra measurements of isomer 1 from Example 1 ;

[0022] FIG. 11 depicts the raw mass spectra for isomer 2 from Example 1;

[0023] FIG. 12 depicts the mass spectra measurements of isomer 1 from Example 1;

[0024] FIG. 13 is a graph of a ThT assay depicting the fluorescence signal of the control insulin and produced monocrosslinked insulin from Example 1, wherein the plotted signal is an average of three determinations;

[0025] FIG. 14 is a graph showing a long term fibrillation assay of the control insulin and produced monocrosslinked insulin from Example 1, wherein the plotted signal is an average of three determinations;

[0026] FIG. 15 depicts activation of naphthalenediols by carbonyldiimidazole (“CDI”);

[0027] FIG. 16 depicts reaction schematics between insulin and the activated naphthalenediols;

[0028] FIG. 17 depicts anHPLC chromatogram for 2,6-monocrosslinked insulin synthesis;

[0029] FIG. 18 depicts an HPLC chromatogram of repurified 2,6-monocrosslinked insulin;

[0030] FIG. 19 depicts a total ion chromatogram from LC-MS (QToF) of 2,6- monocrosslinked insulin;

[0031] FIG. 20 depicts a raw mass spectra of 2,6-monocrosslinked insulin;

[0032] FIG. 21 depicts a reconstructed mass spectra of 2,6-monocrosslinked insulin;

[0033] FIG. 22 depicts an HPLC chromatogram of crude reaction mixture for 2,7- monocrosslink insulin synthesis;

[0034] FIG. 23 depicts an LC-MS (QToF) of monocrosslink insulin 2,7-monocrosslinked insulin;

[0035] FIG. 24 depicts a raw mass spectra of 2,7-monocrosslinked insulin;

[0036] FIG. 25 depicts a reconstructed mass spectra of 2,7-monocrosslinked insulin;

[0037] FIG. 26 depicts an HPLC chromatogram of crude reaction mixture for 1,5- monocrosslink insulin synthesis;

[0038] FIG. 27 depicts a LC-MS (QToF) of monocrosslink insulin 1,5 -monocrosslinked insulin;

[0039] FIG. 28 depicts a raw mass spectra of 1,5-monocrosslinked insulin; and

[0040] FIG. 29 depicts a reconstructed mass spectra of 1,5-monocrosslinked insulin.DETAILED DESCRIPTION

[0041] The present disclosure provides a method for preventing protein self-association without changing the primary structure of the therapeutic protein. Generally, the method involves forming a protein blocking assembly, particularly an anti-fibril protein assembly, by linking a therapeutic protein to a crosslinking material containing a linking moiety. The linking moiety of the crosslinking material creates two or more reversible bonds between the protein. The resulting protein blocking assembly presents a stable form of the protein that inhibits formation of fibrils, hexamers, or other protein-protein complexes and structures. When administered to a subject, the bonds are cleaved to release the native, active protein. As used herein, the terms “anti -fibril protein assembly” and “protein blocking assembly” may be used interchangeably and refer to the protein complex formed between the protein of interest and the crosslinking material described herein.

[0042] More particularly, the present disclosure describes a way of preventing fibril formation in proteins, by introducing a crosslink or crosslinks in the proteins that limit the exposure of key portions of the protein which facilitate fibril formation. Generally, in various embodiments, these crosslinks may be introduced by reacting two or more amino acid side chains in the protein with a crosslinking material containing a linking moiety. The resulting interaction can form an anti-fibril protein assembly that contains at least two bonds between at least two different functional groups on the protein and the crosslinking material. The anti-fibril protein assembly contains a chemical structure, which is formed between the multiple bonds between the protein and the crosslinking material, that prevents the interaction of the anti-fibril protein assembly with a second protein due to steric and / or static interference.

[0043] In various embodiments, and in contrast to the methodology described in U.S. Patent Application Publication No. 2023 / 0025256, the entire disclosure of which is incorporated by reference, the protein blocking assembly of the present disclosure does not utilize an additional blocking molecule with a blocking group. In other words, in such embodiments, the protein blocking assembly of the present disclosure will not contain a separate blocking material / molecule that is distinct and separate from the crosslinking material with the linking moiety. Unlike previous methodologies, which utilize a linking molecule to form a bridge between the protein and a blocking molecule with a blocking group capable of deterring fibril formation, the methodology of the present disclosure does not contain or utilize such blocking compounds. Rather, the crosslinking material, by itself, can form at least two separate bonds with the protein. Though notwishing to be bound by theory, it is believed that the resulting protein blocking assembly between the protein and the crosslinking material forms a complex that prevents the interaction of the protein blocking assembly with a second protein due to steric and / or static interference. Additionally, in various embodiments, the resulting linkages between the protein and the crosslinking material can be cleaved in the body by chemical, enzymatic, photolytic, and / or other mechanisms to release the crosslinking material and protein, after the protein is administered.

[0044] The objective of the crosslinking material is to interfere with the contact of one protein molecule with another. Generally, the crosslinking material is preferably selected to form at least two separate bonds with the protein and thereby interfere with the point of contact of another protein with the bonded protein. This blocking effect may be brought about by different properties of the crosslinking material, including steric factors (size and / or shape) and electrostatic factors (charges and / or partial charges) that cause repulsion between protein blocking assemblies. More particularly, the resulting protein blocking assembly can prevent the formation of undesirable protein-protein complexes, such as fibrils and / or hexamers, via steric and / or electrostatic interference caused by the protein blocking assembly’s resulting size, shape, and / or charge (e.g., partial charge), which is derived from the combination of the protein and the crosslinking material.

[0045] The aim of the incorporated crosslink within the protein blocking assembly is to prevent fibril formation and / or protein aggregation from taking place. Without wishing to be bound by theory, there are two ways in which the crosslinking material and the resulting crosslinks it can form with the protein can be effective. In the first case, as shown in FIG. 1, a protein that has a conformationally flexible or mobile segment, which has the propensity to stick to the same segment in other proteins, is depicted. An example of such a protein is insulin, where the C terminal end of the B strand is both mobile (conformationally flexible) and “sticky” and, therefore, will aggregate with the same segment in other insulin molecules, resulting in the formation of an aggregate or fibril. In this case, that interaction is driven by beta sheet formation. As shown in FIG. 1, the crosslink formed by the crosslinking material can prevent the mobility of this protein segment. Consequently, this then prevents it from being exposed and from then interacting with the same segment in similarly crosslinked insulin molecules, thus blocking aggregation / fibril formation.

[0046] In the second case, as depicted in FIG. 2, a protein is shown that has a propensity to form an extended adherent conformation, which can adhere tightly to other proteins of the same type. Typically, this conformation is an extended, often beta sheet, conformation. An example of this type of protein is glucagon, which easily forms an extended beta conformation that can adhere to other glucagon molecules to form insoluble fibrils and aggregates. In this case, and as shown in FIG. 2, the crosslink of the present disclosure prevents this extended and particularly “sticky” conformation from forming by distorting the protein. Consequently, this then prevents the individual modified proteins from efficiently packing, thereby preventing fibril / aggregate formation.

[0047] Generally, in one or more embodiments, the crosslinking material comprises a linking moiety for bonding to the protein of interest. In various embodiments, the linking moiety comprises two or more reactive groups that can form at least two covalent bonds with side chains of the protein.

[0048] In one or more embodiments, the crosslinking material comprises a peptide, a lipid, a small molecule, a polyol (e.g., diol), a nucleic acid, a saccharide, or a combinations thereof. In certain embodiments, the crosslinking material can be a polyol, such as a diol. For example, the crosslinking material can be an aromatic or non-aromatic activated diol.

[0049] In one or more embodiments, the crosslinking material can be an activated aromatic diol and / or an activated non-aromatic diol, which has been activated with one or more condensing agents such as carbonyldiimidazole (“CDI”), N,N'-dicyclohexylcarbodiimide (“DCC”), l-(3- dimethylaminopropyl)-3-ethylcarbodiimide (“EDC”), uranium salts (“HATU”), and / or other condensing agents. In such embodiments, the diols can comprise isosorbide, 2,6-naphthalenediol, 2,7-naphthalenediol, 1,5 -naphthalenediol, or a combination thereof.

[0050] In certain embodiments, the crosslinking material can be an isosorbide, such as activated D-isosorbide, which has been activated with one or more condensing agents including carbonyldiimidazole (“CDI”), N,N'-dicyclohexylcarbodiimide (“DCC”), l-(3- dimethylaminopropyl)-3-ethylcarbodiimide (“EDC”), uranium salts (“HATU”), and / or other condensing agents. Generally, if an activated diol, such as activated D-isosorbide, is utilized, it can be activated with CDI.

[0051] Generally, the crosslinking material has a linking moiety with functional groups suitable for bonding to the protein. Suitable functional groups associated with the linking moietycan include, for example, amines, hydroxyl groups, alcohols, carboxylic acids, guanidinium, amide, thiols, or a combination thereof. Other suitable functional groups may include vinylsulfone, alkyne, azide, maleimide, isothiocyanate, isocyanate, imidate, alpha-halo-amide, Michael acceptor, hydrazide, oxyamine, hydrazine, alkyl, aryl, alkenyl, alkynyl, cyano, nitro, azido, heterocycles, and combinations thereof or with the previously listed functional groups. Terminal groups may be added to protect and / or modify the charge of crosslinking material. Such modifications are considered to be within the scope of the peptides and other crosslinking materials disclosed herein. As used herein, a “linking moiety” refers to one or more substituent functional groups that forms part of the crosslinking material. For example, the linking moiety can include a plurality of hydroxyl groups, amines, alcohols, carboxylic acids, or combinations thereof that are capable of forming bonds with the protein of interest.

[0052] Generally, the linking moiety of the crosslinking material forms two or more cleavable bonds (i.e., at least a first bond and a second bond) with the protein. As used herein, a cleavable or reversible bond is a bond that can be cleaved by chemical, enzymatic, photolytic, or other mechanisms to release the crosslinking material and protein, preferably without alteration of the native form of the protein. In certain embodiments, the cleavable bonds are sensitive to endogenous chemical and / or enzymatic reactions, i.e., reactions that naturally take place in the body of a subject to which the protein blocking assembly is administered. Examples of such reactions include hydrolysis by esterases, hydrolysis by peptidases, hydrolysis by phosphatases, hydrolysis by other enzymes, reduction, oxidation, and combinations thereof. Alternatively, the linking moiety of the crosslinking material can form photocleavable bonds, which may be removed by application of a light source with a wavelength matched to the photocleavable linking moiety. In certain other embodiments, other chemical or enzymatic reactants can be introduced to the subject to cleave one or more of the bonds of the protein blocking assembly, either before, simultaneously with, or after administration of the protein blocking assembly.

[0053] In one or more embodiments, the linking moiety of the crosslinking material comprises chemical groups that produce bonds sensitive to chemical, enzymatic, and / or photolytic reactions. Bonds formed between the linking moiety and the protein consistent with the present disclosure include, but are not limited to, esters, amides, carbamates, carbonates, phospho-esters, phosphor-amides, di-sulfides, ethers, ketals, aminals, acetals, sulfonamides, imines, and / or hydrazones and as well as other groups known to be useful in forming prodrugs. Chemical groupsmay be added to facilitate formation of desired bonds. Exemplary functional groups in the linking moiety of the crosslinking material that can be used to create the cleavable bonds with the protein can include amino, carboxyl, thiol, phosphate, hydroxyl, and / or alcohol. Suitable chemical groups and bonds will depend in part on the physiological characteristics of the portion of the body into which the protein blocking assembly will be administered.

[0054] In one or more embodiments, the crosslinking material is in the form of small molecules and, therefore, there is no need for further biodegradation of the crosslinking material once it is cleaved from the protein. The crosslinking material can be easily cleared from the body by natural pathways after cleavage. Generally, the crosslinking material does not comprise a long polymer chain and is not a polymer backbone to which multiple drugs are cross-linked. In certain embodiments, the molecular weight of the crosslinking material is less than 5,000, 4,000, 3,000, 2,000, 1,000, or 500 g / mol. For example, the crosslinking material can have a molecular weight in the range of 10 to 5,000, 50 to 3,000, or 100 to 1,000 g / mol.

[0055] In one or more embodiments, the crosslinking material is bioresorbable. As used here, the term “bioresorbable” refers to a material whose degradative products, or the group itself, are metabolized in vivo or excreted from the body via natural pathways. In general, by “bioresorbable,” it is meant that the material will be broken down and absorbed within the human body, for example, by a cell or tissue.

[0056] In one or more embodiments, the crosslinking material is biocompatible. As used herein the term “biocompatible” means that the material will not cause substantial tissue irritation or necrosis when administered. Preferably, the material is approved for use in the body by the Food and Drug Administration.

[0057] In one or more embodiments, the crosslinking material is bioresorbable and biocompatible. In general, the crosslinking material can include any agent that may be linked to the protein, upon exposure to physiological conditions, chemical effectors, enzymes, and / or light, releases the therapeutic peptide in functional form (or a suitable prodrug form). In various embodiments, the crosslinking material may have a formulation that comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 atoms (e.g., carbon atoms). Additionally, or alternatively, the crosslinking material may have a formulation that comprises less than 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, or 23 atoms (e g., carbon atoms).

[0058] In one or more embodiments, the linking moiety of the crosslinking material may be comprised of carbon, nitrogen, oxygen, sulfur, hydrogen, phosphorous atoms, or combinations thereof. For example, the linking moiety may be a hydroxyl group.

[0059] Generally, in various embodiments, the crosslinking material can form multiple bonds to the protein via the linking moiety of the crosslinking material. In such embodiments, the bonding occurs via chemical side chain functional groups on the protein, or a functional group may be added to the protein to facilitate bonding. Suitable side chain functional groups in such embodiments can include amines, alcohols, carboxylic acids, guanidinium, amide, and / or thiol. Functional groups that may be added to the protein include carboxylic halide, vinylsulfone, alkyne, azide, maleimide, isothiocyanate, isocyanate, imidate, alpha-halo-amide, Michael acceptor, hydrazide, oxyamine, and / or hydrazine.

[0060] In one or more embodiments, the ratio of protein to the crosslinking material in the protein blocking assembly can be greater than 2:1, 3: 1, 4: 1, 5:1, 6: 1, 7: 1, 8:1, 9: 1, or 10: 1 (wt:wt) and / or less than 100: 1, 50: 1, 40: 1, or 30: 1 (wt:wt). For example, the ratio of protein to the crosslinking material in the protein blocking assembly can be 2: 1 to 100: 1 or 2: 1 to 50: 1.

[0061] In one or more embodiments, the protein may be any therapeutic protein useful in the treatment or prevention of a disease or condition. A “therapeutic protein” as used herein refers to any peptide or protein that alters the physiology of a patient. The term “therapeutic” may be used interchangeably herein or in the art with the terms “biologically active” and “pharmaceutically active” and includes analogs and derivatives of a therapeutic protein. Thus, the “therapeutic protein” that is cleaved form the protein blocking assembly may be a drug, drug precursor, prodrug, or modified drug that is not fully active or available until converted in vivo to its therapeutically active or available form. The protein may be naturally occurring or synthetic.

[0062] The protein blocking assembly may be particularly useful with therapeutic proteins that are vulnerable to self-association. Representative non-limiting classes of proteins useful in the present invention include those falling into the following therapeutic categories: dACE-inhibitors; anti-anginal drugs; anti-arrhythmias; anti-asthmatics; anti-cholesterolemics; anti-convulsants; anti-depressants; anti-diarrhea preparations; anti-histamines; anti-hypertensive drugs; anti- infectives; anti-inflammatory agents; anti-lipid agents; anti-manics; anti-nauseants; anti-stroke agents; anti-thyroid preparations; anti-tumor drugs; anti-tussives; anti-uricemic drugs; anti-viral agents; acne drugs; alkaloids; amino acid preparations; anabolic drugs; analgesics; anesthetics;angiogenesis inhibitors; antacids; anti -arthri tics; antibiotics; anticoagulants; antiemetics; antiobesity drugs; antiparasitics; antipsychotics; antipyretics; antispasmodics; antithrombotic drugs; anxiolytic agents; appetite stimulants; appetite suppressants; beta blocking agents; bronchodilators; cardiovascular agents; cerebral dilators; chelating agents; cholecystokinin antagonists; chemotherapeutic agents; cognition activators; contraceptives; coronary dilators; cough suppressants; decongestants; deodorants; dermatological agents; diabetes agents; diuretics; emollients; enzymes; erythropoietic drugs; expectorants; fertility agents; fungicides; gastrointestinal agents; growth regulators; hormone replacement agents; hyperglycemic agents; hypnotics; hypoglycemic agents; migraine treatments; mineral supplements; mucolytics; narcotics; neuroleptics; neuromuscular drugs; peripheral vasodilators; polypeptides; prostaglandins; psychotropics; renin inhibitors; respiratory stimulants; stimulants; sympatholytics; thyroid preparations; tranquilizers; uterine relaxants; vaginal preparations; vasoconstrictors; vasodilators; vertigo agents; and wound healing agents.

[0063] In one or more embodiments, the protein is of the type described in U.S. Patent Application Publication No. 2011 / 0166063 and / or U.S. Patent No. 6,858,580, both which are incorporated by reference herein with respect to such disclosures. Exemplary therapeutic peptides and proteins may be selected from the group consisting of insulin; glucagon; calcitonin; gastrin; parathyroid hormones; angiotensin; growth hormones; secretin; luteotropic hormones (prolactin); thyrotropic hormones; melanocyte-stimulating hormones; thyroid-stimulating hormones (thyrotropin); luteinizing-hormone-stimulating hormones; vasopressin; oxytocin; protirelin; peptide hormones such as corticotropin; growth-hormone-stimulating factor (somatostatin); G- CSG, erythropoietin; EGF; physiologically active proteins, such as interferons and interleukins; superoxide dismutase and derivatives thereof; enzymes such as urokinases and lysozymes; and analogues or derivatives thereof. In another aspect, the therapeutic protein is selected from the group consisting of human growth hormone, bovine growth hormone, growth horm one-releasing hormone, an interferon, interleukin- 1, interleukin-II, insulin, calcitonin, erythropoietin, atrial natriuretic factor, an antigen, a monoclonal antibody, somatostatin, adrenocorticotropin, gonadotropin releasing hormone, oxytocin, vasopressin, analogues, or derivatives thereof.

[0064] In one or more embodiments, the therapeutic protein is an anti-diabetic agent already in the clinical practice or in the pipeline of development. The anti-diabetic drug molecules are broadly categorized herein as insulin / insulin analogs and non-insulin anti-diabetic drugs.

[0065] In one or more embodiments, the protein is insulin, glucagon, or immunoglobulin. One preferred therapeutic peptide is glucagon (or an analog or derivative thereof). Another preferred therapeutic peptide is insulin (or an analog or derivative thereof). As used herein, the term insulin embraces analogues or derivatives thereof. Exemplary insulin compounds are described in U.S. Patent Application Publication No. 2011 / 0144010, which is incorporated by reference with respect to such disclosures. In certain embodiments, the therapeutic peptide is insulin (or an analog or derivative thereof) in its hexameric form, typically in the presence of zinc.

[0066] The protein blocking assembly of the present disclosure may be administered to a subject in need of the protein. After administration to the subject, the protein blocking assembly may be cleaved to release the protein, allowing the protein to perform its intended function. Preferably, the protein is in its native state after being cleaved. In certain embodiments, the protein blocking assembly comprises a first bond and a second bond between the linking moiety of the crosslinking material and the protein, and one or both bonds are cleaved after administration.

[0067] In various embodiments, the resulting anti-fibril protein assembly may be administered to a subject in a pharmaceutically acceptable formulation. The anti -fibril protein assembly is preferably stable in the formulation. However, after administration, the crosslinking material may be cleaved from the protein in the body to thereby reveal the native, active protein, In certain embodiments, the crosslinking material may be removed by endogenous chemical or biochemical processes. For example, the crosslinking material may be removed by a chemical effector and / or an enzyme, such as a chemical effector or enzyme present in the body of the subject, more preferably by a chemical effector or enzyme naturally occurring in the body of the subject. As used herein, a “chemical effector” is any chemical that can affect cleavage of the bond connecting the protein and the linking moiety. In other embodiments, the crosslinking material may be linked to the protein via a photocleavable linking moiety, and the crosslinking material may be removed by application of a light source with a wavelength matched to the photocleavable linking moiety.

[0068] Generally, the subject is an animal (e.g., a warm-blooded mammal) and may be either a human or a non-human animal. Exemplary non-human animals include, but are not limited to, non-human primates, rodents, farm animals (e.g., cattle, horses, pigs, goats, and sheep), and pets (e.g., dogs, cats, ferrets, and rodents). The patient is typically a mammal. The term “mammal”refers to organisms from the taxonomy class “mammalian,” including, but not limited to, humans, chimpanzees, apes, orangutans, monkeys, rats, mice, cats, dogs, cows, horses, etc.

[0069] The method of administration will depend on the therapeutic protein used. Suitable methods of administration include cutaneous, subcutaneous, intravenous, or intramuscular injection. The protein blocking assembly can also be delivered nasally, transbuccally, sublingually, or via similar administration routes. In certain embodiments, the protein blocking assembly can be administered in a manner similar to that used for the native peptide. It is further contemplated that the protein-blocking group can be administered through an artificial pancreas system.

[0070] The protein blocking assembly is typically administered to the target site of the subject using a “cannula” or “needle” that can be a part of a drug delivery device, e.g., a syringe, a gun drug delivery device, or any medical device suitable for the application of a drug to a targeted organ or anatomic region. The cannula or needle of the protein blocking assembly is designed to cause minimal physical and psychological trauma to the subject.

[0071] Cannulas or needles include tubes that may be made from materials, such as, for example, polyurethane, polyurea, polyether(amide), PEBA, thermoplastic elastomeric olefin, copolyester, styrenic thermoplastic elastomer, steel, aluminum, stainless steel, titanium, metal alloys with high non-ferrous metal content and a low relative proportion of iron, carbon fiber, glass fiber, plastics, ceramics, or combinations thereof. The cannula or needle may optionally include one or more tapered regions. The cannula or needle may also be beveled. The cannula or needle may also have a tip style vital for accurate treatment of the patient depending on the site for implantation. Examples of tip styles include, for example, Trephine, Coumand, Veress, Huber, Seidinger, Chiba, Francine, Bias, Crawford, deflected tips, Hustead, Lancet, or Tuohey. The cannula or needle may also be non-coring and have a sheath covering it to avoid unwanted needle sticks. The dimensions of the hollow cannula or needle, among other things, will depend on the site for injection.

[0072] In one or more embodiments, the protein blocking assembly of the present invention is formulated in a pharmaceutically acceptable carrier. It will be appreciated to those skilled in the art that the carrier may optionally contain inactive materials such as saline; buffering agents; pH adjusting agents, such as potassium bicarbonate, potassium carbonate, potassium hydroxide, sodium acetate, sodium borate, sodium bicarbonate, sodium carbonate, sodium hydroxide, orsodium phosphate; degradation / release modifiers; drug release adjusting agents; emulsifiers; preservatives, such as benzalkonium chloride, chlorobutanol, phenylmercuric acetate and phenylmercuric nitrate, sodium bisulfate, sodium bisulfite, sodium thiosulfate, thimerosal, methylparaben, polyvinyl alcohol, and / or phenylethyl alcohol; solubility adjusting agents; stabilizers; and / or cohesion modifiers. If the assembly is to be injected the spinal area, the carrier may comprise a sterile preservative free material.

[0073] Upon administration, the protein should be rapidly released from the crosslinking material. For example, the protein may be cleaved from the protein blocking assembly by a chemical effector or enzyme, as described above. In certain embodiments, the multiple bonds between the linking moiety of the crosslinking material and protein are cleavable by hydrolysis by esterases, hydrolysis by peptidases, hydrolysis by phosphatases, hydrolysis by other enzymes, reduction, and / or oxidation.

[0074] In certain embodiments, the protein can be insulin and the crosslinking material can be a crosslinking diol, such as isosorbide, which has been activated using carbonyl di-imidazole (“CDI”). In these embodiments, the crosslinking diol can form a single crosslink between two amines in the insulin via two carbamate linkages. The resulting modified insulin may show complete resistance to fibril formation, with no fibrils forming after 65 hours of heating and agitation.

[0075] Various aspects of the present disclosure are directed to the protein blocking assembly produced using any of the methods, proteins, and / or crosslinking materials discussed herein. Additional aspects of the present disclosure are directed to methods for using any such protein blocking assemblies.

[0076] This invention can be further illustrated by the following examples of embodiments thereof, although it will be understood that these examples are included merely for the purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated.EXAMPLESExample 1

[0077] Using insulin as a model protein, we reacted insulin with a crosslinking diol (isosorbide) using carbonyl di-imidazole (“CDI”). First, isosorbide was activated with the CDI.More particularly, dianhydro-D-glucitol / D-isosorbide (15mg, O.lmmol) and carbonyldiimidazole (CDI) (65.2 mg, 0.4mmol) were mixed in 5 mL of tetrahydrofuran (THF). This activation of isosorbide by CDI was kept at 40°C for 24 hours under continuous stirring. The reaction progress and formation of activated isosorbide were monitored using thin-layer chromatography (“TLC”).

[0078] The reaction scheme for the formation of activated isosorbide is shown in FIG. 3. The activated isosorbide was then extracted by adding 50 mL of dichloromethane (“DCM”) in the reaction mixture. This mixture was washed twice with water (2 x 50 mL) followed by brine and dried over anhydrous sodium sulfate. A rotary evaporator was used to remove the solvent. The end product, activated isosorbide, was confirmed by TLC using a solvent system (10% methanol in DCM). This reaction was performed multiple times to obtain enough activated isosorbide for further reactions.

[0079] As shown in FIG. 3, activated isosorbide contains two imidazole carbonyloxy ester groups, which can react with amines to form carbamate bonds, thereby releasing imidazole. Insulin has three reactive amines that can react with the two activated ester groups of isosorbide (i.e., the linking moiety of isosorbide) to form a crosslink between amines, thereby yielding a monocrosslinked insulin.

[0080] Monocrosslinked insulin was prepared by dissolving insulin (47.04 mg, 8 pmol) in 8 mL of anhydrous dimethyl sulfoxide (“DMSO”). The activated isosorbide (13.98 mg, 40 pmol) was then added to the dissolved mixture and dissolved. This reaction was incubated at 40°C for 5 days. The schematics of this reaction are depicted in FIG. 4. The reaction was repeated as needed to produce additional monocrosslinked insulin.

[0081] This reaction was monitored using analytical high-performance liquid chromatography (“HPLC”) (C4 column, phenomenex, 3.6 pm, 150 * 2.1 mm). FIG. 5 shows the analytical HPLC chromatogram for this reaction. As shown in FIG. 5, reveals two distinct peaks corresponding to the two isomers (isomer 1 and isomer 2) of monocrosslinked insulin. Thus, this reaction generated two isomers, namely isomer 1 and isomer 2, of monocrosslinked insulin as shown in FIG. 5. Both isomers were purified using ultra-high-performance liquid chromatography (“UHPLC”) with a C 14 semi-prep column (250 * 10mm, phenomenex, 5 pm, 300° A). The UHPLC chromatogram is shown in FIG. 6, where both isomers eluted as a single peak. The boxed region in FIG. 6 indicated the collected fraction contained both monocrosslinked insulin isomers.

[0082] The UHPLC fractions were analyzed by analytical HPLC and mass spectrometry (HPLC-MS QToF) to confirm the identity and purity of both isomers.

[0083] The zoomed analytical HPLC chromatogram is shown in FIG. 7, displaying two distinct peaks corresponding to isomer 1 and isomer 2. FIG. 8 shows the mass spectrometry analysis confirming both isomers of monocrosslinked insulin. As the isomers differed only in the site of crosslinking, their expected molecular masses were identical.

[0084] The expected molecular mass of monocrosslinked insulin is 6004.6 amu, with expected m / 4 and m / 5 ions at 1502.4 amu and 1202.1 amu, respectively. Raw mass spectra for isomer 1 and isomer 2 are shown in FIGS. 9 and 11, respectively. The observed m / 4 and m / 5 values matched the theoretical masses, confirming the identities of both isomers. Reconstructed mass spectra (FIGS. 10 and 12) further confirmed the expected molecular weights of 6005.6 amu (isomer 1) and 6004.6 amu (isomer 2).

[0085] The isomer fraction collected from UHPLC was then dialyzed. The dialyzing membrane (pre-wetted regenerated cellulose membrane - MWCO 1 kD) was pre-soaked in deionized water for 30 minutes prior to its use. The fraction was sealed within the membrane using magnetic clips and dialyzed first against phosphate-buffer (20 mM, pH 7.0) for 24 hours (buffer changed after 8 hours), followed by deionized water for another 9 hours (water changed every 3 hours). The dialyzed product was flash-frozen and lyophilized.

[0086] Analytical HPLC was carried out using reversed-phase HPLC (flow rate 0.4 mL / min, run time 60 minutes with 5 minutes post-run) with solvent A (0.1% TFA in H2O), solvent B (0.1% TFA in ACN). The gradient was 3% B isocratic for first 3 minutes, which was increased to 20% B in 20 minutes, increased to 30% B in 40 minutes, increased to 40% B in 45 minutes, increased to 97% B in 50 minutes, decreased to 3% B in 52 minutes, 3% B isocratic until 55 minutes. This was carried out in a C4 Phenomenex column (3.6 pm, 150 x 2.1 mm).

[0087] UHPLC was carried out via reversed-phase UHPLC (flow rate 2.5 mL / min, runtime 60 minutes with 5 minutes pre-equilibration) with solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in ACN). The gradient was 5% B in 0 minutes, increased to 25% B in 7 minutes, increased to 35% B in 15 minutes, increased to 43% B in 40 minutes, 43% B isocratic in 41 minutes, decreased to 100% B in 55 minutes. This was carried out in a C4 semi-prep Phenomenex column (5 pm, 250 * 10 mm).

[0088] Mass spectrometry (LCMS-QToF) was carried out via reversed-phase HPLC (flow rate 0.4 mL / min, runtime 55 minutes) with solvent A (0.1% formic acid in H2O) and solvent B (0.1% formic acid in ACN). The gradient was 5% B isocratic for first 3 minutes, increased to 20% B in 20 minutes, increased to 30% B in 40 minutes, increased to 40% B in next 45 minutes, increased to 97% B in next 50 minutes, decreased to 3% B in 52 minutes, and 3% B isocratic in 55 minutes. This was carried out in a C4 Phenomenex column (3.6 pm, 150 * 2.1 mm).

[0089] Thioflavin T (“ThT”) is a fluorescent dye that binds to amyloid fibrils with P-sheet structures, thereby producing a characteristic fluorescence signal upon excitation. Fibril formation kinetics for monocrosslinked insulin were monitored using both short-term and long-term ThT assays, as described below.

[0090] An insulin control and the formed monocrosslinked insulin were weighed and both the samples were dissolved in buffer (10 mM PBS, 150 mM NaCl, pH 7) at concentrations of 130 pM and 110 pM, respectively. Both the samples were then passed through a 0.22 pm syringe filter. UV spectroscopy was then used to quantify the concentrations of the filtered samples.

[0091] A thioflavin T (“ThT”) solution (8 mg, 25 pmol) was prepared by dissolving in 10 mb buffer (lOmM PBS, 150 mM NaCl, pH 7). The solution was kept at 40°C with intermittent shaking to dissolve the dye. Once dissolved, the solution was filtered using a 0.22 pm filter.

[0092] 20pl of this ThT solution was then separately combined with the insulin control and the monocrosslinked insulin, along with buffer, into clear bottom, black 96 well plates. The volume of the insulin control and monocrosslinked insulin was adjusted in a way that the concentration of insulin control and monocrosslinked insulin in the assay was kept a constant of 25 pM. The volume in each well was 200 pL. The analysis was performed in triplicates. The samples were incubated at 48°C with shaking. Thioflavin T measurements were taken every 15 minutes using a Spectramax multimode microplate reader with an excitation and emission wavelengths of 440 nm and 482 nm, respectively. The concentration of ThT in the assay was 250pM.

[0093] FIG. 13 is a graph showing the result of the ThT assay for the insulin control and monocrosslinked insulin. As shown in FIG. 13, the insulin control showed a higher fluorescence signal within 5 hours of the start of the assay. This indicates that fibril formation has occurred within the insulin control, as ThT bonded to these fibril structure, thereby resulting in a higher fluorescence signal. In contrast, the fluorescence signal for the monocrosslinked insulin remainedconstant even after 65 hours after the start of the assay, indicating the intactness and stability of the protein structure, i.e., there was no fibril formation.

[0094] Subsequently, insulin and monocrosslinked insulin were dissolved in buffer (10 mM PBS, 150 mM NaCl, pH 7.0) to a concentration of 250 pM, filtered (0.22 pm), and quantified by UV spectroscopy. Samples were then diluted to 25 pM and subjected to a longer term fibrillation assay using a ThT solution.

[0095] Another ThT solution (8 mg, 25 pmol) was prepared as above to make a stock solution. For each assay vial (3 mL of insulin or monocrosslinked insulin), the samples were incubated at 48°C with shaking (250 rpm) and analyzed in triplicate. At various time points, 180 pL of sample was mixed with 20 pL of ThT stock solution in a 96-well plate, and fluorescence was recorded using the same reader settings.

[0096] FIG. 14 shows the results of the long term fibrillation assay using the ThT solution. As shown in FIG. 14, the insulin control exhibited increased fluorescence within 10 hours, consistent with fibril formation. In contrast, the monocrosslinked insulin maintained constant fluorescence for up to 70 days, indicating structural stability and absence of fibril formation.

[0097] Thus, this demonstrated that the methodology described herein can effectively install a single crosslink between two amines in insulin via two carbamate linkages. The resulting modified insulin showed complete resistance to fibril formation, with no fibrils forming after 65 hours of heating and agitation (under conditions in which normal insulin fibrillates in less than 5 hours).

[0098] While we have demonstrated this approach with isosorbide as the crosslinking molecule, and amine groups on the insulin forming the crosslink via carbamate bonds, this approach can potentially work with a wide range of crosslinkings, protein functional groups, and bond types in the final crosslink. The benefit of using carbamate linkages is that they can be cleaved in the body by existing enzymes after the protein is administered (i.e. the prodrug approach). Other types of linkages may also be effective in this regard (e.g. ester linkages).Example 2

[0099] For this study, an aromatic activated diol system for monocrosslinking insulin was developed and tested. Specifically, three aromatic diols were tested: 2,6-naphthalenediol, 2,7- naphthalen ediol, and 1,5-naphthalenediol, which differ in the positional arrangement of theirhydroxyl groups. Activation of each diol was achieved through reaction with carbonyldiimidazole (“CDI”) to yield the corresponding activated naphthalenediols. These were then reacted with insulin to make crosslinked insulins, analogous to the method described in Example 1.

[0100] First, activated 2,6-naphthalenediol was created by dissolving 2,6-naphthalenediol (32.9 mg, 0.2 mmol) and CDI (326.6 mg, 2 mmol) in 10 m of tetrahydrofuran (“THF”). The mixture was stirred continuously at 40°C for 24 h, during which a precipitate formed, indicating product formation. The reaction was quenched by adding 10 mL of water, and the precipitate was collected using a 0.22 pm filter and vacuum-dried. The reaction scheme is depicted in FIG. 15, which yielded the activated 2,6-naphthalenediol.

[0101] Second, activated 1,5 -naphthalenediol was created by dissolving 1,5- naphthalenediol (32.7 mg, 0.2 mmol) and CDI (325.1 mg, 2 mmol) in 10 mL THF and stirred at 40°C for 24 h. The resulting precipitate was collected after quenching with 10 mL water, filtered (0.22 pm), and vacuum-dried. The corresponding reaction scheme is shown in FIG. 15, which yielded the activated 1,5 -naphthalenediol.

[0102] Lastly, activated 2,7-naphthalenediol was created by dissolving 2,7- naphthalenediol (32.3 mg, 0.2 mmol) and CDI (325.4 mg, 2 mmol) in 10 mL THF and stirred at 40°C for 24 h. The reaction mixture was then extracted with 200 mL dichloromethane (DCM), washed twice with 100 mL portions of water, followed by brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, yielding activated 2,7-naphthalenediol. The reaction schematics are depicted in FIG. 15.

[0103] Product formation in all cases was confirmed by TLC using a solvent system of 10% methanol in DCM.

[0104] Subsequently, as shown in FIG. 16, monocrosslinked insulin was separately prepared with each of the activated naphthalendiols.

[0105] First, activated 2,6-naphthalenediol (2.3 mg, 6.6 pmol) was dissolved in 1.32 mL of anhydrous DMSO (stock solution). A 0.8 mL aliquot of this stock solution (4 pmol) was combined with insulin (23.8 mg, 4 pmol) in DMSO to a total volume of 4 mL. The reaction was incubated at 40°C for 7 days. The scheme is shown in FIG. 16, yielding 2,6-monocrosslinked insulin.

[0106] Second, activated 1,5 -naphthalenediol (2 mg, 5.7 pmol) was dissolved in 1.149 mL DMSO (stock solution). A 200 pL aliquot of this stock solution (1 pmol) was added to insulin(5.95 mg, 1 pmol) in DMSO, adjusted to a total volume of 1 mL, and incubated at 40°C for 7 days. As shown in FIG. 16, the reaction yielded 1,5 -monocrosslinked insulin.

[0107] Finally, activated 2,7-naphthalenediol (1.89 mg, 5.4 pmol) was dissolved in 1.1 mL DMSO (stock solution ). A 380 pL aliquot of this stock solution (2 pmol) was added to insulin (11.9 mg, 2 pmol) and DMSO to a total volume of 2 mL. The mixture was incubated at 40°C for 7 days, yielding 2,7-monocrosslinked insulin, as shown in FIG. 16.

[0108] All reactions were monitored by analytical HPLC, and product masses were confirmed by LC-MS QToF. The monocrosslinked insulins had identical masses (6019.6 amu) and differ only in the positional isomerism of the crosslinking diol. The expected m / z values were m / 4 = 1505.9 amu and m / 5 = 1204.9 amu.

[0109] Regarding the activated 2,6-naphthalenediol crosslinking, FIG. 17 shows the HPLC chromatogram for the insulin reaction with activated 2,6-naphthalenediol, which yielded a single species of 2,6-monocrosslinked insulin. The product peak was collected, re-purified, and analyzed by mass spectrometry. The observed m / 4 and m / 5 values 1505.9 amu and 1204.9 amu, respectively were in agreement with our expected m / z values (FIGS. 18, 19, and 20), and the reconstructed molecular mass (6019.9 amu; FIG. 21) confirmed product identity.

[0110] Regarding the activated 2,7-naphthalenediol crosslinking, the HPLC chromatogram in FIG. 22 shows a product peak corresponding to 2,7-monocrosslinked insulin. Mass spectrometry analysis (FIGS. 23, 24 and 25) confirmed an observed m / 4 = 1505.9 amu and reconstructed mass = 6018.9 amu, consistent with the expected value (m / 4 = 1505.9 amu and mass 6019.6 amu).

[0111] Regarding the activated 1,5 -naphthalenediol crosslinking, the HPLC chromatogram (FIG. 26) shows one product peak corresponding to 1,5-monocrosslinked insulin. Mass spectrometry (FIGS. 27, 28, and 29) yielded m / 4 = 1505.9 amu and a reconstructed molecular mass of 6018.9 amu, in agreement with the expected m / z and mass (m / 4 = 1505.9 amu and mass 6019.6 amu).

[0112] For the analytical HPLC, reversed-phase HPLC was performed at 0.4 mL / min (runtime = 40 minutes + 5 minutes post-run) using solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in ACN). Gradient: 5% B to 25% B (5 minutes) to 65% B (30 minutes) to 97% B (36 minutes); 97% B isocratic to 37 minutes, returned to 5% B by 40 minutes. Column: C4 Phenomenex (3.6 pm, 150 x 2.1 mm).

[0113] For the mass spectrometry (LC-MS QToF), reversed-phase HPLC was at 0.4 mL / min (runtime = 48 minutes) using solvent A (0.1% formic acid in FLO) and solvent B (0.1% formic acid in ACN). Gradient: 5% B isocratic (3 minutes) to 25% B (8 minutes) to 65% B (33 minutes) to 95% B (38 minutes); 95% B isocratic (39 minutes) to 5% B (40 minutes) to 3% B isocratic (48 minutes). Column: C4 Phenomenex (3.6 pm, 150 x 2.1 mm).DEFINITIONS

[0114] It should be understood that the following is not intended to be an exclusive list of defined terms. Other definitions may be provided in the foregoing description, such as, for example, when accompanying the use of a defined term in context.

[0115] As used herein, the terms “a,” “an,” and “the” mean one or more.

[0116] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and / or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination, B and C in combination; or A, B, and C in combination.

[0117] As used herein, the terms “comprising,” “comprises,” and “comprise” are open- ended transition terms used to transition from a subject recited before the term to one or more elements recited after the term, where the element or elements listed after the transition term are not necessarily the only elements that make up the subject.

[0118] As used herein, the terms “having,” “has,” and “have” have the same open-ended meaning as “comprising,” “comprises,” and “comprise” provided above.

[0119] As used herein, the terms “including,” “include,” and “included” have the same open-ended meaning as “comprising,” “comprises,” and “comprise” provided above.

[0120] Throughout this specification, references to “one embodiment”, “an embodiment”, or “embodiments” mean that the feature or features being referred to are included in at least one embodiment of the technology. Separate references to “one embodiment”, “an embodiment”, or “embodiments” in this description do not necessarily refer to the same embodiment and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, act, etc. described in one embodiment may also be included in other embodiments but is not necessarilyincluded. Thus, the current invention can include a variety of combinations and / or integrations of the embodiments described herein.

[0121] The patent claims at the end of this patent application are not intended to be construed under 35 U.S.C. § 112(f) unless traditional means-plus-function language is expressly recited, such as “means for” or “step for” language being explicitly recited in the claim(s).NUMERICAL RANGES

[0122] The present description uses numerical ranges to quantify certain parameters relating to the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of 10 to 100 provides literal support for a claim reciting “greater than 10” (with no upper bounds) and a claim reciting “less than 100” (with no lower bounds).CLAIMS NOT LIMITED TO DISCLOSED EMBODIMENTS

[0123] Throughout this specification, plural instances may implement components, operations, or structures described as a single instance. Although individual operations of one or more methods are illustrated and described as separate operations, one or more of the individual operations may be performed concurrently, and nothing requires that the operations be performed in the order illustrated. Structures and functionality presented as separate components in example configurations may be implemented as a combined structure or component. Similarly, structures and functionality presented as a single component may be implemented as separate components. These and other variations, modifications, additions, and improvements fall within the scope of the subject matter herein.

[0124] The detailed description of the technology references the accompanying drawings that illustrate specific embodiments in which the technology can be practiced. The embodiments are intended to describe aspects of the technology in sufficient detail to enable those skilled in the art to practice the technology. Other embodiments can be utilized, and changes can be made without departing from the scope of the current invention. The detailed description is, therefore,not to be taken in a limiting sense. The scope of the current invention is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0125] Although the present application sets forth a detailed description of numerous different embodiments, it should be understood that the legal scope of the description is defined by the words of the claims set forth at the end of this patent and equivalents. The detailed description is to be construed as exemplary only and does not describe every possible embodiment since describing every possible embodiment would be impractical. Numerous alternative embodiments may be implemented, using either current technology or technology developed after the filing date of this patent, which would still fall within the scope of the claims.

[0126] The preferred forms of the invention described above are to be used as illustration only, and should not be used in a limiting sense to interpret the scope of the present invention. Modifications to the exemplary embodiments, set forth above, could be readily made by those skilled in the art without departing from the spirit of the present invention.

[0127] The inventors hereby state their intent to rely on the Doctrine of Equivalents to determine and assess the reasonably fair scope of the present invention as it pertains to any apparatus not materially departing from but outside the literal scope of the invention as set forth in the following claims.

Claims

What is claimed is:

1. A method for reversibly preventing a protein from interacting with a second protein comprising:(a) providing the protein;(b) providing a crosslinking material; and(c) producing a protein blocking assembly by forming a first bond and a second bond between the crosslinking material and the protein via two different functional groups on the protein; wherein the protein blocking assembly comprises a chemical structure that prevents interaction of the protein blocking assembly with the second protein due to steric interference or static interference; and wherein the first bond and the second bond are cleavable.

2. The method according to claim 1, wherein the crosslinking material comprises a peptide, a lipid, a diol, a nucleic acid, a saccharide, or a combination thereof.

3. The method according to claim 1, wherein the crosslinking material comprises a diol activated with an activating agent.

4. The method according to claim 3, wherein the diol comprises a non-aromatic diol.

5. The method according to claim 3, wherein the diol comprises an aromatic diol.

6. The method according to claim 3, wherein the diol comprises isosorbide, 2,6- naphthalenediol, 2,7-naphthalenediol, 1,5 -naphthalenediol, or a combination thereof.

7. The method according to claim 3, wherein the diol comprises isosorbide.

8. The method according to claim 3, wherein the activating agent comprises carbonyldiimidazole, N,N'-di cyclohexylcarbodiimide, l-(3-dimethylaminopropyl)-3- ethylcarbodiimide, or uranium salts.

9. The method according to claim 1, wherein the protein is selected from the group consisting of insulin, glucagon, immunoglobulin, a monoclonal antibody, their derivatives or analogs, and combinations thereof.

10. A method for treating an animal with a protein blocking assembly, the method comprising:(a) providing a protein blocking assembly comprising a protein bonded to a crosslinking material via a first bond and a second bond, wherein the first bond and the second bond are associated with two different functional groups on the protein, wherein the protein blocking assembly comprises a chemical structure that prevents interaction of the protein blocking assembly with a second protein due to steric interference or static interference;(b) administering the protein blocking assembly to the animal; and(c) cleaving at least the first bond or the second bond within the animal to thereby release the protein from the protein blocking assembly.

11. The method according to claim 10, wherein the cleaving is carried out by an enzyme or chemical effector endogenous to the animal.

12. The method according to claim 10, wherein the crosslinking material comprises a peptide, a lipid, a diol, a nucleic acid, a saccharide, or a combination thereof.

13. The method according to claim 10, wherein the crosslinking material comprises a diol activated with an activating agent.

14. The method according to claim 13, wherein the diol comprises a non-aromatic diol.1 . The method according to claim 13, wherein the diol comprises an aromatic diol.

16. The method according to claim 13, wherein the diol comprises isosorbide, 2,6- naphthalenediol, 2,7-naphthalenediol, 1,5 -naphthalenediol, or a combination thereof.

17. The method according to claim 13, wherein the diol comprises isosorbide.

18. The method according to claim 13, wherein the activating agent comprises carbonyldiimidazole, N,N'-di cyclohexylcarbodiimide, l-(3-dimethylaminopropyl)-3- ethylcarbodiimide, or uranium salts.

19. The method according to claim 10, wherein the protein is selected from the group consisting of insulin, glucagon, immunoglobulin, a monoclonal antibody, their derivatives or analogs, and combinations thereof.

20. A protein blocking assembly comprising:(a) a protein; and(b) a crosslinking material, wherein the protein and the crosslinking material are bonded via a first bond and a second bond, wherein the first bond and the second bond are associated with two different functional groups on the protein, wherein the protein blocking assembly comprises a chemical structure that prevents interaction of the protein blocking assembly with a second protein due to steric interference or static interference.

21. The protein blocking assembly according to claim 20, wherein the crosslinking material comprises a peptide, a lipid, a diol, a nucleic acid, a saccharide, or a combination thereof.

22. The protein blocking assembly according to claim 20, wherein the crosslinking material comprises a diol activated with an activating agent.

23. The protein blocking assembly according to claim 22, wherein the diol comprises a non-aromatic diol.

24. The protein blocking assembly according to claim 22, wherein the diol comprises an aromatic diol.

25. The protein blocking assembly according to claim 22, wherein the diol comprises isosorbide, 2,6-naphthalenediol, 2,7-naphthalenediol, 1,5-naphthalenediol, or a combination thereof.

26. The protein blocking assembly according to claim 22, wherein the diol comprises isosorbide.

27. The protein blocking assembly according to claim 22, wherein the activating agent comprises carbonyldiimidazole, N,N'-dicyclohexylcarbodiimide, l-(3-dimethylaminopropyl)-3- ethylcarbodiimide, or uranium salts.

28. The protein blocking assembly according to claim 20, wherein the protein is selected from the group consisting of insulin, glucagon, immunoglobulin, a monoclonal antibody, their derivatives or analogs, and combinations thereof.

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