Alkaline phosphatase formulations and uses thereof

Abstract

To provide novel alkaline phosphatase formulations for use in therapeutic intervention.SOLUTION: The present invention provides, in part, formulations comprising an alkaline phosphatase. Particularly, modified-release formulations comprising an alkaline phosphatase are provided, which release a substantial amount of the alkaline phosphatase in the intestines. Therapeutic uses of the alkaline phosphatase formulations are also provided.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention provides, in part, formulations comprising alkaline phosphatase and uses thereof.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 856,309, filed June 3, 2019, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated by reference in their entirety herein: Computer-readable copy of the sequence listing (Filename: SYN-044PC_ST25.txt, Creation Date: June 2, 2020, File Size: 78,381 bytes). [Background technology]

[0004] Alkaline phosphatases are dimeric metalloenzymes that catalyze the hydrolysis of phosphate esters and dephosphorylate various target substrates at physiological and higher pH. Alkaline phosphatases (APs) are found in prokaryotes and eukaryotes (e.g., E. coli and mammals). Mammalian APs have been shown to play important roles in intestinal homeostasis, mucosal barrier function, promotion of commensal bacteria, and defense against pathogens. Mammalian APs exert their properties primarily by targeting lipopolysaccharide (LPS, Toll-like receptor 4 (TLR4) agonist), flagellin (TLR5 agonist), and CpG DNA (TLR9 ​​agonist). APs also degrade intestinal luminal nucleotide triphosphates (NTPs, e.g., ATP, GTP, etc.), which promotes the growth of beneficial bacteria and reverses dysbiosis.

[0005] Treatment of gastrointestinal (GI) disorders increasingly relies on the role of the microbiome as a mediator that maintains healthy function of the GI tract. Thus, the role of alkaline phosphatase (AP) in promoting the proliferation of beneficial bacteria and reversing dysbiosis is an important and growing area of ​​research in advancing treatment options for GI disorders. Thus, AP may be used clinically, for example, as a microbiome protector to treat various gastrointestinal (GI) disorders.

[0006] However, the formulation of protein biologics, including APs, is particularly challenging given the various considerations that must be taken into account regarding the drug's route of administration. Furthermore, providing a layered drug-modified release formulation that is stable and exhibits a sufficient dissolution / release profile to allow targeted release to the GI tract can be difficult due to the complex nature of protein biologics.

[0007] Thus, there remains a need for new alkaline phosphatase preparations for use in therapeutic intervention. Summary of the Invention

[0008] Thus, the present invention provides a modified release formulation comprising an alkaline phosphatase (AP) based drug and / or an additional therapeutic agent. In various embodiments, the formulation releases a substantial amount of the AP based drug to the GI tract. In one embodiment, the formulation comprises at least one core particle and a base coat on the core particle, the base coat comprising the AP based drug. In another embodiment, the formulation comprises at least one core particle, the AP based drug being encapsulated within the core particle. In various embodiments, the formulation comprises a modified release coating, such as a delayed release coating, disposed on the core particle. In some embodiments, the delayed release coating is substantially stable in gastric fluids. In some embodiments, the delayed release coating is substantially stable in intestinal fluids. In one embodiment, the delayed release coating comprises a EUDRAGIT compound. In various embodiments, the formulation may be in the form of a sucrose pellet. In some embodiments, the pellet comprises a plurality of core particles.

[0009] These AP-based drugs are used in many therapies, including the prevention or treatment of CDI and / or C. difficile-associated disease or other antibiotic-induced adverse effects in the GI tract. The AP-based drugs of the present invention are also used in therapies for microbiome-associated disorders, including but not limited to metabolic disorders, neurological disorders, celiac disease, cystic fibrosis, radiation enteropathy, sepsis, and HIV-mediated intestinal dysbiosis.

[0010] For example, AP-based drugs can be used to allow patients to receive antibiotic therapy while being protected from diseases that may result from excessive antibiotics that negatively affect microbiome.Such use does not prevent the systemic usefulness of antibiotics.Without wishing to be bound by theory, rather, AP-based drugs maintain gut microbiota, thereby preventing the destruction of microbiota that is associated with various pathologies described herein. [Brief description of the drawings]

[0011] [Figure 1-1]1 shows sequences for alkaline phosphatase-based agents present in the formulations described herein. [Figure 1-2] 1 shows sequences for alkaline phosphatase-based agents present in the formulations described herein. [Figure 1-3] 1 shows sequences for alkaline phosphatase-based agents present in the formulations described herein. [Figure 1-4] 1 shows sequences for alkaline phosphatase-based agents present in the formulations described herein. [Figure 1-5] 1 shows sequences for alkaline phosphatase-based agents present in the formulations described herein. [Figure 1-6] 1 shows sequences for alkaline phosphatase-based agents present in the formulations described herein. [Figure 1-7] 1 shows sequences for alkaline phosphatase-based agents present in the formulations described herein. [Figure 1-8] 1 shows sequences for alkaline phosphatase-based agents present in the formulations described herein. [Diagram 2] 1 shows IAP activity over a 96 hour period in an IAP-binder assay. Each set of histograms represents various time points, with the leftmost bar representing 0 hours, the middle bar representing 24 hours, and the rightmost bar representing 96 hours. [Diagram 3] 1 shows IAP activity over 16 days in an IAP-vehicle assay, with various time points represented in each set of histograms, with each bar representing 0 hours, 24 hours, 48 ​​hours, day 6, and day 16 from the leftmost bar to the rightmost bar. [Figure 4] An SEM photograph of one coated pellet (30% EUDRAGIT L30 D-55 coated onto the pellet) is shown. [Diagram 5] An SEM photograph of the cross section of one coated pellet (30% EUDRAGIT L30 D-55 coated onto the pellet) is shown. [Figure 6]Figure 1 shows the percent release from 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 only by UV analysis. [Figure 7] The percent activity of FaSSGF / FaSSIF or FaSSIF alone coated pellets by enzyme assay is shown. [Figure 8] A calibration curve was generated to calculate sample concentrations at various time points based on the equation of the linear trend line. "SYN-020" is synonymous with IAP (i.e., SEQ ID NO: 39). [Figure 9] 1 shows stability results using a dissolution test of coated pellet capsules after one month. [Figure 10] The results of a dissolution test in which the coated pellets were stored for about one year are shown. [Figure 11A] 1 shows the profile of non-GMP SYN-020 5 mg and 15 mg capsule doses. [Figure 11B] 1 shows the profile of non-GMP SYN-020 5 mg and 15 mg capsule doses. [Figure 11C] 1 shows the profile of non-GMP SYN-020 5 mg and 15 mg capsule doses. [Figure 11D] 1 shows the profile of non-GMP SYN-020 5 mg and 15 mg capsule doses. [Figure 11E] 1 shows the profile of non-GMP SYN-020 5 mg and 15 mg capsule doses. [Figure 11F] 1 shows the profile of non-GMP SYN-020 5 mg and 15 mg capsule doses. [Figure 11G] 1 shows the profile of non-GMP SYN-020 5 mg and 15 mg capsule doses. [Figure 11H] 1 shows the profile of non-GMP SYN-020 5 mg and 15 mg capsule doses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In various aspects, the present invention provides modified release formulations comprising an alkaline phosphatase (AP)-based drug and / or an additional therapeutic agent. In various embodiments, the formulation releases a substantial amount of the AP-based drug to the GI tract. In one embodiment, the formulation comprises at least one core particle and a base coat on the core particle, the base coat comprising the AP-based drug. In various embodiments, the formulation comprises a modified release coating, such as a delayed release coating, disposed on the core particle. In some embodiments, the delayed release coating is substantially stable in gastric fluids. In one embodiment, the delayed release coating comprises a EUDRAGIT compound.

[0013] Alkaline phosphatase-based drugs The present invention relates, in part, to pharmaceutical compositions, formulations, and uses of one or more alkaline phosphatase-based drugs (AP-based drugs).Exemplary AP-based drugs that can be utilized 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; found primarily 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 that contains alkaline phosphatase activity.

[0014] 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 non-specific alkaline phosphatase (TNAP).

[0015] IAP In some embodiments, the AP-based agent is an IAP. IAPs are produced in the proximal small intestine and are bound to intestinal cells via glycosylphosphatidylinositol (GPI) anchors. Some IAPs are released into the intestinal lumen as soluble proteins that are removed from cells via phospholipases, along with vesicles that are shed from cells. The enzyme then passes through the small and large intestines, which allows some active enzymes to be detected in 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, including bIAP II and bIAP IV, which have a higher specific activity than bIAP I. In one embodiment, the IAP is a cIAP or any one of the 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.

[0016] IAP variants The definition of IAP also includes 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 present invention are 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 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% sequence identity to the IAP variants. Additionally, the IAP variants retain most or all of their biochemical activity as measured as described herein.

[0017] Mammalian alkaline phosphatases are GPI-anchored proteins. They have a signal peptide and are translated into the secretory pathway. Upon entry into the endoplasmic reticulum (ER), the proteins are glycosylated and folded. There are two disulfide bonds and a single free cysteine ​​that appears to be inaccessible at the surface. In the late ER, the carboxy terminus is removed and a GPI anchor is added. Thus, GPI anchoring is a process that occurs at the carboxy terminus of alkaline phosphatase. The inclusion of a stop codon at the anchor site allows for secretion of a biologically active protein (presumably a homodimer). Although there is no consensus sequence, the carboxy terminus contains three amino acids, termed omega, omega+1, and omega+2, followed by a short stretch of hydrophilic amino acids, followed by a stretch of hydrophobic amino acids. Without wishing to be bound by theory, it is believed that the hydrophobicity is crucial for embedding the carboxy terminus into the ER membrane, where an enzymatic reaction replaces the carboxy terminus with a GPI anchor.

[0018] In human placental alkaline phosphatase (hPLAP), the GPI anchor is attached to an aspartic acid of the sequence DAAH. Similarly, this DAAH sequence is conserved in hIAP, bIAP II, and bIAP IV, and may function as the GPI anchor site. Mutation studies with hPLAP indicate that preventing GPI anchoring results in intracellular retention. Furthermore, mutations around the anchor site or in the hydrophobic domain either 1) prevent anchor binding and result in intracellular retention, or 2) do not block anchor binding. Without wishing to be bound by theory, the hydrophobic domain is believed to function as a signal for GPI anchor attachment. Truncation or removal of the hydrophobic domain results in secretion. Finally, there is a single mutation in the hydrophobic domain that, in hPLAP, allows the protein to be secreted without modifying the hydrophobic domain.

[0019] In other embodiments, the AP-based agents of the 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 agents may lack a GPI anchor site, e.g., the DAAH site may be removed, resulting in secretion. Alternatively, in some embodiments, this can be achieved, where 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 of the DAAH consensus site (e.g., amino acid 503 for hIAP and bIAP IV or amino acid 506 for bIAP II). Figure 1 shows 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). In one embodiment, the AP-based agent is bIAP IV, which contains a stop codon after amino acid 508 to mimic the secreted PLAP construct as shown in FIG. 1 (SEQ ID NO:7).

[0020] Human IAPs In various embodiments, the AP-based agent is a hIAP, hi some embodiments, the AP-based agent is a hIAP comprising the amino acid sequence of SEQ ID NO:1 as shown in Figure 1 or a variant described herein, provided that the hIAP variant retains at least 80%, 85%, 90%, 95%, 98% or 100% of the phosphatase activity when compared to the wild-type enzyme using the assays outlined herein.

[0021] Amino acid modifications are included in the definition of hIAP, and amino acid substitutions find particular use in the present invention. For example, without wishing to be bound by theory, it is believed that the carboxy-terminal cysteine ​​of AP-based agents (e.g., at position 500 of SEQ ID NO:1) may disrupt protein folding. Thus, in some embodiments, AP-based agents include a cysteine ​​mutation (e.g., at position 500 of SEQ ID NO:1). In some embodiments, the cysteine ​​is replaced with any amino acid, although in some embodiments, glycine finds particular use. Additionally, the C-terminal cysteine ​​can also be deleted.

[0022] As will be appreciated by those skilled in the art, further amino acid modifications can be made to hIAP as discussed herein. For example, in some embodiments, a stop codon can be inserted after the aspartic acid of the DAAH consensus site (e.g., amino acid 503 of hIAP). Figure 1 shows hIAP (SEQ ID NO: 4) with a stop codon inserted.

[0023] Bovine IAP In some embodiments, the IAP is a bovine IAP (bIAP).

[0024] In various embodiments, the AP-based agent is bovine IAP II (bIAP II) or a variant described herein, provided that the bIAP variant retains at least 80%, 85%, 90%, 95%, 98% or 100% phosphatase activity using the assays outlined herein. In one embodiment, bIAP II comprises the signal peptide and carboxy terminus of bIAP I. In one embodiment, bIAP II comprises an aspartic acid at position 248 (similar to bIAP IV). In one embodiment, bIAP II comprises the amino acid sequence of SEQ ID NO:2. FIG. 1 shows BIAP II with 248D-SEQ ID NO:2. The signal peptide and sequence from 480 onwards are derived from bIAP I.

[0025] The amino acid variants described herein are also included in the definition of bIAP II. For example, in some embodiments, a stop codon can be inserted after the aspartic acid of the DAAH consensus site (e.g., amino acid 506 of bIAP II). Figure 1 shows bIAP II (SEQ ID NO: 5) with a stop codon inserted.

[0026] In various embodiments, bIAP II comprises the amino acid sequence of SEQ ID NO:39. BIAP II with a stop codon and no leader sequence (SYN-020) (SEQ ID NO:39): LIPAEEENPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQC NTTRGNEVTSVINRAKKAGKAVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQKNGCQDIAAQLVYNMDIDVILGGGRMYMFPEGTPDPEYPDDASVNGVRKDKQNLVQEWQAKHQG AQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLAEMTEAALQVLSRNPRGFYLFVEGGRIDHGHHDGKAYMALTEAIMFDNAIAKANELTSELDTLILVTADHSHVFSFG GYTLRGTSIFGLAPGKALDSKSYTSILYGNGPGYALGGGSRPDVNGSTSEEPSYRQQAAVPLASETHGGEDVAVFARGPQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPATATSIPD

[0027] In various embodiments, the AP-based agent is bIAP IV or a variant thereof described herein, provided that the bIAP IV variant retains at least 80%, 85%, 90%, 95%, 98% or 100% phosphatase activity using the assays outlined herein. In one embodiment, bIAP IV comprises the amino acid sequence of SEQ ID NO:3, as shown in FIG.

[0028] Amino acid variants described herein are also included in the definition of bIAP IV. For example, in some embodiments, a stop codon can be inserted after the aspartic acid of the DAAH consensus site (e.g., amino acid 503 of bIAP IV). Figure 1 shows bIAP IV (SEQ ID NO: 6) with a stop codon inserted. In one embodiment, the AP-based agent is bIAP IV and includes a stop codon after amino acid 508 to mimic the secreted PLAP construct, as shown in Figure 1 (SEQ ID NO: 7).

[0029] Bacteria AP In various embodiments, the present invention contemplates the use of bacterial alkaline phosphatase. In some embodiments, the AP-based agent of the present invention is derived from Bacillus subtilis. Bacillus subtilis is a Gram-positive bacterium found in soil and the human GI tract. Bacillus subtilis secretes high levels of properly folded proteins into the environment and into the human GI tract. Without wishing to be bound by theory, it is believed that proteins secreted by Bacillus subtilis in the GI tract may be resistant to degradation by common GI proteases. Bacillus subtilis expresses a multigene family of alkaline phosphatases at high levels. Of these isozymes, alkaline phosphatase IV is responsible for the majority of the total alkaline phosphatase expression and activity in B. subtilis. In some embodiments, the AP-based agents of the present invention are derived from Bacillus In some embodiments, the AP-based agent of the present invention is derived from Escherichia coli.

[0030] Thus, in an exemplary embodiment, the AP-based agent of the invention is derived from Bacillus subtilis alkaline phosphatase IV. In one embodiment, the bacterial alkaline phosphatase may have the nucleotide and amino acid sequences shown in Figure 1, 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 variants described herein (provided that the hIAP variant retains at least 80%, 85%, 90%, 95%, 98% or 100% phosphatase activity using the assays outlined herein).

[0031] In some embodiments, the AP-based agent comprises bacterial alkaline phosphatases with one or more mutations that alter their catalytic activity. In some embodiments, the bacterial alkaline phosphatases comprise one or more mutations such that their catalytic activity is similar to or higher than that of mammalian alkaline phosphatases. In some embodiments, the bacterial alkaline phosphatases comprise one or more mutations that alter their dephosphorylation profile. In one embodiment, the bacterial alkaline phosphatases of the invention exhibit a dephosphorylation profile similar to that of mammalian alkaline phosphatases. In some embodiments, the bacterial alkaline phosphatases comprise one or more mutations that alter their activity at relatively high pH. In one embodiment, the bacterial alkaline phosphatases of the invention exhibit activity similar to that of mammalian alkaline phosphatases at relatively high pH. In some embodiments, the bacterial alkaline phosphatases comprise one or more mutations that alter their metal requirements. In one embodiment, the bacterial alkaline phosphatases of the invention exhibit metal requirements (e.g., Mg) similar to that of mammalian alkaline phosphatases.

[0032] For example, in certain embodiments, the AP-based agent of the invention is derived from Bacillus subtilis JH642 alkaline phosphatase IV and has one or more mutations at positions 101, 328, A330, and 374. For example, the AP-based agent may include one or more of the following mutations: D101A, W328H, A330N, and G374C.

[0033] Fusion proteins In some embodiments, the AP-based agent comprises an alkaline phosphatase fused to a "fusion partner", which is a protein domain that is added to either the N-terminus or C-terminus of the IAP domain, and optionally includes a linker. In some embodiments, the alkaline phosphatase is fused to a protein domain that facilitates protein folding and / or protein purification and / or protein dimerization and / or protein stability. In various embodiments, the fusion protein of the AP-based agent has an extended serum half-life. In various embodiments, the AP-based agent of the invention is an Fc fusion protein.

[0034] In one embodiment, the alkaline phosphatase is fused to an immunoglobulin Fc domain and / or hinge region. In one embodiment, the AP-based drug of the invention comprises an alkaline phosphatase fused to the hinge region and / or Fc domain of an IgG.

[0035] In various embodiments, the AP-based agent is fused to an IgG Fc domain that contains one or more mutations. In some embodiments, the one or more mutations in the IgG Fc domain function to increase serum half-life and longevity. In some embodiments, the IgG Fc domain contains 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 of the amino acid substitutions in the IgG Fc domain is at amino acid residue 252, 254, 256, 309, 311, 433, or 434. In one embodiment, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan, or threonine. In one embodiment, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In one embodiment, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In one embodiment, the amino acid substitution at amino acid residue 309 is a substitution with proline. In one embodiment, the amino acid substitution at amino acid residue 311 is a substitution with serine. In one embodiment, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine, or glycine. In one embodiment, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In one embodiment, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In one embodiment, the amino acid substitution at amino acid residue 389 is a substitution with a proline, serine, or asparagine.In one embodiment, the amino acid substitution at amino acid residue 433 is 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.

[0036] In some embodiments, the IgG Fc domain comprises one or more mutations at amino acid residues 252, 254, 256, 433, 434 or 436. In one embodiment, the IgG Fc domain comprises a M252Y / S254T / T256E triple mutation or a YTE mutation. In another embodiment, the IgG Fc domain comprises a H433K / N434F / Y436H triple mutation or a KFH mutation. In a further embodiment, the IgG Fc domain comprises a combination of YTE and KFH mutations. Additional exemplary 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. Patent No. 7,083,784, the entire contents of which are incorporated herein by reference. In various embodiments, one or more mutations in the Fc domain of IgG increase affinity for the fetal Fc receptor (FcRn). In some embodiments, 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.

[0037] In various embodiments, the alkaline phosphatase is fused to one or more of PEG, XTENylation (e.g., as rPEG), polysialic acid (POLYXEN), albumin, elastin-like protein, elastin like protein (ELP), PAS, HAP, GLK, CTP, and transferrin. In various embodiments, the alkaline phosphatase is fused to one or more of the agents described in BioDrugs (2015) 29:215-239, the entire contents of which are incorporated herein by reference.

[0038] Linker In one embodiment, alkaline phosphatase is fused to a protein domain (e.g., an immunoglobulin Fc domain) via a linker to a GPI anchor site. For example, alkaline phosphatase can be fused to a protein domain via an aspartic acid in a GPI anchor sequence. The present invention contemplates the use of various linker sequences. In various embodiments, the linker can be derived from a naturally occurring multidomain protein or is an empirical linker, for example, 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 incorporated herein by reference). In some embodiments, the linker 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 incorporated herein by reference. In various embodiments, the linker can be functional. For example, but not limited to, the linker can function to improve the folding and / or stability, improve expression, improve pharmacokinetics, and / or improve bioactivity of the AP-based agent of the present invention. In another example, the linker can function to target the AP-based agent to a specific cell type or location.

[0039] In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids in length. For example, the linker 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 length. In some embodiments, the linker is flexible. In another embodiment, the linker is rigid.

[0040] In various embodiments, the linker is substantially composed 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). Additional exemplary linkers include, but are not limited to, linkers having the sequence LE, GGGGS (SEQ ID NO: 19), (GGGGS)n (n = 2 to 4) (SEQ ID NOs: 20 to 22), (Gly)8 (SEQ ID NO: 23), (Gly)6 (SEQ ID NO: 24), (EAAAK)n (n = 1 to 3) (SEQ ID NOs: 25 to 27), A(EAAAK)nA (n = 2 to 5) (SEQ ID NOs: 28 to 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 (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.

[0041] Exemplary Fc fusion constructs of the invention include those shown in FIG. 1 , which comprises BIAP II with an Fc fusion (SEQ ID NO: 8)--Fc domain underlined, and BIAP IV with an Fc fusion (SEQ ID NO: 9)--Fc domain underlined.

[0042] Proenzyme fusions The present invention further provides C-terminal fusions for proenzyme function. Without wishing to be bound by theory, it is believed that mammalian alkaline phosphatase can also be generated as an inactive proenzyme. This is because alkaline phosphatase can dephosphorylate ATP, which allows activity in the ER to drain the ER's main energy source. Without wishing to be bound by theory, it is believed that the inhibitory function is located at the carboxy terminus, which is released upon addition of the GPI anchor. Alternatively, other activities such as folding or inclusion of metals (Zn or Mg) may control activity.

[0043] In various embodiments, the AP-based drug of the present invention is a proenzyme. In one embodiment, the activity of the proenzyme is inhibited by the carboxy terminus. In one embodiment, removal of the carboxy terminus protease reactivates the alkaline phosphatase enzyme activity. In one embodiment, the proenzyme is secreted more efficiently than the enzyme without the carboxy terminus.

[0044] The Saccharomyces alkaline phosphatase, Pho8, is produced as an inactive proenzyme. It is not GPI-anchored, but is a transmembrane protein whose amino terminus extends from the lysosome into the cytoplasm. Within the lysosome, the enzyme PEP4 cleaves the carboxy terminus, activating the enzyme.

[0045] In some embodiments, the native carboxy terminus of alkaline phosphatase is replaced with a similar sequence from hPLAP to generate the proenzyme. In some embodiments, a mutation is made in the hydrophobic carboxy tail to facilitate protein secretion without truncating the carboxy terminus. In an exemplary embodiment, a single point mutation, such as, for example, a substitution of leucine with arginine, occurs in the hydrophobic carboxy terminus (e.g., ALLPLLAGTL is changed to, for example, ALLPLRAGTL), resulting in the secretion of the enzyme without removing the carboxy terminus.

[0046] In one embodiment, the AP-based drug is modified to include a specific enzyme cleavage site that can subsequently remove the carboxy terminus. In one embodiment, the AP-based drug includes a protease cleavage site. Exemplary 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, submandibular gland protease, clostripain, thrombin, collagenase, and other endoproteases. In an alternative embodiment, the AP-based drug includes a cleavage site recognized by digestive enzymes present in the GI tract. In such an embodiment, the AP-based drug can be administered as a prodrug that is subsequently activated in the GI tract.

[0047] In an exemplary embodiment, the proenzyme is a proenzyme of bIAP IV having the sequence shown in Figure 1, including BIAP IV (SEQ ID NO:10) with the hPLAP carboxy terminus and mutations for unprocessed secretion and RV3C cleavage (at ...LEVLFQGP...), and BIAP IV (SEQ ID NO:11) with the hPLAP carboxy terminus and mutations for unprocessed secretion and FXa cleavage (at ...IEGR...).

[0048] Expression variants 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.

[0049] In various embodiments, the AP-based agent is efficiently expressed in the 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 adjacent to the ATG start codon that directs the ribosome to begin translation. There is flexibility in the design of the Kozak sequence, but one canonical sequence is GCCGCCACCATGG (SEQ ID NO: 38). The purine at position -3 and the G at position +4 are the most important bases for translation initiation. For hIAP, bIAP II, and bIAP IV, the second amino acid, the amino acid following the initiating methionine, is glutamine. All codons for glutamine have a C at the first position. This causes all of their Kozak sequences to have an ATGC sequence. Thus, 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 may be compatible with signal peptide function. In an alternative embodiment, an entire signal peptide is used in place of a peptide having a canonical Kozak sequence, where the entire signal peptide is derived from a highly expressed protein, such as an immunoglobulin.

[0050] In various embodiments, the signal peptide of the AP-based agent can be deleted and / or replaced, for example, the signal peptide can be deleted, mutated, and / or replaced (e.g., with another signal peptide) to ensure expression of the protein.

[0051] In some embodiments, the DNA constructs encoding the AP-based agents of the present invention include non-translated DNA sequences. Such sequences include introns (including the native first and / or second introns and / or the native 3'UTR) that may be heterologous to the IAP protein or may be native to the IAP protein. Without wishing to be bound by theory, it is believed that the inclusion of these sequences enhances protein expression by stabilizing the mRNA. Thus, in various embodiments, the DNA constructs encoding the AP-based agents of the present invention include the 5'UTR and / or the 3'UTR. Shown in FIG. 1 are exemplary IAP DNA sequences with first introns and 3'UTRs, including hIAP-SEQ ID NO:12 with the native first intron (shown in bold and underlined), hIAP-SEQ ID NO:13 with the native 3'UTR (shown in bold and underlined), bIAP IV-SEQ ID NO:14 with the first intron from bIAP I (shown in bold and underlined), and bIAP IV-SEQ ID NO:15 with the 3'UTR from bIAP I (shown in bold and underlined).

[0052] In various embodiments, the AP-based agents of the present invention are 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% sequence identity to the nucleic acid sequence.

[0053] In various embodiments, the AP-based agents of the present invention may comprise an amino acid sequence having one or more amino acid mutations compared to any of the protein sequences described herein. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0054] In various embodiments, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolidine, N-formylmethionine β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of 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, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, and common amino acid analogs).

[0055] The AP-based drugs of the present invention can be mutated to select drugs with desired properties. For example, mutations can be made to generate AP-based drugs with enhanced catalytic activity or protein stability. In various embodiments, directed evolution methods can be used to generate AP-based drugs of the present invention. For example, error-prone PCR and DNA shuffling can be used to identify mutations in bacterial alkaline phosphatase that result in enhanced activity.

[0056] Modified Release Formulations and Dosage Forms The modified release formulation of an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) may further comprise a pharma- ceutically acceptable carrier or excipient. As will be appreciated by those skilled in the art, the formulation may be in any suitable form appropriate for the desired use and route of administration.

[0057] In various embodiments, the modified release formulation of the present invention comprises a core particle, a base coat on the core particle, and the base coat comprises an AP-based drug. In further embodiments, the core particle comprises sucrose. In some embodiments, the AP-based drug of the base coat is encapsulated in the core particle and may comprise multiple core particles.

[0058] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing a plurality of pellets containing an enteric coated AP-based drug. In such an embodiment, the plurality of pellets (or each individual pellet) contains an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof), sucrose spheres onto which the AP-based drug (e.g., an IAP or variant) is sprayed, a binding excipient (e.g., hydroxypropylcellulose (HPC)), an enteric polymer (e.g., EUDRAGIT L30 D-55), HTP-20 (e.g., PLASACRYL HTP 20), 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).

[0059] In various embodiments, the formulations of the present invention comprise a modified release coating that is stable in gastric and / or intestinal fluids and is degraded by microbial enzymes present in the intestinal flora. In further embodiments, the solubility of the modified release coating is pH-dependent. In further embodiments, the modified release coating has a time-dependent erosion profile.

[0060] In various embodiments, the formulation of the present invention comprises at least one modified release pellet, each modified release pellet comprising about 1-10% by weight of an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). For example, the AP-based drug (e.g., an IAP, or other AP-based drug 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 pellets (or each individual pellet) comprise about 75-95% by weight of sucrose spheres. For example, the sucrose spheres may 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 some embodiments, the pellets (or each individual pellet) contain about 5-15% hydroxypropyl cellulose (HPC). For example, the hydroxypropyl cellulose may 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 plurality of pellets (or each individual pellet) comprises about 0.5-2% by weight of a buffer salt. The buffer salt may be selected from Tris base, magnesium chloride, and zinc sulfate. For example, the buffer salt may 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%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0% by weight. In some embodiments, the formulation comprises a single layer enteric coating comprising about 20-40%, about 25-40%, about 25-35%, about 30-40%, or about 35-40% weight gain of enteric polymer.In some embodiments, the formulation comprises a two-layer enteric coating comprising about 20-40%, about 25-40%, about 25-35%, about 30-40%, or about 35-40% weight gain of 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% weight gain of hydroxypropylcellulose. Weights stated herein refer to the total weight of all components.

[0061] In various embodiments, the formulations of the invention comprise at least one modified release pellet, each of which comprises about 5% by weight of an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof), about 85% by weight of sucrose spheres, about 9% by weight of hydroxypropylcellulose, and about 1% by weight of a buffer salt. In some embodiments, the formulation comprises a single layer enteric coating comprising about 20-40% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55), or a two-layer enteric coating comprising about 30% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55) and about 7% by weight of hydroxypropylcellulose. Weights described herein refer to the total weight of all components.

[0062] In various embodiments, the formulations of the present invention comprise at least one modified release pellet, each of which comprises about 4.7% by weight of an AP-based drug (e.g., an IAP, or other AP-based drug 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 salt. In some embodiments, the formulation comprises a single layer enteric coating comprising about 20-40% by weight of enteric polymer (e.g., EUDRAGIT L 30 D-55), or a two-layer enteric coating comprising about 30% by weight of enteric polymer (e.g., EUDRAGIT L 30 D-55) and about 7% by weight of hydroxypropylcellulose. Weights described herein refer to the total weight of all components.

[0063] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 25 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In various embodiments, the formulation of the present invention includes at least one modified release pellet, each modified release pellet containing about 1-15% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). For example, the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) may be present at about 1% by weight, about 2% by weight, about 3% by weight, about 4% by weight, about 5% by weight, about 6% by weight, about 7% by weight, about 8% by weight, about 9% by weight, about 10% by weight, about 12% by weight, about 13% by weight, about 14% by weight, or about 15% by weight. In some embodiments, the plurality of pellets (or each individual pellet) comprises 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 some embodiments, the plurality of pellets (or each individual pellet) comprises about 5-25% 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%, 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 plurality of pellets (or each individual pellet) comprises about 20-35% by weight 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) contain about 0.5-11% HTP-20 by weight. For example, HTP-20 may 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 plurality of pellets (or each individual pellet) comprises about 0.5-2.5% by weight of a buffer salt. The buffer salt may be selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate. For example, the 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%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% by weight.

[0064] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 25 mg of an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In such an embodiment, the formulation contains about 10% by weight of an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof), about 38% by weight of sucrose spheres, about 19% by weight of hydroxypropylcellulose (HPC), about 2% by weight of a buffer salt, and about 26% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55).

[0065] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 25 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In such an embodiment, the formulation contains about 9.7% by weight of AP-based drug (e.g., an IAP, or other AP-based drug 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 salt, and about 26.3% by weight of enteric polymer (e.g., EUDRAGIT L 30 D-55).

[0066] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 5 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains multiple pellets containing the enteric coated AP-based drug. In various embodiments, the formulation of the present invention includes at least one modified release pellet, each modified release pellet containing about 1-15% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). For example, the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) may be present at about 1% by weight, about 2% by weight, about 3% by weight, about 4% by weight, about 5% by weight, about 6% by weight, about 7% by weight, about 8% by weight, about 9% by weight, about 10% by weight, about 12% by weight, about 13% by weight, about 14% by weight, or about 15% by weight. In some embodiments, the plurality of pellets (or each individual pellet) comprises 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 some embodiments, the plurality of pellets (or each individual pellet) comprises about 5-25% 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%, 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 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) contain about 0.5-11% HTP-20 by weight. For example, HTP-20 may 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 plurality of pellets (or each individual pellet) comprises about 0.5-2.5% by weight of a buffer salt. The buffer salt may be selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate. For example, the 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%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% by weight.

[0067] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 5 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In such an embodiment, the formulation contains about 9.7% by weight of AP-based drug (e.g., an IAP, or other AP-based drug 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 salt, and about 26.3% by weight of enteric polymer (e.g., EUDRAGIT L 30 D-55).

[0068] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 5 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In such an embodiment, the formulation contains about 9.7% by weight of AP-based drug (e.g., an IAP, or other AP-based drug 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 salt, and about 26.3% by weight of enteric polymer (e.g., EUDRAGIT L 30 D-55).

[0069] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 15 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In various embodiments, the formulation of the present invention includes at least one modified release pellet, each modified release pellet containing about 1-15% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). For example, the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) may be present at about 1% by weight, about 2% by weight, about 3% by weight, about 4% by weight, about 5% by weight, about 6% by weight, about 7% by weight, about 8% by weight, about 9% by weight, about 10% by weight, about 12% by weight, about 13% by weight, about 14% by weight, or about 15% by weight. In some embodiments, the plurality of pellets (or each individual pellet) comprises 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 some embodiments, the plurality of pellets (or each individual pellet) comprises about 5-25% 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%, 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 plurality of pellets (or each individual pellet) comprises about 20-35% by weight 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) contain about 0.5-11% HTP-20 by weight. For example, HTP-20 may 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 plurality of pellets (or each individual pellet) comprises about 0.1-2.5% by weight of a buffer salt. The buffer salt may be selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate. For example, the buffer salts 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.

[0070] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 15 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains multiple pellets containing the enteric-coated AP-based drug. In such an embodiment, the formulation contains about 10% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug 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 salt, and about 26% by weight of an enteric polymer (e.g., EUDRAGIT L30). D-55).

[0071] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 15 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In such an embodiment, the formulation contains about 10.0% by weight of AP-based drug (e.g., an IAP, or other AP-based drug 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 salt, and about 26.3% by weight of enteric polymer (e.g., EUDRAGIT L 30 D-55).

[0072] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 5 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains multiple pellets containing the enteric coated AP-based drug. In various embodiments, the formulation of the present invention includes at least one modified release pellet, each modified release pellet containing about 1-15% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). For example, the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) may be present at about 1% by weight, about 2% by weight, about 3% by weight, about 4% by weight, about 5% by weight, about 6% by weight, about 7% by weight, about 8% by weight, about 9% by weight, about 10% by weight, about 12% by weight, about 13% by weight, about 14% by weight, or about 15% by weight. In some embodiments, the plurality of pellets (or each individual pellet) comprises 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 some embodiments, the plurality of pellets (or each individual pellet) comprises about 5-25% 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%, 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 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) contain about 0.5-11% HTP-20 by weight. For example, HTP-20 may 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 plurality of pellets (or each individual pellet) comprises about 0.1-2.5% by weight of a buffer salt. The buffer salt may be selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate. For example, the buffer salts 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.

[0073] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 5 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In such an embodiment, the formulation contains about 10% by weight of AP-based drug (e.g., an IAP, or other AP-based drug 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 salt, and about 26% by weight of enteric polymer (e.g., EUDRAGIT L30 D-55).

[0074] In various embodiments, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin capsule or an HPMC capsule) containing about 5 mg of AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof). The capsule contains a plurality of pellets containing an enteric-coated AP-based drug. In such an embodiment, the formulation contains about 10.0% by weight of AP-based drug (e.g., an IAP, or other AP-based drug 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 salt, and about 26.3% by weight of enteric polymer (e.g., EUDRAGIT L 30 D-55).

[0075] In some embodiments, the administration of the modified release formulation containing the AP-based drug (and / or additional therapeutic agent) is any one of oral, intravenous, and parenteral. In some embodiments, the administration of the modified release formulation containing the AP-based drug (and / or additional therapeutic agent) is not intravenous, for example to prevent interference with systemically administered antibiotics. In other embodiments, the administration route includes, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical (especially to the ear, nose, eye, or skin).

[0076] In some embodiments, any formulation of the present invention comprises a core particle having a size of about 0.8 mm to about 2.0 mm, about 0.9 mm to about 1.9 mm, about 1 mm to about 1.8 mm, about 1.1 mm to about 1.7 mm, about 1.2 mm to about 1.6 mm, about 1.3 mm to about 1.5 mm, about 1 mm to about 1.3 mm, about 1 mm to about 1.4 mm, about 1 mm to about 1.5 mm, about 1 mm to about 1.6 mm, about 1 mm to about 1.7 mm, about 1 mm to about 1.9 mm, or about 1 mm to 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.

[0077] Any modified release formulation containing an AP-based agent (and / or additional therapeutic agent) described herein may be free of cofactors (e.g., CaCl2 or CoCl2).

[0078] Any modified release formulation containing an AP-based drug (and / or additional therapeutic agent) described herein may be administered orally. Such inventive formulations may also be administered by any other convenient route, such as intravenous infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), or may be administered together with additional therapeutic agents. Administration may be systemic or local.

[0079] Dosage forms suitable 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 a capsule. In another embodiment, the modified release formulation is in the form of a tablet. In yet another embodiment, the modified release formulation is in the form of a soft gel capsule. In a further embodiment, the modified release formulation is in the form of a gelatin capsule or a hydroxypropylmethylcellulose (HPMC) capsule.

[0080] In some dosage forms, the agents described herein may be administered in the form of a pharmaceutical composition comprising at least one inert, pharma- ceutically acceptable excipient or carrier, such as, for example, sodium citrate, dicalcium phosphate, and / or a) fillers or extenders, such as, for example, starch, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and Bakers Special Sugar; b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, gum arabic, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylcellulose (HPC), and hydroxymethylcellulose; c) humectants, such as, for example, glycerol; d) disintegrants, such as, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, cross-linked polymers, such as, for example, crospovidone (cross-linked polyvinylpyrrolidone), croscarmellose sodium, or the like. (crosslinked sodium carboxymethylcellulose), sodium starch glycolate, etc., e) dissolution retarders, such as paraffin, f) absorption promoters, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, glyceryl behenate, etc., j) antioxidants, and mixtures of such excipients. Those skilled in the art will recognize that a particular excipient can have more than one function in oral dosage forms. In the case of oral dosage forms, such as capsules or tablets, the dosage form can also include a buffering agent.

[0081] The controlled release formulation may further comprise a surfactant.Surfactants suitable for use in the present invention include, but are not limited to, any pharma- ceutically acceptable non-toxic surfactant.Surfactant classes suitable for use in the present composition include, but are not limited to, polyethoxylated fatty acid, PEG-fatty acid diester, mixture of PEG-fatty acid monoester and PEG-fatty acid diester, polyethylene glycol glycerol fatty acid ester, alcohol-oil transesterification products, polyglyceryl fatty acid, propylene glycol fatty acid ester, mixture of propylene glycol ester-glycerol ester, monoglycerides and diglycerides, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid ester, polyethylene glycol alkyl ether, sugar ester, polyethylene glycol alkyl phenol, polyoxyethylene-polyoxypropylene block copolymer, sorbitan fatty acid ester, lower alcohol fatty acid ester, ionic surfactant, and mixtures thereof. In some embodiments, the compositions of the present invention may include one or more surfactants, including but not limited to sodium lauryl sulfate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and triethyl citrate.

[0082] The modified release formulations may also contain certain pharma- ceutically acceptable plasticizers to obtain desired mechanical properties such as flexibility and hardness, including, but not limited to, triacetin, citrate esters, triethyl citrate, phthalate esters, dibutyl sebacate, cetyl alcohol, polyethylene glycol, polysorbates, or other plasticizers.

[0083] The modified release formulation may also include one or more application solvents. For example, some of the more common solvents that can be used to apply the delayed release coating composition include isopropyl alcohol, acetone, methylene chloride, etc.

[0084] The controlled release formulation may also include one or more alkaline substances.The alkaline substances suitable for use in the compositions of the present invention include, but are not limited to, the sodium, potassium, calcium, magnesium and aluminum salts of acids such as phosphoric acid, carbonic acid, citric acid, and other aluminum / magnesium compounds.In addition, the alkaline substance may be selected from antacid substances such as aluminum hydroxide, calcium hydroxide, magnesium hydroxide, and magnesium oxide.

[0085] A solid oral dosage form can be prepared, for example, by granulation (e.g., wet or dry granulation) of the agent of the present invention with one or more suitable excipients. Alternatively, the agent of the present invention can be layered onto an inert core (e.g., nonpareil / sugar spheres, such as sucrose or silica spheres) using conventional methods such as fluidized bed or pan coating, or extruded and spheronized into active compound-containing pellets using methods known in the art. In an embodiment, an AP-based agent (e.g., an IAP, or other AP-based agent described herein, and variants thereof) is spray-coated onto a sucrose sphere. Such pellets can then be incorporated into tablets or capsules using conventional methods.

[0086] Suspensions may contain, in addition to the active substance, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and the like, as well as mixtures thereof.

[0087] Besides inert diluents, the oral compositions can also include adjuvants such as sweetening, flavoring, and perfuming agents.

[0088] 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 prepared in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable solvent immediately before use. They can contain, for example, suspending or dispersing agents known in the art.

[0089] The formulations containing the AP-based drug (and / or additional therapeutic agent) may be conveniently provided in unit dosage form and may be prepared by any method known in the art of pharmacy. Such methods generally include the step of combining the therapeutic agent with a carrier that constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately combining the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation (e.g., by wet or dry granulation, powder blending, etc., followed by tableting using conventional methods known in the art).

[0090] In various embodiments, the modified release formulations of the present invention can utilize one or more modified release coatings, such as delayed release coatings, to effectively, delayed yet substantially deliver an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) to the GI tract, optionally together with other additional therapeutic agents.

[0091] In one embodiment, the delayed release coating comprises an enteric agent that is substantially stable in an acidic environment and substantially unstable in a near-neutral to alkaline environment. In one embodiment, the delayed release coating contains an enteric agent that is substantially stable in gastric and / or intestinal fluids. The enteric agent may be selected from, for example, a solution or dispersion of methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, carboxymethylethylcellulose, and EUDRAGIT type polymers (poly(methacrylic acid, methyl methacrylate)), hydroxypropylmethylcellulose acetate succinate, cellulose acetate trimellitate, shellac or other suitable enteric coating polymers. EUDRAGIT type polymers include, for example, EUDRAGIT FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P, RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5, and S 12,5 P. Similar polymers include KOLLICOAT MAE 30 DP and KOLLICOAT MAE 100 P. In some embodiments, one or more of EUDRAGIT FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P, RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5, S 12,5 P, KOLLICOAT MAE 30 DP, and KOLLICOAT MAE 100 P are used. In various embodiments, the enteric agent can be a combination of the aforementioned solutions or dispersions. In one embodiment, the delayed release coating comprises the enteric agent EUDRAGIT L 30 D-55 (poly(methacrylic acid-ethyl acrylate copolymer) 1:1).

[0092] In certain embodiments, one or more coating system additives are used with the enteric drug. For example, one or more PLASACRYL additives can be used as anti-adherent coating additives. Exemplary PLASACRYL additives include, but are not limited to, PLASACRYL HTP20 and PLASACRYL T20. In one embodiment, PLASACRYL HTP20 is formulated with EUDRAGIT L 30 D-55 coating. In another embodiment, PlasACRYLT20 is formulated with EUDRAGIT FS 30 D coating.

[0093] In another embodiment, the delayed release coating may degrade as a function of time when in aqueous solution, independent of pH and / or the presence of enzymes in the solution. Such coatings may include water-insoluble polymers. Thus, their solubility in aqueous solution is independent of pH. The term "pH-independent" as used herein means that the water permeability of the polymer and its ability to release the pharmaceutical ingredient are independent of pH and / or only slightly dependent on pH. Such coatings may be used, for example, to prepare sustained release formulations. Suitable water-insoluble polymers include pharma-ceutically acceptable non-toxic polymers that are substantially insoluble in aqueous media, e.g., water, independent 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 of the hydroxyl groups on the cellulose backbone are replaced with alkyl groups and some are modified with alkanoyl groups. Examples include ethyl cellulose, acetyl cellulose, nitrocellulose, and the like. Other examples of insoluble polymers include, but are not limited to, lacquers, acrylic and / or methacrylic ester polymers, acrylic or methacrylic acid polymers or copolymers with low quaternary ammonium content, or mixtures thereof. 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, polyacrylic esters, butadiene styrene copolymers, and the like. In one embodiment, colonic delivery is achieved by using a slowly eroding wax plug (e.g., various PEGs, including PEG 6000).

[0094] In further embodiments, the delayed release coating may be degraded by microbial enzymes present in the intestinal flora. In one embodiment, the delayed release coating may be degraded by microorganisms present in the small intestine. In another embodiment, the delayed release coating may be degraded by microorganisms present in the large intestine.

[0095] In various embodiments, the present invention provides a formulation comprising a core particle having a base coat comprising one or more AP-based agents (e.g., an 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 fluids, and / or may be substantially unstable in near-neutral to alkaline environments and / or intestinal fluids, thereby exposing the coated core particle to intestinal fluids. For example, in some embodiments, the coated pellets of the present invention do not substantially release the AP-based agent during FaSSGF, but substantially release the AP-based agent during 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 drug into 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.

[0096] In some embodiments, the coated pellets of the invention containing an AP-based drug are stable during 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 further embodiments, the coated pellets of the invention containing an AP-based drug 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% activity over the storage period.

[0097] The base coat containing one or more AP-based agents may further contain one or more additional therapeutic agents. Optionally, multiple base coats may be applied to the core, each of which may contain 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., an IAP, or other AP-based agents described herein and variants thereof) may be sprayed onto an inert core (e.g., a sucrose core or sucrose sphere) and spray-dried with an enteric layer (e.g., EUDRAGIT L30 D-55) to form a pellet containing an AP-based agent (e.g., an IAP, or other AP-based agents described herein and variants thereof).

[0098] 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 AP-based agents may be encapsulated in the core particle, for example, in the form of a microsphere. For example, the AP-based agent may be combined with a polymer (e.g., latex) and then formed into a particulate microencapsulated enzyme preparation without the use of a sucrose core. The microspheres thus formed may optionally be coated with a delayed release coating.

[0099] Various approaches are known for producing particles (such as microspheres, aggregates, and others) that are applicable to the inclusion of enzymes. They usually contain at least two phases, one containing the enzyme and the other containing the polymer that forms the backbone of the particle. The most common are coacervation, where the polymer is separated from the solvent phase by adding a third component, or multiphase emulsions such as water-in-oil-in-water (w / o / w) emulsions, where the inner aqueous phase contains the protein and the middle organic phase contains the polymer and an outer aqueous phase stabilizer that supports the w / o / w double emulsion until the solvent is removed and the microspheres can be formed. Alternatively, the AP-based drug (e.g., IAP, or other AP-based drugs described herein and variants thereof) and stabilizing excipients (e.g., trehalose, mannitol, Tween 80, polyvinyl alcohol) are combined, sprayed from an aqueous solution, and collected. The particles are then suspended in a dry water-immiscible organic solvent containing the polymer and the release-modifying compound, and the suspension is sonicated to disperse the particles. A further approach uses an aqueous phase but no organic solvent. Specifically, the enzyme, buffer components, polymer latex, and stabilizing and release-modifying excipients are dissolved / dispersed in water. The aqueous dispersion is spray-dried, resulting in latex coalescence and incorporation of the protein and excipients into the coalesced latex particles. If the release-modifying agent is insoluble in acidic conditions but soluble at higher pH (such as carboxylic acids), release from the matrix is ​​inhibited in the gastric environment.

[0100] In some embodiments, prior to applying the delayed release coating to the coated core particles, the particles can be optionally coated with one or more separating layers comprising pharmaceutical excipients, e.g., alkaline compounds, such as pH buffering compounds, that substantially separate the coated core particles from the delayed release coating.

[0101] The separating layer can be applied to the coated core particles by commonly used coating or layering procedures using coating equipment such as coating pans, coating granulators, or in fluidized bed equipment using water and / or organic solvents for the coating process. Alternatively, the separating layer can be applied to the core material by using powder coating techniques. Materials for the separating layer are pharma- ceutically acceptable compounds such as sugars, polyethylene glycols, polyvinylpyrrolidones, polyvinyl alcohols, polyvinyl acetates, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and others, used alone or in mixtures. Additives such as plasticizers, colorants, pigments, fillers, anti-adhesives, anti-static agents, for example, magnesium stearate, titanium dioxide, talc, and other additives, can also be included in the separating layer.

[0102] In some embodiments, the particles coated with delayed release coating can be further covered by an overcoat layer. The overcoat layer can be applied as described for other coating compositions. The overcoat material is a pharma- ceutically acceptable compound, such as sugar, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and others, used alone or in mixture. The overcoat material can prevent possible agglomeration of the particles coated with delayed release coating, protect the delayed release coating from cracking during the compression process, or enhance the tableting process.

[0103] In some embodiments, the modified release formulation is a capsule filled with multiple pellets containing the AP-based drug (e.g., pellets containing an IAP (or other AP-based drug as described herein, and variants thereof)) from which the AP-based drug is released. In one embodiment, the capsule is a gelatin capsule, such as a hard gelatin capsule. In another embodiment, the capsule is a hydroxypropyl methylcellulose (HPMC) capsule. For example, the formulation can be in the form of a capsule containing multiple pellets. For example, the formulation can be in the form of a capsule, such as a gelatin capsule or a hydroxypropyl methylcellulose (HPMC) capsule, containing multiple enteric coated pellets containing the AP-based drug (e.g., an IAP, or other AP-based drug as described herein, and variants thereof). In such embodiments, a combination of pellets can be utilized, with each pellet designed to release at a specific time or location. In various embodiments, the pellets (e.g., enteric coated pellets) are designed to pass through the stomach unchanged and then release the AP-based drug (e.g., an IAP, or other AP-based drug as described herein, and variants thereof) into one or more regions of the intestine. In some embodiments, the pellets containing an AP-based drug can be enterically coated to release the AP-based drug (e.g., an IAP, or other AP-based drug described herein and variants thereof) at various intestinal pH values.

[0104] The present invention also provides modified release formulations that release multiple doses of AP-based drugs (e.g., IAP, or other AP-based drugs described herein, and variants thereof) and / or additional therapeutic agents along the gastrointestinal tract. In such embodiments, the overall release profile of such formulations can be tailored, for example, by utilizing multiple particle types or multiple layers. In one embodiment, a first dose of AP-based drug can be formulated for release, for example, in the small intestine (e.g., one or more of the duodenum, jejunum, ileum) or large intestine (e.g., one or more of the cecum, ascending, transverse, descending, or sigmoid portion of the colon, and rectum), while a second dose is formulated for delayed release, for example, in a different region of the small intestine (e.g., one or more of the duodenum, jejunum, ileum) or large intestine (e.g., one or more of the cecum, ascending, transverse, descending, or sigmoid portion of the colon, and rectum). Alternatively, multiple doses are released at different locations along the intestine. For example, in one embodiment, the first dose of the AP-based drug may be formulated for release in the small intestine (e.g., one or more of the duodenum, jejunum, ileum), while the second dose is formulated for delayed release in another part of the small intestine (e.g., one or more of the duodenum, jejunum, ileum). In another embodiment, the first dose of the AP-based drug may be formulated for release in the large intestine (e.g., one or more of the cecum, the ascending, transverse, descending, or sigmoid part of the colon, and the rectum), while the second dose is formulated for delayed release in another part of the large intestine (e.g., one or more of the cecum, the ascending, transverse, descending, or sigmoid part of the colon, and the rectum).

[0105] In various embodiments, the agents described herein may be in the form of pharma- ceutically acceptable salts, i.e., salts suitable for use in contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic responses, 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 can be prepared separately by reacting a free base function with a suitable acid, or by reacting a free acid function with a suitable alkali moiety. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, Examples of suitable salts include 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, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.

[0106] In various embodiments, the formulations provide many advantages. For example, the inventors have successfully formulated proteins (i.e., AP-based drugs), which is a challenge in itself. This is further complicated by the GI tract environment in which the formulations release the drug in various embodiments. Furthermore, in various embodiments, the formulations provide a release in the GI tract that is slow enough to provide good coverage of protection in the GI tract, for example, from the adverse effects of various antibiotics in the small intestine, a benefit that is accentuated by the increased half-life of the AP-based drug commensurate with the slower release. Furthermore, by coating the drug substance layer of the pellets of the invention with HPC rather than EUDRAGIT, for example, the formulations minimize the amount of EUGRAGIT in the formulation, thereby reducing potential dose-limiting toxicity and manufacturing complications.

[0107] Modified Release Profile In one aspect, the present invention provides a modified release formulation comprising at least one alkaline phosphatase (AP)-based drug, the formulation releases a substantial amount of the AP-based drug to one or more regions of the GI tract. In some embodiments, the AP-based drug is an IAP, or other AP-based drug described herein, and variants thereof (e.g., as described above). For example, the formulation can release at least about 60% of the AP-based drug, such as an IAP, past the stomach to one or more regions of the GI tract.

[0108] In various embodiments, the modified release formulation of the present invention is designed for immediate release (e.g., immediately after ingestion). In various embodiments, the modified release formulation may have a sustained release profile, i.e., delayed release of the active ingredient(s) in the body (e.g., the GI tract) over an extended period of time. In various embodiments, the modified release formulation may have a delayed release profile (i.e., does not immediately release the active ingredient(s) immediately after ingestion), but rather postpones release of the active ingredient(s) until the composition has traveled down the gastrointestinal tract, for example, for release in the small intestine (e.g., one or more of the duodenum, jejunum, ileum) or large intestine (e.g., one or more of the cecum, ascending, transverse, descending, or sigmoid portion of the colon, and rectum). For example, the composition may be enterically coated to delay release of the active ingredient(s) until the composition reaches the small intestine or large intestine. In some embodiments, the active ingredient(s) of the formulation are not present in substantial amounts in feces.

[0109] In various embodiments, the modified release formulations of the present invention release at least 60% of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) beyond the stomach into one or more regions of the 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., an IAP, or other AP-based agents described herein and variants thereof) to the intestine.

[0110] In various embodiments, the modified release formulations of the present invention release at least 60% of the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) into 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., an IAP, or other AP-based agents described herein and variants thereof) to the small intestine.

[0111] In one embodiment, the modified release formulation of the present invention releases at least 60% of the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) into 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., an IAP, or other AP-based agents described herein and variants thereof) to the duodenum.

[0112] In one embodiment, the modified release formulation of the present invention releases at least 60% of the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) to 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., an IAP, or other AP-based agents described herein and variants thereof) to the jejunum.

[0113] In one embodiment, the modified release formulation of the present invention releases at least 60% of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) to the ileum and / or ileocecal junction. For example, the modified release formulation may release 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 122%, at least 124%, at least 126%, at least 128%, at least 129%, at least 130%, at least 131 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., an IAP, or other AP-based agent described herein, and variants thereof) is released to the ileum and / or ileocecal junction.

[0114] In various embodiments, the modified release formulations of the present invention release at least 60% of the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) to 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., an IAP, or other AP-based agents described herein and variants thereof) to the large intestine.

[0115] In one embodiment, the modified release formulation of the present invention releases at least 60% of the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) to 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., an IAP, or other AP-based agents described herein and variants thereof) to the cecum.

[0116] In one embodiment, the modified release formulation of the present invention releases at least 60% of the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) to 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., an IAP, or other AP-based agent described herein, and variants thereof) to the ascending colon.

[0117] In one embodiment, the modified release formulation of the present invention releases at least 60% of the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) to 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., an IAP, or other AP-based agents described herein and variants thereof) to the transverse colon.

[0118] In one embodiment, the modified release formulation of the present invention releases at least 60% of the AP-based agent (eg, an IAP, or other AP-based agent described herein and variants thereof) to 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., an IAP, or other AP-based agents described herein and variants thereof) to the descending colon.

[0119] In one embodiment, the modified release formulation of the present invention releases at least 60% of the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) to 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., an IAP, or other AP-based agents described herein and variants thereof) to the sigmoid colon.

[0120] In various embodiments, the modified release formulation does not substantially release the AP-based drug (eg, an IAP, or other AP-based drug described herein, and variants thereof) in the stomach.

[0121] In certain embodiments, the modified release formulation releases the AP-based drug (e.g., an IAP or other AP-based drug described herein and variants thereof) at a particular pH. For example, in some embodiments, the modified release formulation is substantially stable in an acidic environment and substantially unstable (e.g., rapidly dissolves or is physically unstable) in a near-neutral to alkaline environment. In some embodiments, stability indicates substantially no release, whereas instability indicates substantially release. 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 formulation is stable in a relatively low pH range and thus is not substantially 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 less and is substantially unstable at higher pH values. In these embodiments, the modified release formulation is not substantially 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 less and is therefore substantially unstable at higher pH values ​​and is therefore not substantially 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 values ​​described herein may be adjusted, as known in the art, to take into account the state of the subject, for example, whether in a fasted or postprandial state.

[0122] In some embodiments, the modified release formulation is substantially stable in gastric fluids and substantially unstable in intestinal fluids such that it is substantially released 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).

[0123] 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 drug (e.g., an IAP, or other AP-based drug described herein and variants thereof) and / or additional therapeutic agent in the modified release formulation in gastric fluid with a pH of about 4 to about 5 or less or in simulated gastric fluid with a pH of about 4 to about 5 or less in about 15 minutes, or about 30 minutes, or about 45 minutes, or about 60 minutes, or about 90 minutes. The modified release formulation of the present invention may release about 0% to about 30% by weight, about 0% to about 25% by weight, about 0% to about 20% by weight, about 0% to about 15% by weight, about 0% to about 10% by weight, about 5% to about 30% by weight, about 5% to about 25% by weight, about 5% to about 20% by weight, about 5% to about 15% by weight, or about 5% to about 10% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) and / or additional therapeutic agent in gastric fluid having a pH of 4-5 or less or in artificial gastric fluid having a pH of 4-5 or less in about 15 minutes, or about 30 minutes, or about 45 minutes, or about 60 minutes, or about 90 minutes. The modified release formulations of the present invention may 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 amount of AP-based drug (e.g., an IAP, or other AP-based drug described herein and variants thereof) and / or additional therapeutic agent in the modified release formulation in gastric fluid with a pH of 5 or less or in simulated gastric fluid with a pH of 5 or less in about 15 minutes, or about 30 minutes, or about 45 minutes, or about 60 minutes, or about 90 minutes.

[0124] In some embodiments, the modified release formulations are unstable in intestinal fluids. These modified release formulations release about 70% by weight or more of the AP-based drug (e.g., IAP, or other AP-based drug and variants thereof described herein) and / or additional therapeutic agent in the modified release formulation into intestinal fluid or simulated intestinal fluid in about 15 minutes, or about 30 minutes, or about 45 minutes, or about 60 minutes, or about 90 minutes. In some embodiments, the modified release formulations are unstable in near-neutral to alkaline environments. These modified release formulations release about 70% by weight or more of the AP-based drug (e.g., IAP, or other AP-based drug and variants thereof described herein) and / or additional therapeutic agent in the modified release formulation into intestinal fluid with a pH of about 4-5 or more or into simulated intestinal fluid with a pH of about 4-5 or more in about 15 minutes, or about 30 minutes, or about 45 minutes, or about 60 minutes, or about 90 minutes. Modified release formulations that are unstable in near-neutral or alkaline environments include those in which 70% or more by weight of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) and / or additional therapeutic agent in the modified release formulation is dissolved in a liquid having a pH greater than about 5 (e.g., 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.

[0125] Examples of simulated gastric and intestinal fluids include, but are not limited to, those disclosed in Test Solutions at page 2858 of the 2005 Pharmacopoeia 23NF / 28USP, and / or other simulated gastric and intestinal fluids known to those of skill in the art, such as those prepared without enzymes.

[0126] In one embodiment, the modified release formulation may remain nearly intact or nearly insoluble in gastric juice. The modified release formulation may include one or more delayed release coatings that are pH-dependent. The delayed release coatings that are pH-dependent are substantially stable in an acidic environment (pH of about 5 or less) and substantially unstable in near-neutral to alkaline environments (pH of about 5 or more). For example, the delayed release coating may substantially disintegrate or dissolve in near-neutral to alkaline environments such as those 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).

[0127] Alternatively, the stability of the modified release formulation may be enzyme-dependent. In such an embodiment, the modified release formulation may include one or more delayed release coatings that are enzyme-dependent. The delayed release coating that is enzyme-dependent is substantially stable in a liquid that does not contain a particular enzyme, and is substantially unstable in a liquid that contains the enzyme. The delayed release coating substantially disintegrates or dissolves in a liquid that contains a suitable enzyme. Enzyme-dependent control may be achieved by using a material that releases the active ingredient only upon exposure to enzymes in the intestine, such as, for example, galactomannan. The stability of the modified release formulation may also depend on enzyme stability in the presence of microbial enzymes present in the intestinal flora.

[0128] In various embodiments, the modified release formulation comprising an AP-based drug (e.g., an IAP or its variant) is substantially stable in chyme. For example, in some embodiments, the activity of the AP-based drug is reduced by less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10% at about 10 hours, or 9 hours, or 8 hours, or 7 hours, or 6 hours, or 5 hours, or 4 hours, or 3 hours, or 2 hours, or 1 hour after administration.

[0129] In some embodiments, a dual pulse formulation is provided. In various embodiments, the present invention provides a modified release formulation that releases multiple doses of an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) at different locations along the intestine, at different times, and / or at different pHs. In an exemplary embodiment, the modified release formulation includes a first dose of an AP-based drug and a second dose of an AP-based drug, where the first dose and the second dose are released at different locations along the intestine, at different times, and / or at different pHs. 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 may release at least 1 dose, at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, or at least 8 doses of an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) at different locations along the intestine, at different times, and / or at different pHs. Additionally, the dual pulse description herein applies to modified release formulations that release an AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) and an additional therapeutic agent.

[0130] Administration and Dosage It will be understood that the actual dose of AP-based drug (e.g., IAP, or other AP-based drug described herein, and its variants) administered according to the present invention will vary, for example, according to the specific dosage form and mode of administration. Many factors that can change the action of AP-based drug (e.g., body weight, sex, diet, administration time, administration route, excretion rate, subject condition, drug combination, genetic predisposition, and response sensitivity) can be taken into consideration by those skilled in the art. Administration can be performed continuously or in one or more individual doses within the maximum tolerated dose. The optimal administration rate for a given set of conditions can be confirmed by those skilled in the art using conventional dose administration tests.

[0131] Individual doses of AP-based agents (e.g., IAPs, or other AP-based agents described herein, and variants thereof) may range, for example, from about 0.01 mg to about 1,000 mg, from about 0.01 mg to about 950 mg, from about 0.01 mg to about 900 mg, from about 0.01 mg to about 850 mg, from about 0.01 mg to about 800 mg, from about 0.01 mg to about 750 mg, from about 0.01 mg to about 700 mg, from about 0.01 mg to about 650 mg, from about 0.01 mg to about 600 mg, from about 0.01 mg to about 550 mg, from about 0.01 mg to about 500 mg, from about 0.01 mg to about 450 mg, from about 0.01 mg to about 400 mg, from about 0.01 mg to about 350 mg, from about 0.01 mg to about 3 The dosage form may be administered in a unit dosage form (e.g., a tablet or capsule) containing 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 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, unit dosage forms may include 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, about 50 mg, about 65 mg, about 70 mg, about 85 mg, about 90 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 650 mg, about 700 mg, about 850 mg, about 900 mg, about 1000 mg, about 1500 mg, about 2000 mg, about 2500 mg, about 3000 mg, about 4000 mg, about 5000 mg, about 6500 mg, about 7000 mg, about 8500 mg, about 9000 mg, about 10000 mg, about 15000 mg, about 20000 mg, about 25000 mg, about 30000 mg, about 40000 mg, about 50000 mg, about g, 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). In one embodiment, the individual doses of AP-based agent (e.g., IAP, or other AP-based agents described herein and variants thereof) are administered in a unit dosage form containing 25 mg of AP-based agent. In another embodiment, each dose of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) is administered in a unit dosage form containing 50 mg of the AP-based drug. In a further embodiment, each dose of the AP-based drug (e.g., an IAP, or other AP-based drug described herein, and variants thereof) is administered in a unit dosage form containing 75 mg of the AP-based drug.

[0132] In one embodiment, the AP-based drug is administered in an amount of about 0.01 mg to about 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 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 0.1mg to about 100mg, about 0.1mg to about 95mg per day, about 0.1mg to about 90mg per day, about 0.1mg to about 85mg per day, about 0.1mg to about 80mg per day, about 0.1mg to about 75mg per day, about 0.1mg to about 70mg per day, about 0.1mg to about 65mg per day, about 0.1mg to about 60mg per day, about 0.1mg to about 55mg per day, about 0.1mg to about 50mg per day, about 0.1mg to about 4 5 mg, 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.

[0133] In various embodiments, the AP-based agent is 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 ...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 4 mg, about 5 mg, about 5 mg, about and the like. In some embodiments, the compound is administered at a daily dose of about 0 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).

[0134] In some embodiments, a suitable dosage of an AP-based agent (e.g., an IAP, or other AP-based agents described herein, and variants thereof) is within the range of about 0.01 mg / kg body weight to about 100 mg / kg body weight of a subject, e.g., about 0.01 mg / kg body weight, about 0.02 mg / kg body weight, about 0.03 mg / kg body weight, about 0.04 mg / kg body weight, about 0.05 mg / kg body weight, about 0.06 mg / kg body weight, about 0.07 mg / kg body weight, about 0.08 mg / kg body weight, about 0.09 mg / kg body weight, about 10 ... g / kg body weight, about 0.08 mg / kg body weight, about 0.09 mg / kg body weight, about 0.1 mg / kg body weight, about 0.2 mg / kg body weight, about 0.3 mg / kg body weight, about 0.4 mg / kg body weight, about 0.5 mg / kg body weight, about 0.6 mg / kg body weight, about 0.7 mg / kg body weight, about 0.8 mg / kg body weight, about 0.9 mg / kg body weight, about 1 mg / kg body weight, about 1.1 mg / kg body weight, about 1.2 mg / kg body weight, about 1.3 mg / kg body weight, about 1.4 mg / kg body weight, about 1.5 mg / kg body weight, about 1.6 mg / kg body weight, about 1.7 mg / kg body weight, about 1.8 mg / kg body weight, 1.9 mg / kg body weight, about 2 mg / kg body weight, about 3 mg / kg body weight, about 4 mg / kg body weight, about 5 mg / kg body weight, about 6 mg / kg body weight, about 7 mg / kg body weight, about 8 mg / kg body weight, about 9 mg / kg body weight, about 10 mg / kg body weight, about 15 mg / kg body weight, about 20 mg / kg body weight, about 25 mg / kg body weight, about 30 mg / kg body weight, about 35 mg / kg body weight, about 40 mg / kg body weight, about 45 mg / kg body weight, about 50 mg / kg body weight, about 55 mg / kg body weight, about 60 mg / kg body weight, about 65 mg / kg body weight, about 70 mg / kg body weight, about 75 mg / kg body weight, about 80 mg / kg body weight, about 85 mg / kg body weight, about 90 mg / kg body weight, about 95 mg / kg body weight, or about 100 mg / kg body weight (including all values ​​and ranges therebetween).In other embodiments, a suitable dosage of the AP-based drug is 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 about 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.

[0135] According to certain embodiments of the present invention, the AP-based agent may be administered, for example, about once a day, about every other day, about every third day, 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 may 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.

[0136] Additional Therapeutic Agents and Combination Therapies or Co-Formulations The administration of the compositions and formulations of the present invention comprising an AP-based drug can be combined with an additional therapeutic agent. The co-administration of the additional therapeutic agent and the composition / formulation of the present invention can be simultaneous or sequential. In addition, the compositions / formulations of the present invention can include an additional therapeutic agent (e.g., via co-formulation). For example, the additional therapeutic agent and the AP-based drug can be combined in a single formulation. Alternatively, the additional therapeutic agent and the AP-based drug can be formulated separately.

[0137] In one embodiment, the additional therapeutic agent and the AP-based agent are administered to the subject simultaneously. The term "simultaneously" as used herein means that the additional therapeutic agent and the AP-based agent are administered at a time interval of about 60 minutes or less, for example, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, about 5 minutes or less, or about 1 minute or less. The administration of the additional therapeutic agent and the AP-based agent can be simultaneous administration of a single formulation (e.g., a formulation that includes the additional therapeutic agent and the AP-based agent) or separate formulations (e.g., a first formulation that includes the additional therapeutic agent and a second formulation that includes the AP-based agent).

[0138] In further embodiments, the additional therapeutic agent and the AP-based agent are administered to a subject simultaneously, although 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) can occur sequentially.

[0139] Concurrent administration does not require the additional therapeutic agent and the AP-based agent to be administered at the same time, provided that the timing of administration is such that the pharmacological activities of the additional therapeutic agent and the AP-based agent overlap in time. For example, the additional therapeutic agent and the AP-based agent can be administered sequentially. The term "sequentially" as used herein means that the additional therapeutic agent and the AP-based agent are administered at a time interval of more than about 60 minutes. For example, the time 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 depends on the metabolism rate, excretion rate, and / or pharmacodynamic activity rate of the additional therapeutic agent and the AP-based agent administered. Either the additional therapeutic agent or the AP-based agent can be administered first.

[0140] 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 suitable route, e.g., parenterally or non-parenterally.

[0141] In some embodiments, the additional therapeutic agent is an antibacterial agent, including, but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole), fluoroquinolone antibiotics (cipro, levaquin, floxin, tequin, avelox, and norflox), tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline), penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin), monobactam antibiotics (aztreonam), and carbapenem antibiotics (ertapenem, doripenem, imipenem / cilastatin, and meropenem). In some embodiments, the antibacterial agent can be any of the penicillin, cephalosporin, monobactam, and carbapenem antibiotics.

[0142] In some embodiments, the additional therapeutic agent is an adjunctive therapy, e.g., used in the treatment of CDI. In some embodiments, the additional therapeutic agent is metronidazole (e.g., FLAGYL), fidaxomicin (e.g., DIFICID), or vancomycin (e.g., VANCOCIN), rifaximin, charcoal-based binders / adsorbents (e.g., DAV132), fecal bacteriotherapy, probiotic therapy (see, e.g., Intnat'l J Inf Dis, 16(11):e786, the contents of which are incorporated herein by reference).Exemplary probiotic agents include Saccharomyces boulardii; Lactobacillus rhamnosus GG; Lactobacillus plantarum 299v; Clostridium butyricum M588; Clostridioides difficile VP20621 (a non-toxigenic C. difficile strain (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, and Lactobacillus bulgaricus casei, Lactobacillus bulgaricus plantarum, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium breve, and Streptococcus salivarius subsp. thermophilus (VSL#3)), as well as antibodies or other biological therapeutics (e.g., monoclonal antibodies against C. difficile toxins A and B, as described in N Engl J Med. 2010;362(3):197, the contents of which are incorporated herein by reference in their entirety; neutralizing binding proteins arranged as multimers against one or more of the SEQ ID NOs: 59, 60, 95, 67, 68, and 87, as described in U.S. Patent Application Publication No. 2013 / 0058962, the contents of which are incorporated herein by reference); or neutralizing binding proteins against any of the C. difficile binary toxins.

[0143] 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, paregoric, and codeine, synthetic opioids such as diphenoxylate, difenoxin, and loperamide, bismuth subsalicylate, lanreotide, vapreotide, and octreotide, motiln antagonists, COX2 inhibitors (such as celecoxib), glutamine, thalidomide, and traditional antidiarrheal treatments such as kaolin, pectin, berberine, and muscarinic agents.

[0144] 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 (NSAIDS). Steroids, particularly adrenal corticosteroids and their synthetic analogs, are well known in the art. Examples of corticosteroids useful in the present invention include, but are not limited to, hydroxyltriamcinolone, alpha-methyldexamethasone, beta-methylbetamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, desonide, desoximetasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butyl ester, fluocortolone, fluprednidene acetate (fluprednylidene), flurandreno. Examples of acetaminophen include acetaminophen, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolon acetonide, medrysone, amcinafide, amcinafide, betamethasone and the remainder of its esters, chloroprednisone, clocortelone, clesinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, and beclomethasone dipropionate. The (NSAIDS) that may be used in the present invention include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylimide, benzyl 2,5-diacetoxybenzoate, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin. Additional anti-inflammatory agents are described, for example, in U.S. Patent No. 4,537,776, the entire contents of which are incorporated herein by reference.

[0145] In some embodiments, the additional therapeutic agent may be an analgesic. Analgesics useful 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 their derivatives, buprenorphine, piperidine, morphinan, benzomorphan, tetrahydroisoquinoline, thiambutane, benzylamine, tilidine, viminol, nefopam, capsaicin (8-methyl-N-vanillyl-6E-nonenamide), "synthetic" capsaicin (N-vanillylnonamide), and related compounds.

[0146] In some embodiments, the additional therapeutic agent can 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.

[0147] In some embodiments, the additional therapeutic agent can be an agent useful in the treatment of 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 therapeutics.

[0148] In some embodiments, the additional therapeutic agent may be an agent useful in the treatment of celiac disease. Exemplary agents include, but are not limited to, AVX-176 (Avaxia Biologics), Actobiotics (ActoGeniX), CALY-002 (Calypso biotech), HLA-DQ2 antagonists, HLA-DQ2 / DQ8 antagonists, tTG inhibitors (including ERW1041E (GlaxoSmithKline) and ZED-101 / ZED-1227 (Zedira)), larazotide acetate (Alba Therapeutics), latiglutenase (Alvine Pharmaceuticals), BL-7010 (BioLineRx), and NexVax-2 (ImmunsanT).

[0149] In some embodiments, the additional therapeutic agent may be an agent useful in the treatment of cystic fibrosis. Exemplary 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, glycerol phenylbutyrate, riociguat (Bayer), recombinant A1PI (Grifols, SA), cysteamine IR, JBT-101 (Corbus Pharmaceuticals), N-91115 (Nivalis Therapeutics), and vancomycin.

[0150] In some embodiments, the additional therapeutic agent can be a drug useful for treating obesity. Exemplary drugs include, but are not limited to, orlistat, lorcaserin, phentermine-topiramate, naltrexone-bupropion, sibutramine, rimonabant, exenatide, pramlintide, phentermine, benzphetamine, diethylpropion, phendimetrazine, bupropion, and metformin. In various embodiments, the additional drug is a drug that interferes with the body's ability to absorb certain nutrients in food, such as orlistat, glucomannan, and guar gum. The drug that suppresses appetite can also be one of the additional drugs, such as catecholamines and their derivatives (such as phentermine and other amphetamine drugs), various antidepressants and mood stabilizers (e.g., bupropion and topiramate), and appetite suppressants (e.g., dexedrine, digoxin). Drugs that increase the body's metabolism can also be one of the additional drugs. In some embodiments, the additional agent may be selected from among appetite suppressants, neurotransmitter reuptake inhibitors, dopaminergic agonists, serotonergic agonists, modulators of GABAergic signaling, anticonvulsants, antidepressants, monoamine oxidase inhibitors, substance P (NK1) receptor antagonists, melanocortin receptor agonists and antagonists, lipase inhibitors, fat absorption inhibitors, regulators of energy intake or metabolism, cannabinoid receptor modulators, agents for the treatment of addiction, agents for the treatment of metabolic syndrome, peroxisome proliferator-activated receptor (PPAR) modulators, and dipeptidyl peptidase 4 (DPP-4) antagonists.In some embodiments, the additional agent may be selected from among amphetamines, benzodiazepines, sulfonylureas, meglitinides, thiazolidinediones, biguanides, beta blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, phenlermine, sibutramine, lorcaserin, cetilistat, rimonabant, taranabant, topiramate, gabapentin, valproic acid, vigabatrin, bupropion, tiagabine, sertraline, fluoxetine, trazodone, zonisamide, methylphenidate, varenicline, naltrexone, diethylpropion, phendimetrazine, repaglinide, nateglinide, glimepiride, pioglitazone, rosiglitazone, liraglutide, and sitagliptin.

[0151] In one embodiment, the additional therapeutic agent is an agent for treating prediabetes, diabetes, type II diabetes, insulin resistance, impaired glucose tolerance, 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. Additional 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, and 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, and Glumetza. Examples of thiazolidinediones include, but are not limited to, rosiglitazone and pioglitazone. Examples of insulins include, but are not limited to, aspart, detemir, glargine, glulisine, and lispro. Examples of concomitant medications 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.

[0152] Treatment method In various embodiments, the present invention provides methods for treating or preventing radiation-induced disorders, including but not limited to enteritis, radiation-induced enteropathy, colitis, and / or proctitis resulting from cancer radiation therapy. Radiation-induced enteropathy is characterized by mucosal atrophy, vascular sclerosis, and progressive intestinal wall fibrosis. Symptoms of the disorder can include malabsorption of nutrients, altered intestinal transit, motility disorders, and abnormal progression of intestinal contents. In some embodiments, acute radiation-induced enteropathy occurs within the first month, first two months, or first three months after radiation exposure. In some embodiments, symptoms of late radiation enteropathy are chronic and may not appear until at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months after radiation exposure. In some embodiments, symptoms of late radiation enteropathy may not appear until 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 after radiation exposure, in some embodiments, symptoms of late radiation enteropathy may not appear until about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years after radiation exposure.

[0153] In some embodiments, the present invention provides treatment and / or administration of an AP-based agent to a subject exposed to radiation, including but not limited to radiation therapy. In various embodiments, administration of the AP-based agent occurs prior to exposure to radiation, such as, for example, prior to radiation therapy as part of a 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 immediately after exposure to radiation. In some embodiments, administration of the AP-based agent occurs immediately after exposure to radiation. In various embodiments, administration of the AP-based agent occurs at the time of exposure to radiation and continues for an extended period of time after exposure to radiation. In some embodiments, administration of the AP-based agent continues for an extended period of time after exposure to radiation. In various embodiments, administration of the AP-based agent occurs at the onset of late radiation enteropathy. In some embodiments, the present invention provides treatment and / or administration of an AP-based agent to a subject that has been exposed or will be exposed to radiation, where the administration of the AP-based agent occurs for 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.

[0154] In various embodiments, the present invention provides for the treatment and / or administration to a subject suffering from a radiation-related disease or disorder, including but not limited to side effects of radiation therapy or ARS.

[0155] In some embodiments, the present invention provides for the prevention, treatment, and / or administration to a subject suffering from radiation enteritis.For example, the subject may suffer from either acute or chronic radiation enteritis.Symptoms of radiation enteritis include, but are not limited to, nausea, vomiting, stomach cramps, frequent and urgent toilet use, watery diarrhea, mucus discharge from the rectum, rectal pain, rectal bleeding, weight loss, and undulating stomach pain.

[0156] In some embodiments, the present invention relates to a method for treating or preventing radiation-related diseases or disorders in a patient in need thereof, comprising administering to the subject a modified release formulation comprising an AP-based drug, as described herein, for example and without limitation, the AP-based drug of the formulation is an orally administrable IAP.

[0157] In various embodiments, the present invention provides for the treatment and / or administration to a subject suffering from late radiation enteropathy and / or intestinal toxicity. In some embodiments, the late radiation enteropathy occurs at least 3 months after the end of radiation therapy. In various embodiments, the radiation therapy is radiation therapy for treating cancer. In various embodiments, the subject is a cancer patient. In one embodiment, the radiation therapy is directed to a tumor in the pelvis, abdomen, or lower torso. In some embodiments, the treatment of the present invention does not interfere with cancer treatment, including but not limited to radiation therapy.

[0158] In various embodiments, the radiation comprises ionizing radiation, hi various embodiments, the radiation comprises one or more of x-rays, gamma rays, and charged particles.

[0159] In various embodiments, the radiation exposure is at 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, 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.

[0160] In various embodiments, the radiation exposure is local or systemic.

[0161] In some embodiments, the present invention relates to a method for treating or preventing a radiation-related disease or disorder in a patient in need thereof, the method comprising administering to a subject a modified release formulation comprising an AP-based drug (optionally an orally administrable IAP) as described herein, wherein the radiation-related disease or disorder is a consequence or side effect of radiation therapy.

[0162] In some embodiments, the methods relate to preventing or reducing a reduction in gut microbiome diversity (e.g., which is a side effect or consequence of radiation exposure (including radiotherapy) and / or chemotherapy). In some embodiments, the methods relate to repairing and / or repopulating a subject's gut microbiome following radiation exposure (including radiotherapy) and / or chemotherapy.

[0163] In some embodiments, radiation therapy can be part of cancer treatment, either as a primary or adjuvant therapy (e.g., with chemotherapy). In some embodiments, radiation therapy can be used to prevent tumor recurrence after surgery and / or to remove a primary malignant tumor. In various embodiments, the subject is a cancer patient.

[0164] In some embodiments, radiation therapy can be part of a treatment for Dupuytren's disease, Lederhose disease, or part of a post-operative treatment. In various embodiments, the subject has Dupuytren's disease, Lederhose disease, or has recently undergone surgery.

[0165] In various embodiments, the method reduces or eliminates the side effects of radiation therapy, including acute side effects, long-term side effects, or cumulative side effects. In various embodiments, the method reduces or eliminates the local or systemic side effects of radiation therapy. In various embodiments, the side effects of radiation therapy are one or more of fatigue, nausea and vomiting, damage to epithelial surfaces (e.g., but not limited to, moist desquamation), oral, pharyngeal and gastric pain, intestinal discomfort (e.g., but not limited to, pain, diarrhea, and nausea), swelling, infertility, fibrosis, hair loss, dryness (e.g., but not limited to, dry mouth (xerostomia) and dry eyes (xerophthalmia), and dryness of the axillary and vaginal mucosa), lymphedema, cardiac disease, cognitive decline, radiation enteropathy (e.g., atrophy, fibrosis, and vascular changes that may cause malabsorption, diarrhea, steatorrhea, and hemorrhage with bile acid diarrhea), and vitamin B12 malabsorption commonly seen due to ileal lesions. Pelvic radiation illness includes radiation proctitis, incidence of bleeding, diarrhea and urgency, and radiation cystitis.

[0166] In various embodiments, radiation therapy is pelvic radiation therapy.In such embodiments, the modified release formulation comprising AP-based drug (optionally orally administrable IAP) as described herein reduces or eliminates GI-related side effects as described herein.In such embodiments, the AP-based drug (optionally orally administrable IAP) reduces or eliminates lower body-related side effects as described herein.

[0167] 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, bleeding with bile acid diarrhea, 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 the incidence of radiation proctitis, bleeding, diarrhea and urgency, and radiation cystitis.

[0168] In various embodiments, radiation therapy is provided as one or more of external beam radiation therapy, brachytherapy, and systemic radiation therapy.

[0169] In various embodiments, the radiation therapy is external beam radiation therapy selected from three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT, e.g., RAPIDARC), image-guided radiation therapy (IGRT), electromagnetically guided radiation therapy (e.g., CALYPSO) tomotherapy, stereotactic radiosurgery (SRS), stereotactic body radiation therapy (SBRT, e.g., CYBERKNIFE, GAMMAKNIFE, X-KNIFE, CLINAC), intraoperative radiation therapy (IORT), and proton therapy.

[0170] In various embodiments, the radiation therapy is brachytherapy selected from interstitial brachytherapy, intracavitary brachytherapy, and episcleral brachytherapy.

[0171] In various embodiments, the radiation therapy is a systemic radiation therapy selected from radioactive iodine and radioactive biologics. For example, the radiation therapy can be radioactive iodine (131I), ibritumomab tiuxetan (ZEVALIN), tositumomab and iodine I131 tositumomab (BEXXAR), samarium-153-lexidronam (QUADRAMET), and strontium-89 chloride (METASTRON).

[0172] In various embodiments, radiation therapy comprises a dose (optionally fractionated) 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.

[0173] In some embodiments, the present invention relates to a method of treating or preventing a radiation-related disease or disorder in a patient in need thereof, the method comprising administering to a subject a modified release formulation described herein, wherein the radiation-related disease or disorder is acute radiation syndrome.

[0174] In some embodiments, the ARS comprises one or more of a gastrointestinal syndrome, a hematopoietic syndrome, a neurovascular syndrome, an apoptosis-mediated tissue damage (wherein apoptosis may result from cellular stress), and an ionizing radiation-induced apoptotic tissue damage. In some embodiments, the high dose of 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, optionally sufficient to classify the Unit Radiation Exposure Status as RES3. In various embodiments, the high dose of radiation is the result of a radiation disaster, and / or the human patient being treated has been exposed to or is at risk of being exposed to a high dose of radiation as a result of one or more of military or emergency responder actions in a contaminated area, a nuclear explosion, a criticality accident, a radiotherapy accident, a terrorist attack, exposure from space travel, a radioactive waste leak, exposure to open source radiation, and a malfunction of a nuclear reactor.

[0175] In various embodiments, the methods and compositions of the present invention provide treatment or prevention of radiation-related disorders, such as ARS. In various embodiments, the treatments described herein reduce morbidity or mortality in exposed human patient populations and / or promote recovery from symptoms of ARS. ARS often manifests as a series of stages of symptoms, which may vary depending on the individual's radiation sensitivity, the type of radiation, and the amount of radiation absorbed. While not wishing to be bound by theory, generally, the severity of symptoms increases and the duration of each stage decreases with increasing radiation dose. ARS can be divided into three stages: prodromal stage (also known as NVD stage), latent stage, and manifest disease. In various embodiments, the modified release formulation of the AP-based drug of the present invention can be administered to a human patient in any one of these three stages (i.e., the AP-based drug can be administered to a human patient in the prodromal stage, the AP-based drug can be administered to a human patient in the latent stage, or the AP-based drug can be administered to a human patient in the manifest disease stage).

[0176] In the prodromal phase, nausea, vomiting, and fatigue often develop relatively rapidly. The use of antiemetics (e.g., prophylactic oral antiemetics), such as 5-HT3 blockers with or without the combination of granisetron (KYTRIL), ondansetron (ZOFRAN), and dexamethasone, may be required in situations where high doses of radiation exposure may have occurred or are unavoidable. Thus, in various embodiments, the AP-based agent may be administered to a human patient who has been administered an antiemetic agent, or the AP-based agent may be administered to a human patient in combination with an antiemetic agent. For example, the AP-based agent may be added to the following antiemetic regimen: Ondansetron: 0.15 mg / kg IV initially; continuous IV dosing options consist of 8 mg followed by 1 mg / hour for the next 24 hours. Oral doses are 8 mg or granisetron (oral dosage form) every 8 hours as needed, and typically the dose is 1 mg initially, repeated 12 hours after the initial dose. Alternatively, 2 mg can be taken as one dose. IV dose is based on body weight, usually 10 μg / kg (4.5 μg / lb) body weight.

[0177] During the latent phase, human patients may be relatively asymptomatic. The length of this phase varies with dose. The latent phase preceding the hematopoietic syndrome, myelosuppression, is the longest and can vary between approximately 2 and 6 weeks. The latent phase preceding the gastrointestinal syndrome is somewhat shorter, lasting a few days to a week. The one preceding the neurovascular syndrome is the shortest of all, lasting only a few hours. These times are variable and may be altered by the presence of other diseases or injuries. Overt disease shows clinical symptoms related to damaged major organ systems (bone marrow, gut, neurovascular).

[0178] In some embodiments, the present invention relates to reducing or protecting cells from the effects of radiation exposure. In some embodiments, the present invention relates to a method of reducing and / or protecting a human patient from radiation, 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 gastrointestinal or hematopoietic syndrome.

[0179] In some embodiments, the ARS comprises one or more of a gastrointestinal syndrome, a hematopoietic syndrome, a neurovascular syndrome, apoptosis-mediated tissue damage (wherein apoptosis may result from cellular stress), and ionizing radiation-induced apoptotic tissue damage.

[0180] Hematopoietic syndrome (also known as bone marrow syndrome) is characterized by the loss of hematopoietic cells and their precursors, resulting in the inability to regenerate the blood and lymphatic system. This syndrome is often characterized by a low blood cell count, i.e., aplastic anemia. This can lead to infections (e.g., opportunistic infections) due to the low amount of white blood cells, bleeding due to platelet deficiency, and anemia due to the low number of red blood cells in circulation. These changes can be detected by blood tests after receiving an acute dose of radiation throughout the body. Conventional trauma and burns resulting from blast are exacerbated by poor wound healing caused by hematopoietic syndrome, resulting in increased mortality. Death can occur as a result of infection (e.g., as a result of immunosuppression), bleeding, and / or anemia. Hematopoietic syndrome usually spreads with lower doses of radiation and leads to later death than GI syndrome.

[0181] The gastrointestinal syndrome is caused by massive cell death in the intestinal epithelium, primarily in the small intestine, followed by the breakdown of the intestinal wall and death from bacteremia and sepsis. Symptoms of this form of radiation injury include nausea, vomiting, loss of appetite, decreased absorptive capacity, bleeding from exposed areas, and abdominal pain. Exemplary systemic effects of the gastrointestinal syndrome include malnutrition, dehydration, renal failure, anemia, sepsis, and the like. Without treatment (including, for example, bone marrow transplantation), death typically occurs (e.g., via infection from enteric bacteria). In some embodiments, the AP-based agent may be used in combination with bone marrow transplantation. In some embodiments, the AP-based agent may be used in combination with one or more inhibitors of GI syndrome and / or any of the additional agents described herein.

[0182] The neurovascular syndrome presents with neurological symptoms such as dizziness, headache, or decreased level of consciousness, and occurs within minutes to hours without vomiting. Additional symptoms include extreme irritability and confusion; severe nausea, vomiting, and watery diarrhea; loss of consciousness; and a burning sensation of the skin. The neurovascular syndrome is generally fatal.

[0183] In various embodiments, methods and compositions of the present invention provide for the treatment and / or prevention of radiation-induced intestinal fibrosis, which in some embodiments includes one or more of intestinal inflammation, intestinal fibrosis, vascular sclerosis, chronic ulcers, submucosal swelling, increased proliferation of fibroblasts and smooth muscle cells, and excessive deposition of collagen and other extracellular matrix components.

[0184] In some embodiments, the present invention provides a method for reducing the risk of death after exposure to radiation, comprising administering an effective amount of an AP-based agent. In some embodiments, the radiation may be lethal, and in some cases, occurs as a result of a radiation disaster. In various embodiments, the AP-based agent is administered within 24 hours after radiation exposure. In various embodiments, the AP-based agent is administered within 48 hours after radiation exposure.

[0185] In some embodiments, the AP-based drug controlled release formulation is administered in combination with any additional drug described herein, including but not limited to radioprotectants (e.g., antioxidants (e.g., amifostine and vitamin E), cytokines (e.g., stem cell factor), etc.). Damage and death of normal cells by ionizing radiation is a combination of direct radiation-induced damage to exposed cells and an active, genetically programmed cellular response to radiation-induced stress, resulting in suicidal death or apoptosis. Apoptosis plays an important role in the massive cell loss that occurs in several radiation-sensitive organs (e.g., hematopoietic and immune systems, gastrointestinal epithelium, etc.), the failure of which determines the general radiation sensitivity of an organism. In some embodiments, administration of the AP-based drug of the present invention to a human patient in need thereof inhibits cellular apoptosis. In some embodiments, the AP-based drug of the present invention is administered to a human patient to protect healthy cells from the harmful effects of radiation therapy.

[0186] In various embodiments, the present invention provides methods for reducing apoptosis following exposure to irradiation. In one embodiment, the present invention provides methods for reducing apoptosis of hematopoietic cells following irradiation. In another embodiment, the present invention provides methods for reducing apoptosis of gastrointestinal cells following irradiation.

[0187] In various embodiments, 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 cells can be stimulated to proliferate and regenerate. Thus, the present invention provides methods for increasing the number of stem cells, such as hematopoietic or gastrointestinal stem cells, in a patient. In some embodiments, hematopoietic or gastrointestinal progenitor cells are expanded. In various embodiments, the present invention provides methods and compositions for protecting stem or progenitor cells from cell death (e.g., apoptosis or necrosis).

[0188] In various embodiments, the methods and compositions of the invention significantly enhance recovery of the hematopoietic and GI systems following irradiation. For example, the methods and compositions of the invention enhance bone marrow recovery following irradiation. In another example, the methods and compositions of the invention enhance regeneration of GI crypts.

[0189] Exposure to ionizing radiation (IR) may be short-term or long-term, and / or may be experienced as single or multiple doses, and / or may be applied systemically or locally. The present invention relates in some embodiments to nuclear accidents or military attacks that may involve exposure to a single high dose of total body irradiation (occasionally followed by long-term poisoning with radioisotopes), as further described herein. The same applies (with strict control of the applied dose) for conditioning bone marrow transplant patients, for example, when it is necessary to prepare the hematopoietic organ for the donor's bone marrow by "cleaning" it from the host's blood precursors. Cancer treatments may involve multiple doses of localized radiation that would be significantly more than lethal if applied as total body irradiation (e.g., radiotherapy accidents). Poisoning or treatment with radioisotopes results in localized exposure to radiation for a long period of time in the target organ (e.g., the thyroid gland in the case of inhalation of 125I). Furthermore, there are many physical forms of ionizing radiation that vary significantly in the severity of their biological effects.

[0190] Radiation particles can cause breaks and cross-links in DNA, proteins, cell membranes, and other macromolecular structures at the molecular and cellular levels. Ionizing radiation also induces secondary damage to cellular components by generating free radicals and reactive oxygen species (ROS). This damage is reversed by multiple repair systems, including several DNA repair pathways that restore DNA integrity and fidelity, as well as antioxidant chemicals and enzymes that scavenge free radicals and ROS and reduce oxidized proteins and lipids. Cellular checkpoint systems detect defects in DNA and delay progression through the cell cycle until the damage is repaired or a decision is reached to cause the cell to stop growing or undergo programmed cell death (apoptosis).

[0191] Radiation can produce damage in mammalian organisms ranging from mild mutagenic and carcinogenic effects at low doses to almost immediate death at higher doses. The overall radiosensitivity of an organism is determined by the pathological changes that occur in several highly sensitive tissues, including the hematopoietic system, the reproductive system, and various epithelia with high rates of cell turnover.

[0192] The acute pathological outcome of lethal gamma irradiation varies with dose and may be determined by failure of specific organs that define the threshold of sensitivity of the organism to each specific dose. Thus, lethality at low doses results from bone marrow hypoplasia, whereas moderate doses cause earlier death by induction of gastrointestinal (GI) syndrome. Extremely high doses of radiation can cause almost immediate death inducing neuronal degeneration. Organisms that survive a certain acute toxicity period of radiation may suffer long-term indirect consequences, including radiation-induced carcinogenesis and fibrosis occurring in exposed organs (e.g., kidney, liver, or lung) months and years after irradiation. Cellular DNA is the primary target of IR, which induces various types of DNA damage (genotoxic stress) by direct and indirect (e.g., free radical-based) mechanisms. All organisms maintain a DNA repair system capable of effective restoration of radiation-damaged DNA, but errors during the DNA repair process can lead to mutations.

[0193] AP-based drugs have potent survival-promoting activity at the cellular level and in the whole organism. For supra-lethal radiation doses, AP-based drugs can inhibit both the gastrointestinal and hematopoietic syndromes, which are the leading causes of death from acute radiation exposure. As a result of these properties, AP-based drugs can be used to treat the effects of natural radiation events and nuclear accidents. Moreover, AP-based drugs can be used in combination with other radioprotectors, dramatically increasing the magnitude of protection from ionizing radiation.

[0194] AP-based agents can be used as radioprotectants to extend the range of radiation tolerance by increasing the radiation resistance of the human body beyond the levels achievable by currently available means (shielding and application of existing bioprotective agents) and to dramatically increase the chances of crew survival in the event of, for example, a nuclear accident or a large solar particle event.

[0195] AP-based drugs may inhibit radiation-induced programmed cell death or apoptosis in response to damage to DNA and other cellular structures. In some embodiments, AP-based drugs may not address damage at the cellular level 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 are effective in preventing damage caused by free radicals.

[0196] Additionally, in some embodiments, the present invention relates to the prevention or treatment of cutaneous radiation syndrome (CRS), i.e., cutaneous symptoms of radiation exposure (e.g., redness (optionally accompanied by itching), blistering, ulceration, hair loss, damage to sebaceous and sweat glands, atrophy, fibrosis, loss or increase in skin pigmentation, ulceration or necrosis of moist desquamation of exposed tissue resulting in full thickness loss of exposed skin and collapse of the dermal vasculature after two months).

[0197] In various embodiments, administration of AP-based drugs reduces the incidence of wounds, septic complications, and microbial infections in post-irradiation patients.

[0198] In some embodiments, the human patient of the invention experiences leukopenia and / or neutropenia (e.g., absolute neutrophil count (ANC) <100 cells / μL). In some embodiments, the methods and compositions of the invention relate to a human patient that exhibits a reduction in lymphocyte count of about 50% within about 24 to about 48 hours. In some embodiments, the human patient's lymphocyte count is (e.g., within about 24 to about 48 hours) less than about 1000 cells / μL, or less than about 900 cells / μL, or less than about 800 cells / μL, or less than about 700 cells / μL, or less than about 600 cells / μL, or less than about 500 cells / μL, or less than about 400 cells / μL, or less than about 300 cells / μL, or less than about 200 cells / μL, or less than about 100 cells / μL. In some embodiments, the patient's lymphocyte profile 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 incorporated herein by reference). In some embodiments, the methods and compositions of the present invention relate to a human patient exhibiting a reduction in platelet count of about 50% within about 24 to about 48 hours. In some embodiments, the human patient of the present invention experiences thrombocytopenia, anemia, and / or neutropenia. Thrombocytopenia is defined as a platelet count less than 50,000 / μL. For example, thrombocytopenia may be characterized as grade 1 thrombocytopenia (i.e., platelet counts between 75,000 and 150,000 / μL), grade 2 (i.e., platelet counts between 50,000 and <75,000 μL), grade 3 (platelet counts between 25,000 and <50,000 / μL), or grade 4 (i.e., platelet counts less than 25,000 / μL).Anemia may be diagnosed when men have a hemoglobin content less than 13-14 g / dL and women have a hemoglobin content between 12-13 g / dL. For example, anemia may be classified into various grades based on hemoglobin levels: grade 0 (within normal range, ≥12 g / dL), grade 1 (mild, 11.9-10 g / dL), grade 2 (moderate, 9.9-8 g / dL), grade 3 (severe / severe, 7.9-6.5 g / dL), and grade 4 (life-threatening, <6.5 g / dL). Neutropenia may be defined as an absolute neutrophil count (ANC) less than 1,500 cells / mm3. For example, neutropenia may be graded as grade 1 (i.e., ANC ≦1,500 / mm3 to greater than 2,000 / mm3), grade 2 (ANC ≦1,000 / mm3 to greater than 1,500 / mm3), grade 3 (ANC ≦500 / mm3 to greater than 1,000 / mm3), or grade 4 (ANC less than 500 / mm3). In various embodiments, the methods and compositions of the invention reduce the duration and severity of thrombocytopenia, anemia, and / or neutropenia in a patient after irradiation. For example, the methods and compositions of the invention may reduce the duration and severity of grade 4 thrombocytopenia, anemia, and / or neutropenia in a patient after irradiation.

[0199] In various embodiments, the high dose of radiation refers to a whole body dose. In various embodiments, the high dose of radiation may not be uniform. In various embodiments, the ARS is a result of the high dose of radiation. In various embodiments, the high dose of radiation is 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 15 Gy, or about 20 Gy, or about 25 Gy, or about 30 Gy. In various embodiments, the high dose of radiation is about 5 to about 30 Gy, or about 10 to 25 Gy, or about 15 to 20 Gy. In some embodiments, the high dose of radiation is assessed by one or more of physical dosimetry and / or biological dosimetry (e.g., multi-parameter dose assessment), cytogenesis (e.g., chromosome analysis of blood samples, etc., including dicentric analysis as a non-limiting example).

[0200] In various embodiments, total body radiation doses can be divided into sublethal (<2 Gy), potentially lethal (2-10 Gy), and supralethal (>10 Gy).

[0201] The Radiation Exposure Status (RES) of a given unit is based on operational exposure above normal background radiation. The RES is designed to be an average based on unit level dosimeters. In various embodiments, a high dose of radiation is sufficient to classify the unit radiation exposure status as RES3.

[0202] 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 radiation interacts with atoms, energy is deposited, leading to ionization (electron excitation). This ionization can damage certain critical molecules or structures within cells through direct and indirect action. Radiation can directly reach particularly sensitive atoms or molecules within cells. This damage is irreparable, leading to either cell death or cell dysfunction. Radiation can also indirectly damage cells by interacting with water molecules in the body. Energy deposited in water leads to the production of unstable and toxic superoxide molecules, which in turn damage sensitive molecules and affect intracellular structures.

[0203] In some embodiments, the radiation may be produced by one or more of the following radioactive substances: 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).

[0204] 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 a high dose of radiation, which may be the result of one or more of military or emergency responder actions in a contaminated area, a nuclear explosion, a criticality accident, a radiotherapy accident, a terrorist attack, exposure from space travel, a radioactive waste leak, exposure to open source radiation, and a malfunction of a nuclear reactor.

[0205] A method of treating or preventing the side effects of chemotherapy treatment in a patient in need thereof, comprising administering to the subject a modified release formulation described herein.

[0206] In various embodiments, the present invention provides for treatment and / or administration to subjects suffering from, for example, the side effects of chemotherapy treatment.

[0207] In some embodiments, the side effects of chemotherapy treatment are selected from alopecia, bone marrow suppression, nephrotoxicity, weight loss, pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, numbness, altered taste, decreased appetite, thinning or brittle hair, oral sores, memory loss, bleeding, cardiac toxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment.

[0208] A method of treating cancer in a patient in need of treatment by improving the efficacy of chemotherapy treatment, comprising administering to the subject a modified release formulation as described herein. In various embodiments, the modified release formulation of an AP-based drug as described herein acts as an adjuvant to the chemotherapy treatment as described herein. In various embodiments, the modified release formulation of an AP-based drug as described herein improves the anti-cancer effect and / or increases the therapeutic window of any of the chemotherapy treatments as described herein. In various embodiments, administering to the subject a modified release formulation of an AP-based drug does not interfere with the treatment of cancer.

[0209] The present invention further provides, in certain embodiments, a method for improving or reducing and / or treating or preventing frailty in a subject, the method comprising identifying a subject desiring or in need of treatment or prevention of frailty, and administering to the subject a modified release formulation of an AP-based drug as described herein.

[0210] Aging is a gradual systemic pathological change of mammalian organisms that progresses over time. Aging is associated with the accumulation of multiple defects in the function of multiple organs and tissues and the reduction of regenerative capacity, which lead to the development of age-related chronic diseases or disorders, including atherosclerosis, diabetes, pulmonary fibrosis, blindness, dementia, renal dysfunction, osteoarthritis, and low-grade chronic sterile inflammation, as well as other age-related diseases and disorders contemplated herein. These conditions are often accompanied by the gradual development of geriatric syndromes, including frailty, cognitive impairment, and immobility. Aging is a natural and inevitable process. Although the root cause of aging remains uncertain, two characteristics of aging, namely, increased DNA damage and the development of systemic chronic sterile inflammation, are generally accepted as universal and are both considered to be the main causes of age-related pathologies.

[0211] In some embodiments, the present invention provides a method 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 such that the score increases at a slower rate than if the subject had not been administered a modified release formulation of the AP-based drug of the present invention. In some embodiments of the present invention, the patient's PFI score remains approximately the same over time. In further embodiments, the methods of the present invention provide for the reduction of cellular senescence and immunosenescence associated with natural aging and / or accelerated aging (e.g., accelerated aging induced by cancer or cancer treatment, etc.).

[0212] In another aspect, the present invention provides a method for treating or preventing age-related disease or disorder in a subject, the method comprises identifying a subject who desires or needs to treat or prevent age-related disease or disorder, and administering to said subject a controlled release formulation of AP-based drug.In some embodiments, the age-related disease or disorder is characterized by increased cellular senescence or immunosenescence.

[0213] In some embodiments, the age-related disease or disorder is selected from accelerated aging, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, cardiac diastolic dysfunction, benign prostatic hyperplasia, 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 conditions, skin nevi, wrinkled skin, hyperpigmentation, scars, keloids, rosacea, vitiligo, ichthyosis vulgaris, dermatomyositis, actinic keratosis, and sarcopenia.

[0214] In certain embodiments, the methods of the present invention include treating or preventing accelerated aging. In some embodiments, the accelerated aging is a progeroid syndrome or a symptom thereof, including, but not limited to, Hutchinson-Gilford progeroid syndrome (HGPS), Werner syndrome (WS), Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), Cockayne syndrome (CS), xeroderma pigmentosum (XP), trichotillomania difficile (TTD), combined xeroderma pigmentosum-Cocayne syndrome (XP-CS), or restrictive dermatopathy (RD). Subjects with any of these diseases or disorders typically have a shortened life span (i.e., length of life).

[0215] In a further embodiment, the accelerated aging is induced by cancer or cancer treatment.For example, it is contemplated by the present invention that the cancer treatment that induces the acceleration of the natural aging process is selected from one or more of the following therapies: radiation therapy, hormone therapy, tyrosine kinase inhibitors, anthracyclines, alkylating agents, topoisomerase inhibitors, antimetabolites / cytotoxic agents, BRAF inhibitors, antitumor antibiotics, isoquinoline alkaloids, Bcl-2 inhibitors, hematopoietic cell transplantation (HCT), telomerase inhibitors, nucleoside analogue reverse transcriptase inhibitors, DNA crosslinking agents, ribonucleotide reductase inhibitors, microtubule inhibitors, and miRNA.

[0216] In some embodiments, any cancer in which the subject undergoes treatment that may induce accelerated aging is contemplated. In one embodiment, the cancer in which the subject undergoes treatment is a hematological cancer. Furthermore, in some embodiments, the subject undergoes cancer treatment in childhood.

[0217] In some embodiments, frailty includes the accumulation of deficits in key physiological functions, reduced regenerative capacity, impaired wound healing, and increased risk of age-related diseases. For example, in some embodiments, frailty is associated with natural aging or accelerated aging. Frailty can be measured according to any number of indices or tests known to those skilled in the art. For example, one such index, the physiological frailty index (PFI), includes measurements of one or more parameters selected from grip strength, systolic blood pressure, diastolic blood pressure, blood flow, blood neutrophil count, blood neutrophil percentage, blood monocyte count, blood monocyte percentage, lymphocyte count, red blood cell count, hemoglobin level, hematocrit level, mean corpuscular volume, mean corpuscular hemoglobin level, mean corpuscular hemoglobin concentration, and keratinocyte-derived cytokine level. Deviations from the reference standard in any individual are referred to as deficits, and the individual's overall average PFI score is the ratio of deficits to the total number of parameters measured.

[0218] Frailty may manifest as a reduced vulnerability to stressors and ability to resist stress. For example, the disclosure of Buchner and Wagner 1992 Clin Geriatr Med.1992 Feb;8(1):1-17 is incorporated herein by reference in its entirety. Frailty may manifest 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 Mar;57(3):B115-25 is incorporated herein by reference in its entirety. Frailty may also manifest as disuse and / or reduced energy flow through the organism, as described in Bortz 2002, J Gerontol A Biol Sci Med Sci.2002 May;57(5):M283-8, incorporated herein by reference in its entirety. As noted by Ferrucci 2005 J. Gerontol. A Biol. Sci. Med. Sci. 60, 56 (hereby incorporated by reference in its entirety), frailty can also manifest as a homeostatic dysregulation.

[0219] Several comprehensive approaches exist to quantitatively assess the age-related accumulation of deficits and frailty in humans and animals. Individual organisms are heterogeneous in their health status and aging rate. To account for such heterogeneity, the Frailty Index (FI) has been introduced as a numerical score that is the ratio of deficits present in a person to the total number of deficits considered in the study. Changes in the FI characterize the aging rate of an individual. A similar approach has been applied to experimental animals. The Frailty Index is a reliable and widely accepted indicator of "biological age", and the degree of decline in overall health status is considered to indicate a reduced quality of life.

[0220] In certain aspects and embodiments, provided herein include methods for improving and / or treating or preventing frailty and / or reducing frailty index in a subject. Frailty can be assessed by any of a number of methods known in the art. For example, methods for assessing / indexing frailty and frailty are described in Hubbard, et al., Ageing, published electronically November, 2008 pages 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 incorporated herein by reference.

[0221] In various embodiments, the frailty index is calculated as described in US 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 healthy to frailty range of organisms of the same chronological age to address the idea that chronological age does not necessarily reflect biological age. Based on 16 parameters (including measurements of weight, grip strength, blood pressure, total blood count, and cytokine level analysis), the FI is calculated as the ratio of the total number of defects measured, and an FI score between 0 (no defects = healthy) and 1 (all defects present = frail) is assigned. Thus, a higher FI indicates a poorer health status of the organism. In this regard, the FI is provided as a useful tool for assessing the "healthy" to "frail" range of organisms of the same chronological age.

[0222] In certain embodiments, the method of the present invention reduces or prevents frailty in a subject as measured according to the physiological frailty index (PFI), as described in Antoch et al. Aging. 2017;9:1-12, which is incorporated herein by reference in its entirety. For example, the PFI can be determined for each individual subject with reference to a young reference subject. Various parameters are measured for each subject. These parameters include non-invasive measurements such as 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. For each parameter, the mean and standard deviation are calculated. Subjects that differ from the mean of any single parameter by more than one standard deviation (STDEV) are excluded from the reference group. The value of each parameter measured for the elderly subject is compared with the corresponding value of the reference group and assigned a score. Values ​​that differ by less than 1 STDEV are assigned a score of 0 (no deficiency, within the range of the reference group). Values ​​that differ by 1 STDEV are scored as 0.25 (very slight deficiency). Values ​​that differ by 2 STDEV from the corresponding value of the reference group are scored as 0.5, and values ​​that differ by 3 STDEV are scored as 0.75. If the value is more than 3 STDEV, it is scored as 1 (extreme deficiency). The number of deficiencies exhibited by an individual subject is calculated as a ratio of the total number of parameters measured and is called the physiological frailty index (PFI).

[0223] In some embodiments, the method of the present invention reduces or improves and / or treats or prevents frailty in a subject as measured by PFI. For example, administering a modified release formulation of an AP-based drug to a subject to reduce or improve and / or treat or prevent frailty can reduce 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 at least 100%. In some embodiments, the subject's PFI score is reduced by about 25%-75%, about 25%-50%, or about 50%-75%. In further embodiments, the subject's PFI score is reduced by 0.9 or less, 0.85 or less, 0.8 or less, 0.75 or less, 0.7 or less, 0.65 or less, 0.6 or less, 0.55 or less, 0.5 or less, 0.45 or less, 0.4 or less, 0.35 or less, 0.3 or less, 0.25 or less, 0.2 or less, 0.15 or less, 0.1 or less, or 0.5 or less.

[0224] Additionally, frailty as an accumulation of deficits can be measured by the Rockwood Frailty Index, 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. In an embodiment, the method reduces or prevents frailty as assessed by the Rockwood Frailty Index.

[0225] Frailty, as a biological syndrome of reduced reserve capacity due to cumulative decline across 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 is comprised of the Physical Frailty Phenotype (PFP), which measures various parameters such as weight loss of more than 10 pounds, grip-related weakness, self-reported fatigue, 15-foot walking speed, and physical activity in Kcal per week. The Fried Frailty Score introduces a scoring system of 0 (not frail), 1-2 (moderately frail), and 3 or more (frail). In various embodiments, the methods of the present invention reduce or ameliorate and / or treat or prevent frailty in a subject as measured by the Fried Frailty Score. For example, administering to a subject a modified release formulation of an AP-based drug to reduce or ameliorate and / or treat or prevent frailty may reduce the 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. Additionally, in some embodiments, administering to a subject a modified release formulation of an AP-based drug to reduce or ameliorate and / or treat or prevent frailty eliminates an increase in the subject's Fried frailty score.

[0226] Frailty can also be measured by the FRAIL scale 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 by the FRAIL scale include persistent fatigue, endurance (ability to climb a flight of stairs), ambulation (ability to walk one block), more than five illnesses, and weight loss of more than 5%. The FRAIL scale introduces a scoring system of 0 (not frail), 1-2 (moderately frail), and 3 or more (frail). In various embodiments, the methods of the invention reduce or improve the frailty of a subject as measured by the FRAIL scale score. For example, administering to a subject a modified release formulation of an AP-based drug to reduce or ameliorate frailty may reduce 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. Additionally, in some embodiments, administering to a subject a modified release formulation of an AP-based drug to reduce or ameliorate and / or treat or prevent frailty eliminates an increase in the subject's FRAIL scale score.

[0227] In some embodiments, the methods provided herein improve (or reduce) the frailty index or delay or slow the decline in frailty using at least one acceptable indicator of frailty. In some embodiments, the methods provided herein improve (or reduce) the frailty index or delay or slow the decline in frailty using at least one acceptable indicator of frailty selected from the Frailty Index (FI), Physiological Frailty Index (PFI), Fried Frailty Score, Rockwood Frailty Index, FRAIL Scale, and Modified Frailty Index.

[0228] In some embodiments, frailty comprises low lean mass, weakness, fatigue, low energy expenditure, and / or slow walking speed. In embodiments, the methods of the invention reduce or prevent the onset or occurrence of one or more of low lean mass, weakness, fatigue, low energy expenditure, and / or slow walking speed.

[0229] Without wishing to be bound by theory, AP-based drugs, including AP-based drugs (e.g., IAPs), are believed to play an important role in many gastrointestinal and systemic processes, including, for example, participating in intestinal defense, mediating anti-inflammatory functions, maintaining normal gut microbiota profile, maintaining mucosal barrier integrity, and regulating digestion and nutrient (fat) absorption. Thus, the present invention provides for the use of AP-based drugs in a wide range of therapeutic applications to regulate immune, metabolic, and neurological functions. In various embodiments, the present invention provides for the treatment of microbiome-related disorders, GI dysbiosis, GI 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.

[0230] In various aspects, the present invention provides methods for modulating 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 a subject with reduced levels and / or function of gastrointestinal flora by administering an AP-based agent of the present invention to increase or preserve the number of commensal bacteria and the composition of the gastrointestinal microbiome. In other embodiments, the methods of the present invention relate to the treatment of infections caused by pathogenic bacteria in the gastrointestinal tract and / or the inhibition of the growth of pathogenic bacteria or the reduction of the number of pathogenic bacteria.

[0231] In various embodiments, the methods of the present invention include treating or preventing microbiome-mediated disorders. Exemplary microbiome-mediated disorders include, but are not limited to, those found in, for example, Table 3 of WO2014 / 121298, the entire contents of which are incorporated herein by reference. For example, the described methods can be used to treat conditions associated with reduced levels of commensal bacteria and / or function of gastrointestinal flora, such as antibiotic-associated diarrhea (AAD), Clostridioides difficile-associated disease (CDAD), inflammatory disorders, acquired immune deficiency syndrome (AIDS) (including HIV-mediated gut dysbiosis and GI barrier dysfunction), hypothyroidism, and obesity.

[0232] In various aspects, the present invention provides pharmaceutical compositions comprising the AP-based agent of the present invention (and / or additional therapeutic agents) for use in treating antibiotic-induced adverse effects in the GI tract and / or preventing or treating C. difficile infection (CDI) and / or C. difficile-associated diseases in a subject in need thereof. Without wishing to be bound by theory, it is believed that the AP-based agent of the present invention mediates the dephosphorylation of nucleoside triphosphates, which promotes the growth of commensal bacteria in preference to pathological bacteria and speeds recovery from antibiotic-induced dysbiosis. Thus, treatment with the AP-based agent of the present invention may protect against Clostridioides difficile infection and gram-negative pathogens in the intestine. In various embodiments, the antibiotic-induced adverse effects and / or CDI or C. difficile associated disease is one or more of antibiotic-associated diarrhea, C. difficile diarrhea (CDD), C. difficile intestinal inflammatory disease, colitis, pseudomembranous colitis, fever, abdominal pain, dehydration and electrolyte disturbances, megacolon, peritonitis, and colonic perforation and / or rupture.

[0233] In various embodiments, the subject includes, but is not limited to, a subject at particular risk for a microbiome-mediated disorder, such as, by way of non-limiting example, a person who is undergoing or has recently undergone antibiotic treatment. For example, the subject may have taken antibiotics within the past 30 days or so, and / or has a weakened immune system (e.g., resulting from a chronic illness), and / or is female, and / or is elderly (e.g., over about 65 years old), and / or is undergoing (or has undergone) treatment for heartburn or stomach acid disorders (e.g., with drugs such as PREVACID, TAGAMET, PRILOSEC, or NEXIUM and related drugs), and / or has recently been hospitalized (including intensive care unit, or residence in a nursing home). Thus, in some embodiments, the methods and uses of the present invention treat or prevent nosocomial infections and / or acute secondary infections and / or hospital acquired infections (HAIs).

[0234] In various embodiments, the present invention provides a method for treating antibiotic-induced adverse effects in the GI 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 adverse effects in the GI 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).

[0235] 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 adverse 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).

[0236] In various embodiments, the methods of the invention provide compositions and methods for treating infections caused by pathogenic bacteria and / or inhibiting the growth of or reducing the number of pathogenic bacteria in the gastrointestinal tract. In various embodiments, the invention provides compositions and methods for reducing or preventing the overgrowth of various coliforms (including virulent and / or antibiotic-resistant coliforms) in the intestines of a patient. Exemplary coliforms include Citrobacter, Enterobacer, Hafnia, Kelbsiella, and Escherichia. In various aspects, the methods and compositions described herein prevent or reduce secondary infections caused by resistant organisms. In one embodiment, the pathogenic bacteria is an enterobacterium, such as Salmonella.

[0237] In some embodiments, the present invention prevents the expansion of the gut resistome. The gut resistome refers to the reservoir of antibiotic resistance genes that may be carried by the human gut microbiome. Such antibiotic resistance genes confer antibiotic resistance among pathogenic microorganisms that pose a major threat to public health. Bacteria can acquire antibiotic resistance genes by mobilization and transfer of resistance genes from donor strains. In various embodiments, the prevention method reduces the number of antibiotic-resistant bacteria in the gastrointestinal tract, thereby reducing the expansion of the gut resistome. In various embodiments, the present invention reduces or prevents the proliferation of antibiotic-resistant bacteria, thereby preventing or reducing the expansion of the gut resistome.

[0238] In various embodiments, the present invention provides a method for treating or preventing C. difficile infection (CDI) and / or C. difficile associated disease, comprising administering an effective amount of an AP-based agent of the present invention to a subject in need thereof. In one embodiment, the present invention provides a method for preventing C. difficile infection (CDI) and / or C. difficile associated disease, comprising administering an effective amount of an AP-based agent of the present invention to a subject in need thereof (as a non-limiting example, a patient receiving or to be receiving an antibiotic).

[0239] In some embodiments, the present invention relates to a method for preventing C. difficile infection (CDI) and / or C. difficile-associated disease, comprising administering an effective amount of an AP-based agent of the present invention to a subject in need thereof, the subject being treated with a first-line antibiotic. "First-line antibiotic" refers to an antibiotic administered to a patient that may cause CDI and / or C. difficile-associated disease. These include antibiotics that most frequently result in CDI and / or C. difficile-associated disease, such as fluoroquinolones, cephalosporins, clindamycin, and penicillins.

[0240] In various embodiments, CDI and / or C. difficile-associated disease is treated or prevented in the setting of initial onset or relapse / recurrence (e.g., by continuing or reinitiating antibiotic therapy). For example, in patients who have previously suffered from CDI, the AP-based medicament of the present invention may be administered at the first symptom of relapse. As a non-limiting example, symptoms of relapse may include, in mild cases, about 5 to about 10 watery bowel movements per day, no significant fever and only mild abdominal cramps, but blood tests may show a mild elevation of the white blood cell count 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 to 104° F.), rectal bleeding, severe abdominal pain (e.g., with tenderness), abdominal distension, and high white blood cell count (e.g., about 15,000 to about 40,000).

[0241] CDI and / or C. difficile-related disease, whether first onset or relapse / recurrence, may be diagnosed by any of the symptoms described herein (e.g., watery diarrhea about 3 or more times per day for about 2 or more days, mild to severe abdominal cramps and pain, fever, blood or pus in the stool, nausea, dehydration, loss of appetite, weight loss, etc.). CDI and / or C. difficile-related disease, whether first onset or relapse / recurrence, may also be diagnosed by enzyme immunoassays (e.g., to detect C. difficile toxin A or B antigens and / or glutamine dehydrogenase (GDH) produced by C. difficile organisms), polymerase chain reaction (e.g., to detect 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 Toxins A / B, Wampole Toxin A / B Quik Chek, Wampole C.diff Quik Chek Complete, Remel Xpect Clostridioides difficile (formerly Clostridium difficile) Toxin A / B, Meridian Premier Toxins A / B, Wampole C.difficile Tox A / B II, Remel Prospect Toxin A / B EIA, Biomerieux Vidas C.difficile Toxin A&B, BD Geneohm C.difficile, Prodesse Progastro CD, and Cepheld Xpert C.difficile. In various embodiments, the clinical sample is a fecal sample from the patient. Additionally, a flexible sigmoidoscopy "scope" exam and / or abdominal x-rays and / or computed tomography (CT) scans, which provide images of the colon, may be used to evaluate the patient (e.g., looking for characteristic creamy white or yellow plaques attached to the colon wall).Additionally, biopsies (eg, of any area of ​​the GI tract) can be used to evaluate patients for possible CDI and / or C. difficile-associated disease.

[0242] In some embodiments, the methods and uses of the present invention include those in which an initial therapy and / or adjunctive therapy is administered to the subject. Initial therapy and / or adjunctive therapy refers to a therapy used to treat, for example, a microbiome-mediated disorder or disease upon detection of such disorder or disease. In one embodiment, initial therapy and / or adjunctive therapy refers to a therapy used to treat CDI and / or C. difficile-associated disease upon detection of such disease. In some embodiments, the initial therapy and / or adjunctive therapy is one or more of metronidazole, vancomycin, fidaxomicin, rifaximin, charcoal-based binders / adsorbents, fecal bacteriotherapy, probiotic therapy, and antibody therapy. In various embodiments, the methods and uses of the present invention include the use of alkaline phosphatase as an adjunct to any of these initial therapy and / or adjunctive therapies (including simultaneous or sequential administration). In various embodiments, the methods and uses of the present invention include administration of an AP-based agent as described herein to a subject undergoing initial therapy and / or adjunctive therapy.

[0243] In various embodiments, the AP-based medicament of the present invention is administered to a subject suffering from increased mucosal permeability of gastrointestinal tract.In some embodiments, increased mucosal permeability of gastrointestinal tract is the result of reduced intestinal perfusion or ischemia.Ischemia, i.e. lack of oxygen supply by blood flow, can be caused by, for example, heart failure, congenital heart disease, congestive heart failure, coronary heart disease, ischemic heart disease, injury, trauma, or surgery.

[0244] In some embodiments, increased mucosal permeability of the gastrointestinal tract is associated with or caused by autoimmune bowel disease and inflammatory bowel disease (IBD), such as celiac disease, Crohn's disease, and colitis (e.g., ulcerative colitis). Thus, in some embodiments, the present invention provides a method for treating or preventing autoimmune bowel disease and inflammatory bowel disease (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 group of inflammatory conditions of the large intestine, and sometimes 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, infectious colitis, and indeterminate colitis.

[0245] In some embodiments, the present invention provides a method for treating celiac disease. In some embodiments, the present invention provides a method for treating gastrointestinal disorders associated with celiac disease. Celiac disease is an autoimmune disease that can occur in genetically predisposed people in whom ingestion of gluten leads to damage to the small intestine. Individuals with celiac disease have increased intestinal permeability, which allows gluten breakdown products (the antigens that cause celiac disease) to reach gut-associated lymphoid tissues, initiating an inflammatory response including the release of inflammatory cytokines and recruitment of T cells. Celiac disease is characterized by atrophy of the small intestinal villi and chronic inflammation of the small intestinal mucosa, which can cause a wide variety of symptoms, including 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 GI symptoms, abdominal symptoms, and non-GI symptoms.

[0246] Methods for measuring improvement of one or more symptoms of celiac disease include assessment of the lactulose to mannitol (LAMA) ratio, a laboratory biomarker of intestinal permeability (Kelly et al., (2012) Aliment Pharmacol Ther 2013;37:252-262, the disclosure of which is incorporated herein by reference in its entirety); measurement of anti-transglutaminase antibody levels; and assessment of symptoms of celiac disease, such as the Celiac Disease Patient Reported Outcomes (CeD PRO), Gastrointestinal Symptom Rating Scale (GSRS), Celiac Disease Gastrointestinal Symptom Rating Scale (CeD GSRS), Bristol Stool Form Scale (BSFS), General Well-Being Questionnaire, Short Form 12 Health Survey Version 2, as disclosed, for example, in WO / 2015 / 154010, the disclosure of which is incorporated herein by reference in its entirety. 2) (SF12V2), the Celiac Disease Quality of Life Questionnaire (CeD-QoL), and the Clinician Global Assessment of Disease Activity (CGA). In various embodiments, the methods of the invention for treating celiac disease provide a therapeutic effect as assessed by one or more of these measures.

[0247] In some embodiments, the methods treat celiac disease and allow the subject to introduce gluten into their diet without substantial symptoms.

[0248] In various embodiments, increased mucosal permeability of the gastrointestinal tract is associated with or results from 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.

[0249] 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 appetite loss. In some embodiments, the method reduces, eliminates, or prevents one or more of nutritionally irreversible weight loss and loss of adipose tissue storage. In some embodiments, the method treats or prevents sarcopenia.

[0250] In some embodiments, the 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, mercurialism (acropylgia), and hormone deficiencies.

[0251] In various embodiments, the inventive treatment or prevention of cachexia with an AP-based agent of the invention is used in combination with, or in patients undergoing treatment with, one or more of: β-hydroxyβ-methylbutyrate (HMB), various progestins (e.g., megestrol acetate), anabolic androgenic steroids (e.g., oxandrolone), thalidomide and cytokine antagonists, cannabinoids, omega-3 fatty acids (e.g., eicosapentaenoic acid (EPA)), nonsteroidal anti-inflammatory drugs, prokinetic agents, ghrelin and ghrelin receptor agonists, anabolic catabolic transforming agents such as MT-102, selective androgen receptor modulators, cyproheptadine, and hydrazine.

[0252] In some embodiments, increased mucosal permeability of the gastrointestinal tract is associated with or caused by acquired immune deficiency syndrome (AIDS). Thus, in some embodiments, the present invention provides a method for treating gastrointestinal disorders associated with acquired immune deficiency syndrome (AIDS). Gastrointestinal disorders are the most frequent complaint in patients with human immunodeficiency virus 1 (HIV-1) or human immunodeficiency virus 2 (HIV-2) associated AIDS. Gastrointestinal manifestations of HIV disease include diarrhea, dysphagia, odynophagia, 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).

[0253] Progressive HIV infection often results in GI tract damage, microbial translocation, inflammation, and immune activation that promote disease progression to AIDS. The term "HIV enteropathy" has been used to describe the changes in mucosal structure and function associated with gut-mediated immune dysfunction, as well as to indicate a clinical syndrome of chronic diarrhea without an identified infectious source. In addition to chronic diarrhea, HIV enteropathy is often characterized by increased GI inflammation, increased intestinal permeability, and malabsorption of bile acids and vitamin B12, abnormalities believed to result from the direct or indirect effects of HIV on the intestinal mucosa (Brenchley JM, Douek DC. Mucosal Immunol 2008;1:23-30). Clinical consequences include decreased absorption of fats and carbohydrates, a tendency for small intestinal transit time to decrease, and jejunal atrophy. In various embodiments, the methods of the invention effectively treat the symptomatic effects of HIV enteropathy. In various embodiments, the methods of the invention prevent, slow, or reverse the progression of HIV infection to AIDS. In various embodiments, the methods of the invention prevent or slow the fatal progression of AIDS.

[0254] Furthermore, the HIV-1 subtype with which a subject is infected may be a factor in the rate of progression to AIDS. In various embodiments, the methods effectively treat patients infected with HIV-1 subtypes C, D, and G. In another embodiment, the methods effectively treat patients infected with HIV-1 subtype A.

[0255] In some embodiments, the present invention provides methods of treating various GI disorders associated with HIV infection and / or AIDS, for example, the present invention provides methods of treating HIV-mediated intestinal dysbiosis and GI barrier dysfunction, which in various embodiments may be due to HIV, antibiotics administered to a subject infected with HIV, and / or medications being administered to a subject infected with HIV. For example, a subject infected with HIV may be taking one or more nucleoside analogs, such as deoxyadenosine analogs (e.g., didanosine, vidarabine), adenosine analogs (e.g., BCX4430), deoxycytidine analogs (e.g., cytarabine, emtricitabine, lamivudine, zalcitabine), guanosine and deoxyguanosine analogs (e.g., abacavir, acyclovir, entecavir), thymidine and deoxythymidine analogs (e.g., stavudine, telbivudine, zidovudine), and deoxyuridine analogs (e.g., idoxuridine, trifluridine). In some embodiments, a subject infected with HIV may be taking one or more drugs of a highly active antiretroviral therapy (HAART) regimen. Exemplary HAART drugs include entry or fusion inhibitors (e.g., maraviroc, enfuvirtide), nucleoside reverse transcriptase inhibitors (NRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs) 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).

[0256] In various embodiments, the method reduces local inflammation, alters the composition of the GI microbiota, enhances the clearance of products of microbial translocation from the circulation, and restores the enterocyte barrier in subjects infected with HIV and / or subjects with AIDS. In one embodiment, the method of the invention reduces GI tract damage and intestinal dysbiosis in subjects infected with HIV and / or subjects with AIDS. For example, the method of the invention may reverse the changes in the GI microbiota observed in subjects infected with HIV or subjects with AIDS. By way of example, these changes in the GI microbiota that can be reversed by the method of the invention include changes in the microbiota characterized by an increase in pathogenic commensals such as Staphylococcus genera, Psedomonas genera, and Enterobacteriaceae family members with pro-inflammatory capabilities, as well as enteropathogenic bacteria (including Psudemonas, Xanthomonas, Bacillus, and Burkholderia genera) that catabolize tryptophan to kynurenine derivatives. In one embodiment, the method reduces GI barrier dysfunction in subjects infected with HIV and / or subjects with AIDS. For example, the methods of the invention may reverse increased intestinal permeability (e.g., leaky gut syndrome) in subjects infected with HIV and / or subjects with AIDS. In one embodiment, the methods of the invention reduce microbial translocation or translocation of microbial products and inflammatory mediators (e.g., LPS) into the systemic circulation in subjects infected with HIV and / or subjects with AIDS. In such methods, the levels of LPS, EndoCAb, sCD14, and I-FABP in the plasma of the subject may be reduced. In one embodiment, the methods of the invention reduce immune activation and inflammation (e.g., local and systemic immune activation and inflammation) in subjects infected with HIV and / or subjects with AIDS. For example, the methods of the invention may reduce inflammation in the gut-associated lymphoid tissue (GALT) and increase the number of CD4+ cells and Th17 cells. The methods of the invention may 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.

[0257] In some embodiments, the present invention provides methods for treating or preventing dysbiosis and gastrointestinal dysfunction in patients with cystic fibrosis (CF). Cystic fibrosis (CF), a genetic disease, is associated with mutations in the CF transmembrane conductance regulator (CFTR), which regulates ion and water permeability of epithelial cells. In some embodiments, the methods are used to treat subjects who are homozygous for one or more mutations in the CFTR gene. In some embodiments, the subjects are 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 translational mutations. Examples of CFTR mutations include 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, W128 CFTR mutations include, but are not limited to, 2X, 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+10kbC>T, 1898+1G>A, 2183AA>G, and / or 5 / 7 / 9T. In various embodiments, the methods of the invention are used to treat CF patients with one or more of the 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 an F508del mutation. Methods for screening a patient's genotype for CFTR mutations are known and can be performed, for example, by DNA sequencing, such as bidirectional sequencing.

[0258] CF patients often exhibit symptoms such as chronic respiratory infections and dysfunction of the gastrointestinal (GI) mucosal surface, resulting in significant morbidity and mortality. One of the earliest signs of CF is GI dysfunction, including severe recurrent intestinal obstruction and nutrient malabsorption, which results in growth failure. CF patients also exhibit GI dysbiosis, such as an overabundance of E. coli and a reduced relative abundance of Bifidobacterium species in the fecal microbiota. In various embodiments, the methods of the present invention effectively treat one or more GI-related symptoms in CF patients.

[0259] Methods for measuring changes and / or improvements in Gl tract function can include, but are not limited to, endoscopy to directly examine the epithelium and mucosa; histological evaluation and / or tissue procurement to directly assess structural changes and / or immune biomarkers; urinalysis to assess permeability to non-absorbable sugars and LPS levels; stool testing (e.g., by PCR) to assess changes in inflammation and / or microbiota; and / or blood tests to assess specific markers including CD4+ cell count, Th17 cell count, and / or LPS levels.

[0260] In some embodiments, the present invention provides a method 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).In many cases, hypothyroidism can slow down the digestive tract, causing constipation, or even stop digestive tract motility altogether.The method of the present invention can alleviate one or more gastrointestinal symptoms associated with hypothyroidism.

[0261] In some embodiments, the present invention provides methods for preventing or treating necrotizing enterocolitis (NEC).

[0262] In various embodiments, the methods of the invention relate to pediatric subjects for the prevention or treatment of 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 an infant about 1 day old, about 2 days old, about 3 days old, about 4 days old, about 5 days old, about 6 days old, about 1 week old, about 2 weeks old, about 3 weeks old, about 4 weeks old, about 1 month old, about 2 months old, about 3 months old, about 4 months old, about 5 months old, about 6 months old, about 7 months old, about 8 months old, about 9 months old, about 10 months old, about 11 months old, or about 12 months old. In various embodiments, the pediatric subject is formula and / or milk fed, hi various embodiments, the pediatric subject is undergoing or has recently undergone antibiotic treatment.

[0263] In various embodiments, the pediatric subject is a premature infant. In some embodiments, the premature infant is born at a gestational age of less than 37 weeks. In some embodiments, the premature infant is born at a gestational age of 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. In other embodiments, the pediatric subject is a full-term infant, e.g., an infant born after about 37 weeks of gestational age. In some embodiments, the pediatric subject may exhibit one or more of fetal distress, shock, sepsis, or congenital heart disease. In various embodiments, the pediatric subject is of low birth weight. In various embodiments, the pediatric subject weighs less than about 5 pounds, less than about 4 pounds, less than about 3 pounds, or less than about 2 pounds.

[0264] In various embodiments, the methods of the invention relate to a pregnant woman being treated for the prevention or treatment of NEC, hi some embodiments, the pregnant woman is undergoing or has recently undergone antibiotic treatment.

[0265] 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 methods treat disease at any of these stages.

[0266] [ka]

[0267] In various embodiments, the methods of the invention effectively treat one or more symptoms of NEC, including any of the symptoms described above, as well as symptoms known in the art, including GI symptoms, abdominal symptoms, and non-GI symptoms. In various embodiments, the methods of the invention effectively prevent the occurrence of NEC in a subject, such as a pediatric subject. In various embodiments, the methods of the invention effectively prevent the progression of NEC in a subject, such as a pediatric subject, for example, from stage I to stage II, or from stage II to stage III. In various embodiments, the methods of the invention effectively cause regression of NEC in a subject, such as a pediatric subject, for example, from stage III to stage II, or from stage I to complete resolution, or from stage II to stage I or complete resolution.

[0268] Intestinal dysbiosis is associated with the development of NEC and can be detected in a subject before any clinical evidence of disease. In various embodiments, the methods of the invention effectively restore normal microflora in the intestinal tract of a treated subject. In some embodiments, the methods of the invention maintain normal microflora in the intestinal tract. For example, in some embodiments, the methods of the invention maintain a healthy balance (e.g., a healthy ratio and / or a healthy distribution) of gut microflora in a subject. In another embodiment, the methods of the invention treat or prevent overgrowth of one or more pathogenic microorganisms in the GI tract. In certain embodiments, the methods of the invention effectively reduce the levels of Clostridium butyricum and / or Clostridium perfringens in the intestinal tract.

[0269] Methods for measuring the improvement of one or more symptoms of NEC include imaging modalities such as X-ray and ultrasound. Methods for measuring changes and / or improvements in Gl tract function can include, but are not limited to, endoscopy or colonoscopy to directly examine epithelium and mucosa; histological evaluation and / or tissue acquisition to directly evaluate structural changes and / or immune biomarkers; fecal testing (e.g., by PCR) to evaluate inflammation and / or changes in the microbiota; and / or blood testing to evaluate specific markers and cells.

[0270] 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 glucose, elevated fasting insulin, reduced postprandial glucose clearance, insulin resistance, impaired glucose tolerance, obesity, and combinations thereof. For example, the methods of the present invention can be used to treat subjects with metabolic syndrome and abdominal obesity (e.g., waist circumference of 40 inches or more for men or 35 inches or more for women), blood triglyceride levels of 150 mg / dL or more, HDL of less than 40 mg / dL for men or less than 50 mg / dL for women, systolic blood pressure of 130 mmHg or more, or diastolic blood pressure of 85 mmHg or more, and / or fasting blood glucose of 100 mg / dL or more. Additional metabolic diseases that can be treated using the methods of the present invention include those described in US2013 / 0251701, US2011 / 0206654, and US2004 / 0115185, the contents of which are incorporated by reference in their entireties.

[0271] In one embodiment, the metabolic disease is obesity. Early exposure to antibiotics (e.g., within about 2 years of life) can disrupt the microbiome and lead to potential disease. Bailey, et al. JAMA Pediatr. 168(11), Nov 2014 (the entire contents of which are incorporated herein by reference) describes how early exposure to antibiotics is associated with obesity. Thus, in some embodiments, the method protects the pediatric microbiome and prevents diseases such as obesity. Furthermore, an altered ratio of Firmicutes to Bacteroidetes bacterial phyla is often found in obese individuals. Thus, 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 preserves the normal diversity of bacteria in the intestinal tract, such as, for example, Bacteroidetes, Proteobacteria, and Firmicutes, thereby treating or preventing obesity. Furthermore, AP-based agents can affect fat absorption in the gastrointestinal tract. Thus, in some embodiments, the present invention provides methods for treating or preventing obesity by limiting GI fat absorption. In various embodiments, the methods of the present invention are effective in inducing weight loss or preventing weight gain. In some embodiments, the subject may have undergone or is likely to undergo surgery on the digestive system; may be about 80-100 pounds overweight; or has a BMI of about 35 kg / m 2 or may have obesity-related health problems. In some embodiments, the subject may have dyslipidemia, including hyperlipidemia and hyperlipoproteinemia.

[0272] In another embodiment, the metabolic disease is diabetes. In various embodiments, the invention relates to treating diabetes (type 1 or type 2) and / or impaired glucose tolerance. In various embodiments, the invention relates to preventing diabetes (type 1 or type 2) and / or impaired glucose tolerance. In various embodiments, the invention relates to reducing complications due to diabetes (type 1 or type 2) and / or impaired glucose tolerance. In some embodiments, the invention relates to methods for treating diabetes and subjects at risk for one or more of insulin resistance, pre-diabetes, impaired fasting glucose (IFG), and impaired glucose tolerance (IGT).

[0273] In various embodiments, the present invention relates to the treatment of type 1 diabetes with AP-based drugs. Type 1 diabetes, previously known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin. Treatment is often by intensive insulin regimens that attempt to mimic the body's normal insulin secretion pattern, often including basal and bolus insulin coverage. For example, one common regimen is to administer a long-acting insulin (e.g., including glargine / detemir) once or twice daily before or after meals with a fast-acting insulin (e.g., including aspart, glulisine, lispro) and correct hyperglycemia as needed (e.g., by monitoring with a glucometer). Doses administered before or after meals or as needed to correct hyperglycemia are sometimes referred to as bolus doses. Another common regimen involves administration, including continuous administration via an insulin pump (or continuous subcutaneous insulin infusion device (CSII)), of a fast-acting insulin (as described herein, including, e.g., aspart, glulisine, lispro). In various embodiments, the AP-based drug can replace any of the insulins used in various regimens, including when insulin is not providing effective treatment to the patient, and the AP-based drug can improve patient compliance because it can allow easier self-administration compared to various forms of insulin that must be administered at various doses throughout the day even with insulin pumps that require programming. In addition, the AP-based drug can counter common administration frustrations for diabetic patients, such as dawn phenomenon. Alternatively, the AP-based drug can be used as an adjunct to any of the type 1 diabetes treatments described herein, for example, to normalize a patient's regimen and avoid blood glucose "dip" (e.g., hypoglycemia, e.g., blood glucose below about 70 mg / dL) and "spike" (e.g., hyperglycemia, e.g., blood glucose above about 200 mg / dL) that many patients suffer from.Thus, in some embodiments, the AP-based agent may be used to treat symptoms such as, for example, tremors, anxiety, irritability, palpitations, tachycardia, pallor, chills, cold clammy feeling, dilated pupils (mydriasis), hunger, borborygmi, nausea, vomiting, abdominal discomfort, headache, altered mental status, impaired judgment, nonspecific discomfort, paresthesia, rejection, irritability, combativeness, combativeness, agitation, personality changes, emotional lability, fatigue, weakness, blunted affect, lethargy, daydreaming, sleep, confusion, amnesia, loss of consciousness, Symptoms associated with hypoglycemia may be treated or prevented, including: dizziness or dizziness, delirium, staring, "glassy" eyes, blurred vision, double vision, flashes of light in the field of vision, automatism, difficulty speaking, slurred speech, ataxia, incoordination, focal or global motor disorders, 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 may treat or prevent symptoms associated with hyperglycemia, including, for example, hyperphagia, polydipsia, 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, type 1 diabetes patients may be administered additional drugs to supplement insulin therapy. In some embodiments, AP-based drugs are used in this manner. AP-based drugs may provide additional therapeutic benefits to patients who struggle to manage type 1 diabetes with insulin therapy alone. In some embodiments, patients who struggle to manage type 1 diabetes with insulin therapy alone have inadequate glycemic control, as described herein.

[0274] In some embodiments, the AP-based agent is used to reduce a patient's blood glucose level to less than about 10 mM, for example, within the range of about 4 mM to about 7 mM.

[0275] In some embodiments, the present invention provides methods for treating type 1 or type 2 diabetes comprising administering an effective amount of an AP-based agent.

[0276] In many embodiments, including those in which the AP-based agent prevents diabetes and / or treats a pre-diabetic condition, a patient is at risk for diabetes if they are characterized by one or more of the following: being physically inactive; having a parent or sibling with diabetes; having a family history associated with a high incidence of diabetes selected from African American, Alaska Native, American Indian, Asian American, Hispanic / Latin American, or Pacific Islander American; giving birth to an infant weighing more than 9 pounds; being diagnosed with gestational diabetes; having high blood pressure of about 140 / 90 mmHg or greater; being treated for high blood pressure; having HDL cholesterol levels less than about 35 mg / dL and / or triglyceride levels greater than about 250 mg / dL; having polycystic ovary syndrome (PCOS); and having cardiovascular disease.

[0277] In various embodiments, the AP-based drug may be used to treat diabetes in a hospitalized setting. For example, in some embodiments, the AP-based drug may be administered to a patient in a diabetic coma. In some embodiments, the patient may be administered to a patient with one or more of the following: severe diabetic hypoglycemia, advanced diabetic ketoacidosis (e.g., advanced enough to result in loss of consciousness, where contributing factors may include one or more of hyperglycemia, dehydration, shock, and exhaustion), and hyperosmolar nonketotic coma (e.g., where one or more of hyperglycemia and dehydration are contributing factors). In these embodiments, the AP-based drug may be used in conjunction with standard treatment regimens for diabetic coma, including administering one or more of glucose, glucagon, insulin, and fluids (e.g., saline with potassium and / or other electrolytes), all of which are optionally administered intravenously. In some embodiments, the AP-based drug may replace insulin in these treatment regimens, and is optionally administered orally.

[0278] In various embodiments, AP-based drugs can be used to treat pregnant women at high risk for gestational diabetes. Some pregnant women develop gestational diabetes as early as 24 weeks of pregnancy, and if left untreated, gestational diabetes can lead to premature birth and stillbirth. In some embodiments, the present invention provides a method for preventing and / or treating gestational diabetes in pregnant women. In various embodiments, the methods of the present invention can also be utilized to treat pregnant women at high risk for inflammation, such as GI inflammation. In some embodiments, the methods reduce inflammation in pregnant women.

[0279] Additionally, in various embodiments related to diabetes, the patient may be administered or co-administered one or more additional drugs. Exemplary additional drugs include insulin or any antidiabetic agent (e.g., insulin secretagogues such as biguanides, sulfonylureas or meglitinides, inhibitors of α-glucosidase, thiazolidinediones, etc.). The methods of treatment described herein may, in various embodiments, include administering an AP-based drug to a patient who is administered one or more additional drugs and / or non-insulin diabetic drugs.Additional medications include sulfonylureas (e.g., DYMELOR (acetohexamide), DIABINESE (chlorpropamide), ORINASE (tolbutamide), and TOLINASE (tolazamide), GLUCOTROL (glipizide), GLUCOTROL XL (extended release), DIABETA (glyburide), MICRONASE (glyburide), GLYNASE PRESTAB (glyburide), 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 TRADJENTA ( sodium-glucose cotransporter type 2 (SGLT2) inhibitors (e.g., INVOKANA (canaglifozin)); and combination drugs (e.g., GLUCOVANCE, which combines glyburide (a sulfonylurea) with metformin, METAGLIP, which combines glipizide (a sulfonylurea) with metformin, and AVANDAMET, which uses both metformin and rosiglitazone (AVANDIA) in one drug, KAZANO (alogliptin and metformin), and OSENI (alogliptin + pioglitazone)).

[0280] Other additional medications 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, and NOVOLOG Mix. FLEXPEN 70-30 subcutaneous injection, oral GLYBURIDE-METFORMIN, oral Acarbose, SYMLINPEN 60 subcutaneous injection, oral GLUCOTROl XL, NOVOLIN R injection, oral GLUCOTROL, oral DUETACT, oral sitagliptin, SYMLINPEN 120 subcutaneous injection, HUMALOG KWIKPEN subcutaneous injection, oral JANUMET XR, oral GLIPIZIDE-METFORMIN, oral CYCLOSET, HUMALOG MIX 75-25 subcutaneous injection, oral nateglinide, HUMALOG Mix 75-25 KWIKPEN subcutaneous injection, HUMULIN 70 / 30 subcutaneous injection, oral PRECOSE, APIDRA subcutaneous injection, Humulin R injection, oral GentaDuet, Victoza 3-Pak subcutaneous injection, Novolin 70 / 30 subcutaneous injection, NOVOLIN N subcutaneous injection, insulin detemir subcutaneous injection, oral micronized glyburide, 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 / 30 Pen subcutaneous injection, oral colesevelam, oral sitagliptin-metformin, oral DIABETA, human regular insulin 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 These include XR, oral miglitol, recombinant human NPH insulin subcutaneous, NPH insulin and human regular insulin subcutaneous, oral tolazamide, oral mifepristone, insulin aspart protamine-insulin aspart subcutaneous, oral repaglinide-metformin, oral saxagliptin-metformin, oral linagliptin-metformin, oral NESINA, oral OSENI, oral tolbutamide, insulin lispro protamine and lispro subcutaneous, pramlintide subcutaneous, insulin glulizine subcutaneous, oral pioglitazone-glimepiride, oral PRANDIMET, NOVOLOG PenFill subcutaneous, oral linagliptin, exenatide microspheres subcutaneous, oral KORLYM, oral alogliptin, oral alogliptin-pioglitazone, oral alogliptin-metformin, and oral canagliflozin.

[0281] Other additional medications include lispro (HUMALOG), aspart (NOVOLOG), glulisine (APIDRA), regular (NOVOLIN R or HUMULIN R), NPH (NOVOLIN N or HUMULIN N), glargine (LANTUS), detemir (LEVEMIR), HUMULIN or NOVOLIN 70 / 30, and NOVOLOG Mix 70 / 30 HUMALOG Mix 75 / 25 or 50 / 50.

[0282] In various embodiments, the present invention is used to treat or prevent various neurodegenerative diseases. In some embodiments, the neurodegenerative disease is 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 parkinsonism (including but not limited to, idiopathic Parkinson's disease, vascular parkinsonism, drug-induced parkinsonism, dementia with Lewy bodies, hereditary Parkinson's disease, and early-onset 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, Hirayama disease, The present invention relates to a method for treating a variety of conditions, including the following:

[0283] In various embodiments, the present invention is used to treat spinal cord injury and / or spinal cord injury-associated dysbiosis. Spinal cord injury has been shown to increase intestinal permeability and bacterial translocation from the gut. These changes are associated with activation of immune cells in the gut-associated lymphoid tissue (GALT) and alterations in the gut microbiome. Gut dysbiosis associated with spinal cord injury is associated with impaired motor recovery, as well as exacerbated pathology of neuropathology and intraspinal inflammation. In some embodiments, the present invention prevents and / or treats bacterial translocation and gut dysbiosis associated with spinal cord injury. In some embodiments, the methods of the present invention provide 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 the GALT. In some embodiments, the methods of the present invention reduce neurological pathology and intraspinal inflammation associated with spinal cord injury.

[0284] In various embodiments, AP-based drugs can be used to treat traumatic brain injury (TBI) and its symptoms. In various embodiments, AP-based drugs can be used to treat various conditions related to trauma to the brain or spinal cord (e.g., caused by physical forces acting on the skull or spinal column), ischemia, stroke, respiratory arrest, cardiac arrest, cerebral thrombosis or cerebral embolism, neurological problems (caused by AIDS, cerebral hemorrhage, encephalomyelitis, hydrocephalus, postoperative events, brain infections, and concussion), or increased intracranial pressure.

[0285] In various embodiments, AP-based drugs may be used to treat changes in brain function caused by external forces or other evidence of brain pathology.

[0286] In various embodiments, AP-based drugs may be used to treat TBI classified by various methods. For example, clinical severity (e.g., Glasgow Coma Scale (GSC), a 15-point scale that observes the motor, verbal, and eye-opening responses of the patient after resuscitation (see Teasdale & Jennett (1974) Lancet, 2, 81-84)) may be used. In various embodiments, AP-based drugs may be used to treat mild, moderate, or severe head injuries as defined by the GSC. Additionally, TBI may be classified using a pathology-based classification of TBI, for example, based on an assessment of abnormalities that require treatment from an anatomical and physiological perspective. The most common pathophysiological model used to describe TBI is to evaluate whether the injury is either primary or secondary. Primary injury is generally accepted to be due to the initial immediate injury and is unavoidable. Secondary injury refers to avoidable subsequent injuries such as hypoxia, hypertension, or hypercapnia. Additionally, TBI can be assessed by computed tomography (CT) or magnetic resonance imaging (MRI), as well as electroencephalogram (EEG) and intracranial pressure (ICP), among others.

[0287] In various embodiments, AP-based agents can be used to treat or prevent stroke.For example, the stroke being treated can be ischemic or hemorrhagic.In various embodiments, hemorrhagic stroke is a neurological disorder of cerebrovascular origin that lasts for more than 24 hours or is interrupted by death within 24 hours.Hemorrhagic stroke can be cerebral hemorrhage (intracerebral hemorrhage) due to either intraparenchymal or intraventricular hemorrhage.Hemorrhagic stroke can be subarachnoid hemorrhage. In various embodiments, the stroke is ischemic, e.g., classified according to the Oxford Community Stroke Project classification (OCSP, i.e., total anterior circulation infarction (TACI), partial anterior circulation infarction (PACI), lacunar infarction (LACI), or posterior circulation infarction (POCI)), or the TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification (i.e., stroke is classified as due to (1) atherosclerotic thrombosis or embolism of the large arteries, (2) embolism of cardiac origin, (3) complete occlusion of small vessels, (4) other confirmed causes, or (5) cryptogenic (two possible causes, cause not identified, or incomplete investigation)).

[0288] 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 pus-forming and other pathogenic organisms, or their toxins, in the blood or tissues. In some embodiments, the present invention provides methods for treating or preventing sepsis (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).

[0289] 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 an increase in serum creatinine levels. In acute renal failure, glomerular filtration rate decreases over a period of days to weeks. This results in reduced excretion of nitrogenous waste products and failure to maintain fluid and electrolyte balance. Patients with acute renal failure are often asymptomatic, and the condition is diagnosed by observed increases in blood urea nitrogen (BUN) and serum creatinine levels. Complete renal failure is indicated when serum creatinine levels rise by at least 0.5 mg per dL per day and urine output is less than 400 mL per day (oliguria). The AP-based drugs described herein can be used not only to treat renal failure, but also to improve renal cases in which renal function is at least partially impaired or reduced.

[0290] In various embodiments, the AP-based drug may be used to treat or prevent congestive heart failure. In various embodiments, the AP-based drug may be used to treat or prevent one or more symptoms of congestive heart failure, such as shortness of breath, fatigue, and leg swelling. In various embodiments, the AP-based drug may be used to treat or prevent congestive heart failure due to coronary artery disease, including a history of myocardial infarction (heart attack), high blood pressure, atrial fibrillation, valvular heart disease, excessive alcohol use, infection, and cardiomyopathy of unknown etiology. In various embodiments, the AP-based drug may be used to treat or prevent class I congestive heart failure. In various embodiments, the AP-based drug may be used to treat or prevent class II congestive heart failure. In various embodiments, the AP-based drug may be used to treat or prevent class III congestive heart failure. In various embodiments, the AP-based drug may be used to treat or prevent class IV congestive heart failure.

[0291] In various embodiments, AP-based drugs may be used to treat or prevent chronic fatigue syndrome, including myalgic encephalomyelitis (ME) and, more recently, systemic exertion intolerance disorder (SEID). In various embodiments, AP-based drugs may be used to treat or prevent one or more symptoms of chronic fatigue syndrome, including fatigue, loss of memory or concentration, sore throat, enlarged lymph nodes in the neck or armpits, unexplained muscle pain, joint pain, headache, non-restorative sleep, and extreme exhaustion lasting more than 24 hours after physical or mental exertion.

[0292] In various embodiments, the AP-based drug can be used to treat or prevent pancreatitis. In various embodiments, the pancreatitis is mild acute pancreatitis. In various embodiments, the pancreatitis is severe pancreatitis, for example, with organ failure, necrosis, infectious necrosis, pseudocyst, and abscess. In various embodiments, the AP-based drug can be used to treat or prevent one or more symptoms of pancreatitis, such as epigastric abdominal pain or vague abdominal pain that may radiate to the back, serum amylase or lipase levels greater than about 3 times the upper limit of normal, and imaging tests (e.g., CT, MRI, abdominal ultrasound, or endoscopic ultrasound) with characteristic changes.

[0293] In various embodiments, AP-based agents may be used to treat or prevent pancreatitis in subjects with one or more risk factors, such as gallstones, ethanol 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).

[0294] Several scoring systems are used to predict the severity of a pancreatitis attack. Examples include APACHE II, Ranson, BISAP, and Glasgow. In various embodiments, pancreatitis is defined under the modified Glasgow criteria by at least three of the following: pO2<60mmHg or 7.9kPa, age >55, white blood cells >15 with increased neutrophils, calcium <2mmol / L, renal urea >16mmol / L, enzymes lactate dehydrogenase (LDH) >600iu / L, aspartate transaminase (AST) >200iu / L, albumin <32g / L, and sugar glucose >10mmol / L.

[0295] In some embodiments, AP-based agents are used to reduce the incidence and / or severity of complications associated with IV administration of beta-lactam antibiotics to allogeneic HCT recipients, including, but not limited to, aGVHD and / or VRE colonization and / or infection.

[0296] In certain embodiments, the present invention provides compositions and methods for preventing VRE colonization and / or infection, hi some embodiments, the methods of the present invention prevent intestinal dysbiosis associated with VRE colonization and / or infection.

[0297] In some embodiments, methods are provided for preventing VRE colonization and / or infection in a transplant recipient comprising administering a beta-lactamase agent of the invention.

[0298] In certain embodiments, the present 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 an allogeneic 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 receiving or has been receiving IV beta-lactam antibiotics.

[0299] In some embodiments, the methods of the invention prevent intestinal dysbiosis associated with aGVHD. In various embodiments, the methods of the invention prevent loss of gut microbiota diversity and / or intestinal dysbiosis associated with aGVHD. For example, the methods of the invention contemplate administration of compositions to modulate and / or reduce the monodominance of any given bacterial strain (e.g., Enterococcus) associated with aGVHD. Further embodiments of the invention provide compositions and methods for preventing loss of microbiome diversity in transplanted subjects.

[0300] In some embodiments, methods are provided for reducing the incidence and / or severity of aGVHD in a transplant recipient comprising administering a beta-lactamase agent of the invention. For example, reducing the severity of aGVHD can include patients with less than grade IV, which is classified as the most severe form of aGVHD.

[0301] In embodiments, the method relates to reducing the incidence and / or severity of aGVHD in a transplant recipient. In embodiments, the transplant recipient is a cancer patient, e.g., a patient who has undergone or has undergone radiation or chemotherapy and subsequently undergoes allogeneic HCT. In embodiments, the transplant recipient has a blood 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's tumor, chordoma, and chondrosarcoma.

[0302] aGVHD is the alteration of cells or tissues transplanted from a donor to a recipient due to the recognition by the recipient's immune system that the cells or tissues are foreign. Therefore, because class I MHC is present on a relatively large number of cells in the body, it is most desirable to transplant cells and tissues from people with the most matching class I MHC profile, followed by people with the most matching class II MHC profile. Thus, in most transplant recipients, aGVHD is due to activation of the immune system against mismatched class II MHC molecules and other polymorphic proteins (minor histocompatibility antigens).

[0303] One option for treating cancers of the blood and bone marrow is to kill the existing blood and bone marrow cells, for example by radiation or chemotherapy, and transplant similar cells from a healthy donor, called allogeneic hematopoietic stem cell transplant (allo-HCT).

[0304] In an embodiment, the method relates to acute and chronic forms of GVHD. Classical acute or fulminant forms of the disease (aGVHD) are usually observed within the first 100 days after transplantation and are a major challenge to the efficacy of transplantation due to the associated morbidity and mortality. Chronic forms of graft-versus-host disease (cGVHD) traditionally occur after 100 days. The emergence of moderate to severe cases of cGVHD adversely affects long-term survival. After bone marrow transplantation, T cells present in the graft, either as a contaminant or introduced into the host intentionally, attack the tissues of the transplant recipient after recognizing the host tissues as antigenically foreign. These T cells overproduce cytokines, including TNF-alpha and interferon-gamma (IFNγ). Tissue damage in cGVHD is primarily due to fibrosis. A wide range of host antigens can cause graft-versus-host disease, including human leukocyte antigens (HLA). However, GVHD can occur even when the donor is an HLA-identical sibling. aGVHD is characterized by selective damage to the liver, skin, mucosa, and GI tract. Tissue damage in aGVHD is primarily due to apoptosis. The severity of aGVHD is graded on a scale of I (mild) to IV (very severe) based on the extent and type of lesions / rash (skin), amount of diarrhea (GI), and serum bilirubin levels (liver). cGVHD is characterized by a much broader tissue distribution than aGVHD. The skin and lungs are considered the primary target organs of cGVHD, along with the GI tract, liver, eyes, musculoskeletal system, and hematopoietic system. A hyperacute and rapidly fatal form of aGVHD can occur within the first 2 weeks of allogeneic HCT, usually due to significant HLA mismatch or inadequate GVHD prophylaxis. Risk factors associated with cGVHD usually remain unchanged after adjustment for preceding aGVHD, suggesting that cGVHD is not simply an extension of preceding aGVHD.

[0305] In embodiments, the methods relate to reducing the incidence and / or severity of aGVHD. In embodiments, the methods contemplate administration of beta-lactamase to patients who have been treated with intravenous administration of beta-lactam antibiotics and who have one or more risk factors for aGVHD, such as HLA "mismatched" or unrelated donors, advanced patient age, female donor to male recipient, intensity of total body irradiation during conditioning regimen or conditioning regimen, and donor lymphocyte infusion. In embodiments, the methods reduce the incidence and / or severity of aGVHD symptoms, such as skin rash, gastrointestinal (GI) tract disorders, and liver symptoms.

[0306] In embodiments, the methods relate to GVHD defined by one or more of the Billingham criteria: 1) administration of an immunologically competent graft with viable and functional immune cells, 2) the recipient is immunologically histoincompatible, and 3) the recipient is immunocompromised such that the transplanted cells cannot be destroyed or inactivated.

[0307] In various embodiments, the AP-based medicament may be used to treat or prevent allergies. In various embodiments, the AP-based medicament may be used to treat or prevent atopic allergies or IgE-mediated allergies. In various embodiments, the AP-based medicament may 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, the AP-based medicament may be used to treat or prevent one or more of anaphylaxis, drug hypersensitivity, skin allergies, eczema, allergic rhinitis, urticaria, atopic dermatitis, dry eye disease, allergic contact allergies, food hypersensitivity, 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).

[0308] In various embodiments, the AP-based medicament may be used to treat or prevent asthma, including but not limited to allergic asthma. In various embodiments, the AP-based medicament may be used to reduce immunoglobulin E stimulation, airway hyperresponsiveness, mast cell infiltration, pulmonary eosinophilia, and accumulation of alternatively activated macrophages in the lung. In various embodiments, the AP-based medicament may be used to treat or prevent one or more of recurrent wheezing and intermittent airflow limitation.

[0309] 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, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig, a rabbit, a sheep, or a non-human primate, e.g., a monkey, a chimpanzee, or a baboon. In other embodiments, the subject and / or animal is a non-mammal, e.g., a zebrafish.

[0310] In various embodiments, the methods of the invention are useful for treating a human subject. In some embodiments, the human is a pediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. 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.

[0311] In certain embodiments, the human is in the range of ages 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 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.

[0312] kit The present invention provides a kit that can simplify the administration of the modified release formulations described herein.The kit is a collection of materials or components that includes at least one of the modified release formulations described herein.The exact nature of the components that are configured in the kit varies depending on its intended purpose.In one embodiment, the kit is configured for the purpose of treating human subjects.

[0313] Instructions for use may be included in the kit. The instructions will usually include specific language describing techniques to be used in using the components of the kit to affect a desired result, such as to treat a relevant disorder described herein. Optionally, the kit will also include other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring devices, dressings, or other useful paraphernalia readily recognized by those skilled in the art.

[0314] The materials and components assembled in the kit can be provided to the practitioner's store in any convenient and suitable manner that maintains their operability and usefulness. For example, the components can be provided at room temperature, refrigerated, or frozen temperatures. The components are typically contained within suitable packaging materials. In various embodiments, the packaging materials are preferably configured to provide a sterile, contaminant-free environment, by well-known methods. The packaging materials can have an exterior label indicating the contents and / or purpose of the kit and / or its components.

[0315] definition As used herein, "a," "an," or "the" may mean one or more.

[0316] Additionally, the term "about" when used in connection with a stated numerical indication means the stated numerical indication plus or minus up to 10% of the stated numerical indication. For example, the term "about 50%" encompasses a range from 45% to 55%.

[0317] An "effective amount" when used in relation to medical applications is an amount effective to provide a measurable treatment, prevention, or reduction in the rate of pathology of the disorder of interest.

[0318] As used herein, something is "decreased" if the activity and / or effect readout is reduced by a substantial amount, e.g., 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 at least about 100%), in the presence of an agent or stimulus compared to the absence of such modulation. As will be appreciated by one of skill in the art, in some embodiments, when activity is decreased, downstream readouts may decrease or increase.

[0319] Conversely, activity is "increased" if the activity and / or effect readout is increased by a substantial amount, e.g., 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 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, in the presence of an agent or stimulus compared to the absence of such agent or stimulus.

[0320] When referred to herein, all composition percentages are by weight of the total composition unless otherwise specified. As used herein, the word "include" and variations thereof are intended to be non-limiting such that the description of items in a list does not exclude other similar items that may also be useful in the compositions and methods of the present technology. Similarly, the terms "can" and "may" and variations thereof are intended to be non-limiting such that a description that an embodiment can or may include certain elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

[0321] 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, the invention or embodiments thereof may instead be described using alternative terms such as "consisting of" or "consisting essentially of."

[0322] As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under similar or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

[0323] The amount of the compositions described herein necessary to achieve a therapeutic effect can be empirically determined according to conventional procedures for a particular purpose. In general, for administering a therapeutic agent (e.g., a beta-lactamase and / or an additional therapeutic agent described herein) for therapeutic purposes, the therapeutic agent is given in a pharmacologically effective dose. A "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, particularly for the treatment of a disorder or disease. As used herein, an effective amount includes, for example, an amount sufficient to delay the onset of a symptom of a disorder or disease, to alter the course of a symptom of a disorder or disease (e.g., to slow the progression of a symptom of a disease), to reduce or eliminate one or more symptoms or signs of a disorder or disease, and to reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether an improvement is realized.

[0324] Effective doses, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates, or experimental animals, for example, to determine the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). Dosages can vary depending on the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. In some embodiments, compositions and methods that exhibit large therapeutic indices are preferred. Therapeutically effective doses can be estimated initially from in vitro assays, including, for example, cell culture assays. Doses can also be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 determined in cell culture or an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by an appropriate bioassay. Dosages can be determined by the physician and adjusted, if necessary, to the observed therapeutic effects.

[0325] In certain embodiments, the effect results in 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 results in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[0326] As used herein, "method of treatment" is equally applicable to the use of a composition to treat a disease or disorder described herein and / or the use of a composition for use(s) in the manufacture of a medicament for treating a disease or disorder described herein. EXAMPLES

[0327] Example 1: Development of IAP delayed release enteric coated pellets Two batches of IAP-layered sucrose cores were prepared with enzyme amounts of 3.45% w / w (first batch) and 4.65% w / w (second batch). The IAP activity after layering was maintained when compared to the activity of the IAP solution used for layering. The IAP solution showed approximately 70% activity relative to the Sigma standard, while the layered drug core showed 60% activity relative to the Sigma standard.

[0328] To assess the stability of the IAP, batch 1 of the layered drug cores was tested 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 under both conditions. Batch 2 of the layered drug cores was then coated with either a 30% weight gain of EUDRAGIT L30D-55 (single layer) or a 7% weight gain of HPC followed by 30% EUDRAGIT L30D-55 as an inner separating layer. Both coated formulations showed acid resistance in fasted simulated gastric fluid (FaSSGF) for 2 hours and very similar dissolution profiles in fasted simulated intestinal fluid (FaSSIF) without loss of enzyme activity at the end of the dissolution study.

[0329] Coated pellets were stored under refrigerated conditions in the presence of silica and after 6 months there was no decrease in enzyme activity, suggesting that the HPC (binder) and coating layer (acidic polymer EUDRAGIT L30D-55) did not affect enzyme activity over this period.

[0330] Overall, the layered drug pellet formulation is a promising option for formulating IAP due to the obtained dissolution profile and good flowability of the pellets.Another advantage of this formulation is the flexibility to obtain different enzyme strengths based on one formulation.It also provides a simplified process since layering and enteric coating can be performed in the same fluidized bed coater.

[0331] Example 2: Evaluation of excipient-IAP compatibility A selection of candidate binders were combined with IAP to determine the effect of binders on IAP activity. As shown in Table 1 and Figure 2, binders tested for compatibility with IAP in the presence of sucrose or lactose did not significantly alter IAP activity after 96 hours of incubation, showing similar % activity as the initial IAP solution alone (IAP-binder combination #11).

[0332] [Table 1]

[0333] As shown in Table 1 and Figure 2, most of the combinations did not result in significant changes in enzyme activity when compared to the initial values ​​at t=0 hours. The results indicated that under the current test conditions, no incompatibility issues were identified as no loss of IAP activity was observed in the presence of any of the excipients tested. The results suggest that the layered drug core using HPC as a binder does not impair IAP activity and this core was the proposed combination selected for further testing.

[0334] Cofactors tested for compatibility with IAPs are shown in Table 2 and Figure 3. It was found that under the current test conditions, the cofactors did not significantly increase IAP activity.

[0335] [Table 2]

[0336] As shown in Table 2 and Figure 3, the first four combinations without cofactors (#1-#4) did not show a trend towards reduced IAP activity when compared to the four combinations with cofactors (#5-#8). Similar to the IAP-binder compatibility study (results shown in Figure 2), the results of the IAP-cofactor compatibility study confirmed that IAP activity was not impaired in the presence of HPC, sucrose, or a combination of both. The addition of cofactors to the formulations did not significantly increase IAP activity across different time points. For this reason, formulations containing cofactors were not further investigated. The IAP buffer solution contains 1.0 mM MgCl2 and 0.1 mM ZnCl2. The additional metal ions tested in these experiments (CoCl2 and CaCl2) did not alter the enzyme activity of the IAP-binder mixtures.

[0337] Since the excipient / IAP mixtures were incubated at 37°C, the aqueous medium evaporated over time due to the incubation temperature, resulting in a clear loss of solution volume. This led to an increase in enzyme activity in combination #2 at the final time point. The decrease in enzyme activity in combinations #4, #5, #7, and #8 at the final time point may be related to assay variability.

[0338] Example 3: Stability testing of the first batch of cores The 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 utilized for drug layering. To prepare the final drug layering solution, 1.12 g of HPC (Klucel EF) was added to 49.5 mL of IAP solution under slow magnetic stirring (<200 RPM). The subsequent solution was spray layered onto 6.00 g of 600 / 710 μm Suglets in a Caleva mini coater / dryer.

[0339] The IAP-HPC solution was sprayed with an atomizing air pressure of 0.8 bar, an inlet air velocity of 11.5 m / sec, and a chamber agitation rate of 10.5 Hz. The temperature during the spraying process was 45°C for the first 30-45 minutes and then reduced to 40°C. This is because the suglets will easily agglomerate if the temperature is not sufficient to rapidly dry the aqueous solution on the surface of the cores during the initial stages of the coating process. As the coating process progressed the cores had a thin layer of coating which protected them and reduced the chance of agglomeration, resulting in a reduced process temperature.

[0340] The layered drug cores were dried at 35° C. for 30 minutes every hour after spraying, and upon completion of coating, the layered drug cores were further dried at 40° C. for 1 hour before actual weight gain was determined.

[0341] The layered drug cores (AP0361 / 24 / 1) were composed of 87.92 / 3.45 / 7.77 / 0.87 w / w% Suglets / IAP / HPC / Salt (from IAP solution), respectively. This batch of cores had a slightly different ratio of IAP / HPC of 1:2.25 (w / w) instead of the desired ratio of 1:2 (w / w) due to a preparation error. After completion of the drug layering process, the cores were stored at 2-8 °C in sealed glass vials with silica desiccant bags.

[0342] The resulting cores were stored dry in sealed vials at 2-8 °C for the first 12 days after drug layering. To test the effect of non-drying conditions, half of the contents were removed from the glass vials after the first 12 days of drying and stored at 2-8 °C without the silica bag. Enzyme activity results were measured over time in short-term stability studies of these layered drug cores, as shown in Table 3.

[0343] [Table 3]

[0344] Some variation in % IAP activity over time was observed, but these may have been related to assay variability. Based on the results in Table 3, it was concluded that non-dried conditions did not significantly reduce IAP activity when compared to dried conditions.

[0345] For enzyme activity testing, the cores were dissolved in diethanolamine buffer. Specifically, IAP activity assay was performed. A 140 mg / mL solution in purified water with diethanolamine (Sigma D8885) was prepared and the pH of diethanolamine was adjusted to 9.8 with 5 M 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 1 M magnesium chloride solution (Sigma M1028) was added to the solution. The diethanolamine was freshly prepared and protected from direct sunlight.

[0346] An IAP enzyme standard solution was prepared by diluting a 19.4 mg / mL stock solution (concentration may vary with Sigma lot) to 1.00 mg / mL with diethanolamine buffer (5 μL in 92 μL diethanolamine buffer). The enzyme solution was stored on ice before further dilution. This purified intestinal alkaline phosphatase (Sigma#PO114) was used as a control to test the activity of the assayed samples.

[0347] All samples, including Sigma standards and IAP solutions, were loaded into a 96-well plate and diluted to a theoretical 5ng / mL solution in diethanolamine buffer.

[0348] pNPP was reconstituted in 1.86 mg / mL assay buffer for a 5 mM solution (Abcam ab146203) and the pNPP solution was stable on ice for 12 hours.

[0349] Next, a kinetic assay was performed using a plate reader (DYNEX #MRX On a TCII), 80 μL of standards and samples were added to wells of a 96-well plate. All enzymatic reactions were performed in duplicate wells. Enough 5 mM pNPP solution for later addition to the samples was added to each empty well, and the plate was incubated at 37° C. for 5 min. Using a multichannel pipette, 50 μL of pre-heated 5 mM pNPP solution was quickly added to each well containing either standards or samples. During this addition, the formation of air bubbles was avoided. The reaction sequence was started and performed at 37° C. After an additional 10 min initial delay and an initial shaking step of 2 s, the colorimetric results (dephosphorylation of pNPP NPP by enzyme phosphatase activity) were measured as a function of the increase in optical density (OD) at 405 nm every 20 s for 5 min. Blank wells containing no enzyme were performed in parallel on the same plate.

[0350] The kinetic assay provided a readout of enzyme kinetics over this period and was used to compare the slope to the slope generated from the IAP Sigma standard (% activity in sample = slope of sample / slope of Sigma standard x 100%. The blank slope was subtracted from the slopes of the sample and Sigma standard).

[0351] Example 4: Enteric coating and dissolution testing of the second batch of cores A second batch of layered drug cores was prepared for coating testing and manufactured according to the same instrument parameters (Example 3) as the first batch of cores. 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 layering solution was prepared using 0.948 g of HPC (Klucel EF) added to 47.0 mL of IAP solution under slow magnetic stirring (less than 200 RPM) in a 1:2 (w / w) IAP:HPC ratio.

[0352] Two types of coatings were applied to the second batch of layered drug cores: either a 30% weight gain of EUDRAGIT L30D-55 (single enteric layer) or a 7% weight gain layer of HPC followed by an outer layer of 30% polymer weight gain of EUDRAGIT L30D-55 (enteric layer with subcoating). The formulations are shown in Table 4.

[0353] [Table 4]

[0354] The layered drug cores (batch 2) were layered with a final composition of 1:2 (w / w) IAP / HPC solids ratio and 4.65% weight gain of IAP, followed by a monolayer coating (30% weight gain of EUDRAGIT L30D-55) or bilayer coating (7% weight gain of HPC + 30% weight gain of EUDRAGIT L30D-55). As shown in the SEM image in Figure 4, the diameter of the monolayer coated pellets was approximately 800 μm. Figure 4 shows an SEM photograph of one coated pellet (30% EUDRAGIT L30D-55 coated onto pellets from the second batch).

[0355] A cross-section of the pellets of the second batch is also shown in Figure 5, where the sucrose core, the IAP / HPC layer, and the L30D-55 coating layer can be clearly distinguished. The IAP / HPC layer was about 20 μm thick, and the EUDRAGIT L30D-55 coating layer was about 40 μm thick (recommended thickness by the polymer manufacturer Evonik). Figure 5 shows an SEM photograph of the cross-section of one coated pellet (30% L30D-55 coated on the pellets of the second batch).

[0356] The dissolution behavior of these coated pellets was tested. The amount of pellets in each dissolution vessel was 1.14 g for single-layer coated pellets and 1.22 g for double-layer coated pellets, which corresponds to an IAP dose of 40 mg for both formulations. For each dissolution test, one tablet was incubated in 20 mL of FaSSIF pH 1.6 in a 60 mL vial on a heater / shaker for 2 hours at 37°C with orbital shaking at 200 RPM. After the first 2 hours, 20 mL of double-strength FaSSIF was added and the pH was adjusted to 5.5 or 5.8 using 1 M NaOH. After an additional 45 minutes, the pH was further adjusted to 6.5 or 6.8 and the dissolution test was allowed to proceed for an additional 2-3 hours.

[0357] The coated pellets were tested in FaSSGF at pH 1.6 for 2 h, after which FaSSIF was added to reach pH 5.8 for 45 min, which was then increased to pH 6.8 for the next 3 h. Dissolution studies of coated pellets in FaSSIF alone (pH 5.8 for 45 min, followed by pH 6.8 for 3 h) were also performed for comparison purposes.

[0358] The UV protein release profiles are shown in Figure 6. The % release of IAP during dissolution in FaSSIF only is represented as "X" and "Triangle" graphs for monolayer and bilayer coatings, respectively. The % release of IAP during dissolution in FaSSGF and FaSSIF is represented as red and blue graphs for monolayer and bilayer coatings, respectively.

[0359] The profiles indicated that the coated pellets were enterically protected at low pH, as no IAP release was detected in FaSSGF by UV analysis. Furthermore, the dissolution profiles of the one- and two-step approaches were very similar, with release beginning to occur immediately after the pellets were exposed to FaSSIF at pH 5.8, and after 45 min at pH 5.8, the dissolution rate increased when the pH was adjusted to pH 6.8. Complete release was achieved after an additional 3 h at pH 6.8.

[0360] The enzyme activity profiles are shown in Figure 7. The activity % of IAP dissolved in FaSSIF only is shown as "X" and "Triangle" graphs for monolayer and bilayer coatings, respectively. The activity % of IAP dissolved in FaSSGF and FaSSIF is shown as red and blue graphs for monolayer and bilayer coatings, respectively.

[0361] The enzyme activity profile followed the same trend as the % release of IAP by UV in Figure 6. The maximum enzyme activity detected at the end of both dissolution tests was about 45%-55%, which was the same level as the starting material (about 50% of the layered drug core). The results showed that the applied coating was enterically protected at low pH and that the enzyme activity was not reduced in the presence of the coating polymer. Due to the similar dissolution profiles produced by the single and double coated layered cores, it was concluded that a sub-coating HPC to separate and protect the enzyme from the acidic coating polymer was not necessary.

[0362] The % activity achieved by exposing 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 at pH 1.6, confirming that a sufficient enteric coating thickness was applied, as similar levels of enzyme activity were obtained.

[0363] This study demonstrated that enteric coating of a layered core containing an aqueous dispersion of EUDRAGIT L30D-55 at 30% polymer weight gain provided acid resistance for up to 2 hours in FaSSGF and sustained release of IAP over 4 hours in FaSSIF. The presence of a 7% HPC separating layer did not alter the dissolution profile or affect enzyme activity. Therefore, it was determined that a separating sublayer was not necessary.

[0364] Example 5: Evaluation of stability of IAP activity in pellets The uncoated / coated layered drug cores were placed in sealed glass vials with silica desiccant bags for stability evaluation at 2-8°C for up to 12 months. Enzyme activity was monitored for these cores at 0, 1, 3, and 6 months by dissolving the cores in diethanolamine buffer and assaying for IAP activity.

[0365] Both uncoated and coated pellets were stored dry and at refrigerated temperatures. Enzyme activity was measured for up to 6 months and the results are shown in Table 5.

[0366] [Table 5]

[0367] The results show that the enzyme activity of uncoated cores and coated pellets remained at similar levels over 6 months of storage at dry, refrigerated temperatures.

[0368] Example 6: Stability Evaluation of 25 mg and 5 mg IAP Pellet Capsules Size 2 HPMC transparent capsules were filled with the coated IAP pellets. The pellets show a drug load of 9.7 w / w% (analyzed by UV spectroscopy) and an enzyme activity of 78.1% (analyzed by DYNEX microplate reader) compared to a Sigma standard. 90 capsules contained 25 mg of IAP and 90 capsules contained 5 mg of IAP. Pellets were produced by layering IAP / HPC (1 / 2 (w / w)) onto 600 / 710 μm Suglets (Colorcon) followed by coating with EUDRAGIT L30D-55 (Evonik).

[0369] [Table 6]

[0370] The stability and percent activity of the pellet capsules during one year storage in a refrigerator is shown in Table 7 below.

[0371] [Table 7]

[0372] The present inventors have unexpectedly discovered that IAP pellet capsules maintain at least about 80% of the IAP activity when stored at 2-8° C. for up to one year.

[0373] Next, an in vitro dissolution test was performed. Approximately 450 mg of coated pellets were incubated in 25 mL FaSSGF (pH 1.6, 37°C) in a 60 mL glass vial, and the vial was gently shaken on a heater / shaker with an orbital shaking speed of 200 RPM. At 60 and 120 minutes, 1 mL of medium was withdrawn through a 10 μm cannula filter and filtered at 0.2 μm for UV analysis. 50 μL of the filtered solution was frozen for further activity analysis. The solution was then returned to the vial.

[0374] After the first 2 hours of incubation in FaSSGF, 25 mL of FaSSIF was added to the vial to obtain a pH of 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 procedure was the same as described in the paragraph above.

[0375] The filtered samples were analyzed by UV spectrometer at 280 nm and 320 nm. The absorbance value is equal to A280-A320. The absorbance from blank medium was further subtracted from the sample absorbance value, and the resulting value was used to calculate the % release. First, a calibration curve was constructed as shown in Figure 8. The sample concentration at each time point was calculated based on the equation of the linear trend line.

[0376] The results of the dissolution test are shown in Figure 9. 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%. Furthermore, the pellets fully recovered activity at the end of the dissolution test, indicating that acid did not permeate through the coating film during the initial incubation. During the first 45 minutes, the release of IAP was slow in FaSSIF at pH 5.8, as the pH was close to 5.5, the dissolution threshold of EUDRAGIT L30D-55. When the pH was adjusted to 6.8, the coating film began to dissolve rapidly, and the release of IAP increased dramatically.

[0377] Figure 10 shows the results of a dissolution test in which the coated pellets were stored for approximately one year. The coated pellets were resistant to FaSSGF for 2 hours, as no release was detected. The percent release at 60 and 120 minutes (T=0, red line) was approximately 1%. Furthermore, the pellets fully recovered activity at the end of the dissolution test, indicating that acid did not permeate through the coating film during the initial incubation. During the first 45 minutes, the release of IAP was slow in FaSSIF at pH 5.8, as the pH was close to 5.5, the dissolution threshold of EUDRAGIT L30D-55. When the pH was adjusted to 6.8, the coating film began to dissolve rapidly, and the release of IAP increased dramatically.

[0378] Various studies 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 the SYN-020 delayed release capsules (15 mg and 5 mg capsules), and SYN-020 placebo pellets, as well as the results of the various studies, are shown in Tables 8 and 9 below.

[0379] [Table 8] HTP-20 is a premixed formulation of glyceryl monostearate, polysorbate-80, and triethyl citrate, which provides anti-tack, plasticizer, and stabilizer functions.

[0380] [Table 9-1]

[0381] [Table 9-2]

[0382] 11A-H show further analysis of non-GMP SYN-020 5 mg and 15 mg capsule dosages. Results regarding dosage strength and stability were analyzed over a 6 month period, including parameters such as specific activity, purity, water content, and dissolution.

[0383] Equivalent While the invention has been described in relation to particular embodiments thereof, it is capable of further modifications, and it will be understood that this application is intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention in general, including such departures from the disclosure as are known or within the practice in the art to which this invention pertains and which may be applicable to the essential features described above, as well as departures from the disclosure as will fall within the scope of the appended claims.

[0384] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed within the scope of the following claims.

[0385] Incorporation by Reference All patents and publications referenced herein are hereby incorporated by reference in their entirety.

[0386] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0387] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may be equally applicable to all sections.

Claims

1. 1. A modified release formulation comprising at least one modified release pellet comprising a phosphatase (AP)-based drug, the modified release formulation releasing a substantial amount of the AP-based drug into the GI tract, wherein each modified release pellet comprises: About 1-10% by weight of an AP-based agent; About 75-95% by weight sucrose spheres; About 5-15% by weight of hydroxypropyl cellulose; About 0.5-2% by weight of a buffer salt; The modified release formulation.

2. Each modified release pellet comprises: About 5% by weight of an AP-based drug; About 85% by weight of sucrose spheres; About 9% by weight of hydroxypropyl cellulose; About 1% by weight of a buffer salt; 2. The modified release formulation of claim 1, comprising:

3. Each modified release pellet comprises: About 4.7% by weight of an AP-based drug; About 84.9% by weight of sucrose spheres; About 9.3% by weight of hydroxypropyl cellulose; About 1.2% by weight of a buffer salt; 2. The modified release formulation of claim 1, comprising:

4. 4. The modified release formulation of any one of claims 1 to 3, further comprising a single layer enteric coating having a weight gain of about 20-40% enteric polymer, optionally having a weight gain of about 30% enteric polymer.

5. a layer of enteric coating having a weight gain of about 20-40% enteric polymer; a layer of enteric coating having a weight gain of about 5-15% hydroxypropyl cellulose (optionally having a weight gain of about 30% enteric polymer and having a weight gain of about 7% hydroxypropyl cellulose); The modified release formulation of any one of claims 1 to 3, further comprising:

6. 1. A modified release formulation comprising at least one modified release pellet contained in a capsule that releases a substantial amount of an AP-based drug into the GI tract, About 5-15% by weight of an AP-based agent; About 30-40% by weight of sucrose spheres; About 15-25% by weight of hydroxypropyl cellulose; About 2-4% by weight of a buffer salt; about 25-35% by weight of an enteric polymer; The modified release formulation.

7. The modified release formulation comprises: About 10% by weight of an AP-based drug; About 38% by weight of sucrose spheres; About 19% by weight of hydroxypropyl cellulose; About 2% by weight of a buffer salt; about 26% by weight of an enteric polymer; The modified release formulation of claim 6, comprising:

8. The modified release formulation comprises: About 9.7% by weight of an AP-based drug; About 37.7% by weight of sucrose spheres; About 19.4% by weight of hydroxypropyl cellulose; About 2.4% by weight of a buffer salt; about 26.3% by weight of an enteric polymer; The modified release formulation of claim 6, comprising:

9. The modified release formulation of any one of claims 6 to 8, wherein the capsule contains about 25 mg or 5 mg of AP-based drug.

10. 1. A modified release formulation comprising at least one modified release pellet contained in a capsule that releases a substantial amount of an AP-based drug into the GI tract, About 5-15% by weight of an AP-based agent; About 35-45% by weight sucrose spheres; About 15-25% by weight of hydroxypropyl cellulose; About 0.1 to 1% by weight of a buffer salt; about 20-30% by weight of an enteric polymer; The modified release formulation.

11. The modified release formulation comprises: About 10% by weight of an AP-based drug; About 39% by weight of sucrose spheres; About 20% by weight of hydroxypropyl cellulose; About 0.5% by weight of a buffer salt; about 26% by weight of an enteric polymer; The modified release formulation of claim 10, comprising:

12. The modified release formulation comprises: About 10.0% by weight of an AP-based drug; About 38.9% by weight of sucrose spheres; About 20.0% by weight of hydroxypropyl cellulose; About 0.3% by weight of a buffer salt; about 26.3% by weight of an enteric polymer; The modified release formulation of claim 10, comprising:

13. The modified release formulation of any one of claims 10 to 12, wherein the capsule contains about 15 mg or 5 mg of AP-based drug.

14. The modified release formulation of any one of claims 6 to 13, further comprising about 1-10% HTP-20.

15. The modified release formulation of any one of claims 6 to 12, wherein the capsule comprises gelatin or hydroxypropyl methylcellulose.

16. 13. The modified release formulation of any one of claims 1 to 12, wherein the formulation maintains at least about 80% of the IAP activity when stored at 2-8°C for at least 1 month, 3 months, 6 months, or 12 months.

17. The modified release formulation of any one of claims 1 to 16, wherein the AP-based drug comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 1-17 and 39.

18. 18. The modified release formulation of claim 17, wherein the AP-based drug comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:

39.

19. 2. The modified release formulation according to any one of the preceding claims, wherein the AP-based drug is substantially released in the small intestine.

20. The modified release formulation of any one of claims 1 to 18, wherein the AP-based drug is substantially released in the large intestine.

21. 2. The modified release formulation according to any one of the preceding claims, wherein the formulation comprises a core particle and a base coat on the core particle, the base coat comprising the AP-based drug.

22. 22. The modified release formulation of claim 21, wherein the core particle comprises sucrose.

23. The modified release formulation of any one of claims 21 to 22, wherein the formulation comprises a plurality of core particles.

24. 24. The modified release formulation of claim 23, wherein the core particle has a size of about 1 mm to about 1.3 mm in diameter.

25. 10. The modified release formulation of any one of the preceding claims, wherein the formulation further comprises a modified release coating that is substantially stable in gastric fluids.

26. 10. The modified release formulation of any one of the preceding claims, wherein the formulation further comprises a modified release coating that is degraded by microbial enzymes present in the intestinal flora.

27. 10. The modified release formulation of any one of the preceding claims, wherein the formulation comprises a modified release coating having a solubility that is pH dependent.

28. 10. The modified release formulation of any one of the preceding claims, wherein the formulation comprises a modified release coating having a time-dependent erosion profile.

29. 2. The modified release formulation of any one of the preceding claims, wherein the formulation further comprises an additional therapeutic agent.

30. 2. The modified release formulation of any one of the preceding claims, wherein the buffer salt is selected from Tris base, magnesium chloride, magnesium sulfate, zinc chloride, and zinc sulfate.

31. 10. The modified release formulation of any one of the preceding claims, wherein the enteric polymer is selected from EUDRAGIT L30 D-55, FS 30D, L 100-55, L 100, L 12,5, L 12,5 P, RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5, and S 12,5 P.

32. 32. The modified release formulation of claim 31, wherein the enteric polymer is EUDRAGIT L 30 D-55 (poly(methacrylic acid-ethyl acrylate copolymer) 1:1).

33. 13. A method for treating or preventing radiation-induced damage in a patient in need thereof, comprising administering to said subject a formulation according to any one of the preceding claims.

34. 34. The method of claim 33, wherein the radiation-induced injury is selected from enteritis resulting from cancer radiotherapy, radiation-induced enteropathy, colitis, and proctitis.

35. 35. The method of claim 33 or 34, wherein the radiation-induced damage comprises intestinal toxicity.

36. 36. The method of any one of claims 33 to 35, wherein the radiation-induced damage comprises one or more of mucosal atrophy, vascular sclerosis, and progressive intestinal wall fibrosis.

37. 37. The method of any one of claims 33 to 36, wherein the radiation comprises ionizing radiation, optionally said radiation comprises one or more of X-rays, gamma rays, and charged particles.

38. The method according to any one of claims 33 to 37, wherein the radiation-induced damage is a side effect of radiation therapy.

39. 39. The method of claim 38, wherein the radiation therapy comprises a dose (optionally fractionated) 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.

40. 1. A modified release formulation comprising at least one modified release pellet containing an AP-based drug, the modified release formulation releasing a substantial amount of the AP-based drug into the GI tract, Each modified release pellet comprises: About 5% by weight of an AP-based agent comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 39; About 85% by weight of sucrose spheres; About 9% by weight of hydroxypropyl cellulose; About 1% by weight of a buffer salt; a single layer enteric coating having an enteric polymer weight gain of about 30%, said enteric polymer being EUDRAGIT L 30 D-55 (poly(methacrylic acid-ethyl acrylate copolymer) 1:1); The modified release formulation.

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