Alkaline phosphatase formulations and uses thereof

Abstract

The present invention relates to alkaline phosphatase formulations and uses thereof, in particular, the present invention provides, in part, formulations comprising alkaline phosphatase. In particular, improved release formulations comprising alkaline phosphatase are provided that release substantial amounts of alkaline phosphatase in the intestine. Therapeutic uses of the alkaline phosphatase formulations are also provided.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2020800537049, application date June 3, 2020, and invention name “Alkaline phosphatase preparations and uses thereof”. Technical Field

[0002] The present invention provides, in part, formulations comprising alkaline phosphatase and uses thereof.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 62 / 856,309, filed on June 3, 2019, the entire contents of which are hereby incorporated by reference in their entirety.

[0005] Instructions for electronically submitting text documents

[0006] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer readable format copy of the Sequence Listing (File Name: "SYN-044PC_ST25.txt"; Creation Date: June 2, 2020; File Size: 78,381 bytes). Background Art

[0007] Alkaline phosphatases are dimeric metalloenzymes that catalyze phosphate hydrolysis and dephosphorylate a variety of target substrates at physiological and higher pH. Alkaline phosphatases (APs) are found in prokaryotes and eukaryotes (e.g., Escherichia coli (E. coli) and mammals). Mammalian APs have been shown to play an important role in intestinal homeostasis, mucosal barrier function, promotion of commensal bacteria, and protection against pathogens. Mammalian APs exert their properties by primarily targeting lipopolysaccharide (LPS, a toll-like receptor 4 (TLR4) agonist), flagellin (a TLR5 agonist), and CpG DNA (a TLR9 agonist). APs also degrade intestinal lumen nucleoside triphosphates (NTPs, such as ATP, GTP, etc.), thereby promoting the growth of beneficial bacteria and reversing dysbiosis.

[0008] Treatment of gastrointestinal (GI) disorders increasingly relies on the role of the microbiome as a mediator in maintaining healthy function of the GI tract. Thus, the role of alkaline phosphatase (AP) in promoting the growth of beneficial bacteria and reversing dysbiosis is an important and growing area of ​​research to advance treatment options for GI disorders. Therefore, AP can be used clinically as a microbiome preserver, for example, for the treatment of various gastrointestinal (GI) disorders.

[0009] However, formulating protein biologics, including APs, is a particularly challenging task given the various factors that must be considered for drug administration routes. Furthermore, due to the complexity of protein biologics, it can be challenging to provide a modified release formulation of a drug coating that is both stable and exhibits an adequate dissolution / release profile to allow targeted release to the gastrointestinal tract.

[0010] Therefore, there remains a need for new alkaline phosphatase preparations for therapeutic intervention. Summary of the invention

[0011] Accordingly, the present invention provides a modified release formulation comprising an alkaline phosphatase (AP)-based agent and / or an additional therapeutic agent. In various embodiments, the formulation releases a large amount of AP-based agent in the gastrointestinal tract. In one embodiment, the formulation comprises at least one core particle and a base coating on the core particle, wherein the base coating comprises an AP-based agent. In another embodiment, the formulation comprises at least one core particle, wherein the AP-based agent is encapsulated in the core particle. In various embodiments, the formulation comprises a modified release coating, such as a delayed release coating placed on the core particle. In some embodiments, the delayed release coating is substantially stable in gastric fluid. In some embodiments, the delayed release coating is substantially stable in intestinal fluid. In one embodiment, the delayed release coating comprises a EUDRAGIT compound. In various embodiments, the formulation may be in the form of sucrose pellets. In some embodiments, the pellets comprise a plurality of core particles.

[0012] These AP-based agents can be used for a variety of therapies, including the prevention or treatment of CDI and / or C. difficile-related diseases or other antibiotic-induced gastrointestinal adverse reactions. The AP-based agents of the present invention are also used for therapies for microbiome-related disorders, including but not limited to metabolic disorders, neurological disorders, celiac disease, cystic fibrosis, radiation enteropathy, sepsis, and HIV-mediated intestinal dysbiosis.

[0013] For example, AP-based agents can be used to allow patients to receive antibiotic therapy while preventing diseases caused by excessive antibiotics that negatively affect the microbiome. Such use does not interfere with the systemic effectiveness of antibiotics. Instead, without wishing to be bound by theory, AP-based agents maintain the intestinal microflora, thereby preventing the disruption of the microflora associated with various disease states described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Depicted are sequences associated with alkaline phosphatase-based agents present in the formulations described herein.

[0015] Figure 2IAP activity over 96 hours in the IAP-adhesive evaluation is shown. In each set of histograms, different time points are presented, with the leftmost bar representing 0 hours; the middle bar representing 24 hours; and the rightmost bar representing 96 hours.

[0016] Figure 3 IAP activity over 16 days in the IAP-vehicle evaluation is depicted. In each set of histograms, different time points are presented, with the bars from the far left to the far right representing 0 hours, 24 hours, 48 ​​hours, 6 days, and 16 days.

[0017] Figure 4 A SEM picture of a coated pellet (30% EUDRAGIT L30 D-55 coated on the pellet) is depicted.

[0018] Figure 5 A SEM picture of a coated pellet (30% EUDRAGIT L30 D-55 coated on the pellet) is depicted.

[0019] Figure 6 Depicted are the percentage release of two formulations of coated IAP pellets in a dissolution apparatus containing fasted state simulated gastric fluid (FaSSGF) / fasted state simulated intestinal fluid (FaSSIF) or FaSSIF alone, obtained by UV analysis.

[0020] Figure 7 The percentage activity of the coated pellets in FaSSGF / FaSSIF or FaSSIF alone obtained by enzymatic assay is shown.

[0021] Figure 8 A calibration curve generated based on the linear trend line equation for calculating sample concentrations at different time points is shown. "SYN-020" is synonymous with IAP (ie, SEQ ID NO: 39).

[0022] Fig. 9 Depicted are stability results from a dissolution test using coated pellet capsules after one month.

[0023] Fig.10 Results of a dissolution test are shown, in which the coated pellets had been stored for about one year.

[0024] Fig.11 AH depicts the results of analyses of non-GMP SYN-020 5 mg and 15 mg capsule doses. DETAILED DESCRIPTION

[0025] In various aspects, the present invention provides modified release formulations comprising alkaline phosphatase (AP)-based agents and / or additional therapeutic agents. In various embodiments, the formulation releases a large amount of AP-based agents in the gastrointestinal tract. In one embodiment, the formulation comprises at least one core particle and a base coating on the core particle, wherein the base coating comprises an AP-based agent. In various embodiments, the formulation comprises a modified release coating, such as a delayed release coating placed on the core particle. In some embodiments, the delayed release coating is substantially stable in gastric fluid. In one embodiment, the delayed release coating comprises a EUDRAGIT compound.

[0026] Alkaline phosphatase-based agents

[0027] The present invention relates in part to pharmaceutical compositions, formulations and uses of one or more alkaline phosphatase-based agents (AP-based agents). Exemplary AP-based agents that can be used in the present invention include, but are not limited to, intestinal alkaline phosphatase (IAP; e.g., human IAP, calf IAP or bovine IAP, chicken IAP, goat IAP), bovine intestinal alkaline phosphatase (bIAP), recombinant bovine intestinal alkaline phosphatase (rbIAP), placental alkaline phosphatase (PLAP), placental-like alkaline phosphatase, germ cell alkaline phosphatase (GCAP), tissue nonspecific alkaline phosphatase (TNAP; which is mainly found in liver, kidney and bone), bone alkaline phosphatase, liver alkaline phosphatase, kidney alkaline phosphatase, bacterial alkaline phosphatase, fungal alkaline phosphatase, shrimp alkaline phosphatase, modified IAP, recombinant IAP or any polypeptide having alkaline phosphatase activity.

[0028] In various embodiments, the present invention contemplates the use of mammalian alkaline phosphatases including, but not limited to, intestinal alkaline phosphatase (IAP), placental alkaline phosphatase (PLAP), germ cell alkaline phosphatase (GCAP), and tissue nonspecific alkaline phosphatase (TNAP).

[0029] IAP

[0030] In some embodiments, the AP-based agent is an IAP. IAP is produced in the proximal small intestine and binds to intestinal cells via a glycosylphosphatidylinositol (GPI) anchor. Some IAPs are released into the intestinal lumen together with vesicles shed by cells and are stripped from cells via phospholipases as soluble proteins. The enzyme then passes through the small intestine and large intestine, so that some active enzymes can be detected in the feces. In one embodiment, the IAP is a human IAP (hIAP). In one embodiment, the IAP is a calf IAP (cIAP), also known as a bovine IAP (bIAP). There are multiple isozymes of bIAP, for example, bIAP II and IV have a higher specific activity than bIAP I. In one embodiment, the IAP is any one of the cIAP or bIAP isozymes (e.g., bIAP I, II, and IV). In one embodiment, the IAP is bIAP II. In another embodiment, the IAP is bIAP IV.

[0031] IAP variants

[0032] Also included in the definition of IAPs are IAP variants. IAP variants have at least one or more amino acid modifications, typically amino acid substitutions, compared to the parent wild-type sequence. In some embodiments, the IAPs of the invention comprise an IAP that has at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100%, or about 101%, or about 102%, or about 103%, or about 104%, or about 105%, or about 106%, or about 107%, or about 108%, or about 109%, or about 110%, or about 111%, or about 112%, or about 113%, or about 114%, or about 115%, or about 116%, or about 117%, or about 118%, or about 119%, or about 120%, or about 121%, or about 122%, or about 123%, or about 124%, or about 125%, or about 126%, or about 127%, or about or about 98%, or about 99%) sequence identity. In some embodiments, the IAP variants retain most or all of their biochemical activity as measured as described herein.

[0033] Mammalian alkaline phosphatases are GPI-anchored proteins. They have a signal peptide and are translated into the secretory pathway. Once in the endoplasmic reticulum (ER), the protein is glycosylated and folded. There are two disulfide bonds and a single free cysteine ​​that is apparently inaccessible on the surface. In the late ER, the carboxyl terminus is removed and a GPI anchor is attached. Therefore, GPI anchoring is a process that occurs at the carboxyl terminus of alkaline phosphatase. The inclusion of a stop codon at the anchor site makes secretion of biologically active proteins (possibly homodimers) possible. Although there is no consensus sequence, the carboxyl terminus includes three amino acids, called ω, ω+1, and ω+2, followed by a short stretch of hydrophilic amino acids and then a stretch of hydrophobic amino acids. Without wishing to be bound by theory, it is believed that hydrophobicity is critical for embedding the carboxyl terminus into the ER membrane. There, the enzymatic reaction replaces the carboxyl terminus with the GPI anchor.

[0034] In human placental alkaline phosphatase (hPLAP), the GPI anchor is attached to the aspartic acid in the sequence DAAH. Similarly, hIAP, bIAP II and bIAP IV also have this conservative DAAH sequence, which may be used as a GPI anchor. Mutation studies of hPLAP have shown that blocking GPI anchoring can lead to intracellular retention. In addition, mutations around the anchor site or in the hydrophobic domain 1) prevent the anchor from attaching, resulting in intracellular retention or 2) will not block the anchor attachment. Without wishing to be bound by theory, it is believed that the hydrophobic domain serves as a signal for the GPI anchor to attach. Truncating or eliminating the hydrophobic domain leads to secretion. Finally, there is a single mutation in the hydrophobic domain that enables the protein to be secreted in the case of its hydrophobic domain being intact in hPLAP.

[0035] In other embodiments, the AP-based agents of the present invention are secreted proteins; that is, in some embodiments, the AP-based agents are not GPI-anchored, resulting in secretion rather than intracellular retention. This can be achieved in several ways. In some embodiments, the AP-based agent may lack a GPI anchor site, such as the DAAH site is removed, resulting in secretion. Alternatively, this can be achieved in some embodiments, and the AP-based agent includes a stop codon inserted immediately before the GPI anchor site. In one embodiment, the AP-based agent includes a stop codon after the aspartic acid in the DAAH consensus site (e.g., at amino acid 503 of hIAP and bIAP IV or amino acid 506 of bIAP II). Figure 1 HIAP with a stop codon (SEQ ID NO: 4), bIAP II with a stop codon (SEQ ID NO: 5), and bIAP IV with a stop codon (SEQ ID NO: 6) are depicted. In one embodiment, the AP-based agent is bIAP IV and includes a stop codon after amino acid 508 to mimic Figure 1The secreted PLAP construct depicted (SEQ ID NO: 7).

[0036] Human IAP

[0037] In various embodiments, the AP-based agent is hIAP. In some embodiments, the AP-based agent is a Figure 1 hIAP having the amino acid sequence of SEQ ID NO: 1 as depicted, or a variant as described herein, as long as the hIAP variant retains at least 80%, 85%, 90%, 95%, 98% or 100% of the phosphatase activity compared to the wild-type enzyme using the assays outlined herein.

[0038] Amino acid modifications are included in the definition of hIAP, with amino acid substitutions being particularly useful in the present invention. For example, without wishing to be bound by theory, it is believed that a cysteine ​​at the carboxyl terminus of an AP-based agent (e.g., at position 500 of SEQ ID NO: 1) can interfere with protein folding. Thus, in some embodiments, an AP-based agent includes a mutation of a cysteine ​​(e.g., at position 500 of SEQ ID NO: 1). In some embodiments, the cysteine ​​is replaced with any amino acid, but glycine is particularly useful in some embodiments. In addition, the C-terminal cysteine ​​may also be deleted.

[0039] As will be appreciated by those skilled in the art, additional amino acid modifications may be made in hIAP as discussed herein. For example, in some embodiments, a stop codon may be inserted after the aspartic acid in the DAAH consensus site (eg, at amino acid 503 of hIAP). Figure 1 Depicted is hIAP (SEQ ID NO: 4) with an inserted stop codon.

[0040] Cow IAP

[0041] In some embodiments, the IAP is bovine IAP (bIAP).

[0042] In various embodiments, the AP-based agent is IAP II (bIAP II) or a variant as described herein, provided that the bIAP variant retains at least 80%, 85%, 90%, 95%, 98% or 100% of the phosphatase activity using the assays outlined herein. In one embodiment, bIAP II comprises the signal peptide and carboxyl terminus of bIAP I. In one embodiment, bIAP II comprises aspartic acid at position 248 (similar to bIAP IV). In one embodiment, bIAP II comprises the amino acid sequence of SEQ ID NO: 2. Figure 1Depicted is BIAP II with 248D - SEQ ID NO: 2. The signal peptide and sequence after 480 are derived from blAP I.

[0043] Also included within the definition of bIAP II are the amino acid variants described herein. For example, in some embodiments, a stop codon can be inserted after the aspartic acid in the DAAH consensus site (eg, at amino acid 506 of bIAP II). Figure 1 Depicted is blAP II (SEQ ID NO: 5) with an inserted stop codon.

[0044] In various embodiments, blAP II comprises the amino acid sequence of SEQ ID NO:39.

[0045] BIAP II (SYN-020) with a stop codon and no leader sequence (SEQ ID NO: 39):

[0046] LIPAEEENPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFP

[0047] YVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQCNTTRGNEVTSVINRAKKAGKAVGVVTTT

[0048] RVQHASPAGAYAHTVNRNWYSDADLPADAQKNGCQDIAAQLVYNMDIDVILGGGRMYMFPEGTPDPEYPDDAS

[0049] VNGVRKDKQNLVQEWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLAEMTE

[0050] AALQVLSRNPRGFYLFVEGGRIDHGHHDGKAYMALTEAIMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTL

[0051] RGTSIFGLAPGKALDSKSYTSILYGNGPGYALGGGSRPDVNGSTSEEPSYRQQAAVPLASETHGGEDVAVFARG

[0052] PQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPATATSIPD

[0053] In various embodiments, the AP-based agent is a bIAP IV as described herein, or a variant thereof, provided that the bIAP IV variant retains at least 80%, 85%, 90%, 95%, 98%, or 100% of the phosphatase activity using the assays outlined herein. In one embodiment, the bIAP IV comprises Figure 1 The amino acid sequence of SEQ ID NO: 3 is depicted.

[0054] Also included within the definition of bIAP IV are the amino acid variants described herein. For example, in some embodiments, a stop codon can be inserted after the aspartic acid in the DAAH consensus site (eg, at amino acid 503 of bIAP IV). Figure 1 bIAP IV (SEQ ID NO: 6) with an inserted stop codon is depicted. In one embodiment, the AP-based agent is bIAP IV and includes a stop codon after amino acid 508 to mimic Figure 1 The secreted PLAP construct depicted (SEQ ID NO: 7).

[0055] Bacterial AP

[0056] In various embodiments, the present invention contemplates the use of bacterial alkaline phosphatases. In some embodiments, the AP-based agents of the present invention are derived from Bacillus subtilis. Bacillus subtilis is a Gram-positive bacterium found in soil and the human gastrointestinal tract. Bacillus subtilis secretes high levels of correctly folded proteins into the environment and the human gastrointestinal tract. Without wishing to be bound by theory, it is believed that the proteins secreted by Bacillus subtilis in the gastrointestinal tract can resist degradation by common gastrointestinal proteases. Bacillus subtilis expresses a multigene family of alkaline phosphatases at high levels. Among these isozymes, alkaline phosphatase IV is responsible for the expression and activity of most alkaline phosphatases in Bacillus subtilis. In some embodiments, the AP-based agents of the present invention are derived from Bacillus licheniformis. In some embodiments, the AP-based agents of the present invention are derived from Escherichia coli.

[0057] Thus, in one illustrative embodiment, the AP-based agent of the invention is derived from alkaline phosphatase IV of Bacillus subtilis. In one embodiment, the bacterial alkaline phosphatase may have a Figure 1The nucleotide and amino acid sequences depicted, including Bacillus subtilis JH642 alkaline phosphatase IV, mature protein nucleotide sequence - SEQ ID NO: 16; and Bacillus subtilis JH642 alkaline phosphatase IV, mature protein amino acid sequence - SEQ ID NO: 17, or a variant as described herein, so long as the hIAP variant retains at least 80%, 85%, 90%, 95%, 98% or 100% of the phosphatase activity using the assays outlined herein.

[0058] In some embodiments, the AP-based agent includes a bacterial alkaline phosphatase having one or more mutations that change the catalytic activity. In some embodiments, the bacterial alkaline phosphatase includes one or more mutations so that their catalytic activity is similar to or higher than that of mammalian alkaline phosphatases. In some embodiments, the bacterial alkaline phosphatase includes one or more mutations that change its dephosphorylation properties. In one embodiment, the bacterial alkaline phosphatase of the present invention exhibits dephosphorylation properties similar to those of mammalian alkaline phosphatases. In some embodiments, the bacterial alkaline phosphatase includes one or more mutations that change their activity at higher pH. In one embodiment, the bacterial alkaline phosphatase of the present invention exhibits activity similar to that of mammalian alkaline phosphatases at higher pH. In some embodiments, the bacterial alkaline phosphatase includes one or more mutations that change its metal requirement. In one embodiment, the bacterial alkaline phosphatase of the present invention exhibits metal requirements (e.g., Mg) similar to those of mammalian alkaline phosphatases.

[0059] For example, in certain embodiments, the AP-based agents of the invention are derived from Bacillus subtilis JH642 alkaline phosphatase IV and have one or more mutations at positions 101, 328, A330, and 374. For example, the AP-based agent can include one or more of the following mutations: D101A, W328H, A330N, and G374C.

[0060] Fusion Protein

[0061] In some embodiments, the AP-based agent comprises alkaline phosphatase fused to a "fusion partner", which is a protein domain added to the N-terminus or C-terminus of the IAP domain, optionally including a linker. In some embodiments, alkaline phosphatase is fused to a protein domain that promotes protein folding and / or protein purification and / or protein dimerization and / or protein stability. In various embodiments, the AP-based agent fusion protein has an extended serum half-life. In various embodiments, the AP-based agent of the invention is an Fc fusion protein.

[0062] In one embodiment, the alkaline phosphatase is fused to an immunoglobulin Fc domain and / or hinge region.In one embodiment, an AP-based agent of the invention comprises alkaline phosphatase fused to the hinge region and / or Fc domain of an IgG.

[0063] In various embodiments, the AP-based agent is fused to the Fc domain of an IgG comprising one or more mutations. In some embodiments, the one or more mutations in the Fc domain of an IgG serve to increase serum half-life and lifespan. In some embodiments, the Fc domain of an IgG comprises one or more mutations at amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, DC). In some embodiments, at least one amino acid substitution in the Fc domain of an IgG is located at amino acid residues 252, 254, 256, 309, 311, 433, or 434. In one embodiment, the amino acid substitution at amino acid residue 252 is substituted with tyrosine, phenylalanine, tryptophan, or threonine. In one embodiment, the amino acid substitution at amino acid residue 254 is substituted with threonine. In one embodiment, the amino acid substitution at amino acid residue 256 is substituted with serine, arginine, glutamine, glutamic acid, aspartic acid or threonine. In one embodiment, the amino acid substitution at amino acid residue 309 is substituted with proline. In one embodiment, the amino acid substitution at amino acid residue 311 is substituted with serine. In one embodiment, the amino acid substitution at amino acid residue 385 is substituted with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine. In one embodiment, the amino acid substitution at amino acid residue 386 is substituted with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine or methionine. In one embodiment, the amino acid substitution at amino acid residue 387 is substituted with arginine, proline, histidine, serine, threonine or alanine. In one embodiment, the amino acid substitution at amino acid residue 389 is substituted with proline, serine or asparagine. In one embodiment, the amino acid substitution at amino acid residue 433 is substituted with arginine, serine, isoleucine, proline or glutamine. In one embodiment, the amino acid substitution at amino acid residue 434 is with histidine, phenylalanine or tyrosine.

[0064] In some embodiments, the Fc domain of IgG comprises one or more mutations at amino acid residues 252, 254, 256, 433, 434 or 436. In one embodiment, the Fc domain of IgG comprises triple M252Y / S254T / T256E mutations or YTE mutations. In another embodiment, the Fc domain of IgG comprises triple H433K / N434F / Y436H mutations or KFH mutations. In yet another embodiment, the Fc domain of IgG comprises a combination of YTE and KFH mutations. Other illustrative mutations in the Fc domain of IgG are described, for example, in Robbie et al., Antimicrobial Agents and Chemotherapy (2013), 57(12):6147-6153, Dall'Acqua et al., JBC (2006), 281(33):23514-24, Dall'Acqua et al., Journal of Immunology (2002), 169:5171-80, and U.S. Pat. No. 7,083,784, the entire contents of which are hereby incorporated by reference. In various embodiments, the one or more mutations in the Fc domain of IgG increase affinity for neonatal Fc receptor (FcRn). In some embodiments, the one or more mutations in the Fc domain of IgG increase affinity for FcRn at a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

[0065] In various embodiments, alkaline phosphatase is fused to one or more of PEG, XTEN base (e.g., as rPEG), polysialic acid (POLYXEN), albumin, elastin-like protein, elastin-like protein (ELP), PAS, HAP, GLK, CTP, and transferrin. In various embodiments, alkaline phosphatase is fused to one or more agents described in BioDrugs (2015) 29: 215–239, the entire contents of which are hereby incorporated by reference.

[0066] Connectors

[0067] In one embodiment, alkaline phosphatase is fused to a protein domain (e.g., an immunoglobulin Fc domain) via a linker connecting a GPI anchor site. For example, alkaline phosphatase can be fused to a protein domain via an aspartic acid at a GPI anchor sequence. The present invention contemplates the use of a variety of linker sequences. In various embodiments, the linker can be derived from a naturally occurring multidomain protein or an empirical linker such as described in Chichili et al., (2013), Protein Sci. 22(2): 153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369, the entire contents of which are hereby incorporated by reference. In some embodiments, linkers can be designed using linker design databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference. In various embodiments, the linker can be functional. For example, but not limited to, the linker can serve to improve the folding and / or stability of the AP-based agents of the present invention, improve expression, improve pharmacokinetics, and / or improve biological activity. In another example, the linker can serve to target the AP-based agent to a specific cell type or location.

[0068] In some embodiments, the joint is a polypeptide. In some embodiments, the joint length is less than about 100 amino acids. For example, the joint length can be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3 or about 2 amino acids. In some embodiments, the joint is flexible. In another embodiment, the joint is rigid.

[0069] In various embodiments, the linker consists essentially of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 18). Other illustrative linkers include, but are not limited to, linkers having the sequences LE, GGGGS (SEQ ID NO: 19), (GGGGS)n(n=2-4) (SEQ ID NOs: 20-22), (Gly)8 (SEQ ID NO: 23), (Gly)6 (SEQ ID NO: 24), (EAAAK)n(n=1-3) (SEQ ID NOs: 25-27), A(EAAAK)nA(n=2-5) (SEQ ID NOs: 28-31), AEAAAKEAAAKA SEQ ID NO: 32), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 33), PAPAP (SEQ ID NO: 34), KESGSVSSEQLAQFRSLD (SEQ ID NO: 35), EGKSSGSGSESKST (SEQ ID NO: 36), GSAGSAAGSGEF (SEQ ID NO: 37), and (XP)n, wherein X represents any amino acid, e.g., Ala, Lys, or Glu. In some embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In some embodiments, the linker is a synthetic linker, such as PEG.

[0070] Illustrative Fc fusion constructs of the invention include Figure 1 Those depicted in , including BIAP II with Fc fusion (SEQ ID NO: 8)—the Fc domain is underlined; and BIAP IV with Fc fusion (SEQ ID NO: 9)—the Fc domain is underlined.

[0071] Proenzyme Fusion

[0072] The present invention additionally provides C-terminal fusions for zymogen function. Without wishing to be bound by theory, it is believed that mammalian alkaline phosphatase can also be produced as an inactive zymogen. This is because alkaline phosphatase can dephosphorylate ATP, so that activity in the ER can consume the main energy source of the ER. Without wishing to be bound by theory, it is believed that the inhibitory function is located at the carboxyl terminus, which will be released when the GPI anchor is added. Alternatively, other activities, such as folding or metal (Zn or Mg) inclusions can control activity.

[0073] In various embodiments, the AP-based agent of the invention is a zymogen. In one embodiment, the activity of the zymogen is suppressed by the carboxyl terminus. In one embodiment, protease removal of the carboxyl terminus reactivates the enzymatic activity of alkaline phosphatase. In one embodiment, the zymogen is secreted more efficiently than the enzyme without the carboxyl terminus.

[0074] The yeast alkaline phosphatase Pho8 is produced as an inactive zymogen. It is not GPI-anchored but is a transmembrane protein with its amino terminus extending from the lysosome into the cytoplasm. Within the lysosome, an enzyme PEP4 cleaves the carboxyl terminus to activate the enzyme.

[0075] In some embodiments, to produce zymogen, the native carboxyl terminus of alkaline phosphatase is replaced by a similar sequence from hPLAP. In some embodiments, a mutation is produced in the hydrophobic carboxyl tail to promote protein secretion without cleavage of the carboxyl terminus. In an illustrative embodiment, a single point mutation is produced at the hydrophobic carboxyl terminus, such as replacing leucine with, for example, arginine (e.g., ALLPLLAGTL becomes, for example, ALLPLRAGTL) to cause secretion of the enzyme without removing the carboxyl terminus.

[0076] In one embodiment, the AP-based agent is modified to include a specific enzymatic cleavage site that allows for subsequent removal of the carboxyl terminus. In one embodiment, the AP-based agent includes a protease cleavage site. Illustrative protease cleavage sites include, but are not limited to, cleavage sites recognized by furin, rhinovirus 16 3C protease, factor Xa protease, trypsin, chymotrypsin, elastase, pepsin, papain, subtilisin, thermolysin, V-8 protease, submaxillaris protease, clostripain, thrombin, collagenase, and any other endoprotease. In an alternative embodiment, the AP-based agent includes a cleavage site recognized by digestive enzymes present in the gastrointestinal tract. In such embodiments, the AP-based agent can be administered as a prodrug that is subsequently activated in the gastrointestinal tract.

[0077] In an illustrative embodiment, the zymogen is Figure 1Proenzymes of bIAP IV of the depicted sequences include BIAPIV (SEQ ID NO: 10) with an hPLAP carboxyl terminus and mutations for unprocessed secretion and RV3C cleavage (at ...LEVLFQGP ...); BIAP IV (SEQ ID NO: 11) with an hPLAP carboxyl terminus and mutations for unprocessed secretion and FXa cleavage (at ...IEGR ...).

[0078] Expression variants

[0079] In various embodiments, the AP-based agents of the invention are efficiently expressed and secreted from the host cell. In one embodiment, the AP-based agents of the invention are efficiently transcribed in the host cell. In another embodiment, the AP-based agents exhibit enhanced RNA stability and / or transport in the host cell. In another embodiment, the AP-based agents are efficiently translated in the host cell. In another embodiment, the AP-based agents exhibit enhanced protein stability.

[0080] In various embodiments, the AP-based agent is effectively expressed in a host cell. In one embodiment, the Kozak sequence of the DNA construct encoding the AP-based agent is optimized. The Kozak sequence is a nucleotide sequence flanking the ATG start codon that instructs the ribosome to start translation. The design of the Kozak sequence is flexible, but a standard sequence is GCCGCCACCATGG (SEQ ID NO: 38). The purine at the -3 position and the G at the +4 position are the most important bases for translation initiation. For hIAP, bIAP II and bIAP IV, the second amino acid, the amino acid after the start methionine, is glutamine. The codon for glutamine all has a C at the first position. Therefore, their Kozak sequences all have an ATGC sequence. Therefore, in various embodiments, the ATGC sequence is changed to ATGG. This can be achieved by changing the second amino acid to glycine, alanine, valine, aspartic acid or glutamic acid, all of which have a G at the first position. These amino acids are compatible with signal peptide function. In an alternative embodiment, the entire signal peptide is replaced by a peptide having a canonical Kozak sequence and derived from a highly expressed protein, such as an immunoglobulin.

[0081] In various embodiments, the signal peptide of the AP-based agent can be deleted and / or substituted. For example, the signal peptide can be deleted, mutated and / or substituted (eg, replaced with another signal peptide) to ensure protein expression.

[0082] In some embodiments, the DNA construct encoding the AP-based agent of the present invention comprises an untranslated DNA sequence. Such sequences include introns, which may be heterologous to the IAP protein or native to the IAP protein, including the native first and / or second introns and / or the native 3'UTR. Without wishing to be bound by theory, it is believed that including these sequences enhances protein expression by stabilizing the mRNA. Thus, in various embodiments, the DNA construct encoding the AP-based agent of the present invention comprises a 5'UTR and / or a 3'UTR. Figure 1 Illustrative IAP DNA sequences having a first intron and a 3'UTR are provided in, including hIAP having a native first intron (shown in bold and underlined) - SEQ ID NO: 12; hIAP having a native 3'UTR (shown in bold and underlined) - SEQ ID NO: 13; bIAP IV having a first intron from bIAP I (shown in bold and underlined) - SEQ ID NO: 14; and bIAP IV having a 3'UTR from bIAP I (shown in bold and underlined) - SEQ ID NO: 15.

[0083] In various embodiments, the AP-based agents of the invention comprise a sequence that is at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100%, or about 101%, or about 102%, or about 103%, or about 104%, or about 105%, or about 106%, or about 107%, or about 108%, or about 109%, or about 110%, or about 111%, or about 112%, or about 113%, or about 114%, or about 115%, or about 116%, or about 117%, or about 118%, or about 119%, or about 120%, or about 121%, or about 122%, or about 123%, or about 124%, or about 125%, or about 126%, or about 127%, or about 128%, or about 129%, or about or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity.

[0084] In various embodiments, the AP-based agents of the invention may comprise an amino acid sequence having one or more amino acid mutations relative to any protein sequence described herein. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0085] In various embodiments, substitutions can also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids (such as β-methyl amino acids), Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general).

[0086] The AP-based agents of the invention can be mutated to select agents with desired characteristics. For example, mutations can be made to generate AP-based agents with enhanced catalytic activity or protein stability. In various embodiments, directed evolution can be used to generate AP-based agents of the invention. For example, error-prone PCR and DNA shuffling can be used to identify mutations in bacterial alkaline phosphatases that confer enhanced activity.

[0087] Modified release formulations and dosage forms

[0088] The modified release formulation of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) may also comprise a pharmaceutically acceptable carrier or excipient. As will be appreciated by those skilled in the art, the formulation may be in any suitable form suitable for the intended use and route of administration.

[0089] In various embodiments, the modified release formulation of the present invention comprises a core particle, a base coating on the core particle, and wherein the base coating comprises an AP-based agent. In other embodiments, the core particle comprises sucrose. In some embodiments, the AP-based agent of the base coating is encapsulated within the core particle and may include multiple core particles.

[0090] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) comprising a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the pellets (or each individual pellet) comprise an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof), sucrose spheres (the AP-based agent, e.g., IAP or variant, is sprayed on the sucrose spheres), a binder excipient (e.g., hydroxypropylcellulose (HPC)), an enteric polymer (e.g., EUDRAGIT L30 D-55), HTP-20 (e.g., PLASACRYL HTP 20) (which is an additive that improves coating efficiency and reduces processing time), and a buffer salt (e.g., Tris base, magnesium chloride, magnesium sulfate, zinc chloride, or zinc sulfate).

[0091] In various embodiments, the formulations of the invention comprise a modified release coating that is stable in gastric and / or intestinal fluids and degraded by microbial enzymes present in the intestinal flora. In other embodiments, the modified release coating solubility is pH dependent. In other embodiments, the modified release coating has time-dependent erosion characteristics.

[0092] In various embodiments, the formulation of the invention comprises at least one modified release pellet, wherein each modified release pellet comprises about 1-10% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). For example, the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) may be present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight. In some embodiments, the pellet (or each individual pellet) comprises about 75-95% by weight of sucrose spheres. For example, the sucrose spheres can be present at about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% or about 95% by weight. In various embodiments, the pellets (or each separate pellet) contain about 5-15% by weight of hydroxypropyl cellulose (HPC). For example, hydroxypropyl cellulose can be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% or about 15% by weight. In some embodiments, the pellets (or each separate pellet) contain about 0.5-2% by weight of buffer salts. Buffer salts can be selected from Tris base, magnesium chloride and zinc sulfate. For example, buffer salts can be present at about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, 1.6%, about 1.7%, about 1.8%, about 1.9% or about 2.0% by weight. In some embodiments, the preparation comprises a monolayer enteric coating, and the monolayer enteric coating comprises about 20-40%, about 25-40%, about 25-35%, about 30-40% or about 35-40% enteric polymer weight gain. In some embodiments, the formulation comprises a double enteric coating comprising about 20-40%, about 25-40%, about 25-35%, about 30-40%, or about 35-40% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55) and about 5-15%, about 5-10%, about 7-15%, about 7-10%, about 10-15%, about 6-9%, or about 7-8% by weight of hydroxypropylcellulose. The weights described herein refer to the total weight of all components.

[0093] In various embodiments, the formulations of the present invention comprise at least one modified release pellet, wherein each modified release pellet comprises about 5% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 85% by weight of sucrose spheres; about 9% by weight of hydroxypropylcellulose; and about 1% by weight of buffer salts. In some embodiments, the formulation comprises a single enteric coating containing about 20-40% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55) or a double enteric coating containing about 30% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55) and about 7% by weight of hydroxypropylcellulose. The weights as described herein refer to the total weight of all components.

[0094] In various embodiments, the formulations of the present invention comprise at least one modified release pellet, wherein each modified release pellet comprises about 4.7% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 84.9% by weight of sucrose spheres; about 9.3% by weight of hydroxypropylcellulose; and about 1.2% by weight of buffer salts. In some embodiments, the formulation comprises a single enteric coating containing about 20-40% by weight gain of an enteric polymer (e.g., EUDRAGIT L 30D-55) or a double enteric coating containing about 30% by weight gain of an enteric polymer (e.g., EUDRAGIT L 30D-55) and about 7% by weight gain of hydroxypropylcellulose. The weights as described herein refer to the total weight of all components.

[0095] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 25 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In various embodiments, the formulation of the present invention comprises at least one modified release pellet, wherein each modified release pellet comprises about 1-15% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). For example, the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) may be present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 13%, about 14%, or about 15% by weight. In some embodiments, the pellets (or each individual pellet) comprise about 30-55% by weight of sucrose spheres. For example, the sucrose spheres can be present at about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55% by weight. In various embodiments, the pellets (or each individual pellet) contain about 5-25% by weight hydroxypropyl cellulose (HPC). For example, hydroxypropylcellulose can be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight. In some embodiments, the pellets (or each individual pellet) contain about 20-35% by weight of EUDRAGIT L30 D-55. For example, EUDRAGIT L30 D-55 can be present at about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% by weight. In some embodiments, the pellets (or each individual pellet) comprise about 0.5-11% by weight HTP-20. For example, HTP-20 can be present at about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, or about 11.0% by weight.In some embodiments, the pellet (or each individual pellet) comprises about 0.5-2.5% by weight of a buffer salt. The buffer salt can be selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate. For example, the buffer salt can be present at about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% by weight.

[0096] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 25 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the formulation comprises about 10% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 38% by weight of sucrose spheres; about 19% by weight of hydroxypropylcellulose (HPC); about 2% by weight of buffer salts; and about 26% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55).

[0097] In various embodiments, the formulation of the invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 25 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the formulation comprises about 9.7% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 37.7% by weight of sucrose spheres; about 19.4% by weight of hydroxypropylcellulose (HPC); about 2.4% by weight of buffer salts; and about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT L 30D-55).

[0098] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In various embodiments, the formulation of the present invention comprises at least one modified release pellet, wherein each modified release pellet comprises about 1-15% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). For example, the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) may be present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 13%, about 14%, or about 15% by weight. In some embodiments, the pellets (or each individual pellet) comprise about 30-55% by weight of sucrose spheres. For example, the sucrose spheres can be present at about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55% by weight. In various embodiments, the pellets (or each individual pellet) contain about 5-25% by weight hydroxypropyl cellulose (HPC). For example, hydroxypropylcellulose can be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight. In some embodiments, the pellets (or each individual pellet) contain about 20-35% by weight of EUDRAGIT L30 D-55. For example, EUDRAGIT L30 D-55 can be present at about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% by weight. In some embodiments, the pellets (or each individual pellet) comprise about 0.5-11% by weight HTP-20. For example, HTP-20 can be present at about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, or about 11.0% by weight.In some embodiments, the pellet (or each individual pellet) comprises about 0.5-2.5% by weight of a buffer salt. The buffer salt can be selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate. For example, the buffer salt can be present at about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% by weight.

[0099] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the formulation comprises about 9.7% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 37.7% by weight of sucrose spheres; about 19.4% by weight of hydroxypropylcellulose (HPC); about 2.4% by weight of buffer salts; and about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT L 30D-55).

[0100] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the formulation comprises about 9.7% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 37.7% by weight of sucrose spheres; about 19.4% by weight of hydroxypropylcellulose (HPC); about 2.4% by weight of buffer salts; and about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT L 30D-55).

[0101] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 15 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In various embodiments, the formulation of the present invention comprises at least one modified release pellet, wherein each modified release pellet comprises about 1-15% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). For example, the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) may be present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 13%, about 14%, or about 15% by weight. In some embodiments, the pellets (or each individual pellet) comprise about 30-55% by weight of sucrose spheres. For example, the sucrose spheres can be present at about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55% by weight. In various embodiments, the pellets (or each individual pellet) contain about 5-25% by weight hydroxypropyl cellulose (HPC). For example, hydroxypropylcellulose can be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight. In some embodiments, the pellets (or each individual pellet) contain about 20-35% by weight of EUDRAGIT L30 D-55. For example, EUDRAGIT L30 D-55 can be present at about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% by weight. In some embodiments, the pellets (or each individual pellet) comprise about 0.5-11% by weight HTP-20. For example, HTP-20 can be present at about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, or about 11.0% by weight.In some embodiments, the pellet (or each individual pellet) comprises about 0.1-2.5% by weight of a buffer salt. The buffer salt can be selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate. For example, the buffer salt can be present at about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% by weight.

[0102] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 15 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the formulation comprises about 10% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 39% by weight of sucrose spheres; about 20% by weight of hydroxypropylcellulose (HPC); about 0.5% by weight of buffer salts; and about 26% by weight of an enteric polymer (e.g., EUDRAGIT L30D-55).

[0103] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 15 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the formulation comprises about 10.0% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 38.9% by weight of sucrose spheres; about 20.0% by weight of hydroxypropylcellulose (HPC); about 0.3% by weight of buffer salts; and about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55).

[0104] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In various embodiments, the formulation of the present invention comprises at least one modified release pellet, wherein each modified release pellet comprises about 1-15% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). For example, the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) may be present at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 13%, about 14%, or about 15% by weight. In some embodiments, the pellets (or each individual pellet) comprise about 30-55% by weight of sucrose spheres. For example, the sucrose spheres can be present at about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55% by weight. In various embodiments, the pellets (or each individual pellet) contain about 5-25% by weight hydroxypropyl cellulose (HPC). For example, hydroxypropylcellulose can be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight. In some embodiments, the pellets (or each individual pellet) contain about 20-35% by weight of EUDRAGIT L30 D-55. For example, EUDRAGIT L30 D-55 can be present at about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% by weight. In some embodiments, the pellets (or each individual pellet) comprise about 0.5-11% by weight HTP-20. For example, HTP-20 can be present at about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, or about 11.0% by weight.In some embodiments, the pellet (or each individual pellet) comprises about 0.1-2.5% by weight of a buffer salt. The buffer salt can be selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate. For example, the buffer salt can be present at about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% by weight.

[0105] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the formulation comprises about 10% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 39% by weight of sucrose spheres; about 20% by weight of hydroxypropylcellulose (HPC); about 0.5% by weight of buffer salts; and about 26% by weight of an enteric polymer (e.g., EUDRAGIT L30D-55).

[0106] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). The capsule includes a plurality of enteric-coated pellets containing an AP-based agent. In such embodiments, the formulation comprises about 10.0% by weight of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof); about 38.9% by weight of sucrose spheres; about 20.0% by weight of hydroxypropylcellulose (HPC); about 0.3% by weight of buffer salts; and about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55).

[0107] In some embodiments, the administration of the modified release formulation comprising an AP-based agent (and / or an additional therapeutic agent) is any of oral, intravenous, and parenteral. In some embodiments, the administration of the modified release formulation comprising an AP-based agent (and / or an additional agent) is not intravenous, for example, to prevent interference with systemically administered antibiotics. In other embodiments, routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intracerebral, intravaginal, transdermal, rectal, by inhalation, or topically, particularly to the ear, nose, eye, or skin.

[0108] In some embodiments, any formulation of the present invention comprises a core particle having a size between about 0.8 mm and about 2.0 mm, about 0.9 mm and about 1.9 mm, about 1 mm and about 1.8 mm, about 1.1 mm and about 1.7 mm, about 1.2 mm and about 1.6 mm, about 1.3 mm and about 1.5 mm, about 1 mm and about 1.3 mm, about 1 mm and about 1.4 mm, about 1 mm and about 1.5 mm, about 1 mm and about 1.6 mm, about 1 mm and about 1.7 mm, about 1 mm and about 1.9 mm, about 1 mm and about 2.0 mm in diameter. In some embodiments, the formulation comprises a core particle having a size of about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, or about 2.0 mm in diameter.

[0109] Any modified release formulation comprising an AP-based agent as described herein (and / or an additional therapeutic agent) may be free of a cofactor (eg, CaCl 2 or CoCl 2 ).

[0110] Any modified release formulation containing an AP-based agent (and / or an additional therapeutic agent) as described herein may be administered orally. Such formulations of the invention may also be administered by any other convenient route, such as by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with an additional therapeutic agent. Administration may be systemic or local.

[0111] Suitable dosage forms for oral use include, for example, solid dosage forms, such as tablets, dispersible powders, granules, and capsules. In one embodiment, the modified release formulation is in the form of capsules. In another embodiment, the modified release formulation is in the form of tablets. In yet another embodiment, the modified release formulation is in the form of soft gel capsules. In another embodiment, the modified release formulation is in the form of gelatin or hydroxypropyl methylcellulose (HPMC) capsules.

[0112] In some dosage forms, the agents described herein are mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate, dicalcium phosphate, and the like, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and Bakers Special Sugar, and the like; b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, acacia, polyvinyl alcohol, polyvinyl pyrrolidone, methylcellulose, hydroxypropyl cellulose (HPC), and hydroxymethyl cellulose, and the like; c) humectants, such as glycerol, and the like; d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, sodium carbonate, cross-linked polymers, such as crospovidone (cross-linked polyvinyl pyrrolidone), cross-linked sodium carboxymethyl cellulose (cross-linked sodium carboxymethyl cellulose), sodium starch glycolate, and the like; e) solution retarding agents, such as glycerol, and the like. agents), such as paraffin, etc.; f) absorption promoters, such as quaternary ammonium compounds, etc.; g) wetting agents, such as cetyl alcohol and glyceryl monostearate, etc.; h) adsorbents, such as kaolin and bentonite clay, etc.; i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, glyceryl behenate, etc.; j) and mixtures of such excipients. Those skilled in the art will recognize that a particular excipient may have two or more functions in an oral dosage form. In the case of an oral dosage form such as a capsule or tablet, the dosage form may also contain a buffer.

[0113] The modified release formulation may additionally include a surfactant. Surfactants suitable for use in the present invention include, but are not limited to, any pharmaceutically acceptable nontoxic surfactant. Surfactant species suitable for use in the compositions of the present invention include, but are not limited to, polyethoxylated fatty acids, PEG-fatty acid diesters, PEG-fatty acid monoesters and diester mixtures, polyethylene glycol glycerol fatty acid esters, alcohol-oil transesterification products, polyglycerol fatty acids, propylene glycol fatty acid esters, mixtures of propylene glycol ester-glycerides, monoglycerides and diglycerides, sterols and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar esters, polyethylene glycol alkylphenols, polyoxyethylene-polyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants and mixtures thereof. In some embodiments, the compositions of the present invention may include one or more surfactants, including, but are not limited to, sodium lauryl sulfate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 and triethyl citrate.

[0114] The modified release formulation may also contain a pharmaceutically acceptable plasticizer to obtain desired mechanical properties, such as flexibility and hardness. Such plasticizers include, but are not limited to, triacetin, citrate esters, triethyl citrate, phthalates, dibutyl sebacate, cetyl alcohol, polyethylene glycol, polysorbate or other plasticizers.

[0115] The modified release formulation may also include one or more application solvents. Some of the more common solvents that can be used to apply, for example, a delayed release coating composition include isopropyl alcohol, acetone, methylene chloride, and the like.

[0116] The modified release formulation may also include one or more alkaline materials. Alkaline materials suitable for use in the compositions of the present invention include, but are not limited to, sodium salts, potassium salts, calcium salts, magnesium salts and aluminum salts of acids such as phosphoric acid, carbonic acid, citric acid and other aluminum / magnesium compounds. In addition, the alkaline material may be selected from antacid materials such as aluminum hydroxide, calcium hydroxide, magnesium hydroxide and magnesium oxide.

[0117] Solid oral dosage forms can be prepared, for example, by granulating the agent of the invention with one or more suitable excipients (e.g., wet or dry granulation). Alternatively, the agent of the invention can be coated onto an inert core (e.g., nonpareil / sugar spheres, such as sucrose spheres or silica spheres) using conventional methods such as fluidized bed or pan coating, or extruded and spheronized into pellets containing the active compound using methods known in the art. In an embodiment, an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) is spray coated onto sucrose spheres. Such pellets can then be incorporated into tablets or capsules using conventional methods.

[0118] In addition to the active agent, the suspension may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like, and mixtures thereof.

[0119] Besides inert diluents, the oral compositions can also include adjuvants such as sweetening, flavoring, and perfuming agents.

[0120] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, etc. They can also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable medium immediately before use. They may contain, for example, suspending agents or dispersing agents known in the art.

[0121] The formulations containing the AP-based agent (and / or additional therapeutic agent) can be conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical art. Such methods generally include the step of combining the therapeutic agent in a carrier consisting of one or more additional ingredients. Generally, the formulation is prepared by uniformly and intimately combining the therapeutic agent with a liquid carrier, a fine-grained solid carrier, or both, and then, if necessary, forming the product into the dosage form of the desired formulation (e.g., wet or dry granulation, powder blending, etc., followed by tableting using conventional methods known in the art).

[0122] In various embodiments, the modified release formulations of the invention may utilize one or more modified release coatings such as delayed release coatings to provide effective, delayed bulk delivery of an AP-based agent (e.g., IAP or other AP-based agents described herein, and variants thereof) along with optional other additional therapeutic agents to the gastrointestinal tract.

[0123] In one embodiment, the delayed release coating is included in an enteric solvent that is basically stable in an acidic environment and is unstable in a nearly neutral to alkaline environment. In one embodiment, the delayed release coating is included in an enteric solvent that is basically stable in gastric juice and / or intestinal juice. Enteric solvents can be selected from solutions or dispersions of, for example, methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, carboxymethyl ethyl cellulose and EUDRAGIT type polymers (poly (methacrylic acid, methyl methacrylate), hydroxypropyl methylcellulose acetate succinate), cellulose acetate trimellitate, shellac or other suitable enteric coating polymers. EUDRAGIT type polymers include, for example, EUDRAGIT FS 30D, L 30D-55, L 100-55, L 100, L 12,5, L 12,5P, RL 30D, RL PO, RL 100, RL12,5, RS 30D, RS PO, RS100, RS12,5, NE 30D, NE 40D, NM 30D, S100, S12,5 and S12,5P. Similar polymers include KOLLICOAT MAE 30DP and KOLLICOAT MAE 100P. In some embodiments, one or more of EUDRAGIT FS 30D, L 30D-55, L 100-55, L 100, L 12,5, L 12,5P RL 30D, RL PO, RL 100, RL 12,5, RS30D, RS PO, RS100, RS12,5, NE 30D, NE 40D, NM 30D, S100, S12,5S12,5P, KOLLICOAT MAE30DP, and KOLLICOAT MAE 100P are used. In various embodiments, the enteric solvent can be a combination of the aforementioned solutions or dispersions. In one embodiment, the delayed release coating includes the enteric solvent EUDRAGIT L 30D-55 (poly(methacrylic acid-ethyl acrylate copolymer) 1:1).

[0124] In certain embodiments, one or more coating system additives are used with an enteric solvent. For example, one or more PLASACRYL additives can be used as anti-tacking agent coating additives. Exemplary PLASACRYL additives include, but are not limited to, PLASACRYL HTP20 and PLASACRYL T20. In one embodiment, PLASACRYL HTP20 is formulated with a EUDRAGIT L 30D-55 coating. In another embodiment, PlasACRYL T20 is formulated with a EUDRAGIT FS 30D coating.

[0125] In another embodiment, the delayed release coating can degrade over time in an aqueous solution, regardless of the pH and / or presence of enzymes in the solution. Such coatings may include water-insoluble polymers. Therefore, its solubility in aqueous solution is independent of pH. As used herein, the term "pH-independent" refers to the water permeability of a polymer and its ability to release a drug ingredient that does not change with pH and / or only depends very slightly on pH. Such coatings can be used to prepare, for example, sustained-release formulations. Suitable water-insoluble polymers include pharmaceutically acceptable nontoxic polymers that are substantially insoluble in aqueous media (e.g., water) regardless of the pH of the solution. Suitable polymers include, but are not limited to, cellulose ethers, cellulose esters, or cellulose ether-esters, i.e., cellulose derivatives in which some hydroxyls on the cellulose backbone are substituted by alkyl groups and some hydroxyls are modified by alkanoyl groups. Examples include ethyl cellulose, acetyl cellulose, nitrocellulose, etc. Other examples of insoluble polymers include, but are not limited to, lacquer and acrylic and / or methacrylate polymers, polymers or copolymers of acrylic or methacrylates with low quaternary ammonium content, or mixtures thereof, etc. Other examples of insoluble polymers include EUDRAGIT RS, EUDRAGIT RL and EUDRAGIT NE. Insoluble polymers useful in the present invention include polyvinyl esters, polyvinyl acetals, polyacrylates, butadiene styrene copolymers, etc. In one embodiment, colonic delivery is achieved by using a slowly eroding wax plug (e.g., various PEGS, including, for example, PEG6000).

[0126] In another embodiment, the delayed release coating is degradable by microbial enzymes present in the intestinal flora. In one embodiment, the delayed release coating is degradable by bacteria present in the small intestine. In another embodiment, the delayed release coating is degradable by bacteria present in the large intestine.

[0127] In various embodiments, the present invention provides a formulation comprising: a core particle having a base coating comprising one or more AP-based agents (e.g., IAP, or other AP-based agents described herein, and variants thereof), and a delayed release coating disposed on the coated core particle. The delayed release coating may be substantially stable in an acidic environment and / or gastric fluid, and / or substantially unstable in a near-neutral to alkaline environment and / or intestinal fluid, thereby exposing the coated core particle to intestinal fluid. For example, in some embodiments, the coated pellets of the present invention do not substantially release the AP-based agent in FaSSGF, but substantially release the AP-based agent in FaSSIF. In some embodiments, the coated pellets of the present invention release at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the AP-based agent in simulated gastric and / or intestinal fluid in about 20 minutes, about 40 minutes, about 1 hour, about 80 minutes, about 100 minutes, about 2 hours, about 140 minutes, about 160 minutes, about 3 hours, about 200 minutes, about 220 minutes, or about 4 hours.

[0128] In some embodiments, the coated pellets of the present invention comprising an AP-based agent are stable in storage for at least about 3 days, at least about 9 days, at least about 12 days, at least about 15 days, at least about 20 days, at least about 23 days, at least about 27 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 1.5 years, at least about 2 years. In some embodiments, the pellets are stored at 2-8° C. under dry conditions. In other embodiments, the coated pellets of the present invention comprising an AP-based agent retain at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of their activity over the storage duration.

[0129] The base coating comprising one or more AP-based agents may also comprise one or more additional therapeutic agents. Optionally, multiple base coatings may be applied to the core, each of which may comprise an AP-based agent and / or an additional therapeutic agent. In one embodiment, the core particle comprises sucrose. The formulation may be prepared by methods known in the art. For example, an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) may be sprayed onto an inert core (e.g., sucrose cores or sucrose spheres) and spray dried with an enteric layer (e.g., EUDRAGIT L30 D-55) to form pellets containing an AP-based agent (e.g., IAP or other AP-based agents described herein, and variants thereof).

[0130] Optionally, the core particle may contain one or more AP-based agents (e.g., IAP, or other AP-based agents described herein, and variants thereof) and / or one or more additional therapeutic agents. In one embodiment, one or more doses of an AP-based agent may be encapsulated in the core particle, for example, in the form of microspheres. For example, an AP-based agent may be combined with a polymer (e.g., latex) and then formed into microparticles, microencapsulated enzyme preparations, without using a sucrose core. The microspheres thus formed may optionally be coated with a delayed release coating.

[0131] A variety of methods are known for producing microparticles such as microspheres, aggregates, etc., which are suitable for the inclusion of enzymes. They generally involve at least two phases, one containing the enzyme and one containing the polymer that forms the microparticle backbone. The most common are coacervation, in which the polymer is separated from its solvent phase by the addition of a third component; or multiphase emulsions, such as water-in-oil-in-water (w / o / w) emulsions, in which the inner aqueous phase contains the protein, the middle organic phase contains the polymer, and an outer aqueous phase stabilizer that supports the w / o / w double emulsion until the solvent can be removed to form microspheres. Alternatively, an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) and a stabilizing excipient (e.g., trehalose, mannitol, Tween 80, polyvinyl alcohol) are combined and sprayed and collected from an aqueous solution. The particles are then suspended in an anhydrous, water-immiscible organic solvent containing the polymer and the release-modifying compound, and the suspension is sonicated to disperse the particles. Another method uses an aqueous phase but no organic solvent. Specifically, enzymes, buffer components, polymer latex, and stabilizing and release-modifying excipients are dissolved / dispersed in water. The aqueous dispersion is spray dried, resulting in latex agglomeration, and the protein and excipients are incorporated into the agglomerated latex particles. When the release modifier is insoluble under acidic conditions but soluble under higher pH (such as carboxylic acids), release from the matrix is ​​inhibited in the gastric environment.

[0132] In some embodiments, before applying the delayed release coating to the coated core particles, the particles may be optionally coated with one or more separation layers comprising pharmaceutical excipients, including alkaline compounds such as pH buffering compounds. The separation layer substantially separates the coated core particles from the delayed release coating.

[0133] Separating layer can be applied to the coated core particles by the coating or coating procedure used in the fluidized bed device using water and / or organic solvent to carry out coating process by using coating equipment such as coating pan, coating granulator usually.As an alternative, separating layer can be applied to the core material by using powder coating technology.The material used for separating layer is pharmaceutically acceptable compound, for example sugar, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methylcellulose, ethyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose etc., alone or in combination.Additives such as plasticizer, coloring agent, pigment, filler, antitackifier and antistatic agent (such as magnesium stearate, titanium dioxide, talcum) and other additives also can be included in the separating layer.

[0134] In some embodiments, the coated particles with delayed release coating can be further coated with an outer coating layer (overcoat layer). The outer coating layer can be applied as described for other coating compositions. The outer coating material is a pharmaceutically acceptable compound, such as sugar, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, etc., used alone or in combination. The outer coating material can prevent the potential agglomeration of the particles coated with the delayed release coating, and the delayed release coating is protected from cracking or enhancing the tableting process during compaction.

[0135] In some embodiments, the modified release formulation is a capsule filled with a plurality of AP-based agent-containing pellets (e.g., IAP (or other AP-based agents described herein, and variants thereof), from which the AP-based agent is released. In one embodiment, the capsule is a gelatin capsule, such as a hard gelatin capsule. In another embodiment, the capsule is a hydroxypropylmethylcellulose (HPMC) capsule. For example, the formulation can be in the form of a capsule comprising a plurality of pellets. For example, the formulation can be in the form of a capsule, such as a plurality of enteric-coated pellets containing an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof). of gelatin or hydroxypropyl methylcellulose (HPMC) capsules. In such embodiments, a combination of pellets may be utilized, wherein each pellet is designed to release at a specific time point or location. In various embodiments, the pellets (e.g., enteric coated pellets) are designed to pass through the stomach as is and then release the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) to one or more regions of the intestine. In some embodiments, the pellets containing the AP-based agent may have an enteric coating to release the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) at different intestinal pH values.

[0136] The present invention also provides modified release formulations that release multiple doses of an AP-based agent (e.g., an IAP, or other AP-based agents described herein, and variants thereof) and / or an additional therapeutic agent along the gastrointestinal tract. In such embodiments, the overall release characteristics of such formulations can be adjusted by utilizing, for example, multiple particle types or multiple layers. In one embodiment, a first dose of an AP-based agent can be formulated for release in, for example, the small intestine (e.g., one or more of the duodenum, jejunum, ileum) or the large intestine (e.g., one or more of the cecum, the ascending, transverse, descending or sigmoid portion of the colon, and the rectum), while a second dose is formulated for delayed release in, for example, a different region of the small intestine (e.g., one or more of the duodenum, jejunum, ileum) or the large intestine (e.g., one or more of the cecum, the ascending, transverse, descending or sigmoid portion of the colon, and the rectum). Alternatively, multiple doses are released at different locations along the intestine. For example, in one embodiment, a first dose of an AP-based agent may be formulated for release in, for example, the small intestine (e.g., one or more of the duodenum, jejunum, ileum), while a second dose is formulated for delayed release in, for example, another portion of the small intestine (e.g., one or more of the duodenum, jejunum, ileum). In another embodiment, a first dose of an AP-based agent may be formulated for release in, for example, the large intestine (e.g., one or more of the cecum, the ascending, transverse, descending, or sigmoid portion of the colon, and the rectum), while a second dose is formulated for delayed release in, for example, another portion of the large intestine (e.g., one or more of the cecum, the ascending, transverse, descending, or sigmoid portion of the colon, and the rectum).

[0137] In various embodiments, the agents described herein are in the form of pharmaceutically acceptable salts, i.e., those salts suitable for use in contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic reaction, etc., and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Salts can be prepared in situ during the final isolation and purification of the therapeutic agent, or prepared separately by reacting a free base functional group with a suitable acid or a free acid functional group with a suitable base moiety. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0138] In various embodiments, the formulations of the present invention provide many advantages. For example, the inventors have successfully formulated proteins (i.e., AP-based agents), which are inherently challenging. In various embodiments, this is further exacerbated by the gastrointestinal environment in which the formulations of the present invention release the drug. In addition, in various embodiments, the formulations of the present invention provide a sufficiently slow gastrointestinal release to allow for good protective coating in the gastrointestinal tract from the adverse effects of various antibiotics, such as in the small intestine (a benefit highlighted by the increase in the half-life of AP-based agents commensurate with the slower release). In addition, for example, unlike EUDRAGIT, by coating the drug substance layer of the pellets of the present invention with HPC, the formulations of the present invention minimize the amount of EUGRAGIT in the formulation and thereby mitigate possible dose-limiting toxicities and manufacturing complications.

[0139] Improved release properties

[0140] In one aspect, the invention provides a modified release formulation comprising at least one alkaline phosphatase (AP)-based agent, wherein the formulation releases a substantial amount of the AP-based agent into one or more regions of the gastrointestinal tract. In some embodiments, the AP-based agent is an IAP, or other AP-based agents described herein, and variants thereof (e.g., as described above). For example, the formulation can release at least about 60% of the AP-based agent (e.g., IAP) into one or more regions of the gastrointestinal tract after the stomach.

[0141] In various embodiments, the modified release formulations of the present invention are designed to release immediately (e.g., upon ingestion). In various embodiments, the modified release formulations may have sustained release characteristics, i.e., the active ingredient is slowly released in the body (e.g., the gastrointestinal tract) over an extended period of time. In various embodiments, the modified release formulations may have delayed release characteristics, i.e., the active ingredient is not released immediately upon ingestion; instead, the release of the active ingredient is postponed until the composition is in the lower gastrointestinal tract; for example, for release in the small intestine (e.g., one or more of the duodenum, jejunum, ileum) or the large intestine (e.g., one or more of the ascending, transverse, descending or sigmoid colon portions of the cecum, colon, and rectum). For example, the composition may be enteric coated to delay the release of the active ingredient until it reaches the small intestine or large intestine. In some embodiments, there is no significant amount of the active ingredient of the formulation of the present invention in feces.

[0142] In various embodiments, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) to one or more regions of the intestine after the stomach. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the intestine.

[0143] In various embodiments, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the small intestine. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the small intestine.

[0144] In one embodiment, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the duodenum. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the duodenum.

[0145] In one embodiment, the modified release formulation of the invention releases at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the jejunum. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the jejunum.

[0146] In one embodiment, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the ileum and / or ileocecal junction. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the ileum and / or ileocecal junction.

[0147] In various embodiments, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the large intestine. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the large intestine.

[0148] In one embodiment, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the cecum. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the cecum.

[0149] In one embodiment, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the ascending colon. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the ascending colon.

[0150] In one embodiment, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the transverse colon. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the transverse colon.

[0151] In one embodiment, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the descending colon. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the descending colon.

[0152] In one embodiment, the modified release formulations of the invention release at least 60% of the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the sigmoid colon. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) in the sigmoid colon.

[0153] In various embodiments, the modified release formulations of the invention do not substantially release the AP-based agent (eg, IAP, or other AP-based agents described herein, and variations thereof) in the stomach.

[0154] In various embodiments, the modified release formulations of the present invention release an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) at a specific pH. For example, in some embodiments, the modified release formulation is substantially stable in an acidic environment and substantially unstable (e.g., rapidly dissolving or physically unstable) in a near neutral to alkaline environment. In some embodiments, stable means substantially not releasing, while unstable means substantially releasing. For example, in some embodiments, the modified release formulation is substantially stable at a pH of about 7.0 or less, or about 6.5 or less, or about 6.0 or less, or about 5.5 or less, or about 5.0 or less, or about 4.5 or less, or about 4.0 or less, or about 3.5 or less, or about 3.0 or less, or about 2.5 or less, or about 2.0 or less, or about 1.5 or less, or about 1.0 or less. In some embodiments, the formulations of the present invention are stable in lower pH regions and are therefore substantially not released, for example, in the stomach. In some embodiments, the modified release formulation is substantially stable at a pH of about 1 to about 4 or lower, and is substantially unstable at a higher pH value. In these embodiments, the modified release formulation is substantially not released in the stomach. In these embodiments, the modified release formulation is substantially released in the small intestine (e.g., one or more of the duodenum, jejunum and ileum) and / or the large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon and sigmoid colon). In some embodiments, the modified release formulation is substantially stable at a pH of about 4 to about 5 or lower, and is therefore substantially unstable at a higher pH value, and is therefore substantially not released in the stomach and / or the small intestine (e.g., one or more of the duodenum, jejunum and ileum). In these embodiments, the modified release formulation is substantially released in the large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon and sigmoid colon). In various embodiments, the pH value described herein can be adjusted as known in the art to illustrate the state of the subject, for example, whether it is in a fasting state or a postprandial state.

[0155] In some embodiments, the modified release formulation is substantially stable in gastric fluid and substantially unstable in intestinal fluid, and thus releases substantially in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) and / or the large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon).

[0156] In some embodiments, the modified release formulations are stable in gastric fluid or stable in an acidic environment. These modified release formulations release about 30% by weight or less of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) and / or the additional therapeutic agent in the modified release formulation in gastric fluid having a pH of about 4 to about 5 or less, or simulated gastric fluid having a pH of about 4 to about 5 or less, within about 15, or about 30, or about 45, or about 60, or about 90 minutes. The modified release formulations of the present invention can release about 0% to about 30%, about 0% to about 25%, about 0% to about 20%, about 0% to about 15%, about 0% to about 10%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10% by weight of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) and / or the additional therapeutic agent in the modified release formulation in gastric fluid at a pH of 4-5 or less, or simulated gastric fluid at a pH of 4-5 or less, in about 15, or about 30, or about 45, or about 60, or about 90 minutes. The modified release formulations of the invention can release about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% by weight of the total AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) and / or the additional therapeutic agent in the modified release formulation in gastric fluid at a pH of 5 or less, or simulated gastric fluid at a pH of 5 or less, within about 15, or about 30, or about 45, or about 60 or about 90 minutes.

[0157] In some embodiments, the modified release formulation is unstable in intestinal fluid. These modified release formulations release about 70% by weight or more of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) and / or the additional therapeutic agent in the modified release formulation in intestinal fluid or simulated intestinal fluid within about 15, or about 30, or about 45, or about 60, or about 90 minutes. In some embodiments, the modified release formulation is unstable in a nearly neutral to alkaline environment. These modified release formulations release about 70% by weight or more of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) and / or the additional therapeutic agent in the modified release formulation in intestinal fluid or simulated intestinal fluid at a pH of about 4-5 or higher within about 15, or about 30, or about 45, or about 60, or about 90 minutes. A modified release formulation that is unstable in a near neutral or alkaline environment can release 70% by weight or more of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variations thereof) and / or an additional therapeutic agent in the modified release formulation in a fluid having a pH greater than about 5 (e.g., a fluid having a pH of about 5 to about 14, about 6 to about 14, about 7 to about 14, about 8 to about 14, about 9 to about 14, about 10 to about 14, or about 11 to about 14) in about 5 minutes to about 90 minutes, or about 10 minutes to about 90 minutes, or about 15 minutes to about 90 minutes, or about 20 minutes to about 90 minutes, or about 25 minutes to about 90 minutes, or about 30 minutes to about 90 minutes, or about 5 minutes to about 60 minutes, or about 10 minutes to about 60 minutes, or about 15 minutes to about 60 minutes, or about 20 minutes to about 60 minutes, or about 25 minutes to about 90 minutes, or about 30 minutes to about 60 minutes.

[0158] Examples of simulated gastric fluid and simulated intestinal fluid include, but are not limited to, those disclosed in Test Solutions on page 2858 of the 2005 Pharmacopoeia 23NF / 28USP and / or other simulated gastric fluid and simulated intestinal fluid known to those skilled in the art, for example, simulated gastric fluid and / or intestinal fluid prepared in the absence of enzymes.

[0159] In one embodiment, the modified release formulation can remain substantially intact in gastric fluid, or can be substantially insoluble. The modified release formulation may include one or more pH-dependent delayed release coatings. The pH-dependent delayed release coating will be substantially stable in an acidic environment (pH of about 5 or less), and substantially unstable in a near neutral to alkaline environment (pH greater than about 5). For example, the delayed release coating may substantially disintegrate or dissolve in a near neutral to alkaline environment, as found in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) and / or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon).

[0160] Alternatively, the stability of the modified release formulation can be enzyme dependent. In such embodiments, the modified release formulation can include one or more enzyme-dependent delayed release coatings. The enzyme-dependent delayed release coating will be substantially stable in a fluid that does not contain a specific enzyme and is substantially unstable in a fluid that contains an enzyme. The delayed release coating will substantially disintegrate or dissolve in a fluid containing an appropriate enzyme. Enzyme-dependent control can be achieved, for example, by using a material that releases the active ingredient only when exposed to an enzyme in the intestine, such as galactomannan. In addition, in the presence of microbial enzymes in the intestinal flora, the stability of the modified release formulation can depend on the stability of the enzyme.

[0161] In various embodiments, the modified release formulation comprising an AP-based agent (e.g., an IAP or a variant thereof) is substantially stable in the chyme. For example, in some embodiments, the loss of activity of the AP-based agent is less than about 50%, or about 40%, or about 30%, or about 20%, or about 10% within about 10, or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 hour after administration.

[0162] In some embodiments, dual pulse formulations are provided. In various embodiments, the present invention provides a modified release formulation that releases multiple doses of an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) at different times and / or different pHs at different locations along the intestinal tract. In an illustrative embodiment, the modified release formulation comprises a first dose of an AP-based agent and a second dose of an AP-based agent, wherein the first dose and the second dose are released at different times and / or different pHs at different locations along the intestinal tract. For example, the first dose is released in the duodenum and the second dose is released in the ileum. In another example, the first dose is released in the jejunum and the second dose is released in the ileum. In other embodiments, the first dose is released at a location along the small intestine (e.g., the duodenum) and the second dose is released along the large intestine (e.g., the ascending colon). In various embodiments, the modified release formulation can release at least one dose, at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, or at least eight doses of an AP-based agent (e.g., IAP or other AP-based agents described herein, and variants thereof) at different locations along the intestinal tract at different times and / or different pHs. In addition, the dual pulse description herein is applicable to modified release formulations that release an AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) and an additional therapeutic agent.

[0163] Administration and Dosage

[0164] It should be understood that the actual dosage of the AP-based agent (e.g., IAP, or other AP-based agents described herein, and variants thereof) administered in accordance with the present invention will vary depending, for example, on the particular dosage form and mode of administration. One skilled in the art can consider many factors that may alter the effects of the AP-based agent (e.g., body weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combination, genetic predisposition, and reaction sensitivity). Administration may be performed continuously within the maximum tolerated dose or in one or more discrete doses. One skilled in the art can use routine dosing tests to determine the optimal administration rate under a given set of conditions.

[0165] Individual doses of an AP-based agent (e.g., IAP or other AP-based agents described herein, and variants thereof) can be administered in a unit dosage form (e.g., tablet or capsule) containing, for example, about 0.01 mg to about 1,000 mg, about 0.01 mg to about 950 mg, about 0.01 mg to about 900 mg, about 0.01 mg to about 850 mg, about 0.01 mg to about 800 mg, about 0.01 mg to about 750 mg, about 0.01 mg to about 700 mg, about 0.01 mg to about 650 mg, about 0.01 mg to about 600 mg, about 0.01 mg to about 550 mg, about 0.01 mg to about 500 mg, about 0.01 mg to about 450 mg, about 0.01 mg to about 400 mg per unit dosage form. mg, about 0.01 mg to about 350 mg, about 0.01 mg to about 300 mg, about 0.01 mg to about 250 mg, about 0.01 mg to about 200 mg, about 0.01 mg to about 150 mg, about 0.01 mg to about 100 mg, about 0.1 mg to about 90 mg, about 0.1 mg to about 80 mg, about 0.1 mg to about 70 mg, about 0.1 mg to about 60 mg, about 0.1 mg to about 50 mg, about 0.1 mg to about 40 mg of active ingredient, about 0.1 mg to about 30 mg, about 0.1 mg to about 20 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 3 mg, about 0.1 mg to about 1 mg, or about 5 mg to about 80 mg per unit dosage form. For example, the unit dosage form can be about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 3 about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1500 mg, about 2000 mg, about 2500 mg, about 3000 mg, about 3500 mg, about 4000 mg, about 4500 mg, about 5000 mg, about 5500 mg, about 6000 mg, about 6500 mg, about 7000 mg, about 7500 mg, about 8000 mg, about 8500 mg, about 9000 mg, about 9500 mg or about 1,000 mg, including all values ​​and ranges therebetween.In one embodiment, a single dose of an AP-based agent (e.g., IAP or other AP-based agents described herein, and variants thereof) is administered in a unit dosage form containing 25 mg of the AP-based agent. In another embodiment, a single dose of an AP-based agent (e.g., IAP or other AP-based agents described herein, and variants thereof) is administered in a unit dosage form containing 50 mg of the AP-based agent. In yet another embodiment, a single dose of an AP-based agent (e.g., IAP or other AP-based agents described herein, and variants thereof) is administered in a unit dosage form containing 75 mg of the AP-based agent.

[0166] In one embodiment, the composition is in an amount of about 0.01 mg to 100 mg per day, about 0.01 mg to about 1,000 mg per day, about 0.01 mg to about 950 mg per day, about 0.01 mg to about 900 mg per day, about 0.01 mg to about 850 mg per day, about 0.01 mg to about 800 mg per day, about 0.01 mg to about 750 mg per day, about 0.01 mg to about 700 mg per day, about 0.01 mg to about 650 mg per day, about 0.01 mg to about 800 mg per day, about 0.01 mg to about 800 mg per day, about 0.01 mg to about 900 mg per day, about 0.01 mg to about 1000 mg per day, about 0.01 mg to about 15 ...500 mg per day, about 0.01 mg to about 1500 mg per day, about 0.01 mg to about 1500 mg per day, about 0.01 mg to about 1500 mg per day, about 0.01 mg to about 1500 mg per day, about 0.01 mg to about 1500 mg per day, about 0.01 mg 0.01 mg to about 600 mg per day, about 0.01 mg to about 550 mg per day, about 0.01 mg to about 500 mg per day, about 0.01 mg to about 450 mg per day, about 0.01 mg to about 400 mg per day, about 0.01 mg to about 350 mg per day, about 0.01 mg to about 300 mg per day, about 0.01 mg to about 250 mg per day, about 0.01 mg to about 200 mg per day, about 0.01 mg to about 150 mg per day, about 0.1 mg per day g to about 100 mg per day, about 0.1 mg to about 95 mg per day, about 0.1 mg to about 90 mg per day, about 0.1 mg to about 85 mg per day, about 0.1 mg to about 80 mg per day, about 0.1 mg to about 75 mg per day, about 0.1 mg to about 70 mg per day, about 0.1 mg to about 65 mg per day, about 0.1 mg to about 60 mg per day, about 0.1 mg to about 55 mg per day, about 0.1 mg to about 50 mg per day, about 0.1 mg to about 45 mg per day g, about 0.1 mg to about 40 mg per day, about 0.1 mg to about 35 mg per day, about 0.1 mg to about 30 mg per day, about 0.1 mg to about 25 mg per day, about 0.1 mg to about 20 mg per day, about 0.1 mg to about 15 mg per day, about 0.1 mg to about 10 mg per day, about 0.1 mg to about 5 mg per day, about 0.1 mg to about 3 mg per day, about 0.1 mg to about 1 mg per day, or about 5 mg to about 80 mg per day.

[0167] In various embodiments, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg The AP-based agent is administered at a daily dose of about 100 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg or about 1,000 mg (including all values ​​and ranges therebetween).

[0168] In some embodiments, a suitable dosage of an AP-based agent (e.g., IAP or other AP-based agents described herein, and variants thereof) is in the range of about 0.01 mg / kg to about 100 mg / kg of subject body weight, for example, about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about or about 100 mg / kg body weight, including all values ​​and ranges therebetween. In other embodiments, suitable dosages of AP-based agents are in the range of about 0.01 mg / kg body weight to about 10 mg / kg body weight, in the range of about 0.01 mg / kg body weight to about 9 mg / kg body weight, in the range of about 0.01 mg / kg body weight to about 8 mg / kg body weight, in the range of about 0.01 mg / kg body weight to about 7 mg / kg body weight, in the range of 0.01 mg / kg body weight to about 6 mg / kg body weight, in the range of about 0.05 mg / kg body weight to about 5 mg / kg body weight, in the range of about 0.05 mg / kg body weight to about 4 mg / kg body weight, in the range of about 0.05 mg / kg body weight to about 3 mg / kg body weight, in the range of about 0.05 mg / kg body weight to about 2 mg / kg body weight, in the range of about 0.05 mg / kg body weight to about 1.5 mg / kg body weight, or in the range of about 0.05 mg / kg body weight to about 1 mg / kg body weight.

[0169] According to certain embodiments of the present invention, the AP-based agent can be administered, for example, about once a day, about once every other day, about once every three days, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year. In certain embodiments, the AP-based agent can be administered more than once a day, for example, about twice, about three times, about four times, about five times, about six times, about seven times, about eight times, about nine times, or about ten times a day.

[0170] Additional therapeutic agents and combination therapies or co-formulations

[0171] Administration of compositions and formulations of the invention comprising AP-based agents can be combined with additional therapeutic agents. The combined administration of additional therapeutic agents and compositions / formulations of the invention can be simultaneous or sequential. In addition, compositions / formulations of the invention can comprise additional therapeutic agents (e.g., via co-formulation). For example, additional therapeutic agents and AP-based agents can be combined into a single formulation. Alternatively, additional therapeutic agents and AP-based agents can be formulated separately.

[0172] In one embodiment, the additional therapeutic agent and the AP-based agent are administered to the subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the AP-based agent are administered with a time interval of no more than about 60 minutes, such as no more than about 30 minutes, no more than about 20 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. Administration of the additional therapeutic agent and the AP-based agent can be performed by simultaneously administering a single formulation (e.g., a formulation comprising the additional therapeutic agent and the AP-based agent) or separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the AP-based agent).

[0173] In another embodiment, the additional therapeutic agent and the AP-based agent are administered to the subject simultaneously, but release of the additional therapeutic agent and the AP-based agent from their respective dosage forms (or a single unit dosage form if co-formulated) occurs sequentially.

[0174] If the administration of the additional therapeutic agent and the AP-based agent is scheduled so that the pharmacological activities of the additional therapeutic agent and the AP-based agent overlap in time, then the combined administration does not require the additional therapeutic agent and the AP-based agent to be administered at the same time. For example, the additional therapeutic agent and the AP-based agent can be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the AP-based agent are administered with a time interval of more than about 60 minutes. For example, the time interval between the sequential administration of the additional therapeutic agent and the AP-based agent can be more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, or more than about 1 week. The optimal administration time will depend on the metabolic rate, excretion rate, and / or pharmacodynamic activity of the additional therapeutic agent and the AP-based agent being administered. Either the additional therapeutic agent or the AP-based agent can be administered first.

[0175] Co-administration also does not require that the additional therapeutic agent and the AP-based agent be administered to the subject by the same route of administration. Rather, each therapeutic agent can be administered by any appropriate route (eg, parenteral or non-parenteral).

[0176] In some embodiments, the additional therapeutic agent is an antibacterial agent, including but not limited to cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefmandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, levofloxacin, ofloxacin, tequin, avelox, and norflox); tetracycline antibiotics (cephalosporin, cefadroxil, cefuroxime, cefadroxil, cefazolin, cefadroxil, ceftriaxone ... Cycline antibiotics (tetracycline, minocycline, oxytetracycline and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin and methicillin); monoamine antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem / cilastatin and meropenem). In some embodiments, the antibacterial agent can be any one of penicillin, cephalosporin, monoamine and carbapenem antibiotics.

[0177] In some embodiments, the additional therapeutic agent is an adjunctive therapy, for example, to treat CDI. In some embodiments, the additional therapeutic agent is metronidazole (e.g., FLAGYL), fidaxomicin (e.g., DIFICID), or vancomycin (e.g., VANCOCIN), rifaximin, a charcoal-based binder / adsorbent (e.g., DAV132), fecal bacterial therapy, probiotic therapy (see, e.g., Intnat'l J Inf Dis, 16(11):e786, the contents of which are hereby incorporated by reference, illustrative probiotics include Saccharomyces boulardii; Lactobacillus rhamnosus GG; Lactobacillus plantarum 299v; Clostridium butyricum M588; Clostridium difficile VP20621 (a non-toxigenic strain of Clostridium difficile, formerly known as Clostridium difficile); a combination of Lactobacillus casei and Lactobacillus acidophilus (Bio-K+CL1285); a combination of Lactobacillus casei, Lactobacillus bulgaricus, and Streptococcus thermophilus (Actimel); a combination of Lactobacillus acidophilus and Bifidobacterium bifidum (Florajen3); a combination of Lactobacillus acidophilus, Lactobacillus bulgaricus delbrueckii subsp.bulgaricus, Lactobacillus bulgaricus casei, Lactobacillus bulgaricus plantarum, Bifidobacterium longum longum), Bifidobacterium infantis, Bifidobacterium breve, Streptococcus salivarius subsp. thermophilus (VSL#3), and antibodies or other biological therapies (e.g., monoclonal antibodies against Clostridium difficile, such as those described in N Engl J Med.2010;362(3):197, the contents of which are hereby incorporated by reference in their entirety; neutralizing binding proteins, such as neutralizing binding proteins arranged as multimers, directed against one or more of the SEQ ID NOs recited in U.S. Patent Publication No. 2013 / 0058962 (e.g., one or more of SEQ ID NOs: 59, 60, 95, 67, 68, and 87), the contents of which are hereby incorporated by reference in their entirety); or any neutralizing binding protein directed against Clostridium difficile binary toxin. .

[0178] In some embodiments, the additional therapeutic agent is an antidiarrheal agent. Antidiarrheal agents suitable for use in the present invention include, but are not limited to, DPP-IV inhibitors, natural opioids (such as opium tincture, analgesics, and codeine), synthetic opioids (such as diphenoxylate, difenoxin, and loperamide), bismuth subsalicylate, lanreotide, vapreotide, and octreotide, motilin antagonists, COX2 inhibitors (such as celecoxib), glutamine, thalidomide, and traditional antidiarrheals (such as kaolin, pectin, berberine, and muscarinics).

[0179] In some embodiments, the additional therapeutic agent is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a nonsteroidal anti-inflammatory agent (NSAID). Steroids, particularly adrenocortical steroids and synthetic analogs thereof are well known in the art. Examples of corticosteroids that can be used in the present invention include, but are not limited to, hydroxytriamcinolone, α-methyldexamethasone, β-methylbetamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, dexamethasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone valerate, fluocortol ... acetonide), flumethasone pivalate, fluocinolone acetate, fluocinolone, fluocinolone butyl, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolide, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, fluocinolone acetonide, fludrocortisone, difluroxolone diacetate, flurazepam, medrysone, amcinolone, ancifet, betamethasone and its ester balance, cloprednisolone, clocortolone, decilone, diclofenac, difluprednate, fluclofenac, flunisolide, fluorometholone, fluperone, fluprednisolone, hydrocortisone, methylprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate. (NSAID) that can be used in the present invention includes but is not limited to salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylamide, benzyl-2,5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone and indomethacin. Additional anti-inflammatory agents are described, for example, in U.S. Pat. No. 4,537,776, the entire contents of which are incorporated herein by reference.

[0180] In some embodiments, the additional therapeutic agent can be an analgesic. Analgesics that can be used in the compositions and methods of the present invention include, but are not limited to, morphine, codeine, heroin, methadone and related compounds, thebaine, orpiavine and its derivatives, buprenorphine, piperidine, morphinan, benzomorphan, tetrahydroisoquinoline, thiobutane, benzylamine, tilidine, viminol, nefopam, capsaicin (8-methyl-N-vanillyl-6E-nonenamide), "synthetic" capsaicin (N-vanillylnonenamide) and related compounds.

[0181] In some embodiments, the additional therapeutic agent may be an antiviral agent, including but not limited to Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet.

[0182] In some embodiments, the additional therapeutic agent can be an agent for treating inflammatory bowel disease. For example, the agent can be used to treat colitis (e.g., ulcerative colitis) and Crohn's disease, including but not limited to vedolizumab (ENTYVIO), tofacitinib (XELJANZ), DIMS 0150 (KAPPAPROCT), golimumab (SIMPONI), adalimumab (HUMIRA) and other anti-TNF therapeutic agents.

[0183] In some embodiments, the additional therapeutic agent may be an agent for treating celiac disease. Illustrative agents include, but are not limited to, AVX-176 (Avaxia Biologics), Actobiotics (ActoGeniX), CALY-002 (Calypsobiotech), HLA-DQ2 antagonists, HLA-DQ2 / DQ8 antagonists, tTG inhibitors including ERW1041E (GlaxoSmithKline) and ZED-101 / ZED-1227 (Zedira), Larazotide actate (Alba Therapeutics), Latiglutenase (Alvine Pharmaceuticals), BL-7010 (BioLineRx), and NexVax-2 (ImmunsanT).

[0184] In some embodiments, the additional therapeutic agent can be an agent for treating cystic fibrosis. Illustrative agents include, but are not limited to, ivacaftor (KALYDECO; Vertex), lumacaftor / ivacaftor (ORKAMBI; Vertex), VX-152 (Vertex), VX-440 (Vertex), VX-371 (Vertex), nitric oxide, glyceryl phenylbutyrate, riociguat (Bayer), recombinant A1PI (Grifols, SA), cysteamine IR, JBT-101 (Corbus Pharmaceuticals), N-91115 (Nivalis Therapeutics), and vancomycin.

[0185] In some embodiments, the additional therapeutic agent is an agent for treating obesity. Illustrative agents include but are not limited to orlistat, lorcaserin, phentermine-topiramate, naltrexone-bupropion, rimonabant, exenatide, pramlintide, phentermine, benzphetamine, diethylpropion, phendimetrazine, bupropion and metformin. In various embodiments, additional agents are agents that interfere with the body's ability to absorb specific nutrients in food, such as orlistat, glucomannan and guar gum. Agents that suppress appetite also belong to additional agents, such as catecholamines and their derivatives (such as phentermine and other amphetamine-based drugs), various antidepressants and mood stabilizers (such as bupropion and topiramate), anorexia agents (such as dextroamphetamine, digoxin). Agents that increase body metabolism also belong to additional agents. In some embodiments, additional agents can be selected from appetite suppressants, neurotransmitter reuptake inhibitors, dopaminergic agonists, serotonergic agonists, GABAergic signaling regulators, anticonvulsants, antidepressants, monoamine oxidase inhibitors, substance P (NKI) receptor antagonists, melanocortin receptor agonists and antagonists, lipase inhibitors, fat absorption inhibitors, energy intake or metabolism regulators, cannabinoid receptor modulators, addiction treatment agents, metabolic syndrome treatment agents, peroxisome proliferator-activated receptor (PPAR) modulators; and dipeptidyl peptidase 4 (DPP-4) antagonists.In some embodiments, the additional agent can be selected from amphetamines, benzodiazepines, sulfonylureas, meglitinides, thiazolidinediones, biguanides, beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, phentermine, sibutramine, lorcaserin, cetilistat, rimonabant, taranabant, topiramate, gabapentin, valproate, vigabatrin, bupropion, tiagabine, sertraline, line), fluoxetine, trazodone, zonisamide, methylphenidate, varenicline, naltrexone, diethylpropion, phendimetrazine, repaglinide, nateglinide, glimepiride, pioglitazone, rosiglilazone, liraglutide, and sitagliptin.

[0186] In one embodiment, the additional therapeutic agent is an agent for treating prediabetes, diabetes, type II diabetes, insulin resistance, glucose intolerance, or hyperglycemia. Examples of drugs include, but are not limited to, alpha-glucosidase inhibitors, amylin analogs, dipeptidyl peptidase-4 inhibitors, GLP1 agonists, meglitinides, sulfonylureas, biguanides, thiazolidinediones (TZDs), and insulin. Other examples of such agents include bromocriptine and Welchol. Examples of alpha-glucosidase inhibitors include, but are not limited to, acarbose and miglitol. An example of an amylin analog is pramlintide. Examples of dipeptidyl peptidase-4 inhibitors include, but are not limited to, saxagliptin, sitagliptin, vildagliptin, linagliptin, and alogliptin. Examples of GLP1 agonists include, but are not limited to, liraglutide, exenatide, exenatide extended release. Examples of meglitinides include, but are not limited to, nateglinide and repaglinide. Examples of sulfonylureas include, but are not limited to, chlorpropamide, glimepiride, glipizide, glyburide, tolazamide, and tolbutamide. Examples of biguanides include, but are not limited to, metformin, Riomet, Glucophage, Glucophage XR, Glumetza. Examples of thiazolidinediones include, but are not limited to, rosiglitazone and pioglitazone. Examples of insulin include, but are not limited to, Aspart, Detemir, Glargine, Glulisine, and Lispro. Examples of combination drugs include, but are not limited to, glipizide / metformin, glyburide / metformin, pioglitazone / glimepiride, pioglitazone / metformin, repaglinide / metformin, rosiglitazone / glimepiride, rosiglitazone / metformin, saxagliptin / metformin, sitagliptin / simvastatin, sitagliptin / metformin, linagliptin / metformin, alogliptin / metformin, and alogliptin / pioglitazone.

[0187] Treatment

[0188] In various embodiments, the present invention provides a method for treating or preventing radiation-induced conditions, including but not limited to enterocolitis, radiation-induced enteropathy, colitis and / or proctitis caused by cancer radiotherapy. Radiation-induced enteropathy is characterized by mucosal atrophy, vascular sclerosis and progressive intestinal wall fibrosis. The symptoms of this condition may include malnutrition absorption, intestinal transit changes, dyskinesia and abnormal advancement of intestinal contents. In some embodiments, radiation-induced acute enteropathy occurs within the first month, first 2 months or first 3 months after radiation exposure. In some embodiments, delayed radiation enteropathy symptoms are chronic and may not present until at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 months after radiation exposure. In some embodiments, delayed radiation enteropathy symptoms may not present until about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 months after radiation exposure. In some embodiments, delayed radiation enteropathy symptoms may not appear until about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years after radiation exposure.

[0189] In some embodiments, the present invention provides for the treatment and / or administration of an AP-based agent to a subject that has been exposed to radiation, including but not limited to radiotherapy. In various embodiments, administration of the AP-based agent occurs prior to exposure to radiation, for example, prior to radiotherapy as part of cancer treatment. In certain embodiments, administration of the AP-based agent occurs at the time of radiation exposure. In various embodiments, administration of the AP-based agent occurs at the time of exposure to radiation and shortly after exposure to radiation. In some embodiments, administration of the AP-based agent occurs shortly after exposure to radiation. In various embodiments, administration of the AP-based agent occurs at the time of exposure to radiation and continues long-term after exposure to radiation. In some embodiments, administration of the AP-based agent continues long-term after exposure to radiation. In various embodiments, administration of the AP-based agent occurs at the onset of delayed radiation enteropathy. In some embodiments, the present invention provides for treatment and / or administration of an AP-based agent to a subject that has been exposed or will be exposed to radiation, wherein administration of the AP-based agent occurs at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least 3 years, at least 3.5 years, 4 years, at least 4.5 years, at least 5 years, at least 5.5 years, at least 6 years, at least 6.5 years, or at least 7 years after exposure to radiation.

[0190] In various embodiments, the present invention provides for the treatment and / or administration of a subject suffering from a radiation-related disease or condition, such as, but not limited to, side effects of radiation therapy or ARS.

[0191] In some embodiments, the present invention provides for the prevention, treatment and / or administration of a subject suffering from radiation enteritis. For example, the subject may suffer from acute or chronic radiation enteritis. Symptoms of radiation enteritis include, but are not limited to, nausea, vomiting, stomach cramps, frequent toileting, watery diarrhea, rectal mucus discharge, rectal pain, rectal bleeding, weight loss, and wavy stomach pain.

[0192] In some embodiments, the present invention relates to a method of treating or preventing a radiation-related disease or condition in a subject in need thereof, comprising administering to the subject a modified release formulation comprising an AP-based agent as described herein. For example, but not limited to, the AP-based agent of the formulation is an orally administrable IAP.

[0193] In various embodiments, the present invention provides treatment and / or administration to a subject suffering from delayed radiation enteropathy and / or enterotoxicity. In some embodiments, delayed radiation enteropathy occurs at least 3 months after the end of radiotherapy. In various embodiments, radiotherapy is radiotherapy for treating cancer. In various embodiments, the subject is a cancer patient. In one embodiment, radiotherapy is directed to a tumor in the pelvis, abdomen, or lower trunk. In some embodiments, the present treatment of the present invention does not interfere with cancer treatment, including but not limited to radiotherapy.

[0194] In various embodiments, the radiation comprises ionizing radiation.In various embodiments, the radiation comprises one or more of X-rays, gamma rays, and charged particles.

[0195] In various embodiments, the radiation exposure is a dose of about 2 Gy, or about 2.5 Gy, or about 3 Gy, or about 3.5 Gy, or about 4 Gy, or about 4.5 Gy, or about 5 Gy, or about 10 Gy, or about 20 Gy, or about 30 Gy, or about 40 Gy, or about 50 Gy, or about 60 Gy, or about 70 Gy, or about 80 Gy, or about 90 Gy, or about 100 Gy.

[0196] In various embodiments, radiation exposure is local or systemic.

[0197] In some embodiments, the present invention is directed to a method for treating or preventing a radiation-related disease or condition in a subject in need thereof, comprising administering to the subject a modified release formulation described herein, comprising an AP-based (optionally an IAP) agent that can be orally administered when the radiation-related disease or condition is a result or side effect of radiation therapy.

[0198] In some embodiments, the present method relates to preventing or reducing a decrease in gut microbiome diversity, for example, a decrease in gut microbiome diversity is a side effect or result of radiation exposure (including radiotherapy) and / or chemotherapy. In some embodiments, the present method relates to repairing and / or rebuilding a subject's gut microbiome after radiation exposure (including radiotherapy) and / or chemotherapy.

[0199] In some embodiments, radiation therapy can be part of cancer treatment, as a primary therapy or adjuvant therapy (e.g., with chemotherapy). In some embodiments, radiation therapy can be used to prevent tumor recurrence after surgery and / or remove primary malignancies. In various embodiments, the subject is a cancer patient.

[0200] In some embodiments, radiation therapy can be part of the treatment of Dupuytren's disease, Ledderhose disease, or as part of postoperative treatment. In various embodiments, the subject has Dupuytren's disease, Ledderhose disease, or has recently undergone surgery.

[0201] In various embodiments, the present method reduces or eliminates the side effects of radiotherapy, including acute side effects, long-term side effects, or cumulative side effects. In various embodiments, the present method reduces or eliminates local or systemic side effects of radiotherapy. In various embodiments, the side effects of radiotherapy and / or radiation exposure are one or more of the following: fatigue, nausea and vomiting, damage to epithelial surfaces (such as but not limited to moist peeling), oral, throat and stomach pain, intestinal discomfort (such as but not limited to soreness, diarrhea and nausea), swelling, infertility, fibrosis, hair loss, dryness (such as but not limited to dry mouth (xerostomia) and dry eyes (xerophthalmia), and dry axillary and vaginal mucosa), lymphedema, heart disease, cognitive decline, radiation enteropathy (such as but not limited to atrophy, fibrosis and vascular changes, which can produce malabsorption, diarrhea, steatorrhea and bleeding, accompanied by bile acid diarrhea and vitamin B12 malabsorption usually found due to ileal involvement). Pelvic radiation disease includes radiation proctitis, bleeding, diarrhea and urinary urgency, and radiation cystitis.

[0202] In various embodiments, the radiation therapy is pelvic radiation therapy. In such embodiments, the modified release formulations described herein comprising an orally administrable AP (optionally IAP) based agent reduce or eliminate gastrointestinal related side effects as described herein. In such embodiments, the orally administrable AP (optionally IAP) based agent reduces or eliminates lower extremity related side effects as described herein.

[0203] In various embodiments, the radiation therapy is pelvic radiation therapy, and the modified release formulations described herein reduce or eliminate one or more of radiation enteropathy, atrophy, fibrosis and vascular changes, malabsorption, diarrhea, steatorrhea, bile acid diarrhea bleeding, malabsorption (e.g., vitamin malabsorption, e.g., vitamin B12 malabsorption). In various embodiments, the radiation therapy is pelvic radiation therapy, and the modified release formulations described herein reduce or eliminate radiation proctitis, bleeding, diarrhea and urinary urgency, and radiation cystitis.

[0204] In various embodiments, radiation therapy is delivered as one or more of external-beam radiation therapy, brachytherapy, and systemic radiation therapy.

[0205] In various embodiments, the radiation therapy is external beam radiation therapy selected from 3-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT, such as RAPIDARC), image guided radiation therapy (IGRT), electromagnetic guided radiation therapy (such as CALYPSO), helical tomotherapy, stereotactic radiosurgery (SRS), stereotactic body radiation therapy (SBRT, such as CYBERKNIFE, GAMMAKNIFE, X-KNIFE, CLINIC), intraoperative radiation therapy (IORT) and proton therapy.

[0206] In various embodiments, the radiation therapy is brachytherapy selected from interstitial brachytherapy, intracavitary brachytherapy, episcleral brachytherapy.

[0207] In various embodiments, the radiation therapy is systemic radiation therapy selected from radioiodine and radioactive biological agents. For example, the radiation therapy can be radioiodine (131I), ibritumomab tiuxetan (ZEVALIN), tositumomab and iodine I 131 tositumomab (BEXXAR), lexidrone samarium 153 (QUADRAMET) and strontium chloride-89 (METASTRON).

[0208] In various embodiments, radiation therapy comprises a dose of about 20 Gy, or about 30 Gy, or about 40 Gy, or about 50 Gy, or about 60 Gy, or about 70 Gy, or about 80 Gy, or about 90 Gy, or about 100 Gy, optionally fractionated.

[0209] In some embodiments, the invention relates to a method of treating or preventing a radiation-related disease or condition in a subject in need thereof, wherein the radiation-related disease or condition is acute radiation syndrome, the method comprising administering to the subject a modified release formulation described herein.

[0210] In some embodiments, ARS includes one or more of the following: gastrointestinal syndrome; hematopoietic syndrome; neurovascular syndrome; apoptosis-mediated tissue damage, wherein apoptosis is optionally attributable to cell stress; and ionizing radiation-induced apoptotic tissue damage. In some embodiments, high-dose radiation (e.g., ionizing radiation) is about 5 to about 30 Gy, or about 10 to about 25 Gy, or about 15 to about 20 Gy, and is optionally sufficient for classification of RES 3 unit radiation exposure state. In various embodiments, high-dose radiation is the result of a radiation disaster and / or a human patient being treated has been exposed to high-dose radiation or is at risk of exposure to high-dose radiation due to one or more of the following reasons: military operations or first responder operations in contaminated areas; nuclear explosions; criticality accidents; radiotherapy accidents; terrorist attacks; exposure to space travel; radioactive waste escape; exposure to open source radiation; and nuclear reactor failures.

[0211] In various embodiments, the methods and compositions of the present invention provide treatment or prevention of radiation-related conditions such as ARS. In various embodiments, the treatments described herein reduce the morbidity or mortality of exposed human patient populations or accelerate recovery from ARS symptoms. ARS typically presents as a series of staged symptoms, which may vary depending on individual radiation sensitivity, radiation type, and absorbed radiation dose. Generally, without wishing to be bound by theory, as the radiation dose increases, the extent of the symptoms will increase, and the duration of each stage will shorten. ARS can be divided into three stages: the prodromal phase (also known as the NVD phase), the latent phase, and the manifest disease. In various embodiments, the modified release formulation of the AP-based agent of the present invention can be administered to human patients at any of these three stages (i.e., the AP-based agent can be administered to human patients during the prodromal phase, the AP-based agent can be administered to human patients during the latent phase, or the AP-based agent can be administered to human patients during the manifest disease phase).

[0212] During the prodromal period, there is often a relatively rapid onset of nausea, vomiting, and malaise. In situations where high-dose radiation exposure has occurred, is possible, or is unavoidable, the use of antiemetics (e.g., oral prophylactic antiemetics) such as granisetron (KYTRIL), ondansetron (ZOFRAN), and 5-HT3 blockers, with or without dexamethasone, may be indicated. Thus, in various embodiments, an AP-based agent may be administered to a human patient while receiving an antiemetic, or an AP-based agent may be administered to a human patient in combination with an antiemetic. For example, an AP-based agent may also be added to the following antiemetic regimen: Ondansetron: Initial 0.15 mg / kg IV; Continuous IV dose options include 8 mg, then 1 mg / h over the next 24 hours. The oral dose is 8 mg every 8 hours, or Granisetron (oral dosage form): The initial dose is typically 1 mg, and is repeated 12 hours after the first dose. Alternatively, 2 mg may be taken as one dose. IV doses are based on body weight; typically 10 μg / kg (4.5 μg / lb) body weight.

[0213] During the incubation period, human patients may be relatively asymptomatic. The length of this stage varies with dosage. The incubation period is the longest before the bone marrow suppression of hematopoietic syndrome and can vary between about 2 and 6 weeks. The incubation period is slightly shorter before the gastrointestinal syndrome, lasting several days to a week. It is the shortest of all before the neurovascular syndrome, lasting only a few hours. These times are variable and can be changed by the presence of other diseases or injuries. Manifested disease presents as clinical symptoms associated with major organ system damage (bone marrow, intestinal tract, neurovascular).

[0214] In some embodiments, the present invention relates to mitigating or protecting cells from the effects of exposure to radiation. In some embodiments, the present invention relates to a method of mitigating and / or protecting a human patient from radiation, the method comprising administering an AP-based agent as described herein. In some embodiments, the radiation is ionizing radiation. In some embodiments, the ionizing radiation is sufficient to cause a gastrointestinal syndrome or a hematopoietic syndrome.

[0215] In some embodiments, ARS includes one or more of: gastrointestinal syndrome; hematopoietic syndrome; neurovascular syndrome; apoptosis-mediated tissue damage, wherein apoptosis is optionally attributable to cellular stress; and ionizing radiation-induced apoptotic tissue damage.

[0216] Hematopoietic syndrome (also known as bone marrow syndrome) is characterized by the loss of hematopoietic cells and their progenitors, making the blood and lymphatic system unable to regenerate. This syndrome is usually characterized by a drop in the number of blood cells, i.e., aplastic anemia. This can lead to infections (e.g., opportunistic infections) due to low numbers of white blood cells, bleeding due to lack of platelets, and anemia due to too few circulating red blood cells. These changes can be detected by blood tests after receiving an acute dose of the whole body. Conventional trauma and burns caused by bomb blasts are complicated by poor wound healing caused by hematopoietic syndrome, thereby increasing mortality. Death can occur due to infection (e.g., as a result of immunosuppression), bleeding and / or anemia. Hematopoietic syndrome usually prevails at lower doses of radiation and leads to more delayed death than gastrointestinal syndrome.

[0217] Gastrointestinal syndrome is caused by massive cell death in the intestinal epithelium (mainly the small intestine), followed by collapse of the intestinal wall and death from bacteremia and sepsis. Symptoms of this form of radiation damage include nausea, vomiting, loss of appetite, loss of absorptive capacity, bleeding from exposed areas, and abdominal pain. Illustrative systemic effects of gastrointestinal syndrome include malnutrition, dehydration, renal failure, anemia, sepsis, etc. Without treatment (including, for example, bone marrow transplantation), death is common (e.g., by intestinal bacterial infection). In some embodiments, AP-based agents may be used in combination with bone marrow transplantation. In some embodiments, AP-based agents may be used in combination with one or more gastrointestinal syndrome inhibitors and / or any additional agents described herein.

[0218] Neurovascular syndrome presents with neurologic symptoms, such as dizziness, headache, or decreased level of consciousness, that develop over minutes to hours and without vomiting. Other symptoms include extreme nervousness and confusion; severe nausea, vomiting, and watery diarrhea; loss of consciousness; and a burning sensation on the skin. Neurovascular syndrome is usually fatal.

[0219] In various embodiments, the methods and compositions of the present invention provide treatment or prevention of radiation-induced intestinal fibrosis. In some embodiments, radiation-induced intestinal fibrosis includes one or more of intestinal inflammation, intestinal fibrosis, vascular sclerosis, chronic ulcers, submucosal enlargement, enhanced proliferation of fibroblasts and smooth muscle cells, and excessive deposition of collagen and other extracellular matrix components.

[0220] In some embodiments, the present invention provides a method of reducing the risk of death after exposure to radiation, comprising administering an effective amount of an AP-based agent. In some embodiments, radiation can be fatal and optionally occurs as a result of a radiation disaster. In various embodiments, the AP-based agent is administered within 24 hours after the radiation exposure. In various embodiments, the AP-based agent is administered within 48 hours after the radiation exposure.

[0221] In some embodiments, modified release formulations of AP-based agents are administered in combination with any of the additional agents described herein, including, but not limited to, radioprotectants, such as antioxidants (e.g., amifostine and vitamin E), cytokines (e.g., stem cell factor), and the like. Damage and death of normal cells due to ionizing radiation is a combination of radiation-induced direct damage to exposed cells and an active genetically programmed cellular response to radiation-induced stress, resulting in suicidal death or apoptosis. Apoptosis plays a key role in the massive cell loss that occurs in several radiation-sensitive organs (e.g., hematopoietic and immune systems, digestive tract epithelium, etc.), and failure of apoptosis determines the general radiation sensitivity of an organism. In some embodiments, administration of an AP-based agent of the present invention to a human patient in need thereof inhibits apoptosis of cells. In some embodiments, an AP-based agent of the present invention is administered to a human patient to protect healthy cells from the damaging effects of radiation therapy.

[0222] In various embodiments, the present invention provides a method for reducing apoptosis of cells after exposure to radiation. In one embodiment, the present invention provides a method for reducing apoptosis of hematopoietic cells after radiation. In another embodiment, the present invention provides a method for reducing apoptosis of gastrointestinal cells after radiation.

[0223] In various embodiments, the administration of AP-based agents stimulates and protects stem cells. For example, the present invention and compositions can stimulate and protect hematopoietic stem cells, including various hematopoietic progenitor cells. In another example, the present invention and compositions can stimulate and protect gastrointestinal stem cells, such as intestinal crypt stem cells. In some embodiments, stem cell proliferation and regeneration can be stimulated. Therefore, the present invention provides a method for expanding the number of stem cells in a patient's body, such as hematopoietic stem cells or gastrointestinal stem cells. In some embodiments, hematopoietic progenitor cells or gastrointestinal progenitor cells are expanded. In various embodiments, the present invention provides methods and compositions for protecting stem cells or progenitor cells from cell death (e.g., apoptosis or necrosis).

[0224] In various embodiments, the methods and compositions of the present invention significantly enhance the recovery of the hematopoietic system and the gastrointestinal system after irradiation. For example, the methods and compositions of the present invention enhance bone marrow recovery after irradiation. In another example, the methods and compositions of the present invention enhance the regeneration of gastrointestinal crypts.

[0225] Exposure to ionizing radiation (IR) can be short-term or long-term, and / or it can be experienced as a single dose or multiple doses and / or it can be applied systemically or locally. In some embodiments, the present invention relates to nuclear accidents or military attacks, which may involve exposure to a single high dose of whole-body irradiation (sometimes followed by long-term poisoning with radioisotopes), as further described herein. For example, for pretreatment of bone marrow transplant patients, the same is true when it is necessary to prepare the hematopoietic organs for donor bone marrow by "clearing" them from host blood precursors (strictly controlling the dose applied). Cancer treatment may involve multiple doses of local irradiation, which, if applied as whole-body irradiation, greatly exceeds the lethal dose (e.g., chemotherapy accidents). Radioisotope poisoning or treatment results in long-term local exposure of the targeted organ (e.g., the thyroid gland in the case of inhaled 125I) to radiation. In addition, there are many physical forms of ionizing radiation that differ significantly in the severity of the biological effects.

[0226] At the molecular and cellular level, radiation particles can produce breaks and cross-links in DNA, proteins, cell membranes, and other macromolecular structures. Ionizing radiation also induces secondary damage to cellular components by generating free radicals and reactive oxygen species (ROS). A variety of repair systems can counteract this damage, such as several DNA repair pathways that restore the integrity and fidelity of DNA, and antioxidant chemicals and enzymes that scavenge free radicals and ROS and reduce oxidized proteins and lipids. Cellular checkpoint systems detect DNA defects and delay cell cycle progression until the damage is repaired or decide to arrest cell growth or reach programmed cell death (apoptosis).

[0227] Radiation can cause damage to mammalian organisms ranging from mild mutagenicity and carcinogenicity at low doses to almost immediate killing at high doses. The overall radiation sensitivity of an organism is determined by pathological changes that develop in several sensitive tissues including the hematopoietic system, reproductive system, and different epithelia with high cell turnover rates.

[0228] The acute pathological consequences of gamma irradiation leading to death vary with different doses and may be determined by the failure of certain organs that define the threshold of sensitivity of the organism to each specific dose. Thus, lethality at lower doses occurs due to bone marrow dysplasia, while moderate doses are more lethal by inducing gastrointestinal (GI) syndrome. Very high doses of radiation can lead to almost immediate death, inducing neuronal degeneration. Organisms that survive the acute toxicity of radiation may suffer long-term remote consequences, including radiation-induced carcinogenesis and fibrosis that develops in exposed organs (e.g., kidneys, liver, or lungs) months and years after irradiation. Cellular DNA is the primary target of IR, which causes various types of DNA damage (genotoxic stress) through direct and indirect (e.g., free radical-based) mechanisms. All organisms maintain a DNA repair system that can effectively restore radiation-damaged DNA; errors in the DNA repair process can lead to mutations.

[0229] AP-based agents have strong prosurvival activity at the cellular level and on the whole organism. In response to supralethal doses of radiation, AP-based agents can suppress gastrointestinal syndrome and hematopoietic syndrome, which are the main causes of death due to acute radiation exposure. Due to these properties, AP-based agents can be used to treat the effects of natural radiation events and nuclear accidents. Moreover, AP-based agents can be used in combination with other radioprotectants to significantly increase the level of protection against ionizing radiation.

[0230] AP-based agents can be used as radioprotectants to extend the range of tolerable radiation doses by, for example, increasing the radiation resistance of the human organism to a level beyond that achievable with currently available measures (shielding and application of existing bioprotective agents) and significantly increase the chances of survival of the crew, for example in the event of a nuclear accident or a large-scale solar particle event.

[0231] AP-based agents can inhibit radiation-induced programmed cell death or apoptosis in response to damage to DNA and other cellular structures. In some embodiments, AP-based agents may not address damage to cellular water and may not prevent mutations. Free radicals and reactive oxygen species (ROS) are the main causes of mutations and other intracellular damage. Antioxidants and free radical scavengers can effectively prevent damage caused by free radicals.

[0232] In addition, in some embodiments, the present invention relates to the prevention or treatment of cutaneous radiation syndrome (CRS), which is skin symptoms two months after radiation exposure (e.g., redness (optionally accompanied by itching), blistering, ulceration, hair loss, damage to sebaceous and sweat glands, atrophy, fibrosis, decreased or increased skin pigmentation, ulceration or necrosis of exposed tissue, moist desquamation and collapse of the dermal vasculature, leading to full-thickness loss of exposed skin.

[0233] In various embodiments, administration of AP-based agents reduces the incidence of post-irradiation wounds, septic complications, and microbial infections in patients.

[0234] In some embodiments, the human patient of the invention experiences leukopenia and / or neutropenia (e.g., an absolute neutrophil count (ANC) <100 cells / μL. In some embodiments, the methods and compositions of the invention relate to a human patient who presents with a lymphocyte count reduction of about 50% within about 24 to about 48 hours. In some embodiments, the lymphocyte count of the human patient is less than about 1000 cells / μL or about 900 cells / μL or about 800 cells / μL or about 700 cells / μL or about 600 cells / μL or about 500 cells / μL or about 400 cells / μL or about 300 cells / μL or about 200 cells / μL or about 100 / cell μL (e.g., within about 24 to about 48 hours). In some embodiments, the lymphocyte profile of the patient is assessed by the Andrews lymphocyte nomogram (see Andrews GA, Auxier JA, Lushbaugh CC. The Importance of Dosimetry to the Medical Management of Persons Exposed to High Levels of Radiation. In Personal Dosimetry for Radiation Accidents. Vienna: International Atomic Energy Agency; 1965, the contents of which are hereby incorporated by reference). In some embodiments, the methods and compositions of the present invention relate to human patients who present with a reduction in thrombocyte count of about 50% within about 24 to about 48 hours. In some embodiments, the human patients of the present invention experience thrombocytopenia, anemia, and / or neutropenia. Thrombocytopenia is defined as a platelet count of less than 50,000 / μL. For example, thrombocytopenia can be characterized as Grade 1 thrombocytopenia (i.e., a platelet count of 75,000 to 150,000 / μL), Grade 2 (i.e., a platelet count of 50,000 to <75,000 μL), Grade 3 (a platelet count of 25,000 to <50,000 / μL), or Grade 4 (a platelet count of 25,000 to <50,000 / μL). Anemia can be diagnosed as a hemoglobin level of less than 13 to 14 g / dL in men and 12 to 13 g / dL in women. For example, anemia is divided into different grades based on hemoglobin levels: grade 0 (within normal limits, ≥12 g / dL); grade 1 (mild, 11.9 to 10 g / dL); grade 2 (moderate, 9.9 to 8 g / dL); grade 3 (severe / severe, 7.9 to 6.5 g / dL); and grade 4 (life-threatening, <6.5 g / dL). Neutropenia can be defined as an absolute neutrophil count (ANC) of less than 1,500 cells / mm3.For example, neutropenia is graded as Grade 1 (i.e., ANC of 1,500 / mm3 or less to greater than 2,000 / mm3), Grade 2 (ANC of 1,000 / mm3 or less to greater than 1,500 / mm3), Grade 3 (ANC of 500 / mm3 or less to greater than 1,000 / mm3), or Grade 4 (ANC less than 500 / mm3). In various embodiments, the methods and compositions of the present invention reduce the duration and severity of thrombocytopenia, anemia, and / or neutropenia in patients after irradiation. For example, the methods and compositions of the present invention can reduce the duration and severity of Grade 4 thrombocytopenia, anemia, and / or neutropenia in patients after irradiation.

[0235] In various embodiments, high-dose radiation refers to whole-body dose. In various embodiments, high-dose radiation may be uneven. In various embodiments, ARS is the result of high-dose radiation. In various embodiments, high-dose radiation is about 2Gy, or about 2.5Gy, or about 3Gy, or about 3.5Gy, or about 4Gy, or about 4.5Gy, or about 5Gy, or about 10Gy, or about 15Gy, or about 20Gy, or about 25Gy, or about 30Gy. In various embodiments, high-dose radiation is about 5 to about 30Gy, or about 10 to 25Gy, or about 15 to 20Gy. In some embodiments, high-dose radiation is assessed by one or more of physical dosimetry and / or biological dosimetry (e.g., multi-parameter dose assessment), cytogenetics (e.g., for chromosome analysis (including, by non-limiting example, dicentric analysis) of, for example, blood samples.

[0236] In various embodiments, whole body radiation doses can be divided into sublethal doses (<2 Gy), potentially lethal doses (2-10 Gy), and supralethal doses (>10 Gy).

[0237] The radiation exposure state (RES) for a given unit is based on operational exposure above normal background radiation. It is designed as an average based on unit-level dosimeters. In various embodiments, a high dose of radiation is sufficient for classification of the RES 3 unit radiation exposure state.

[0238] In various embodiments, the radiation is ionizing radiation (e.g., one or more of alpha particles, beta particles, gamma rays, and neutrons). In various embodiments, when the radiation interacts with atoms, energy is stored, resulting in ionization (excitation of electrons). This ionization can damage certain key molecules or structures in cells through direct and indirect effects. The radiation can directly hit particularly sensitive atoms or molecules in the cell. The damage caused is irreparable; the cell dies or causes dysfunction. The radiation can also indirectly damage cells by interacting with water molecules in the body. The energy stored in the water can lead to the production of unstable toxic superoxide molecules; these can then damage sensitive molecules and affect subcellular structures.

[0239] In some embodiments, radiation may be caused by one or more of the following radioactive materials: americium (e.g., 241Am), cesium (e.g., 137Cs), cobalt (e.g., 60Co), uranium (e.g., depleted uranium), iodine (e.g., 131, 132, 134, 135I), phosphorus (e.g., 32P), plutonium (e.g., 238, 239Pu), radium (e.g., 226Ra), strontium (e.g., 90Sr), tritium (e.g., 3H), and uranium (e.g., 235U, 238U, 239U).

[0240] In various embodiments, the high dose of radiation is the result of a radiation disaster. In various embodiments, the human patient has been exposed to or is at risk of being exposed to high doses of radiation, which may be the result of one or more of the following: military operations or first responder operations in contaminated areas; nuclear explosions; criticality accidents; radiation therapy accidents; terrorist attacks; exposure in space travel; radioactive waste escapes; exposure to open source radiation; and nuclear reactor failures.

[0241] A method of treating or preventing a side effect of chemotherapeutic treatment in a subject in need thereof comprises administering to the subject a modified release formulation described herein.

[0242] In various embodiments, the present invention provides for the treatment and / or administration of a subject suffering from side effects of chemotherapeutic treatment.

[0243] In some embodiments, the side effects of chemotherapeutic treatment are selected from alopecia, bone marrow suppression, nephrotoxicity, weight loss, pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, numbness, taste changes, loss of appetite, thinning or burnt hair, mouth sores, memory loss, bleeding, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment.

[0244] A method of treating cancer in a subject in need thereof by increasing the effectiveness of a chemotherapeutic treatment, comprising administering to the subject a modified release formulation described herein. In various embodiments, the modified release formulation of the AP-based agent described herein acts as an adjuvant to the chemotherapeutic treatment described herein. In various embodiments, the modified release formulation of the AP-based agent described herein improves the anti-cancer effect and / or increases the therapeutic window of any chemotherapeutic treatment described herein. In various embodiments, administration of a modified release formulation of an AP-based agent to a subject does not interfere with the treatment of cancer.

[0245] In certain aspects, the present invention further provides a method for improving or reducing and / or treating or preventing frailty in a subject, wherein the method comprises: identifying a subject who desires or needs treatment or prevention of frailty, and administering to the subject a modified release formulation of an AP-based agent described herein.

[0246] Aging is a progressive systemic pathological transformation of mammalian organisms that progresses over time. It is associated with the accumulation of multiple functional defects and reduced regenerative capacity of multiple organs and tissues, leading to the development of age-related chronic diseases, including atherosclerosis, diabetes, pulmonary fibrosis, blindness, dementia, renal dysfunction, osteoarthritis and low-grade chronic sterile inflammation and other age-related diseases and conditions considered herein. These diseases often occur simultaneously with the gradual development of geriatric syndromes (including weakness, cognitive impairment and mobility impairment). Aging is a natural and inevitable process. The root cause of aging remains controversial; however, two characteristics of aging are generally considered to be ubiquitous: an increase in DNA damage and the development of systemic sterile chronic inflammation, both of which are considered to be the main contributing factors to age-related pathology.

[0247] In some embodiments, the present invention provides methods for improving or reducing and / or treating or preventing frailty in a subject, as measured by a reduction in the subject's PFI score. In some embodiments, the methods and compositions of the present invention for improving or reducing and / or treating or preventing frailty in a subject include maintaining the PFI score over time so that the score increases at a slower rate than when the subject is not administered a modified release formulation of an AP-based agent of the present invention. In some embodiments of the present invention, the patient's PFI score remains almost the same over time. In other embodiments, the methods of the present invention provide a reduction in cellular aging and immune aging associated with natural aging and / or accelerated aging (e.g., accelerated aging induced by, for example, cancer or cancer treatment).

[0248] In another aspect, the present invention provides a method for treating or preventing an age-related disease or condition in a subject, wherein the method comprises: identifying a subject for whom treatment or prevention of an age-related disease or condition is desired or required, and administering to the subject a modified release formulation of an AP-based agent. In some embodiments, the age-related disease or condition is characterized by increased cellular senescence or immune senescence.

[0249] In some embodiments, the age-related disease or disorder is selected from accelerated aging, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, diastolic insufficiency, benign prostatic hypertrophy, aortic aneurysm, emphysema, atherosclerosis, diabetes, pulmonary fibrosis, blindness, dementia, Alzheimer's disease, renal dysfunction, osteoarthritis, low-grade chronic sterile inflammation, herniated disc, frailty, hair loss, hearing loss, vision loss, muscle fatigue, skin disorders, skin moles, wrinkled skin, hyperpigmentation, scars, keloids, rosacea, vitiligo, ichthyosis vulgaris, dermatomyositis, actinic keratosis, and sarcopenia.

[0250] In a specific embodiment, the method of the present invention includes treating or preventing accelerated aging. In some embodiments, accelerated aging is a progeria syndrome or its symptoms, including but not limited to Hutchinson-Gilford progeria syndrome (HGPS), Werner syndrome (WS), Bloom syndrome (BS), Rosemont-Thomson syndrome (RTS), Cockayne syndrome (CS), xeroderma pigmentosum (XP), hair hyposulfur dystrophy (TTD), combined xeroderma pigmentosum and Cockayne syndrome (XP-CS) or restrictive dermatosis (RD). Subjects suffering from one of these diseases or conditions usually have a shortened life span (i.e., life span).

[0251] In other embodiments, accelerated aging is induced by cancer or cancer treatment. For example, the present invention contemplates cancer treatments that induce acceleration of the natural aging process selected from one or more therapies consisting of radiation therapy, hormones, tyrosine kinase inhibitors, anthracyclines, alkylating agents, topoisomerase inhibitors, antimetabolites / cytotoxic drugs, BRAF inhibitors, antitumor antibiotics, isoquinoline alkaloids, Bcl-2 inhibitors, hematopoietic cell transplantation (HCT), telomerase inhibitors, nucleoside analog reverse transcriptase inhibitors, DNA cross-linking agents, ribonucleotide reductase inhibitors, microtubule inhibitors, and miRNA.

[0252] In some embodiments, any cancer for which the subject receives treatment that induces accelerated aging is contemplated. In one embodiment, the cancer for which the subject receives treatment is a hematological cancer. Additionally, in some embodiments, the subject received cancer treatment during childhood.

[0253] In some embodiments, weakness includes the accumulation of major physiological function defects, the reduction of regenerative capacity, the increased risk of impaired wound healing and age-related diseases. For example, in some embodiments, weakness is related to natural aging or accelerated aging. Weakness can be measured according to many indexes or experiments known to those skilled in the art. For example, a kind of such index, i.e., physiological frailty index (PFI), includes the measurement of one or more parameters, which are selected from grip strength, systolic blood pressure, diastolic blood pressure, blood flow, the number of blood neutrophils, the percentage of blood neutrophils, the number of blood monocytes, the percentage of blood monocytes, the number of lymphocytes, the number of red blood cells, hemoglobin level, hematocrit level, mean corpuscular volume, mean corpuscular hemoglobin level, mean corpuscular hemoglobin concentration and keratinocyte-derived cytokine levels. Any individual deviation from the reference standard is called a defect, and the overall average PFI score of the individual is the ratio of the defect to the total number of measured parameters.

[0254] Weakness can be manifested as reduced vulnerability to stressors and the ability to withstand stress. For example, the disclosure of Buchner and Wagner 1992 Clin Geriatr Med. 1992 February; 8(1): 1-17 is hereby incorporated by reference in its entirety. Weakness can be manifested as a loss of complexity in homeostatic mechanisms (e.g., interconnectedness and / or feedback or feedforward). For example, the disclosure of Lipsitz 2002 J Gerontol A Biol Sci Med Sci. 2002 March; 57(3): B115-25 is hereby incorporated by reference in its entirety. As described in Bortz 2002, J Gerontol A Biol Sci Med Sci. 2002 May; 57(5): M283-8, which is hereby incorporated by reference in its entirety, weakness can also be manifested as loss of use and / or reduced energy flow through the organism. Frailty can also manifest as a disturbance in homeostasis as described in Ferrucci 2005 J. Gerontol. A Biol. Sci. Med. Sci. 60, 56, which is hereby incorporated by reference in its entirety.

[0255] There are several comprehensive methods for quantitatively assessing the accumulation of aging-related defects and frailty in humans and animals. The health status and aging rate of individual organisms vary. To account for this heterogeneity, the frailty index (FI) was introduced as a numerical score, which is the ratio of the defects present in humans to the total number of defects considered in the study. Changes in the FI characterize the rate of individual aging. Similar methods have been applied to laboratory animals. The frailty index is considered to be a reliable and widely accepted measure of "biological age" and the degree of overall health decline that indicates a decline in quality of life.

[0256] In certain aspects and embodiments, provided herein are methods including for improving and / or treating or preventing weakness and / or reducing weakness index in a subject. Frailty can be assessed by any of a number of methods known in the art. For example, frailty and methods for assessing frailty / measuring frailty index are described in Hubbard et al., Ageing, published electronically November, 2008, pp. 115-118; Cesari et al., Age and Ageing, 43: 10-12, 2014; and Mohler et al., Experimental Gerontology, 54: 6-13, 2014, all of which are hereby incorporated by reference.

[0257] In various embodiments, the frailty index is calculated as described in U.S. Patent Application Publication No. 2015 / 0285823, which is incorporated herein by reference. For example, a description of the determination of the frailty index is provided. The frailty index was developed to assess the range of health to frailty of organisms of the same actual age, to address the view that actual age does not always reflect biological age. Based on 16 parameters (including measurements of body weight, grip strength, blood pressure, complete blood count, cytokine level analysis), the FI is calculated as the ratio of the total number of defects measured and the FI score between 0 (no defects = health) and 1 (all defects = frailty) is assigned. Therefore, the higher the FI, the worse the health of the organism. In this regard, FI is provided as a range from "health" to "frail" for assessing the same actual age organism.

[0258] In certain embodiments, as described in Antoch et al. Aging. 2017; 9: 1-12 (hereby incorporated by reference in its entirety), the methods of the present invention reduce or prevent frailty in subjects measured according to a physiological frailty index (PFI). For example, the PFI of an individual subject can be determined with reference to a young reference subject. For each subject, various parameters are measured. These parameters include non-invasive measurements, including age, weight, grip strength, and diastolic blood pressure. Additional blood chemistry measurements can also be determined, including white blood cell count, neutrophil count, neutrophil percentage, lymphocyte percentage, monocyte percentage, eosinophil percentage, red blood cell count, hemoglobin level, hematocrit level, mean corpuscular volume, mean corpuscular hemoglobin level, mean corpuscular hemoglobin concentration, platelet count, and mean platelet volume. The mean and standard deviation of each parameter are calculated. Subjects whose mean value differs from any single parameter by more than one standard deviation (STDEV) are excluded from the reference group. The values ​​of each parameter measured for older subjects are compared with the corresponding values ​​of the reference group and a score is assigned. The value that is less than 1 STDEV is assigned a score of 0 (no defect, within the reference group). The value that differs by one STDEV is scored as 0.25 (minimal defect). The value that differs by 2 STDEVs from the corresponding value in the reference group is scored as 0.5, and those that differ by 3 STDEVs are scored as 0.75. If the value is higher than 3 STDEVs, it is scored as 1 (extreme defect). The number of defects that the individual subject exhibits is calculated as the ratio of the total number of measured parameters and is called the Physiological Frailty Index (PFI).

[0259] In some embodiments, the methods of the present invention reduce or improve and / or treat or prevent weakness in a subject, as measured by PFI. For example, administering a modified release formulation of an AP-based agent to a subject to reduce or improve and / or treat or prevent weakness can result in a decrease in the PFI score. In some embodiments, the subject's PFI score is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, the subject's PFI score is reduced by about 25%-75%, about 25%-50%, or about 50% to 75%. In other embodiments, the subject's PFI score is reduced to no greater than 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, or 0.5.

[0260] Further, as described in Rockwood et al., J Gerontol A Biol Sci Med Sci. 2007 Jul; 62(7):722-727, which is incorporated by reference in its entirety, frailty as an accumulation of deficits can be measured by the Rockwood Frailty Index. In embodiments, the present method reduces or prevents frailty as assessed by the Rockwood Frailty Index.

[0261] Frailty, as a biological syndrome of reduced reserve resulting from cumulative declines in multiple physiological systems, can be measured by the Fried frailty score, as described in Fried et al., J Gerontol A Biol Sci Med Sci. 2001 Mar; 56(3): M146-56, which is incorporated by reference in its entirety. The Fried frailty score includes a physical frailty phenotype (PFP), which measures various parameters such as weight loss greater than 10 pounds; weakness associated with grip strength; self-reported fatigue; 15-foot walking speed; and physical activity in Kcal per week. The Fried frailty score includes scores of 0 (not frail), 1-2 (moderate frailty), and greater than or equal to 3 (frailty). In various embodiments, the methods of the present invention reduce or improve and / or treat or prevent frailty in a subject, as measured by the Fried frailty score. For example, administration of a modified release formulation of an AP-based agent to a subject to reduce or improve and / or treat or prevent frailty may result in a decrease in Fried frailty score from 3 to 2, from 3 to 1, from 3 to 0, from 2 to 1, from 2 to 0, or from 1 to 0. Further, in some embodiments, administration of a modified release formulation of an AP-based agent to a subject to reduce or improve and / or treat or prevent frailty results in no increase in the subject's Fried frailty score.

[0262] Frailty can also be measured by the FRAIL scale, as described in Abellean Van Kan et al., J Am Med Dir Assoc. 2008 Feb;9(2):71-2. doi:10.1016 / j.jamda.2007.11.005, which is incorporated by reference in its entirety. Parameters measured in the FRAIL scale include persistent fatigue; resistance (ability to climb a flight of stairs); walking (ability to walk a block); more than five illnesses; and weight loss of more than 5%. The FRAIL scale incorporates scores of 0 (not frail), 1-2 (moderately frail), and greater than or equal to 3 (frail). In various embodiments, the methods of the invention reduce or improve frailty in a subject as measured by the FRAIL scale score. For example, administration of a modified release formulation of an AP-based agent to a subject to reduce or improve frailty may result in a decrease in the FRAIL scale score from 3 to 2, from 3 to 1, from 3 to 0, from 2 to 1, from 2 to 0, or from 1 to 0. Further, in some embodiments, administration of a modified release formulation of an AP-based agent to a subject to reduce or improve and / or treat or prevent frailty results in no increase in the subject's FRAIL scale score.

[0263] In some embodiments, the method as provided herein uses at least one accepted frailty measurement to improve (or reduce) frailty index, or delays or slows the decline of frailty. In some embodiments, the method as provided herein uses at least one accepted frailty measurement selected from frailty index (FI), physiological frailty index (PFI), Fried frailty score, Rockwood frailty index, FRAIL scale and modified frailty index to improve (or reduce) frailty index, or delays or slows the decline of frailty.

[0264] In some embodiments, weakness includes low lean body mass, weakness, fatigue, low energy expenditure and / or slow walking speed. In embodiments, the method reduces or prevents the occurrence or development of one or more of low lean body mass, weakness, fatigue, low energy expenditure and / or slow walking speed.

[0265] Without wishing to be bound by theory, it is believed that AP-based agents, including AP-based agents (e.g., IAPs), play a key role in many gastrointestinal and systemic processes, including, for example, participation in intestinal defense, mediating anti-inflammatory functions, maintaining normal intestinal flora distribution, maintaining the integrity of the mucosal barrier, and regulating digestion and nutrient (fat) absorption. Thus, the present invention provides for the use of AP-based agents in a wide range of therapeutic applications for modulating immune, metabolic, and neural functions. In various embodiments, the present invention provides treatments for microbiome-related disorders, gastrointestinal disorders, gastrointestinal inflammation, colitis (e.g., ulcerative colitis), metabolic diseases (e.g., metabolic syndrome, obesity, and diabetes), neurological diseases (e.g., multiple sclerosis and spinal cord injury), cystic fibrosis, sepsis, and renal failure.

[0266] In various aspects, the present invention provides methods for regulating and protecting the gastrointestinal microbiome of a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an AP-based agent (and / or an additional therapeutic agent). In some embodiments, the methods of the present invention can be used to treat subjects with reduced gastrointestinal flora levels and / or function by administering an AP-based agent of the present invention so as to increase or maintain the number of commensal bacteria and the composition of the gastrointestinal microbiome. In other embodiments, the methods of the present invention relate to treating infections caused by pathogenic bacteria and / or inhibiting the growth of pathogenic bacteria in the gastrointestinal tract or reducing their number.

[0267] In various embodiments, the methods of the present invention include treating or preventing microbiome-mediated disorders. Illustrative microbiome-mediated disorders include, but are not limited to, those found, for example, in Table 3 of WO 2014 / 121298, the entire contents of which are incorporated herein by reference. For example, the method can be used to treat symptoms associated with reduced levels of symbiotic bacteria and / or reduced gastrointestinal flora function, such as antibiotic-associated diarrhea (AAD), Clostridium difficile-related diseases (CDAD), inflammatory conditions, acquired immunodeficiency syndrome (AIDS) (including HIV-mediated intestinal dysbiosis and gastrointestinal barrier dysfunction), hypothyroidism, and obesity.

[0268] In various aspects, the present invention provides pharmaceutical compositions comprising an AP-based agent of the present invention (and / or an additional therapeutic agent) for use in treating antibiotic-induced side effects in the gastrointestinal tract and / or preventing or treating Clostridium difficile infection (CDI) and / or Clostridium difficile-associated diseases in a subject in need thereof. Without wishing to be bound by theory, it is believed that the AP-based agents of the present invention mediate dephosphorylation of nucleoside triphosphates, which promotes the growth of commensal bacteria in preference to pathogenic bacteria and promotes recovery from antibiotic-induced dysbiosis. Therefore, treatment with the AP-based agents of the present invention has the potential to prevent Clostridium difficile infection and intestinal Gram-negative pathogens. In various embodiments, the antibiotic-induced side effects and / or CDI or Clostridium difficile-associated diseases are one or more of the following: antibiotic-associated diarrhea, Clostridium difficile diarrhea (CDD), Clostridium difficile intestinal inflammatory disease, colitis, pseudomembranous colitis, fever, abdominal pain, dehydration and electrolyte imbalance, megacolon, peritonitis, and colonic perforation and / or rupture.

[0269] In various embodiments, subjects include, but are not limited to, subjects at particular risk for microbiome-mediated conditions, such as, by way of non-limiting example, those who are currently being treated for or have recently been treated with antibiotics. For example, the subject may have taken antibiotics in the past approximately 30 days or so and / or have a weak immune system (e.g., caused by a chronic disease) and / or be female and / or elderly (e.g., over about 65 years old) and / or are currently being treated (or have been treated) for heartburn or gastric acid disease (e.g., with agents such as PREVACID, TAGAMET, PRILOSEC, or NEXIUM and related medications) and / or have recently been hospitalized, including in an intensive care unit, or have lived in a nursing home. Thus, in some embodiments, the methods and uses of the present invention treat or prevent nosocomial infections and / or secondary emergency infections and / or hospital-acquired infections (HAIs).

[0270] In various embodiments, the present invention provides a method for treating antibiotic-induced side effects in the gastrointestinal tract, comprising administering to a subject in need thereof an effective amount of an AP-based agent of the present invention (and / or an additional therapeutic agent). In another embodiment, the present invention provides a method for preventing antibiotic-induced side effects in the gastrointestinal tract, comprising administering to a subject in need thereof an effective amount of an AP-based agent of the present invention (and / or an additional therapeutic agent).

[0271] In various embodiments, the AP-based agents of the present invention protect the gut microbiome from antibiotic-induced damage. In one embodiment, the AP-based agents protect the gut microbiome from cephalosporin-induced damage. In some embodiments, the AP-based agents of the present invention protect the gut microbiome from ceftriaxone (CRO)-induced damage. In some embodiments, the methods of the present invention treat or prevent antibiotic-associated side effects, including but not limited to diarrhea, nausea, vomiting, dysgeusia, colitis, and pseudomembranous colitis diseases and / or symptoms. In one embodiment, the methods of the present invention can be used to treat or prevent antibiotic-associated diarrhea (AAD).

[0272] In various embodiments, the present invention provides compositions and methods for treating infections caused by pathogens and / or inhibiting the growth of pathogens in the gastrointestinal tract or reducing their number. In various embodiments, the present invention provides compositions and methods for alleviating or preventing the overgrowth of various coliform bacteria (including coliform bacteria with toxicity and / or antibiotic resistance) in the intestinal tract of patients. Illustrative coliform bacteria include Citrobacter, Enterobacter, Hafnia, Kelbsiella and Escherichia. In various aspects, the methods and compositions described herein prevent or reduce secondary infections of resistant organisms. In one embodiment, the pathogen is an enterobacterium, such as Salmonella.

[0273] In some embodiments, the present invention prevents the amplification of the intestinal resistome. The intestinal resistome refers to a reservoir of antibiotic resistance genes that may be hidden in the human intestinal microbial flora. Such antibiotic resistance genes confer antibiotic resistance in bacterial pathogens that pose a major threat to public health. Bacteria can acquire antibiotic resistance genes by mobilizing and transferring resistance genes from donor strains. In various embodiments, the present method reduces the number of antibiotic-resistant bacteria in the gastrointestinal tract, thereby reducing the amplification of the intestinal resistome. In various embodiments, the present invention reduces or prevents the growth of antibiotic-resistant bacteria, thereby preventing or reducing the amplification of the intestinal resistome.

[0274] In various embodiments, the present invention provides methods for treating or preventing Clostridium difficile infection (CDI) and / or Clostridium difficile-associated diseases, comprising administering to a subject in need thereof an effective amount of an AP-based agent of the present invention. In various embodiments, the present invention provides methods for preventing Clostridium difficile infection (CDI) and / or Clostridium difficile-associated diseases, comprising administering to a subject in need thereof (by way of non-limiting example, a patient who is being administered antibiotics or is to be administered antibiotics) an effective amount of an AP-based agent of the present invention.

[0275] In various embodiments, the present invention relates to a method for preventing Clostridium difficile infection (CDI) and / or Clostridium difficile-associated disease, comprising administering an effective amount of an AP-based agent of the present invention to a subject in need thereof, wherein the subject is receiving therapy with a primary antibiotic. "Primary antibiotics" refers to antibiotics that are administered to a patient and that may cause CDI and / or Clostridium difficile-associated disease. These include the antibiotics that most commonly cause CDI and / or Clostridium difficile-associated disease: for example, fluoroquinolones, cephalosporins, clindamycin, and penicillin.

[0276] In various embodiments, CDI and / or C. difficile-associated diseases are treated or prevented in the setting of an initial attack or a relapse / recurrence (e.g., due to continuation or restarting of antibiotic therapy). For example, in a patient who previously suffered from CDI, an AP-based agent of the invention may be administered at the first symptom of a relapse. By way of non-limiting example, symptoms of a relapse include, in mild cases, about 5 to about 10 watery bowel movements per day, no significant fever, only mild abdominal cramping, and a blood test may show a mildly elevated white blood cell count of up to about 15,000 (normal levels are up to about 10,000), and in severe cases, more than about 10 watery bowel movements per day, nausea, vomiting, high fever (e.g., about 102-104°F), rectal bleeding, severe abdominal pain (e.g., with tenderness), bloating, and a high white blood cell count (e.g., about 15,000 to about 40,000).

[0277] Whether it is an initial episode or a recurrence / relapse, CDI and / or C. difficile-related disease can be diagnosed by any of the symptoms described herein (e.g., about 3 or more episodes of watery diarrhea per day for about 2 days or longer, mild to severe abdominal cramping and pain, fever, blood or pus in the stool, nausea, dehydration, loss of appetite, weight loss, etc.). CDI and / or C. difficile-associated disease, whether an initial episode or a relapse / recurrence, can also be diagnosed via enzyme immunoassays (e.g., detection of C. difficile toxin A or B antigens and / or glutamine dehydrogenase (GDH), which are produced by C. difficile organisms), polymerase chain reactions (e.g., detection of C. difficile toxin A or B genes or portions thereof (e.g., tcdA or tcdB) (including ILLUMIGENE LAMP assays), cytotoxicity assays. For example, any of the following tests can be used: Meridian ImmunoCard Toxin A / B; Wampole Toxin A / B Rapid Test; Wampole C. difficile Complete Rapid Test; Remel Xpect C. difficile (formerly C. difficile) Toxin A / B; Meridian Premier Toxin A / B; Wampole C. difficile Toxin A / B II; Remel Foreground Toxin A / B EIA; Biomerieux Vidas C. difficile Toxins A and B; BD Geneohm C. difficile; Prodesse Progastro CD; and Cepheld Xpert C. difficile. In various embodiments, the clinical sample is a stool sample from the patient. Patients may also be evaluated using a flexible sigmoidoscopy "scope" test and / or an abdominal X-ray and / or a computed tomography (CT) scan (which provides an image of your colon) (e.g., looking for characteristic milky or yellow plaques attached to the colon wall). Further, a biopsy (e.g., a biopsy of any area of ​​the gastrointestinal tract) may be used to evaluate patients for potential CDI and / or C. difficile-associated disease.

[0278] In some embodiments, the methods and uses of the present invention include methods and uses of administering initial and / or adjunctive therapy to a subject. Initial and / or adjunctive therapy refers to a therapy for treating, for example, such a condition or disease when a microbiome-mediated condition or disease is detected. In one embodiment, initial and / or adjunctive therapy refers to a therapy for treating such a disease when CDI and / or Clostridium difficile-related diseases are detected. In some embodiments, the initial and / or adjunctive therapy is one or more of metronidazole, vancomycin, fidaxomicin, rifaximin, charcoal-based adhesives / adsorbents, fecal bacterial therapy, probiotic therapy, and antibody therapy. In various embodiments, the methods and uses of the present invention include the use of alkaline phosphatase as an adjuvant for any of these initial and / or adjunctive therapies (including co-administration or sequential administration). In various embodiments, the methods and uses of the present invention include administering an AP-based agent as described herein to a subject receiving initial and / or adjunctive therapy.

[0279] In various embodiments, the AP-based agents of the invention are administered to subjects suffering from increased gastrointestinal mucosal permeability. In some embodiments, increased gastrointestinal mucosal permeability is the result of decreased intestinal perfusion or ischemia. Ischemia or insufficient blood flow oxygen can be caused by, for example, heart failure, congenital heart disease, congestive heart failure, coronary heart disease, ischemic heart disease, injury, trauma or surgery.

[0280] In some embodiments, increased gastrointestinal mucosal permeability is associated with or caused by autoimmune and inflammatory bowel diseases (IBD), such as celiac disease, Crohn's disease, and colitis (e.g., ulcerative colitis). Therefore, in some embodiments, the present invention provides a method for treating or preventing autoimmune and inflammatory bowel diseases (IBD), such as celiac disease, Crohn's disease, and colitis (e.g., ulcerative colitis), comprising administering an effective amount of an AP-based agent of the present invention to a subject in need thereof. Inflammatory bowel disease (IBD) is a type of inflammatory disorder of the large intestine and, in some cases, of the small intestine. The main forms of IBD are Crohn's disease and ulcerative colitis (UC). IBD also includes collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome ( syndrome), infectious colitis, and indeterminate colitis.

[0281] In some embodiments, the present invention provides methods for treating celiac disease. In some embodiments, the present invention provides methods for treating gastrointestinal disorders associated with celiac disease. Celiac disease is an autoimmune disorder that can occur in people with a genetic predisposition, in which the ingestion of gluten causes damage to the small intestine. Individuals with celiac disease have increased intestinal permeability, which allows gluten breakdown products (the triggering antigen for celiac disease) to reach the gut-associated lymphoid tissue, thereby triggering an inflammatory response, including the release of inflammatory cytokines and the recruitment of T cells. Celiac disease is characterized by chronic inflammation of the small intestinal mucosa, which can lead to villous atrophy of the small intestine and a variety of symptoms such as malabsorption, diarrhea, abdominal pain, bloating, fatigue, and nausea. In various embodiments, the methods of the present invention effectively treat one or more symptoms of celiac disease, including gastrointestinal symptoms, abdominal symptoms, and non-gastrointestinal symptoms.

[0282] Methods for measuring improvement in one or more symptoms of celiac disease may include assessing the ratio of lactulose to mannitol (LAMA), which is an experimental biomarker of intestinal permeability (Kelly et al., (2012) Aliment Pharmacol Ther 2013; 37: 252–262, the entire disclosure is hereby incorporated by reference); measuring anti-transglutaminase antibody levels; and assessing clinical symptoms using, for example, the celiac disease patient-reported outcomes (CeD PRO), the Gastrointestinal Symptom Rating Scale (GSRS), the Celiac Disease Gastrointestinal Symptom Rating Scale (CeDGSRS), the Bristol Stool Shape Scale (BSFS), the General Health Questionnaire, the Short Form 12 Health Survey Version 2 (SF12V2), the Celiac Disease Quality of Life Questionnaire (CeD-QoL), and the Clinician Global Disease Activity Assessment (CGA) disclosed in WO / 2015 / 154010 (the entire disclosure of which is hereby incorporated by reference). In various embodiments, the present methods of treating celiac disease provide a therapeutic effect assessed by one or more of these measurements.

[0283] In some embodiments, the present methods treat celiac disease and allow a subject to introduce gluten into their diet without substantial symptoms.

[0284] In various embodiments, increased gastrointestinal mucosal permeability is associated with or caused by cachexia, such as cachexia associated with one or more of cancer, AIDS, heart failure, and / or chronic obstructive pulmonary disease (COPD). In some embodiments, the present invention treats or prevents increased intestinal permeability and / or intestinal dysbiosis associated with cachexia.

[0285] In some embodiments, the method treats or prevents cachexia or wasting syndrome. In some embodiments, the method reduces, eliminates or prevents one or more of weight loss, muscle atrophy, fatigue, weakness, and loss of appetite. In some embodiments, the method reduces, eliminates or prevents one or more of nutritionally irreversible weight loss and reduction in adipose tissue accumulation. In some embodiments, the method treats or prevents sarcopenia.

[0286] In some embodiments, cachexia treated by the present invention is found in patients suffering from one or more of cancer, HIV or AIDS, celiac disease, chronic obstructive pulmonary disease, multiple sclerosis, rheumatoid arthritis, congestive heart failure, tuberculosis, familial amyloid polyneuropathy, mercury poisoning (amelodynia), and hormone deficiency.

[0287] In various embodiments, treatment or prevention of cachexia with the AP-based agents of the invention may be used in combination with one or more of, or in a patient receiving one or more of, β-hydroxy β-methylbutyrate (HMB), various progestins (e.g., megestrol acetate), androgenic anabolic steroids (e.g., oxandrolone), thalidomide and cytokine antagonists, cannabinoids, omega-3 fatty acids (e.g., eicosapentaenoic acid (EPA)), nonsteroidal anti-inflammatory drugs, prokinetics, ghrelin and ghrelin receptor agonists, anabolic-catabolic converters (such as MT-102), selective androgen receptor modulators, cyproheptadine, and hydrazine.

[0288] In some embodiments, increased gastrointestinal mucosal permeability is associated with or caused by acquired immunodeficiency syndrome (AIDS). Therefore, in some embodiments, the present invention provides a method for treating gastrointestinal disorders associated with acquired immunodeficiency syndrome (AIDS). Gastrointestinal disorders are one of the most common complaints in AIDS patients associated with human immunodeficiency virus 1 (HIV-1) or human immunodeficiency virus 2 (HIV-2). Gastrointestinal manifestations of HIV disease include diarrhea, dysphagia, dysphagia, nausea, vomiting, weight loss, abdominal pain, anorectal disease, jaundice, hepatomegaly, gastrointestinal bleeding, and gastrointestinal tumors (e.g., Kaposi's sarcoma and non-Hodgkin's lymphoma).

[0289] Progressive HIV infection often leads to gastrointestinal damage, microbial translocation, inflammation and immune activation, thereby promoting the progression of the disease to AIDS. The term "HIV enteropathy" has been used to describe changes in mucosal structure and function associated with intestinal-mediated immune dysfunction, as well as to refer to a clinical syndrome of chronic diarrhea without a determined cause of infection. In addition to chronic diarrhea, HIV enteropathy is often characterized by increased gastrointestinal inflammation, increased intestinal permeability, and poor absorption of bile acids and vitamin B12 - these abnormalities are believed to be due to direct or indirect effects of HIV on the intestinal mucosa (Brenchley JM, Douek DC. Mucosal Immunol 2008; 1: 23-30). Clinical consequences include reduced absorption of fat and carbohydrates, a tendency to reduced small intestinal transit time and jejunal atrophy. In various embodiments, the method of the present invention effectively treats the symptomatic effects of HIV enteropathy. In various embodiments, the method of the present invention prevents, slows or reverses the progression of HIV infection to AIDS. In various embodiments, the method of the present invention prevents or slows the progression of AIDS to death.

[0290] In addition, the HIV-1 subtype with which the subject is infected may be a factor in the rate of progression to AIDS. In various embodiments, the present method is effective in treating patients infected with HIV-1 subtypes C, D, and G. In another embodiment, the present method is effective in treating patients infected with HIV-1 subtype A.

[0291] In some embodiments, the present invention provides methods for treating gastrointestinal disorders associated with HIV infection and / or AIDS. For example, the present invention provides methods for treating HIV-mediated intestinal dysbiosis and gastrointestinal barrier dysfunction, which in various embodiments may be caused by HIV, antibiotics administered to HIV-infected subjects, and / or drugs being administered to HIV-infected subjects. For example, HIV-infected subjects may be taking one or more nucleoside analogs, such as deoxyadenosine analogs (e.g., didanosine, adenosine), adenosine analogs (e.g., BCX4430), deoxycytidine analogs (e.g., cytarabine, emtricitabine, lamivudine, zalcitabine), guanosine and deoxyguanosine analogs (e.g., abacavir, aciclovir, entecavir), thymidine and deoxythymidine analogs (e.g., stavudine, telbivudine, zidovudine), and deoxyuridine analogs (e.g., iodoxuridine, trifluridine). In some embodiments, HIV-infected subjects may be taking one or more drugs of a highly active antiretroviral therapy (HAART) regimen. Exemplary HAART drugs include entry inhibitors or fusion inhibitors (e.g., maraviroc, enfuvirtide), nucleoside reverse transcriptase inhibitors (NRTI) and nucleotide reverse transcriptase inhibitors (NtRTI) (such as the nucleoside and nucleotide analogs described herein), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine, efavirenz, etravirine, rilpivirine), integrase inhibitors (e.g., raltegravir), and protease inhibitors (e.g., lopinavir, indinavir, nelfinavir, amprenavir, ritonavir, darunavir, atazanavir).

[0292] In various embodiments, the method reduces local inflammation in HIV infected subjects and / or subjects suffering from AIDS, changes the composition of gastrointestinal microflora, enhances the removal of microbial translocation products from the circulation, and repairs the intestinal cell barrier. In one embodiment, the method reduces gastrointestinal damage and intestinal dysbiosis in HIV infected subjects and / or subjects suffering from AIDS. For example, the method can reverse the changes in the gastrointestinal microflora observed in HIV infected subjects or subjects suffering from AIDS. For example, the changes in these gastrointestinal microflora that can be reversed by the method include microflora changes, which are characterized by an increase in pathogens, such as Staphylococcus, Pseudomonas, Enterobacteriaceae members with proinflammatory potential, and enteric pathogenic bacteria (including Pseudomonas, Xanthomonas, Bacillus and Burkholderia) that metabolize tryptophan into kynurenine derivatives. In one embodiment, the present method reduces the GI barrier dysfunction of HIV infected subjects and / or subjects with AIDS. For example, the present method can reverse the increase in intestinal permeability (for example, leaky gut syndrome) in HIV infected subjects and / or subjects with AIDS. In one embodiment, the present method reduces the translocation of microorganisms or microbial products and inflammatory mediators (for example, LPS) in HIV infected subjects and / or subjects with AIDS into the systemic circulation. In such methods, the levels of LPS, EndoCAb, sCD14 and I-FABP in subject plasma can be reduced. In one embodiment, the present method reduces immune activation and inflammation (for example, local and systemic immune activation and inflammation) in HIV infected subjects and / or subjects with AIDS. For example, the present method can reduce inflammation in intestinal associated lymphoid tissue (GALT) and increase the number of CD4+ cells and Th17 cells. The present method can further inhibit the release of cytotoxic T cells and the production of inflammatory mucosal cytokines and markers such as interferon-α, tumor necrosis factor-α, CRP, IL-1β, IL-2, IL4, IL-6 and IL-13.

[0293] In some embodiments, the present invention provides methods for treating or preventing dysbiosis and gastrointestinal dysfunction in patients with cystic fibrosis (CF). The genetic disease cystic fibrosis (CF) is associated with mutations in the CF transmembrane conductance regulator (CFTR) that regulates the permeability of epithelial cells to ions and water. In some embodiments, the present method is used to treat subjects who are homozygous for one or more mutations in the CFTR gene. In some embodiments, the subject is heterozygous for one or more mutations in the CFTR gene. In some embodiments, the one or more CFTR mutations are nonsense mutations. In some embodiments, the one or more CFTR mutations are gating mutations. In some embodiments, the one or more CFTR mutations are protein processing mutations. In some embodiments, the one or more CFTR mutations are conductance mutations. In some embodiments, the one or more CFTR mutations are translation mutations. Examples of CFTR mutations include, but are not limited to, F508del, G542X, G85E, R334W, Y122X, G551D, R117H, A455E, S549R, R553X, V520F, R1162X, R347H, N1203K, S549N, R347P, R560T, G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549R, S1255X, Add9T, Y1092X, M1191K, W 1282X, 3659delC, 394delTT, 3905insT, 1078delT, delta 1507, 3876delA, 2184delA, 2307insA, 711+1G>T, 1717-1G>A, 2789+5G>A, 1898+5G>T, 3120+1G>A, 621+1G>T, 3849+lOkbC>T, 1898+1G>A, 2183AA>G and / or 5 / 7 / 9T. In various embodiments, the methods of the present invention are used to treat CF patients with one or more CFTR mutations disclosed herein. In one embodiment, the patient has one or more of the following CFTR mutations: G551D, G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549N, S549R and / or R117H. In one embodiment, the patient has a F508del mutation. Methods for screening a patient's genotype for a CFTR mutation are known and can be performed, for example, by DNA sequencing such as bidirectional sequencing.

[0294] CF patients often exhibit symptoms including chronic respiratory infections and gastrointestinal (GI) mucosal surface dysfunction, resulting in substantial morbidity and mortality. One of the earliest manifestations of CF is gastrointestinal dysfunction, including severe and recurrent intestinal obstruction and nutrient malabsorption, which can lead to growth failure. CF patients also exhibit gastrointestinal dysbiosis, such as an overabundance of Escherichia coli (E. coli) in the fecal microflora, and a decrease in the relative abundance of species of the genus Bifidobacterium. In various embodiments, the methods of the present invention are effective in treating one or more gastrointestinal-related symptoms in CF patients.

[0295] Methods for measuring changes and / or improvements in gastrointestinal function may include, but are not limited to: endoscopy for direct examination of the epithelium and mucosa; histological evaluation and / or tissue acquisition for direct evaluation of structural changes and / or immune biomarkers; urine tests for assessment of permeability to nonabsorbable sugars and LPS levels; stool tests (e.g., by PCR) for assessment of inflammation and / or changes in microflora; and / or blood tests for assessment of specific markers, including CD4+ cell counts, Th17 cell counts, and / or LPS levels.

[0296] In some embodiments, the present invention provides methods for treating gastrointestinal disorders associated with hypothyroidism. Hypothyroidism is a condition in which the thyroid gland does not produce enough thyroid hormone (thyroxine or T4). Often, hypothyroidism slows down the activity of the digestive tract, leading to constipation, or the digestive tract may stop moving completely. The methods of the present invention can alleviate one or more gastrointestinal symptoms associated with hypothyroidism.

[0297] In some embodiments, the present invention provides methods for preventing or treating necrotizing enterocolitis (NEC).

[0298] In various embodiments, the method of the present invention relates to a pediatric subject for preventing or treating NEC. In various embodiments, the pediatric subject can be about 1 day to about 1 week old, about 1 week to about 1 month old, about 1 month to about 12 months old, about 12 months to about 18 months old, about 18 to about 36 months old, about 1 to about 5 years old, about 5 to about 10 years old, about 10 to about 15 years old or about 15 to about 18 years old. In some embodiments, the pediatric subject is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months of age. In various embodiments, the pediatric subject is fed with formula milk and / or milk. In various embodiments, the pediatric subject is receiving antibiotic treatment or has recently received antibiotic treatment.

[0299] In various embodiments, the pediatric subject is a premature infant. In some embodiments, the premature infant is born when being less than 37 weeks of gestational age. In some embodiments, the premature infant is born when about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks or about 37 weeks of gestational age. In other embodiments, the pediatric subject is a full-term infant, such as a baby born later than about 37 weeks of gestational age. In some embodiments, the pediatric subject can show one or more of antenatal asphyxia, shock, sepsis or congenital heart disease. In various embodiments, the pediatric subject has a low birth weight. In various embodiments, the weight of the pediatric subject is less than about 5 pounds, about 4 pounds, about 3 pounds or about 2 pounds.

[0300] In various embodiments, the methods of the invention relate to preventing or treating NEC in pregnant women. In some embodiments, the pregnant woman is currently receiving antibiotic therapy or has recently received antibiotic therapy.

[0301] The presence and severity of NEC is graded using the staging system of Bell et al., J. Ped. Surg., 15:569 (1980) as follows: In various embodiments, the present methods treat the disease at any of these stages.

[0302]

[0303] In various embodiments, the methods of the present invention are effective in treating one or more symptoms of NEC, including any of the above symptoms and those known in the art, including gastrointestinal symptoms, abdominal symptoms, and non-gastrointestinal symptoms. In various embodiments, the methods of the present invention are effective in preventing the development of NEC in a subject, such as a pediatric subject. In various embodiments, the methods of the present invention are effective in preventing the progression of NEC in a subject, such as a pediatric subject, e.g., from stage I to stage II or from stage II to stage III. In various embodiments, the methods of the present invention are effective in causing regression of NEC in a subject, such as a pediatric subject, e.g., from stage III to stage II or stage I to complete cure, or from stage II to stage I or to complete cure.

[0304] Intestinal dysbiosis is associated with the development of NEC and can be detected in a subject before any clinical evidence of the disease. In various embodiments, the methods of the present invention effectively restore the normal microflora in the intestine of the treated subject. In some embodiments, the methods of the present invention maintain the normal microflora in the intestine. For example, in some embodiments, the methods of the present invention maintain a healthy balance (e.g., a healthy ratio and / or a healthy distribution) of the subject's intestinal microflora. In another embodiment, the methods of the present invention treat or prevent overgrowth of one or more pathogenic microorganisms in the gastrointestinal tract. In certain embodiments, the methods of the present invention effectively reduce the levels of Clostridium butyricum and / or Clostridium perfringens in the intestine.

[0305] Methods for measuring improvement in one or more NEC symptoms include diagnostic imaging modalities such as X-rays and ultrasonography. Methods for measuring changes and / or improvements in gastrointestinal function may include, but are not limited to: endoscopy or colonoscopy for direct examination of the epithelium and mucosa; histological evaluation and / or tissue acquisition for direct evaluation of structural changes and / or immune biomarkers; stool tests (e.g., by PCR) for evaluation of inflammation and / or changes in microflora; and / or blood tests for evaluation of specific markers and cells.

[0306] In some embodiments, the present invention provides methods for treating or preventing metabolic syndrome, diabetes, hypertension, cardiovascular disease, non-alcoholic fatty liver disease and other metabolic diseases. In various embodiments, metabolic syndrome is associated with elevated triglycerides, elevated low-density lipoprotein, reduced high-density lipoprotein, reduced lipoprotein index, elevated fasting blood glucose levels, elevated fasting insulin, reduced glucose clearance after eating, insulin resistance, impaired glucose tolerance, obesity and combinations thereof. For example, the present method can be used to treat subjects with metabolic syndrome and abdominal obesity (e.g., a waist circumference of 40 inches or more for men or 35 inches or more for women), a blood triglyceride level of 150 mg / dL or more, a male HDL lower than 40 mg / dL or a female lower than 50 mg / dL, a systolic blood pressure of 130 mm Hg or more or a diastolic blood pressure of 85 mm Hg or more and / or a fasting blood glucose of 100 mg / dL or more. Other metabolic diseases that may be treated using the methods of the present invention include those described in US2013 / 0251701, US2011 / 0206654, and US2004 / 0115185, the entire contents of which are hereby incorporated by reference.

[0307] In one embodiment, the metabolic disease is obesity. Early exposure to antibiotics (e.g., within the first 2 years of life) can disrupt the microbiome and ultimately lead to disease. Bailey et al. JAMA Pediatr. 168 (11), November 2014 (the entire contents of which are hereby incorporated by reference), describe the link between early exposure to antibiotics and obesity. Therefore, in some embodiments, the present method protects the child's microbiome and prevents diseases such as obesity. In addition, changes in the ratio between the bacterial divisions Firmicutes and Bacteroidetes are often observed in obese individuals. Therefore, in some embodiments, the present invention provides a method for treating or preventing obesity by administering an AP agent of the present invention. The method of the present invention maintains normal bacterial diversity in the intestine, such as Bacteroidetes, Proteobacteria, and Firmicutes, thereby treating or preventing obesity. In addition, AP-based agents may affect fat absorption in the gastrointestinal tract. Therefore, in various embodiments, the present invention provides a method for treating or preventing obesity by limiting gastrointestinal fat absorption. In various embodiments, the method of the present invention is effective for inducing weight loss or preventing weight gain. In some embodiments, the subject may have undergone or will undergo digestive surgery; be overweight by more than about 80-100 pounds; have a BMI greater than about 35 kg / m 2 ; or have health problems related to obesity. In some embodiments, the subject may suffer from dyslipidemia, including hyperlipidemia and hyperlipoproteinemia.

[0308] In another embodiment, the metabolic disease is diabetes. In various embodiments, the present invention relates to the treatment of diabetes (type 1 or type 2) and / or glucose intolerance. In various embodiments, the present invention relates to the prevention of diabetes (type 1 or type 2) and / or glucose intolerance. In various embodiments, the present invention relates to the reduction of complications of diabetes (type 1 or type 2) and / or glucose intolerance. In some embodiments, the present invention relates to a method for treating a subject at risk of one or more of diabetes, insulin resistance, prediabetes, impaired fasting glucose (IFG) and impaired glucose tolerance (IGT).

[0309] In various embodiments, the present invention relates to treating type 1 diabetes with an AP-based agent. Type 1 diabetes, once known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin. Treatment is often performed via an intensive insulin regimen that attempts to mimic the body's normal insulin secretion pattern and often involves basal and bolus insulin coverage. For example, a common regimen is to administer long-acting insulin (including, for example, glargine / detemir) once or twice a day and administer rapid-acting insulin (including, for example, aspart, glulisine, lispro) before or after meals and as needed to correct hyperglycemia (e.g., monitored by a blood glucose meter). Doses administered before or after meals or as needed to correct hyperglycemia can be referred to as bolus administration. Another common regimen involves administration, including continuous administration of, for example, rapid-acting insulin (as described herein and including, for example, aspartate, glutamate, lispro) via an insulin pump (or a continuous subcutaneous insulin infusion device (CSII)). In various embodiments, an AP-based agent can replace any insulin used in a variety of regimens, including situations where insulin is not able to provide effective treatment in a patient, and an AP-based agent can result in increased patient compliance because it can allow for easier self-administration relative to various forms of insulin that must be administered in varying doses throughout the day - even in situations where a programmed insulin pump is required. Further, an AP-based agent can counteract common setbacks to dosing in diabetic patients, such as the dawn phenomenon. Alternatively, an AP-based agent can be used as an adjuvant to any of the type 1 diabetes treatments described herein, for example, to standardize a patient's regimen and avoid blood glucose "drops" (e.g., hypoglycemia, e.g., blood glucose below about 70 mg / dL) and "spikes" (e.g., hyperglycemia, e.g., blood glucose above about 200 mg / dL) that afflict many patients. Thus, in some embodiments, AP-based agents can treat or prevent symptoms associated with hypoglycemia, including, for example, trembling, anxiety, nervousness, palpitations, tachycardia, pallor, coldness, clammy, dilated pupils (mydriasis), hunger, borborygmi, nausea, vomiting, abdominal discomfort, headache, mental abnormalities, impaired judgment, nonspecific restlessness, paresthesias, aggression, irritability, belligerence, aggressiveness, anger, personality changes, mood swings, fatigue, weakness, apathy, lethargy, daydreaming, sleep, confusion, amnesia, lightheadedness or vertigo, delirium, staring, "glassy" appearance, blurred vision, double vision, flashes of light in the field of vision, automatisms, difficulty speaking, slurred speech, ataxia, incoordination, focal or general motor deficits, paralysis, hemiplegia, paresthesias, headache, stupor, coma, respiratory abnormalities, generalized or focal seizures, memory loss, CNS damage (e.g., cognitive impairment), amnesia, and death.Thus, in some embodiments, AP-based agents can treat or prevent symptoms associated with hyperglycemia, including, for example, bulimia, thirst, polyuria, blurred vision, fatigue, weight loss, poor wound healing, dry mouth, dry or itchy skin, tingling in the feet or heels, erectile dysfunction, recurrent infections, external ear infections (e.g., swimmer's ear), cardiac arrhythmias, stupor, coma, and seizures. In various regimens, patients with type 1 diabetes may receive an additional agent to supplement insulin therapy. In some embodiments, AP-based agents are used in this manner. AP-based agents can provide additional therapeutic benefits for patients with type 1 diabetes that is difficult to manage with insulin therapy alone. In some embodiments, as described herein, patients with type 1 diabetes that is difficult to manage with insulin therapy alone have poor glycemic control as described herein.

[0310] In some embodiments, AP-based agents can be used to lower a patient's blood glucose levels to below about 10 mM, such as in the range of about 4 mM to about 7 mM.

[0311] In some aspects, the invention provides a method for treating type 1 or type 2 diabetes comprising administering an effective amount of an AP-based agent.

[0312] In many embodiments, including those in which AP-based agents are used to prevent diabetes and / or treat a prediabetic condition, a patient is at risk for diabetes if the patient is characterized by one or more of the following: being physically inactive; having a parent or sibling with diabetes; having a family background associated with a high incidence of diabetes, selected from African American, Alaska Native, American Indian, Asian American, Hispanic / Latino, or U.S. Pacific Islander; giving birth to an infant weighing more than 9 pounds; being diagnosed with gestational diabetes; having hypertension of about 140 / 90 mmHg or above; being treated for hypertension; having an HDL cholesterol level of less than about 35 mg / dL and / or a triglyceride level of greater than about 250 mg / dL; having polycystic ovary syndrome (PCOS); and having cardiovascular disease.

[0313] In various embodiments, AP-based agents can be used to treat diabetes in an inpatient setting. For example, in some embodiments, an AP-based agent can be administered to a patient in a diabetic coma. In some embodiments, the patient to whom it can be administered is a patient suffering from one or more of the following: severe diabetic hypoglycemia, advanced diabetic ketoacidosis (e.g., advanced enough to cause unconsciousness, contributing factors can include one or more of hyperglycemia, dehydration, shock, and exhaustion), hyperosmolar non-ketotic coma (e.g., one or more of hyperglycemia and dehydration are contributing factors). In these embodiments, the AP-based agent can be used in conjunction with standard treatment regimens for diabetic coma, including administration of one or more of glucose, glucagon, insulin, fluids (e.g., saline containing potassium and / or other electrolytes), any of which is optionally administered intravenously. In some embodiments, the AP-based agent can replace insulin in these treatment regimens and is optionally administered orally.

[0314] In various embodiments, AP-based agents can be used to treat pregnant women at increased risk for gestational diabetes. Some pregnant women begin to develop gestational diabetes around 24 weeks of pregnancy, and if untreated, gestational diabetes can lead to premature birth and stillbirth. In some embodiments, the present invention provides methods for preventing and / or treating gestational diabetes in pregnant women. In various embodiments, the methods of the present invention can also be used to treat pregnant women who are at increased risk for inflammation, such as gastrointestinal inflammation. In some embodiments, the present methods reduce inflammation in pregnant women.

[0315] Further, in various embodiments related to diabetes, the patient may be receiving or may be co-administered with one or more additional agents. Illustrative additional agents include insulin or any antidiabetic agent (e.g., biguanides, insulin secretagogues (such as sulfonylureas or meglitinides), alpha-glucosidase inhibitors, thiazolidinediones, etc.). In various embodiments, the treatment methods described herein may include administering an AP-based agent to a patient who is receiving one or more additional agents and / or non-insulin diabetes agents. Additional agents include one or more of the following: sulfonylureas (e.g., DYMELOR (acetohexamide), DIABINESE (chlorpropamide), ORINASE (tolbutamide), and TOLINASE (tolazone), GLUCOTROL (glipizide), GLUCOTROL XL (extended release), DIABETA (glibenclamide), MICRONASE (glibenclamide), GLYNASE PRESTAB (glibenclamide), and AMARYL (glimepiride)); biguanides (e.g., metformin (GLUCOPHAGE, GLUCOPHAGE XR, RIOMET, FORTAMET, and GLUMETZA); thiazolidinediones (e.g., ACTOS (pioglitazone) and AVANDIA (rosiglitazone); alpha-glucosidase inhibitors (e.g., PRECOSE (acarbose) and GLYSET (miglitol); meglitinides (e.g., PRANDIN (repaglinide) and STARLIX (nateglinide)); dipeptidyl peptidase IV (DPP-IV) inhibitors (e.g., JANUVIA (sitagliptin), NESINA (alogliptin), ONGLYZA (saxagliptin), and sodium-glucose co-transporter 2 (SGLT2) inhibitors (e.g., INVOKANA (canagliflozin)); and combination pills (e.g., GLUCOVANCE, which combines glyburide (a sulfonylurea) and metformin, METAGLIP, which combines glipizide (a sulfonylurea) and metformin, and AVANDAMET, which combines both metformin and rosiglitazone (AVANDIA) in one pill, KAZANO (alogliptin and metformin), and OSENI (alogliptin plus pioglitazone).

[0316] Other additional agents include oral metformin, oral ACTOS, BYETTA subcutaneous injection, oral JANUVIA, oral WELCHOL, oral JANUMET, oral glipizide, oral glimepiride, oral GLUCOPHAGE, LANTUS subcutaneous injection, oral glyburide, oral ONGLYZA, oral AMARYl, LANTUS SOLOSTAR subcutaneous injection, BYDUREON subcutaneous injection, LEVEMIR FLEXPEN subcutaneous injection, oral ACTOPLUS MET, oral GLUMETZA, oral TRADJENTA, oral bromocriptine, oral KOMBIGLYZE XR, oral INVOKANA, oral PRANDIN, LEVEMIR subcutaneous injection, oral PARLODEL, oral pioglitazone, NOVOLOG subcutaneous injection, NOVOLOG FLEXPEN subcutaneous injection, VICTOZA 2-PAK subcutaneous injection, HUMALOG subcutaneous injection, oral STARLIX, oral FORTAMET, oral GLUCOVANCE, oral GLUCOPHAGE XR, NOVOLOG MIX 70-30 FLEXPEN subcutaneous injection, oral glibenclamide-metformin, oral acarbose, SYMLINPEN 60 subcutaneous injection, oral GLUCOTROL XL, NOVOLIN R injection, oral GLUCOTROL, oral DUETACT, oral sitagliptin, SYMLINPEN 120 subcutaneous injection, HUMALOGKWIKPEN subcutaneous injection, oral JANUMET XR, oral GLIPIZIDE-METFORMIN, oral CYCLOSET, HUMALOGMIX 75-25 subcutaneous injection, oral nateglinide, HUMALOG mixed 75-25 KWIKPEN subcutaneous injection, HUMULIN70 / 30 subcutaneous injection, oral PRECOSE, APIDRA subcutaneous injection, Humulin R injection, oral Jentadueto, Victoza3-Pak subcutaneous injection, Novolin 70 / 30 subcutaneous injection, NOVOLIN N subcutaneous injection, insulin detemir subcutaneous injection, oral micronized glibenclamide, oral GLYNASE, HUMULIN N subcutaneous injection, insulin glargine subcutaneous injection, oral RIOMET, oral pioglitazone-metformin, APIDRA SOLOSTAR subcutaneous injection, insulin lispro subcutaneous injection, oral GLYSET, HUMULIN 70 / 30Pen subcutaneous injection, colesevelam oral, sitagliptin-metformin oral, DIABETA oral, insulin regular human injection, HUMULIN NPen subcutaneous injection, exenatide subcutaneous injection, HUMALOG mix 50-50 KWIKPEN subcutaneous injection, liraglutide subcutaneous injection, oral KAZANO, oral repaglinide, oral chlorpropamide, insulin aspart subcutaneous injection, NOVOLOG mix 70-30 subcutaneous injection, HUMALOG mix 50-50 subcutaneous injection, oral saxagliptin, oral ACTOPLUS Met XR, oral miglitol, human recombinant NPH insulin subcutaneous injection, insulin NPH and regular human subcutaneous injection, oral tolazamide, oral mifepristone, insulin aspart protamine-insulin aspart subcutaneous injection, oral repaglinide-metformin, oral saxagliptin-metformin, oral linagliptin-metformin, oral NESINA, oral OSENI, oral tolbutamide, insulin lispro protamine and insulin lispro injection, pramlintide subcutaneous injection, insulin glulisine subcutaneous injection, oral pioglitazone-glimepiride, oral PRANDIMET, NOVOLOG PenFill subcutaneous injection, oral linagliptin, exenatide microspheres subcutaneous injection, oral KORLYM, oral alogliptin, oral alogliptin-pioglitazone, oral alogliptin-metformin, and oral canagliflozin.

[0317] Other additional agents include insulin lispro (HUMALOG); insulin aspart (NOVOLOG); insulin glulisine (APIDRA); regular (NOVOLIN R or HUMULIN R); NPH (NOVOLIN N or HUMULIN N); insulin glargine (LANTUS); insulin detemir (LEVEMIR); HUMULIN or NOVOLIN 70 / 30; and NOVOLOG mix 70 / 30 HUMALOG mix 75 / 25 or 50 / 50.

[0318] In various embodiments, the present invention is used to treat or prevent various neurodegenerative diseases. In some embodiments, the neurodegenerative disease is selected from multiple sclerosis (MS; including but not limited to benign multiple sclerosis, relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), progressive relapsing multiple sclerosis (PRMS), and primary progressive multiple sclerosis (PPMS)), Alzheimer's disease (including but not limited to early-onset Alzheimer's disease, late-onset Alzheimer's disease, and familial Alzheimer's disease (FAD), Parkinson's disease and Parkinson's disease (including but not limited to idiopathic Parkinson's disease, vascular Parkinson's disease, drug-induced Parkinson's disease, Lewy body dementia, hereditary Parkinson's disease, juvenile Parkinson's disease), Huntington's disease, and amyotrophic lateral sclerosis (ALS, including but not limited to sporadic ALS, familial ALS, western Pacific ALS, juvenile ALS, Hiramaya Disease).

[0319] In various embodiments, the present invention is used to treat spinal cord injury and / or dysbiosis associated with spinal cord injury. Spinal cord injury has been shown to increase intestinal permeability and intestinal bacterial translocation. These changes are associated with immune cell activation and changes in the intestinal microbiome in the gut-associated lymphoid tissue (GALT). Intestinal dysbiosis associated with spinal cord injury has been implicated in impairing motor recovery and exacerbating neuropathy pathology and intraspinal inflammation. In some embodiments, the present invention prevents and / or treats bacterial translocation and intestinal dysbiosis associated with spinal cord injury. In some embodiments, the methods of the present invention confer neuroprotection and / or improve motor recovery after spinal cord injury. In some embodiments, the methods of the present invention induce an anti-inflammatory immune phenotype in GALT. In some embodiments, the methods of the present invention reduce neurological lesions and intraspinal inflammation associated with spinal cord injury.

[0320] In various embodiments, AP-based agents can be used to treat traumatic brain injury (TBI) and its symptoms. In various embodiments, AP-based agents can be used to treat various conditions associated with trauma to the brain or spinal cord, such as caused by physical forces acting on the skull or spine, ischemia, stroke, respiratory arrest, cardiac arrest, cerebral thrombosis or embolism, neurological problems caused by AIDS, cerebral hemorrhage, encephalomyelitis, hydrocephalus, postoperative events, brain infection, and concussion or increased intracranial pressure.

[0321] In various embodiments, AP-based agents can be used to treat changes in brain function or other evidence of brain pathology caused by external forces.

[0322] In various embodiments, AP-based agents can be used to treat TBI classified by various methods. For example, clinical severity can be used (e.g., the Glasgow Coma Scale (GSC), see Teasdale and Jennett (1974) Lancet, 2, 81-84, a 15-point scale that studies the movement, sound, and eye opening responses of patients after resuscitation). In various embodiments, AP-based agents can be used to treat mild, moderate, or severe head injuries as defined by the GSC. In addition, TBI can be classified using a pathology-based classification of TBI, such as based on an assessment of the abnormalities that require treatment from an anatomical and physiological perspective. The most common patho-physiological model used to describe TBI is to evaluate whether the injury is a primary injury or a secondary injury. It is generally believed that primary injuries are injuries that are caused by the initial direct injury and are unavoidable. Secondary injuries refer to subsequent injuries that can be avoided, such as hypoxia, hypertension, or hypercapnia. Further assessment of TBI may be done with, for example, computed tomography (CT) or magnetic resonance imaging (MRI), as well as electroencephalography (EEG) and intracranial pressure (ICP).

[0323] In various embodiments, AP-based agents can be used to treat or prevent stroke. For example, the stroke treated can be ischemic or hemorrhagic. In various embodiments, a hemorrhagic stroke is a neurological deficit of cerebrovascular origin that persists for more than 24 hours or is interrupted by death within 24 hours. A hemorrhagic stroke can be an cerebral hemorrhage (intracranial hemorrhage) due to intraparenchymal hemorrhage or intraventricular hemorrhage. A hemorrhagic stroke can be a subarachnoid hemorrhage. In various embodiments, the stroke is ischemic, for example according to the Oxford Community Stroke Project classification (OCSP, i.e., classified as total anterior circulation infarction (TACI), partial anterior circulation infarction (PACI), lacunar infarction (LACI), or posterior circulation infarction (POCI)) or TOAST (Organization for Acute Stroke Treatment 10172 Trial) classification (i.e., classifying stroke as caused by (1) thrombosis or embolism due to atherosclerosis of the large arteries, (2) embolism originating from the heart, (3) complete occlusion of a small vessel, (4) other determined causes, (5) undetermined causes (two possible causes, no determined cause, or incomplete investigation)).

[0324] In various embodiments, the present invention provides methods for treating or preventing sepsis. Sepsis is characterized by a systemic inflammatory state caused by infection. Sepsis includes the presence of various purulent and other pathogenic organisms or their toxins in the blood or tissues. In some embodiments, the present invention provides methods for treating or preventing septicemia (blood poisoning), bacteremia, viremia and / or fungemia. In various embodiments, the present invention treats various end-organ pathologies associated with sepsis, such as hypotension, acute tubular necrosis (ATN), and acute respiratory distress syndrome (ARDS).

[0325] In various embodiments, the present invention provides methods for treating or preventing renal failure such as acute renal failure (ARF). Acute renal failure involves an acute loss of renal function, resulting in elevated serum creatinine levels. In acute renal failure, the glomerular filtration rate decreases over a period of days to weeks. As a result, the excretion of nitrogenous waste products is reduced, and fluid and electrolyte balance cannot be maintained. Patients with acute renal failure are often asymptomatic, and the condition can be diagnosed by observing elevated blood urea nitrogen (BUN) and serum creatinine levels. Complete renal shutdown occurs when serum creatinine levels increase by at least 0.5 mg / dL / day and urine output is less than 400 mL / day (oliguria). The AP-based agents described herein can be used not only to treat renal failure, but also to improve kidney conditions in which renal function is at least partially impaired or reduced.

[0326] In various embodiments, AP-based agents are useful for treating or preventing congestive heart failure. In various embodiments, AP-based agents are useful for treating or preventing one or more symptoms of congestive heart failure, such as shortness of breath, feeling tired, and leg swelling. In various embodiments, AP-based agents are useful for treating or preventing congestive heart failure caused by coronary artery disease, including old myocardial infarction (heart attack), high blood pressure, atrial fibrillation, valvular heart disease, excessive drinking, infection, and unexplained cardiomyopathy. In various embodiments, AP-based agents are useful for treating or preventing Class I congestive heart failure. In various embodiments, AP-based agents are useful for treating or preventing Class II congestive heart failure. In various embodiments, AP-based agents are useful for treating or preventing Class III congestive heart failure. In various embodiments, AP-based agents are useful for treating or preventing Class IV congestive heart failure.

[0327] In various embodiments, AP-based agents are useful for treating or preventing chronic fatigue syndrome, including myalgic encephalomyelitis (ME) and more recently, systemic exertion intolerance (SEID). In various embodiments, AP-based agents are useful for treating or preventing one or more symptoms of chronic fatigue syndrome, including fatigue, loss of memory or concentration, sore throat, swollen lymph nodes in the neck or armpits, unexplained muscle pain, joint pain, headaches, lack of sleep, and extreme exhaustion that lasts for more than 24 hours after physical or mental exertion.

[0328] In various embodiments, the AP-based agents are useful for treating or preventing pancreatitis. In various embodiments, the pancreatitis is mild acute pancreatitis. In various embodiments, the pancreatitis is severe pancreatitis, such as associated with organ failure, necrosis, infected necrosis, pseudocysts, and abscesses. In various embodiments, the AP-based agents are useful for treating or preventing one or more symptoms of pancreatitis, such as upper abdominal pain that may radiate to the back or vague abdominal pain, serum amylase or lipase levels > about 3 times the upper limit of normal, and imaging studies (e.g., CT, MRI, abdominal ultrasound, or endoscopic ultrasound) with characteristic changes.

[0329] In various embodiments, AP-based agents can be used to treat or prevent pancreatitis in subjects with one or more risk factors, such as: gallstones, alcohol consumption, trauma, steroid consumption, mumps infection, autoimmune disease, scorpion stings, hyperlipidemia, hypothermia, hyperparathyroidism, endoscopic retrograde cholangiopancreatography, and use of certain medications (e.g., azathioprine, valproic acid).

[0330] Several scoring systems are used to predict the severity of pancreatitis attacks. Examples include APACHE II, Ranson, BISAP, and Glasgow. In various embodiments, pancreatitis is defined by at least three of the following according to the Glasgow Modified Criteria: pO2<60 mmHg or 7.9 kPa; age>55 years; neutrophils>15; calcium<2 mmol / L; renal urea>16 mmol / L; lactate dehydrogenase (LDH)>600 iu / L; aspartate aminotransferase (AST)>200 iu / L; albumin<32 g / L; and sugar glucose>10 mmol / L.

[0331] In some embodiments, AP-based agents are used to reduce the incidence and / or severity of complications associated with administration of IV β-lactam antibiotics to allo-HCT recipients, including but not limited to aGVHD and / or VRE colonization and / or infection.

[0332] In certain embodiments, the present invention provides compositions and methods for preventing VRE colonization and / or infection.In some embodiments, the methods of the present invention prevent intestinal dysbiosis associated with VRE colonization and / or infection.

[0333] In some aspects, methods of preventing VRE colonization and / or infection in a transplant recipient are provided, comprising administering a β-lactamase agent of the invention.

[0334] In certain embodiments, the invention provides compositions and methods for reducing the incidence and / or severity of aGVHD. In various embodiments, the subject at risk of developing aGVHD is a transplant recipient. In further embodiments, the subject is a recipient of allo-HCT. In some embodiments, the subject is a recipient of one or more of bone marrow cells, peripheral blood cells, and umbilical cord cells. In various embodiments, the subject at risk of developing aGVHD is being administered or has been administered IV β-lactam antibiotics.

[0335] In some embodiments, the method of the present invention prevents intestinal dysbiosis associated with aGVHD. In various embodiments, the method of the present invention prevents loss of intestinal microflora diversity and / or intestinal dysbiosis associated with aGVHD. For example, the method of the present invention contemplates administering a monodominant composition for regulating and / or reducing any given bacterial strain (e.g., Enterococcus) associated with aGVHD. Additional embodiments of the present invention provide compositions and methods for preventing reduced diversity of microbiota in subjects who have received transplantation.

[0336] In some aspects, a method for reducing the incidence and / or severity of aGVHD in a transplant recipient is provided, the method comprising administering a beta-lactamase agent of the present invention. For example, reducing the severity of aGVHD may include receiving a patient with a grade lower than grade IV (classified as the most severe form of aGVHD).

[0337] In embodiments, the present method relates to reducing the incidence and / or severity of aGVHD in a transplant recipient. In embodiments, the transplant recipient is a cancer patient, such as a patient who has received or is receiving radiation or chemotherapy and has subsequently received allo-HCT. In embodiments, the transplant recipient suffers from blood cancer or bone marrow cancer. In embodiments, the transplant recipient is a recipient of a hematopoietic stem cell transplant. In certain embodiments, the cancer is selected from leukemia, lymphoma, myeloma and myelodysplasia. In certain embodiments, the cancer is selected from osteosarcoma, Ewing tumor, chordoma and chondrosarcoma.

[0338] aGVHD is the degradation of cells or tissues transplanted from the donor to the recipient due to the recipient's immune system recognizing the cells or tissues as foreign. Therefore, since class I MHC is on more cells in the body, it is ideal to transplant cells and tissues from people with the highest matching class I MHC properties, followed by the highest matching class II MHC properties. Therefore, in most transplant recipients, aGVHD is caused by the activation of the immune system to mismatched class II MHC molecules and other polymorphic proteins (minor histocompatibility antigens).

[0339] One option for treating blood and bone marrow cancers is to kill existing blood and bone marrow cells, such as through radiation therapy or chemotherapy, and transplant similar cells from a healthy donor, known as allogeneic hematopoietic stem cell transplantation (allo-HCT).

[0340] In embodiments, the present method relates to acute and chronic forms of GVHD. The typical acute or fulminant form of the disease (aGVHD) is usually observed within the first 100 days after transplantation, and is a major challenge to the effectiveness of transplantation due to the associated morbidity and mortality. The chronic form of graft-versus-host disease (cGVHD) traditionally occurs after 100 days. The emergence of moderate to severe cGVHD cases has an adverse effect on long-term survival. After bone marrow transplantation, T cells present in the graft, whether as a contaminant or intentionally introduced into the host, attack the tissue of the transplant recipient after considering the host tissue as a foreign antigen. T cells produce excessive cytokines, including TNFα and interferon-γ (IFNγ). Tissue damage in cGVHD is mainly due to fibrosis. A variety of host antigens can cause graft-versus-host disease, including human leukocyte antigen (HLA). However, even if the same sibling of HLA is a donor, GVHD may also occur. aGVHD is characterized by selective damage to the liver, skin and mucous membranes and the gastrointestinal tract. Tissue damage in aGVHD is mainly due to apoptosis. aGVHD severity is graded on a scale from I (mild) to IV (very severe) based on the extent and type of lesions / rash (skin), the amount of diarrhea (gastrointestinal), and serum bilirubin levels (liver). cGVHD is characterized by a wider tissue distribution than aGVHD. The skin and lungs are considered the main target organs of cGVHD, as well as the gastrointestinal tract, liver, eyes, musculoskeletal system, and hematopoietic system. Hyperacute and rapidly fatal forms of aGVHD can occur within the first 2 weeks of allogeneic HCT, usually due to significant HLA mismatch or inadequate GVHD prevention. Risk factors associated with cGVHD generally do not change after adjustment for prior aGVHD, indicating that cGVHD is not just an evolution of prior aGVHD.

[0341] In embodiments, the methods of the invention relate to reducing the incidence and / or severity of aGVHD. In embodiments, the methods contemplate administering beta-lactamase to patients treated with IV beta-lactam antibiotics and having one or more risk factors for aGVHD, such as HLA "mismatched" or unrelated donors, older patient age, female donors to male recipients, intensity of conditioning regimens or whole body irradiation during conditioning regimens, and donor lymphocyte infusions. In embodiments, the methods reduce the incidence and / or severity of aGVHD symptoms such as rash, gastrointestinal (GI) tract disorders, and liver symptoms.

[0342] In embodiments, the method involves GVHD as defined by one or more of the Billingham criteria: 1) administration of an immunocompetent transplant with viable and functional immune cells; 2) the recipient is immunohistoincompatible; and 3) the recipient is immunocompromised and therefore unable to destroy or inactivate the transplanted cells.

[0343] In various embodiments, AP-based agents can be used to treat or prevent allergies. In various embodiments, AP-based agents can be used to treat or prevent atopic allergies or IgE-mediated allergies. In various embodiments, AP-based agents can be used to treat or prevent one or more of hay fever, fur allergies, dust mite allergies, insect venom allergies, extrinsic asthma, and many types of food allergies. In various embodiments, AP-based agents can be used to treat or prevent allergic reactions, drug allergies, skin allergies, eczema, allergic rhinitis, urticaria, atopic dermatitis, dry eyes, allergic contact allergies, food allergies, allergic conjunctivitis, insect venom allergies, bronchial asthma, allergic asthma, intrinsic asthma, occupational asthma, atopic asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). One or more.

[0344] In various embodiments, AP-based agents are useful for treating or preventing asthma, including but not limited to allergic asthma. In various embodiments, AP-based agents are useful for reducing immunoglobulin E stimulation, airway hyperresponsiveness, mast cell infiltration, pulmonary eosinophilia, and accumulation of alternatively activated macrophages in the lungs. In various embodiments, AP-based agents are useful for treating or preventing one or more of recurrent wheezing and intermittent airflow limitation.

[0345] In some embodiments, the terms "patient" and "subject" are used interchangeably. In some embodiments, the subject and / or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or a non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and / or animal is a non-mammal, such as a zebrafish.

[0346] In various embodiments, the methods of the present invention can be used to treat human subjects. In some embodiments, the human is a pediatric human. In other embodiments, the human is an adult. In other embodiments, the human is an elderly. In other embodiments, the human may be referred to as a patient. In some embodiments, the human is a female. In some embodiments, the human is a male.

[0347] In certain embodiments, the age of the human ranges from about 1 to about 18 months, about 18 to about 36 months, about 1 to about 5 years, about 5 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, about 20 to about 25 years, about 25 to about 30 years, about 30 to about 35 years, about 35 to about 40 years, about 40 to about 45 years, about 45 to about 50 years, about 50 to about 55 years, about 55 to about 60 years, about 60 to about 65 years, about 65 to about 70 years, about 70 to about 75 years, about 75 to about 80 years, about 80 to about 85 years, about 85 to about 90 years, about 90 to about 95 years, or about 95 to about 100 years.

[0348] Pill Box

[0349] The present invention provides kits that can simplify the administration of modified release formulations described herein. The kit is a collection of materials or components, including at least one modified release formulation described herein. The exact nature of the components configured in the kit depends on its intended purpose. In one embodiment, the kit is configured to treat human subjects.

[0350] Instructions for use may be included in the kit. Instructions for use typically include a tangible statement describing the techniques to be employed in using the components of the kit to achieve the desired results (e.g., treating the conditions associated with the invention as described herein). Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandage materials, or other useful paraphernalia as will be readily recognized by those skilled in the art.

[0351] The material or component that is assembled in the medicine box can maintain any convenient and suitable mode of its operability and practicality and be provided to the practitioner's store.For example, the component can be provided at room temperature, refrigerated or frozen temperature.The component is usually contained in a suitable packaging material.In various embodiments, the packaging material is constructed by the method known to all, preferably to provide an aseptic, non-contaminated environment.The packaging material has an outer label, which shows the content and / or purpose of the medicine box and / or its components.

[0352] definition

[0353] As used herein, "a", "an" or "the" may mean one or more than one.

[0354] In addition, when used in conjunction with a reference number, the term "about" means the reference number plus or minus up to 10% of the reference number. For example, the language "about 50%" covers a range of 45% to 55%.

[0355] When used in connection with medical uses, an "effective amount" is an amount effective to provide a measurable treatment, prevention, or reduction in the incidence of the target condition.

[0356] As used herein, something is "reduced" if the readout of activity and / or effect in the presence of an agent or stimulus is reduced by a significant amount relative to the absence of such modulation, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more up to and including at least about 100%. As will be appreciated by one of ordinary skill in the art, in some embodiments, activity is reduced and some downstream readouts will decrease but others may increase.

[0357] In contrast, an activity is "increased" if the readout of the activity and / or effect in the presence of an agent or stimulus is increased by a significant amount relative to the absence of such agent or stimulus, e.g., by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold.

[0358] As mentioned herein, unless otherwise specified, all composition percentages are by weight of the total composition. As used herein, the word "includes" and variations thereof are intended to be non-restrictive, so that the detailed description of the items in the list does not exclude other similar items that may also be applicable to the compositions and methods of this technology. Similarly, the terms "may" and "can" and variations thereof are intended to be non-restrictive, so that the detailed description of an embodiment that can or may include certain elements or characteristics does not exclude other embodiments of the present technology that do not include those elements or characteristics.

[0359] Although the open-ended term "comprising," as a synonym for terms such as including, containing, or having, is used herein to describe and claim the invention, alternative terms such as "consisting of" or "consisting essentially of" may alternatively be used to describe the invention or embodiments thereof.

[0360] As used herein, the words "preferred" and "preferably" refer to embodiments of the present technology that provide certain benefits in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the detailed description of one or more preferred embodiments does not mean that other embodiments are not applicable, and is not intended to exclude other embodiments from the scope of the present technology.

[0361] The amount of the composition required for achieving the therapeutic effect described herein can be determined empirically according to routine procedures for a specific purpose. Typically, a therapeutic agent (e.g., beta-lactamase and / or additional therapeutic agent described herein) is administered for therapeutic purposes, and the therapeutic agent is administered in a pharmacologically effective dose. "Pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount" or "effective amount" refers to an amount sufficient to produce a desired physiological effect or to achieve a desired result, especially an amount for treating a disorder or disease. As used herein, an effective amount includes an amount sufficient to, for example, delay the development of a symptom of a disorder or disease, change the course of a symptom of a disorder or disease (e.g., slowing down the progress of a symptom of a disease), reduce or eliminate one or more symptoms or manifestations of a disorder or disease, and reverse the symptom of a disorder or disease. Therapeutic benefit also includes stopping or slowing down the progress of a potential disease or disorder, regardless of whether improvement is achieved.

[0362] The effective amount, toxicity and therapeutic efficacy can be determined in cell culture, tissue samples, tissue homogenates or experimental animals by standard pharmaceutical procedures, such as for determining LD50 (a dose lethal to about 50% of the population) and ED50 (a dose effective for treatment in about 50% of the population). The dosage can be changed according to the dosage form adopted and the route of administration utilized. The dose ratio between toxic effects and therapeutic effects is the therapeutic index, and it can be expressed as a ratio LD50 / ED50. In some embodiments, compositions and methods showing a large therapeutic index are preferred. The therapeutically effective dose can be initially assessed by in vitro assays, including, for example, cell culture assays. In addition, the dosage can be formulated in an animal model to achieve a circulating plasma concentration range including an IC50 as determined in cell culture or in an appropriate animal model. The level of the composition in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dose can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, if necessary, to suit the observed therapeutic effect.

[0363] In certain embodiments, the effect will cause a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will cause a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes stopping or slowing the progression of the underlying disease or condition, regardless of whether improvement is achieved.

[0364] As used herein, "therapeutic method" also applies to the use of a composition for treating a disease or condition described herein and / or use of a composition for treating a disease or condition described herein and / or in the manufacture of a medicament for treating a disease or condition described herein.

[0365] Example

[0366] Example 1: Development of IAP delayed-release enteric-coated pellets

[0367] Two batches of IAP coated sucrose cores were prepared at enzyme loadings of 3.45% w / w (first batch) and 4.65% w / w (second batch). The IAP activity after coating was maintained compared to the IAP solution used for coating. The IAP solution exhibited approximately 70% activity compared to the Sigma standard, while the drug coated cores exhibited 60% activity compared to the Sigma standard.

[0368] Drug coated core batch 1 was studied to evaluate the stability of the IAP by activity assay. The cores were stored under refrigerated conditions in the presence and absence of silica desiccant and the IAP activity was found to remain stable over 8 weeks in both cases. Drug coated core batch 2 was then coated to 30% EUDRAGIT L30D-55 WG (monolayer) or 7% HPC WG as an inner barrier layer followed by 30% EUDRAGIT L30D-55. Both coating formulations exhibited acid resistance in fasted state simulated gastric fluid (FaSSGF) for 2 hours and the dissolution profiles in fasted state simulated intestinal fluid (FaSSIF) were very similar with no loss of enzyme activity at the end of the dissolution test.

[0369] In the presence of silica, the coated pellets were stored under refrigerated conditions and after 6 months, the enzyme activity did not decrease, indicating that the HPC (binder) and coating layer (acidic polymer EUDRAGIT L30D-55) had no effect on the enzyme activity during this 6 month period.

[0370] Overall, drug-coated pellet formulation is a promising option for formulating IAPs due to the obtained dissolution profile and good flow properties of the pellets. Another advantage is the flexibility to obtain various enzyme strengths based on one formulation. It also offers a simplified process as coating and enteric coating can be performed in the same fluid bed coater.

[0371] Example 2: Evaluation of excipient-IAP compatibility

[0372] IAPs were combined with selected potential binders to determine the effect of the binders on IAP activity. Figure 2 As shown, adhesives tested for compatibility with IAPs in the presence of sucrose or lactose did not significantly alter IAP activity after 96 hours of incubation and showed similar % activity to the initial IAP solution alone (IAP-adhesive combination #11).

[0373] Table 1. IAP-adhesive combination numbers and compositions.

[0374]

[0375]

[0376] As shown in Table 1 and Figure 2 As shown, most combinations did not result in significant changes in enzyme activity compared to the initial value at t=0 hours. The results show that under the current test conditions, no loss of IAP activity was observed in the presence of any of the excipients tested, so no incompatibility was determined. The results suggest that drug-coated cores using HPC as a binder do not compromise IAP activity and this is the suggested combination to select for further studies.

[0377] The cofactors tested for compatibility with IAP are shown in Table 2 and Figure 3 It was found that the cofactor did not significantly increase IAP activity under the current test conditions.

[0378] Table 2. IAP-excipient combination numbers and compositions.

[0379]

[0380] As shown in Table 2 and Figure 3 As shown, the first four combinations without any cofactor (#1-#4) did not show a decreasing trend in IAP activity compared to the four combinations with cofactor (#5-#8). Figure 2Similar to the results provided in , the results of the IAP cofactor compatibility studies have demonstrated that IAP activity is not compromised in the presence of HPC, sucrose, or a combination of both. Addition of cofactors to the formulations did not significantly increase IAP activity at different time points. Therefore, formulations containing cofactors were not studied further. The IAP buffer solution contained 1.0 mM MgCl2 and 0.1 mM ZnCl2. Other metal ions (CoCl2 and CaCl2) tested in these experiments did not alter the enzymatic activity of the IAP-binder mixtures.

[0381] The excipient / IAP mixed solutions were incubated at 37°C and therefore, due to the incubation temperature, the aqueous medium evaporated over time and a significant loss of solution volume was observed. This resulted in an increase in enzyme activity at the final time point for combination #2. The decrease in enzyme activity in combinations #4, #5, #7, and #8 at the final time point may be related to assay variability.

[0382] Example 3: Stability test of the first batch of cores

[0383] IAP solution was supplied frozen as 10.1 mg / mL IAP (intestinal alkaline phosphatase) in 20 mM Tris, 0.1 mM ZnSO4 and 1 mM MgCl2 buffer at pH 7.5 and used for drug coating. To prepare the final drug coating solution, 1.12 g of HPC (Klucel EF) was added to 49.5 mL of IAP solution under slow magnetic stirring (less than 200 RPM). The subsequent solution was spray coated onto 6.00 g of 600 / 710 μm Suglet in a Caleva Mini Coater / Dryer.

[0384] The IAP-HPC solution was sprayed at an atomizing air pressure of 0.8 bar with an air inlet velocity of 11.5 m / s and a chamber stirring rate of 10.5 Hz. The spraying process temperature was 45°C for the initial 30-45 minutes and then decreased to 40°C. This is because in the early stage of the coating process, if the temperature is not high enough to quickly dry the aqueous solution on the core surface, the Suglet is easily aggregated. As the coating process proceeds, the core has a thin coating layer, which can protect the core and cause a reduction in the chance of aggregation, so the process temperature is reduced.

[0385] After each hour of spraying, the drug coated cores were dried at 35°C for 30 minutes and once coating was complete, the drug coated cores were dried at 40°C for an additional hour before confirming the actual weight gain.

[0386] The drug coated cores (AP0361 / 24 / 1) consisted of 87.92 / 3.45 / 7.77 / 0.87% w / w of Suglet / IAP / HPC / salt (from IAP solution), respectively. Due to a manufacturing error, this batch of cores had a slightly different IAP / HPC ratio of 1:2.25 w / w instead of the expected 1:2 w / w ratio. After the drug coating process was completed, the cores were stored at 2-8°C in a sealed glass vial with a silica desiccant bag.

[0387] The resulting cores were stored in sealed vials, dried at 2-8°C for the first 12 days after drug coating. To study the effect of non-drying conditions, half of the contents were removed from the glass vials after the initial drying for 12 days and stored at 2-8°C in the absence of the silica bag. The results of enzyme activity measured over time in the short-term stability study of these drug-coated cores are shown in Table 3.

[0388] Table 3. IAP activity of drug coated cores in refrigerator under dry / non-dry conditions.

[0389]

[0390] Some fluctuations in % IAP activity over time were observed; however, these may be related to assay variability. Based on the results in Table 3, it can be concluded that non-drying conditions did not significantly reduce IAP activity compared to drying conditions.

[0391] For enzyme activity testing, the core was dissolved in diethanolamine buffer. Specifically, an IAP activity assay was performed. A 140 mg / mL solution was prepared in purified water using diethanolamine (Sigma D8885), and the pH of the diethanolamine was adjusted to 9.8 with 5M HCl at 37°C. The pH-adjusted solution was diluted with purified water to a final diethanolamine concentration of 105 mg / mL. Finally, 500 μL / L of 1M magnesium chloride solution (Sigma M1028) was added to the solution. The diethanolamine was freshly prepared and protected from direct sunlight.

[0392] Prepare the IAP enzyme standard solution by diluting the 19.4 mg / mL stock solution (concentration may vary from lot to lot from Sigma) to 1.00 mg / mL (5 μL in 92 μL diethanolamine buffer) in diethanolamine buffer. Keep the enzyme solution on ice before further dilution. This purified intestinal alkaline phosphatase (Sigma #PO114) was used as a control to test the activity of the samples assayed.

[0393] All samples (including Sigma standards and IAP solution) were loaded onto a 96-well plate and diluted with diethanolamine buffer to a theoretical 5 ng / mL solution.

[0394] pNPP (Abcam ab146203) was reconstituted to a 5 mM solution at 1.86 mg / mL assay buffer, and the pNPP solution was stable on ice for 12 hours.

[0395] Then carry out kinetic assay.On the plate reader (DYNEX#MRX TC II), 80 μL of standards and samples are added to the wells of 96-well plates.All enzyme reactions are run in duplicate wells.Add enough 5mM pNPP solution in each empty well to prepare for adding to the sample later and incubate the plate at 37 ° C for 5 minutes.Use a multichannel pipette to quickly add 50 μL of preheated 5mM pNPP solution to each well containing standards or samples.During this addition, avoid bubble formation.The reaction sequence starts and proceeds at 37 ° C.After an additional 10-minute initial delay and a 2-second initial oscillation step, the colorimetric output (pNPP NPP dephosphorylation via enzyme phosphatase activity) is measured every 20 seconds within 5 minutes as a function of the increase in optical density (OD) at 405nm.Blank wells without enzymes are run in parallel on the same plate.

[0396] The kinetic assay provides a readout of the enzyme kinetics over this time period and the slope is used to compare to the slope generated by the IAP Sigma standard (sample % activity = sample slope / Sigma standard slope * 100%; the slope of the blank is subtracted from the slopes of the sample and Sigma standard).

[0397] Example 4: Enteric coating and dissolution testing of the second batch of cores

[0398] A second batch of drug coated cores was prepared for coating trials and manufactured using the same equipment parameters as the first batch of cores (Example 3). The second batch of cores consisted of 84.9 / 4.7 / 9.3 / 1.2% w / w Suglets / IAP / HPC / salt (from IAP solution). The drug coating solution was prepared using 0.948 g of HPC (Klucel EF) added to 47.0 mL of IAP solution at a 1:2 w / w IAP:HPC ratio under slow magnetic stirring (less than 200 RPM).

[0399] Two types of coatings were applied to the second batch of drug coated cores, either a 30% WG EUDRAGIT L30D-55 (single enteric layer) or a 7% WG HPC layer followed by an outer layer of 30% WG EUDRAGIT L30D-55 polymer (enteric layer with primer). The formulations are listed in Table 4.

[0400] Table 4. Formulation composition of the second batch of drug coated cores with enteric coating.

[0401]

[0402] Drug coated cores (Batch 2) were coated with IAP / HPC at a 1:2 w / w solids ratio, with a final composition of 4.65% WG of IAP followed by either a single layer coating (30% WG of EUDRAGIT L30D-55) or a double layer coating (7% WG of HPC + 30% WG of EUDRAGIT L30D-55). Figure 4 As shown in the SEM image in , the diameter of the single-layer coated pellets is approximately 800 μm. Figure 4 A SEM picture of one coated pellet (30% EUDRAGIT L30D-55 coated on pellets from the second batch) is depicted.

[0403] A cross section of the second batch of pellets is also shown. Figure 5 In the embodiment of the present invention, the sucrose core, the IAP / HPC layer and the L30D-55 coating layer can be clearly distinguished. The thickness of the IAP / HPC layer is about 20 μm and the thickness of the EUDRAGIT L30D-55 coating layer is about 40 μm (thickness recommended by the polymer manufacturer Evonik). Figure 5 Depicted is a SEM picture of a cross section of one coated pellet (30% L30D-55 coated on pellets from the second batch).

[0404] The dissolution behavior of these coated pellets was studied. The amount of pellets in each dissolution vessel was 1.14 g single-layer coated pellets and 1.22 g double-layer coated pellets, which corresponds to an amount of 40 mg IAP for both formulations. In each dissolution test, one tablet was incubated in 20 mL FaSSIF (pH 1.6) in a 60 mL vial on a heater / shaker for 2 hours at 37°C and 200 RPM shaking. After the initial 2 hours, 20 mL double strength FaSSIF was added and the pH was adjusted to 5.5 or 5.8 using 1 M NaOH. After a further 45 minutes, the pH was further adjusted to 6.5 or 6.8 and the dissolution test was performed for another 2-3 hours.

[0405] The coated pellets were tested in FaSSGF at pH 1.6 for 2 hours, then FaSSIF was added to reach pH 5.8, held for 45 minutes, then increased to pH 6.8, and held for another 3 hours. Dissolution testing of the coated pellets in FaSSIF alone (pH 5.8 for 45 minutes, then pH 6.8 for 3 hours) was also performed for comparison.

[0406] UV protein release curve Figure 6The % release of IAP during dissolution in FaSSIF alone is presented as the "x" and "triangle" curves for the monolayer and bilayer coatings, respectively. The % release of IAP during dissolution in FaSSGF and FaSSIF is presented as the red and blue curves for the monolayer and bilayer coatings, respectively.

[0407] The curves indicate that the coated pellets are enteric protected at low pH, as no IAP release was detected in FaSSGF by UV analysis. Moreover, the dissolution profiles between the single-stage and bi-stage processes were very similar - release started immediately after pellets were exposed to FaSSIF at pH 5.8, and after 45 minutes at pH 5.8, the dissolution rate increased when the pH was adjusted to pH 6.8. Complete release was achieved after another 3 hours at pH 6.8.

[0408] Enzyme activity curve Figure 7 The % activity of IAP during dissolution in FaSSIF alone is presented as the "x" and "triangle" curves for the monolayer and bilayer coatings, respectively. The % activity of IAP during dissolution in FaSSGF and FaSSIF is presented as the red and blue curves for the monolayer and bilayer coatings, respectively.

[0409] Enzyme activity curve and Figure 6 The % release of IAP due to UV in the 20% and 20% scavenger cores followed the same trend. The maximum enzyme activity detected at the endpoint of both dissolution tests was approximately 45%-55%, which was at the same level as the raw material (approximately 50% for drug coated cores). The results indicate that the applied coatings were enteric protected at low pH and that the enzyme activity was not reduced in the presence of the coating polymer. Since the dissolution profiles produced by the mono- and bi-layer coated cores were similar, it can be concluded that a bottom layer HPC is not required to isolate and protect the enzyme from the acidic coating polymer.

[0410] The % activity obtained by exposing the pellets to FaSSIF alone was similar to that of FaSSGF / FaSSIF, indicating that the IAP in the coated pellets was not affected or denatured by FaSSGF pH 1.6. This confirms that sufficient enteric coating thickness was applied to obtain the same level of enzyme activity.

[0411] The study showed that an enteric coating of a core coated with a 30% polymer weight gain EUDRAGIT L30D-55 aqueous dispersion provided acid resistance for up to 2 hours in FaSSGF and sustained release of IAP in FaSSIF over 4 hours. The presence of a 7% HPC barrier layer did not alter the dissolution profile nor affect enzyme activity. Therefore, it was judged that a barrier sublayer was not required.

[0412] Example 5: Stability Assessment of IAP Activity in Pellets

[0413] Stability assessment was performed by placing uncoated / coated drug coated cores in sealed glass vials with silica desiccant bags at 2-8°C for up to 12 months. Enzyme activity of the cores was monitored at 0, 1, 3 and 6 months by dissolving the cores in diethanolamine buffer and analyzing IAP activity.

[0414] Both the uncoated pellets and the coated pellets were stored under dry conditions at refrigerated temperature. Enzyme activity was measured over a period of up to 6 months and the results are shown in Table 5.

[0415] Table 5. Stability of IAP activity of uncoated / coated drug-layered pellets when stored in dry conditions at 2-8°C.

[0416]

[0417]

[0418] The results showed that the enzyme activities of uncoated cores and coated pellets remained at the same level during storage at refrigerated temperature under dry conditions for 6 months.

[0419] Example 6: Stability Evaluation of 25 mg and 5 mg IAP Pellets Capsules

[0420] HPMC transparent capsules No. 2 were filled with coated IAP pellets, which exhibited a drug loading of 9.7% w / w (by UV spectrophotometer analysis) and an enzyme activity of 78.1% (by DYNEX microplate reader analysis) relative to Sigma standards. 90 capsules contained 25 mg IAP and 90 capsules contained 5 mg IAP. Pellets were manufactured by coating IAP / HPC (1 / 2 w / w) onto 600 / 710 μm Suglet (Colorcon) followed by EUDRAGIT L30D-55 (Evonik) coating.

[0421] Table 6. Composition of capsules containing 5 mg and 25 mg IAP

[0422]

[0423] The stability and percent activity of the pellet capsules during storage in a refrigerator for one year are shown in Table 7 below.

[0424] Table 7. Pellet capsule activity over the course of one year.

[0425] Storage duration at 2-8°C Activity % (compared to IAP standard) 0(Date of manufacture) 93.0 1 month 93.8 3 months 87.3 6 months 83.4 12 months 79.7

[0426] The present inventors surprisingly found that IAP pellet capsules maintained about at least 80% of the IAP activity when stored at 2-8°C for a period of one year.

[0427] Then carry out in vitro dissolution test.About 450mg coated pellets are incubated in 25mL FaSSGF (pH 1.6, 37 ℃) in 60mL glass vials, and the vials are gently shaken at 200RPM orbital shaking speed on a heater / oscillator. At 60 minutes and 120 minutes, 1mL of medium is extracted through a 10μm cannula filter and filtered through a 0.2μm filter for UV analysis. The 50μL filtered solution is frozen for further activity analysis. The solution is returned to the vial afterwards.

[0428] After the initial incubation in FaSSGF for 2 hours, 25 mL of FaSSIF was added to the vial to obtain pH 5.8. Samples were taken every 15 minutes. After 45 minutes, the pH was adjusted to 6.8 and samples were taken every 15 minutes. The sampling method was the same as described in the previous paragraph.

[0429] The filtered samples were analyzed in a UV spectrometer at 280 nm and 320 nm and the absorbance was equal to A280-A320. The absorbance of the blank medium was further subtracted from the sample absorbance and the subsequent values ​​were used to calculate the release %. Figure 8 As shown, a calibration curve was first generated. The sample concentration at each time point was calculated based on the linear trend line equation.

[0430] Fig. 9 The results of the dissolution test are depicted in . The coated pellets were resistant to FaSSGF for 2 hours as no release was detected. The % release at 60 and 120 minutes (T=0, red line) was approximately 1%. In addition, at the end of the dissolution test, the activity of the pellets was fully recovered, indicating that the acid did not penetrate the coating during the initial incubation period. The IAP release was slow during the first 45 minutes in FaSSIF at pH 5.8 because the pH was close to 5.5, the dissolution threshold of EUDRAGIT L30D-55. Once the pH was adjusted to 6.8, the coating began to dissolve rapidly and the amount of IAP released increased dramatically.

[0431] Fig.10 Depicted are the results of a dissolution test in which the coated pellets had been stored for about a year. The coated pellets were resistant to FaSSGF for 2 hours, as no release was detected. The percentage release at 60 and 120 minutes (T=0, red line) was approximately 1%. In addition, at the end of the dissolution test, the activity of the pellets was fully recovered, indicating that the acid did not penetrate the coating during the initial incubation period. The IAP release was slow during the first 45 minutes in FaSSIF at pH 5.8, as the pH was close to 5.5, the dissolution threshold of EUDRAGIT L30D-55. Once the pH was adjusted to 6.8, the coating began to dissolve rapidly and the amount of IAP released increased dramatically.

[0432] Various tests were performed on separate batches of IAP pellets, including: non-GMP SYN-020 pellets; non-GMP SYN-020 pellets (HPD514-A-001); non-GMP SYN-020 capsules (5 mg); non-GMP SYN-020 capsules (15 mg); and GMP SYN-020 pellets. The composition of SYN-020 delayed-release capsules, i.e., 15 mg and 5 mg capsules, and SYN-020 placebo pellets and the results of various tests are depicted in Tables 8 and 9 below.

[0433] Table 8. Composition of SYN-020 delayed-release capsules, 15 mg and 5 mg capsules.

[0434]

[0435] HTP-20 is a premixed formulation of glyceryl monostearate, polysorbate-80, and triethyl citrate, which provides the functions of anti-sticking, plasticizer, and stabilizer.

[0436] Table 9. Batch analysis of SYN-020IAP coated pellets and capsules

[0437]

[0438]

[0439] Fig.11 AH depicts additional analysis of non-GMP SYN-020 5 mg and 15 mg capsule doses. Results regarding dose strength and stability, including parameters such as specific activity, purity, water content, and dissolution, were analyzed over a 6-month period.

[0440] Equivalent solutions

[0441] Although the invention has been described in conjunction with specific embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is intended to cover any variations, uses, or modifications of the invention which are generally consistent with the principles of the invention and which come within the known or customary practice in the art to which the invention pertains and which departure from the present disclosure which may be applied to the basic features set forth above and within the scope of the appended claims.

[0442] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments explicitly described herein. Such equivalents are intended to be encompassed within the scope of the appended claims.

[0443] Incorporated by Reference

[0444] All patents and publications cited herein are incorporated by reference in their entirety.

[0445] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0446] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of any individual section may apply equally to all sections.

Claims

1. A modified release formulation comprising at least one modified release pellet comprising an alkaline phosphatase (AP)-based agent contained in a capsule, said modified release pellet releasing a substantial amount of the AP-based agent in the gastrointestinal tract, wherein said modified release formulation comprises: about 10 wt % of an AP-based agent; about 39% by weight sucrose spheres; about 20% by weight hydroxypropyl cellulose; about 0.5% by weight of a buffer salt; and About 26% by weight of an enteric polymer, wherein the formulation retains at least about 80% of the activity of the AP-based agent when stored at 2-8°C for at least 1 month, 3 months, 6 months or 12 months.

2. A modified release formulation comprising at least one modified release pellet comprising an alkaline phosphatase (AP)-based agent contained in a capsule, said modified release pellet releasing a substantial amount of the AP-based agent in the gastrointestinal tract, wherein said modified release formulation comprises: about 10.0 wt % of an AP-based agent; about 38.9% by weight sucrose spheres; about 20.0% by weight hydroxypropyl cellulose; about 0.3% by weight of a buffer salt; and About 26.3% by weight of enteric polymer, wherein the formulation retains at least about 80% of the activity of the AP-based agent when stored at 2-8°C for at least 1 month, 3 months, 6 months or 12 months.

3. The modified release formulation of claim 1 or 2, wherein the capsule contains about 15 mg or 5 mg of the AP-based agent.

4. A modified release formulation comprising at least one modified release pellet comprising an alkaline phosphatase (AP)-based agent contained in a capsule, said modified release pellet releasing a substantial amount of the AP-based agent in the gastrointestinal tract, wherein said modified release formulation comprises: about 10 wt % of an AP-based agent; about 38% by weight sucrose spheres; about 19% by weight hydroxypropyl cellulose; about 2% by weight of buffer salts; and About 26% by weight of an enteric polymer, wherein the formulation retains at least about 80% of the activity of the AP-based agent when stored at 2-8°C for at least 1 month, 3 months, 6 months or 12 months.

5. A modified release formulation comprising at least one modified release pellet comprising an alkaline phosphatase (AP)-based agent contained in a capsule, said modified release pellet releasing a substantial amount of the AP-based agent in the gastrointestinal tract, wherein said modified release formulation comprises: about 9.7 wt % of an AP-based agent; about 37.7% by weight sucrose spheres; about 19.4% by weight hydroxypropyl cellulose; about 2.4% by weight buffer salts; and About 26.3% by weight of enteric polymer, wherein the formulation retains at least about 80% of the activity of the AP-based agent when stored at 2-8°C for at least 1 month, 3 months, 6 months or 12 months.

6. The modified release formulation of claim 4 or 5, wherein the capsule contains about 25 mg or 5 mg of the AP-based agent.

7. The modified release formulation of any one of claims 1, 2, 4 or 5, further comprising 1-10% HTP-20.

8. The modified release formulation of any one of claims 1, 2, 4 or 5, wherein the capsule comprises gelatin or hydroxypropylmethylcellulose.

9. A modified release formulation comprising at least one modified release pellet comprising an alkaline phosphatase (AP)-based agent, said modified release pellet releasing a substantial amount of the AP-based agent in the gastrointestinal tract, wherein each modified release pellet comprises: about 5 wt % of an AP-based agent; about 85% by weight sucrose spheres; about 9% by weight hydroxypropyl cellulose; and About 1% by weight buffer salt.

10. A modified release formulation comprising at least one modified release pellet comprising an alkaline phosphatase (AP)-based agent, said modified release pellet releasing a substantial amount of the AP-based agent in the gastrointestinal tract, wherein each modified release pellet comprises: about 4.7 wt % of an AP-based agent; about 84.9% by weight sucrose spheres; about 9.3% by weight hydroxypropyl cellulose; and About 1.2% by weight buffer salts.

11. The modified release formulation of claim 9 or 10, further comprising a single layer enteric coating having a weight gain of 20-40% enteric polymer, optionally having a weight gain of about 30% enteric polymer.

12. The modified release formulation of claim 9 or 10, further comprising an enteric coating layer having a weight gain of 20-40% enteric polymer and an enteric coating layer having a weight gain of 5-15% hydroxypropyl cellulose, optionally having a weight gain of about 30% enteric polymer and having a weight gain of about 7% hydroxypropyl cellulose.

13. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the AP-based agent comprises an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 39 and 1-17.

14. The modified release formulation of claim 13, wherein the AP-based agent comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

39.

15. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the AP-based agent is released substantially in the small intestine.

16. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the AP-based agent is released substantially in the large intestine.

17. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the formulation comprises a core particle and a base coating on the core particle, wherein the base coating comprises the AP-based agent.

18. The modified release formulation of claim 17, wherein the core particle comprises sucrose.

19. The modified release formulation of claim 17, wherein the formulation comprises a plurality of core particles.

20. The modified release formulation of claim 19, wherein the core particles have a diameter ranging from 1 mm to 1.3 mm.

21. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the formulation further comprises a modified release coating that is substantially stable in gastric fluid.

22. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the formulation comprises a modified release coating that is degraded by microbial enzymes present in the intestinal flora.

23. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the formulation comprises a modified release coating having a pH-dependent solubility.

24. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the formulation comprises a modified release coating having a time-dependent erosion profile.

25. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the formulation further comprises an additional therapeutic agent.

26. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the buffer salt is selected from the group consisting of Tris base, magnesium chloride, magnesium sulfate, zinc chloride and zinc sulfate.

27. The modified release formulation of any one of claims 1, 2, 4, 5, 9 or 10, wherein the enteric polymer is selected from EUDRAGIT L30 D-55, FS 30D, L 100-55, L 100, L 12,5, L 12,5P, RL 30D, RL PO, RL100, RL 12,5, RS 30D, RS PO, RS100, RS12,5, NE 30D, NE 40D, NM 30D, S100, S12,5 and S12,5P.

28. The modified release formulation of claim 27, wherein the enteric polymer is EUDRAGIT L 30D-55 (poly(methacrylic acid-co-ethyl acrylate) 1:1).

29. Use of the formulation of any one of claims 1, 2, 4, 5, 9 or 10 in the preparation of a medicament for treating or preventing a radiation-induced disorder in a subject in need thereof.

30. The use according to claim 29, wherein the radiation-induced condition is selected from enterocolitis caused by cancer radiotherapy, radiation-induced enteropathy, colitis and proctitis.

31. The use of claim 29, wherein the radiation-induced disorder comprises intestinal toxicity.

32. The use of claim 29, wherein the radiation-induced disorder comprises one or more of mucosal atrophy, vascular sclerosis, and progressive intestinal wall fibrosis.

33. The use of claim 29, wherein the radiation comprises ionizing radiation, optionally wherein the radiation comprises one or more of X-rays, gamma rays and charged particles.

34. The use of claim 29, wherein the radiation-induced condition is a side effect of radiation therapy.

35. The use of claim 34, wherein the radiation therapy comprises a dose of about 20 Gy, or about 30 Gy, or about 40 Gy, or about 50 Gy, or about 60 Gy, or about 70 Gy, or about 80 Gy, or about 90 Gy, or about 100 Gy, optionally fractionated.

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