Treatment or prevention of gastrointestinal immunotherapy side effects

Abstract

The present disclosure relates, inter alia, to methods for preventing and / or reducing gastrointestinal side effects from checkpoint inhibitors by administering a therapeutic intestinal alkaline phosphatase. The disclosure further relates to compositions comprising a combination of an immune checkpoint therapy and a therapeutic alkaline phosphatase, and the use of said compositions in preventing and / or treating gastrointestinal side effects from immune checkpoint inhibitors, such as colitis.

Description

[Technical Field]

[0001] The present disclosure relates, inter alia, to methods for preventing and / or reducing gastrointestinal side effects of immune checkpoint immunotherapy by administering therapeutic intestinal alkaline phosphatase.

[0002] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 396,398, filed August 9, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Description of electronically submitted XML files This application contains a Sequence Listing that has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML file, created on August 1, 2023, has the file name "SYN-060_Sequence_Listing.xml" and is 28,615 bytes in size. [Background technology]

[0004] The administration of immune checkpoint inhibitors (CPIs) has revolutionized the field of cancer treatment, as these treatments offer improved efficacy compared to standard cancer treatments such as chemotherapy and radiation therapy. However, CPI treatment can be limited by the occurrence of gastrointestinal (GI) disorders associated with CPI treatment, such as diarrhea and / or colitis.

[0005] Alkaline phosphatase ("AP," EC 3.1.3.1) is a hydrogenase enzyme that can remove phosphate groups from a variety of targets, including nucleotides and proteins. Specifically, mammalian APs exert these properties by primarily targeting LPS (a TLR4 agonist), flagellin (a TLR5 agonist), and CpG DNA (a TLR9 agonist). APs also degrade intestinal luminal NTPs (e.g., ATP, GTP, etc.), which promotes the growth of beneficial bacteria and reverses dysbiosis. Therefore, APs may find clinical applications, for example, in the treatment of various GI disorders.

[0006] Given the need for improved cancer treatments and reduced adverse side effects to allow for improved cancer patient care, there is an increasing need for therapeutic compositions that prevent and / or reduce the side effects associated with CPIs. Summary of the Invention

[0007] Thus, in some aspects, the present disclosure provides methods for preventing or reducing CPI-mediated GI side effects in patients experiencing such side effects, such as cancer patients experiencing such side effects, by administering one or more intestinal alkaline phosphatase (IAP) constructs comprising variants thereof. In embodiments, the IAP constructs are mammalian IAPs, including but not limited to human IAPs (hIAPs), bovine IAPs (cIAPs), and bovine IAPs (bIAPs). In embodiments, the IAPs are secreted from host cells. In embodiments, the IAPs are administered orally. In embodiments, the IAPs are administered prior to the CPI.

[0008] In some embodiments, the patient is undergoing treatment with immune checkpoint inhibitor immunotherapy, such as an agent that modulates one or more of programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2), inducible T-cell costimulatory molecule (ICOS), inducible T-cell costimulatory molecule ligand (ICOSL), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0009] In some aspects, the present disclosure provides methods for improving and / or increasing the efficacy of immune checkpoint inhibitor immunotherapy (e.g., used in tumor treatment). For example, in embodiments, the methods of the present disclosure improve the efficacy of CPIs by widening the therapeutic window of the CPIs. In embodiments, the efficacy of CPI therapy is not hindered by administration of an IAP construct. In embodiments, administration of the IAP construct does not reduce the efficacy of CPI therapy. In further embodiments, administration of the IAP construct improves the anti-tumor efficacy of CPI therapy.

[0010] In another aspect, the present disclosure provides methods for preventing GI side effects from CPIs by administering an additional therapeutic agent, such as, but not limited to, a composition comprising a therapeutic IAP and an immune checkpoint immunotherapy selected from an agent that modulates one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4.

[0011] In another aspect, the disclosure provides methods of treating cancer by administering an additional therapeutic agent, such as, but not limited to, a composition comprising a therapeutic IAP and an immune checkpoint immunotherapy selected from an agent that modulates one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, wherein the treatment produces fewer CPI-related GI side effects than treatment without the IAP.

[0012] In another aspect, the present disclosure provides methods for the therapeutic use of an IAP. In some aspects, the IAP is administered to a patient undergoing treatment with an immune checkpoint inhibitor immunotherapy selected from agents that modulate one or more of programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), inducible T cell costimulatory molecule (ICOS), inducible T cell costimulatory molecule ligand (ICOSL), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In other aspects, a therapeutically effective amount of a composition comprising an immune checkpoint inhibitor immunotherapy is administered to a patient undergoing treatment with an IAP.

[0013] In embodiments, the present disclosure provides methods for preventing or treating GI side effects caused by CPIs. In certain embodiments, the present disclosure provides methods for treating GI side effects caused by CPIs, such as diarrhea and / or colitis. In embodiments, the present disclosure provides for patients undergoing treatment with corticosteroids and / or TNF-α targeting agents to treat mediated GI side effects. In embodiments, the present disclosure provides methods for reducing and / or eliminating the dose or frequency of administration of corticosteroids and / or TNF-α targeting agents required to treat mediated GI side effects. [Brief explanation of the drawings]

[0014] [Figure 1-1] FIG. 1 shows sequences for alkaline phosphatase agents present in the compositions and / or formulations described herein. [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 1-4] Same as above [Figure 1-5] Same as above [Figure 1-6] Same as above [Figure 1-7] Same as above [Figure 1-8] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure is based, inter alia, on the discovery that alkaline phosphatases, such as IAPs, can be used to prevent, reduce, or eliminate CPI-induced GI side effects, such as diarrhea and / or colitis, in patients. Such use of alkaline phosphatases (e.g., IAPs) allows for more effective cancer treatment because CPI therapy is not constrained by dose- and regimen-limiting GI side effects. In embodiments, the use of alkaline phosphatases improves the efficacy (e.g., antitumor efficacy) of CPI therapy. Furthermore, the present discovery includes methods for sparing patients from steroid therapy, which is currently used to reduce CPI-induced GI side effects.

[0016] Cancer immunotherapy involves the use of naturally occurring or synthetically produced components to stimulate or enhance the immune system to fight cancer. Immune checkpoint inhibitor immunotherapy is effective in fighting cancer by priming and activating the immune system to produce antitumor effects, often with highly specific targeting. Along with the promise of cancer immunotherapy, there is also a need to maintain the immune system's complex balance between identifying and eradicating foreign antigens, a process necessary to suppress uncontrolled immune responses. Indeed, there is a need to mitigate potentially harmful side effects associated with cancer immunotherapy, such as immune checkpoint inhibitor immunotherapy. Such side effects may include, but are not limited to, diarrhea and / or colitis. Common practice today involves minimizing checkpoint inhibitor side effects, or immune-related adverse events, by discontinuing checkpoint inhibitor immunotherapy and administering agents that temporarily induce immunosuppression, such as corticosteroids and / or tumor necrosis factor alpha (TNF-α) antagonists.

[0017] IAPs are endogenous proteins expressed by the intestinal epithelium that can be used to reduce inflammation and maintain intestinal homeostasis. For example, loss of IAP expression or function is associated with increased intestinal inflammation, dysbiosis, bacterial translocation, and systemic inflammation. Their key functions in maintaining gastrointestinal homeostasis are generally recognized as regulating bicarbonate secretion and duodenal surface pH, long-chain fatty acid absorption, and reducing intestinal inflammation by detoxifying pathogen-associated molecular patterns, as well as regulating the intestinal microbiota. Some substrates acted upon by the phosphatase function of IAPs include lipopolysaccharide (LPS), flagellin, CpG DNA, and nucleotide diphosphates and triphosphates. Specifically, IAPs are targets for therapeutic agents due to their ability to downregulate inflammation, modulate the microbiota, strengthen the intestinal barrier by enhancing the expression of claudins and occludins, and affect adenosine triphosphate and adenosine diphosphate (ATP and ADP) metabolism. In one aspect, the present disclosure provides compositions comprising IAP that do not interfere with cancer treatment for patients. Indeed, according to the present disclosure, the methods described herein broaden the therapeutic window of CPIs.

[0018] The present disclosure is directed, in part, to pharmaceutical compositions, formulations, and uses of a combination of one or more IAPs and a composition comprising one or more CPIs, including but not limited to, one or more agents that modulate one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4. In certain aspects, a patient is treated with a composition comprising an immune checkpoint inhibitor, including but not limited to, one or more agents that modulate one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4.

[0019] (Alkaline phosphatase (AP)) The present disclosure is directed, in part, to pharmaceutical compositions, formulations, and uses of one or more alkaline phosphatases. Alkaline phosphatases are dimeric metalloenzymes that catalyze the hydrolysis of phosphate esters and dephosphorylate various target substrates at physiological pH and above. Exemplary APs that can be utilized in the present disclosure include, but are not limited to, IAPs (e.g., calf IAP or bovine IAP, chicken IAP, caprine IAP), 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 IAPs, recombinant IAPs, or any polypeptide containing alkaline phosphatase activity.

[0020] In embodiments, the present disclosure contemplates the use of mammalian alkaline phosphatases, including but not limited to IAP, placental alkaline phosphatase (PLAP), germ cell alkaline phosphatase (GCAP), and tissue-nonspecific alkaline phosphatase (TNAP).

[0021] IAP In embodiments, alkaline phosphatase is an IAP. IAPs are produced in the proximal small intestine and are bound to enterocytes by glycosylphosphatidylinositol (GPI) anchors. Some IAPs are released into the intestinal lumen along with vesicles released by cells as soluble proteins detached from cells by phospholipases. The enzyme then travels beyond the small and large intestine, and some active enzymes can be detected in feces. In embodiments, the IAP is a human IAP (hIAP). In embodiments, the IAP is a calf IAP (cIAP), also known as a bovine IAP (bIAP). There are multiple isozymes of bIAP, including bIAP II and IV, which have higher specific activity than bIAP I. In embodiments, the IAP is a cIAP or any one of the bIAP isozymes (e.g., bIAP I, II, and IV). In embodiments, the IAP is bIAP II. In embodiments, the IAP is bIAP IV.

[0022] In embodiments, an IAP of the present disclosure has a specific enzymatic activity that is greater than a commercially available AP, e.g., bovine IAP (cIAP). In non-limiting embodiments, an IAP of the present disclosure has a specific enzymatic activity that is about 1-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold, 100,000-fold, or 1,000,000-fold greater than the specific enzymatic activity of a commercially available AP, e.g., bovine IAP (cIAP).

[0023] IAP variants IAP variants are also included within the definition of IAP. IAP variants have at least one or more amino acid modifications, generally amino acid substitutions, compared to the parent wild-type sequence. In embodiments, an IAP of the present disclosure has an IAP sequence that is at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%) identical to any of the sequences disclosed herein. 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 variant. In addition, the IAP variant retains most or all of these biochemical activities when measured as described herein. In embodiments, the IAP comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 1-6, or 10-14. In embodiments, the IAP comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11.

[0024] GPI-anchored proteins Mammalian alkaline phosphatase is a GPI-anchored protein. It has a signal peptide and is translated into the secretory pathway. In the endoplasmic reticulum (ER), the protein is glycosylated and folded. There is one free cysteine ​​that is not clearly accessible on the surface, as well as two disulfide bonds. In the late ER, the carboxy terminus is removed and the GPI anchor is added. Thus, GPI anchoring 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 no consensus sequence exists, the carboxy terminus contains three amino acids, terminus ω, ω+1, and ω+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 thought that hydrophobicity is important for embedding the carboxy terminus in the ER membrane. An enzymatic reaction replaces the carboxy terminus with the GPI anchor.

[0025] In embodiments, the IAPs of the present disclosure are secreted proteins; i.e., in embodiments, the IAPs are not GPI-anchored and are secreted rather than retained intracellularly. This can be achieved in several ways. In embodiments, the IAP can lack a GPI anchor site, e.g., the DAAH site is removed and secreted. Alternatively, in embodiments, this can be achieved by the IAP including a stop codon inserted immediately before the GPI anchor site. In embodiments, the IAP includes a stop codon after the aspartate in the DAAH consensus site (e.g., amino acid 503 for hIAP and bIAP or amino acid 506 for bIAP II). Figure 1 illustrates HIAP (SEQ ID NO: 3) including a stop codon and bIAP II (SEQ ID NO: 4) including a stop codon.

[0026] Human IAP In embodiments, the IAP is a human IAP (hIAP). In embodiments, the IAP is a hIAP comprising the amino acid sequence of SEQ ID NO: 1, as depicted in Figure 1, or a variant described herein, wherein the hIAP variant retains at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the phosphatase activity compared to the wild-type enzyme using the assays outlined herein.

[0027] Amino acid modifications, including amino acid substitutions, that find particular use in the present disclosure are included within the definition of hIAP. For example, without wishing to be bound by theory, it is believed that a cysteine ​​at the carboxy terminus of an AP-based agent (e.g., position 500 of SEQ ID NO: 1) may interfere with protein folding. Thus, in embodiments, an AP-based agent includes a mutation of the cysteine ​​(e.g., position 500 of SEQ ID NO: 1). In embodiments, any amino acid may be substituted for the cysteine, although in embodiments, glycine is particularly used. Additionally, the C-terminal cysteine ​​may be deleted.

[0028] In embodiments, the hIAP is a recombinant glycosylated hIAP. In embodiments, the hIAP is glycosylated with one or more terminal sialic acids. In embodiments, the recombinant hIAP is glycosylated at the N-terminus, C-terminus, and / or glycosylated on one or more residues located internally within the primary amino acid sequence. In embodiments, the recombinant hIAP is terminally sialylated, e.g., where the sialylation is at the terminus of one or more glycosylated structures.

[0029] As one skilled in the art would appreciate, additional amino acid modifications can be made in the hIAPs disclosed herein. For example, in embodiments, a stop codon can be inserted after the aspartic acid at the DAAH consensus site (e.g., amino acid 503 of hIAP). Figure 1 illustrates a hIAP containing an inserted stop codon (SEQ ID NO: 3).

[0030] fusion proteins In embodiments, the present disclosure provides chimeric proteins. In embodiments, the present disclosure provides chimeric fusion proteins. For example, in embodiments, the present disclosure provides an isolated or recombinant alkaline phosphatase comprising a crown domain and a catalytic domain, wherein the crown domain and the catalytic domain are obtained from separate alkaline phosphatases (e.g., human alkaline phosphatase and bovine alkaline phosphatase). In embodiments, the alkaline phosphatases are both human APs. In certain embodiments, the present disclosure provides a recombinant fusion protein comprising domains of a human IAP and human placental alkaline phosphatase. In certain embodiments, the present disclosure provides a chimeric hIAP placental fusion protein. In embodiments, the IAP is a human recombinant fusion protein, such as a human placental AP / intestinal AP fusion protein (e.g., Ilofotase alfa).

[0031] In embodiments, the AP-based drug of the present disclosure is a fusion protein. In embodiments, the AP-based drug comprises alkaline phosphatase fused to a protein domain that replaces a GPI anchor sequence. In 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 embodiments, the AP-based drug fusion protein has an extended serum half-life.

[0032] In embodiments, the alkaline phosphatase is fused to an immunoglobulin Fc domain and / or hinge region. In embodiments, the immunoglobulin Fc domain and / or hinge region is derived from the Fc domain and / or hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subpopulations (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In embodiments, an AP-based agent of the present disclosure comprises alkaline phosphatase fused to the hinge region and / or Fc domain of an IgG.

[0033] In embodiments, the AP-based drug of the present disclosure is a proenzyme. In embodiments, the activity of the proenzyme is inhibited by the carboxy terminus. In embodiments, protease removal of the carboxy terminus restores alkaline phosphatase enzymatic activity. In embodiments, the proenzyme is secreted more efficiently than an enzyme lacking the carboxy terminus.

[0034] In embodiments, to generate the proenzyme, the native carboxy terminus of the alkaline phosphatase is replaced with a similar sequence from hPLAP. In embodiments, the hydrophobic carboxy terminus is mutated to promote protein secretion without truncating the carboxy terminus. In embodiments, a single point mutation, e.g., substitution of leucine with arginine, is made in the hydrophobic carboxy terminus (e.g., changing allpllagtl to, e.g., allplragtl; e.g., changing Leu515 to, e.g., Arg515 in SEQ ID NO: 1), resulting in secretion of the enzyme without removing the carboxy terminus.

[0035] Bovine IAP In embodiments, the IAP is a bovine IAP (bIAP). In embodiments, the bIAP is selected from bIAP I, bIAP II, and bIAP IV.

[0036] In embodiments, the IAP comprises an amino sequence having at least about 90%, or about 95%, or about 97%, or about 98%, or about 99% sequence identity to any one of SEQ ID NOs: 1 through 14. In embodiments, the IAP comprises an amino sequence having at least about 97% sequence identity to SEQ ID NO: 11. In embodiments, the IAP comprises an amino sequence having at least about 99% sequence identity to SEQ ID NO: 11.

[0037] Wild-type bIAP produced in calf intestine is not naturally sialylated. In embodiments, the bIAP is a recombinant sialylated bIAP. In embodiments, the bIAP is a recombinant glycosylated bIAP. In embodiments, the bIAP is glycosylated with one or more terminal sialic acids. In embodiments, the recombinant bIAP is glycosylated at the N-terminus, C-terminus, and / or at one or more residues located internally within the primary amino acid sequence. In embodiments, the recombinant hIAP is terminally sialylated, e.g., where the sialylation is at the terminal end of one or more glycosylated structures.

[0038] In embodiments, the recombinant sialylated bIAP comprises an amino acid sequence having about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% or more sequence identity to SEQ ID NO: 11. In embodiments, the recombinant sialylated bIAP comprises the amino acid sequence of SEQ ID NO: 11.

[0039] In embodiments, a recombinant IAP (e.g., sialylated bIAP) of the present disclosure has a specific activity that is substantially the same as or greater than that of a commercially available AP, e.g., bovine IAP (cIAP). In embodiments, a recombinant IAP (e.g., sialylated bIAP) of the present disclosure has a half-life that is substantially the same as or greater than that of a commercially available IAP.

[0040] a.bIAP II In embodiments, the IAP is bovine IAP II (bIAP II) or a variant described herein, wherein the bIAP variant retains at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of phosphatase activity using the assays outlined herein. In embodiments, the bIAP II comprises the signal peptide and carboxy terminus of bIAP I. In embodiments, the bIAP II comprises an aspartic acid at position 248 (similar to bIAP IV). In embodiments, the bIAP II comprises the amino acid sequence of SEQ ID NO: 2, or a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Figure 1 illustrates SEQ ID NO: 2, which is BIAP II, to which 248D is assigned. The signal peptide and sequence from 480 onward are derived from bIAP I.

[0041] In embodiments, the bIAP II comprises SEQ ID NO: 11, or a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In embodiments, the bIAP II comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 11.

[0042] Amino acid variants described herein are also included within the definition of bIAP II. For example, in embodiments, a stop codon may be inserted after the aspartic acid at the DAAH consensus site (e.g., amino acid 506 of bIAP II). Figure 1 illustrates bIAP II with an inserted stop codon (SEQ ID NO: 4).

[0043] Expression variants In embodiments, an IAP of the present disclosure is efficiently expressed and secreted from a host cell. In embodiments, an IAP of the present disclosure is efficiently transcribed within a host cell. In embodiments, the IAP exhibits enhanced RNA stability and / or trafficking within a host cell. In embodiments, the IAP is efficiently translated within a host cell. In embodiments, the IAP exhibits enhanced protein stability.

[0044] In embodiments, the IAP is efficiently expressed in a host cell. In embodiments, the Kozak sequence of a DNA construct encoding the AP-based drug is optimized. A Kozak sequence is a nucleotide sequence adjacent to the ATG start codon that directs ribosomes to initiate translation. While there is flexibility in the design of Kozak sequences, one canonical sequence is GCCGCCACCATGG (SEQ ID NO: 15). 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, i.e., the amino acid after the initiator methionine, is glutamine. All codons for glutamine have a C at position 1. Thus, all of these Kozak sequences have the sequence ATGC. Thus, in embodiments, the ATGC sequence is changed to ATGG. This can be done by changing the second amino acid to glycine, alanine, valine, aspartic acid, or glutamic acid (all of which have a G at position 1). These amino acids may be adapted for signal peptide function. In an alternative embodiment, the entire signal peptide is replaced with a peptide having a canonical Kozak sequence, derived from a highly expressed protein such as an immunoglobulin.

[0045] In embodiments, the signal peptide of the IAP may be deleted and / or substituted, for example, the signal peptide may be deleted, mutated, and / or substituted (e.g., with an alternative signal peptide) to ensure optimal protein expression.

[0046] In embodiments, DNA constructs encoding IAPs of the present disclosure include non-translated DNA sequences. Such sequences include introns, which may be heterologous to the IAP protein or derived from an IAP containing the native first and / or second introns and / or the native 3' UTR. 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 embodiments, DNA constructs encoding IAPs of the present disclosure include a 5' UTR and / or a 3' UTR. Shown in Figure 1 are exemplary IAP DNA sequences containing the first intron and 3' UTR, including SEQ ID NO:7, which contains hIAP with the native first intron (shown in bold and underlined), and SEQ ID NO:8, which contains hIAP with the native 3' UTR (shown in bold and underlined).

[0047] In embodiments, the IAPs of the present disclosure 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 129%, or about 130%, or about 131%, or about 132%, or about 133%, or about 134%, or about or about 8%, 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.

[0048] In embodiments, an IAP of the present disclosure may comprise an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences described herein, which may, in embodiments, be independently selected from substitutions, insertions, deletions, and truncations.

[0049] In embodiments, the substitutions are made with non-classical amino acids (e.g., selenocysteine, pyrrolysine, 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, designed amino acids such as fluoroamino acids, β-methylamino acids, C α -methyl amino acids, N α -methylamino acids, and amino acid analogs in general).

[0050] IAPs of the present disclosure may be mutated to select agents with desired characteristics. For example, mutations may be performed to generate IAPs with enhanced catalytic activity or protein stability. In embodiments, directed evolution methods may be used to generate IAPs of the present disclosure. For example, error-prone PCR and DNA shuffling may be used to identify mutations in bacterial alkaline phosphatase that confer enhanced activity.

[0051] (Immune checkpoint inhibitor (CPI) immunotherapy) The present disclosure provides, in part, pharmaceutical compositions, formulations, and uses of immune checkpoint inhibitor immunotherapy. Cancer immunotherapy involves utilizing naturally occurring or synthetically produced components to stimulate or enhance the immune system to fight cancer. Immune checkpoint inhibitor immunotherapy is effective in fighting cancer by priming and activating the immune system to achieve antitumor effects, often involving highly specific targeting. Along with the promise of cancer immunotherapy, there is also the need to maintain the immune system's complex balance between identifying and eradicating foreign antigens, a process necessary to suppress uncontrolled immune responses. While providing important clinical benefits, checkpoint inhibition is associated with a unique spectrum of side effects, or immune-related adverse events, including, but not limited to, dermatological, GI, hepatic, endocrine, and other less common inflammatory events. In various embodiments of the present disclosure, GI side effects associated with CPIs include diarrhea and / or colitis. Overall, treatment of these moderate or severe side effects from immune checkpoint inhibitor immunotherapy may require interruption of checkpoint inhibitor immunotherapy and the use of corticosteroid immunosuppression.

[0052] In some aspects, the present disclosure contemplates methods of improving and / or increasing and / or enhancing the efficacy of immune checkpoint inhibitor immunotherapy (e.g., used in tumor treatment). For example, in embodiments, the methods of the present disclosure improve the efficacy of CPIs by widening the therapeutic window of the CPI. In embodiments, the efficacy of CPI therapy is not hindered by administration of an IAP construct. In embodiments, administration of the IAP construct does not reduce the efficacy of CPI therapy. In further embodiments, administration of the IAP construct improves the anti-tumor efficacy of CPI therapy. In embodiments of the present disclosure, immune checkpoint inhibitor immunotherapy is enhanced, e.g., as measured by an increased anti-tumor effect of the immunotherapy.

[0053] In some aspects, the disclosure provides pharmaceutical compositions, formulations, and uses of immune checkpoint inhibitor immunotherapy, including, but not limited to, one or more agents that modulate one or more of programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), inducible T cell costimulatory molecule (ICOS), inducible T cell costimulatory molecule ligand (ICOSL), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In embodiments, the patient is undergoing treatment with an immune checkpoint inhibitor immunotherapy selected from an agent that modulates one or more of programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), inducible T cell costimulatory molecule (ICOS), inducible T cell costimulatory molecule ligand (ICOSL), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4).

[0054] In another aspect, the present disclosure contemplates a method for preventing GI side effects from a CPI by administering a combination of a CPI selected from an agent that modulates one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4 and an IAP.

[0055] In embodiments, the agent that modulates one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4 is an antibody or antibody format specific for one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4. The antibody or antibody format specific for one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4 is selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, a single-chain Fv, a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

[0056] For example, in embodiments, the present disclosure provides a CPI that is an agent that modulates PD-1, wherein the agent is an antibody or antibody format specific for PD-1. In embodiments, the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab. In other embodiments, the present disclosure provides a CPI that is an agent that modulates PD-L1, wherein the agent is an antibody or antibody format specific for PD-L1. In embodiments, the antibody or antibody format specific for PD-L1 is selected from BMS-936559, atezolizumab, avelumab, and durvalumab. In other embodiments, the present disclosure provides a CPI that is an agent that modulates PD-L2, wherein the agent is an antibody or antibody format specific for PD-L2. In other embodiments, the present disclosure provides a CPI that is an agent that modulates ICOS, wherein the agent is an antibody or antibody format specific for ICOS. In embodiments, the antibody or antibody format specific for ICOS comprises JTX-2011. In other embodiments, the disclosure provides a CPI that is an agent that modulates ICOSL, wherein the agent is an antibody or antibody format specific for ICOSL. In other embodiments, the disclosure provides a CPI that is an agent that modulates CTLA-4, wherein the agent is an antibody or antibody format specific for CTLA-4. In embodiments, the antibody or antibody format specific for CTLA-4 is selected from tremelimumab and ipilimumab.

[0057] Add-on therapies for treating immune checkpoint inhibitor-induced GI side effects The present disclosure provides, in part, methods for preventing and / or reducing GI side effects from CPIs by further administering to the patient a corticosteroid and / or a tumor necrosis factor alpha (TNF-α) targeting agent to provide temporary immunosuppression. In embodiments, the patient is treated with one or more corticosteroids. In embodiments, the patient is treated with one or more TNF-α targeting agents. In embodiments, the one or more TNF-α targeting agents are antibodies or fusion proteins. In embodiments, the one or more TNF-α targeting agents are selected from infliximab (Remicade), infliximab-dyyb (Inflectra), infliximab-abda (Renflexis), and Flixabi. In embodiments, administration of a corticosteroid and / or a TNF-α targeting agent to the patient experiencing a CPI interrupts immunotherapy.

[0058] In embodiments, the present disclosure provides methods for preventing and / or reducing GI side effects caused by CPIs by administering an IAP to a patient who is also undergoing treatment with a corticosteroid and / or a TNF-α-targeting agent. In embodiments, the IAP treatment reduces the dose or frequency of corticosteroid administration required to treat the GI side effects caused by immunotherapy. In embodiments, the IAP treatment obviates the need for corticosteroid administration to treat the GI side effects caused by immunotherapy. In embodiments, the IAP treatment reduces the dose or frequency of administration of one or more agents that target TNF-α required to treat the GI side effects caused by immunotherapy. In embodiments, the IAP treatment obviates the need for administration of one or more agents that target TNF-α to treat the GI side effects caused by immunotherapy.

[0059] In embodiments, the patient is a cancer patient. In embodiments, the cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including GI cancer), glioblastoma, hepatic carcinoma, hepatoma, carcinoma in situ, kidney or renal cancer, laryngeal cancer, leukemia, liver cancer, and the like. cancer); lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer cancer); testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NH L; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other adenocarcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and vascular proliferative disorders associated with phacomatosis; edema (such as that associated with brain tumors), and Meigs' syndrome.

[0060] (Method of Producing IAP of the Present Disclosure) The IAPs of the present disclosure are produced using standard molecular biology techniques. For example, nucleic acid compositions encoding the IAPs of the present disclosure are provided, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and / or expression vector compositions. As will be appreciated by one of skill in the art, the protein sequences illustrated herein can be encoded by any number of possible nucleic acid sequences due to the degeneracy of the genetic code.

[0061] As is known in the art, nucleic acids encoding the components of the present disclosure can be incorporated into expression vectors known in the art and, depending on the host cell, used to produce the IAP compositions of the present disclosure. Generally, the nucleic acid will be operably linked to a number of regulatory elements (promoter, origin of replication, selectable marker, ribosome binding site, inducer, etc.). Expression vectors can be extrachromosomal or integrating vectors.

[0062] The nucleic acids and / or expression vectors of the present disclosure are then transformed into a variety of host cells known in the art, including mammalian, bacterial, yeast, insect and / or fungal cells, although mammalian cells (e.g., CHO cells) find use in many embodiments.

[0063] In embodiments, the cell is a mammalian cell. In embodiments, the mammalian cell is a human cell. In embodiments, the cell is an insect cell. In embodiments, the cell is immortalized.

[0064] In embodiments, the cells are Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, human embryonic kidney (HEK293T) cells, Vero cells, or Spodoptera frugiperda 9 (Sf9) cells. In embodiments, the CHO cells are CHO-K1, CHO-DHB11, CHO-DXB1, CHO-S, or CHO-DG44 cells. In embodiments, the CHO cells include or are selected from CHO-K1 (ATCC CCL-61) cells, SURE CHO-M cells (a derivative of CHO-K1), and baby hamster kidney cells (BHK, ATCC CCL-10). In embodiments, the Vero cells include or are selected from Vero, Vero 76, and Vero E6. In embodiments, the cells are the Per C6 cell line, e.g., a human fetal retinal cell line transformed with adenovirus type 5 (Ad5) E1A and E1B genes. In embodiments, the cells are immortalized cell lines based on primary human amniotic cells (e.g., including amniotic fluid stem cells, somatic fetal stem cells), generated by transfection with a vector carrying adenovirus type 5 (Ad5) E1 and pIX functions, e.g., in the CAP cell line (CEVEC Pharmaceuticals, amniotic cell production cell line).

[0065] In embodiments, the cells are, but are not limited to, human cervical cancer cells (HELA, ATCC CCL-2), 293 (ATCC CRL-1573), 3T3 (ATCC CCL-163), or monkey kidney CV1 line (ATCC CCL-70), which can be transformed with SV40 (COS-7, ATCC CRL-1587).

[0066] The IAPs of the present disclosure are produced by culturing host cells containing expression vectors well known to those of skill in the art. Once produced, traditional purification steps are performed. In embodiments, the IAPs of the present disclosure are produced by: i) introducing into host cells a nucleic acid (e.g., an expression vector) encoding one or more APs described herein; ii) culturing the host cells under conditions appropriate for expression (e.g., under antibiotic selection); and iii) isolating the IAP (e.g., using various purification techniques, such as affinity chromatography, size exclusion, etc.).

[0067] (formulation) The present disclosure provides compositions comprising the IAP and / or one or more CPIs (and / or additional therapeutic agents) in various formulations. Compositions comprising any of the IAPs and / or one or more CPIs (and / or additional therapeutic agents) described herein can be in the form of a tablet, pill, pellet, capsule, liquid-containing capsule, multiparticulate-containing capsule, powder, liquid, emulsion, drop, suppository, emulsion, aerosol, spray, suspension, delayed-release formulation, sustained-release formulation, controlled-release formulation, or any other form suitable for use. In embodiments, the IAP of the present disclosure is formulated in a delayed-release capsule.

[0068] In embodiments, the IAPs described herein are formulated into compositions suitable for any of the methods of administration described herein.

[0069] In embodiments, the IAP is formulated for release substantially within the GI tract. In embodiments, the IAP is formulated for release substantially within the small intestine. In embodiments, the IAP is formulated for release substantially within the large intestine. In embodiments, the IAP is formulated for release substantially free of systemic release.

[0070] Formulations comprising the IAP and / or one or more CPI-containing compositions (and / or additional therapeutic agents) may conveniently be presented in unit dosage form. For example, such dosage forms may be prepared by a process comprising the step of bringing a therapeutic agent into association with a carrier which constitutes one or more accessory ingredients. For example, the formulation is prepared by uniformly and intimately bringing a therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if desired, shaping the product into the desired dosage form (e.g., wet or dry granulation, powder blending, etc., followed by tableting).

[0071] In embodiments, a composition comprising an IAP and / or one or more CPIs described herein (and / or additional therapeutic agents) is formulated as a composition compatible with the methods of administration described herein.

[0072] In some embodiments, the recombinant IAP comprises an amino acid sequence having about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% or more sequence identity to any one of SEQ ID NOs:1-14.

[0073] In embodiments, the formulations of the present invention are in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 15 mg of the AP-based drug (e.g., an IAP, or other AP-based drug, drugs described herein, and variants thereof). The capsule contains pellets containing multiple AP-based drugs. In embodiments, the formulations of the present invention include at least one modified-release pellet, each containing about 5% to 25% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug, drugs described herein, and variants thereof). For example, the AP-based agent (e.g., an IAP described herein or other AP-based agent, and variants thereof) can be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, 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 embodiments, the pellet (or each individual pellet) comprises about 45% to 65% sucrose spheres by weight. For example, the sucrose spheres can be present at about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, or about 65% by weight. In embodiments, the pellets (or each individual pellet) comprise about 20% to 40% hydroxypropyl cellulose (HPC) by weight. For example, the hydroxypropyl cellulose 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%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% by weight. In embodiments, the pellets (or each individual pellet) comprise between about 0.2% and 2% 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 salt may be present at about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% by weight. In embodiments, each modified-release pellet comprises about or at least about 5%-25% (wt / w) recombinant IAP, about or at least about 45%-65% (wt / w) sucrose spheres, about or at least about 20%-40% (wt / w) hydroxypropyl cellulose, and about or at least about 0.2%-2% (wt / w) buffering agent. In embodiments, each modified-release pellet comprises about 10%-15% (wt / w) recombinant IAP, about 55%-60% (wt / w) sucrose spheres, about 25%-30% (wt / w) hydroxypropyl cellulose, and about 0.2%-1% (wt / w) buffering agent. In embodiments, each modified-release pellet comprises about 15% (wt / w) recombinant IAP, about 55% (wt / w) sucrose spheres, about 30% (wt / w) hydroxypropyl cellulose, and about 0.5% (wt / w) buffering agent. In embodiments, each modified-release pellet comprises about 14.5% (wt / wt) recombinant IAP, about 56.2% (wt / wt) sucrose spheres, about 28.9% (wt / wt) hydroxypropyl cellulose, and about 0.4% (wt / wt) buffering agent.

[0074] In embodiments, the pellets (or each individual pellet) contain about 10% to 40% by weight EUDRAGIT L30 D-55. For example, the EUDRAGIT L30 D-55 may be present at 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%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% by weight. In embodiments, the pellets (or each individual pellet) contain about 0.5% to 11% by weight of HTP-20. For example, the HTP-20 can be present at about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, or about 11.0% by weight.

[0075] In an embodiment, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 15 mg of the AP-based drug (e.g., an IAP, or other AP-based drug, drugs described herein, and variants thereof). The capsule contains a plurality of enteric-coated pellets containing the AP-based drug. In an embodiment, the capsule contains about 5% to 15% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug, drugs described herein, and variants thereof). For example, the AP-based drug may be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 13%, about 14%, or about 15% by weight. In an embodiment, the capsule contains about 35% to 45% by weight of sucrose spheres. For example, the sucrose spheres may be present at about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45% by weight. In embodiments, the capsule contains about 15% to 25% by weight of hydroxypropyl cellulose (HPC). For example, the HPC may be present at 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 embodiments, the capsule contains about 0.1% to 1.5% by weight of a buffer salt. In embodiments, the capsule contains about 20% to 30% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55). For example, the enteric polymer can be present at about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% by weight. In embodiments, the capsule contains about 1% to 10% by weight of HTP-20 (e.g., PLASACRYL HTP 20).For example, the HTP-20 can be present at about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5%, or about 10 wt%.

[0076] In an embodiment, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 15 mg of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof). The capsule contains multiple enteric-coated pellets containing the 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, 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 a buffer salt; about 26% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55), and about 4.5% by weight of HTP-20 (e.g., PLASACRYL HTP 20).

[0077] In an embodiment, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 15 mg of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof). The capsule contains multiple enteric-coated pellets containing the AP-based drug. In such an embodiment, the formulation contains about 10.0% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof); about 38.9% by weight of sucrose spheres; about 20.0% by weight of hydroxypropyl cellulose (HPC); about 0.3% by weight of a buffer salt; about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT 30 D-55), and about 4.5% by weight of HTP-20 (e.g., PLASACRYL HTP 20).

[0078] In embodiments, the formulations of the present invention are in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of the AP-based drug (e.g., an IAP, or other AP-based drug, drugs described herein, and variants thereof). The capsule contains pellets containing multiple AP-based drugs. In embodiments, the formulations of the present invention include at least one modified-release pellet, each containing about 5% to 25% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug, drugs described herein, and variants thereof). For example, the AP-based agent (e.g., an IAP described herein or other AP-based agent, and variants thereof) can be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, 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 embodiments, the pellet (or each individual pellet) comprises about 45% to 65% sucrose spheres by weight. For example, the sucrose spheres can be present at about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, or about 65% by weight. In embodiments, the pellets (or each individual pellet) comprise about 20% to 40% hydroxypropyl cellulose (HPC) by weight. For example, the hydroxypropyl cellulose 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%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% by weight. In embodiments, the pellets (or each individual pellet) comprise between about 0.2% and 2% 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 salt may be present at about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% by weight. In embodiments, each modified-release pellet comprises about or at least about 5%-25% (wt / w) recombinant IAP, about or at least about 45%-65% (wt / w) sucrose spheres, about or at least about 20%-40% (wt / w) hydroxypropyl cellulose, and about or at least about 0.2%-2% (wt / w) buffering agent. In embodiments, each modified-release pellet comprises about 10%-15% (wt / w) recombinant IAP, about 55%-60% (wt / w) sucrose spheres, about 25%-30% (wt / w) hydroxypropyl cellulose, and about 0.2%-1% (wt / w) buffering agent. In embodiments, each modified-release pellet comprises about 15% (wt / w) recombinant IAP, about 55% (wt / w) sucrose spheres, about 30% (wt / w) hydroxypropyl cellulose, and about 0.5% (wt / w) buffering agent. In embodiments, each modified-release pellet comprises about 14.5% (wt / wt) recombinant IAP, about 56.2% (wt / wt) sucrose spheres, about 28.9% (wt / wt) hydroxypropyl cellulose, and about 0.4% (wt / wt) buffering agent.

[0079] In embodiments, the pellets (or each individual pellet) contain about 10% to 40% by weight EUDRAGIT L30 D-55. For example, the EUDRAGIT L30 D-55 may be present at 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%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% by weight. In embodiments, the pellets (or each individual pellet) contain about 0.5% to 11% by weight of HTP-20. For example, the HTP-20 can be present at about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, or about 11.0% by weight.

[0080] In an embodiment, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of the AP-based agent (e.g., an IAP, or other AP-based agent, agents described herein, and variants thereof). The capsule contains a plurality of enteric-coated pellets containing the AP-based agent. In an embodiment, the capsule contains about 5% to 15% by weight of the AP-based agent (e.g., an IAP, or other AP-based agent, agents described herein, and variants thereof). For example, the AP-based agent may be present at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 13%, about 14%, or about 15% by weight. In an embodiment, the capsule contains about 35% to 45% by weight of sucrose spheres. For example, the sucrose spheres may be present at about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45% by weight. In embodiments, the capsule contains about 15% to 25% by weight of hydroxypropyl cellulose (HPC). For example, the HPC may be present at 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 embodiments, the capsule contains about 0.1% to 1.5% by weight of a buffer salt. In embodiments, the capsule contains about 20% to 30% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55). For example, the enteric polymer can be present at about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% by weight. In embodiments, the capsule contains about 1% to 10% by weight of HTP-20 (e.g., PLASACRYL HTP 20).For example, the HTP-20 can be present at about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5%, or about 10 wt%.

[0081] In an embodiment, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of the AP-based agent (e.g., an IAP, or other AP-based agents described herein, and variants thereof). The capsule contains a plurality of enterically coated pellets containing the AP-based agent. In such an embodiment, the formulation contains about 10% by weight of the AP-based agent (e.g., an IAP, or other AP-based agent, agent 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 a buffer salt; about 26% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55), and about 4.5% by weight of HTP-20 (e.g., PLASACRYL HTP 20).

[0082] In an embodiment, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof). The capsule contains multiple enteric-coated pellets containing the AP-based drug. In such an embodiment, the formulation contains about 10.0% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof); about 38.9% by weight of sucrose spheres; about 20.0% by weight of hydroxypropyl cellulose (HPC); about 0.3% by weight of a buffer salt; about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55), and about 4.5% by weight of HTP-20 (e.g., PLASACRYL HTP 20).

[0083] In embodiments, the IAP is combined with a composition comprising one or more CPIs.

[0084] In embodiments, the formulation comprising the IAP is compression resistant and therefore suitable for tableting. In embodiments, the formulation comprising the one or more CPIs is compression resistant and therefore suitable for tableting. The IAP can be provided in the form of a powder that is then tableted, for example, by physical compression of the dried material.

[0085] In embodiments, the formulation includes one or more enzyme cofactors, such as zinc and / or magnesium. In embodiments, the enzyme cofactor zinc is used. In embodiments, the zinc is provided as zinc sulfate heptahydrate. In embodiments, the enzyme cofactor magnesium is used. In embodiments, the magnesium is provided as magnesium sulfate heptahydrate.

[0086] In embodiments, the formulation includes a protein stabilizer such as trehalose, sucrose, lactose, mannitol, Tween 80, and / or polyvinyl alcohol. In embodiments, the stabilizer is arginine. In embodiments, the stabilizer is sucrose. In embodiments, the stabilizer is lactose.

[0087] In embodiments, the formulation includes one or more surfactants, which may be used as solubilizers or emulsifiers. Exemplary surfactants include, but are not limited to, vitamin E polyethylene glycol succinate, sorbitan monostearate-60 / 80, polysorbate 20, polysorbate 80, and polyoxyl 40 hydrogenated castor oil.

[0088] In embodiments, compositions comprising an IAP and / or one or more CPIs of the present disclosure are stable and / or active in the GI tract, e.g., one or more of the mouth, esophagus, stomach, duodenum, small intestine, duodenum, jejunum, ileum, large intestine, transverse colon, descending colon, ascending colon, sigmoid colon, cecum, and rectum. In certain embodiments, compositions comprising the IAP and / or one or more CPIs are optionally stable in the large intestine, selected from one or more of the transverse colon, descending colon, ascending colon, sigmoid colon, and cecum. In certain embodiments, compositions comprising the IAP and / or one or more CPIs are optionally stable in the small intestine, selected from one or more of the duodenum, jejunum, and ileum. In embodiments, compositions comprising the IAP and / or one or more CPIs are resistant to proteases in the GI tract, e.g., including the small intestine. In embodiments, the IAP composition comprising one or more CPIs is substantially active at a pH of about 5.0 or greater. For example, the IAP and / or one or more CPI-containing compositions can be substantially active at a pH of about 6.0 to about 12, e.g., about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, or about 6.7, or about 6.8, or about 6.9, or about 7.0, or about 7.1, or about 7.2, or about 7.3, or about 7.4, or about 7.5, or about 8.0, or about 8.5, or about 9.0, or about 9.5, or about 10.0, or about 10.5, or about 11.0, or about 11.5, or about 12.0 (e.g., by formulation as described herein, inclusive). In embodiments, stable refers to an enzyme that has a sufficiently long half-life and maintains sufficient activity for therapeutic efficacy.

[0089] In embodiments, compositions comprising an IAP and / or one or more CPIs of the present disclosure are stable in chyme, gastric fluid, and / or bile salts. To evaluate compositions comprising an IAP and / or one or more CPIs in chyme, samples of the composition comprising an IAP and / or one or more CPIs are incubated in human chyme at 37°C. Stability is then assessed by evaluating aliquots removed from the incubated samples at 0, 0.5, 1, 2, 3, 4, 5, and 6 hours for AP activity using para-nitrophenyl phosphate (pNPP) AP substrate. Different chyme specimens can be used for stability assessment, including mixed chyme samples. The chyme samples are characterized for pH, liquid content, and protease activity.

[0090] In embodiments, the IAPs described herein include modified derivatives, i.e., derivatives modified by the covalent attachment of any type of molecule to alkaline phosphatase such that the covalent attachment does not interfere with the activity of the enzyme. For example, derivatives include, but are not limited to, alkaline phosphatase modified by, among others, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, derivatives can include one or more non-classical amino acids. In embodiments, the IAPs are glycosylated to ensure proper protein folding.

[0091] In embodiments, the formulations and dosage forms herein, and those used in the methods herein, include combinations, simultaneous administration, and / or formulation components of those described in U.S. Patent No. 10,987,410, U.S. Patent Application Publication No. 2021 / 0030686, and U.S. Patent Application Publication No. 2022 / 0323367 (e.g., alkaline phosphatase formulations and uses thereof), each of which is incorporated herein by reference in its entirety.

[0092] Pharmaceutically acceptable salts The compositions containing an IAP and / or one or more CPIs described herein may have sufficiently basic functional groups or carboxyl groups that are reactive with inorganic or organic bases to form pharmaceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids as is well known in the art. Examples of such salts include those listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts: Properties, Selection, and Use, PH Stahl and CG Wermuth (eds.), Verlag, Zurich (Switzerland), 2002, which are incorporated herein by reference in their entirety.

[0093] The term "pharmaceutically acceptable salt" also refers to a salt of alkaline phosphatase having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, and organic amines such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines) such as mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-lower alkyl)amine such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

[0094] In embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt. In embodiments, the formulation comprises 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% by weight of a pharmaceutically acceptable salt.

[0095] Pharmaceutical Excipients Additionally, compositions containing any of the IAPs and / or one or more CPIs described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle, such compositions optionally containing a suitable amount of a pharmaceutically acceptable excipient to provide a form for proper administration.

[0096] The pharmaceutical excipient may be a liquid, such as water or oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut, soybean, mineral, sesame, and the like. The pharmaceutical excipient may be, for example, saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary substances, stabilizers, thickeners, lubricants, and coloring agents may be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. When any of the agents described herein are administered intravenously, water is a useful excipient. Saline and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for water for injection. Suitable pharmaceutical excipients also include starch, glucose, hypromellose, lactose, sucrose, trehalose, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, povidone, crospovidone, ethanol, and the like. Any of the formulations described herein can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0097] A suitable pharmaceutical excipient for tableting purposes is Ludipress (lactose, povidone, crospovidone; CAS numbers: 5989-81-1 + 9003-39-8).

[0098] If necessary, the compositions and / or pharmaceutical compositions containing the IAP and / or one or more CPIs (and / or additional therapeutic agents) can include a solubilizing agent. Furthermore, the agents can be delivered using a suitable vehicle or delivery device. The combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.

[0099] In one embodiment, a composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) described herein is formulated as a composition adapted for oral administration. Oral delivery compositions can be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, sprinkles, emulsions, capsules, syrups, or elixirs. Orally administered compositions can contain one or more agents to provide a pharmaceutically palatable formulation, such as sweeteners such as fructose, aspartame, or saccharin; flavorings such as peppermint, wintergreen oil, or cherry; coloring agents; and preservatives. Furthermore, when in tablet or pill form, the compositions can be coated to delay disintegration for sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active agent driving any of the IAPs (and / or additional therapeutic agents) described herein are also suitable for orally administered compositions. In these advanced platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which expands and displaces the agent or agent composition through an opening. These delivery platforms can provide an essentially zero-order delivery profile, as opposed to the spike-and-drop profile of immediate-release formulations. Time-delay agents such as glycerol monostearate or glycerol stearate may also be useful. Oral compositions can include excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, ethacrynic acid and its derivative polymers, and magnesium carbonate. In one embodiment, the excipients are pharmaceutical grade. In addition to the active compound, the suspending agent can include a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like, as well as mixtures thereof.

[0100] In embodiments, the composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) is formulated as a solid dosage form, such as a tablet, a dispersible powder, a granule, or a capsule. In one embodiment, the composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) is formulated as a capsule. In embodiments, the composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) is formulated as a tablet. In a further embodiment, the composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) is formulated as a hard gel capsule. In embodiments, the composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) is formulated as a gelatin capsule.

[0101] In embodiments, the formulation of the composition comprising the IAP and / or one or more CPIs may further comprise a pharmaceutically acceptable carrier or excipient. As one of skill in the art will recognize, the formulation may be in any form appropriate for the desired use and route of administration.

[0102] In some dosage forms, the agents described herein are combined with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate, dicalcium phosphate, and the like, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and Bakers Special Sugar, and the like; b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, gum acacia, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropyl cellulose (HPC), and hydroxymethylcellulose, and the like; c) humectants, such as glycerol, and the like; d) agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, cross-linked polymers such as crospovidone (cross-linked polyvinylpyrrolidone), croscarmellose sodium, and the like. e) disintegrants such as cellulose acetate (crosslinked sodium carboxymethylcellulose), sodium starch glycolate, etc.; f) solution retarders such as paraffin, etc.; f) absorption accelerators such as quaternary ammonium compounds, etc.; g) wetting agents such as cetyl alcohol and glycerol monostearate, etc.; h) absorbents such as kaolin and bentonite clay, etc.; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, glyceryl behenate, etc., and mixtures of such excipients. Those skilled in the art will recognize that a particular excipient may have two or more functions in an oral dosage form. In the case of oral dosage forms, such as capsules or tablets, the dosage form may also include a buffering agent.

[0103] surfactants Any formulation described herein may further comprise a surfactant.Suitable surfactants for use in the present disclosure include, but are not limited to, any pharmaceutically acceptable non-toxic surfactant.Classes of surfactants suitable for use in the compositions of the present disclosure include, but are not limited to, polyethoxylated fatty acids, PEG-fatty acid diesters, PEG-fatty acid mono- and di-ester mixtures, polyethylene glycol glycerol fatty acid esters, alcohol oil transesterification products, polyglyceridized fatty acids, propylene glycol fatty acid esters, propylene glycol ester-glycerol ester mixtures, mono- and diglycerides, sterols and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar esters, polyethylene glycol alkylphenols, polyoxyethylene-polyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants, and mixtures thereof. In embodiments, compositions of the present disclosure 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.

[0104] The formulations may also contain pharmaceutically acceptable plasticizers to achieve 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.

[0105] The formulation may also include one or more coating solvents. For example, some of the more common solvents that can be used to apply the release-delayed coating composition include isopropyl alcohol, acetone, methylene chloride, and the like.

[0106] The formulation may also contain one or more alkaline substances. Suitable alkaline substances for use in the compositions of the present disclosure include, but are not limited to, sodium, potassium, calcium, magnesium, and aluminum salts of acids such as phosphate, carbonate, and citric acid, and other aluminum / magnesium compounds. In addition, the alkaline substance may be selected from substances with antacid effects, such as aluminum hydroxide, calcium hydroxide, magnesium hydroxide, and magnesium oxide.

[0107] In embodiments, the formulation may further comprise magnesium and / or zinc. Without wishing to be bound by theory, the inclusion of magnesium and / or zinc in the formulation promotes protein folding (e.g., dimerization) and biological activity of the IAP. In embodiments, the formulation may comprise magnesium at a concentration of about 1 μM to greater than 5 mM (e.g., about 1 μM to more than 5 mM), including all ranges and values ​​therein. In embodiments, the magnesium is present in the formulation at 1.0 mM. In embodiments, the formulation may comprise zinc at a concentration of about 1 μM to greater than 1 mM (e.g., about 1 μM to more than 1 mM), including all ranges and values ​​therein. In embodiments, the zinc is present in the formulation at 0.1 mM. In embodiments, the formulations of the present disclosure are substantially free of metal chelators.

[0108] In embodiments, the pH of the formulation ensures that the IAP is properly folded (e.g., dimerized) and biologically active. In embodiments, the formulation is maintained at a pH such that amino acids that modulate magnesium and / or zinc binding within the IAP-based drug are not protonated. Protonation of such modulating amino acids can result in loss of metal ions and biological activity and dimer dissociation. In embodiments, the pH of the formulation is greater than about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, or about 12.

[0109] Besides inert diluents, the oral compositions may also include adjuvants such as sweetening, flavoring, and perfuming agents.

[0110] Delivery Various methods can be used to formulate and / or deliver the agents described herein to a desired location. For example, a composition comprising an IAP and / or one or more CPIs described herein (and / or an additional therapeutic agent) can be formulated for delivery to the GI tract. The GI tract includes organs of the digestive system, such as the mouth, esophagus, stomach, duodenum, small intestine, large intestine, and rectum, including all subsections thereof (e.g., the small intestine can include the duodenum, jejunum, and ileum; the large intestine can include the transverse colon, descending colon, ascending colon, sigmoid colon, and cecum). For example, a composition comprising an IAP and / or one or more CPIs described herein (and / or an additional therapeutic agent) can be formulated for delivery to one or more of the stomach, small intestine, large intestine, and rectum, and all subsections thereof (e.g., the duodenum, jejunum, and ileum, the transverse colon, descending colon, ascending colon, sigmoid colon, and cecum). In embodiments, a composition described herein can be formulated for delivery to the intestine. In embodiments, the compositions described herein may be formulated for delivery to the upper or lower GI tract, and in embodiments, the compositions comprising the IAP and / or one or more CPIs (and / or additional therapeutic agents) may be administered, for example, by direct or indirect contact with mucosal tissue of the GI tract.

[0111] In embodiments, the administration of the composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) to the GI tract is by, for example, oral delivery, a nasogastric tube, intestinal intubation (e.g., an enteral tube or feeding tube, such as a jejunal tube or gastro-jejunal tube, etc.), direct injection (e.g., duodenal infusion), endoscopy, colonoscopy, sigmoidoscopy, or enema.

[0112] For example, in embodiments, the present disclosure provides modified-release formulations comprising at least one of the IAP (and / or additional therapeutic agent), which release a significant amount of the IAP (and / or additional therapeutic agent) to one or more regions of the GI tract. For example, the formulation may release at least about 60% of the AP-based agent to one or more regions of the GI tract after the stomach.

[0113] In embodiments, the IAP is formulated for release substantially within the GI tract. In embodiments, the IAP is formulated for release substantially within the small intestine. In embodiments, the IAP is formulated for release substantially within the large intestine. In embodiments, the IAP is formulated for release substantially free of systemic release.

[0114] In embodiments, the modified release formulations of the present disclosure release at least 60% of the IAP (or additional therapeutic agent) in one or more regions of the intestine after the stomach. For example, the modified release formulation releases at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the IAP (or additional therapeutic agent) in the intestine.

[0115] In embodiments, the modified release formulations of the present disclosure release at least 60% of the IAP (or additional therapeutic agent) in the small intestine. For example, the modified release formulations 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 80%, 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 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 1 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%.

[0116] In embodiments, the modified release formulations of the present disclosure release at least 60% of the IAP (or additional therapeutic agent) in the large intestine. 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%, or at least 76% of the IAP (or additional therapeutic agent) in the large intestine (e.g., one or more of the cecum, the ascending, transverse, descending, or sigmoid portion of the colon, and the rectum). 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%.

[0117] In embodiments, the modified release formulation does not substantially release the IAP (or additional therapeutic agent) in the stomach.

[0118] In certain embodiments, the modified-release formulation releases the composition comprising the IAP and / or one or more CPIs (or additional therapeutic agents) above a certain pH. For example, in embodiments, the modified-release formulation is substantially stable in an acidic environment and substantially unstable (e.g., rapidly dissolving or physically unstable) in a near-neutral to alkaline environment. In embodiments, unstable indicates substantial release, while stable indicates substantially no release. For example, in 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 embodiments, the formulation is stable at lower pH ranges and therefore does not substantially release, for example, in the stomach. In embodiments, the modified release formulation is substantially stable at a pH of about 1 to about 5 or less and substantially unstable at higher pH values. In these embodiments, the modified release formulation does not substantially release in the stomach. In these embodiments, the modified release formulation substantially releases in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) and / or large intestine (e.g., the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). In embodiments, the modified release formulation is substantially stable at a pH of about 4 to about 7 or less and, as a result, is substantially unstable at higher pH values ​​and, therefore, does not substantially release in the stomach and / or proximal small intestine (e.g., one or more of the duodenum and jejunum). In these embodiments, the modified release formulation substantially releases in the distal small intestine or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). In 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 they are in a fasting or postprandial state.

[0119] In embodiments, the modified release formulation is substantially stable in gastric fluids and substantially unstable in intestinal fluids, and therefore 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).

[0120] In embodiments, the modified release formulations are stable in gastric fluid or stable in an acidic environment, and contain no more than about 30% by weight of alkaline phosphatase and / or additional therapeutic agent in gastric fluid having a pH of about 4 to about 5 or less, or simulated gastric fluid having a pH of about 4 to about 5 or less, and release the alkaline phosphatase and / or additional therapeutic agent in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. The modified-release formulation of the present disclosure can release alkaline phosphatase and / or additional therapeutic agent in gastric fluid having a pH of 4 to 5 or less, or simulated gastric fluid having a pH of 4 to 5 or less, in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes, and the alkaline phosphatase and / or additional therapeutic agent in the modified-release formulation is 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. The modified release formulations of the present disclosure may be released in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes in gastric fluid having a pH of 5 or less or simulated gastric fluid having a pH of 5 or less, and the total alkaline phosphatase and / or additional therapeutic agent in said modified release formulation is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight.

[0121] In embodiments, the modified release formulations are unstable in intestinal fluid. These modified release formulations release about 70% or more by weight of the alkaline phosphatase and / or additional therapeutic agent in the modified release formulation in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes in intestinal fluid or simulated intestinal fluid. In embodiments, the modified release formulations are unstable in a near-neutral to alkaline environment. These modified release formulations release about 70% or more by weight of the alkaline phosphatase and / or additional therapeutic agent in gastric fluid having a pH of about 4 to about 5 or higher, or simulated gastric fluid having a pH of about 4 to about 5 or higher, in about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. A modified-release formulation that is unstable in a nearly neutral to alkaline environment may release about 70% by weight or more of the alkaline phosphatase and / or additional therapeutic agent in a body fluid having a pH greater than about 5 (e.g., a body fluid having a pH of about 5 to about 14, about 6 to about 14, about 7 to about 14, about 8 to about 14, about 9 to about 14, about 10 to about 14, or about 11 to about 14) in about 5 to about 90 minutes, or about 10 to about 90 minutes, or about 15 to about 90 minutes, or about 20 to about 90 minutes, or about 25 to about 90 minutes, or about 30 to about 90 minutes, or about 5 to about 60 minutes, or about 10 to about 60 minutes, or about 15 to about 60 minutes, or about 20 to about 60 minutes, or about 25 to about 90 minutes, or 30 to about 60 minutes.

[0122] Examples of simulated gastric and simulated intestinal fluids include, but are not limited to, those disclosed in Test Solutions' 2005 Pharmacopeia 23NF / 28USP on page 2858 and / or other simulated gastric and simulated intestinal fluids known to those skilled in the art, such as simulated gastric and / or intestinal fluids prepared without enzymes.

[0123] In embodiments, the modified release formulations of the present disclosure are substantially stable in chyme, for example, in embodiments, there is less than about 50%, or about 40%, or about 30%, or about 20%, or about 10% loss of AP-based drug activity 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.

[0124] In embodiments, the modified release formulations of the present disclosure are designed for immediate release (e.g., upon ingestion). In embodiments, the modified release formulations may have a sustained release profile, i.e., slow release of the active ingredient in the body (e.g., the GI tract) over an extended period of time. In embodiments, the modified release formulations may have a delayed release profile, i.e., do not immediately release the active ingredient upon ingestion; rather, release is delayed until the composition is lower in the GI tract, for example, in the small intestine (e.g., one or more of the duodenum, jejunum, and ileum) or the large intestine (e.g., one or more of the cecum, the ascending, transverse, descending, or sigmoid portion of the colon, and the rectum). For example, the composition may be enterically coated to delay release of the active ingredient until it reaches the small intestine or large intestine.

[0125] Enteric coating In embodiments, the formulations of the present disclosure (e.g., IAP as a powder or tablet) are coated to provide protection of the active agent in the GI tract, including the stomach. For example, in embodiments, the formulations can be encapsulated in enteric-coated capsules. Additionally, in embodiments, the formulations (e.g., IAP as a powder or tablet) are themselves coated with one or more coatings, such as one or more modified-release coatings described herein (e.g., after a powder granulation step). Further, in embodiments, the powder formulations (e.g., IAP as a powder) can be compressed into enteric-coated tablets.

[0126] In embodiments, the modified release formulations of the present disclosure may use one or more modified release coatings, such as delayed release coatings, to provide effective and substantially delayed delivery of the alkaline phosphatase to the GI tract, optionally along with an additional therapeutic agent.

[0127] In embodiments, the modified release formulations of the present disclosure may employ one or more modified release coatings, such as delayed release coatings, to provide effective and substantially delayed delivery of the IAP to the intestine, optionally along with other additional therapeutic agents.

[0128] In one embodiment, the release-retarding coating includes an enteric drug that is substantially stable in an acidic environment and substantially unstable in a near-neutral to alkaline environment. In one embodiment, the release-retarding coating includes an enteric drug that is substantially stable in gastric fluids. The enteric coating may be selected from, for example, solutions or dispersions of methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, carboxymethylethylcellulose, and EuDRAGIT®-type polymers (poly(methacrylic acid, methyl methacrylate), hydroxypropyl methylcellulose acetate succinate, cellulose acetate trimellitate, shellac, and other suitable enteric coating polymers. Such polymers are described in international pharmacopoeias such as Ph.Eur., USP / NF, DMF, and JPE. 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 various KOLLICOAT (e.g., polyvinyl alcohol, PEG, and colloidal anhydrous silica) (e.g., Kollicoat® MAE 30 DP or Kollicoat® MAE 100 P) and EUDRAGIT (e.g., polymethacrylate-based copolymer) polymers and formulations. In embodiments, 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 embodiments, the enteric agent may be a combination of the foregoing solutions or dispersions. In embodiments, the release-delaying coating comprises the enteric agent EUDRAGIT® L 100.

[0129] As a non-limiting example, there are various EUDRAGIT formulations that dissolve with increasing pH, including formulations that dissolve at pH > 5.5 (EUDRAGIT L30 D-550), pH > 6.0 (EUDRAGIT L12.5), and pH > 7.0 (EUDRAGIT FS 30D). Because the ileum has the highest pH in the small intestine, ranging from 7.3 to 7.8, the use of EUDRAGIT FS 30D as an enteric-coated formulation can delay dissolution until the ileum, thereby localizing release of the AP-based drug to the ileum. However, the jejunum has a pH that can range from 6.6 to 7.4, and therefore various EUDRAGIT formulations can be used to target release to this segment of the small intestine. Different types of EUDRAGIT can be combined with each other, or multiple different types of EUDRAGIT coatings can be combined to fine-tune the dissolution profile and target delivery for optimal function. For example, EUDRAGIT L100, EUDRAGIT S100, and triethyl citrate may be mixed, e.g., in a ratio of about 72.7 / 18.2 / 9.1, to produce a coating that substantially releases at a pH above about 6.2. In another example, EUDRAGIT L100, EUDRAGIT S100, and triethyl citrate may be mixed, e.g., in a ratio of about 30 / 60.9 / 9, to produce a coating that substantially releases at a pH above about 6.7. In a further example, DuoCoat™ (Kuecept, Ltd.) may be used, which uses two coatings: an outer layer of enteric polymer (such as EUDRAGIT), and an inner layer of partially neutralized enteric polymer and buffer. DuoCoat™ technology allows for a more rapid release of therapeutic agents initiated at a target pH compared to a single coating of enteric polymer (Liu et al., 2010, European J. Pharmaceutics and Biopharmaceuticals 47:311, the entire contents of all of which are incorporated herein by reference).Targeted release to the ileum and / or ileocecal tract has been demonstrated in 10 healthy volunteers (Varum et al., 2013, European J. Pharmaceuticals and Biopharmaceuticals 84:573, the entire contents of which are incorporated herein by reference).

[0130] In certain embodiments, one or more coating system additives are used in conjunction with the enteric coated drug. For example, one or more PLASACRYL (e.g., various ratios of anti-adherent mono- and di-glycerides (glyceryl monostearate (GMS)), plasticizers (triethyl citrate (TEC)), and / or stabilizers (polymethacrylate copolymers) additives may be used as anti-adherent coating additives, plasticizers, and / or stabilizers. Exemplary PlasACRYL™ additives include, but are not limited to, PlasACRYL™ HTP20 and PlasACRYL™ T20.

[0131] In embodiments, the release-retarding coating may degrade over time in aqueous solution, regardless of pH and / or the presence of enzymes in the solution. Such coatings may include water-insoluble polymers. Therefore, their solubility in aqueous solution is independent of pH. As used herein, the term "pH-independent" means that the polymer's water permeability and its ability to release pharmaceutical ingredients are not a function of pH and / or are only slightly dependent on pH. Such coatings may be used, for example, to prepare sustained-release formulations. Suitable water-insoluble polymers include pharmaceutically acceptable, non-toxic polymers that are substantially insoluble in aqueous media, e.g., water, that are 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 hydroxy 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, and acrylic and / or methacrylic ester polymers, acrylate or methacrylate polymers or copolymers with low quaternary ammonium content, or mixtures thereof, and the like. Other examples of insoluble polymers include EUDRAGIT RS®, EUDRAGIT RL®, and EUDRAGIT NE®. Insoluble polymers useful in the present disclosure include polyvinyl esters, polyvinyl acetals, polyacrylic esters, butadiene-styrene copolymers, and the like. In one embodiment, colonic delivery is achieved through the use of a slowly eroding wax plug (e.g., various PEGs, including PEG 6000) or pectin. In an embodiment, the present disclosure contemplates the use of a release-delaying coating that degrades over time and includes an expanding layer containing croscarmellose sodium and hydroxypropyl cellulose.In such embodiments, the formulation may further comprise an osmotic burst coating comprising ethylcellulose, such as an ethylcellulose dispersion.

[0132] Alternatively, the stability of the modified-release formulation may be enzyme-dependent. An enzyme-dependent release-retarding coating will be substantially stable in body fluids that do not contain a particular enzyme and substantially unstable in body fluids that contain the enzyme. The release-retarding coating will essentially disintegrate or dissolve in body fluids that contain the appropriate enzyme. For example, enzyme-dependent control can be achieved by using a material, such as galactomannan, that only releases the active ingredient upon exposure to enzymes in the small intestine. Furthermore, the stability of the modified-release formulation may depend on enzyme stability in the presence of microbial enzymes present in the intestinal flora. For example, in embodiments, the release-retarding coating may be degraded by microbial enzymes present in the intestinal flora. In embodiments, the release-retarding coating may be degraded by bacteria present in the small intestine. In embodiments, the release-retarding coating may be degraded by bacteria present in the large intestine.

[0133] In embodiments, the modified-release formulation is designed for release in the colon. Various colon-specific delivery approaches may be utilized. For example, the modified-release formulation may be formulated using a colon-specific drug delivery system (CODES), e.g., as described in Li et al., AAPS PharmSciTech (2002), 3(4):1-9, the entire contents of which are incorporated herein by reference. Drug release in such systems is triggered by colonic microflora combined with a pH-sensitive polymer coating. For example, the formulation may be designed as a core tablet with three layers of polymers: a first coating of an acid-soluble polymer (e.g., EUDRAGIT E), with a hydroxypropyl methylcellulose barrier layer sandwiched between them, and an outer coating that is enteric. In embodiments, colonic delivery may be achieved by formulating the alkaline phosphatase (and / or additional therapeutic agent) with a specific polymer that degrades in the colon, such as pectin. Pectin may be further gelled or crosslinked with cations, such as zinc cations. In embodiments, the formulation is in the form of ionically cross-linked pectin beads further coated with a polymer (e.g., a EUDRAGIT polymer). Additional colon-specific formulations include, but are not limited to, pressure-controlled drug delivery systems (e.g., prepared with ethylcellulose) and osmotic-controlled drug delivery systems (i.e., ORDS-CT).

[0134] Formulations for colon-specific delivery of compositions (and / or additional therapeutic agents) comprising an IAP and / or one or more CPIs described herein can be evaluated, for example, using in vitro dissolution tests. For example, parallel dissolution tests in different buffers can be performed to characterize the behavior of the formulation at different pH levels. Alternatively, in vitro enzymatic tests can be performed. For example, the formulation can be incubated in a fermenter containing an appropriate medium for bacteria, and the amount of drug released at different time intervals can be determined. Drug release tests can also be performed in a buffered medium containing enzymes or rat, guinea pig, or rabbit cecal contents to determine the amount of drug released at a specific time. In embodiments, in vivo evaluations can be performed using animal models such as dogs, guinea pigs, rats, and pigs. Furthermore, clinical evaluation of colon-specific drug delivery formulations can be evaluated by calculating the drug delivery index (DDI), which is the relative ratio of RCE (relative colon tissue exposure to the drug) to RSC (relative drug concentration in the blood, i.e., relative systemic exposure to the drug). A higher drug DDI indicates better colon drug delivery. Drug absorption from the colon can be monitored by colonoscopy and intubation.

[0135] In embodiments, the formulations provide substantially uniform dissolution of the IAP (and / or additional therapeutic agent) in the region of release in the GI tract, hi embodiments, the formulations minimize patchy or heterogeneous release of the AP-based drug.

[0136] In embodiments, the present disclosure provides a modified release formulation that releases multiple doses of the AP-based drug at different times and / or at different pH levels along the intestine. In embodiments, the modified release formulation comprises a first dose of the IAP and a second dose of the IAP, and the first and second doses are released at different times and / or at different pH levels along the intestine. 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 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 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 the AP-based drug at different times and / or at different pHs at different locations along the intestine.

[0137] In embodiments, the formulations of the present disclosure are disclosed in U.S. Pat. Nos. 8,535,713 and 8,9117,777, and U.S. Patent Application Publication Nos. 2012 / 0141585, 2012 / 0141531, 2006 / 001896, 2007 / 0292523, 2008 / 0020018, 2008 / 0113031, 2010 / 0203120, 201 Nos. 2010 / 0255087, 2010 / 0297221, 2011 / 0052645, 2013 / 0243873, 2013 / 0330411, 2014 / 0017313, and 2014 / 0234418, the contents of which are incorporated herein by reference in their entirety.

[0138] In embodiments, the formulations of the present disclosure may be prepared using any of the methods disclosed in U.S. Patent Nos. 4,196,564; 4,196,565; 4,247,006; 4,250,997; 4,268,265; 5,317,849; 6,572,892; 7,712,634; 8,074,835; 8,398,912; 8,440,224; 8,557,294; 8,646,591; 8,739,812; 8,810,259; Nos. 8,852,631; and 8,911,788, and one or more of U.S. Patent Application Publication Nos. 2014 / 0302132; 2014 / 0227357; 2014 / 0088202; 2013 / 0287842; 2013 / 0295188; 2013 / 0307962; and 2013 / 0184290, the contents of which are incorporated herein by reference in their entirety.

[0139] In embodiments, the method for formulating the IAP is sufficiently gentle so that the tertiary structure (e.g., dimeric structure) of the IAP remains substantially intact. In embodiments, the method for formulating the IAP includes a step of refolding the IAP. In such embodiments, the step of refolding the IAP can include adding magnesium and / or cyclodextrin.

[0140] In embodiments, the modified release formulation is a modified release powder formulation.

[0141] In embodiments, the modified release formulations comprising an IAP, and variations thereof, and / or additional therapeutic agents described herein are administered orally.

[0142] Suitable dosage forms for oral use include solid dosage forms such as, for example, tablets, capsules, powders, and granules. In embodiments, the modified-release formulation is in the form of a powder. In embodiments, the powder formulations of the present disclosure can be added to foods (e.g., juices, strained and / or pureed foods (e.g., fruits, vegetables), sauces, infant formula, milk, etc.). In embodiments, the modified-release formulation is packaged in the form of a sachet. In embodiments, the modified-release formulation is in the form of a tablet. In embodiments, the modified-release formulation is in the form of a tablet containing a powder. In embodiments, the modified-release formulation is in the form of a capsule. In embodiments, the modified-release formulation is in the form of a capsule containing a powder.

[0143] In embodiments, the modified-release formulations of the present disclosure are in the form of a powder. In embodiments, the powder is formed by spray drying and / or spray-dried dispersion (SDD) techniques. In embodiments, a powder containing an AP-based drug is formed by dissolving the AP-based drug and a polymer in a solvent and then spray-drying the solution. The resulting powder contains the AP-based drug dispersed within a solid polymer matrix.

[0144] Various types of polymers may be used for the modified release formulations of the present disclosure. In embodiments, the polymer is an enteric polymer that is substantially stable in acidic environments and substantially unstable in near-neutral to alkaline environments. In embodiments, the enteric polymer is substantially stable in gastric fluids.

[0145] Exemplary polymers include, but are not limited to, copovidone, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, polyvinylpyrrolidinone (PVP), hydroxypropyl methylcellulose or hypromellose (HPMC), hypromellose phthalate (HPMCP), hydroxypropyl methylcellulose or hypromellose acetate succinate (HPMCAS), methacrylate / methacrylic acid copolymer, and mixtures thereof. In embodiments, the polymer is HPMCAS. In embodiments, the polymer is HPMCAS LF, LG, MF, MG, HF, or HG. In embodiments, the polymer is HPMCAS-LF.

[0146] In embodiments, the modified release formulation further comprises a single layer enteric coating that adds about 20% to 40% to the enteric polymer weight, and optionally adds about 26.3% to the enteric polymer weight. In embodiments, the capsule comprises gelatin or hydroxypropyl methylcellulose.

[0147] buffer In embodiments, various types of solvents / buffers are used to prepare the powder formulations of the present disclosure. In embodiments, the solvent / buffer is an organic solvent / buffer. Exemplary solvents / buffers that can be used to dissolve the AP-based drug and polymer before spray drying include, but are not limited to, ethanol, methanol, acetone, IPA, tetrahydrofuran, dichloromethane, and mixtures thereof. In embodiments, the solvent used is water, such as distilled DI water. In embodiments, the buffer used is monosodium phosphate monohydrate.

[0148] In embodiments, various buffers, such as Good's buffers (e.g., MES, ADA, PIPES, ACES, MOPSO, chloramine chloride, MOPS, BES, TES, HEPES, DIPSO, TAPSO, acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Tris, glycinamide, glycylglycine, bicine, and / or TAPS), and salts of these buffers are used. In embodiments, buffers include, but are not limited to, amino acid buffers such as histidine, arginine, and / or cysteine. In embodiments, buffers include phosphate buffered saline (PBS). In embodiments, various salts are used, such as, but not limited to, potassium chloride, sodium chloride, calcium chloride, magnesium chloride, sodium sulfate, calcium sulfate, and / or magnesium sulfate. In embodiments, the buffers and / or salts include any combination of potassium chloride, phosphoric acid, calcium chloride, magnesium sulfate, potassium phosphate (monobasic and / or dibasic), and / or sodium phosphate (monobasic and / or dibasic). In embodiments, the buffering agent used is monobasic sodium phosphate monohydrate. In embodiments, the buffering agent used includes arginine and phosphate.

[0149] In embodiments, an enzyme cofactor comprising zinc and magnesium is used. In embodiments, the enzyme cofactor zinc is used. In embodiments, the zinc is provided as zinc sulfate heptahydrate. In embodiments, the enzyme cofactor magnesium is used. In embodiments, the magnesium is provided as magnesium sulfate heptahydrate.

[0150] In embodiments, the formulation includes a protein stabilizer such as trehalose, sucrose, lactose, mannitol, Tween 80, or polyvinyl alcohol. In embodiments, the stabilizer is sucrose. In embodiments, the stabilizer is lactose.

[0151] In embodiments, the powder formulations of the present disclosure may include a surfactant. The surfactant may be used as a solubilizer or emulsifier. Exemplary surfactants include, but are not limited to, vitamin E polyethylene glycol succinate, sorbitan monostearate-60 / 80, polysorbate 20, polysorbate 80, and polyoxyl 40 hydrogenated castor oil.

[0152] In embodiments, the powder containing the AP-based drug forms a gel. In embodiments, the powder containing the AP-based drug forms a gel in the intestine. In embodiments, the AP-based drug is released from the gel into one or more regions of the intestine. In embodiments, at a pH value greater than about 5 (e.g., about 5, or 6, or 7, or 8, or 9), the gel converts to a solution phase and releases the AP enzyme. In embodiments, the gel is used to control the release of the AP-based drug in the intestine. In embodiments, the AP-based drug is released from the gel into one or more of the group consisting of the small intestine, duodenum, jejunum, ileum, large intestine, transverse colon, descending colon, ascending colon, sigmoid colon, cecum, and rectum.

[0153] In embodiments, the formulations of the present disclosure are in the form of a powder comprising the AP-based drug dispersed within a solid polymer matrix. In embodiments, the powder is formed by dissolving the AP-based drug and polymer in a solvent to form a solution, which is then spray-dried. In embodiments, the solution for spray drying contains about 0.1% to 1% by weight of the AP-based drug. For example, the AP-based drug may be present at about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1.0% by weight. In embodiments, the solution includes about 1% to 10% by weight of a polymer (e.g., HPMCAS-LF). For example, the polymer can 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 embodiments, the solution includes about 0.05% to 0.5% by weight of a buffering agent (e.g., monosodium phosphate monohydrate). For example, the buffering agent can be present at about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, or about 0.50% by weight. In embodiments, the solution can be present at about 0.001% to 0.01% zinc (e.g., zinc sulfate heptahydrate) by weight. For example, the zinc can be present at about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, or about 0.01% by weight. In embodiments, the solution can be present at about 0.01% to 0.1% magnesium (e.g., magnesium sulfate heptahydrate) by weight.For example, the magnesium may be present at about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% by weight. In embodiments, the solution includes about 0.1% to 1% by weight of a protein stabilizer (e.g., trehalose). For example, the protein stabilizer may be present at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% by weight. In embodiments, the solution includes about 90% to 99.9% by weight of a solvent (e.g., water). For example, the solvent can be present at about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% by weight.

[0154] In embodiments, the IAP is formulated into a modified-release formulation comprising at least one modified-release pellet. In embodiments, the modified-release formulation of the present disclosure is in the form of a tablet or capsule. In embodiments, the modified-release formulation is in the form of a tablet or capsule comprising the powder of the present disclosure. Various approaches to making tablets or capsules may be utilized to contain the powder of the present disclosure. In embodiments, the tablets of the present disclosure are made by granulation, such as dry granulation. In such embodiments, the powder is pre-compressed and the resulting tablets or slugs are milled to obtain granules. Alternatively, the powder is pre-compressed using pressure rolls to obtain granules. In further embodiments, the powder is encapsulated in a capsule. In embodiments, the capsule is a gelatin capsule, such as a hard gelatin capsule. In embodiments, the capsule is a hydroxypropyl methylcellulose (HPMC) capsule.

[0155] In embodiments, the tablet or capsule comprises a release-retarding coating comprising an enteric drug that is substantially stable in an acidic environment and substantially unstable in a near-neutral to alkaline environment, hi embodiments, the release-retarding coating comprises an enteric drug that is substantially stable in gastric fluids. The enteric coating may be selected from, for example, methacrylic acid copolymer solutions or dispersions, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, carboxymethylethylcellulose, and EUDRAGIT®-type polymers (poly(methacrylic acid, methyl methacrylate), hydroxypropyl methylcellulose acetate succinate, cellulose acetate trimellitate, shellac, and other suitable enteric coating polymers. Such polymers are described in international pharmacopoeias, such as Ph.Eur., USP / NF, DMF, and JPE. 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 Examples of suitable polymers include 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. In embodiments, 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 In embodiments, the enteric agent may be a combination of the aforementioned solutions or dispersions. In embodiments, the release-delaying coating comprises the enteric agent EUDRAGIT® L 100.In embodiments, the tablet or capsule is coated with the enteric agent at about 1% to 20% coating weight, such as about 1%, about 2%, about 3%, about 4%, 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%, or about 20% coating weight.

[0156] (Administration and Dosage) It will be understood that the actual dose of a composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) administered according to the present disclosure will vary depending on the particular compound, the particular dosage form, and the method of administration. Many factors that may modify the action of the AP-based drug (e.g., body weight, sex, diet, administration time, administration route, excretion rate, condition of the subject, concomitant use of drugs, genetic predisposition, and reaction susceptibility) can be taken into consideration by those skilled in the art. Administration can be carried out 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 determined by those skilled in the art using conventional dosage and administration tests.

[0157] The individual dose of the composition containing the IAP and / or one or more CPIs (and / or additional therapeutic agent) is, for example, about 0.01 mg to about 1,000 mg, about 0.01 mg to about 900 mg, about 0.01 mg to about 800 mg, about 0.01 mg to about 700 mg, about 0.01 mg to about 600 mg, about 0.01 mg to about 500 mg, about 0.01 mg to about 400 mg, about 0.01 mg to about 300 mg, about 0.01 mg to about 200 mg, about 0.1 mg It can be administered in a unit dosage form (e.g., tablet or capsule) containing about 0.1 mg to about 100 mg, about 0.1 mg to about 90 mg, about 0.1 mg to about 80 mg, about 0.1 mg to about 70 mg, about 0.1 mg to about 60 mg, about 0.1 mg to about 50 mg, about 0.1 mg to about 40 mg, 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, or about 0.1 mg to about 1 mg of the active ingredient.For example, unit dosage forms may contain 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 11 mg, about 12 mg, about 13mg, about 14mg, about 15mg, about 16mg, about 17mg, about 18mg, about 19mg, about 20mg, about 21mg, about 22mg, about 23mg, about 24mg, about 25mg, about 26mg, about 27mg, about 28mg, about 29mg, about 30 mg, about 31mg, about 32mg, about 33mg, about 34mg, about 35mg, about 36mg, about 37mg, about 38mg, about 39mg, about 40mg, about 41mg, about 42mg, about 43mg, about 44mg, about 45mg, about 46mg, about 47mg, About 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg , about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1,000 mg, including all values ​​and ranges therebetween.

[0158] In one embodiment, the composition comprising the IAP and / or one or more CPIs (and / or additional therapeutic agent) is administered in an amount of about 0.01 mg to about 1,000 mg per day, about 0.01 mg to about 900 mg per day, about 0.01 mg to about 800 mg per day, about 0.01 mg to about 700 mg per day, about 0.01 mg to about 600 mg per day, about 0.01 mg to about 500 mg per day, about 0.01 mg to about 400 mg per day, about 0.01 mg to about 300 mg per day, about 0.01 mg to about 200 mg per day, about 0.01 mg to about 100 mg per day, about 0.1 mg to about 100 mg per day, about 0.1 mg to about 95 mg per day, about 0.1 mg to about 90 mg per day, about 0.1 mg to about 85 mg per day, about 0.1 mg to about 100 mg per day, about 0.1 mg to about 100 mg per day, about 0.1 mg to about 100 mg per day, about 0.1 mg to about 105 mg per day, about 0.1 mg to about 150 ... Approximately 0.1mg to approximately 80mg per day, approximately 0.1mg to approximately 75mg per day, approximately 0.1mg to approximately 70mg per day, approximately 0.1mg to approximately 65mg per day, approximately 0.1mg to approximately 60mg per day, approximately 0.1mg to approximately 55mg per day, approximately 0.1mg to approximately 50mg per day, approximately 0.1mg to approximately 45mg per day, approximately 0.1mg to approximately 40mg per day, approximately 0.1mg to approximately The dose is administered in an amount of about 35 mg, 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.In embodiments, the IAP is administered in an amount of 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 11 mg, about 12 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 2mg, about 13mg, about 14mg, about 15mg, about 16mg, about 17mg, about 18mg, about 19mg, about 20mg, about 21mg, about 22mg, about 23mg, about 24mg, about 25mg, about 26mg, about 27mg, about 28mg, about 29m g, about 30mg, about 31mg, about 32mg, about 33mg, about 34mg, about 35mg, about 36mg, about 37mg, about 38mg, about 39mg, about 40mg, about 41mg, about 42mg, about 43mg, about 44mg, about 45mg, about 46mg 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, and about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1,000 mg, including all values ​​and ranges therebetween.

[0159] In embodiments, a suitable dosage of a composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) is from about 0.01 mg / kg to about 100 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 90 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 80 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 70 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 60 mg / kg of the subject's body weight, from about 0.01 mg / kg to about 50 mg / kg of the subject's body weight, The range is about 0.01 mg / kg to about 40 mg / kg of the subject's body weight, about 0.01 mg / kg to about 30 mg / kg of the subject's body weight, about 0.01 mg / kg to about 20 mg / kg of the subject's body weight, or about 0.01 mg / kg to about 10 mg / kg of the subject's body weight, for example, about 0.01 mg / kg of body weight, about 0.02 mg / kg of body weight, about 0.03 mg / kg of body weight, about 0.04 mg / kg of body weight, about 0.05 mg / kg of body weight, about 0.06 mg / kg of body weight, about 0.07 mg / kg of body weight, or about 0.08 mg / kg of body weight. kg, approximately 0.09 mg / kg body weight, approximately 0.1 mg / kg body weight, approximately 0.2 mg / kg body weight, approximately 0.3 mg / kg body weight, approximately 0.4 mg / kg body weight, approximately 0.5 mg / kg body weight, approximately 0.6 mg / kg body weight, approximately 0.7 mg / kg body weight, approximately 0.8 mg / kg body weight, approximately 0.9 mg / kg body weight, approximately 1 mg / kg body weight, approximately 1.1 mg / kg body weight, approximately 1.2 mg / kg body weight, approximately 1.3 mg / kg body weight, approximately 1.4 mg / kg body weight, approximately 1.5 mg / kg body weight, approximately 1.6 mg / kg body weight, approximately 1.7 mg / kg body weight, approximately 1.8 mg / kg body weight kg, 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 20 mg / kg body weight, about 30 mg / kg body weight, about 40 mg / kg body weight, about 50 mg / kg body weight, about 60 mg / kg body weight, about 70 mg / kg body weight, about 80 mg / kg body weight, about 90 mg / kg body weight, or about 100 mg / kg body weight, including all values ​​and ranges therebetween.In embodiments, a suitable dosage of the AP-based drug is in the range of about 0.01 mg / kg to about 10 mg / kg of body weight, about 0.01 mg / kg to about 9 mg / kg of body weight, about 0.01 mg / kg to about 8 mg / kg of body weight, about 0.01 mg / kg to about 7 mg / kg of body weight, about 0.01 mg / kg to about 6 mg / kg of body weight, about 0.05 mg / kg to about 5 mg / kg of body weight, about 0.05 mg / kg to about 4 mg / kg of body weight, about 0.05 mg / kg to about 3 mg / kg of body weight, about 0.05 mg / kg to about 2 mg / kg of body weight, about 0.05 mg / kg to about 1.5 mg / kg of body weight, or about 0.05 mg / kg to about 1 mg / kg of body weight.

[0160] According to certain embodiments of the present disclosure, the composition comprising the IAP and / or one or more CPIs (and / or additional therapeutic agents) may be administered, for example, more than once daily (e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times per day), about once daily, about once every other day, about once every 3 days, about once per week, about once every 2 weeks, about once per month, about once every 2 months, about once every 3 months, about once every 6 months, or about once per year.

[0161] In embodiments, the IAP is administered multiple times. In embodiments, the IAP is administered orally multiple times. In embodiments, the dose of the IAP is tailored to the amount of IAP recovered in a biological sample of the subject being treated (e.g., present in stool, blood, etc.). In embodiments, the dose of the IAP is tailored to the severity and / or presence of one or more symptoms.

[0162] (Treatment method) In embodiments, compositions comprising one or more CPIs (and / or additional therapeutic agents) including, but not limited to, an IAP of the present disclosure and / or one or more agents that modulate one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4 are co-administered. The co-administration can be simultaneous or sequential. In embodiments, the IAP is administered before the CPI.

[0163] In embodiments, the present disclosure provides methods for treating or preventing GI side effects from CPIs, such as, but not limited to, diarrhea and / or colitis. Colitis can refer to inflammation of the lining of the colon. In embodiments, colitis can be accompanied by abdominal pain, cramping, diarrhea (with or without blood in the stool), and combinations thereof. For example, a patient experiencing colitis may find blood in their stool, have a constant urge to defecate (e.g., tenesmus), and experience persistent abdominal pain, fever, chills, and / or other signs of infection and inflammation.

[0164] Thus, in embodiments, the disclosed method provides for the prevention and / or treatment of colitis as a GI side effect of a CPI by administering a composition comprising a CPI selected from an agent that modulates IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4. In embodiments, the patient is a cancer patient and may have undergone treatment with an immune checkpoint inhibitor immunotherapy selected from an agent that modulates one or more of programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), inducible T-cell costimulatory molecule (ICOS), inducible T-cell costimulatory molecule ligand (ICOSL), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0165] In embodiments, the methods and uses of the present disclosure include the use of a composition comprising an IAP and / or one or more CPIs (and / or additional therapeutic agents) as an adjuvant to any of these initial and / or adjunctive therapies (including concomitant or sequential administration). In embodiments, the methods and uses of the present disclosure include administration of a composition comprising one or more CPIs (and / or additional therapeutic agents), including, but not limited to, an agent that modulates said IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, as described herein, to a subject undergoing initial and / or adjunctive therapy.

[0166] In embodiments, the IAP is recombinant bIAP II containing the stop codon annotated in SEQ ID NO: 11 and lacking a leader sequence. In embodiments, the IAP is produced in a mammalian cell line, e.g., a non-bovine cell line such as a CHO cell. In embodiments, the IAP is glycosylated on one or more residues. In embodiments, the IAP is terminally sialylated on one or more glycosylated structures. In embodiments, the IAP has structural / functional characteristics that allow it to be effectively administered at lower doses than required for other APs.

[0167] In embodiments, an IAP of the present disclosure is administered in combination with one or more additional therapeutic agents, for example, as described herein. The combination administration can be simultaneous or sequential. In embodiments, an IAP of the present disclosure can be administered with or without an additional agent. In embodiments, the methods and uses of the present disclosure include the use of an IAP as an adjuvant (including combined or sequential administration) to any of these initial and / or adjunctive therapies. In embodiments, the methods and uses of the present disclosure include administration of an IAP described herein to a subject undergoing initial and / or adjunctive therapy.

[0168] In embodiments, the method comprises administering an IAP formulated for release substantially within the GI tract. In embodiments, the method comprises administering an IAP formulated for release substantially within the small intestine. In embodiments, the method comprises administering an IAP formulated for release substantially within the large intestine. In embodiments, the method comprises administering an IAP formulated for substantially no systemic release. In embodiments, an IAP formulated for substantially no systemic release represents no detectable IAP found in biological samples such as blood, serum, etc., except for stool and intestinal luminal samples.

[0169] In embodiments, the method comprises administering a recombinant IAP having an amino acid sequence having about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% or more sequence identity to any one of SEQ ID NOs: 1-14.

[0170] In an embodiment, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 15 mg of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof). The capsule contains multiple enteric-coated pellets containing the AP-based drug. In such an embodiment, the formulation contains about 10.0% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof); about 38.9% by weight of sucrose spheres; about 20.0% by weight of hydroxypropyl cellulose (HPC); about 0.3% by weight of a buffer salt; about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55), and about 4.5% by weight of HTP-20 (e.g., PLASACRYL HTP 20).

[0171] In an embodiment, the formulation of the present invention is in the form of a capsule (e.g., a hard gelatin or HPMC capsule) containing about 5 mg of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof). The capsule contains multiple enteric-coated pellets containing the AP-based drug. In such an embodiment, the formulation contains about 10.0% by weight of the AP-based drug (e.g., an IAP, or other AP-based drug, drug described herein, and variants thereof); about 38.9% by weight of sucrose spheres; about 20.0% by weight of hydroxypropyl cellulose (HPC); about 0.3% by weight of a buffer salt; about 26.3% by weight of an enteric polymer (e.g., EUDRAGIT L30 D-55), and about 4.5% by weight of HTP-20 (e.g., PLASACRYL HTP 20).

[0172] In embodiments, the one or more pharmaceutically acceptable excipients comprise about or at least about 1%-10% glyceryl monostearate (GMS), triethyl citrate (TEC), and / or polymethacrylate copolymers. In embodiments, the capsule comprises gelatin or hydroxypropyl methylcellulose.

[0173] In embodiments, the IAP and / or additional agent is administered in combination with one or more additional agents, e.g., intended to address a symptom of an IAP or a disease or disorder treated by an IAP, as described herein.

[0174] In embodiments, the terms "patient" and "subject" are used interchangeably. In embodiments, the subject and / or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or a non-human primate such as a monkey, chimpanzee, or baboon. In embodiments, the subject and / or animal is a non-mammal, e.g., a zebrafish, zebra finch, turtle, or iguana.

[0175] In embodiments, the methods of the present disclosure are useful in treating a human patient. In embodiments, the human is a pediatric human. In embodiments, the human is an adult human. In embodiments, the human is an elderly human. In embodiments, the human is a female. In embodiments, the human is a male.

[0176] In certain embodiments, the human patient is of the age of less than 1 year old to about 1 year old, including newborns, 1 year to about 18 months old, about 18 months to about 36 months old, about 1 year to about 5 years old, about 5 years to about 10 years old, about 10 years to about 15 years old, about 15 years to about 20 years old, about 20 years to about 25 years old, about 25 years to about 30 years old, about 30 years to about 35 years old, about 35 years to about 40 years old, about 40 years to about 45 years old, about 45 years to about 50 years old, about 50 years to about 55 years old, about 55 years to about 60 years old, about 60 years to about 65 years old, about 65 years to about 70 years old, The age ranges are about 70 to about 75 years old, about 75 to about 80 years old, about 80 to about 85 years old, about 85 to about 90 years old, about 90 to about 95 years old, about 95 to about 100 years old, about 100 to about 105 years old, about 105 to about 110 years old, about 110 to about 115 years old, about 115 to about 120 years old, about 120 to about 125 years old, about 125 to about 130 years old, about 130 to about 135 years old, about 135 to about 140 years old, about 140 to about 145 years old, and about 145 to about 150 years old.

[0177] (Additional and combination therapies) The compositions and formulations, including compositions comprising one or more CPIs, including, but not limited to, an agent that modulates one or more of the IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, may be administered in combination with an additional therapeutic agent. The combined administration of the additional therapeutic agent and the compositions / formulations may be simultaneous or sequential. Furthermore, the compositions / formulations may include an additional therapeutic agent (e.g., in a combination formulation). For example, the additional therapeutic agent and a composition comprising one or more CPIs, including, but not limited to, an agent that modulates one or more of the IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, may be combined in a single formulation. Alternatively, the additional therapeutic agent and a composition comprising one or more CPIs, including, but not limited to, an agent that modulates one or more of the IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, may be formulated separately.

[0178] In one embodiment, the additional therapeutic agent and a composition comprising one or more CPIs, including but not limited to, an agent that modulates an IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, are administered to a subject simultaneously. As used herein, the term "simultaneously" refers to administration of the additional therapeutic agent and a composition comprising one or more CPIs, including but not limited to, an agent that modulates an IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, at intervals of about 60 minutes or less, such as 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 a composition comprising one or more CPIs, including but not limited to, an agent that modulates one or more of an IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4 can be by simultaneous administration in a single formulation (e.g., a composition comprising the additional therapeutic agent and one or more CPIs, including but not limited to, an agent that modulates one or more of an IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4) or in separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising a composition comprising one or more CPIs, including but not limited to, an agent that modulates one or more of an IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4).

[0179] In embodiments, the additional therapeutic agent and a composition comprising one or more CPIs, including but not limited to, an agent that modulates one or more of an IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, are administered to a subject simultaneously, although release of the additional therapeutic agent and a composition comprising one or more CPIs, including but not limited to, an agent that modulates one or more of an IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4 from their respective dosage forms (or, if combined, a single unit dosage form) may occur sequentially.

[0180] When the timing of their administration is such that the pharmacological activities of the additional therapeutic agent and the composition comprising one or more CPIs, including but not limited to, an agent that modulates an IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4 overlap in time, co-administration does not require that the additional therapeutic agent and the composition comprising one or more CPIs, including but not limited to, an agent that modulates an IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, be administered simultaneously. For example, the additional therapeutic agent and the composition comprising one or more CPIs, including but not limited to, an agent that modulates an IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, can be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the composition comprising one or more CPIs, including but not limited to, an agent that modulates an IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, are administered more than about 60 minutes apart. For example, the time between the sequential administration of the additional therapeutic agent and the composition comprising one or more CPIs, including but not limited to, an agent that modulates an IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, is 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 apart. The optimal administration time will depend on the rate of metabolism, excretion, and / or pharmacodynamic activity of the additional therapeutic agent and the composition comprising one or more CPIs, including, but not limited to, an agent that modulates one or more of IAP and / or PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, being administered.The additional therapeutic agent and a composition containing one or more CPIs, including but not limited to, an agent that modulates the IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, may be administered first.

[0181] Co-administration also does not require that the additional therapeutic agent and the composition comprising one or more CPIs, including, but not limited to, an agent that modulates an IAP and / or one or more of PD-1, PD-L1, PD-L2, ICOS, ICOSL, and CTLA-4, be administered to the subject by the same route of administration. Rather, each therapeutic agent can be administered by any suitable route, such as parenterally or non-parenterally.

[0182] In embodiments, the additional therapeutic agent is a corticosteroid. In embodiments, the additional therapeutic agent is an agent that targets tumor necrosis factor alpha (TNF-α).

[0183] In embodiments, the additional therapeutic agent is a cephalosporin antibiotic (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); a fluoroquinolone antibiotic (cipro, levaquin, floxin, tequin, avelox, and norfloxacin); lox); 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 embodiments, the antibacterial agent can be, but is not limited to, a penicillin antibiotic, a cephalosporin antibiotic, a monobactam antibiotic, or a carbapenem antibiotic.

[0184] Non-limiting examples of additional therapeutic agents include analgesics such as nonsteroidal anti-inflammatory drugs, corticosteroid anti-inflammatory drugs, antipruritics / topical analgesics, opiate agonists, and salicylates; anti-infectives such as anthelmintics, antianemic drugs, antibiotics, aminoglycosides, antifungals, cephalosporins, macrolides, mixed B-lactam antibiotics, penicillins, quinolones, sulfonamides, tetracyclines, antimycobacteria, antituberculous antimycobacteria, antiprotozoans, antimalarials, antivirals, antiretrovirals, and scabicides; acidifiers, alkalinizers, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, and electrolyte replenishers. enzymes such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal drugs such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory drugs, salicylate gastrointestinal anti-inflammatory drugs, antacid antiulcer drugs, gastric acid pump inhibitor antiulcer drugs, gastric mucosal antiulcer drugs, H2 blocker antiulcer drugs, gallstone dissolvers, digestive drugs, emetics, laxatives and stool softeners, and prokinetic drugs; gastrointestinal hormones and hormone modulating agents such as GLP-1, GLP-2, abortifacients, adrenal agonists, corticosteroids, adrenal agonists, androgens, antiandrogens; nutritional fortifiers such as minerals and / or vitamins such as water soluble or fat soluble vitamins, vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and / or vitamin K, and metabolic carbohydrates, amino acids, and / or fats.

[0185] In embodiments, the one or more additional therapeutic agents include fecal microbiota, e.g., from a fecal transplant to repopulate the gut microbiota for therapeutic purposes. In embodiments, the one or more additional therapeutic agents include any therapeutic agent approved for one or more diseases and / or disorders described herein.

[0186] (definition) As used herein, "a," "an," or "the" may mean one or more than one.

[0187] Furthermore, when used in connection with a referenced numerical indication, the term "about" means the referenced numerical indication plus or minus up to 10% of the referenced numerical indication. For example, the term "about 50%" covers a range of 45% to 55%.

[0188] When used in the context of medical applications, an "effective amount" is an amount effective to obtain a measurable treatment, prevention, or reduction in the rate of pathogenesis of the disorder of interest.

[0189] As referred to herein, unless otherwise specified, all composition percentages are percentages based on the weight of the total composition. As used herein, the word "include" and its variations are intended to be non-limiting, and the description of items in a list does not exclude other similar items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and their variations are intended to be non-limiting, and a description that an embodiment can or may include certain elements or features does not exclude other embodiments of this technology that do not include these elements or features.

[0190] As used herein, something is "decreased" if the readout of the activity and / or effect is reduced by a significant amount, including 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, less, and at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of skill in the art, in some embodiments, activity is decreased and some downstream readouts are decreased while others are increased.

[0191] Conversely, activity is "increased" if the activity and / or effect readout increases by a significant amount, e.g., by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 100% in the presence of an agent or stimulus relative to the absence of such agent or stimulus, including by about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, or about 50-fold or more.

[0192] As referred to herein, unless otherwise specified, all composition percentages are percentages based on the weight of the total composition. As used herein, the word "include" and its variations are intended to be non-limiting, and the description of items in a list does not exclude other similar items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and their variations are intended to be non-limiting, and a description that an embodiment can or may include certain elements or features does not exclude other embodiments of this technology that do not include these elements or features.

[0193] As synonymous with terms such as including, containing, or having, the open-ended term "comprising" is used herein to describe the disclosure, the present disclosure, or embodiments thereof, or may be described using alternative terms such as "consisting of" or "consisting essentially of."

[0194] As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that may provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same 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.

[0195] The amount of a composition described herein required to achieve a therapeutic effect may be empirically determined according to conventional procedures for a particular purpose. Generally, for administering a therapeutic agent (e.g., a microbiota-modulating agent and / or an additional therapeutic agent described herein) for therapeutic purposes, the therapeutic agent is given at a pharmacologically effective dose. A "pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount," or "effective amount" refers to an amount capable of achieving a desired physiological effect or desired result, particularly for treating a disorder or disease. As used herein, an effective amount may include, for example, an amount sufficient to delay the onset of symptoms of a disorder or disease, alter the course of symptoms of a disorder or disease (slow the progression of symptoms of a disease), reduce or eliminate one or more symptoms or signs of a disorder or disease, and reverse symptoms of a disorder or disease. A therapeutic effect may also include halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is achieved.

[0196] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates, or experimental animals to determine, for example, the LD50 (the dose lethal to approximately 50% of the population) and the ED50 (the dose therapeutically effective in approximately 50% of the population). Dosages may vary depending on the dosage form used and the route of administration used. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In embodiments, compositions and methods exhibiting large therapeutic indices are preferred. Therapeutically effective doses can be initially estimated from in vitro assays, for example, cell culture assays or measurements of methane production in fecal samples. Further, doses can 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 composition in plasma can be measured, for example, by high-performance liquid chromatography. The effects of any particular dosage can be monitored by an appropriate bioassay. The dosage can be determined by a physician and adjusted, if necessary, to accommodate the observed therapeutic effect.

[0197] In certain embodiments, the effect will result 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 embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. A therapeutic effect also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether an improvement is achieved.

[0198] 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 composition for use and / or use in the manufacture of a medicament to treat a disease or disorder described herein.

[0199] Hereinafter, the present disclosure will be described in more detail with reference to examples. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention. In addition, various modifications and variations can be made without departing from the technical scope of the present invention. [Example]

[0200] Example 1: Treatment of gastrointestinal side effects from immune checkpoint inhibitors (CPIs) A clinical trial of treatment for CPI-mediated gastrointestinal side effects will be conducted. The trial will include subjects undergoing treatment with immune checkpoint inhibitor immunotherapy, where the therapy includes an agent that modulates programmed cell death protein 1 (PD-1) (e.g., an antibody or antibody format specific for PD-1, e.g., nivolumab, pembrolizumab, or pidilizumab), an agent that modulates programmed cell death ligand 1 (PD-L1) (e.g., an antibody or antibody format specific for PD-L1, e.g., BMS-936559, atezolizumab, avelumab, or durvalumab), or an agent that modulates programmed cell death ligand 2 (PD-L2) (e.g., In some embodiments, the therapeutic agent is selected from one or more of: an agent that modulates inducible T-cell costimulatory molecule (ICOS) (e.g., an antibody or antibody format specific for PD-L2), an agent that modulates inducible T-cell costimulatory molecule ligand (ICOSL) (e.g., an antibody or antibody format specific for ICOSL), and an agent that modulates cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (e.g., an antibody or antibody format specific for CTLA-4, e.g., tremelimumab or ipilimumab).

[0201] Any of the subjects may be cancer patients, including basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain cancer and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including GI cancer); glioblastoma; hepatic carcinoma; hepatoma; carcinoma in situ; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer cancer); lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma; and B-cell lymphomas (low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and vascular proliferative disorders associated with phacomatosis; edema (such as that associated with brain tumors), or Meigs' syndrome.

[0202] Subjects are divided into one or more test groups and a control group, both of which are administered an immune checkpoint inhibitor immunotherapy, and the one or more test groups are further orally administered a therapeutically effective amount of an IAP having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:11 before or after administration of the immune checkpoint inhibitor immunotherapy.

[0203] Subjects are monitored for gastrointestinal side effects, diarrhea and / or colitis due to the CPI, for example: Anti-PD-1 / anti-CTLA-4: Inflammatory infiltrate in the lamina propria containing lymphocytes, neutrophils, eosinophils, and plasma cells; neutrophilic crypt abscess formation; increased apoptotic activity within the crypt epithelium; crypt epithelial atrophy and crypt shedding. Chronic inflammatory changes including crypt twisting, basal plasmacytosis, and Paneth cell metaplasia. Granulomas. Lymphocytic colitis and collagenous colitis. Anti-PD-1: acute colitis; chronic colitis (basal lymphoplasmacytosis and abnormal crypt architecture, Paneth cell metaplasia); crypt abscesses; apoptosis; inflammatory infiltrate in the lamina propria containing lymphocytes, neutrophils, eosinophils, and plasma cells. Lymphocytic colitis and collagenous colitis. Anti-CTLA-4: acute colitis; chronic colitis (basal lymphoplasmacytosis and abnormal crypt architecture, Paneth cell metaplasia); neutrophilic inflammation only; lymphocytic inflammation only; combined neutrophilic and lymphocytic inflammation; intraepithelial neutrophilic lymphocytes; crypt inflammation; crypt abscesses; apoptosis; inflammatory infiltrate in the lamina propria composed of lymphocytes, neutrophils, eosinophils, and plasma cells; granulomas. Lymphocytic colitis.

[0204] The test group will show improvement over the control group, for example, one or more of the following is expected: Reduction or prevention of one of the gastrointestinal side effects of CPIs listed above. Prevention or reduction of diarrhea and / or colitis. · Reduction in the dose or frequency of corticosteroid administration required to treat mediated GI side effects. Eliminate the need for corticosteroids to treat GI side effects. A reduction in the dose or frequency of administration of one or more agents targeting TNF-α required to treat mediated GI side effects. Eliminate the need for administration of one or more drugs targeting TNF-α for the treatment of GI side effects. · Extending the therapeutic range of CPIs.

[0205] equivalent While the present disclosure has been described in connection with particular embodiments thereof, it will be understood that further modifications are possible and that this application is intended to cover any variations, uses, or adaptations of the present disclosure generally in accordance with the principles of the disclosure and within the scope of known or customary practice in the art to which the disclosure pertains, as may be applied to the essential features described above, and including such departures from the disclosure, in accordance with the scope of the appended claims.

[0206] 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 by the following claims.

[0207] Incorporation by Reference All patents and publications referenced herein are hereby incorporated by reference in their entirety.

[0208] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0209] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of any individual section may be equally applicable to all sections.

Claims

1. 1. A method for preventing or reducing gastrointestinal side effects from an immune checkpoint inhibitor (CPI) in a patient in need thereof, comprising administering intestinal alkaline phosphatase (IAP) to said patient; wherein the patient is undergoing treatment with an immune checkpoint inhibitor immunotherapy selected from agents that modulate one or more of programmed death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), inducible T-cell costimulatory molecule (ICOS), inducible T-cell costimulatory molecule ligand (ICOSL), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

2. 1. A method for preventing gastrointestinal (GI) side effects from an immune checkpoint inhibitor (CPI) in a patient in need thereof, comprising administering to the patient a CPI selected from an agent that modulates intestinal alkaline phosphatase (IAP) and one or more of programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2), inducible T-cell costimulatory molecule (ICOS), inducible T-cell costimulatory molecule ligand (ICOSL), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

3. 3. The method of claim 2, wherein the IAP is administered before the CPI.

4. The method of any one of claims 1 to 3, wherein the CPI-induced GI side effect is diarrhea and / or colitis.

5. The method according to any one of claims 1 to 4, wherein said IAP is selected from human IAP and calf / bovine IAP.

6. 6. The method of any one of claims 1 to 5, wherein the IAP comprises an amino acid sequence having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100% identity to any one of SEQ ID NOs: 1 to 6 or SEQ ID NOs: 10 to 14.

7. 7. The method of any one of claims 1 to 6, wherein the IAP comprises an amino acid sequence having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100% identity to SEQ ID NO:

2.

8. 8. The method of any one of claims 1 to 7, wherein the IAP comprises an amino acid sequence having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100% identity to SEQ ID NO:

11.

9. The method according to any one of claims 1 to 8, wherein the IAP is administered orally.

10. The method of any one of claims 1 to 9, wherein the IAP is formulated for GI release.

11. The method of any one of claims 1 to 10, wherein the agent that modulates PD-1 is an antibody or antibody format that is specific for PD-1.

12. 12. The method of claim 11, wherein the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, a single-chain Fv, a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

13. 12. The method of claim 11, wherein the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.

14. The method of any one of claims 1 to 13, wherein the agent that modulates PD-L1 is an antibody or antibody format that is specific for PD-L1.

15. 15. The method of claim 14, wherein the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

16. 15. The method of claim 14, wherein the antibody or antibody format specific for PD-L1 is selected from BMS-936559, atezolizumab, avelumab, and durvalumab.

17. The method of any one of claims 1 to 16, wherein the agent that modulates PD-L2 is an antibody or antibody format that is specific for PD-L2.

18. 18. The method of claim 17, wherein the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, a single-chain Fv, a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

19. 19. The method of any one of claims 1 to 18, wherein the agent that modulates ICOS is an antibody or antibody format that is specific for ICOS.

20. 20. The method of claim 19, wherein the antibody or antibody format specific for ICOS is selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

21. 20. The method of claim 19, wherein the antibody or antibody format specific for ICOS comprises JTX-2011.

22. 22. The method of any one of claims 1 to 21, wherein the agent that modulates ICOSL is an antibody or antibody format that is specific for ICOSL.

23. 23. The method of claim 22, wherein the antibody or antibody format specific for ICOSL is selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

24. The method of any one of claims 1 to 23, wherein the agent that modulates CTLA-4 is an antibody or antibody format that is specific for CTLA-4.

25. 25. The method of claim 24, wherein the antibody or antibody format specific for CTLA-4 is selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

26. 25. The method of claim 24, wherein the antibody or antibody format specific for CTLA-4 is selected from tremelimumab and ipilimumab.

27. The method according to any one of claims 1 to 26, wherein the patient is a cancer patient.

28. The cancers include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain cancer and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; Head and neck cancer; gastric cancer (including GI cancer); glioblastoma; hepatic carcinoma; hepatoma; carcinoma in situ; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; Cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle 28. The method of any one of claims 1 to 27, wherein the leukemia is selected from: hairy cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorders (PTLDs), and vascular proliferative disorders associated with phacomatosis; edema (such as that associated with brain tumors), and Meigs' syndrome.

29. 29. The method of any one of claims 1 to 28, wherein the method further comprises administering one or more corticosteroids.

30. 30. The method of any one of claims 1 to 29, wherein the IAP treatment reduces the dose or frequency of corticosteroid administration required to treat the mediated GI side effect.

31. 31. The method of any one of claims 1 to 30, wherein the IAP treatment obviates the need for corticosteroid administration for the treatment of GI side effects.

32. 32. The method of any one of claims 1 to 31, further comprising administering one or more agents that target tumor necrosis factor alpha (TNF-α).

33. 33. The method of any one of claims 1 to 32, wherein said IAP treatment reduces the dosage or frequency of administration of one or more agents targeting TNF-α required to treat the mediated GI side effect.

34. 34. The method of any one of claims 1 to 33, wherein said IAP treatment obviates the need for administration of one or more agents that target TNF-α for the treatment of GI side effects.

35. The method of any one of claims 31 to 33, wherein the agent targeting TNF-α is an antibody or a fusion protein.

36. 35. The method of claim 34, wherein the TNF-α targeting agent is infliximab (Remicade), infliximab-dyyb (Inflectra), infliximab-abda (Renflexis), or Flixabi.

37. 37. The method of any one of claims 1 to 36, wherein the method increases the therapeutic window of the CPI.

38. 38. The method of any one of claims 27 to 37, wherein the IAP does not interfere with cancer treatment in the patient.

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